WELCOME, EVERYBODY.
I'M DAN GALLAHAN THE DIRECTOR OF
THE DIVISION OF CANCER BIOLOGY.
ONE OF THE FOUR MAJOR EXTRAMURAL
DIVISIONS WITHIN THE NCI.
ON BEHALF OF MYSELF, OUR
DIRECTOR, MONICA BERTAGNOLLI WHO
WILL REMAIN OUR DIRECTOR FOR A
WHILE AND THANK YOU FOR
ATTENDING HERE AND VIRTUALLY.
I THINK THIS IS AN EXCITING
MEETING.
I'M GLAD YOU CAN MAKE IT AND
GIVE THESE OPENING REMARKS.
I WASN'T SO SURE.
IT WASN'T THE FACT THAT I WAS IN
IRELAND YESTERDAY.
IT WAS THE FACT I WAS ON 270 FOR
THOSE LOCAL THIS MORNING AND
THERE WAS A LOT OF TRAFFIC BUT
PERSEVERED.
SO ANYWAY, I WANT TO AND I'LL
THANK ANDY FEINBERG FOR SETTING
THE FIELD FOR THIS AND ALLOWING
US TO COME TOGETHER IN THE
MEETING AND THE ORGANIZATION OF
THAT.
WHAT I'D LIKE TO DO I ALREADY
WELCOMED EVERYBODY AND THANK YOU
FOR ATTENDING.
I THINK IN AN AREA THAT I THINK
HAS HAD GREAT IMPACT ALREADY AND
I THINK IS GOING TO REMAIN TO
HAVE IMPACT AND I THINK THE MORE
MEETINGS LIKE THIS WILL BRING IT
MORE FROM UNDERSTANDING OF
EPIGENETICS AND EPIGENETICS TO
TRANSLATIONAL DRUGS.
A YEAR AGO WE FINISHED A 50 YEAR
ANNIVERSARY FOR THE NCI.
THAT MARK WHERE WE ACHIEVED A
LOT BUT AN A LOT WELL STILL TO
BE DONE.
AT THAT TIME I REMEMBER IN TERMS
OF WHERE WE WERE AT AND I WAS
THERE AND LET US IN THE
MOLECULAR AGE COME WOULD CALL IT
WHERE WE ARE STUDYING NOW AND
IDENTIFY GENES.
AND ABLE TO HAVE DISCOVERY OF ON
CA GENES AND TUMOR SUPPRESSER
GENES LEADING TO PERSONALIZED
MEDICINE WHERE THERE WAS A
COMMON DOGMA AT THE TIME FOR
CANCER WHERE WE HAD ONE GENE
ABERRATION, ONE CELL TRANSFORMED
AND THAT WAS CANCER AND IF WE
COULD CURE THAT, IF WE COULD
TREAT THAT OR ANY OTHER WAY
TREAT THAT MUTATION AND THAT
CELL WE COULD CURE CANCER.
AND OBVIOUSLY IT WASN'T THAT
SIMPLE.
WE LEARNED THERE'S THE TUMOR
ENVIRONMENT GROWING FROM ONE
CELL TO A SYSTEM AND THE MODERN
ERA BROUGHT IN NEW TECHNOLOGIES
AND ALSO GAVE US NEW INSIGHT AND
ALONG THIS PATH WE UNDERSTOOD
THAT IT WASN'T JUST SOMATIC AND
GERM LINE MUTATIONS BUT
EPIGENETICS AND OTHER
CONTROLLING EVENTS AND GENES WE
HAD TO PAY ATTENTION TO AS WE
WENT ALONG.
AT THE START OF THIS AND THIS
COINCIDES WITH THE 40 YEARS AND
ANDY FEINBERG OBSERVED THERE WAS
IN CERTAIN TUMORS THEY SAW A
CASE OF HYPOMETHYLATION AND
THERE WERE FOUR PAPERS
ASSOCIATION PAPERS IN 1983 HERE.
THERE WAS ANOTHER PAPER THEY
REFERENCED IN THAT PAPER THAT
SPOKE TO HYPOMETHYLATION -- I
THINK SOMEONE'S SUPPOSED TO BE
MUTING.
ANYWAY, SO ANYWAY, THIS SORT OF
BROUGHT EVERYONE'S ATTENTION TO
THE FACT THAT A FIELD OF
EPIGENETICS IN CANCER WOULD BE
FOREVER WEDDED TOGETHER
OBVIOUSLY, THAT SLIDE EVERYONE
SEES AND WE SEE IN PUB MED, I
DON'T KNOW, I CUT OFF THE Y
AXIS.
THERE WERE A LOT OF PUBLICATIONS
THAT EMANATED FROM THIS FIELD
DURING THIS PERIOD WHERE THE
STUDY OF HERITABLE CHANGES AND
GENE EXPRESSION OCCURRED
INDEPENDENT OF THE PRIMARY DNA
CONSEQUENCE AND STOOD IN THE
FACE OF CENTRAL DOGMA AND MADE
IT MORE COMPLICATED BUT AS
BIOLOGY IS COMPLICATED THERE'S
ALWAYS -- THE WAY I LOOK AT IT
THERE'S NEW OPPORTUNITIES FOR
NOT ONLY UNDERSTANDING BUT
TREATING THE DISEASE.
THIS IS FROM A REVIEW SHOWING
GENETIC MUTATIONS AND MODIFIERS
IN CANCER AND SPECIFIC GENES ARE
AFFECTING -- CAN AFFECT CERTAIN
PROCESSES CRITICAL IN CANCER.
EACH ONE OF THESE.
I HAD ANOTHER VERSION OF THE
SLIDE AND HALLMARKS OF CANCER IN
THE MIDDLE OF THIS BECAUSE I
THINK IT CAN AFFECT ALL THOSE
THINGS.
THE VERSION DIDN'T MAKE IT OUT
OF IRELAND.
SO ANYWAY, I THINK AS WE GO
FORWARD WE'RE INTO THE PHRASE
AND WE ALWAYS HAVE BEEN TO A
CERTAIN EXTENT OF TRANSLATIONAL
EFFORTS FROM EARLY USE OF DNA
METHYLATION AND LOOKING AT THE
HVAV INHIBITORS.
THIS LED US TO NOT ONLY DIRECTLY
BUT NOW AS A WAY USING THE
ADVANTAGE OF EPIGENETIC CONTROL
TO TRY TO DRUG WHAT WE
CONSIDERED AT THE TIME
UNDRUGGABLE PRODUCT AND HAVE
EPIGENETIC THERAPIES AND IT'S AN
AREA WE PUT GREAT HOPE IN IN
TERMS OF WHAT WE'LL BE ABLE TO
DO IN THE FUTURE AS WE
UNDERSTAND MORE OF THE PROCESSES
INVOLVED.
IN IRELAND I MENTIONED ABOUT
SYSTEMS BIOLOGY IN CANCER.
THE HTAN PROGRAM IS LOOKING AT
SPATIAL AWARENESS AMONG THE
ATLAS AND UNDERSTANDING THE
RELATIONSHIP BETWEEN CHANGES IN
CANCER AND HOW IT WILL BE
CRITICAL IN OUR UNDERSTANDING
LEADS TO CANCER TREATMENTS AS WE
GO FORWARD.
SO I THINK THIS HAS BEEN A WELL
DESIGN MEETING WITH ALL THE
IMPORTANT TOPICS.
MY CREDIT TO THE ORGANIZERS AND
ALL AND I THINK OF THE MAJOR
ISSUES AND THIS IS IN THE
MEETING AND THEMES AND GENETIC
LAND SCAPES AND TRANSLATIONAL
GENETICS AND WHERE ELSE ARE THEY
AFFECTED AND THE NEW FRONTIER.
I'M EXCITED ABOUT THIS MEETING.
I THINK IT'S SORT OF AMAZING AND
MY HATS OFF TO ANDY AND THE
OTHERS TO GET THE CALIBER OF
SPEAKERS WE HAVE IN THIS
ENCOURAGING AND GROWING FIELD OF
CANCER EPIGENETICS.
I ALREADY THANKED MONICA.
I'D LIKE TO THANK DENA SINGER
DEPUTY DIRECTOR AT THE NCI.
ANDY WORKED ON WITH DENA AND INE
-- IAN AND MY DEPUTY AND
ADMINISTRATIVELY AS WITH ALL OUR
MEETINGS THANK TIFFANY AND
TOMIKA FROM DCB FOR THIS.
PART OF MY JOB IS TO MAKE SURE
WE STAY ON TIME AND STEFON
DIDN'T TIME ME BUT THAT'S OKAY.
AND SO WITH THAT, I THINK OUR
FIRST SPEAKER IS ANDY FEINBERG.
>> THANK YOU VERY MUCH.
I APPRECIATE THIS AND ALSO THANK
NORM WHO SITTING -- SUGGESTED
THIS AND DENA AND I'M HUMBLED
THIS IS HAPPENING BUT GREAT TO
SEE COLLEAGUES FROM THE OLD DAYS
AND THE YOUNG PEOPLE DOING
AMAZING THINGS.
I'LL START WITH CANCER AS A
DISEASE OF EPIGENETIC PLASTICITY
AND GIVE AN EXAMPLE.
SO THIS IS WHAT WE LOOKED AT
BACK THEN AND INSIDE OF OUR
BRAINS -- I'M ONLY GOING TO
SPEAK FOR MYSELF BUT WHAT I
THINK WE STILL LOOK LIKE UNTIL
YOU LOOK IN THE MIRROR.
AND I WANTED TO SAY, I WANT TO
START BY THANKING HIM AND HOW
MUCH I VALUE HIM.
I WENT TO HIM TO DO A SECOND
POSTDOC AND WORKED IN
DEVELOPMENT BIOLOGY BEFORE AND
WANTED TO DO THIS EPIGENETIC AND
TAUGHT ME ABOUT MOLECULAR
BIOLOGY AND THE SECOND THING IS
I LOVE BURT AND OUR FAMILIES ARE
INCREDIBLY CLOSE.
I CAN'T IMAGINE A WORLD I WENT
THROUGH WITHOUT HIM THERE AND
EILEEN AND HIS KIDS, ONE OF WHOM
IS A COLLEAGUE OF MINE NOW,
JOSH, AND THIRD IS THIS IS A BIT
OF JEWISH HUMOR BUT I'M GRATEFUL
HE DIDN'T THINK WHAT I WORK ON
IS PARTICULARLY IMPORTANT.
WHAT I MEAN IS HE'S NOT BEEN ALL
THAT INTERESTED IN PURSUING THE
KIND OF EPIGENETIC HYPOTHESIS I
HAVE WHICH IS GREAT BECAUSE HE'S
SO GOOD AND THEN HAVE TO COMPETE
WITH HIM AS I WAS GOING ON MY
OWN WAY.
SO I WANT TO JUST SHOW A SLIDE
FROM A REVIEW I WROTE SEVERAL
YEARS AGO ON THE CLONAL GENETIC
MODEL OF CANCER.
SO THE EXTERNAL HYPOTHESIS WHICH
WAS THE REIGNING THING AND
THERE'S A SERIES OF GENETIC
MUTATIONS FROM A NORMAL CELL TO
BENIGN TUMOR AND BURT IS THE
CHAMPION OF THE MODEL AND
PROBABLY MADE THE LEADING
CONTRIBUTION TO INDIVIDUALIZED
THERAPY, TO MOST THE MEDICINES.
YOU MENTION THE EPIGENETIC
MEDICINES AND I HAVE A LOT OF
HOPE FOR THEM.
SOME ARE PRETTY USEFUL BUT IF
YOU THINK OF TARGETED MUTATION
DRUGS IT'S AMAZING AND BURT HAS
LED THE WAY THROUGH THIS.
WHAT I'VE BEEN INTERESTED IN
SINCE THE BEGINNING IS SOMEWHAT
DIFFERENT IDEA THAT CANCER IS A
DISEASE OF EPIGENETIC
PLASTICITY.
I USED TO GET ASKED ALL THE TIME
WHY DON'T YOU THINK GENETICS IS
IMPORTANT.
YOU HEARD ME NOT SAY THIS.
THIS IS ONE OF THE THING YOU
HAVE TO THINK AND INTEGRATING
THE TWO IS VERY IMPORTANT.
THERE ARE A COUPLE OTHER THINGS
I THINK ARE IMPORTANT I
SUGGESTED IN THIS PAPER.
ONE IS THAT THERE MIGHT BE GENES
THAT ARE PROGENITOR GENES FOR
CANCER.
THEY AREN'T MUTATED THAT ARE
ALTERED EPIGENETICALLY IN THE
NORMAL--MAYBE IN RESPONSE TO
INFLAMMATION OR INJURY AND SET
THE STAGE FOR CANCER TO EMERGE
LATER WHEN MUTATIONS TAKE PLACE.
BUT THAT CAN LEAD TO AN EXPANDED
EPIGENETIC PROGENITOR CELL AND
CAME FROM A DOUBLE DOSE OF IGF2
THE CAUSE OF CERTAIN LUMPS TUMOR
ASSOCIATED WITH KIDNEY
OVERGROWTH IN THE SYNDROME AND
YOU HAVE ONCA GENES AND TUMOR
SUPPRESSER GENES BUT IN LATER
STAGES THEY MAY REAR THEIR UGLY
HEADS GOOD-BYE AND LEAD TO
EPIGENETIC PLASTICITY AROUND HOW
DO YOU GET RESISTANCE TO
SOMETHING AND THEN YOU USE
ANOTHER DRUG AND GET RESISTANCE
TO THAT AND THEN YOU HAVE
SENSITIVITY AGAIN WITH THE FIRST
DRUG?
THERE'S AN EXAMPLE IN PANCREATIC
CANCER AND GET RESISTANCE.
THERE'S A SEQUEL PAPER WITH A
COLLEAGUE WHERE I SUGGESTED THE
CLASSIFICATION SCHEME YOU HAVE
EPIGENETIC MODULATORS THINGS
FROM THE ENVIRONMENT THAT AFFECT
THE GENES THAT CODE FOR THE
GENOME RELATED TO AGING AND
ENVIRONMENTAL FACTORS AND THEN
THE MODIFIERS.
THAT'S A NICE REVIEW.
THERE ARE MUTATIONS THAT OCCUR
IN MODIFIERS AND THE MOST COMMON
MUTATIONS IN CANCER FROM THE
CANCER GENOME ATLAS.
THEY MODIFY THE MODULATORS.
THEY DEFINE WHAT YOU CALL THE
EPIGENETIC LANDSCAPE AND DEFINE
THE PLURIPOTENCY MODIFIERS AND
THINGS LIKE DNTs AND THEY
DETERMINE THE LANDSCAPE AND THE
MEDIATORS ARE THINGS THAT I
SUGGESTED TUMOR PROGENITOR GENES
BUT ALSO THE PLURIPOTENT GENES
AND THE YAMIAKA FACTORS AND
FACTORS OF DIFFERENTIATION.
I GOT THIS IDEA.
THIS IS WHAT I'LL TALK ABOUT
MOSTLY.
I GOT THIS IDEA ABOUT YEARS AGO.
I WAS ACTUALLY -- WE JUST
PUBLISHED A PAPER ON THE FIRST
COMPREHENSIVE LOOK AT THE
EPIGENOME.
THERE WASN'T A WAY TO REALLY
MEASURE METHYLATION ACROSS THE
WHOLE GENOME EXCEPT FOR LIBRARY
OF CPGI LUNGS AND WE INVENTED
THIS RAY-BASED METHOD AND THIS
COMPARES NORMAL COLON TO COLON
CANCER AND I CAN'T POINT BECAUSE
I DON'T THINK PEOPLE ON TV CAN
SEE IT BUT ON THE LEFT HAND
PANEL THE LEFT SIDE HAVE THE
NORMAL COLONS AND THE RIGHT SIDE
ARE COLON CANCERS.
THERE'S GENES DOWN THE LEFT AND
THE SAMPLES ON THE X AXIS AND
BESIDES THE DIFFERENCES THERE'S
A HUGE DEGREE OF HETEROGENEITY
OR VARIABILITY IN THE CANCERS.
MAYBE THAT'S RELATED TO TUMOR
HETEROGENEITY.
WHAT'S AMAZING IS IF YOU DO WITH
THE TOP DIFFERENTIALLY METHYL
EIGHTED REGIONS AND DO TISSUES
OF SPLEEN, LIVER AND COLON THEY
DISCRIMINATE FROM EACH OTHER
WHICH MEANS IT'S SORT OF
SHOCKING THE VERY EPIGENETIC
CHANGES THAT TELL COLON CANCER
IT'S NOT NORMAL ARE THE SAME
GENES AND THE CANCER DMRs AND IN
WORK WITH GEORGE DAILY THEY'RE
THE SAME IN PROGRAMMING.
THE DIFFERENCES IN THE TISSUES
AND WITH CANCER INVOLVE KIND OF
THE SAME GENES IT'S JUST THE
QUESTION OF WHICH ONES AND WHICH
ONES TO DISTURB AND SO FORTH.
WHEN WE DID THIS WORK WE WERE
ASKED TO PROVE THERE'S INHERENT
VARIABILITY BY REVIEWERS AND
WENT BACK TO -- I SHOULD SAY,
YOU COULD SEE THIS IN THE NORMAL
TISSUE AS WELL THE VARIABILITY.
THIS IS UNSUPERVISED TISSUES OF
MICE FROM THE SAME LITTER AND
IDENTICAL GENOMES AND THE
TISSUES SEGREGATE COMPLETELY.
NOT THE SPECIES THOUGH WE'RE 50
MILLION YEARS A PART.
IF YOU LOOK THERE AT THE ONES
LABELLED THE MOUSE, THERE'S
ENORMOUS VARIABILITY IN THE
GENES DETERMINING THE
DIFFERENCES FROM ONE TISSUE FROM
ONE MOUSE TO ANOTHER THAT'S ISO
GENIC.
IN OTHER WORDS THEY HAVE
INHERENT VARIABILITY AND WE CALL
THEM VMRs, VARIABLY METH LATED
REGIONS AND IS THERE A CONSERVED
DRUG FOR EPIGENETIC VARIABILITY
AND THE REASON WE WERE ABLE TO
DO THE NEW ASSAYS WE GOT A GRANT
FROM THE NIH WHICH I SHOULD SAY
WE HAVE ALL THESE THANKS FOR
CONTRIBUTIONS FROM PEOPLE AND
THE VALUE OF NIH AND ALLOWING US
TO DO WHAT WE DO IS SO DEEP AND
I'M SO GRATEFUL.
WE HAD THIS GENOME CENTER TO
DEVELOP THESE TECHNIQUES AND THE
PURPOSE OF THE GENOME CENTER WAS
TO EXPLORE, HOW'S EPIGENETICS
INVOLVED IN COMMON DISEASES.
THE COMMON VARIANCE THAT WE'RE
HYPOTHESIZED TO CONTROL HUMAN
DISEASE OCCUR RELATIVELY
COMMONLY BUT CONTRIBUTE
RELATIVELY LITTLE TO THE OVER
ALL EXPLAIN VARIANTS OF COMMON
DISEASE.
EVEN NOW AFTER ALL THE WORK DONE
MY FAVORITE IS THIS ONE FROM
BURT AND JOSH, HIS SON, SHOWED
THAT BASED ON TWIN STUDIES THAT
EVEN IF YOU KNEW EVERYTHING
ABOUT BIOLOGY AND HAD THE
COMPLETE SEQUENCE OF A PERSON
YOU CAN ONLY PREDICT 20% OF THE
RISK OF DISEASE.
AND THE ENVIRONMENT INDUCES A
CHANGE.
INDUCES A CHANGE THAT'S GOING TO
GIVE YOU A SELECTIVE ADVANTAGE
THAT'S GOING TO BE TRANSMITTED
EPIGENETICALLY FROM GENERATION
TO GENERATION AND SHOULD BE
SPORADIC AND RESPONSE TO STRESS.
WHILE THERE'S VARIANTS ACROSS
GENERATIONS THEY'RE DELETERIOUS
AND THAT MAKES FUN OF LAMAR
BECAUSE HE SAID THE FOREST
TURNED INTO A GIRAFFE BECAUSE HE
HAD TO STRETCH TO GET THE LEAVES
OR SOMETHING LIKE THAT.
IF THAT'S NOT THE EXPLANATION
AND STILL DARWINIAN MAYBE WE
SHOULD THINK OF WHAT ELSE CAUSES
DISEASE.
SHE ENVIRONMENT LIKE SMOKING AND
THE SUN AND EATING CHEESE
BURGERS AND DRINKING ALCOHOL AND
THIS MAKES YOU WANT TO GO TO THE
BEACH BUT DON'T HAVE A CIGARETTE
WHEN YOU DO IT.
HOW IS THAT SOMEHOW DARWINIAN
AND I WOULD HAVE EPISODES OF
READING AT NIGHT AND STUFF LIKE
THAT SO I HAPPEN TO BE CHASING
DOWN SAMPLES I WANTED TO GET
FROM SOMEONE IN HOLLAND LIKE
TWINS.
THAT WAS UNSUCCESSFUL AND I FLEW
THERE THROUGH LONDON AND MY SON,
JASON, WHO I PUBLISHED WITH TOO
AND I'M HAPPY ABOUT, HE WAS
WORKING IN SPAIN AND HADN'T SEEN
LONDON SO WE SPENT A WEEKEND
THERE AS I WAS FLYING BACK FROM
HOLLAND ON MY UNSUCCESSFUL
MISSION AND WE LIKE TO GO TO
TALL PLACES AND GET A VIEW SO WE
WENT ON THE LONDON EYE AND WE
SAW BIG BEN AND SAID WE WANT TO
GO SEE THAT SO WE WALKED ACROSS
WESTMINSTER BRIDGE AND THERE'S A
SIGN AT BIG BEN AND THE BRITISH
ARE INSULTING IN A POLITE WAY
AND IT'S AN ART FORM AND IT SAYS
YOU'RE NOT WELCOME UNLESS YOU'RE
ONE OF SO AND IF SO GO AWAY.
NEXT DOOR IS WESTMINSTER ABBY
AND THIS IS NOVEMBER 2009 SAYING
THE 150th ANNIVERSARY OF ORIGIN
SPECIES COME IN AND SEE DARWIN
HE'S HERE.
SO WE WENT IN THERE INSTEAD AND
FOUND OURSELVES STANDING ON
DARWIN'S GRAVE AND NEXT TO
DARWIN'S GRAVE IS NEWTON'S
GRAVE.
NOW, THERE'S A VELVET ROPE SAME
KIND THAT WAS OUTSIDE OF BIG BEN
AND SAYS YOU MAY NOT CROSS BUT
YOU CAN PUT YOUR FILTHY FEET ON
DARWIN'S GRAVE, THAT'S FINE.
AND BEHIND NEWTON'S GRAVE
THERE'S THE CHERUBS AND BIG ARCH
AND NOTHING LIKE THAT FOR DARWIN
AND ON TOP OF NEWTON AND IT'S BY
THE FOUNDER OF A QUANTUM THEORY
AND A THOUGHT WHAT IS THERE AN
URN IN THERE OR BURIED STANDING
UP.
HE'S BURIED SOMEWHERE ELSE AND
NO DISRESPECT TO THE CHURCH BUT
IT'S A FUNNY THING TO SAY AND
THE EPIPHANY WAS WELL WE DON'T
HAVE SOMETHING LIKE THAT IN
BIOLOGY.
THE FOUNDER THIS WAS FLUID IN MY
HEAD OUT OF BODY EXPERIENCE AND
I WALKED WITH JASON A PUB NEXT
TO THE TOWER OF LONDON WHERE
THEY WOULD EXECUTE PEOPLE AND I
WROTE A PAPER BUT THE IDEA IS
THE GENETIC VARIATION SAY
DRIVING FORCE OF DEVELOPMENT OF
ADAPTATION AND IDEA THAT WOULD
FIT THE EVOLUTION OF COMPLEX
SPECIES.
STOCHASTIC VARIATION IS
EPIGENETICALLY BUT THE CONTROL
OF DEGREE -- CONTROLLED BY GENE
SEQUENCES AND GENETIC VARIANCES
THAT INDUCE PLASTICITY OVER
REVOLUTION FOR TRACE AFFECTED BY
A FLUCTUATING ENVIRONMENT.
IF YOU HAVE A LITTLE LESS
ROBUSTNESS BUT HAVE OFFSPRING
OPPOSITE TO THE TRAIT YOU HAVE
IT WON'T BE PASSED ALONG BUT IF
THE ENVIRONMENT SUDDENLY CHANGES
OVER ALL YOU'LL HAVE A SELECTIVE
ADVANTAGE AND WE PROVED THIS
RIGOROUSLY AND MATHEMATICALLY.
IT COULD BE IMPORTANT IN CANCER
PROGRESSION.
HERE ARE THE VARIABLY METHYLATED
REGIONS AND SEE THE HIGH DEGREE
OF VARIATION IN A PARTICULAR
REGION.
IF YOU ASK WHERE THEY ARE
THEY'RE EVERYTHING RELATED TO
DEVELOPMENT.
GUT, MORPH GENESIS AND THAT
MAKES SENSE WITH WHAT I SHOWED
YOU EARLIER FROM THE ARRAY BASED
PLOT AND A FRIEND OF MINE AT
UCSD PROVED THEY'RE TRANSMITTED
THROUGH MULTIPLE GENERATIONS SO
THEY DO EXIST.
A LOT OF PEOPLE STUDIED THEM
NOW.
WHILE THAT MIGHT BE IMPORTANT IN
CANCER AND ONE OF THE BIG THINGS
IS CYCLES OF INJURY AND REPAIR,
EXPOSURE, HEALING, EXPOSURE.
YOU CAN IMAGINE SELECTIVE
ENVIRONMENTAL PRESSURES AND THEN
THAT WILL SELECT SAY FOR
MUTATIONS OR NATIVE EPIGENETIC
CHANGES HAVING INHERENT
VARIABILITY AND WE DISCOVERED
THE MECHANISM FOR THIS.
SO WE DID THE FIRST SEQUENCING
OF PAIRED NORMAL AND CANCER FOR
COLON AND ONE THING WE SAW RIGHT
AWAY HAVE THE BLOCKS WHERE THE
NORMAL'S IN BLUE AND CANCER'S IN
RED AND LOOK HOW VARIABLE THE
CANCERS ARE.
THEY'RE NOT JUST BIG.
THAT'S WHAT WE SAW IN THE PAPER.
SOME ARE AS SMALL AS FIVE KB OR
THREE OR FOUR KB.
WITHIN THE REGIONS THERE'S
ISLANDS.
THEY TEND TO BE HYPERMETHYLATED
AND THIS IS A BAR CODE GENE
EXPRESSION PAIR SET.
THE MORE HIGHLY VARIABLY
EXPRESSED GENES IN THE GENOME
AND INVOLVED IN CANCER
PROGRESSION.
THE ENVIRONMENT ALSO CAN HAVE A
MAJOR AFFECT ON THE GENERATION
OF THESE REGIONS OF VARIABLE
METHYLATION WE LOOKED AT PEOPLE
OLDER OR SUN EXPOSED OR BOTH.
LIKE IN MEN THE LEFT ARM AND
WOMEN THE RIGHT ARM JUST BECAUSE
OF THE HABITS OF PEOPLE DRIVING.
IF YOU LOOK AT SPECIMENS YOU SEE
THE SAME THING ENVIRONMENTALLY
INDUCED.
THE AREAS OF HYPERVARIABILITY
THAT HAVE CHANGES IN ISLANDS AND
SHORES AND THE LONG REGIONS.
THAT'S A DISTRIBUTION OF
METHYLATION ON THE LEFT WITHIN
WHAT WE CALL THE PHOTO AGING
BLOCKS AND THE PINK IS THE
CANCERS.
THE EPIGENETIC CHANGES IN THE
CANCERS ARE AN ACCELERATED
VERSION OF WHAT HAPPENS AFTER
THE EXPOSURE AND OUTSIDE THE
BLOCKS YOU DON'T SEE THE SAME
THING.
HOW DO WE THEN MEASURE THIS
STOCHASTICITY.
I WENT BACK TO SCHOOL AND GOT A
PIONEER AWARDS AND THE FIRST
YEAR I SPENT MY 50% ON
STOCHASTIC MARK-UP PROCESSES AND
I DEVELOPED THIS MODEL WITH A
FRIEND IN THE COURSE AND YOU CAN
GENERATE TRUE STOCHASTIC MODELS
OF LANDSCAPES WHERE YOU CAN FOR
EXAMPLE SEE THE DIFFERENCE
BETWEEN MONOSTABLE METHYLATION
OR BISTABLE METHYLATION.
THERE'S THE SAME MEAN VALUE BUT
THERE'S A DIFFERENCE AND YOU
COULD NOT DETECT THAT USING
OTHER METHODS.
YOU CAN SEE AN EXAMPLE OF A
LANDSCAPE OF COLON NORMAL VERSUS
COLON CANCER AT THE ONE GENE AND
LOOK HOW FLAT THE LANDSCAPE IS
IN THE CANCER SHOWED ENTROPY AND
STEM CELL ON THE LEFT AND
STOCHASTIC AND IN BRAIN
DEVELOPMENT HAS BECOME MUCH LESS
RANDOM AND MEASURE OF ENTROPY.
I WANT TO SHOW HOW USEFUL THIS
IS FOR UNDERSTANDING CANCER.
THIS LOOKED AT ACUTE
LYMPHOBLASTIC LEUKEMIA AND HIGH
ENTROPY THROUGHOUT THE GENOME
DEPENDING ON DIFFERENT GENETIC
BACKGROUNDS BECAUSE THEY HAVE
VARIOUS KINDS OF CHROMOSOMAL
LOCATIONS LEADING TO ALL.
IF YOU LOOK AT ALL THE GENETIC
TYPES THEY CONVERGE ON A SET OF
GENES THAT ARE ENTROPIC AND
COMMON ACROSS THE GROUP.
AND IF YOU THEN LOOK AT THE
GENES AS SINGLE CELL GENE
EXPRESSION AND METHODS OF
MEASURING ENTROPY OPPOSED TO
MEAN EXPRESSION THEY FORM IN A
PARALLEL UNIVERSE SORT OF.
ENTROPY GENE EXPRESSION NETWORK
CONVENING ON A GENE NOT
DESCRIBED MUCH IN CANCER.
IT'S INVOLVED UP PROSTATE CANCER
BUT NOT OTHERS CALLED UFGR1
BETWEEN CHROMATIN AND WE'RE
FINDING A SIMILAR EXAMPLE IN AML
AND IT'S ANOTHER WAY WE CAN SEE
THIS HIDDEN EPIGENOME LOOK AT
STOCHASTICITY AND ANOTHER STUDY
I DID GENERATING XENO GRAPHS
FROM PATIENTS WITH PANCREATIC
CANCER AND THAT'D METASTASES
AFTER THE PATIENT PASSED AND YOU
CAN SEE WHAT GAVE RISE TO THIS
VERSUS THOSE THAT SHOWED LOCAL
SPREAD.
ON THE RIGHT IT SHOWS THERE'S
BLOG LIKE REGIONS OF VARIABLE
SIZE.
SOME SMALL AND SOME LARGE AND
YOU SEE THIS DISORDER CHROMATIN
AND METHYLATION.
THOSE ARE THE REGIONS THAT GIVE
RISE TO THE DISTANT METASTASES.
THERE'S A MECHANISM WE THINK
MIGHT BE DRIVING THESE BUT THESE
DON'T HAVE ADDITIONAL MUTATIONS
DRIVING THE METASTASES SO WE'RE
RIGHT ABOUT THIS IN SOME CASES
NOT BEING DRIVEN BY ADDITIONAL
MUTATIONS BUT BEING DRIVEN BY
EPIGENETIC CHANGE AND AROUND THE
EDGES OF THESE BLOCK LIKE
REGIONS THIS CAME OUT IN CANCER
RESEARCH FROM MY STUDENT AT THE
BOUNDARIES OF THESE THERE'S
TRANSCRIPTION FOR BINDER SITES
AND DOWN BELOW ARE THE GENES
THAT ARE MEDIATORS AND TUMOR
PROGENITOR GENES.
I'LL BE DONE IN A MINUTE.
THEY'RE THE THINGS THAT DRIVE
THE VMRs IN NORMAL DEVELOPMENT
AND IN CANCER AND IN ANOTHER
PAPER THEY CAME OUT THIS YEAR,
OUR COLLEAGUES IN BIO
STATISTICS, USING A
COMPUTATIONAL METHOD ON DATA
SETS LOOKED AT THE DNA
RESPONSIBLE FOR METHYLATION IN
THE FIRST PLACE WHICH HADN'T
BEEN ANSWERED AND SEQUENCE
VARIANTS IN THE BINDING SITES OF
TRANSCRIPTION FACTORS.
SO IT LOOKS LIKE THIS IS RELATED
TO TRANSCRIPTION REGULATORY
NETWORKS AND SUBTLE NETWORKS AND
HOW THEY BIND ACROSS THE GENOME
IN MANY SITES AND PARTICULARLY
IF THE BOUNDARIES BE REGULATING
NOT JUST INDIVIDUAL GENES BUT
BLOCKS OF GENES AND GLAD YOU
MENTIONED THERAPY.
I THINK IT'S RELEVANT.
SOME DRUGS BEING USED MAY BE
AFFECTING THE REGIONS MORE
BROADLY RATHER THAN SAY A
SPECIFIC GENE AND THERE'S
REASONS WE THINK THAT MIGHT BE
THE CASE.
JUST WORTH LOOKING AT.
AND SAME REGIONS CONFIRM GROWTH
ADVANTAGE AND RESISTANCE AND
MEDIATED BY THESE REGIONS.
THERE'S TRICKS YOU CAN USE TO
REVERSE THEM AND THAT GOES AWAY.
IT SEEMS TO UNDER LIE ENT
TRANSITION.
THE MODEL I WOULD HAVE AND I'M
GLAD YOU MENTIONED THE HALLMARKS
PAPER.
THE ORIGINAL DIDN'T MENTION
EPIGENETICS AND THE 10 YEAR
ANNIVERSARY MENTIONED IT TWICE
AND SAID WE DON'T THINK
EPIGENETICS IS PARTICULARLY
IMPORTANT AND SAID IT TWICE.
I THINK IT'S AT THE CENTER,
ACTUALLY, OF THE HALLMARKS.
I THINK WHAT HAPPENS IN THE
REVIEW I DID IS THAT THE
EPIGENETIC PLASTICITY AND I
GUESS MATHEMATICALLY ENTROPY
ALLOWS FOR SELECTION OF THESE
INDIVIDUAL TRAITS THAT ARE
CONSIDERED THE HALLMARKS NOT
THAT MUTATIONS CAN'T DO THAT
INDEPENDENTLY BUT IN TERMS OF
TUMOR PROGRESSION METASTASES THE
SELECTION FOR THESE TRAITS AND
CONTINUED SELECTION AS THE
ENVIRONMENT CHANGES AS THE CELL
METASTASESES DRIVES CANCER
PLASTICITY AND THE KEY THING WE
NEED TO ADDRESS WE HAVEN'T MET.
THE KEY THING I WANT TO SHOW IS
WHEN THAT MODEL CAME OUT I
SUGGESTED AN ART THING.
I MADE THIS AND MY MOM WAS AN
ARTIST.
A COUPLE COVERS I'VE HAD AND
ONLY THING I EVER DID SHE
UNDERSTOOD WERE THESE THINGS.
THIS IS FROM SOME FAMOUS PICTURE
AND HAVE PICTURES OF STOCHASTIC
THINGS AND MADE THIS OUT OF THIS
AND I SNUCK SOMETHING IN THERE.
IF YOU LOOK AT THE JOURNAL
YOURSELF YOU FIND IT AND YOU GO,
JUST THE LOWER LEFT OF THE
SECOND E THERE'S A PICTURE OF MY
WIFE PLAYING MAJONG WITH HER
FRIENDS AND THANK YOU VERY MUCH.
I APPRECIATE IT.
>> THANK YOU.
WE HAVE TIME FOR ONE QUESTION.
>> I'M LIKE THE MODERATOR SO I
COULDN'T TELL MYSELF TO SHUT UP
WHICH IS KIND OF UNIQUE.
JUST USE THE MIC.
>> THE DOUBLE CHANGES.
>> FOR PEOPLE ONLINE -- I'M
GOING TO DISAPPEAR FROM THE
SCREEN BECAUSE I'M GOING TO GO
OUT AND SHARE THE MIC WITH THE
PERSON SO I CAN HEAR THEM
BECAUSE THE ECHO IS SO MUCH IN
THE ROOM.
I'M COMING DOWN TO WHERE YOU
ARE.
>> YOU TALKED ABOUT CANCER HAS
ANYBODY LOOKED AT IS ANY PART OF
THAT EXPLAINED BY NON-CANCER
CELLS IN THE TUMOR
MICROENVIRONMENT OR 100%
CONTRIBUTED BY --
>> THAT'S A GOOD QUESTION.
I DON'T KNOW.
I THINK THERE'S BEEN PAPERS FROM
SUE CLARK ON THIS FOR EXAMPLE ON
FIBROBLAST AND WE'RE DOING PAPER
ON PANCREAS TO LOOK AT THIS IN
DETAIL AND MAYBE THE EXTRA
CELLULAR NOT THE CELLS BUT WE
HAVE DATA AND SOME CAME OUT IN A
RECENT PAPER THE EXTRA CELLULAR
ENVIRONMENT CAN CHANGE THE
EPIGENOME VISCOSITY.
WE'RE DOING OTHER WORK RELATED
TO THAT AND I THINK THE ANSWER
IS CLEARLY YES BUT HASN'T BEEN
PAID ENOUGH ATTENTION TO AND
IT'S A GREAT QUESTION.
THANK YOU.
SHOULD TALK TO THIS GUY ABOUT
WORKING YOUR SLIDES.
I'M REALLY BAD AT THAT.
>> THANK YOU, ANDY FOR THE
INTRODUCTION AND THANKS IAN AND
DAN FOR GIVING ME THIS
OPPORTUNITY.
I'M HONORED AND HUMBLED TO BE IN
THIS ROOM WITH PIONEERS IN THE
FIELD OF EPIGENETICS IN CANCER
RESEARCH.
SO WHAT I'M GOING TO SHARE WITH
YOU TODAY IS LARGELY TECHNOLOGY
RESEARCH IN MY LABORATORY.
THIS IS MY DISCLAIMER.
I WOULD LIKE TO EMPHASIZE MY
VERY EARLY MOMENT OF BIOMEDICAL
RESEARCH IN MY CAREER WHEN I
SWITCHED MY CAREER FROM ENERGY
SCIENCE AND PHYSICAL FUNDAMENTAL
PHYSICS TO BIOMEDICAL RESEARCH.
I WAS INSPIRED BY THE CANCER
BIOLOGY IN PARTICULAR TUMOR
MICRO ENVIRONMENT.
WHEN I READ THE ARTICLE IN 2006
REGARDING A COMPLEX OF TUMOR
MICRO ENVIRONMENT I REALIZED WE
NEEDED NEW TOOLS TO RESOLVE THE
MICROENVIRONMENT IN A SINGLE
RESOLUTION FOR THE CELLS.
AND IF YOU CAN'T MAP OUT THE
CELLS AND WHAT A THAT DO IN THE
TISSUE CONTEXT THAT WOULD BE A
HOME RUN.
LET'S DO THIS STEP BY STEP.
I DEVELOPED THE TECHNOLOGY WHILE
PROTEINS IS PRODUCED BY
DIFFERENT CELLS AND HOW THEY
INTERACT IN A TUMOR MICRO
ENVIRONMENT.
THAT WAS MY EARLY RESEARCH IN
THE IMMUNOINTERACTION AND IN THE
CONTEXT OF EME AND WE CONTINUED
TO DEVELOP NEW TOOLS AND ADAPT
NEW TOOLS IN A SPACE OF SINGLE
CELL BIOLOGY TO LOOK AT CANCER,
IMMUNOTHERAPY AND THE ONCOLOGY.
WE'RE STILL WORKING ON THAT.
YOU SEE THE NUMBER OF
PUBLICATIONS BUT NOW THOSE WILL
ADDRESS MY SECOND QUESTION, CAN
WE LOOK TO AT THE INTERACTION IN
THE CONTEXT OF THE
NATIVE--ENVIRONMENT.
SO PEOPLE REALIZE THAT CHALLENGE
ABOUT 10 YEARS AND DEVELOPED NEW
TECHNOLOGIES TO ADDRESS THIS
SPATIAL BIOLOGY OR SPATIAL
CANCER BIOLOGY.
SO BROADLY SPEAKING THERE'S TWO
DIRECTIONS TO DO THOSE.
MICROSCOPY IS PUSHING THE
BOUNDARY TO A LEVEL UNBELIEVABLE
AND IN THE PAST IF YOU IMAGED
TWO OR THREE PROTEIN MARKERS AND
MAYBE TWO OR THREE TO LOOK AT
DIFFERENT GENES YOU ARE ADVANCED
AND PEOPLE ARELIO JEALOUS YOU'RN
EXPECT AND YOU CAN IMAGE GENES
AND TO LOOK AT THE PROTEINS YOU
CAN EITHER USE
IMMUNOFLUORESCENCE AND A PAPER
IN GENE BIO TECHNOLOGY FROM A
GRUP IN AUSTRALIA THAT
DEMONSTRATE THEY CAN IMAGE UP TO
101 DIFFERENT PROTEIN MARKERS
SETTING A NEW WORLD RECORD IN
IMAGING.
WHAT I'M GOING TO TALK ABOUT IS
A DIFFERENT DIRECTION TO DO
SPATIAL OMICS BASED ON HIGH
THROUGHPUT SEQUENCING.
AND IN THE LANDMARK PAPER THAT
CAME OUT 2006 FROM A LAB IN
STOCKHOLM USING THE DNA MICROWAY
TO CAPTURE THE TISSUE SECTION
AND IF YOU LOOK AT THE TISSUE
SECTION YOU PLACE THE TISSUE
SECTION TO ABUT TRANSCRIPTS TO
BE RELEASED AND THEN CAPTURE THE
PLOTS AND EACH HAS A UNIQUE
SPATIAL ADDRESS CODE AND YOU DO
SYNTHESIS AND YOU'LL INCORPORATE
THE ADDRESS CODES AND WHEN YOU
WILL DECODE WHERE THE DIFFERENT
TRANSCRIPTS ARE COMING FROM IN
THE SPATIAL DEFINED MANNER AND
THE COMMERCIALIZED LABORATORIES
PUSHING THE BOUNDARIES TO PUSH
IT FROM THE BEGINNING IN A LAB
THAT WAS LIKE 100 MICRONS AND
THEY ALL FOLLOW THE SAME
FUNDAMENTAL AND LOOK AT SURFACES
FOR CAPTURE IN THE SPATIAL
TRANSCRIPTOMICS.
ONE OF MY LABORATORY CAME UP
WITH OPPOSITE.
AND THERE'S DIFFERENT WAYS BUT
WE'RE GOOD AT MICRO FLUID
DELIVERY AND LOOSE THE FLUIDIC
SYSTEMS TO DO BAR CODING AND
IT'S VERSATILE.
YOU CAN BAR CODE NOT JUST THE
TRANSCRIPT AND POTENTIALLY BAR
CODE EPIGENETICS.
THAT'S WHY I'M HERE TODAY.
WHEN WE PUBLISHED THAT PAPER IT
TENDED TO SHOW THE BAR CODING TO
LOOK AT WHOLE TRANSCRIPTOME AND
PROTEIN SETTINGS AND A LOT OF
RESEARCH GROUPS AROUND THE WORLD
WERE ABLE TO ADOPT THE
TECHNOLOGY AND SOME ARE ACTUALLY
EVEN DOING BETTER JOB MAKING
THIS MUCH HIGHER THROUGHPUT.
FOR EXAMPLE THE GROUP FROM
GERMANY IN MUNICH WERE ABLE TO
MAP OUT NINE DIFFERENT TISSUES
IN A SINGLE EXPERIMENT AND TAKE
ON THE MICROFLUIDIC TO CREATE
YOUR OWN MARK ARRAY FOR SPATIAL
ANALYSIS.
HOW THE TECHNOLOGY WORKS TO KEEP
IT BRIEF YOU USE THE MICROFLUID
DEVICE TO DELIVER DNA BAR CODES
ONE DIRECTION BUT IT DOES NOT
GIVE THE 2-D SPATIAL ADDRESS
CODE SO YOU USE ANOTHER
MICROFLUID ADVICE TO DELIVER IN
A HORIZONTAL DIRECTION AND BEAD
TOGETHER TO CREATE THE PIXEL
WITH THE COMBINATION AND THAT'S
HIGH LEVEL IDEA TO DO IN TISSUE
BAR CODING AND NOW IT DEPENDS ON
WHAT THEY'RE TRYING TO BAR CODE,
TRANSCRIPT PROTEINS OR SOMETHING
ELSE.
AND SO AFTER WE PUBLISHED THAT
PAPER I THINK A COUPLE MONTHS
LATER, SPATIAL TRANSCRIPTOMICS
WAS CHOSEN AND EVERYONE WAS SO
EXCITED ABOUT IT AND ALSO IN THE
SAME ISSUE OF PAPER AND METHODS
OUR PAPER WAS HIGHLIGHTED WITH
MULTIOMIC SPATIAL NOW.
I HAD TO CONFESS, THAT WAS BACK
TO THAT TIME WE WERE ABLE TO
DETECT PROTEINS IN CONJUNCTION
WITH TRANSCRIPTOME TO MY
STANDARD I WOULD NOT CALL THAT
PROTEOME AND WHETHER OR NOT YOU
CAN LOOK AT HUNDREDS OF PROTEINS
OR EVEN THOUSANDS OF PROTEINS IN
CONJUNCTION WITH
PROTRANSCRIPTOME AND TO
DEMONSTRATE HOW IT WORKS AND WE
WERE ABLE TO MAP 300 PROTEINS
FROM HUMAN TISSUES AND PIXEL BY
PIXEL COMPARING TISSUE SECTIONS
INCLUDING I THINK SOME NORM JULY
AND MALIGNANT TISSUE SAMPLES IN
HUMAN TUMORS.
HOWEVER, WHAT I WANT TO SHARE
WITH YOU IS WHAT WE BELIEVE THE
REAL POTENTIAL IS WAY BEYOND THE
SPATIAL GENE EXPRESSION ANALYSIS
TO SOME DEGREE IS STILL -- SO
MAJORITY OF THE WORK IN THE
SPACE MOST SPATIAL TECHNOLOGIES
IS TO MAPPING THE SPATIAL
AGAINST AND PROTEINS.
WE'RE VERY EXCITED HOW THE
EXPRESSION OF PROTEINS WAS
REGULATED IN AN EPIGENETIC LEVEL
AND THERE'S MANY REGULATIONS
FROM THE ENLIGHTENING TALK FROM
ANDY ABOUT THE MECHANISMS AND
HOW THEY'RE RELATED TO CANCER
AND DECIDED TO START WITH THIS
CHROMATIN ACCESSIBILITY AND
HISTO MODIFICATIONS AND WE
BELIEVE OUR APPROACH CAN BE
ADAPTED AND FURTHER MODIFIED TO
MAP SPATIAL METHYLOME AND 3-D
GENOME AND THERE'S ONGOING WORK
IN MY LABORATORY AND MY TRAINEES
LABORATORIES AND SO WE USED A
TAXIC DEVELOPED BY ELEGANT
CHEMISTRY TREATING THE ISOLATED
NUCLEI AND TRANS POSE THIS WITH
THE SEQUENCE AND IT WILL BE
INSERTED INTO GENOMIC DNA AND
CLAMP IT IN THE SEQUENCE AND
YOU'LL MAP WHERE THE RATINGS ARE
LOCATED IN THE GENOME.
BUT IF YOU DO THIS EXPERIMENT
PEOPLE ALWAYS ADVISE ALL RIGHT,
FIRST STEP IS THE NUCLEI BUT WE
FOUND IF YOU HAVE THIN TISSUE
SECTION YOU CAN GO IN THE
PROCESS WITH TRANSPOSES.
WITH OUR EXPERIMENT TO TREAT
THE--TISSUE SECTIONS AND WE USE
THE MICROFLUID DEVICE FOR BAR
CODE A AND B AND HAVE NO SPATIAL
RESULT ATAXIC DATA PIXEL BY
PIXEL AND IN ONE EXPERIMENT YOU
DO THOUSANDS OF EXPERIMENTS AT
THE SAME TIME AND EACH RESPONDS
TO A TINY TISSUE AND NOW WHERE
THEY ARE IN THE SECTION.
SO FROM THE PIXELS, FOR EXAMPLE,
ASSOCIATED WITH THE EMBRYONIC
LEVER YOU SEE THE DATA TRACE YOU
CAN ALSO AGGREGATE ALL THE
PIXELS FROM LEVER TO PRODUCE
THIS CONCORDANCE WITH A
REFERENCE DATA.
WHEN YOU LOOK AT PIXELS YOU CAN
LOOK AT THE CLUSTER ANALYSIS USE
ROUTINE LEVEL ATAXIC TO KIND OF
AGGREGATE DIFFERENT CAST
DIFFERENT PIXELS INTO DIFFERENT
CLUSTERS.
AND HERE YOU CAN FIGURE OUT
WHERE THE PIXELS ARE IN THE
CLUSTERS ARE LOCATED IN THE
TISSUE AND CLUSTERS IDENTIFIED
IN THE UMAB IN THE FIGURE B
HERE.
YOU'LL SEE WHERE THEY ARE AND
THE CLUSTER IS INDEED FROM THE
ANALYSIS AND YOU CAN DO YOUR OWN
ANNOTATION AND THAT'S THE LIVER
AND SPINE AND YOU CAN PROJECT
ALL THE TISSUE PIXELS AND ALSO
VISUALIZE INDIVIDUAL GENES BY
KEEPING IN MIND WHAT YOU'RE
SEEING IS NOT GENE EXPRESSION
BUT IT'S ASSOCIATED WITH THE
GENES.
NOW YOUR ABLE TO DIRECTLY
VISUALIZE THE DATA IN THE TISSUE
THAT HAS NEVER BEEN POSSIBLE IN
THE PAST.
WHAT'S INTERESTING IN MY LAB
NOWADAYS AND THE IMMUNOLOGY WE
CONTINUE TO LOOK AT THE IMMUNE
SYSTEM AND IN PARTICULAR HOW THE
IMMUNOSENESCENCE IS CONNECTED TO
THE DIFFERENT DISEASE INCLUDING
CANCER.
YOU CAN LOOK AT THE IMMUNOCELL
HALLMARKS AND SOME REASON WE'RE
LOOKING TO EPIGENETICS DATA FOR
IMMUNOSENESCENCE AN THIS IS
BASED ON THE ATAXIC DATA AND THE
HALLMARKS AND MARKERS OF THE
ZONES YOU CAN'T IDENTIFY THE
MARKER GENES AND WE CAN IDENTIFY
THE MARKERS AND THEY DON'T
ALWAYS OVERLAP FURTHER
EMPHASIZE.
EVEN SENESCENT CELLS FROM THE
SAME CELL TYPES ARE HETEROGENOUS
FROM THE DISEASE STATE AND
CHANGE IN THE LOCAL TISSUE
ENVIRONMENT AND OTHER CELLS NEED
TO BE ELUCIDATED.
WE NEED TO LOOK TO THE
MODIFICATIONS AND THERE'S A
SIMILAR CHEMISTRY BUT USE
PROTEIN A TO LOOK AT THE SITES
WITH THE SPECIFIC MODIFICATIONS.
WE DEMONSTRATED THIS IN MOUSE
EMBRYO AND USING DIFFERENT MARKS
AND EVEN WITHOUT DOING THEO
HISTO MODIFICATIONS YOU CAN SEE
HOW THEY CONTROL TISSUE TYPES AT
THIS STAGE OF MOUSE EMBRYONIC
DEVELOPMENT.
WHEN YOU CLUSTER ALL THE PIXELS
TOGETHER AND COMPARE AGAINST THE
REFERENCE DATA YOU DO SEE OVER
ALL CONCORDANCE BETWEEN OUR DATA
AND REFERENCE DATA.
OUR DATA GIVES YOU MORE GRANULAR
INFORMATION BECAUSE WE HAVE
PIXEL BY PIXEL AND HAVE THE SAME
LEVER I THINK DIFFERENT WITHIN
THE LEVER YOU SHOULD BE ABLE TO
SEE EPIGENETICS AS WELL.
SO AND IN THE INTRODUCTION SLIDE
WE WERE ABLE TO FURTHER COMBINE
EPIGENETIC TRANSCRIPTOME IN THE
SAME CELL PIXEL BY PIXEL ACROSS
THE ENTIRE TISSUE AND WHAT
YOU'RE SEEING IS THREE DIFFERENT
HISTO MARKED FROM EVERY SINGLE
HISTO MARK EXPERIMENT WE'RE ABLE
TO PERFORM WHOLE TRANSCRIPTOME
SPATIAL SEQUENCING.
YOU CAN THINK IN THE EPIGENETIC
MECHANISM TO THE OUTCOME IN THE
GENE EXPRESSION.
I DID SHOW YOU ONE EXAMPLE WITH
THE EMBRYONIC MOUSE DEVELOPMENT
PARTICULARLY IN THE BRAIN FROM
EITHER ATAXIC OR RNA.
YOU'RE ABLE TO CLUSTER THE
PIXELS INTO DIFFERENT TISSUE
TYPES.
EITHER ONE WORKED OUT VERY WELL.
YOU CAN ALSO FURTHER INTEGRATE
YOUR DATA WITH SINGLE CELL
ANNOTATION TO SPATIAL AND WE'RE
IN A DIFFERENT CELLS ARE AND
HERE'S A FEW EXAMPLE FOR EXAMPLE
THE INHIBITORY INTERNEURONS AND
PROGENITOR NEURONS LIKE THE
RADIAL GLIA AND IT'S LOCALIZED
IN THE VENTRICLE IS WHAT I
ANTICIPATED AND NOW WHERE THE
MECHANISM CONTROL THE
DEVELOPMENT OF THE PARTICULAR
CELL OR MECHANISM OF THE CELL
FATE.
WITH THE TUMOR DATA YOU CAN LOOK
AT THE GENE REGULATORY NETWORK.
ANOTHER EXAMPLE SHOWING THE
POWER TO HAVE THE SAME CELL
ACROSS THE WHOLE TISSUE IS WE
CAN LOOK AT THE PROCESS FOR THE
PLASTICITY AND THE CANCER CELL
SO TRANSITION FOR EXAMPLE IN
EMT.
WHAT I'M SHOWING IS RADIAL GLIA
DIFFERENTIATION NEURONS AND THE
GENERAL HYPOTHESIS OPENING UP
FIRST AND LATER IN THE GENE
EXPRESSED YOU SEE IT'S TRUE IN
MOST THE CASES BUT YOU'LL SEE
CHROMATIN OPENED UP AND THIS IS
THE GROUP OF GENES.
AND REMAINS OPEN BUT IN THE RNA
QUICKLY DIMINISH AND WE FOUND
THOSE GENES ARE NOT FOR NEURONS
NEURONAL CELLS COMMITTED TO
LINEAGES.
THOSE GENES ARE STRONGLY
ASSOCIATED WITH OLIGO DENDRO
LINEAGE AND THE CELLS DECIDED TO
BE BECOME NEURONS BUT STILL
THEIR POTENTIAL TO BECOME OLIGO
DENDROCYTES IS INTRIGUING SO ANY
ENVIRONMENTAL STIMULATION THAT
MAY TRIGGER THE TRANS
DIFFERENTIATION THOSE CELLS DO
HAVE THE POTENTIAL TO BECOME
OLIGO DENDROCELL LINEAGE AND
THEY HAVE THE POTENTIAL AND
COMMITTED TO THE OLIGO
DENDROLINEAGE AND THE TWO
PATHWAYS THEY MIGHT TAKE AND YOU
CAN VISUALIZE WHERE THEY ARE IN
THE SPATIAL CONTEXT.
I WANTED TO SHOW OUR ONGOING
WORK IN THE CANCER RESEARCH
SPACE.
WE MAPPED OUT THE SPATIAL
PROFILE THE HUMAN GLI AL BLASTOA
AND YOU SEE THE SECOND ROW AND
THIRD AND FOURTH ARE FROM
ANOTHER TUMOR SAMPLE AND RNA AND
PROTEIN AND CHROMATIN ACCESS.
I'LL WRAP UP.
ONE LAST SLIDE.
I CAN DIRECTLY VISUALIZE THE
EPIGENETIC PLASTICITY OF HUMAN
BLASTOMA AND BASED ON THE
SIGNATURES IDENTIFIED FOR MPC,
OPC THROUGH ASTROCYTE.
YOU PERFORM THE TIME TO LOOK AT
THE CELLS AND YOU CAN DIRECTLY
VISUALIZE THE CELL CAPSULE WITH
THE DIFFERENTIATION OR THE
PLASTIC TRANSITION IN THE SPACE
IN THE BOUNDARY OF THE TUMOR
SUBTYPES IN THE TUMOR.
JUST TO WRAP UP HERE, ANOTHER
GAM IS NEUROENDOCRY TUMOR IN THE
HUMAN PANCREAS AND I'LL WRAP UP
BY THANKING MY OWN GROUP MEMBERS
AND MY AMAZING COLLABORATORS AND
SUPPORT FROM NCI.
THANK YOU FOR YOUR ATTENTION.
>> THANK YOU SO MUCH.
IT'S AMAZING WORK.
INCREDIBLY POWERFUL.
I THINK BECAUSE OF THE TIME
WE'LL TAKE ONE QUESTION IF
THAT'S OKAY IF THERE'S A
QUESTION.
GO AHEAD.
I'LL GO DOWN TO MAKE SURE --
>> QUICK QUESTION.
GREAT TALK.
I WAS WONDERING IN THE
SENESCENCE MARKERS YOU WERE
SHOWING THE HETEROGENEITY OF
CDKN WHERE OTHER MARKERS LIKE
IL6 AND SO ON DO YOU SEE A
DIFFERENCE IN DEGRADATION IN
MARKERS?
MY NAME IS HARI ISHWAN FROM
JOHNS HOPKINS UNIVERSITY.
>> THAT'S THE MESSAGE I WANT TO
CONVEY IF I DIDN'T MAKE THAT
CLEAR.
IT'S STILL TOO EARLY TO DRAW ANY
CONCLUSION BUT WE OBSERVED THE
HETEROGENEITY BETWEEN THE
MARKERS P16 AND MGB1 AND NOT
ALWAYS THERE'S SENESCENCE
MARKERS AND ONCE YOU SEE IT
POSITIVE FOR P16 BUT NOT
POSITIVE FOR OTHERS.
SOMETIMES WE SEE THERE'S
MUTUALLY EXCLUSIVE.
WE STILL DON'T HAVE CLEAR ANSWER
BUT THE GENERAL OBSERVATION SO
FAR ACROSS SEVERAL TISSUE TYPES
IF THEY BECOME CELLS DRIVING
DISEASE OR DRIVING AGING AND YOU
SEE THOSE MARKERS REGULARLY
CONVERGE BUT EARLY STAGE YOU SEE
THE SENESCENCE OR SENESCENT LAG
CELLS THEY DEVELOP THROUGH
DIFFERENT BIOLOGICAL PATHWAYS.
AND WE CHOOSE THE MARKERS
BECAUSE THEY REPRESENT PROCESSES
TO BECOME SENESCENT AND YOU SEE
IT COME FROM DIFFERENT PATH AND
IF THEY CONVERGE THEY'RE MAKING
TROUBLE.
BUT I THINK IT'S EARLY
PHILOSOPHICAL THINK.
I THINK WE NEED MORE DATA TO
DRAW CONCLUSIONS.
THANK YOU VERY MUCH.
>> THE NEXT SPEAKER IS STEVE
BAYLIN FROM JOHNS HOPKINS
TALKING ABOUT EPIGENETIC STATES
OF TUMOR PARENT CELLS AND HOW
EPIGENETIC ABNORMALITIES CAN
DRIVE PRE-MALIGNANT TUMOR
PROGRESSION.
>> THANK YOU.
I WANT TO THANK THE ORGANIZERS.
I'M HONORED TO BE PART OF THIS
ANNIVERSARY AND CONTRIBUTE SOME.
THE FIRST ACKNOWLEDGEMENT IS A
WHIMSICAL ONES AND UNDERSTANDING
HOW EMBRYOS FORM ALL THE WAY
THROUGH WE HAVE TRANSPOSE
ELEMENT.
EPIGENETICS WRIT LARGE IS A
BUILDING FIELD AND HERE HAVE YOU
CANCER PIONEERS AND STEVEN JONES
AND ANDY FINEBERG DOWN HERE AND
IT'S QUITE A HISTORY OF
CONTRIBUTIONS FROM THE SHOULDERS
WE STAND ON.
THE ONLY REASON I'M HERE IS I
STAND ON ALL THESE SHOULDERS AND
THESE ARE SOME OF A GROUP OF
PEOPLE THAT TRAINED WITH ME AND
CONTRIBUTED TO THE FIELD AND
SOME IN THE AUDIENCE.
I'LL ONLY INTRODUCE A FEW DURING
THE TALK BUT THEY'VE TAKEN THE
FIELD INTO FIELD I NEVER WOULD
HAVE IMAGINED AND AREAS I NEVER
WOULD HAVE IMAGINED.
HERE'S WHAT I'LL TALK ABOUT
TODAY.
I HAVE REMARKS ANDY MADE AND
TALK ABOUT THE EVOLUTION OF
CANCER FROM THE CELLS OF ORIGIN
AND THEY'RE EPIGENETIC STATE
SETS THE STAGE AND THEY CAN
DEPEND ON PROGRESSION OF
ABNORMALITIES DURING THE PRE
MALIGNANT PHASE AND BRING THEM
TO A POINT THAT ALLOW THE CELL
WHEN IT SEES THE MUTATION TO
FORM CANCER AND HAVE CANCER
INITIATION.
AT THE END I'LL TALK ABOUT VERY
BRIEFLY SOME DRUG DEVELOPMENT
FOR NEW TARGETS THAT HAVE COME
OUT OF SOME OF THE THINGS THAT
I'LL TALK TO YOU ABOUT.
SO THIS IS A SLIDE FROM PETER
JONES.
A GOOD ONE THE THREE DIMENSIONAL
STRUCTURE OF THE EPIGENOME WE'VE
BEEN HEARING ABOUT AND HOW THE
MICROCOSM STARTED IN THE MID
'80s AND A GRANT GAVE US A
CHANCE TO START PLAYING THIS OUT
WHERE YOU HAVE DURING TUMOR
PROGRESSION AT SOME POINT A
TRANSITION WHERE DNA METHYLATION
CREEPS INTO GENES THAT DON'T
HAVE THIS CHANGE FOR THE MOST
PART.
THEY REMAIN UNMETHYLATED AND
MANY GENES THAT MAKE THE CHANGE
ARE CONTROLLED DURING NORMAL
DEVELOPMENT WITHOUT DNA
METHYLATION AND BY THE
PLASTICITY OF THE REPRESSER
UNIT.
I WANT TO ALSO POINT OUT THIS
SAY PROGRESSIVE CHANGE.
THIS IS A REVIEW PETER AND I
WROTE BACK IN 2002 IF YOU HAVE
THIS YOU GENERALLY HAVE ONE AND
YOU KNOCK THE PROTEIN OUT.
THE PROGRESSIVE CHANGES IN CPGI
LINK WHERE IT GETS DENSER AND
DENSER AND BEGINS TO SILENCE THE
GENE IS A HAPLO FLUORESCENT
STATE AND INTERFERE IN THE
PATHWAY.
BACK IN 2012 HARI HAD A PAPER
WHERE HE SHOWED HOW THE
PROGRESSION TO THE DNA
METHYLATION CORRELATED WITH THE
RELATIVE LOCKING IN OF THE STEM
CELL LIKE PHENOTYPE.
AND THROUGH THE WORK IN THIS
PAPER CAME UP WITH THE
HYPOTHESIS WHICH I'M GOING TO
SHOW YOU ONE PROBABLY PLAYING
OUT IN THE DATA THAT THAT STEM
CELL STATE THEN BECAME SENSITIVE
TO THE ONCA GENIC AFFECTS OF
EXPRESSION OR ONCA GENE AND SET
UP THE STAGE FOR CANCER
INITIATION.
AGAIN THE GROUP OF GENES
SUSCEPTIBLE TO THIS CHANGE AND
FOLLOW THIS THROUGH TO CANCER
PROGRESS AND SOME OF THE PAPERS
THAT HAVE COME OUT ON IT DON'T
SAY THE WORD EPIGENETICS.
THAT'S TAKING THE STATE OF THAT
PARENT CELL AND FOLLOWING IT
THROUGH LIKE IN THE COLON CANCER
TO PREINVASIVE CHANGES AND
CANCER AND NOTING THE CHANGE OF
THE ENVIRONMENT ANDY AND THE
PREVIOUS SPEAKER POINTED OUT
THAT CAN IMPOSE ON THIS PARENT
CELL THE START OF A CHRONIC
INFLAMMATION CHANGE THAT STOKES
THIS PROGRESSION ALONG THE
COURSE THAT WE'RE GOING TO TALK
ABOUT.
I WANT TO POINT OUT A PAPER FROM
JOHNS HOPKINS AND THE POINT I'M
GOING TO MAKE IS THE CHANGES
AGAIN STARTING FOR CANCER.
WHAT THEY HAVE DEFINED IN THIS
PAPER WHICH YOU CAN READ IS THIS
FOR THE CPGI METHYLATION
PHENOTYPE AND A QUANTITATIVE
MEASURE AND THE YELLOW ACROSS
THE ARRAY IS THE DENSE DNA
METHYLATION THE NORMAL IS IN
BLUE.
WHAT THEY HAVE CLEVERLY DONE IN
THE PCGA HERE MOST THE PURPOSE
IN THE BAR ARE CANCERS BUT
THEY'VE WOVEN IN AND HARD TO SEE
POLYPS AND IT POINTS OUT YOU'VE
GOT POLYPS THAT HAVE ALREADY
DEVELOPED IN THE DENSE CHANGE IN
THE PHASE AND WHAT THEY
RECOGNIZE A LOW EXPRESSION OF
TRANSCRIPTION FACTORS MANY OF
THEM THE POLY COMB TRANSCRIPTION
FACTORS WHEN THEY WERE LOW, THIS
CORRELATED WITH THE EMERGENCE OF
THIS PHENOTYPE BY SEWING THE
COGNATE BINDING SITES IN THE
ENHANCERS OF GENES AND COGNATE
SITES IN THE PROMOTERS WERE
LINKED TO THIS SUCH THAT YOU HAD
IN THE ONLY CEMP BUT GAINED DNA
METHYLATION IN THE ENHANCERS AND
THE IMPORTANT THING IS LOOK HOW
IT MIMICS THE POLYPS AGAIN ARE
FACTORS IN HERE.
THERE'S LOW EXPRESSION.
THE CHANGES IN THE ENHANCERS
ALMOST VIRTUALLY PARALLEL THE
DEVELOPMENT OF THOSE PROMOTER
CHANGES IN CEMP.
A PLASIA ARISING.
I DON'T KNOW WHAT THEY'RE
REQUESTING OF ME.
HERE'S THE MODEL I WANT TO POINT
OUT WE'VE BEEN USING.
HARI MYSELF AND PEOPLE I'LL TELL
YOU ABOUT.
THIS IS A MODEL TAKING PARENT
CELLS OF ORIGIN IN THE ELEGANS
REGIONS AND DURING PREMALIGNANCY
YOU DEVELOP CEMP AND WHEN THAT
ARISES THE ORIGINAL MUTATION ONE
STEP RIGHT SIDE IN COLON CANCERS
IN THE MODEL.
THE OTHER MODEL IS TO IMPOSE THE
CHRONIC INFLAMMATION
ENVIRONMENTAL CHANGE AND THEN
INDUCING MUTATION AT THAT POINT
AND YOU GET ONE STEP ON THIS.
THIS PUBLICATION FROM
HARIISHWAN'S LAB WAS THE FIRST
IN THE COLON CANCER MODEL AND
YOU HAVE THIS IN THE MOUSE AND
INDUCE IT IN THE ORGANOIDS.
IT TAKES FIVE MONTHS TO GET
AROUND CELL DEATH AND OXYGEN
SPECIES AND DAMAGE -- SO FIVE
MONTHS AT THE MUTATION THAT AT
THAT TIME CAUSES COLON CANCER
BUT IF YOU LET THE ORGANOIDS AGE
THEY DEVELOP AND IF YOU INDUCE
THE MUTATION NOT FIVE MONTHS IT
ONLY TAKES WEEKS AND TO SHOW THE
REQUISITE CONTRIBUTION, A
STUDENT CRISPRED GENETICALLY
SILENCED THE GENES SILENCED HERE
AND SET UP THE MUTATION AND ONLY
TAKES WEEKS.
THE GENES THAT WERE SILENCED
REQUISITE IN THE MODEL TO SET UP
THE CANCER INITIATION RESPONSE
TO THE MUTATION.
LET'S TAKE THIS FORWARD ONE
STEP.
THE MICE CAME FROM INDUCIBLE B
RAP IN THE MOUSE AND WHEN IT'S
DONE IN THE MOUSE YOU GET RIGHT
SIDED COLON CANCER THE RIGHT
SIDE OF THE GUT IS DIFFERENT
THAN THE LEFT SIDE.
AND WHEN YOU INDUCE THIS IN THE
MICE -- I HAVE TO BOOGIE HERE
WITH THE TIME.
WHAT I'LL SHOW YOU NEXT I'LL
TELL YOU WHAT IT IS.
THIS TIME THE MUTATION WAS
INDUCED IN THE MOUSE STARTING IN
OUR LAB AND WORKING WITH OTHERS
AND WHEN THEY INDUCED THIS IN
THE MOUSE AND THEN CHALLENGED
THAT MOUSE WITH THE
MICROBIOME
MICROBIOMEDICAL --
MICROENVIRONMENT THEY SAW
CHANGES THEY'VE NEVER SEEN AND
THIS TIME THEY SAW HYPERPLASIA
ON THE RIGHT SIDE OF THE
PRECURSOR CELLS AND THE MUTATION
PICKED ON PARENT CELLS FOR THE
SENSITIVITY ON THE RIGHT SIDE OF
THE COLON TO INDUCE THAT TUMORO
GENIC CHANGE.
SO WHAT WHERE I'LL TAKE YOU NEXT
IS THE MODEL AND TELL YOU ABOUT
THE WORK OF A YOUNG
INVESTIGATOR, MICHELLE VAS AT
HOPKINS WHO STARTED HER WORK AS
A POSTDOC IN OUR LAB.
A TALENTED POST DOCK WITH HER
AND TAKE IT TO LONG ORGANOIDS
THE ELEGANS MODEL AND START WITH
NORMAL AIRWAY CELLS IN THE LUNG
AND I'LL SHOW YOU WHAT HAPPENS
THERE IS THE SAME PROGRESSION TO
CEMP IN A WAY AT THAT TIME THAT
SOME OF THE GENES THAT GET
SILENCED WOULD OTHERWISE HAVE
VE
PREVENTED THE K RAS PATHWAY FROM
COMING UP.
AT THAT POINT IF YOU INTRODUCE
THE K RAS MUTATION YOU GET ONE
STEP LUNG ADENO CARCINOMA AND A
WANT TO SLOW YOU IF I CAN THE
BEAUTIFUL MODEL AND PICTURES OF
HOW THEY MARK THESE STEM CELLS
AND FOLLOW THEM THROUGH AND -- I
DON'T KNOW.
IS THERE ANY WAY I CAN GET THEM?
I'LL BOOGIE TO THAT SLIDE
BECAUSE -- THERE WE GO.
THAT'S THE RIGHT SIDED MODEL AND
THE ROS MODEL I'LL COME BACK TO
THE WAY WE THINK ABOUT THESE
CHANGES.
THE LUNG IS A PSEUDO STRATIFIED
EPITHELIUM WITH AIRWAY CELLS IN
THE PROXIMAL REGION WITH STEM
CELLS AND THE CARCINOMA COMES
ALONG AND THIS MODEL FOR THE
MOUSE YOU CAN GET ALL THE WAY
DOWN TO THE STEM CELLS AND THE
ATC AND ATP CELLS IN THE ALVEOLI
AND YOU CAN MARK THE CELLS AS
I'LL SHOW IN THE ORGANOID MODEL
WITH DIFFERENTIATION AND I'LL
SHOW YOU A COUPLE CHANGES IN
EXAMPLE.
IN THE UPPER AIRWAYS IN THE
HUMAN AND MOUSE CAPTURE THIS
STRUCTURE WITH THE AIRWAY IN THE
MIDDLE AND BASAL CELL STEM CELLS
AROUND THE EDGE BUT THE AIRWAY
IS SURROUNDED BY GRAPE LIKE
STRUCTURES.
THESE ARE STEM CELLS.
SO KEEP YOU'RE EYE ON THE STEM
CELLS.
WHAT THE MODEL DOES IS THIS.
YOU'VE EXPOSED THEM TO TWO
DOSES, CIGARETTE SMOKE EXTRACT.
THIS IS THE ENVIRONMENTAL
INFLAMMATORY CHALLENGE IN THIS
MODEL.
AND WHEN THIS HAPPENS, ONE
MONTH, THREE MONTHS, SIX MONTHS.
THE ORGANOIDS AFTER CELL DEATH
BEGIN TO GROW.
RAPIDLY.
YOU'LL NOTICE THEY GET DENSER
AND DENSER.
AND STRUCTURES AROUND THE
ORGANOID WHICH ARE THE STEM
CELLS.
YOU CAN MARK THESE STEM CELLS.
THERE'S BEAUTIFUL ARTWORK.
ONE OF THE CHANGES THAT OCCURS.
THERE'S MARKERS OF
DIFFERENTIATION BUT THERE'S AN
IMAGE HERE, WHAT YOU SEE OVER
SIX MONTHS OF THE TWO EXPOSURES
TO THE CIGARETTE EXTRACT IS THE
DIFFERENTIATED ONE STAYS AROUND
AND LOOK AT THE IMAGE IN THE
SIGN OF STEMIS IN THE LUNG AND
YOU SEE THIS WAY AND THIS WAY
THERE'S VIRTUALLY NO KRT14 IN
THE CONTROL BUT AS YOU EXPOSE
THE CIGARETTE SMOKE YOU CAN
TRACK THIS.
MOREOVER, IF YOU FOLLOW THESE
CELLS YOU NOTICE THE CONTROL YOU
SEE THIS NICE MARKER FROM CELLS
DOWN IN THE AIRWAYS IN THE DITCH
STATION MARK.
YOU LOSE DIFFERENTIATION AND
GETTING THIS IN THE MODEL WITH
THE CIGARETTES.
SO THIS IS AN INCREASE IN
PROLIFERATION THAT OCCURS MAKING
IT IMMUNOEXPRESSIVE.
AND THESE GENES TEND TO BE
EFFECTED AND HERE'S THE DNA
METHYLATION PROFILE FOR THE
GENOMES AND THE HYPERMETH LATE
HYPERMETHYLATED GENES AND YOU
SEE EVIDENCE FOR PRE MALIGNANT
IMMUNE EVICTION AND THE RESPONSE
IS EXPRESSED AND THE INTERFERON
ALPHA AND BETA ARE SUPPRESSED
AND THESE ARE MARKERS THAT IF
THEY'RE UP WOULD BE PART OF THE
TUMOR CELL INFLAMMATORY SIGNAL
GETTING EXPRESSED PRE MALIGNANT
IN THE MODEL.
AT THE SAME TIME YOU CAN
ENGINEER THESE ORGANOIDS FOR THE
MICROENVIRONMENT AND THE PURIFY
MACROPHAGES AND AT THE START THE
MORE COMBINANT AND OVER SIX
MONTHS A MORE PROTUMORO GENIC
PHASE.
AND THE KEY IS WHAT ABOUT TUMOR
GENICITY?
WHAT YOU CAN DO WITH THE
ORGANOID MODELS DONE IN THE
COLON ONES TOO TAKE THE CLEAVER
FACTORS OUT THAT SUPPORT THE
NORMAL ORGANOIDS AND THEIR
ABILITY TO GROW SHOWS IT'S A
SIGN OF INITIATION.
AND WHEN YOU EXPOSE THEM THEY
GET BIGGER AND GROW RAPIDLY.
INDEPENDENCE OF THE SUPPORT
FACTORS FOR NORMAL, AT THAT
POINT YOU CAN PUT IN A GENE
MUTATION.
IF YOU PUT THE KRAS IN YOU GET
AN AGGRESSIVE METASTATIC LUNG
CARCINOMA.
IF YOU TAKE OUT THE EXPRESSER
GENE YOU GET A CARCINOMA AND ONE
STEP INITIATION PRE EXISTENT
EPIGENETIC CHANGES FROM THERE.
I WANT TO COME BACK A QUICK
STORY.
THE SLIDE I DIDN'T GET TO SHOW
YOU BEFORE SHOWING HYDROGEN
PEROXIDE IS A GREAT MODEL AND
THIS SYSTEM WAS STARTED WAY BACK
HERE AND THE COLLABORATION WITH
MY WIFE OVER THE PAST DECADE AND
THIS TALENTED INVESTIGATOR
VISITING SCIENTIST AT THE TIME
FROM CHINA AND HER GROUP IN BIG
LUNG CANCER COHORTS WE CAN STUDY
AND THAT TRIGGERS THE OGG1 AND
WHEN IT SEES THAT IT REPRESS THE
COMPLEX TO C-G MODEL AND WITHIN
SIX HOURS IT CAN START TO
INSIGHT THE METHYLATION AND IF
YOU INHIBIT THESE AND NOT YOU
CAN REVERSE REEXPRESS THE
SILENCE GENES AND TUMOROGENIC
PROPERTIES.
AND IT TIGHTLY BINDS MANY OF THE
PROTEINS THE TRANSFERASES
AND BINDS UHFR1.
AND THEY COMPLETED THIS SO THIS
A FASCINATING PROTEIN.
AND THIS RECOGNIZES METHYLATED
CELLS AND IT TRACKS WITH AND
MAPS ACROSS THE BIG COHORTS.
SO MY FINAL SLIDES I'M GOING TO
SHOW YOU THERE'S A NATURAL
INHIBITOR OF THE PROTEINS DPPA1
IT WORKS IN THE HOUSE TO GO GET
UHFR1 AND TAKE IT OFF THE
CHROMATIN, TRANSPORT IT AND KEEP
IT OUT IN THE CYTOPLASM.
IN THIS WORK WE DECIDED TO OVER
EXPRESS AND GOT A REMARKABLE
SERIES OF CHANGES.
WHEN WE TOOK COLON CANCER CELLS
AND PUT THIS IN THE MOUSE ONLY
THE MOUSE WOULD REPLICATE TAKING
UHFR1 OUT OF THE NUCLEUS AND
TRANSFERRING IT TO THE
CYTOPLASM.
NOT THE HUMAN.
HERE'S WHY.
ONLY 31% AMINO ACID IDENTITY
BETWEEN THE MOUSE UHFR IS AND
HUMANS AND1 IS AND
HUMANS AND HERE'S THE AFFECT.S
HUMANS AND HERE'S THE AFFECT. A
HUMANS AND HERE'S THE AFFECT.
AND IT'S ALL DUE TO AN LYSINE
PULLS THEM TOGETHER IN THIS
DIRECTION WHERE THE HUMAN WILL
NOT DO THAT AND REMAINS FURTHER
APART AND LINEAR.
WE PINNED THIS DOWN TO A SMALL
REGION HEMOLOGY.
A SMALL REGION OF 85 TO ABOUT
115 AMINO ACIDS.
THEY'VE BEEN ABLE TO DELIVER
THIS IN HIS WORK IN CHINA WITH
THE NANO PARTICLE IN CELLS AND
IN VIVO AND MIMIC THE INHIBITION
OF THE BLOCK OF THE DNA
METHYLATION.
SO WE ARE HOPEFUL WE HAVE A DRUG
DELIVERY HERE AND IT'S AN
EXAMPLE OF THE NEWER DRUG THAT
COMES OUT UNDERSTANDING THE
REPRESSIVE COMPLEXES.
I'M SORRY FOR THE LAX -- LACK
OF MECHANICS AND THANK YOU FOR
INVITING ME.
ANY QUESTIONS?
>> THANK YOU.
SO THE LAST TALK OF THE
SESSION --
>> SOMEBODY HAS A QUESTION.
GO AHEAD.
>> WHERE ARE THEY?
GO AHEAD, PLEASE.
>> INTRIGUING TALK.
I WAS WONDERING IN THE MODEL --
>> YOU HAVE TO BEND OVER.
YOU'RE A TALL GUY.
>> THE LUNG CANCER ORGANOID
EXPERIMENT US LOOKED AT DNA
METHYLATION CHANGES AFTER
TREATMENT.
HAVE YOU LOOKED AT OTHER
CHROMATIN FEATURES OF HISTO
MODIFICATIONS AND SEE IF ANY OF
THESE ALSO ASSOCIATED WITH GENE
EXPRESSION CHANGES?
>> IN THE PAPER THEY INDEED DID.
THEY'VE DONE IN THE HUMAN TAC
SEQ AND WITH THE REVERSION TO
THE BLOCK THE DNA METHYLATION IS
GIVEN YOU SEE INCREASING
REPRESSIVE METHYLATION AND ALLOW
THE DNA METHYLATION TO OBSERVE
THE REPRESSIVE EFFECTS AND THEY
SHOULD BE SUBMITTING THIS SOON.
IT'S A DEEP PAPER ON GOING
THROUGH THE CHROMATIN AS WELL AS
GOING THROUGH SOME METHYLATION
CHANGES THEY SHOULD.
>> THANK YOU.
>> THE LAST SPEAKER OF THE
SESSION IS CHERYL ARROWSMITH
FROM THE PRINCESS MARGARET
CANCER CENTER UNIVERSITY OF
TORONTO TALKING ABOUT
THERAPEUTIC EPIGENETICS.
>> THANK YOU, IT'S A PLEASURE TO
BE AT THE CELEBRATION.
40 YEARS OF EPIGENETICS.
MY STORY IS IN 1980s WHEN I WAS
A STUDENT I READ SOMEWHERE
METHYLATION HAD SOMETHING TO DO
WITH CANCER.
I WAS A CHEMISTRY STUDENT AND
DIDN'T KNOW WHAT A METHYL GROUP
WAS.
I FOUND TA EXCITING.
I DID KNOW WHAT CANCER WAS
BECAUSE WE HAVE IT IN OUR
FAMILY.
LONG CIRCUITOUS ROUTE BUT BY THE
EARLY 2000s IN THE MEANTIME I'D
BEEN WORKING ON TRANSCRIPTION
FACTORS AND THE RISE OF MANY OF
THE GIANTS ON WHOM SHOULDERS WE
STAND HAD DONE MUCH OF THE
WORK -- SOME OF THE WORK YOU
HEARD ABOUT AND IT WAS BECOMING
CLEAR EPIGENETICS WAS AN
IMPORTANT DRIVER OF CANCER AND I
KNEW MORE ABOUT THE BIOLOGY BY
THAT TIME.
SO OUR WORK IS I'M VERY MUCH
INTERESTED IN METHYLATION
SIGNALLING PARTICULARLY IN
METHYLATION OF PROTEIN HISTONES.
WE CALL THEM CHEMICAL PROBES.
IT'S A TOOL COMPOUND POTENT FOR
ITS TARGET IN CELLS SELECTIVE
ESPECIALLY WITH RESPECT TO OTHER
MEMBERS OF THE PROTEIN FAMILY
AND METHYL TRANSFERASES MEANT TO
BE USED AS A TOOL TO UNDERSTAND
THE FUNCTION IN THE CELL OF THE
PROTEIN OF INTEREST BY
INHIBITING IT.
COMPLIMENTARY GENETIC METHODS.
WE DO IT IN COLLABORATION WITH
INDUSTRY AND ACADEMIC CHEMISTS
AND THESE TOOLS ARE ALL PUBLICLY
AVAILABLE, UNINCUMBERED AND WE
HOPE BY USING THESE THE BIOLOGY
THAT'S DISCOVERED WILL INSPIRE
AND IDENTIFY THERAPEUTIC TARGETS
AND THE MOLECULES COULD BE
FURTHER DEVELOPED OR THE TARGET
IS IDENTIFIED FOR THERAPEUTIC
DEVELOPMENT.
ONE ON TOOL SET AND BRODDER
DOMAINS AND WE'VE WORKED ON
METHYL READER DOMAINS AND
DEMETHYLASE ENZYMES.
ONE THING WE WANT TO DO WITH
THESE IS USE THEM TO IDENTIFY
VULNERABILITIES IN CANCER CELLS
AND CANCER ORGANOIDS.
THIS SUMMARIZES THE CONCEPT OF
THE WORK WE'VE BEEN DOING IN MY
LAB THOUGH AN OLD SLIDE.
ON THE Y AXIS YOU CAN SEE THE
CHEMICAL PROBES WE'VE USED AND
ACROSS THE SLIDE ARE DIFFERENT
TUMOR MODELS.
IN MANY CASES, CERTAIN CASES
SUCH AS THE BROMA DOMAIN
INHIBITORS ALMOST ALL FAST
GROWING CANCER CELLS ARE
SENSITIVE TO THESE INHIBITORS
AND OTHERS LIKE THE TRANSFERASE
INHIBITORS THE CELLS ARE ONLY
SENSITIVE TO THEM IN CERTAIN SUB
TYPES OF TUMOR TYPE AND WHAT WE
SET OUT TO UNDERSTAND IN A
NUMBER OF PAPERS.
WE'VE DONE THESE TYPES OF
SCREENS AND THEN INVESTIGATED
FURTHER WHAT MAKES THE CELL
SUSCEPTIBLE AND WHAT'S THE
MOLECULAR SIGNALLING INTERNALLY
GOING ON WITH INHIBITION OF
USUALLY METHYLATION SOMETIMES
OTHER CHEMICAL BIOLOGY AND HAVE
IDENTIFIED CASES OF OVERCOMING
STEMNESS AND TRIGGERING
DIFFERENTIATION.
A NUMBER OF CASES AND STRESS
RESPONSE AND THIS IS A
METABOLOMICS STORY.
I'LL TELL YOU ABOUT A RECENTLY
PUBLISHED PAPER WHERE WE
IDENTIFIED SUBSETS OF CELLS
WHOSE TRANSCRIPTAL STATE
DETERMINES THE SENSITIVITY TO
ONE OF THE INHIBITORS AND TALK
ABOUT UNMILITIPUBLISHED WORK.
THIS WAS WORK WAS LED BY A
TALENTED POSTDOC WITH HER OWN
LAB NOW IN CHINA AND THEY
SCREENED AGAINST THE CHEMICAL
PROBE LIBRARY.
WE'RE LOOKING AT CELL
PROLIFERATION AND VIABILITY AND
IDENTIFIED A TYPE 1 PROTEIN
ARGININE TRANSFERASE INHIBITOR
IN SUBSET OF THE CELLS ROBUSTLY
STOPS CELL GROWTH.
THE INACTIVE CONTROL COMPOUND
NOT ACTIVE ON THE ENZYME DID NOT
EXPRESS GROWTH.
WHAT THE INHIBITORS INHIBIT THE
ARGININE ON HISTONES AND THEN
IT'S OFTEN GROUPED WITH
EPIGENETIC REGULATORS.
PROBABLY THE MORE SIGNIFICANT
SUBSTRATE ARE RNA BINDING
PROTEINS PARTICULARLY THE
SPLICING MACHINERY AND I'LL COME
BACK TO THAT IN A MOMENT.
HERE WE HAVE A SET OF CELL LINES
THAT ALL HAVE INHIBITION OF THE
ENZYME.
YOU CAN TELL THAT BECAUSE THE
ASYMMETRIC MARK IS INHIBITED
WITH THE ENZYME.
THEY RESPOND IN A DIFFERENT WAY.
THE ONES IN RED ARE SENSITIVE TO
THEY DON'T GROW ANY MORE IN THE
PRESENCE OF THE INHIBITORS AND
OTHERS ARE SENSITIVE.
I WON'T GO INTO DETAILS BUT LONG
STORY SHORT IT'S MOST LIKELY
PRNT1 MOSTLY AFFECTED BY THIS
INHIBITOR AND IT'S AN ESSENTIAL
GENE TRIPLE NEGATIVE BREAST
CANCER IF YOU LOOK IN THE TCGA.
AND THIS JUST SHOWS THAT YOU CAN
PHENOCOPY THIS IN MOUSE MODELS
WITH KNOCK DOWN OF THIS AND YOU
CAN SEE THIS AFFECT IN THE
ORGANOIDS.
WHAT IS IT ABOUT THE SENSITIVE
CELL LINES THAT MAKES THEM
SENSITIVE TO TYPE 1 INHIBITION?
WE FOUND THROUGH USING RNA SEQ
THE SENSITIVE CELL LINES HAVE A
HIGHER LEVEL OF SIGNALLING
PRETREATMENT OF THE INHIBITOR
AND HAVE HIGH INTERFERON
SIGNALLING FEATURE IN THE CELL
LINES AND TUMOR ORGANOIDS.
IF WE TREAT THOSE CELLS WITH THE
INHIBITOR YOU GET A FURTHER
INDUCTION OF INFLAMMATORY SIGNAL
AS SHOWN HERE.
SO CHANGE IN GENE EXPRESSION.
SOME OF THE GENES MODULATED ARE
SHOWN IN THE MIDDLE PANEL HERE
AND VALIDATED WITH PCR ON THE
RIGHT.
WHAT IS IT ABOUT THE
INFLAMMATORY SIGNAL AND WHERE'S
IT COMING FROM?
INSPIRED BY MY COLLEAGUE DANIEL
DI CARDELO HE SAID WHAT IS
HAPPENING TO THE GENES THAT ARE
BEING EXPRESSED HERE?
BECAUSE WE KNOW THAT PRNT
INHIBITERS SPLICE BINDING
PROTEINS AND METHYLATION OF
SPLICING FACTORS IS IMPORTANT
FOR PROPER SPLICING.
WE LOOKED AT THE RNA SEQ WE
LOOKED INTO THE SPLICING EFFECTS
AND THERE ARE LOTS OF
DISRUPTIONS OF SPLICING
PARTICULARLY RETENTIONS TO
IDENTIFY 152 OF THESE.
IF WE LOOKED AT THESE RETAINED
ENTRONS ALMOST -- A VERY LARGE
PROPORTION OF THEM HAVE THIS
POTENTIAL FOR PERFORMING
DOUBLE-STRANDED RNA ANDALO
SEQUENCES THAT HAVE INVERTED
REPEATS NORMALLY NOT EXPRESSED
IN THE CELLS BUT IN THE PRESENCE
OF INHIBITORS AND YOU CAN DETECT
THE DOUBLE-STRANDED RNA WITH
THIS ANTIBODY AND WE CAN ALSO
MIMIC THIS WITH PRNT1 KNOCK DOWN
AND TRIGGERING AN INNATE
RESPONSE WE BELIEVE IT'S
RETAINED WITH THE INVERTED
RETREATS.
IN SUMMARY FOR THIS PROJECT WE
HAVE TWO STATES OF CELLS.
ON THE LEFT WE ARE THE
INSENSITIVE CELLS THAT HAVE WHO
STILL GET RETAINED INTRONS IN
THE RNA SEQ DATA UPON MSO23
TREATMENT AND THE EXPRESSION
PROGRAM DOESN'T HAVE THE
CAPABILITY TO PERFORM INVERTED
REPEATS.
SO YOU DON'T GET THE INTERFERON
SIGNALLING.
THE SENSITIVE CELLS ARE
EXPRESSING TRANSCRIPTS WHO'S
INTRONS WHO NOW GET RETAINED
HAVE THE PROPENSITY TO PERFORM
REPEATS AND THIS SIGNALS CELL
DEATH AND CONTEMPORANEOUS WITH
THIS GROUP AND BOEING ET AL. HAD
SIMILAR RESULTS USING SPLICING
KINASES AND A SIMILAR AFFECT.
WE THINK REMEMBER THE SENSITIVE
CELLS ALSO HAD THIS UNDERLYING
LOW-LEVEL INTERFERON FROM THE
RNA SEQ AND HAD MUTATIONS
ASSOCIATED WITH DNA DAMAGE.
MAYBE THAT WAS LEADING TO THE
UNDERLYING SIGNAL AND NOW YOU
COME IN AND YOU INCREASE IT EVEN
MORE PERHAPS ABOVE A CERTAIN
THRESHOLD THE CELL COULD
TOLERATE.
EPIGENETICS DRUGS HAVE BEEN
SHOWN TO STIMULATE MIMICRY AND
THIS IS MECHANISMS OF GROWTH
EXPRESSION AND HIS OPENS UP
EXCITING AVENUES FOR FURTHER
INVESTIGATION.
SO THE NEXT QUICK LAST STOREY I
WANT TO SHOW MUCH OF IT IS
UNPUBLISHED.
SO WE HAVE THIS COLLECTION OF
COMPOUNDS THAT MODULATE
METHYLATION ON THE HISTONES,
ARGININE AND LYSINE.
WE'VE NOT BEEN ABLE TO IDENTIFY
AN INHIBITOR OF NSD2 OR THE NSD
PROTEINS THAT METHYLATE LYSINE
36.
NOW, WE HAVE BEEN ABLE TO HAVE
ANTAGONIST OF THE READER DOMAINS
OF NST2 AND 3.
WE NOW LAUNCHED INTO USING THESE
AS HANDLES ON THE NST2 PROTEIN
IT CREATE TARGETED DEGRADER
COMPOUNDS.
LET ME FIRST EXPLAIN THE
RATIONALE FOR WHY WE DO THIS AND
THE POLY COMB PROTEINS ARE
DRIVERS OF EPIGENETIC PLASTICITY
AND COORDINATED WITH THIS IS THE
CHANGES IN THE HDK36 METHYLATION
DOMAINS BOTH OCCURRING IN SPREAD
OUT DOMAINS OVER THE EPIGENOME
AND THEY'RE ANTAGONISTIC.
AND NST2 IS SHOWN HERE ON THE
LOWER RIGHT WHERE MUTATIONS
WHERE ACTIVATING MUTATIONS CAN
OCCUR.
AND WE ALSO KNOW ABOUT ONCO
ONCOHISTONES THAT CAN AFFECT THE
PATHWAYS.
THE MUTATIONS AND TRANS
LOCATIONS IN NTS2 OR ACTIVATING
MUTATIONS CAN LEAD TO
ONCO-GENESIS IN MULTIPLE MYELOMA
AND ACUTE LYMPHOBLASTIC LEUKEMIA
AND CAUSE POOR PROGNOSIS AND
OUTCOME.
AND THE DOMAIN WE IDENTIFIED A
COMPOUND THAT DISRUPTS ITS
INTERACTION WITH METHYL LYSINE
IN THE NUCLEOSOMAL CONTEXT AND
THE COMPOUND OVER HERE.
IT DOESN'T HAVE -- IT'S NOT
ENOUGH TO KNOCK NST2 OFF
CHROMATIN BECAUSE IT HAS ALL
THESE OTHER READER DOMAINS SHOWN
HERE AND SO WE DIDN'T SEE MUCH
OF A PHENOTYPE IN THE ONCOLOGY
SETTING WITH THIS.
SO WE THOUGHT WHAT IF WE CAN
JUST GET RID OF THE ENTIRE
PROTEIN.
SO WE'RE USING THE NEW MODALITY
CALLED TARGETED PROTEIN
DEGRADATION OR PROTAX AND YOU
CAN BRING IT TOGETHER WITH AN E3
LIGASE TO CALL DEGRADATION AND
THIS IS THE WORK OF MY TALENTED
STUDENT IN COLLABORATION WITH
LINDSEY JAMES AT THE UNIVERSITY
OF NORTH CAROLINA AND THIS IS
PUBLISHED EARLIER THIS YEAR.
THIS MOLECULE WHICH CAN REDUCE
OR TODAY GRADES NST2 IN CELLS
AND REDUCE METHYLATION MARK
SHOWN HERE OF K36 DIMYETHYL AND
THIS IS THE INHIBITION OR THE
DEGRADATION CURVE SHOWN HERE IN
A DOSE DEPENDENT MANNER.
WE DIDN'T KNOW WHAT THE E3
LIGASE WAS AT THE TIME BECAUSE
IT'S A NOVEL COMPOUND USUALLY
ONE TRIES -- THEY RECRUIT THE
LIGASE AND THIS IS NOT -- THIS
LIGAND DOES NOT RECRUIT EITHER
OF THOSE SO WE HAD TO FIGURE IT
OUT WHAT THE COMPOUND WAS I'LL
SHOW IN A MOMENT AND HAS A MILD
GROWTH EXPRESSIVE ACTIVITY IN A
MULTIPLE MYELOMA CELL LINE SHOWN
HERE BUT SIGNIFICANT AFFECTS ON
ADHESION IN KMS11 CELL LINES.
WE WERE ENCOURAGED TO GO ON AND
TRY TO DEVELOP THIS FURTHER.
WE NOW HAVE A SECOND GENERATION
DEGRADER WHICH HAS SIMILAR CHEMO
TYPE.
IT'S JUST RANGED BY THIS AND AN
UNUSUAL WAR HEAD COMPOUND AND
HAS AN ORDER OF MAGNITUDE DC50
WHERE IT'S 50% DEGRADED USING
BIO I.D. PROTEOMICS WITH THE F
OX COLUMN 1FCS COMPLEX AND CAN
SHOW THE COMPOUND BRINGS NST2
TOGETHER IN CELLS.
I WANT TO END GOING BACK TO THIS
ANTAGONISTIC AND PLASTICITY
ASPECT OF THE POLY COMB MARKS.
THIS IS A TIME SCALE OF
TREATMENT AND MEASURING THE
GLOBAL LEVELS IN A CELL THE K36
AND K27 MARK.
THERE'S A SLOW -- WE DEGRADE
NST2 ALMOST VERY QUICKLY IN A
MATTER OF FOUR HOURS IT'S GONE.
IT TAKES DAYS FOR THE MARKS TO
START TO CHANGE AND IT'S OVER IN
WEEKS FOR THE MARK TO GO AWAY
AND YOU START TO SEE THE KH27
BIMETHYL MARK INCREASING.
AND ONE IS ASSOCIATED WITH
TRANSCRIPTION ACTIVATION AND ONE
WITH TRANSCRIPTIONAL REPRESSION.
AND THEN FURTHER ONCE THE MARK'S
ON THE CHROMATIN CHANGE THERE'S
LIKELY A REPROGRAMMING AND
CHANGE IN GENE EXPRESSION
PROGRAM THAT CAN HAPPEN OVER
FURTHER TIME.
VERY INVESTIGATED THIS AND
IDENTIFIED AND GENERATED A NICE
ISO GENIC PAIR OF CELL LINE AND
HAVE THE ACTIVATING MUTATION AND
THE CELLS ARE SOMEWHAT DEPENDENT
ON THE HIGH EXPRESSION OR HIGH
ACTIVITY OF NST2.
WHEN I THE INHIBITOR WE INHIBIT
EXPRESSION AND IT INCREASES WITH
TIME AND GOING FROM 12 DAYS, 15,
18, 21 YOU SEE A REMARKABLE
DECREASE IN CELL VIABILITY AND
THESE ARE THE CELLS THAT HAVE --
ACTIVATING THE MUTATION.
THE RED.
AND THE ACTIVE COMPOUND AND THE
RED YOU WANT TO FOCUS ON AND
THEN IF WE TEST IN COMBINATION
WITH DEXOMETHOSONE AND THE
REOCCURRENCE IS MET WITH
RESISTANCE TO THIS AND SO IS THE
MUTATION WITH NSD2 AND WE SEE A
SIGNIFICANT INCREASE IN RECOVERY
OF THE DEXOMETHOSONE RESISTANCE.
SO LET ME SUMMARIZE BY SAYING
THE CHEMICAL PROBES AND CHEMICAL
BIOLOGY ARE AMONG THE MOST
USEFUL TOOLS IN DRUG BIOLOGY AND
DRUG DISCOVERY.
EPIGENETIC CHEMICAL PROBES
IDENTIFIED MULTIPLE SUB TYPE AND
WE'VE GIVEN OUR COLLECTION TO
MANY OF YOU IN THE AUDIENCE AND
ALSO MANY OTHERS AND IT IS
INSPIRING NOVEL THERAPEUTIC
HYPOTHESES AND PROCESSES.
GOOD CHEMICAL PROBES ARE VERY
IMPORTANT TO THE BIOLOGY AND OUR
ORGANIZATION HAS INITIATED A
NUMBER OF INITIATIVES TO PROMOTE
BEST PRACTICES IN USING CHEMICAL
PROBES.
THE CHEMICAL PROBES PORTAL HELPS
YOU DETERMINE THE BEST PROBE AND
HELP DRUG DISCOVERY GO FASTER IN
THE FUTURE.
I WANT TO THANK ALL THE GIANTS
ON WHOSE SHOULDERS I STAND AND
IN MY LAB AND MANY COLLABORATORS
PARTICULARLY FOR THE STORIES I
TALKED ABOUT AND LINDSEY JAMES'
LAB AT THE UNIVERSITY OF NORTH
CAROLINA AND MY COLLEAGUES AT
PRINCESS MARGARET.
THE SGC SITE IN TORONTO I LEAD
AND CHEMISTS WHO MAKE THEIR
CHEMICAL PROBES AVAILABLE FOR
EVERYONE TO USE.
OUR FUNDING FROM THE SGC AND
I'LL END WITH A QUICK PLUG FOR
TORONTO.
>> THIS IS INCREDIBLY VALUABLE.
WE HAVE TIME FOR MAYBE A COUPLE
QUICK QUESTIONS BUT THEY HAVE TO
BE QUICK QUESTIONS.
THANKS.
>> HI, THANK YOU FOR A GREAT
TALK.
I'M FROM GEORGE WASHINGTON
UNIVERSITY.
IN YOUR FIRST STORY ABOUT THE
PRMT ONE INHIBITION, DID YOU
LOOK AT WHAT CHARACTERIZED THE
SENSITIVE CELL LINES IN TERMS OF
MORE OF THE ALOS THAT WERE IN
THE SPLICING VARIANTS?
WAS THERE A DIFFERENT IN THE
BASELINE LIKE OTHER EPIGENETIC
MODIFICATIONS?
>> DID WE CHARACTERIZE THE GENES
THAT CONTAINED THE ALOES.
>> YOU NOTICE THE THE SENSITIVE
ONES HAD THE MORE ALLO IRs.
I WAS CURIOUS ABOUT WHY.
>> THEY HAD THEY BOTH HAD THEM
INADVERTENTLY EXPRESSED.
THE SENSITIVE ONES THOSE HAD THE
INVERTED REPEATS IN THEM WHERE
THE OTHER SENSITIVE LINES DID
NOT.
>> I'M IN EPIGENETICS.
GREAT TALK.
I HAD A MORE GENERAL QUESTION
ABOUT THE PROBE COLLECTION WHICH
OBVIOUSLY HASN'T BEEN ENRICHED
WITH INHIBITERS AND HASN'T GROWN
TO INCLUDE THE DE GRID
GRADERS -- DEGRADERS AND WITH
BREAKTHROUGHS IN THE FIELD WHERE
DO THEY STAND?
>> A NUMBER OF OUR CHEMICAL
PROBES AND OUR READERS AND
METHYL TRANSFERASES HAVE BEEN
TURNED INTO TARGETED DEGRADERS.
SOME ARE INCLUDED IN OUR
COLLECTION LIST.
AND WE HAVE SOME OF THESE IN
FRANKFURT AND THEY'RE ALL IN
ENCUMBERED COMPOUNDS.
WE ENCOURAGE ANYBODY WHO WANTS
TO TO MAKE DEGRADERS OUT OF THEM
BUT THEY'RE COMING.
IT'S FAST AND FURIOUS NEW
DEGRADERS COMING FOR A LOT OF
THESE.
>> THANK YOU VERY MUCH.
I APPRECIATE IT.
AND SO WE'RE GOING TO HAVE THE FIRST BREAK.
>> THANK YOU.
IT'S MY PLEASURE TO INTRODUCE
OWE NEXT SPEAKER AND WE'RE IN
FOR A REAL TREAT TO HERE HIS
STORY ABOUT 5 AZACYTIDINE FROM
MYOGENESIS TO MDS.
HE WAS IN SOUTH AFRICA BEFORE
COMING A CHIEF SCIENTIFIC
OFFICER IN GRAND RAPIDS IN
MICHIGAN.
HE'S FROM SOUTHERN CALIFORNIA
AND BECOMING A DISTINGUISHED
PROFESSOR AND DIRECTOR.
THANK YOU.
>> THANK YOU.
IT'S REALLY GREAT TO BE HERE.
THANK YOU FOR PUTTING TOGETHER
THIS SYMPOSIUM.
I'M GOING TO TALK ABOUT 5
AZACYTIDINE AND DESCRIBE MY
STORY HOW I GOT INTO EPIGENETICS
AND HOPEFULLY BRING YOU UP TO
SPEED WITH THE COMPOUND.
MY STORY STARTS WITH THIS 5
AZACYTIDINE DEVELOPED IN PRAGUE
IN THE '60 AS A CHEMO
THERAPEUTIC AGENT AND DID A FEW
CLINICAL TRIALS IN EUROPE AND
THEN SENT THE DRUG TO THE
U.S. WHERE IT UNDERWENT CLINICAL
TESTING AND THIS PAPER WAS A
REVIEW PAPER ON 5 AZACYTIDINE
FROM 1976 AND YOU'LL NOTICE THE
ADDRESS IS BETHESDA, MARYLAND
WHERE WE ARE.
THIS CARRIED OUT CLINICAL
STUDIES DONE HERE AT THE
NATIONAL CANCER INSTITUTE.
THIS DRUG WAS FOUND TO BE NOT
THAT EFFECTIVE IN TUMORS AND HAD
SOME PROMISE WITH AML.
WHEN WE PUT THESE ON MOUSE CELLS
WE WERE SHOCKED TO SEE THE
DEVELOPMENT OF FUNCTIONAL
CONTRACTILE MUSCLE CELLS SEVERAL
DAYS OR WEEKS AFTER EXPOSURE.
THIS PAPER WAS PUBLISHED IN
NATURE IN 1977.
YOU COULD TREAT FOR THE DRUG FOR
24 HOURS, REMOVE THE DRUGS AND
THE CELLS WOULD THEN GROW AND AT
THE TIME WE SAW THE DEVELOPMENT
WITHOUT PLANT OR MUSCLE AND ONE
OF THE FIRST EXAMPLES OF
CELLULAR REPROGRAMMING AND
PROGRAMMING WITH A SMALL
MOLECULE.
NATURALLY I WAS VERY INTERESTED
IN THAT.
I'VE NEVER HEARD OF DNA
METHYLATION.
NEVER HEARD OF EPIGENETICS.
SOMEBODY MENTIONED TO ME MAYBE
WE SHOULD THINK WITH THE FACT
THIS IS ROBERT STALWAGGIN IT MAY
BE INHIBITOR OF DNA METHYLATION
AND SO WE TESTED THAT AND FOUND
OUT VERY QUICKLY -- IN FACT IT
WAS A VERY POWERFUL INHIBITOR OF
DNA METHYLATION AND NOT ONLY
THAT COULD INDUCE CELLULAR
DIFFERENCES AND THIS STIMULATED
ARTHUR RIGS WAS ONE OF THE FIRST
PEOPLE TO SUGGEST DNA
METHYLATION WAS AN IMPORTANT
PROCESS IN CELLS AND MIGHT BE
INVOLVED IN EPIGENETICS.
HE WAS IN DUARTE, CALIFORNIA AND
I ESTABLISHED A LONG-TERM
INTERACTION WITH ART.
WE MEASURED THE AFFECT ON DNA
METHYLATION AND INDUCING MUSCLE
FORMATION.
YOU CAN SEE YOU CAN INHIBIT DNA
METHYLATION BY A LARGE AMOUNT
MORE THAN 50% AND THE INCREASING
DOSES DIDN'T HAVE MUCH MORE OF
AN AFFECT AND ONCE YOU INHIBIT
YOU CAN'T INHIBIT FURTHER
BECAUSE YOU REMOVE THE
TRANSFERASE FROM THE CELL AND
THE RESPONSE CURVE WITH RESPECT
TO DIFFERENTIATION YOU INCREASE
THE DOSE AND THE AFFECT GOES
DOWN.
THIS ACTUALLY, I BELIEVE, HELD
UP THE CLINICAL DEVELOPMENT OF
THIS DRUG FOR MANY MANY YEARS
BECAUSE MANY STUDIES SHOULD FOR
THE MAXIMUM TOLERATE DOSE AND
THIS DOESN'T AND WHEN IT WAS
PEARED BACK THE VALIDITY BECAME
APPARENT.
WE FIGURED OUT HOW THE DRUG
WORKS TO SOME EXTENT.
IT REQUIRES THE INCORPORATION OF
DNA AND WE DIDN'T KNOW ABOUT THE
DIFFERENT DNA TRANSFERASES AT
THE TIME AND FORM A BOND WITH
THE TRANSFERASE AND CAUSES AN
IMMEDIATE REDUCTION IN THE LEVEL
OF PROTEIN IN THE CELL.
SO IT'S A MECHANISM BASED
INHIBITION WHICH LEADS TO THE
DEGRADATION OF THE ENZYME.
IT ALSO CAUSES DNA BREAKS WHICH
COMPLICATE SOME OF THE
INTERPRETATION.
HOW'S IT WORK?
WHAT CAUSE THE CHANGES?
WE SPENT A LOT OF TIME TRYING TO
FIGURE THIS OUT.
WHEN YOU TAKE MOUSE EMBRYO CELLS
THE CELLS GROW IN THE CULTURE
DISH AND WHEN THEY BECOME
IMMORTAL THEY KNOW THEIR
METHYLATION RESULTS IN THE
FORMATION OF IMMORTAL CELLS AND
WHEN THEY'RE TREATED THEY'RE
TURNED BACK TO STEM CELLS AND
CAN DIFFERENTIATE INTO THE THREE
LINEAGES AND HERALD WINETRAUL
LOOKED AT THE DETERMINATION
FACTOR CALLED MYODE1.
AND MY FIRST Ph.D. STUDENT AND I
CELEBRATED THE WORK.
THIS WAS TAKEN PROBABLY IN '83
OR SO.
YOU CAN SEE I HAVE UNDER GONE
EPIGENETIC AGING AS ANDY
FEINBERG POINTED OUT EARLIER ON
AS WELL.
BUT THIS WAS AN EXCITING TIME
AND WE WERE HOOKED ON
EPIGENETICS AND I'VE NEVER
WAVERED FROM EPIGENETICS EVER
SINCE.
NOW, I'M ACTUALLY A CANCER GUY.
I'M NOT A DEVELOPMENT BIOLOGIST
BUT I NOTICED AT THAT TIME THERE
WAS INTEREST IN DNA METHYLATION.
MELANIE ORNIK AND FRED BECKER AT
THE INDIANA CANCER CENTER,
PEOPLE STARTED MEASURING DNA
METHYLATION IN DIFFERENT TUMORS.
I DON'T THINK THIS WENT VERY FAR
BECAUSE NO ONE WAS INTERESTED IN
IT AND NOT ONLY WAS THERE NO
SPECIFICITY.
CANCER CELLS HAVE LOTS OF
DIFFERENT CHANGES BUT THEY WROTE
A REVIEW AND I HAVEN'T LOOKED AT
IT FOR 40 YEARS SINCE I WROTE IT
WITH HIM AND A WAS STUNNED
BECAUSE THERE'S A FIGURE IN
THERE AND I THINK ONE OF THE
VERY FIRST REVIEWS EVER OF DNA
METHYLATION AND CANCER IN 1983
IN WHICH WE PROPOSED THE
METHYLATION THAT CAUSED INSULT
AND YOU CAN GET MUTATIONS AN
DECREASED METHYLATION BASED ON
ALL THIS STUFF AND OF COURSE
THESE COULD BECOME MORE
PROGRESSIVE.
I THINK WE SORT OF HAD THE RIGHT
IDEA BY GUESSWORK AND OF COURSE
WE DID MANAGE -- WHEN ANDY'S
PAPER CAME OUT WE ACTUALLY
REALLY FELT, WELL, THIS REALLY
HAS LEGS BECAUSE ANDY TOOK IT
FROM A GENERAL MODEL TO SHOWING
SPECIFICALLY IN HUMAN TUMORS
WHICH ARE UNCULTURED YOU COULD
SEE CHANGES IN DNA METHYLATION
AT THE GENE LEVEL.
SO WE ALL KNOW METHYLATION LOOKS
LIKE THIS.
AND DURING THE FORMATION OF
CANCER YOU GET THESE CHANGES.
OF COURSE ANDY AND STEVE BAYLIN
PLAYED MAJOR ROLES IN
UNDERSTANDING THESE TWO
PROCESSES.
SO THE QUESTION IS THEN IS HOW
DOES THIS HAPPEN.
WHY DO CELLS CHANGE DNA
METHYLATION PATTERNS?
WELL, EARLY ON, FRED BECKER
ACTUALLY LOOKED TO SEE WHETHER
THE DNA METHYL TRANSFERASE WAS
TO BLAME AND WE DID THIS AND
FOUND THAT IN FACT THOUGH THE
DNA WAS HYPERMETHYLATE THE LEVEL
OF ENZYME WAS INCREASED.
THAT DIDN'T MAKE MUCH SENSE SO
WHY WAS THAT?
AGAIN, BECAUSE HISTORICAL
SYMPOSIUM WE DIDN'T KNOW AT THAT
TIME ABOUT THE EXISTENCE OF
MULTIPLE DNA METHYL TRANSFERASE
THE DI -- DE NOVO TRANSFERASES
AND THE ENZYMES CAN DO A
BEAUTIFUL JOB WITH NAKED DNA AND
NOT SO GOOD WHEN IT COMES TO DNA
IN THE NUCLEOSOME AND EVERYBODY
KNOWS ABOUT EHRF1 AND THE
ACCESSORY PROTEIN EXPRESSED
EXCLUSIVELY IN EMBRYONIC STEM
CELLS AND A SPLICING WHICH IS
ALSO AN ACCESSORY PROTEIN THORR
NOT REALLY ACKNOWLEDGED IN THE
FIELD.
-- I'M NOT GOING TO TALK ABOUT
DNA T1 BECAUSE WE HEARD
SOMETHING ABOUT THAT ALREADY BUT
DNA 302 HAS A SHORT VERSION WITH
A PROMOTER AND A VARIANT OF
SPLICE 3D1 A CATALYTICALLY
ACTIVE DOMAIN AND IT ALLOWS IT
TO BIND TO A CELL AND HERE IN
NORMAL TISSUES THE KEY GUY IS
THE 3A1 AND IN CANCERS THESE ARE
ALL DIFFERENT CANCERS YOU CAN
SEE 3A2 THE SHORT VERSION COMES
UP THE SAME TIME AS THE
ACCESSORY PROTEIN.
THESE ARE SEQ EXPERIMENTS AND
WHAT IS IMPORTANT IS TO REALIZE
IS THEY WORK AS A HETRO DIMER.
WE SOLVED THE STRUCTURE OF THE
HET
HETROTETROMIN AND THE SO-CALLED
ACIDIC PATCH AND IT ANCHORS THE
COMPLEX IN THE ACIDIC PATCH AND
HAVE YOU TWO ACTIVE ENZYMES HERE
AND ANOTHER 3B3 THERE.
THERE'S A CONSISTENT FINDING AND
THIS SEEMS TO BE A PRETTY
UNIVERSAL AS TO WHAT'S GOING ON
HERE.
THE UPREGULATE THE SHORT TERM
EXPRESSED IN THE EMBRYO AND DOWN
REGULATE THE 3B3.
WE THINK MAYBE THE RATIO BETWEEN
THE ENZYME AND THE ACCESSORY
PROTEIN CHANGES AND MAYBE THIS
CONTRIBUTES TO THE FORMATION OF
THE DIFFERENT METHYLATION
PATTERNS.
WE DON'T KNOW THAT YET BUT WE'RE
CERTAINLY TRYING TO FIGURE OUT
WHETHER THAT'S REALLY THE CASE.
WE'VE GONE FROM MEASURING CRUDE
EXTRACTS IN CELLS TO LOOKING AT
THE CRYO EM STRUCTURE AND HOW
THE RATIO CHANGES IN THE
FORMATION OF HUMAN CANCER.
AND IT TOOK 40 YEARS TO GET
TESTED WITH THE SYNDROME AND
THERE WAS A PIVOTAL STUDY
PUBLISHED IN 2009 SHOWING 5
AZACYTIDINE COMBINED WITH
CONVENTIONAL CARE IN A TREATMENT
OF HIGH RISK MDS WAS AN
EFFECTIVE TREATMENT.
AND THE MDS IS A PRE-LEUKEMIC
CONDITION.
MOST PEOPLE DON'T GET CURED.
MOST PEOPLE HAVE AN EXTENDED
LIFE SPAN AND ALSO HAVE AN
EXTENDED QUALITY OF LIFE.
AND SHE RECOGNIZED MY LICENSE
PLATE AND SHE CAME UP TO ME AND
SAID THANK YOU, DOCTOR.
I'M NOT REALLY A DOCTOR BUT
EVERY YEAR I GET A CHRISTMAS
CARD FROM HER.
SHE WAS GOING TO GET A BONE
MARROW TRANSPLANT AND COULDN'T
DO IT BUT SHE GOT ON 5
AZACYTIDINE AND IS STILL ALIVE.
IF ONLY WE COULD DO THAT FOR
EVERY PATIENT.
WHY DO PATIENTS RESPOND TO THESE
THERAPIES.
THIS IS FROM A REVIEW STEVE AND
I WROTE.
ONE MORE THING I THINK IS
INTERESTING IS IT THE IDEA OF
VIRAL MIMICRY WHICH I KNOW
POSTDOCS CAME UP WITH THIS
BEAUTIFUL IDEA THAT MAYBE ONE
WAY THEY WORK IS BY INDUCING THE
STATE OF VIRAL MIMICRY COULD
TRIGGER THE IMMUNE RESPONSE.
THAT IS A COMMON THOUGHT MAYBE
THAT'S SOME OF THE WAY THE
EPIGENETIC DRUGS WORK AND YOU
HEARD TALK ABOUT THAT TODAY AS
WELL.
SO VERY EXCITING.
MAYBE WE CAN NOW FIND OUT HOW
THESE DRUGS WORK.
NOW, WE'RE PUSHING THE ENVELOPE
WITH OUR STAND UP TO CANCER
EPIGENETICS DREAM TEAM TO TAKE
THIS TO THE NEXT LEVEL.
I WANT TO TELL YOU ABOUT AN
EXCITING TRIAL WE JUST
COMPLETED.
THESE ARE THE TWO PHYSICIANS
THAT REALLY COMPLETED IT.
WE TOOK PATIENTS WHO FAILED 5
AZACYTIDINE TREATMENT AND
TREATED THEM WITH A NEW
EPIGENETIC DRUG DNA METHYLATION
INHIBITOR TO SEE WHETHER WE
COULD CHANGE THE PATIENTS AND
MAKE THEM SENSITIVE TO THE
THERAPIES.
AGAIN THE RESULTS ARE ACTUALLY
VERY INTERESTING.
IT'S A VERY SMALL TRIAL AND SO
THE NUMBERS OF PATIENTS ARE VERY
SMALL BUT THESE ARE THE SURVIVAL
PATIENTS IN THE TRIAL AND YOU
CAN SEE THERE'S ONE GROUP OF
THEM THAT DID REALLY WELL, FIVE
OF THEM, WHEREAS THE OTHER ONES
DID NOT DO SO WELL.
THE INTERESTING THING WAS THOSE
PATIENTS THAT HAD MUTATIONS IN
AL601 IN ONE OF CHERYL AS A
SLIDE WAS INTERESTING TO THINK
ABOUT.
THEY HAVE A MUTATION IN THE T
CELL, IT MAKES THAT PATIENT HAVE
A BETTER SURVIVAL AT LEAST IN
THE SMALL CLINICAL TRIAL AND
ONLY FIVE PATIENTS BUT THEY ALL
DID REALLY WELL.
SO WHAT WE THINK IS HAPPENING IS
THE MUTATION MAY BE INCREASING
THE LIFE SPAN OF THE T CELLS.
AND IN CONTEXT OF THIS
IMMUNOTHERAPY.
SO WE ARE SEEING THIS ACTIVELY
AND I FIND IT INTERESTING
BECAUSE WE THINK MUTATIONS FOCUS
ON MUTATIONS IN THE MALIGNANT
CLONE AND FORGET ABOUT THOSE
MUTATIONS THAT MIGHT OCCUR IN
THE IMMUNE CELLS AS IN THIS
PARTICULAR TRIAL.
SO WHERE ARE WE WITH THE
DEVELOPMENT OF DNA METHYLATION
INHIBITORS?
THIS GOES BACK TO 1977.
WE TRIED OTHER DRUGS WHICH
DIDN'T MAKE IT.
I TALKED ABOUT THE NEW DRUG AND
BUT NOT BETTER THAN 5
AZACYTIDINE.
ONE OF THE EXCITING DEVELOPMENTS
BEEN THE DEVELOPMENT OF THIS GSK
DRUG WHICH ACTUALLY -- SHOULDN'T
BE 2003.
THERE'S SOMETHING WRONG WITH THE
DRUG.
IT DOESN'T HAVE TO GET IN THE
DNA TO INHIBIT METHYLATION AND'
SPECIFIC INHIBITOR OF DNA 1 AND
THERE'S A TRIALIVE SICKLE CELL
ANEMIA PATIENTS WITH THE HOPE OF
USING THE EPIGENETIC APPROVE --
APPROACH TO TURN ON THE GENES
AND ONE WAS DONE TO TEST WHETHER
THE DRUG COULD IN FACT TURN ON
THE FETAL GLOBIN PATIENT GENE IN
PATIENTS.
SO THINKING BEYOND MALIGNANCY
THESE DRUGS HAVE TREMENDOUS
POTENTIAL.
SICKLE CELL DISEASE IS NOT A
NICE DISEASE.
INCREDIBLY PAINFUL.
I REALLY HOPE THIS PARTICULAR
DRUG WILL WORK.
WHAT'S INTERESTING NOW IS ALL
THE BIG GUNS IN THE TRIALS AND
NOW CAN SEE IN FACT ALL THE BIG
ONES ARE IN AND THE THERAPIES
ARE NOW MORPHING INTO THE USE OF
COMBINATIONS AND AVAILABLE DRUG.
WHAT IS INHIBITED THE
DEVELOPMENT OF THESE DRUGS TO
SOME EXTENT.
-- I GOT A YELLOW.
I DIDN'T EVEN GET A YELLOW IN
WHAT ARE YOU DOING TO ME?
I DON'T HAVE TO GO THROUGH BUT
WE'RE AT A VERY EXCITING AGE.
I SHARED SOME OF THE PEOPLE AT
THE BEGINNING OF THIS JOURNEY
WHICH IS GOING ON FOR ACTUALLY
MORE THAN 40 YEARS.
THIS IS MY CURRENT LAB BUT I
WANT TO END WITH A VERY
IMPORTANT THANK YOU.
I WANT TO THANK THE NATIONAL
CANCER INSTITUTE FOR 43 YEARS OF
CONTINUOUS SUPPORT.
I'VE BEEN SUPPORTED BY THE NCI
FOR 40 YEARS AND HAVE ANOTHER 7
YEARS TO GO, BY THE WAY.
I'M EXCITED ABOUT THAT AND A
SHOUT OUT TO PAUL O'CONNOR MY
PROGRAM OFFICER.
PAUL HAS BEEN WITH ME NOT
THROUGH THE WHOLE JOURNEY FOR
MOST OF IT AND SOMETIMES I THINK
HE HAS MORE CONFIDENCE IN ME
THAN I HAVE AND WITHOUT HIM
PUSHING ME I DON'T KNOW I WOULD
BE HERE TODAY TALKING TO YOU.
I WANT TO THANK YOU AND THE
WHOLE NCI.
THANK YOU.
>> FOR THE THERAPY WITH PATIENTS
IN CLINICAL AND CAN YOU EXPLAIN
WHY THOSE CELLS SURVIVE LONGER
WITH THE TREATMENT WITH THE
INHI
INHIBITER.
>> I THINK IT'S PREVENTING
EXHAUSTION.
PROPER T CELL BIOLOGY REQUIRES T
CELLS TO BECOME EXHAUSTED AND
STOP CONTINUOUSLY EXPANDING AND
THAT'S REGULATED AND PEOPLE
CONCENTRATED ON DNA METHYLATION
AND DE NOVO DNA METHYLATION IS
PART OF EXHAUSTING CELLS.
I'M NOT AN IMMUNOLOGIST BUT I
THINK WHAT'S HAPPENING IS AL6L1
IS ALLOWING THE T CELLS TO AVOID
BECOMING EXHAUST.
THAT'S THE HYPOTHESIS.
>> WOULD YOU COMMENT ON THE FULL
GENETIC DRUGS ON RESISTANCE AND
RECURRENCE.
>> THE QUESTION IS ABOUT
RESISTANCE AND RECURRENCE.
I GUESS THAT HAPPENS WITH ALL
DRUGS, RIGHT.
I THINK WITH THE EPIGENETIC
DRUGS MAYBE THE RESISTANCE IS
DUE TO, FOR EXAMPLE, NOT
PHOSPHORYLATING THE DRUG TO
INCORPORATE DNA OR ENHANCED
DE-AMINATION BECAUSE WE DON'T
KNOW.
SO USING ADDITIONAL THERAPIES,
FOR EXAMPLE, AS I'VE SHOWED YOU
WITH IMMUNOTHERAPY MIGHT BE
HELPFUL.
>> I'M WITH NCI.
SO HEMATOLOGY MALIGNANCIES THE
TUMOR CELLS ALSO CARRY A 6L1
MUTATION DID YOU LOOK
IN COMPARISON TO THE T-CELLS.
>> WE HAVEN'T GONE THAT FAR,
BUT AL6L1 IS A -- IN OTHER
WORDS, AL6L1 IS VERY OFTEN
ASSOCIATED WITH THE DEVELOPMENT
OF MDS AND AML.
I THINK WHAT'S HAPPENING IS IN
THESE PARTICULAR PATIENTS THE
MUTATION HAS OCCURRED AT A VERY
EARLY STEM CELL, AND AS THE
CELLS ARE BRANCHED INTO THE
MYOLOID AND T-CELL LINEAGE, MOST
OF THE TIME YOU DON'T GET AL6L1
MUTATION IN THE T-CELLS, BUT
WHEN THEY ARE PRESENT IN THE
CONTEXT OF IMMUNOTHERAPY --
BECAUSE, REMEMBER, THESE
PATIENTS DIDN'T RESPOND BEFORE
OR STOPPED RESPONDING.
I THINK IT'S IN THE CONTEXT OF
IMMUNOTHERAPY WHERE YOU ARE
CHANGING THE BIOLOGY OF THE
T-CELLS, WHICH MAYBE IS HAVING
THIS IMPACT.
I HOPE THAT ANSWERS THE
QUESTION.
THANK YOU.
>> SO OUR NEXT SPEAKER COULDN'T
BE HERE BECAUSE OF THE TRAGEDY
THAT HAPPENED.
SHE'S FROM WEIZMANN INSTITUTE,
AND SHE'S GOING TO TALK ABOUT
USING HER VERY INNOVATIVE
TECHNOLOGY FOR DECODING THE
CANCER EPIGENOME FOR RESEARCH
AND DIAGNOSIS, AND SHE'S GOING
TO LISTEN IN, BUT SHE'S ABLE TO
ANSWER QUESTIONS AFTERWARD.
>> I WOULD LIKE TO START BY
THANKING THE ORGANIZERS FOR
INVITING ME TO PRESENT MY LATEST
WORKS IN THIS MEETING.
I WILL SAY THAT I'M SURE ALL OF
YOU ARE AWARE THAT THESE ARE
ESPECIALLY DIFFICULT TIMES IN
ISRAEL.
MY HEART IS BROKEN FOR ALL THE
FRIENDS AND FAMILIES THAT HAVE
LOST LOVED ONES IN THE TERROR
ATTACKS THAT WE HAVE SUFFERED,
AND I THANK ALL OF YOU FOR YOUR
SUPPORT.
TO THE SCIENTIFIC PART, I WILL
TELL YOU ABOUT MY LIFE'S WORK ON
DECODING THE EPIGENOME FOR
CANCER IN RESEARCH AND
DIAGNOSTICS.
OBVIOUSLY I DON'T NEED TO
INTRODUCE YOU TO THE OBVIOUS
WHERE EPIGENETICS IS IMPORTANT,
AND WE KNOW WE HAVE THE T-CELLS
THAT RECRUIT FROM THE GLOBAL
NUCLEOZOME AND SPECIFICATIONS.
IT IS THOUGHT THAT THESE
SPECIFICATIONS AND PATTERNS ARE
ESSENTIAL FOR REGULATING
ESSENTIALLY EVERY DNA TEMPLATE
PROCESS IN THE CELL.
THIS NETWORK OF MODIFICATIONS IS
HOW THEY CAN CREATE SO MANY
TYPES, AND IT'S OFTEN
DEREGULATED IN CANCER.
WE KNOW THAT MORE THAN 50% OF
HUMAN CANCER IS MUTATIONS AND
COMMUNOREGULATORS.
MY LAB IS DEDICATED TO NEW
TECHNOLOGIES TO DECODE GENOMES
IN EPI REGULATION.
ABOUT TWO OF THESE TECHNOLOGIES
THAT ARE VERY DIFFERENT.
THE FIRST ONE IS SINGLE MOLECULE
BAITSED ASSAY THAT ALLOWS YOU TO
CAPTURE NUCLEOZOMES AND DECODE
THEIR PATTERNS OF NOTIFICATIONS.
WHILE THE OTHER TECHNOLOGY, A
SINGLE-CELL-BASED ASSAY THAT
ALLOWS YOU TO ADDRESS QUESTIONS,
SUCH AS EPIGENETIC HYPERGENEITY
IN CANCER, WHICH IS SOMETHING
WE'RE VERY PASSIONATE ABOUT.
SINGLE CELL MOLECULE TECHNOLOGY,
WE TAKE INDIVIDUAL NUCLEOZOMES,
AND WE FRAGMENT THE MICRONUCLEUS
FROM PLASMA, AND I'LL TELL YOU
MORE ABOUT IT IN A BIT.
WE TAKE THESE NUCLEOZOMES AND
ADD IT TO THE DNA END AND ALSO
TELL THEM DID, THIS ENTIRE
TECHNOLOGY IS BUILT ON INTERNAL
REFLECTION MICROSCOPY.
ALL YOU NEED TO KNOW ABOUT IT IS
THAT ESSENTIALLY ALL THE LIGHT
ON THE SURFACE IS REFLECTED BACK
INTO A NARROW OBJECTIONIZATION
WEIGHT.
ALL THE MOLECULES ARE ABOUT 1
HNK NANOMETERS ARE EXCITED, AND
THIS IS HOW YOU DON'T HAVE ANY
BACK FROM THE SOLUTION, AND IT
ALLOWS YOU TO IMAGE INDIVIDUAL
FLOOR FOR US.
YOU SEE THERE ON THE IMAGE EVERY
SPOT IS A SINGLE NUCLOESOME THAT
WAS AT THE SURFACE, AND WE ARE
IMAGING MILLIONS OF THESE.
MILLIONS OF NUKE SYNDCLEONUCLEO.
WE DON'T ACTUALLY THINK ABOUT IT
OFTEN, BUT ANTI-BODIES OFTEN WE
WANT TO KNOW FOR EVERY
NUCLEOSOME HOW IT IS MODIFIED.
SO ESSENTIALLY THIS IS WHERE WE
LEVERAGE.
WE JUST TAKE SEVERAL IMAGES OVER
TIME, SO WE CAN THEN SUM ALL
THESE EVENTS AND COLLOQUELIZE
THEM TO WHERE THE NUCLEOSOMES
ARE.
NOW WE CAN KNOW HOW MANY
NUCLEOSOMES HAVE THIS
MODIFICATION OR ANOTHER, OR IT
CAN BE EFFECTIVE FOR BOTH OF
THESE MODIFICATIONS.
THIS IS REALLY CRITICAL BECAUSE
NOW THAT WE HAVE ALL THE
INFORMATION, WE CAN ALSO WASH
THESE ANTIBODIES AND ADD NEW
ONES AND ACTUALLY GENERATE
HIGHER ORDER COMBINATIONS OF
THESE SYSTEM MODIFICATIONS ON
INDIVIDUAL NUCLEOSOMES.
SO AFTER WE DO THAT, NOW WE WANT
TO KNOW, OF COURSE, WHAT IS THE
SEQUENCE ASSOCIATED WITH THIS
NUCLEOSOME.
THIS IS STILL VERY EXCITING SORT
OF, LIKE, JUST A MASS DATA OF
HOW NUCLEOSOMES MODIFY, BUT WE
ALSO WANT TO IDENTIFY THE
ASSOCIATED SEQUENCE, AND WE DO.
WE JUST TAKE THE PROTEINS FROM
THE SURFACE.
WE DON'T DO ANY FIXATION, AND
THEN WE FOLLOW UP WITH SINGLE
MOLECULE DNA SEQUENCING, WHICH
IN TERM OF CHEMISTRY, IS VERY
SIMILAR TO GENOME SEQUENCING.
WE JUST ADD ONE OF THE
NUCLEOTYPES AND IMAGE WHERE IT'S
INCORPORATED WITH THE MIXTURE.
YOU CLEAN THE FLOOR UNTIL THE
NEXT ONE UNTIL YOU BUILD A
SEQUENCE, WHICH IS LARGE ENOUGH
AND LONG ENOUGH SO YOU CAN ALIGN
TO THE GENOME.
THIS TECHNOLOGY WE USE IN THE
BODY A LOT TO ADDRESS BASIC
QUESTIONS OF EPIGENETIC
VARIATIONS IN CANCER.
TODAY WE'LL TALK ABOUT ONLY ONE,
AND IT'S QUITE DIFFERENT, WHICH
IS HOW WE'RE USING IT FOR LIQUID
BIOSIS FOR A CHANCE OF
DIAGNOSIS.
JUST AS AN INTRODUCTION TO THIS
FIELD, PROBABLY ALL OF YOU KNOW
THAT IF I JUST TAKE AN EXAMPLE
FROM ANY OF YOU, BLOOD EXAMPLE,
YOU WILL HAVE SELF-GENIOATED IN
YOUR BLOODSTREAM.
IF YOU ARE HEALTHY THIS ALSO
COMES FROM BLOOD CELLS THAT DIED
AND DUMPED THEIR CONTENT INTO
THE BLOODSTREAM.
SOMEONE, FOR EXAMPLE, THAT HAS
COLORECTAL CANCER, THEN SOME OF
THE DNA WOULD COME FROM COLON
CELLS THAT DIED AS PART OF THE
HEMOROGENIS PROCESS.
MAYBE THE DNA HAS SOME
MUTATIONS, BUT TRYING TO
IDENTIFY THESE MUTATIONS ARE A
WAY TO IDENTIFY CANCER THAT HAVE
FAILED TO A LARGE EXTENT BECAUSE
ALL OF THE MUTATION, WHETHER WE
HAVE CANCER OR NOT, BUT THEN,
YOU KNOW, PEOPLE REALIZE IF WE
CAN SAY WHERE THE DNA IS COMING
FROM, THEN WE MIGHT BE ABLE TO
DETECT WHETHER THERE IS DEATH
ASSOCIATED WITH CANCER IN THE
SPECIFIC TISSUE.
AND I THINK THAT, YOU KNOW, ONE
OF THE GREAT INFLUENCES IN THE
FIELD CAME WITH THE
UNDERSTANDING THAT THIS DNA,
IT'S NOT JUST NAKED DNA, BUT
IT'S ACTUALLY STILL WRAPPED
AROUND THE PROTEINS IN THE
NUCLEOSOME FORM.
OBVIOUSLY THIS NUCLEUS WILL
STILL HAVE THEIR EPI GENETIC
MODIFICATIONS AND ARE TYPICAL OF
THEIR ORIGIN.
EVERY TISSUE IN OUR BODY HAS ITS
OWN FOOTPRINTING.
IF WE CAN DETECT THE
MODIFICATIONS, THEN WE KNOW
WHERE THE NUCLEOSOMES ARE COMING
FROM.
SO WE DEVELOPED A TECHNOLOGY WE
CALLED FOR -- IT'S ESSENTIALLY
BASED ON A SINGLE MOLECULE
IMAGING BECAUSE WE REALIZED THAT
ONE OF THE MAJOR CHALLENGES IS
THAT THE PLASMA IS FULL OF
PROTEINS.
NUCLEOSOMES ARE JUST A TINY
FRACTION, BUT WE ARE USING
SINGLE MOLECULE IMAGING.
ESSENTIALLY WE NEED ALMOST
NOTHING IN TERMS OF MATERIAL.
IF WE CAN CAPTURE THIS
NUCLEOSOMES ON THE SURFACE FROM
THE PLASMA, THEN WE'LL BE ABLE
TO GET A LOT OF INFORMATION AND
DETECT MANY MODIFICATIONS IN THE
COMMUNITORIAL PATTERNS.
THAT IS WHAT I'M GOING TO SHOW
YOU TODAY.
THIS IS EXACTLY WHAT WE SET UP
TO DO.
OF COURSE, OUR FIRST CHALLENGE
WAS PROVING THAT WE CAN ACTUALLY
CAPTURE THE NUCLEOSOMES FROM THE
PLASMA.
WHAT WE DID IS WE WANTED TO MAKE
SURE THAT WE HAVE SPECIFIC
BINDING, SO AS A CONTROL -- SO
WE EITHER DID THESE POLYATELLING
OR TELL THE NUCLEOSOMES WITH
DTB, AND OBVIOUSLY THEY GENERATE
A POLYT-CELL, THEY CAN'T ANCHOR
TO THE SURFACE.
INDEED, YOU CAN SEE HERE THAT
ONLY WHEN YOU HAVE THE ATLE,
THEN YOU REALLY GET THESE
SPECIFIC SPOTS ON THE SURFACE
CORRESPONDING TO THE NUCLEOSOMES
ON THE PLASMA, AND THEN ONLY
WHEN YOU HAVE NUCLEOSOMES, YOU
ACTUALLY GET SIGNALS FROM THE
ANTIBODIES.
IN THIS CASE I'M SHOWING YOU IN
THESE PICTURES A FORM, WHICH IS
A MARK OF ACTIVE TRANSCRIPTION
AND K-27, WHICH IS A MARK OF
REPRESSIVE TRANSCRIPTION, BUT
WE'VE ACTUALLY MEASURED ALL 60
MODIFICATIONS THAT ARE LISTED
HERE, AND ALL OF THAT FROM A FEW
HUNDRED MICROLITERS OF PLASMA.
IT'S LESS THAN 1 ML, WHICH IS
ESSENTIALLY ALMOST NOTHING.
JUST TO SHOW YOU HOW THE DATA
LOOKS LIKE.
SO, FOR EXAMPLE, NOW WE CAN TELL
YOU EXACTLY WHAT IS A PERCENTAGE
OF K27 NUCLEOSOMES IN A PERSON'S
BLOOD.
HERE IS JUST AN EXAMPLE OF ONE
COLORECTAL CANCER PATIENT AND
ONE HEALTHY INDIVIDUAL.
WE CAN SPECIFICALLY COUNT HOW
MANY K27MEL NUCLEOSOMES ARE
THERE?
IT'S LIKE A DIGITAL ASSAY.
THERE ARE SO MANY THINGS WE'RE
MEASURING.
WE CAN ALSO ASK NOW WHAT IS THE
RATIO BETWEEN K4 AND K27
REPRESSIVE NUCLEOSOMES?
IS IT AFFECTED WHEN SOMEONE HAS
CANCER?
MAYBE THE MOST IMPORTANT PART,
WE CAN LOOK AT COMBINATIONS.
HOW MANY NUCLEOSOMES AT THESE
PEOPLE'S BLOOD HAD BOTH K4ME3
AND K27ME3, SO FROM THOSE OF YOU
FROM THE EPIGENETIC FIELD, THIS
IS THE COMBINATION THAT YOU
OFTEN FIND IN STEM CELLS, BUT
IT'S ALSO DEREGULATED IN CANCER.
THIS IS ESSENTIALLY THE ONLY
TECHNOLOGY THAT CAN QUANTIFY
VERY ACCURATELY AND SENSITIVELY
ALL THESE DIFFERENT
COMBINATIONS.
HERE I JUST SHOWED YOU AN
EXAMPLE, BUT WHAT WE DID IS WE
ACTUALLY LOOK AT, AGAIN, THE 60
MODIFICATIONS OR COMBINATIONS
AND RATIOS.
I DO NOT HAVE TIME TO GO INTO IT
TODAY, BUT WE ARE ALSO MEASURING
AT SINGLE MOLECULE LEVEL DNA
DEREGULATION, AND MEASURING
PROTEIN BIOMARKERS WITH VERY
HIGH SENSITIVITY.
I'M SKIPPING THAT, BUT IT'S ALL
IN THE PAPER.
AT THE END OF THE DAY WE ARE
GETTING A LOT OF DATA FROM EACH
PLASMA SAMPLE, AND THIS IS JUST
TO SHOW YOU, YOU KNOW, SOME OF
THE EPIGENETIC PARAMETERS THAT
ARE SIGNIFICANTLY DIFFERENT
BETWEEN COLORECTAL CANCER AND
HEALTHY.
HERE WE ARE TALKING ABOUT A
COHORT OF 60 COLORECTAL CANCER
PATIENTS VERSUS ABOUT 40 HEALTHY
INDIVIDUALS, AND YOU CAN SEE THE
SIGNIFICANT CHANGES, BUT AT THE
END OF THE DAY NO SINGLE
PARAMETER WOULD BE ENOUGH TO
ACTUALLY BE ABLE TO DIAGNOSE
CANCER.
I THINK THIS IS REALLY WHERE THE
POWER OF OUR TECHNOLOGY IS OUR
ABILITY TO MEASURE MANY, MANY
THINGS, AND AT THE END OF THE
DAY YOU CAN SEE THAT EVEN JUST
USING ALL OF THE INFORMATION IN
THE PTA PLOT, YOU CAN SEE THE
COLORECTAL CANCER PATIENTS IN
THE RED HERE ARE CLUSTERED
SEPARATELY FROM THE HEALTHY
INDIVIDUALS.
WHAT I THINK IS MORE STRIKING --
SO WITHIN THE REVISIONS FOR THIS
PAPER, THE REVIEWERS ASKED US TO
TRY TO LOOK AT AT LEAST ONE
OTHER CANCER, SO WE'VE ANALYZED
TEN SAMPLES OF PANCREATIC
CANCER, AND YOU CAN SEE THAT
THESE SAMPLES ACTUALLY CLUSTER
ALSO SEPARATELY FROM THE
COLORECTAL ON THIS BCA PLOT.
THE REASON WHY THE FINDINGS ARE
AMAZING IS CONSIDER THE FACT
THAT UNTIL NOW WE HAVEN'T
SEQUENCED A SINGLE MOLECULE.
SO ESSENTIALLY THIS IS ALL JUST
BASED ON VERY QUANTITATIVE
INFORMATION ABOUT THE
SIGNIFICANT FICTIONS AND THE
COMBINATIONS AND RATIOS, BUT
THERE IS NO SET SEQUENCING.
THE REASON WHY IT'S IMPORTANT IN
THE BIOIS THIS RENDERS THIS
TECHNOLOGY VERY, VERY CHEAP.
WE CAN DO THIS ENTIRE ANALYSIS
WITHIN A COUPLE OF HOURS, SO
IT'S VERY CHEAP AND SIMPLE TO
DO.
ESSENTIALLY, WHEN WE FEED ALL
THE PARAMETERS TO THE
CLASSIFIER, AGAIN, WITHOUT
SEQUENCING, WE CAN ALREADY VERY
ACCURATELY SEPARATE THE HEALTHY
VERSUS THE COLORECTAL CANCER
PATIENTS, BUT OF COURSE, WHILE
WE CAN SEPARATE, WE DID WANT TO
BE ABLE TO SAY THESE ARE COMING
FROM COLON.
FOR THAT WE DID A SEQUENCE, AND
THERE ARE THREE DIFFERENT
PATIENTS.
THE COLORECTAL CANCER AND YOU
CAN SEE HERE THAT ONCE WE ARE
SEQUENCING, THEN WE CAN CLEARLY
SEE THAT THESE NUCLEOSOMES ARE
COMING FROM COLON, AND
SPECIFICALLY THIS RED AND ORANGE
PATIENTS, THEY ALSO HAD
METASTASIS, WHICH WE CAN DETECT
AS WELL USING THIS TECHNOLOGY.
I HOPE I'VE BEEN ABLE TO
CONVINCE YOU THAT THIS
EPIGENETIC TECHNOLOGIES ALLOWS
YOU TO LOOK AT MANY
MODIFICATIONS AND THEIR
COMBINATIONS AND RATIOS WITH
SINGLE MOLECULE IMAGING, SO IT'S
VERY HIGH RESOLUTION AND VERY
QUANTITATIVE INFORMATION, SO
IT'S REALLY UNIQUE IN ITS
CAPACITY TO GENERATE THIS
MULTI-LAYERED INFORMATION FROM
VERY LIMITED BIO MATERIAL, AND
IT REVEALS SIGNIFICANT
DIFFERENCES BETWEEN HEALTHY
INDIVIDUALS VERSUS COLORECTAL
CANCER PATIENTS.
COMBINING WITH SINGLE MOLECULE
SEQUENCING, SO WE CALL IT
EPINOSIC, ALLOWS THE POTENTIAL
OF THE SPECIFIC ORIGIN.
I'LL SHOW YOU FOR COLORECTAL,
BUT WE'VE ACTUALLY DONE THAT
ALSO FOR BREAST CANCER,
PANCREATIC CANCER, AND LUNG
CANCER AND SO ON.
WE CAN VERY EASILY DETECT WHERE
THESE NUCLEOSOMES ARE COMING
FROM ONCE WE ARE SEQUENCING.
I HAVEN'T MENTION IT, BUT WE'RE
TAKING OUR SEQUENCING DATA, AND
WE OVERLAID THE CHIEF DATA WHICH
HAS ALL THE DIFFERENT TISSUES,
AND THAT'S HOW WE CAN DEDUCE THE
TISSUE OF ORIGIN.
I'M SHIFTING GEARS NOW BECAUSE I
DO WANT TO USE THE LAST FEW
MINUTES OF THE TALK TO TELL YOU
ABOUT THE OTHER TECHNOLOGY.
THE SINGLE MOLECULE TECHNOLOGY
REALLY IS GREAT IN GIVING YOU
THE HIGH RESOLUTION OF HOW
INDIVIDUAL NUCLEOSOMES ARE
MODIFIED, BUT IT'S NOT SINGLE
CELL.
I WAS ALWAYS INTRIGUED BY THE
QUESTION IN CANCER.
MOREOVER, IN THE SINGLE MOLECULE
ASSAY, WE'RE FOCUSED ON THE
GENOME MODIFICATIONS THAT
BASICALLY EVERYONE ELSE IS
STUDYING BECAUSE THEY'RE KNOWN
TO HAVE REGULATORY ROLES, BUT
WHAT ABOUT ALL THE OTHER
MODIFICATIONS?
HOW DO WE KNOW THAT THESE ARE
NOT IMPORTANT AS WELL?
IF YOU LOOK AT THE RECENT YEARS
IN THE EPIGENETIC COMMUNITY, YOU
YOU WOULD SEE THAT IT'S VERY
MUCH BIASED TO STUDY THE SAME
MODIFICATIONS, AND THIS IS
SOMETHING THAT WE REALLY WANTED
TO ADDRESS USING THESE NEW
ASSAYS, SO WE DID.
WE TOOK -- I'M SURE MANY OF YOU
AT LEAST IF YOU'RE FROM
IMMUNOLOGY BACKGROUND, YOU'RE
FAMILIAR WITH THAT.
YOU TAKE A PANEL OF ANTIBODIES,
AND YOU CONJUGATE THEM.
IT'S ABOUT 40 ANTIBODIES.
YOU CAN CONJU GATE TLEM TO
DIFFERENT METALS.
WHEN YOU STAIN YOUR ANTIBODIES
AND WHEN A CELL GOES INTO THE
CYTO MACHINE IT GETS BLOWN OFF,
AND ALL THE METALS ARE DETECTED.
YOU GET QUANTITATIVE INFORMATION
FOR EACH CELL, FOR THE 40
ANTIBODIES THAT YOU MEASURED.
WHEN YOU LOOK AT COMMERCIAL
ANTIBODIES, ALMOST ALL OF THEM
START WITH SOMETHING, SO IT'S
VERY MUCH IMMUNOLOGY.
WHAT WE DECIDED TO DO IS TRY TO
DO A CUSTOM-MADE EPIGENETIC
BIOME.
WE GENERATE THIS EPIGENETIC
CUSTOM TO BE ABLE TO LOOK AT
MANY OF THESE MODIFICATIONS AT
LOW RESOLUTION, SO YOU ONLY GET
THE LOWER LEVELS OF THESE
MODIFICATIONS PER CELL, BUT YOU
CAN ACTUALLY GET INFORMATION
ABOUT THOUSANDS OF CELLS IN EACH
EXPERIMENT.
SO THIS IS WHAT WE SET UP TO DO,
AND THE FIRST IMPLEMENTATION OF
THAT WAS WE DID IT FOR PEDIATRIC
GLIOMAS, AND IT WAS FEATURED ON
THE COVER OF MOLECULAR CELL, AND
WHAT WE'RE TRYING TO ILLUSTRATE
IS HOW WHEN YOU TAKE A STEP
BACK, YOU CAN MAYBE SEE THESE
PATTERNS THAT YOU WOULDN'T HAVE
SEEN OTHERWISE.
SORT OF LIKE A BIRD'S VIEW.
SO THE FIRST IMPLEMENTATION WAS
FOR PEDIATRIC GLIOMAS, AND
SPECIFICALLY THE MIDLINE
GLIOMAS.
THEY ARE AGGRESSIVE TUMORS OF
CHILDREN.
MAYBE SOME OF YOU ARE FAMILIAR
WITH THEM BECAUSE ONE OF THE
MAJOR CHARACTERISTICS OF THESE
TUMORS IS THAT THEY HAVE THE
MUTATION OF THYAZINE.
THE AK327M MUTATION.
THIS MUTATION, YOU KNOW, DESPITE
BEING JUST MINOR OF THE
NUCLEOSOMES BECAUSE THERE ARE
MANY GENES IN ENCODING THE WILD
IP ZONES, IT'S COMPLETELY MESSED
UP WITH THE EPIGENETIC
REGULATION OF THESE CELLS AND
LEADING TO THE CANCER.
WHAT WE DID IS WE TOOK -- WE HAD
A PRIMARY CELL LINE FROM ONE OF
THESE PATIENTS, AND WE HAVE
ANALYZED THIS.
THIS IS ONE OF THE FIRST
EXPERIMENTS THAT WE DID, AND
WHAT WE SAW IS THAT WE ACTUALLY
GET TWO EPIGENETIC
SUBREGULATIONS IN THESE CELLS.
THIS IS JUST ALL THE EPIGENETICS
THAT WE HAVE MEASURED.
THE REASON WHY THIS WASN'T IN
THIS TEST IS JUST CONSIDER THE
FACT THAT THESE CELLS HAVE BEEN
EXTENTIVELY STUDIED IN THE
EPIGENETIC CONTEXT.
PEOPLE ALWAYS ANALYZE THIS, AND
WHAT YOU CAN SEE HERE IS THAT
YOU HAVE TWO SUBREGULATIONS THAT
ARE VERY DISTINCT, AND THEY HAVE
DIFFERENT PATTERNS AND
MODIFICATIONS.
WE THINK THAT WE GENERATE THIS
TO SUBPOPULATIONS.
THEY HAVE HIGHER AND LOWER
LEVELS OF THIS MUTE ANT T-CELLS,
WHICH PUSHES DIFFERENT
EPIGENETIC FATES, AND THIS --
FIRST WE THOUGHT THAT THIS COULD
BE MAYBE SOMETHING THIS IS JUST
WEIRD ABOUT THESE SPECIFIC CELL
LINES, BUT WHAT WE FOUND IS THAT
IN EVERY PATIENT WE ARE TELLING
THAT WE HAVE ANALYZED, WE'VE
ACTUALLY SEEN THIS TO EPIGENETIC
SUBREGULATIONS.
IT'S THE SAME SUBREGULATIONS IN
EVERY PATIENT.
WE HAVE THIS INTERTUMOR
HEPEAGENEITY.
IT MEANS THAT THIS IS SOMETHING
THAT YOU COULD POTENTIALLY
TARGET, WHICH WE THINK IS A VERY
CRITICAL ASPECT OF IT.
I WILL NOT HAVE TIME TO GO INTO
THE DETAILS, BUT I WILL TELL YOU
THAT WE VERIFIED THIS H EPA
GENEITY IN A MOUSE MODEL, AND
YOU CAN ACTUALLY INJECT PATIENT
CELLS TO THE KINDS OF MOUSE TO
GENERATE THE TUMORS.
IT'S A COMPLICATED MOUSE MODEL,
BUT WE'VE SEEN THIS HEPAGENEITY
IN HUMAN BIOPSIES FROM CHILDREN.
AGAIN, I DON'T HAVE ENOUGH TIME
TO TELL YOU ABOUT ALL OF THAT,
BUT THESE TWO SUBREGULATIONS
ACTUALLY HAVE DISTINCT FEATURES
BOTH IN TERMS OF PROLIFERATION
CAPACITY AND STEM CELL FEATURES,
AND WE SOMEHOW -- THE TUMOR --
AND I DON'T KNOW -- I DON'T HAVE
AN EXPLANATION FOR THAT, BUT
SOME OF THE TUMORS SEEM TO
BENEFIT FROM MAINTAINING THIS
HEPAGENEITY.
WE'RE NOW DOING FOLLOW-UP
STUDIES TO TRY TO UNDERSTAND IT
BETTER.
JUST IN CONTEXT OF THE GLIOMAS
AND THEN ALSO CONNECTED TO THE
BIOPSY, SO NOW WHAT WE DO IS WE
ARE ALSO COLLABORATING WITH
DOCTORS AND RESEARCHERS IN THE
HOSPITAL OF MICHIGAN, AND WHAT
WE ARE DOING IS USING THE SINGLE
MOLECULE IMAGING.
WE CAN ACTUALLY CAPTURE
NUCLEOSOMES, MUTANT NUCLEOSOMES
FROM THE CHILDREN'S BLOOD AND
SEE WHETHER THEIR RESPONSE TO
TREATMENT AND SO ON.
THIS IS PRETTY CRITICAL IN ALL
OF THESE KIDS BECAUSE THIS IS
SUCH A DEADLY DISEASE.
THERE ARE MULTIPLE CLINICAL
TRIALS, BUT HOW DO YOU EVEN
MEASURE WHETHER A CHILD IS
RESPONDING TO THE TREATMENT?
SO YOU COULD DO AN MRI, BUT THIS
IS USUALLY DIAGNOSED AT EARLY
AGES, SO IT'S NOT SO EASY TO PUT
A 5-YEAR-OLD IN AN MRI MACHINE
EVERY TWO OR THREE MONTHS, OR
YOU CAN TAKE SPINAL FLUID, BUT
THIS IS AN INVASIVE OF
PROCEDURE.
WHAT WE CAN SHOW IS THAT THE
SINGLE MOLECULE TECHNOLOGY IS SO
SENSITIVE THAT YOU CAN ACTUALLY
CAPTURE THIS MUTANT, THIS K27
MUTANT NUCLEOSOMES FROM THE
BLOOD AND ANALYZE IT WITH A
LIQUID BIOPSY TECHNOLOGY.
JUST TO SUMMARIZE, THE SINGLE
CELL ASSAY WE ADAPTED TO
IDENTIFY THE -- AND IDENTIFY
THESE TWO EPIGENTLY DISTINCT
POPULATIONS.
I DIDN'T HAVE TIME TO GO INTO
IT, BUT THE SINGLE CELL DATA CAN
ACTUALLY REVEAL MANY CONNECTIONS
BETWEEN THESE TWO MODIFICATIONS.
WE'VE SEEN THIS NETWORK, AND
IT'S SOMETHING THAT WE'RE REALLY
INTERESTED IN.
I JUST WANT TO CONCLUDE BY
THANKING MY LAB.
WE'VE DONE THIS WORK, AND FROM
ESPECIALLY NOW MANY OF MY
STUDENTS ARE ACTUALLY DRAFTED TO
THE ARMY, AND I'M REALLY PROUD
OF THEM AND I WOULD LIKE TO
THANK I DON'T FEEL THE DOCTORS
AND THE PATIENTS FOR CONJUGATING
TO THIS WORK AND ALSO THE
FUNDING.
THANK YOU SO MUCH FOR LISTENING.
>> EFRAT, ARE YOU THERE?
>> CAN YOU HEAR ME?
>> THANK YOU FOR YOUR WONDERFUL
PRESENTATION, AND I HOPE YOU AND
YOUR LOVED ONES ARE DOING WELL.
DO YOU MIND FOR QUESTION AND
ANSWERS?
>> YES, OF COURSE.
>> ANY QUESTIONS?
IF I MAY ASK YOU TO TALK ABOUT,
I WONDER WITH YOUR LIQUID
BIOPSY, HAVE YOU EVER TRIED
URINE SAMPLES?
>> THAT'S A GOOD QUESTION.
I THINK THAT IN CONTEXT OF
SEVERAL CANCERS IT COULD
ACTUALLY BE VERY BENEFICIAL TO
LOOK AT URINE SAMPLES.
WE HAVEN'T ACTUALLY TRIED THIS
IN THE LAB, BUT I DON'T SEE ANY
REASON WHY IT SHOULDN'T WORK.
RIGHT NOW WE ARE FOCUSING ON LAB
SAMPLES, BOTH FOR DIAGNOSTICS,
BUT ALSO FOR TREATMENT
MONITORING AND IDENTIFYING
RESIDUAL DISEASE AND SO ON.
>> EFRAT, WE MET IN GERMANY A
COUPLE OF MONTHS AGO.
I HOPE EVERYTHING GOES WELL WITH
YOUR FAMILY.
MAYBE YOU CAN TALK ABOUT THE
SINGLE MOLECULE IMAGING.
YOU WERE ABLE TO LOOK AT
SPECIFIC MODIFICATIONS IF YOU
ARE ABLE TO LOOK AT
MULTIAVAILABILITY AND ALSO
COMBINED WITH THE ISSUES --
>> THAT'S A GREAT QUESTION, AND
WE ARE IN THE PROCESS OF DOING
THAT.
FIRST OF ALL, FOR DNA
MANIPULATION, THE WAY WE ARE
EXACTING IT WITH SINGLE
MOLECULE, WE DIDN'T HAVE TIME TO
GET INTO IT, BUT WHAT WE ARE
DOING IS REUSING THE MBD2 DOMAIN
WHICH BINDS SPECIFICALLY TO MAP
THE DNA.
WE CONJUGATE THE FLOOR, SO WE
ARE USING IT SORT OF LIKE AN
ANTI-BODY, AND IT BINDS
UNMETHYLATED DNA, AND WE ARE
FOLLOWING THE BINDING OF THE TWO
DOMAINS TO THE MOLECULES ON THE
SURFACE TO TRY TO USE THE DNA
MANIPULATION PATTERNS.
OBVIOUSLY THE RESOLUTION THAT WE
HAVE IS NOT A BASE RESOLUTION
LIKE YOU HAVE BY SEQUENCING, BUT
IT'S MORE THE ENTIRE PIECE OF
115 BASE OF DNA THAT YOU HAVE ON
THE SURFACE, BUT WE DEFINITELY
SEE VERY INTERESTING PATTERNS OF
THESE COMBINATIONS BETWEEN
KEYSTONE MODIFICATIONS AND DNA
MANIPULATIONS, AND WHICH
COMBINATIONS SEEM TO BE ENRICHED
OR DEPLETED.
>> THANK YOU.
>> THANK YOU, AGAIN.
HOPE YOU ARE SAFE.
BE WELL.
THANK YOU.
OUR NEXT SPEAKER OR GONZANI, HE
CAME DOWN WITH ACUTE DISEASE.
HE CAN'T BE HERE, AND I HOPE HE
IS DOING WELL.
SO OUR NEXT SPEAKER WILL BE
DENNIS LO.
HE'S RECORDED FROM HONG KONG.
GAINING INSIGHTS INTO CANCER
EPIGENOMICS THROUGH CFDNA.
>> I WOULD LIKE TO THANK THE
ORGANIZERS FOR INVITING ME TO
PARTICI
PARTICIPATE IN THIS CONFERENCE
CELEBRATING 40 YEARS OF CANCER
EPIGENETICS.
I WOULD LIKE TO TALK ABOUT SOME
WORK.
THE TOPIC OF MY TALK IS GAINING
INSIGHTS INTO CANCER EPIGENOMICS
THROUGH CFDNA FRAGMENTOMICS.
OUR GROUP HAS BEEN WORKING IN
TWO OF THESE AREAS.
FIRST, IT'S THE DETECTION OF
PHYTO-DNA IN PREGNANT WOMAN, AND
THE DETECTION OF THE PLASMA OF
CANCER PATIENTS.
THESE TWO AREAS HAVE MANY
SIMILARITIES, AND SO THEY'RE
SINS
SYNERGINISTC IN WORKING
TOGETHER.
SO WE ACHIEVED THIS BACK IN 2013
IN WHICH WE DESCRIBE A GENOME
APPROACH TO DETECT MULTIPLE
TYPES OF CANCER, BUT BY USING
ONE BLOOD TEST.
THEN BY 2015 WE DEVELOPED A
TECHNOLOGY CALLED PLASMA DNA
TISSUE MAPPING IN WHICH WE USED
TISSUE MANIPULATION PROFILES TO
LOCALIZE THE ORIGIN OF
PARTICULAR CANCERS.
THIS INVENTION IS NOW PART OF
THE COMPONENTS OF THE GALLERY
TESTS.
FOR THIS SORT OF TEST AND MANY
OF THE OTHERS WHICH ARE BASED IN
EPIGENETICS BECAUSE THE DNA
TECHNOLOGY IS NORMALLY DESIGNED
TO ANALYZE GENETICS RATHER
EPIGENETICS PHENOMENON.
SO USUALLY WE WILL REQUIRE SOME
SORT OF CONVERSION PROCESS, SUCH
AS -- IN THE CASE OF THE
EPISUFFIC CONVERSION THIS COULD
BE DESTRUCTIVE ON THE DNA.
FOR THIS REASON IN THE LAST FEW
YEARS WE'VE BEEN INTERESTED TO
EXPLORE METHODS WHICH DO NOT
REQUIRE SUCH CONVERSION.
IN ONE AREA THAT WE ARE WORKING
ON IT'S IN THE FRAGMENTATION OF
DNA.
SO BASICALLY IF WE ARE LOOKING
AT DNA INSIDE THE CELL, IT'S
VERY LONG MOLECULES.
IT COULD BE UP TO 2 METERS LONG
IF YOU EXTEND A WHOLE GENOME
TOGETHER.
HOWEVER, THE PLASMA, THE DNA, IS
GENERALLY FRAGMENTS OF DNA.
BY LOOKING AT, FOR EXAMPLE, THE
FRAGMENT SIZE, WE CAN
DIFFERENTIATE CANCER AND
NONCANCER BECAUSE CANCER DNA IS
GENERALLY SHORTER THAN NONCANCER
DNA.
YOU CAN ALSO LOOK AT THE END
MOTIF, THE SUBSTRETCH OF
SEQUENCES OF THE END OF THE
MOLECULES, AND THIS CAN
DIFFERENTIATE CANCER AND
NONCANCER DNA.
YOU CAN ALSO LOOK AT THE END OF
THE FRAGMENT SINGLE STRANDED OR
DOUBLE STRANDED, SO THESE
COMBINATIONS ARE NOW REFERRED TO
AS FRAGMENTOMICS THAT ARE THE
FRAGMENTATION PATTERNS IN BODILY
FLUID.
ONE VERY POWERFUL METHOD TO LOOK
AT THIS FRAGMENTOMICS IS BY THE
USE OF OUR MODEL IN WHICH THE
SELECTED NUCLEOIC GENES IN MICE,
AND THEN WE CAN LOOK AT THE DNA
PROFILE.
FOR EXAMPLE HERE IN THIS
EXPERIMENT WE LOOK AT WILD TYPE
MICE, AND THEN WE ACTUALLY DO
THE SEQUENCING ON THE PLASMA,
AND THEN WE MEASURE THE
MANIPULATION PROFILE.
THEN IF YOU KNOCK OUT IN NUCLEUS
GENE LIKE DNA1 GENE, AND THEN
YOU SEE THE METHYLATION PROFILE
REMAINS UNCHANGED.
IF YOU CONTINUE, YOU COME ACROSS
THE STRAIN OF DNA, WHICH SOME
PEOPLE REFER AS DNA SCAM, WHICH
IS BASICALLY DNA WHICH CUT
CHROMATON, AND IT'S CIRCLING IN
CHROMATON.
YOU CAN SEE THE MANIPULATION
PROFILE IN CREATION OF A LOT OF
THOSE HYPER METHYLATED FRAGMENTS
IN PLASMA.
WHEN WE LOOK AT THE DATA LIKE
THIS, THERE ARE DIFFERENT
POTENTIAL EXPLANATIONS, AND ONE
OF THE QUESTIONS THAT WE
INITIALLY ASKED IS WHETHER
KNOCKING OUT THE DNA GENE WOULD
SOMEHOW CHANGE THE
CONSTITUTIONAL METHYLOMIC
PROFILE OF CELLS, AND THEN WE
SHOW THAT THAT IT DOESN'T HAPPEN
BECAUSE HERE YOU CAN SEE WILD
TYPE DNA KNOCK OUT AND DNA1
KNOCKOUT, AND BASICALLY THERE'S
NO CHANGE IN THE CELLULAR
METHYLOMIC PROFILE.
HOWEVER, CAN YOU LOOK AT PLASMA,
AND THIS IS WHERE THAT CHANGE
OCCURS.
WHEN YOU SEE THAT KNOCKING OF
DNA1 LIVE 3 GENE WILL BASICALLY
RESULT IN HYPER METHYLATION IN
PLASMA.
THIS WORK HAS RESULTED IN THIS
PUBLICATION IN WHICH WE CONCLUDE
THAT NUCLEUS DEFICIENCY WILL
AFFECT THE PLASMA DNA
METHYLATION PROFILES.
THE QUESTION IS WHAT ARE THE
IMPLICATIONS OF THIS WORK?
SO THAT TIME WE HAVE THOUGHT
LONG AND HARD FOR THAT, AND ONE
POTENTIAL IMPLICATION IS THEN,
WELL, INDIVIDUALS WITH DNA1 LIVE
FREE DEFICIENCY MAY GIVE FALSE
POSITIVE RESULTS IN LIQUID
BIOPSY TESTS.
WHILE THIS PARTICULAR RESULT
IS -- THIS CONCLUSION IS VALID,
BUT THEN THE BROADNESS OF THE
BREADTH OF THIS IMPLICATION
COULD BE RATHER NARROW BECAUSE
INDIVIDUALS WITH DNA1L FREE
DEFICIENCY COULD FEEL.
WE THOUGHT ABOUT THIS FOR A
WHILE, AND THEN WE FINALLY
ARRIVE AT A CONCLUSION THAT A
GOOD WAY TO ACTUALLY THINK ABOUT
THIS PROBLEM IS TO ACTUALLY ASK
THE OPPOSITE QUESTION, AND THAT
IS CAN FRAGMENTOMICS PROFILE BE
USED TO DEDUCE THE MANIPULATION
STATUS OF CELL-FREE DNA?
TO DO THIS WE HAVE TO ACTUALLY
LOOK AT FRAGMENTOMIC PROFILE IN
RELATION TO THE METHYLATION
STATUS IN DETAIL.
SO THIS GRAPH HERE ON THE X AXIS
BASICALLY IS GENOMIC COORDINATES
IN WHICH WE PUT THE C ON A CG
SITE AND PUTS IT AT ZERO.
G WOULD BE NUMBER ONE.
THEN THE RED LINE HERE IS WHEN
WE LOOK AT METHYLATED DNA IN
PLASMA.
CAN YOU SEE THAT METHYLATED DNA,
THAT THERE IS A HIGH
PREPONDERANCE OF THE DNA BEING
CLEAVED EXACTLY AT THIS POSITION
ZERO WHERE THE C IS.
WHEN YOU LOOK AT THIS
MANIPULATION, THEN THIS
PROBABILITY REDUCES GREATLY.
IF YOU LOOK AT THE HYPER
METHYLATED CELL FREE DNA, THEN
THE PATTERN IS VERY DIFFERENT.
YOU CAN SEE THAT ACTUALLY THIS
AT ZERO.
THERE'S NO PREPONDERANCE, AND
THEN IT'S NOT A LOT OF
DIFFERENCE IN THIS DISPOSITION
AND THE MINUS ONE POSITION.
WHEN WE THINK ABOUT THIS, THEN
OUR THOUGHT ABOUT THIS CONCEPT
CALLED FRAGMENT WHICH IS
FRAGMENTOMICS-BASED ANALYSIS, SO
IN WHICH YOU HAVE METHYLATED DNA
ON THE LEFT-HAND SIDE, AND
METHYLATED DNA ON THE RIGHT-HAND
SIDE.
YOU CAN ACTUALLY LOOK AT THIS
FREE NUCLEO TYPE CGN.
IF YOU LOOK AT THE FRAGMENT
ENDING IN CGN VERSUS THOSE
ENDING IN HAD CGD IT'S MANY
METHYLATED, IT WILL BE HIGH.
ON THE RIGHT-HAND SIDE IF IT'S
UNMETHYLATED, THE CGN WOULD BE
LOW, AND THE NCG WOULD BE HIGH.
FOR EXAMPLE, YOU SCAN ACROSS A
WHOLE GENOME AND FIND THAT THE
CGN AND NCG RATIO FOR METHYLATED
FRAGMENTS ARE BASICALLY ABOUT
4.5 OR SO, BUT ON THE OTHER HAND
IF IT IS HYPERMETHYLATED, IT'S
JUST ABOVE ONE.
HERE WHEN WE COMPARE THE DATA
WITH GOAL STANDARD SEQUENCING,
FOR EXAMPLE, ACROSS THE WHOLE
GENOME IN OTHER REGIONS AND IN
CVG ISLANDS, THEN YOU HAVE A
NUMBER OF DIFFERENT METHYLATION
DENSITIES.
ON THE RIGHT-HAND SIDE HERE,
WHEN YOU GO TO FRAGMENT DATA,
FRAGMENT WILL MIRROR THE
SEQUENCING RESULT.
IN OTHER WORDS, YOU DON'T HAVE
TO DO THE CONVERSION.
ANOTHER WAY IN WHICH WE CAN
DEMONSTRATE THE FRAGMENT WORKS
IS A LOOK AT -- FOR EXAMPLE, WE
LOOK AT THE GNA AT LOCUSTS IN
THIS PARTICULAR INDIVIDUAL.
THIS ALU HERE IS METHYLATED AS
INDICATED BY THE BLACK DOTS.
THE GL IS HYPERMETHYLATED, AS
INDICATED BY THE WHITE DOTS.
ON THE RIGHT-HAND SIDE HERE YOU
BASICALLY LOOK AT THE CGN TO NCG
RATIO.
YOU FOR G ON THE RIGHT-HAND
SIDE, THE CGN IS LOW, AND THE
NCG IS HIGH.
THAT DEMONSTRATES THE FRAGMENT
CONCEPT WORKS FOR IMPRINTING.
AS WE MENTIONED ABOUT THE
FRAGMENT CONCEPT IS INTIMATELY
RELATED TO THE ACTIVITY OF DNA1
LIVE FREE.
WE ACTUALLY LOOK AT THE
INDIVIDUALS WITH INDIVIDUALS
WITH DNA 1 LIVE FREE
INDIVIDUALS, AND WE LOOK AT THE
FRAGMENT PHENOMENON DOESN'T
EXIST.
YOU CAN SEE THERE'S NO
DIFFERENCE BETWEEN THE
METHYLATED AND UNMETHYLATED
FRAGMENTS IN TERMS OF THIS M
MODE OF RATIOS.
WITH NORMAL INDIVIDUALS WITH THE
FULL XLIM OF THE NUCLEUS GENES,
YOU CAN SEE WE CAN DIFFERENTIATE
THE TWO TYPES OF SEQUENCES.
THEN WE WONDER WHERE CAN WE USE
FRAGMENTS IN TERMS OF DETECTION?
WE DECIDED TO USE IT FOR HEPA
CANCER.
WE USE COPY NUMBER PRAYINGS AS A
MARKER FOR TUMOR DNA, AND Y AXIS
IS THE FRAGMENT RATIO.
CAN YOU SEE THERE'S A
CORRELATION BETWEEN WHAT WE
MEASURE AND THE CNA IN THE
PLASMA.
AS YOU LOOK AT THE DIFFERENT
STAGE OF XCC, WE USE THE
STAGING, AND WE LOOK AT THE
PERILILE STAGE AND IN THE
ADVANCED STAGE.
YOU CAN SEE HERE THAT BASICALLY
AS THE STAGING CHANGES, THE
FRAGMENT RATIOS ALSO
PROGRESSIVELY CHANGE.
THEN WE CAN ALSO USE A SUPPORT
VECTOR MACHINE MODEL TO FURTHER
DIFFERENTIATE.
IN THIS MODEL WE LOOK AT THE
FREE MOTIVE AT THE END OF THE
FRAGMENTS, WHICH CONTAIN THE CG.
NOW WE CAN HAVE CG, THE FIRST
TWO POSITIONS, AND THEN WITH ATC
OR G IN THE FIRST POSITION OR WE
CAN HAVE CG IN THE SECOND OR
THIRD POSITION.
THEN CHANGE THE FIRST POSITION
FROM A, T, C, AND G, AND PUT ALL
THE FRAGMENTS WITH ALL OF THOSE
MOTIFS INTO THIS SBM MODEL.
WE CAN SEE HERE IN THE GRAPH
THAT BASICALLY WE HAVE TO
DIFFERENTIATE THE LIVER CANCER
GROUP, WHICH IS THE THREE PLOTS
HERE VERSUS CONTROLS, AN
INDIVIDUAL WHO H EPA TIZED B.
THE POLICY CURVE IS 0.98.
THAT IS SUPERIOR TO ANOTHER
MOTIVE MEASUREMENT METHOD THAT
WE PREVIOUSLY PUBLISHED IN 2020
CALLED THE MOTIF DIVERSITY SCORE
IN WHICH THE ERROR CURVE IS
0.86.
SO BASICALLY I THINK THAT THE
FRAGMENT IS, THEREFORE, A LOW
COST WAY IN WHICH WE CAN UNLOCK
THE POWER OF THE PLASMA
METHOMES.
BASICALLY ALL WE NEED IS CELL
FREE DNA SEQUENCING RESULTS, AND
THEN FROM THAT WE CAN ALREADY
DEDUCE THE METHYLOMIC STATUS OF
THE SAMPLE.
I THINK THIS HAS MANY
APPLICATIONS BOTH IN CANCER AND
IN OTHER AREAS IN WHICH LIQUID
BIOPSY HAS BEEN APPLIED.
SO UP UNTIL NOW WE'VE
DEMONSTRATED SOME APPLICATIONS
OF CELL FREE DNA GENOMICS, AND
AS WE TALK ABOUT THE DNA ONE
LIVE FREE, THE ROLE IT PLAYS IN
THIS FRAGMENTATION.
WE'RE NOT VERY INTERESTED TO SEE
IF WE CAN ACTUALLY DEVELOP A
GENERIC METHOD IN WHICH WE CAN
EXPAND THE KNOWLEDGE OF CELL
FREE DNA GENOMICS.
FOR EXAMPLE, HOW CAN WE DISCOVER
PREVIOUSLY A NON-FRAGMENTOMICS
METHOD?
ONE METHOD THAT WE'VE BEEN
INVESTIGATING RECENTLY IS THIS
APPROACH BASED ON NON-NEGATIVE
MATE RICKS VECTORIZATION.
BASICALLY IT'S AN APPROACH IN
WHICH WE MATHEMATICALLY TRY TO
DEDUCE COMPONENTS OF A
PARTICULAR SYSTEM.
FOR EXAMPLE, WHICH HAS BEEN
USED, FOR EXAMPLE, IN AUDIO
ENGINEERING.
BASICALLY HERE WE HAVE THE
SYSTEM IN WHICH WE LIKE TO FIND
VARIOUS COMPONENTS AND THEN
THROUGH THE NMF PROCESS WE CAN
SPLIT THEM INTO THE COMPONENTS.
ONE ANALYSIS OF THIS WOULD BE,
LIKE, YOU HAVE COLOR LIGHT, AND
WHEN YOU PASS A LIGHT THROUGH A
PRISM, THE PRISM WOULD THEN
SPLIT IT INTO THE COMPONENT
COLORS.
WHAT WE WOULD LIKE TO KNOW IS
ABOUT WHERE WE CAN USE NMF TO
LOOK AT THE DIFFERENT COMPONENTS
OF THE FRAGMENTATION PROFILES OF
CELL FREE DNA.
WE START THIS WITH MIRROR IN
PLASMA IN WHICH WE HAVE WILD
TYPE MICE AND MICE WITH
DIFFERENT NUCLEUS KNOCK-OUT.
THEN BECAUSE WE ACTUALLY WANT
THE SYSTEM TO HAVE A BIT MORE
RESOLUTION, SO WE LOOK AT THREE,
BUT WE LOOK AT FOUR FOUR.
WE HAVE FOUR POSSIBILITIES,
WHICH IS 256 DIFFERENT MOTIFS.
WHEN WE PASS THIS NMF APPROACH
WE FIND ACTUALLY THAT THERE ARE
SIX MOTIF PROGRAMS.
WE CALL THEM F PROGRAMS.
FOR THIS THE X AXIS IS THE
DIFFERENT FORM OF MOTIF IN WHICH
WE ARRANGE FOR THEM IN DIFFERENT
ORDERS.
SO IN F ORDER.
AN A GROUP HERE, THE C GROUP
HERE, AND G AND T.
THEN Y AXIS IS THE FREQUENCY OF
DIFFERENT MOTIFS.
THIS IS PROFILE ONE, TWO, THREE,
AND THEN FOUR, FIVE, AND SIX.
YOU CAN SEE THAT THE BASIC ISSUE
OF THOSE PROFILES HAS RELATIVE
DIFFERENT RELATIVE WEIGHTINGS OF
THE DIFFERENT COLORS.
FOR EXAMPLE, WE SEE PROFILE ONE.
IT IS PREPONDERANCE OF THIS
WHICH IS CELL FREE FRAGMENTS
WITH CN.
YOU CAN SEE HERE, FOR EXAMPLE,
IN VIAL TWO THAT YOU HAVE A BIT
MORE TN.
EACH PROFILE HAS A
CHARACTERISTIC FEATURE.
THEN WE CAN ACTUALLY USE THIS
APPROACH IN PLASMA, WHICH IS ON
THE LEFT-HAND SIDE, AND THEN
URINE ON THE RIGHT-HAND SIDE.
THEN DIFFERENT COLORS HERE ONCE
AGAIN INDICATE THE DIFFERENT
PROFILES, WHICH IS INDICATED IN
THIS KEY.
SO ONE THROUGH SIX.
ONE THING WE CAN SEE, FOR
EXAMPLE, IS THAT IF YOU LOOK AT
THE WILD TYPE MICE, YOU LOOK AT
PLASMA VERSUS URINE, IT'S THAT
YOU CAN SEE THAT IN PLASMA
THERE'S A LOT OF THIS RED
SIGNAL, WHICH IN URINE IS
SUPPRESSED.
BUT THEN IN REPLACEMENT, THEN WE
HAVE A LOT OF THIS YELLOW
SIGNAL, WHICH IS PROFILE TWO.
BUT THEN INTERESTING WHEN YOU
LOOK AT THE URINE, YOU CAN FIND
IF WE KNOCK OUT THE DNA1 GENE,
THEN THIS YELLOW SIGNAL
DIMINISHES A LOT.
SO FROM THIS WORK THAT WE CAN
CONCLUDE THAT ACTUALLY THIS
PROFILE TOO IS RELATED TO DNA'S
1 ACTIVITY.
NOW LOOK BACK TO THE LEFT-HAND
SIDE IN THE PLASMA.
AGAIN, SEE THAT THE RED SIGNAL
PREPONDERANCE IN WILD TYPE
PLASMA, BUT WHEN WE KNOCK OUT
DNA1 LIVE FREE GENE, IT'S RESTED
GREATLY.
BY DOING THIS, THEN WE CAN
ACTUALLY ENTER THIS PROFILE 1 TO
DNA'S 1 LIVE FREE.
NOW WE'LL LOOK AT MORE DETAIL
HERE.
YOU CAN SEE THIS PROFILE 1 WITH
A LOT OF THE RED SIGNAL.
NORMALLY IF YOU LOOK AT WILD
TYPE MICE, THIS RED SIGNAL
REPRESENTS ABOUT 35% OF THE
PLASMA DNA.
THEN IF WE KNOCK OUT THE 1 LIVE
DNA GENE, THIS IS GOES TO LESS
THAN 5%.
SO BY USING THIS APPROACH, THEN
WE ACTUALLY CAN ENTER PROFILE 1
INTO DNA1 LIVE FREE.
PROFILE 2 TO DNA1, AND PREFILE 3
TO THE NUCLEUS CALLED THE FFB,
WHICH IS DNA'S FRAGMENTATION
FACTOR BETA.
BUT CURRENTLY WE HAVE NO IDEA
WHAT DOES 4, 5, AND 6 DO JUST
YET, BUT WE ARE PROCEEDING WITH
THE WORK TO ILLUCIDATE THEM.
THAT SAID, EVEN WITH OUR FULL
CHARACTERIZATION OF THE
GENERATING 4, 5, AND 6, WE CAN
ACTUALLY STILL USE THIS VARIOUS
PROFILE AS POTENTIAL TO MARKERS.
FOR EXAMPLE, YOU CAN LOOK AT THE
RELATIVE AMOUNT OF THOSE
PROFILES IN RELATION TO, FOR
EXAMPLE, DETECTING HEPA ITCYLUS
CANCER.
INTERESTING ACTIVE PROFILE 6
LOOKS REMARKABLY GOOD WITH THIS
PINK LINE HERE WITH THE AREA AT
0.97.
SO NOW WHEN YOU LOOK AT CONTROL
INDIVIDUALS, PEOPLE WITH CHRONIC
HEPATITIS AND PEOPLE WITH HEPATO
CANCER, CAN YOU SEE THE DEGREE
IN THE REPRESENTATION OF PROFILE
6.
APART FROM HEPA CANCER WE
DECIDED TO LOOK AT COLORECTAL
CANCER.
YOU CAN SEE THAT THERE'S A
CONTROL AND COLORECTAL CANCER
PATIENTS WITHOUT LIVER
METASTASIS TO COLORECTAL CANCER
PATIENTS WITH LIVER METASTASIS
THERE'S A GRADUAL --
WE DECIDED TO TAKE A LOOK IN
MORE DETAIL IN PROFILE 6, AND
YOU CAN SEE THAT PROFILE 6 HAS
THIS RATHER INTERESTING
PHENOMENON IN WHICH THERE IS A,
E, C, G, N, T.
IT WORKS IN KNOCKING OUT THE --
THIS ONE GOES WITH T.
INTERESTING FOR PROFILE 6 WE
DON'T HAVE THAT PREFERENCE.
SO WE STARTED TO WONDER, IS IT
POSSIBLE THERE IS ACTUALLY A
NONENZYMATIC PROCESS?
WE WONDER WHETHER IT COULD BE
RELATED TO SOME PHYSICAL
MECHANISMS, WHICH BREAK UP THE
DNA, FOR EXAMPLE, LIKE RADIATION
OR REACTIVE SPECIES.
IN ORDER TO TEST THAT HYPOTHESIS
WE WILL LOOK AT CANCER PATIENTS
UNDERGOING CHEMO RADIOTHERAPY
AND YOU CAN SEE THAT, FOR
EXAMPLE, BEFORE AND AFTER
THERE'S AN INCREASING
CONTRIBUTION TO PROFILE 6.
BASICALLY BEFORE CHEMO
RADIOTHERAPY PROFILE 6
REPRESENTS ABOUT 6% OF THE
PLASMA DNA PROFILE, WHICH GOES
TO 10% AFTER THIS PROCESS.
I THINK THAT WE SHOULD LOOK AT
MORE CLINICAL SITUATIONS IN
WHICH THERE'S AN INCREASE IN
THOSE PHYSICAL PHENOMENON OF THE
SPECIES WITH RADIATION DAMAGE TO
FULL CONSOLIDATE THIS WORK.
SO IN SUMMARY, WE HAVE BEEN
INTERESTED IN LOOKING AT THE
RELATIONSHIP BETWEEN CELL FREE
DNA AND FRAGMENTOMICS.
WE FIND THERE IS DEFINITELY A
LINK, AND THROUGH THAT LINK WE
HAVE ACTUALLY INVENTED THIS
FRAGMENT CONCEPT, WHICH IS A
RELATIVELY LOW COST WAY TO
ACTUALLY LOOK AT CELL FREE DNA
ANALYSIS.
BASICALLY OPENING THE POWER OF
THE LEVEL TO DO ANY DNA
SEQUENCING HERE.
THEN WE ALSO EXPLORE THE USE OF
THE CONVOLUNTARTION OF OTHERS U
THE FRAGMENTATION THAT WE'LL
PURSUE IN THE COMING YEARS.
FINALLY, I WOULD LIKE TO THANK
OUR GROUP FOR GENERATING THE
DATA WHICH I PRESENTED TO YOU
TODAY.
THANK YOU VERY MUCH FOR YOUR
TIM
TIME.
>> THAT CONCLUDES THIS SESSION.
I WANT TO THANK ALL THE
SPEAKERS, INCLUDING PETER.
I THINK IT'S GOING TO BE LUNCH
BREAK.
I HOPE YOU ALL HAD A NICE
LUNCH.
THE NEXT SESSION WILL BE ON
SYSTEMIC IMPACTS OF HHEPAGENICS.
OUR FIRST SPEAKER IS EVA
HERNANDO-MONGE.
>> IT'S A PLEASURE TO BE HERE
WITH ALL THE PIONEERS IN THIS
FIELD.
I'M VERY GRATEFUL TO BE HERE.
I'M GOING TO PRESENT THE
STUDY -- LET'S SEE HOW I DO
THIS.
THIS IS THE STUDY THAT IS ARE
UNPUBLISHED AND IS THE WORK OF
MY LAB.
IT'S, IN PART, A COLLABORATION
WITH A PROFESSOR IN MY
DEPARTMENT AND A LONG-TERM
COLLABORATOR AND FRIEND, JAMES
COOK, AND ASSISTANT PROFESSOR
K
KATHLEEN DOLE IN HER LAB.
WE WERE ASKED IN PREPARING THIS
TALK TO PRESENT ON HOW THE FIELD
OF CANCER GENETICS HAS AFFECTED
OUR PARTICULAR PROGRAMS AND OUR
PARTICULAR QUESTIONS.
I THOUGHT OF THE COVER OF THE
MAGAZINE "TIME" AND THE TITLE
WAS "WHY YOUR DNA ISN'T YOUR
DESTINY."
I THINK THIS REFLECTS EXACTLY
WHAT IS THE CASE FOR OUR
STUDIES, WHICH ARE FOCUSED ON --
OR MY PARTICULAR FIELD, WHICH IS
THE FIELD OF CANCER METASTASIS.
WE USED THE GENOMA AS A MODEL OF
METASTASIS BECAUSE THESE TUMORS
CAN GO ON AND METASTASIZE IN
DIFFERENT ORGANS.
WHAT IS REALLY INTERESTING ABOUT
THESE TUMORS IS THAT TWO
PATIENTS THAT CARRIED TUMORS
THAT HAVE THE SAME CLINICAL
BIOLOGICAL CHARACTERISTICS AND
EVEN THE SAME GENETIC MAKEUP CAN
HAVE COMPLETELY DIFFERENT
OUTCOMES WITH A PATIENT THAT CAN
BE CURED AFTER RECESSION.
IN ANOTHER PATIENT THAT WILL
HAVE RECURRENCE IN METASTASIS
POST-RESECTION.
AGAIN, THE GENETIC ALTERATIONS
OR COMBIN AINGS OF GENETIC
CHARACTERIST HEICS OF THE
TUMORS, WHICH HAVE BEEN
EXTENSIVELY CHARACTERIZED IN THE
CASE OF MELANOMA CANNOT PREDICT
WHICH PATIENTS WILL REOCCUR AND
METASTASIZE.
MY LAB HAS TURNED INTO EXAMINING
NON-GENETIC MECHANISMS AS
POTENTIAL DRIVERS OF METASTATIC
BEHAVIOR.
IN PARTICULAR, OVER THE PAST FEW
YEARS OUR LAB AND OTHERS HAVE
FOCUSED ATTENTION OF THESE
INTERESTING FINDINGS, WHICH IS
THE PRESENCE OF INTERTUMOR
HETEROGENEITY.
OUR SIMILAR STATES PRODUCE
CANCER CELLS WITH A LOT OF
ADVANTAGES.
STILL, HOWEVER, IT ISN'T CLEAR
HOW THIS INTERTUMOR
HETEROGENEITY IS CURED.
TO REMIND YOU ALL WE MUST
ORIGINATE FROM THE PIGMENTED
CELLS IN THE SKIN AS A
CONSEQUENCE OF UV RADIATION, AND
IMMEDIATELY AT THOSE VERY AREAS
TUMOR REGENESIS, ONE CAN
APPRECIATE ALREADY THE PRESENCE
OF THESE INTERTUMOR
HETEROGENEITY AND
CHARACTERISTICS MORE PIGMENTED
AND DIFFERENTIATED AND ALSO SOME
OTHER POPULATIONS THAT, FOR
EXAMPLE, ARE MORE --
POPULATIONS.
ONE CAN BE MORE GRANULAR AND
IDENTIFY IN OTHER STATES, BUT
FOR THE PURPOSE OF THIS TALK, I
WILL BE TALKING ABOUT THESE TWO
EXTREMES, THE MELANOCYTIC OR H
EPA STATIC POPULATION.
THERE'S A POPULATION THAT WE
CONSIDER INTERMEDIATE.
THESE CELLULAR STATES HAVE BEEN
SEEN TO BE EXTREMELY PLASTIC.
CELLS CAN NAVIGATE OR CAN GO
BACK AND FORTH FROM THESE
DIFFERENT CELLULAR STATES.
TWO TRANSCRIPTION FACTORS HAVE
BEEN SHOWN TO PLAY A VERY
IMPORTANT ROLE IN THE
PREPARATION FROM ONE STATE TO
ANOTHER.
ONE IS THE SAME IDEA, WHICH IS
THE MASTER REGULATOR OF
MELANOMACYTE REGULATION, AND THE
OTHER ONE WHICH ARE IN CONTROL
OF THE BASIC PHENOTYPE.
THE INTERCONVERSION BETWEEN THE
CELLULAR STATES, WHICH IS VERY
PLASTIC, VERY DYNAMIC, IS
CONTROLLED BY INTERACTIONS
BETWEEN THE TUMOR CELLS AND THE
MICROENVIRONMENTS.
ONE OF THE CUE CENTERS FOR THE
STIMULI FOR THESE CONVERSIONS
CAN FROM THE INTERACTIONS
BETWEEN THE CANCER CELLS AND THE
STROMA AS WELL AS THE
INTERABBINGS WITH THE
IMMUNO-CELLS, AND IN SOME CASES
THERAPY CAN BE A STRONG
REGULATOR OF THESE TRANSITIONS.
WE ARE ASKING HOW IS THIS
INTERTUMOR HETERGENEITY
PUBLISHED.
IN PARTICULAR, TODAY'S TALK I'M
GOING TO FOCUS ON THE
TRANSCRIPTIONAL PROGRAMS
DOWNSTREAM OF THESE CHANGES IN
CONFIRMATION.
WITH THE STUDY WE DECIDED TO
CHARACTERIZE BY THE THREE
CHROMATIC CONFIRMATION OF THREE
TYPES OF MELANOMA CELL LINES.
ALTHOUGH I TOLD YOU THAT TUMORS
ARE VERY HETEROGENEROUS, AND
THEY HAVE DIFFERENT STATES.
WHEN CELL LINES ARE ESTABLISHED
FROM THESE TUMORS, THEY ACQUIRE
VERY OFTEN FEATURES OF THE
DIFFERENT STATES.
SO THERE ARE CELL LINES THAT ARE
MORE REPRESENTATIVE OF THE
MELANOCYTIC STATE.
WE CHARACTERIZE THREE OF THESE
TYPES, OR STATES, BUT WE ALSO
TOOK THE 501 CELL, WHICH IS A
MELANOCYTIC CELL LINE AND THE
TRANSCRIPTION FACTOR IN ITF IN
ORDER TO SEE WHAT WOULD BE THE
IMPACT OF TARGETING OR SILENCING
THESE MASTER REGULATOR OF THE
DIFFERENTIATED IMMUNOCYTIC
PHENOTYPE.
WOULD IT BE POSSIBLE THAT A CELL
THAT IS MORE MELANOCYTIC WOULD
BE MORE FROM THE PERSPECTIVE OF
THE CHROMATINE.
THE FIRST ANALYSIS AT THE VERY
HIGH -- AT THE VERY HIGH PART
THAT I MENTIONED SHOWS THAT IN
THE CONVERSION BETWEEN
INTERMEDIATE TO MELANOCYTIC, ONE
CAN SEE THAT THERE IS AN
INCREASE IN THE NUMBER OF
INTERACTIONS.
THIS IS PERCEIVED HERE WITH THIS
RED SIGNAL.
IN THE TRANSITION BETWEEN
INTERMEDIATE TUMORS ONE CAN SEE
THAT THERE ARE NOW MORE INTACT
INTERACTIONS.
THIS IS DEPICTED IN THIS CARTOON
WHERE YOU CAN SEE THAT, AGAIN,
THERE ARE AN INCREASE INITIALLY
OF THE INTRUKS AND THEN
INCREASED OF THESE MORE
LONG-RANGING INTERABBINGS.
WE ARE STARTING TO WONDER WHAT
IS THE IMPACT OF THIS EFFECT?
FIRST OF ALL, WE SEE THAT THE
MISSING KYMA CELLS HAVE AN
ADVANTAGE WHICH IS CONSISTENT
WITH INTERTARGETED INTERACTION,
WHEREAS THERE'S NO CHANGE WHEN
WE COMPARED THE INTERMEDIATE TO
THE MELANOCYTIC.
IF WE ZOOM IN IN THIS PARTICULAR
CASE, ONE CAN SEE THAT INITIALLY
IN THE GENOCYTIC, ONE CAN SEE
THAT THERE ARE SHORT LOOPS.
IT'S CHARACTERISTIC OF THIS, AND
THEY LOCALIZE.
THEN AS YOU MOVE TO THE CHYME
STATE, ONE CAN SEE HOW THIS
DISAPPEARS AND STARTS TO FADE.
ONE CAN SEE NOW THE INCREASE OF
THIS MORE LONG-RANGE
INTERACTIONS.
NOW, IF WE ANALYZE THE
COMPARTMENTS AND, AGAIN, TO
REMIND US ALL COMPARTMENT A IS
THE ONE THAT HAS MORE ACTIVE
TRANSCRIPTION AND IT HAS MORE
PRESENCE OF ACYTL, WHICH
COMPARTMENT B IS ONE THAT IS
INACTIVE.
ONE CAN SEE WHEN WE COMPARE THE
DIFFERENT STATES, THERE ARE
SIGNIFICANT CHANGES IN THIS
COMPARTMENT.
FOR INSTANCE, I WANT TO CALL
YOUR ATTENTION TO THE COMPARISON
BETWEEN THE -- ONE CAN SEE THAT
THERE IS A REGION HERE THAT IS
COMPLETELY COMPARTMENT E HERE ON
THE UPPER PART THAT BECOMES NOW
LESS REPRESSED OR MORE ACTIVE IN
THE MISSING CHYMAL CELLS.
THIS IS CHROMOSOME ONE.
ONE CAN GO CHROMOSOME BY
CHROMOSOME IDENTIFYING CHANGES
IN CHARACTERISTICS IN THE
CHARACTERIST OF THE TRANSITION
OF THE DIFFERENT CELLULAR
STATES.
SO, AGAIN, FOR INSTANCE IN THE
OTHER EXAMPLE IN CHROMOSOME 12
AND CHROMOSOME 2, ONE CAN SEE
ANOTHER AREA THAT HAS
COMPARTMENT A AND B INTEGRATED
TOGETHER, WHEREAS IT BECOMES
COMPLETELY ACTIVE IN COMPARTMENT
A IN THE MISSING CELL LINES.
WHAT TRIGGERS THIS MAJOR
CONFIRMATION?
WE SEE THAT CELLS AS WE MOVE
BETWEEN THESE DIFFERENT STATES
HAVE CHROMATIC CHANGES, BUT WHAT
ARE THE FACTORS RESPONSIBLE FOR
THESE CHANGES?
ARE THERE MECHANICAL FACTORS,
SUCH AS PREPARATION AS THE CELLS
MOVE THROUGH THE MATRIX OR THE
SHEER STRESS AS THEY ENCOUNTER
CIRCULATION?
IS IT THE ABILITY OR HYPOXIA
INFLUENCING THESE CHANGES?
WHAT IS THE ROLE, FOR INSTANCE,
IN THERAPY OF ALL OF THIS?
NOW, WE ALSO WONDER HOW THESE
CHROMATINE CHANGES AND
TOPOLOGICAL CHANGES ARE
EXPRESSED DOWNSTREAM, AND HOW
THIS RESULTS IN THE ADAPTATIONS
THAT WE MENTIONED?
FIRST OF ALL, WE WONDER THE
TRANSCRIPTION PATHS THAT CAN
ORCHESTRATE THE EXPRESSION
CHANGES DOWNSTREAM OF THESE
CONFIRMATIONAL CHANGES.
WE MENTIONED MITF AND AP1 AS THE
TRANSCRIPTION FACTORS THAT SEEM
TO BE CRITICAL FOR THE
CONVERSION BETWEEN THE
METASPECIFIC AND METASTATIC
TYPES.
AS WE LOOK AT THE ANALYSIS, YOU
CAN SEE THAT THREE ARE ABLE TO
CLASSIFY THE CELLS SPEW THE
DIFFERENT PHENOTYPES OR
DIFFERENT STATES.
HERE IN GREEN YOU CAN SEE THE
CELL LINES.
IN BLUE, THE INTERMEDIATE.
IN GREEN, YOU CAN SEE THE MORE
INVASIVE WITH ONE CELL LINE IT
SEEMS TO BE NOT WELL-CLASSIFIED
FOR REASONS THAT WE CAN'T
UNDERSTAND.
IF NOW WE ZOOM INTO THE CELL
LINES, ONE CAN SEE THAT THE
SILENCING OF MITF DID NOT HAVE A
MAJOR AFFECT IN CHROMATING
CONFIRMATION.
IF WE ZOOM INTO THIS REGION, ONE
CAN SEE MITF IS ONLY ABLE TO
PARTIALLY MOVE THE CELLS TOWARDS
THE RIGHT, AND THEY SEEM TO NOW
MIMIC MORE THE BEHAVIOR OF THE
INTERMEDIATE CELL LINES, BUT
STILL, IT'S INCOMPLETE TO FULLY
TRIGGER A CONVERSION, A
CHROMATINE CONFIRMATION CHANGE
CHARACTERISTIC OF THE
INTERMEDIATE AND INVASIVE CELL
LINES.
IF WE LOOK AT THE PROFILES,
SIMILARLY ONE CAN SEE THAT THESE
PROFILES CAN CLUSTER THE CELLS
INTO THE INVASIVE, INTERMEDIATE,
AND ACIDIC, BUT IF WE LOOK AT
THE PROFILES IN THESE CLUSTERS
AND PARTICULARLY AT THE TOP, ONE
CAN SEE THAT THE SILENCING OF
MITF IN THE MELANOCYTIC CELL
LINES HAD SOME IMPACT ON THE
CLUSTERS SO YOU CAN SEE HOW SOME
OF THESE CLUSTERS AND SOME OF
THESE THINGS NOW ARE STARTING TO
CLOSE OR THEY'RE NOT ACTIVE
ANYMORE, BUT THIS SCIENCE IS
ENABLING THE OPENING OF THE
CLUSTERS CHARACTERISTIC OF THE
INTERMEDIAL OR CHYMAL CELL
LINES.
THE MASTER OF THE
DIFFERENTIATION IS STILL UNABLE
TO TRIGGER THE CONVERSION AND
THE ACTIVATION OF ENHANCERS IN
ACTIVE MEDIANSISTIC OF THE
INTERMEDIATE AND INVASIVE CELLS.
IF WE LOOK AT CTCF, FOR
INSTANCE, ONE CAN SEE, AGAIN,
THE SAME PHENOTYPE.
PRESENTLY ONE CAN SEE THAT CTCF
IS ABLE TO CLASSIFY THE CELL
LINES BASED ON THE OPENING OR
THE REGIONS THAT ARE ACCESSIBLE
TO CTCF.
AGAIN, WE SEE THAT THE
INACTIVATION OF MITF CONVERTS
THE ACIDIC, BUT NEVER ABLE TO
CHANGE THEM TO BECOME MORE
>> CONTRIBUTING TO THESE
CHANGES.
AND FOR THAT, WE WILL LOOK AT
ESTABLISHED SET OF MELANOMAS AND
HE LOOK AT EXPRESSION OF THE
TRANSCRIPTION FACTORS OF MORE
THAN 1,000 TRANSCRIPTION
FACTORS.
AND YOU CAN SEE THAT MORE THAN
280 WERE FOUND ARTICULATE IN A
MELANOMA.
AND MORE SPECIFICALLY, HE FOUND
THAT A SUBSET OF THEM, A SUBSET
OF TRANSCRIPTIONAL FACTORS SEEM
TO BE ENRICHED IN MELANOMA
COMPARED TO ANY OTHER CANCER
TYPE.
THESE ARE INDICATED ON THE
RIGHT.
I LX6.
FOR THIS TALK I WILL CONTINUE
NOW TALKING ABOUT EN TWO.
WE FOUND THIS A TRANSCRIPTIONAL
FACTOR THAT A MELANOMA AS YOU
COMPARE TO ANY OTHER CALIFORNIA
CANCER TYPE.
NOT ONLY WE CONFIRM IN BUT ALSO
IN METASTATIC COMPARED TO
PRIMARY MELANOMA.
EXPRESSION OF EN CORRELATES WITH
OUTCOME, HIGHER LEVELS OF
ASSOCIATED WITH SHORTER
SURVIVAL.
SO WHAT EN2.
EN TWO A HOW MOJ JANE OUTS
FAMILY OF TRANSCRIPTIONAL
FACTORS WHICH ARE INVOLVED IN A
PROCESS AND A FOR PARTICULAR
DEVELOPMENT.
YOU CAN SEE ON THE RIGHT.
EN HAS AN ATROPHIC SET OF, AND.
AND ALSO IMPORTANT FOR EM2 TO BE
ASSOCIATED WITH AUTISM.
SO EN2, IT HAS ALSO BEEN FOUND
IN OVARIAN CANCER AND ASSOCIATED
WITH A POOR PROGNOSIS.
WE FOUND THAT EN2 ALTHOUGH COS
HAS ENHANCER EXCLUSIVE OF
MELANOMA.
NOT FOUND IN MELANOCYTIC OR OR
CELL LINES.
LEVERAGING THE ESGA DATA ONE CAN
SEE IT A VERY GOOD CORRELATION
BETWEEN EXPRESSION OF EN2 AND
ACTIVATION OF THE CENTERS YOU
CAN SEE ON THE RIGHT THE
ACTIVITY OF THESE INCIDENCE
HANSERS SEEM TO BE PARTICULARLY
ACTIVE IN MELANOMA SAMPLES.
NOW IF WE LOOK AT CELL LINES AND
WE COMPARE CELL LINES THAT HAVE
HIGH LEVELS OF EN2, WITH
INTERMEDIATE AND LOW ONE CAN SEE
THAT THOSE THAT HAVE HIGH LEVELS
OF EN2 ARE USUALLY THE ONE THAT
MITF HIGH EN2 LOW HAVE HIGH
WHICH HAVE MORE INVASIVE.
THE CELL OF EN2 HAS NO EFFECT IN
PRO LIVE REEGS IN VITRO.
INVIVO IT REDUCED THE ABILITY OF
CELLS TO GROW INVIVO.
WHEN WE DID TRANSCRIPTION OF
THESE CELLS WE LOOK AT THE CELLS
SCIENCE E N2, THEY THE CELLS
THAT WERE MORE SIGNIFICANTLY UP
REGULATED, WHEN WE EN2 IS ON
CONTRARY ON THE UPPOP SID SIDE
THEY ARE DOWN REGULATED
ASSOCIATED TO MORE TILT.
EN2 SEEMS TO BE PARTICULARLY
ACTIVE IN CELLS MISSING CHYMAL
AND.
AND IT SEEMS THAT THEY
EXPRESSION OF EM2 NEGATIVELY
ASSOCIATES WITH RESPONSE TO
THERAPY, MEANING THAT THOSE HAVE
HIGHER LEVELS EN2 USUAL NOT
RESPONDERS.
THE SAME EFFECT CAN BE SEEN IF
WE PATIENTS, INTO HIGH AND LOW.
EN2 LEVELS.
THOUGH WITH EN2 LOW HAVE A MORE
ACTIVITY OF PATHWAYS.
AND AGAIN, HIGHER LEVEL EN2
ASSOCIATED WITH LOWER LEVELS OF
THE SIGNATURE THAT PREVIOUS
ASSOCIATED WITH POOR RESPONSE IN
WITH NOT GOOD PROGNOSIS
MELANOMA.
ALL OF THESE INDICATE THE
POSSIBILITY EN2 PLAY AS ROLE IN
ANTI TUMOR IMMUNE RESPONSE.
SO WE TOOK THE CELLS ENGINIC
CELL LINE AND TRANS FUSED IT
WITH EN2.
AS YOU CAN SEE ON THE LEFT, THEY
OVER EXPRESSION OF EN2 HAD
MINIMAL EFFECT ON THE GROWTH OF
THE EN CELLS WHEN THEY ARE
INJECTED ANY IMMUNE DEFICIENT
MICE.
IF YOU SIMULTANEOUS IN AN IMMUNE
COMPROMISED MOUSE ONE CAN SEE A
VERY SIGNIFICANT ENHANCEMENT OF
GROWTH.
THIS ASSOCIATES WITH A
PRACTICALLY TOTAL EXCLUSION OF
BOTH CDA AND CD4 POSITIVE T
CELLS.
SAME RESULTS WERE AN IN ANOTHER
INDEPENDENT CELL LINE WITH
EXPRESSION OF EN2 ALSO ENHANCES
GROWTH.
AND SUPPRESSES INFILTRATION.
HOW EN2 AFFECTING ALL AND FOUND
EN2 BINDS IN GENIC REGIONS AND
IS A PARTICULARLY OR A MORE
FOUND IN ENHANCERS ARE OH
IPRODUCTION MALENHANCERS WITH
CO-LOCALIZING PROBLEMS WITH
CONVERSION.
BUT IT REALLY COLOCALIZES WITH
AS WELL AS ATAXIC OPENING.
BUT WHAT IS STRANGE TO US IS
THAT THE NUMBER OF PEAKS IS
LIKELY LOW.
DOESN'T SEEM TO ECONOMIC
ACTUALLY BIND.
SO WHAT WE FOUND IS THAT EN2 IS
ABLE TO ORCHESTRATE LONGER
INTERACTIONS.
SO ONE CAN FIND A LOCATION OF A
27 DISABILITY THAT STUDIES LONG
RANGE INTERACTIONS BASED ON
DATA.
AND THE THESE INTERACTIONS ARE
OUT THERE WHEN WE SILENCE EN2.
SO THIS OPENS POSSIBILITY THAT
EN2 IS CONTROLLING A LONG RANGES
INTERACTIONS IN ESTABLISHING
TRANSCRIP
TRANSCRIPT
TRANSCRIPTIONAL HALVES TO
FARTHER INVESTIGATE THIS
POSSIBILITY, WE LOOK AT FIRST
LOCATION OF THE NECHLT 2 BY
DOING AFRACTION NATION.
WE FOUND THAT EN2 IS AS EXPECTED
IN NUCLEUS BUT MATURITY OF EN2
ISN'T NOUN SOLUBLE FORM MEANING
NOT BOUNDED TO CHROMATIN.
ONLY ONE FRACTION ATTACHED TO
CHROMATIN.
IT MAY BE SCHULTZING IN AND OUT
OF CHROMATIN.
AND IN.
E. 2 FOR MASS SPECK TOMORROW
TREE.
EN2 INTERACT WITH A COMPONENTS,
COMPLEX.
THAT IS AND IS A SUPPRESSIVE
COMPLEX THAT DOESN'T BIND.
SO WE FOUND WE CONFIRMED THIS
INTERACTION BY CHROMATIN.
THIS OPENS THE POSSIBILITY THAT
EN2 INTERACTS WITH IN CHROMATIN
OR THAT THE THERE IS A
COMPETITION BETWEEN EN2 AND
OTHER TRANSCRIPTIONAL FACTORS
FOR BINDING AND ACCESS TO
CHROMATIN.
TO FINALIZE, THIS IS OUR MODEL.
MELANOMA CELLS INITIALLY ARE A
RECOGNIZED BY T CELLS IN THE
INITIAL STEPS OF BUT WHEN EN2
LEVELS GO THESE MELANOMA CELLS
START TRANSCRIPT FACTORS THAT
MAKE THEM NOW MORE, MORE IMMUNE
OR MORE ANAL TO AN EVADE IMMUNE
RESPONSE.
AND THIS IS DUE TO THE ABILITY
OF EN2 TO FROM SORRY FROM
CYTOPLASM INTO NUCLEUS.
WE NO FROM DATA THAT I HADN'T
HAD A CHANCE TO PRESENT IN THIS
UP UP REGULATED WHEN, THEY
SUPPRESS GENES.
AT THE SAME TIME EN2 CAN BE AN
ACTIVE REGULATOR OF EN WENT 2
INNOVATION GENES.
I WILL NOT READ THE BECAUSE I'M
RUNNING OUT OF TIME.
BUT JUST TO PUT TOGETHER THE TWO
PARTS THE PRESENTATION AS I
MENTIONED ABLE TO DIFFERENT
STATES AND WE ARE HYPOTHESIZING
DOWN STREAM OF THESE MAY ACCEPT
MAYR YOUR CHANGES IN
TRANSCRIPTIONAL HALVES AND
ORCHESTRATE SOME OF THIS
TRANSCRIPTIONAL CHANGES THAT
CONFIRM MELANOMA CELLS WITH
ADAPTATION AND ABILITY TO ME TAZ
SIZE AND EVADE IMMUNE SYSTEM.
AGAIN I WANT TO THANK THE
MEMBERS OF MY LAB WHO WORK WITH
US ON THE THIS PROJECT.
AND I WILL STOP HERE.
I APPRECIATE YOUR TIME AND STOP
HERE AND TAKE QUESTIONS, THANK
YOU.
[ APPLAUSE ]
>> A BEAUTIFUL TALK.
THERE WE GO.
OKAY.
I HAVE A QUESTION ABOUT I THINK
YOU MENTIONED EN2 WAS INVOLVED
IN BRAIN DEVELOPMENT.
I WONDERED IF THERE IS ANY
SPECIFIC RELATIONSHIP TO METS IN
THE PLAIN TBRAIN THERE
>> AS YOU KNOW WE HAVE DON'T NOT
HAVE A TRANSCRIPTION UP DATA AS
WE HAVE FOR SITE OF ME TAZ CIS.
PRIMARILY ANALYSIS OF A
COMPUTATION, PREMA TAZ SEES
DOESN'T SHOW A DIFFERENCE OF
EXPRESSION IN EN2.
WE DIDN'T SEE THAT THERE IS
INCREASED EXPRESSION.
BUT I THINK THAT'S A VERY
IMPORTANT QUESTION.
WE UNDERSTAND IN WHETHER
TRANSCRIPTIONAL PROGRAM THATTASE
ESTABLISHING IN BRAIN.
SO WE DEFINITELY LOOKING INTO
IT.
AND I HOPE I WILL PROVIDE
UPDATES, THANK YOU.
>> NEXT SPEAKER IS CHRISTINA
LESLIE.
CANCER EPIGENETICS IN 3D.
SHE WILL GIVE IT VIRTUALLY.
>> OKAY.
I HOPE YOU CAN SEMI SLIDES.
THANKS SO MUCH FOR THE
ORGANIZERS FOR INVITING ME.
I'M VERY SORRY THAT I COULDN'T
BE THERE IN PERSON.
I AM CURRENTLY GROUNDED IN NEW
YORK WITH COVID.
SO I WANT TO START WITH A
VIGNETTE TO REMIND EVERYONE THAT
SOMATIC ALTERATIONS IN EPIGENTIC
REGULATORS CAN A CAUSE GLOBAL
CHANGES AND SHORT VIGNETTE IN
COLLABORATION WITH AN EMILY
CHANG THAT DID ALL THE
EXPERIMENTS WORK AND WE DID
COMPUTATIONAL ANALYSIS.
SO THIS WAS A STUDY OF A LOSS OF
FUNCTION MUTATION IN RENAL CELL
CARCINOMA.
SO IT IS THE NONREDONE DANTS
HISTONE METHYL TRANSFER RACE
THAT CO-TRANSCRIPTIONALLY
DEPOSITS A33 K TRY METHYL.
EVEN THOUGH IT IS ASSOCIATED
WITH ACTIVELY TRANSCRIBED GENES,
IT IS A REPRESSIVE MARK.
IT'S SUPPRESS CRIPPIC INITIATION
OF TRANSCRIPTION.
IT IS MUTATED IN 13 PERCENT OF
CLEAR CELL REENLT CELL CARCINOMA
ASSOCIATED WITH PROGRESSION AND
ME TAZ CIS.
EMILY.
AND AN EMILY CHANG HAS NIGHT
PATIENT MODEL WITH A MUTANT IN
THE XENOGRAPH YOU CAN
RECAPITULATE THIS METASTASIS TO
A BRAIN, LUNG AND LIVER.
AND, THEN SHE CAN ENGINEER THE
CELLS TO RESTORE ACTIVITY OF AND
THAT REDUCES THE METASTASIS.
OKAY.
SO WHAT WE DID IN THIS PAPER AND
AGAIN JUST VERY QUICKLY, WE
TRIED TO UNDERSTAND E
EPIGENOMECALLY WHAT IS HAPPENING
WITH A FUNCTION.
AND THE FIRST THING WE COULD SEE
IS THAT YOU HAVE MASSIVE CHANGES
IN CHROMATIN ACCESSIBILITY.
SO OVER HERE THIS IS YOU CAN SEE
A YOU GET OPENING OF MANY PEAKS.
AND FROM THE TORNADO PLOTS HERE
YOU CAN SEE BOTH ERNT GENIC
REGIONS YOU, WHEN YOU LOSS H3 K
36 TRY METHYL, LOSS THE
ACTIVITY, YOU GET OPENING OF
PEAKS AND IT'S ALSO ASSOCIATED
WITH ACTIVE HISTONE MARKS.
AND THAT MEANS THAT GENETIC
PROGRAMS ARE AMPLIFIED.
SO FOR EXAMPLE THIS IS A PRO
METASTASIS GENE.
AND YOU CAN SEE WITH LOSS OF
ACTIVITY OF 2 INCREASE IN
ACCESSIBILITY OF ENHANCERS AND
INCREASE OF EXPRESSION.
OKAY.
THERE'S MORE ON THE PAPER.
THERE'S ALSO INTERESTING
DEPENDENCIES IN THESE CELLS WHEN
YOU LOSE H K 36 YOU HAVE GREATER
HISTONE TURN OVER.
AND IT SENSITIZES THE CELLS TO
INHIBITERS OF HISTONE CHAPERONES
AND TRANSCRIPTION ELONGATION.
OKAY.
SO THAT IS MY OPEN VIGNETTE.
BUT WHAT I REALLY WANT TO TALK
TO YOU TODAY IS THE IDEA THAT
SOMATIC ALTERATIONS CAN ALSO
LEAD TO GLOBAL CHANGES IN 3D
ORGANIZATION.
OKAY.
I'M GOING TO START WITH SOME
PRELIMINARIES ON HI C
ANALYSISIKE DO THAT BECAUSE HIGH
C WAS INTRODUCED IN THE PREVIOUS
TALK.
I'M GOING TO VERY BRIEFLY
MENTION SOME OF THE WORK WE DO
AS COMPUTATIONAL BIEGS
BIOLOGISTS DEVELOPING MACHINE
LEARNING MODELS TO PREDICT 3D
THE CONTACT MATRIX FROM ONE D
ESPECIALLY GENOMIC TRACKS.
THEN I WILL TELL YOU A STORY
THAT UNPUBLISHED IN
COLLABORATION WITH IN MELANOMA
V
VIA DEFICIENCY THAT A DRIVES 3D
CHANGES AND A -- SO THAT'S THE
PLAN.
ALL RIGHT.
SO, UM, VERY QUICKLY, SINCE
WE'VE ALREADY SEEN HIGH C IN THE
PREVIOUS TALK, THIS IS AN ASSAY
THAT IS BASED ON CROSSLINKING
DNA N C 2 IN THE NUCLEUS AND
RESTRICTION ENZYME DYE TEST
PROXIMITY LIGATION PULL DOWN.
IN THE END WE HAVE THEE CHI
MERIC READS.
WE SEQUENCE ANCHORS THE ANCHORS
SOMETIMES COME FROM REGION THAT
IS VERY FAR IN THE 1 D GENOME
BUT MUST HAVE BEEN CLOSE IN
CONTACT IN SOME CELLS IN THE
INPUT.
SO WE CALL THESE CONTACTS.
AND WE BUILD A CONTACT MATRIX SO
A MATRIX WHERE WE'RE JUST
COUNTING THE NUMBER OF PARADIGM
READS WITH ANCHORS AOKAY.
AND IT HAS ALREADY BEEN
DISCUSSED THAT YOU CAN YOU CAN
LOOK AT CONTACT MATRIX AT
DIFFERENT LEVELS.
IT IS VERY INTERESTING AND
COMPLEX DATA EVEN THOUGH YOU'RE
ONLY CAPTURING CONTACTS THAT ARE
CLOSE, IT IS GIVING INFORMATION
ON MORE GLOBAL AND HIERARCHICAL
3D STRUCTURE.
BUT WE'RE GOING TO FOCUS MORE AT
THIS LEVEL OF TABS THAT TOX
LOGICALLY ASSOCIATING DOMAINS
THAT WE'VE ALREADY TALKED ABOUT,
THE IDEA THAT YOU HAVE REGIONS
MEGA BASE AND LENGTHS THAT ARE
MORE SELF INTERACTING THAN
INTERACTING WITH ANOTHER JAVENT
TAB.
AND YOU CAN ALSO SOMETIMES BY
EYE SEE THESE STRONGS LOOPS THAT
ARE MEDIATED .
SO ONE THING I JUST WANT TO
POINT OUT IS THAT IN THE FIELD
MANY AGAIN, VERY QUICKLY.
THIS IS LOT OF INTEREST IN MANY
LEARNING FOR GENOMIC COMMUNITY
IN PREDICTING MATRIX FROM DNA
SEQUENCE.
WE WERE INTERESTED IN DEVELOPING
A MODEL THAT COULD PREDICT FROM
ESPECI
E
EPIGENOMIC MARKS.
BECAUSE THEN, YOU COULD IMAGINE
THE MODEL GENERALIZING TO NEW
CELL TYPES.
AND THIS MODEL CALLED EPIPHANY,
IS LOCATIONAL CONTACT MATRIX
FROM A SET OF HISTONE MARKS WAS
JUST PUBLISHED THIS YEAR.
AND AN IDEA
WHERE WE'RE GOING WITH THIS AND
CONNECTING WITH AT STORY, WE
ALSO HAVE A HI C IN OUR R C C
CLEAR CELL CARCINOMA MODEL WITH
LOSS OF FUNCTION OF SET D2 AND
SET D 2 RESTORED.
SO YOU SEE FOR EXAMPLE, THIS IS
H3 K 36 TRY METHYL THESE DOMAINS
ONCE ACTIVITY IS RESTORED.
LOST IN THE MUTANT.
HD F IS ONE GENES THAT MOST
HIGHLY UPREGULATED IN THE SET D
2 MUTANT.
THERE ARE SUBTLE BUT
REPRODUCIBLE DIFFERENCES IN HI
C.
IF YOU LOOK AT THE A LAB CHANGES
IT LOOKS QUITE COMPLICATED.
IF YOU SEPARATE THE POSITIVE
FROM NEGATIVE LOG FULL CHANGES
YOU START TO SEE THAT THERE ARE
STRUCT
STRUCTURES ASSOCIATED WITH SET D
2 ACTIVITY VERSE SET D2 LOSS.
AND WE CAN PREDICT THESE
POSITIVE AND NEGATIVE CHANGES
FROM HISTONE MARKS.
SO USING THIS MODEL BUILT ON
EPIPHANY.
SO WE'RE GOING FORWARD TRYING TO
USE THE FACT THAT WE CAN PREDICT
THE CONTACT MATRIX AND THESE
CHANGES FROM HISTONE MARKS TO
TRY TO UNDERSTAND THE CHANGES
THAT WE'RE SEEING.
OKAY.
I'M GOING SWITCH GEARS AND TALK
ABOUT AT SECOND STORY.
NI PBL HAPPEN ALTHOUGH DIVISION
INC.
THIS IS A COLLABORATION.
AND IT IS STARTED WITH ONE GENE.
IT STARTED WITH AN ALKAGENE
CALLED AT LKATI.
AND PING DESCRIBED THAT THROUGH
ALTERNATIVE TRANSCRIPTION
INITIATION IN THE LOCUS OF THE
ALK YOU LOSE THESE EXTRA
CELLULAR MEMBRANE DOMAINS.
YOU ARE LEFT WITH CHI MACE
DOMAIN AND IT IS AN ALKAGENE.
IF YOU LOOK AT THE A LOCUS AND
YOU ZOOM IN AND SEE WHERE THIS
INITIATION, WHERE THIS
ALTERNATIVE TRANSCRIPTION
INITIATION HAPPENS, IT ACTUALLY
HAPP
HAPPENS, OKAY, SO THIS IN, THIS
IS HI C IN A CELL LINE WHERE ALK
IS NOT EXPRESSED.
IT IS INSIDE SUBTEXT.
SO JUST WITHIN THE BOUNDARY OF A
SUBTAT.
AND YOU CAN ALSO SEE IF YOU DO 4
C WHICH IS WHICH A VERY POINT
CHROMOSOME CAPTURE.
I'M SHOWING HERE IN THE MELANOMA
CELL LINES WHERE ALKATI IS
EXPRESSED VERSUS NOT EXPRESSED,
THIS ALTERNATIVE TRANSCRIPTION
INITIATION, TRANSCRIPTION START
SITE IS MAKING CONTACT WITH DOWN
-- UPSTREAM AND DOWN STREAM
REGIONS AND YOU DON'T SEE THOSE
KAK
CONTACTS WHEN ALKATI IS NOT
EXPRESSED.
SO SOME INTERESTING ORGANIZATION
OF ALKATI BEING JUST WITHIN A
SUBTAT IS IT ASSOCIATED WITH
COHESS INDYSREGULATION.
AND WHAT ONE COULD SEE, WE KIND
OF OR PING CHI ZOOMED IN ON IT
IS TCOHESIN LOADER AND IS
IMPORTANT FOR SORT OF INITIATING
THIS PROCESS OF LOADING COHESIN
AND GETTING LOOP EXTRUSION WHICH
AS ASSOCIATED WITH TATS.
AND WHAT YOU CAN SEE IS THAT IN
MELANOMA LINES THAT DON'T
EXPRESS ALKATI, IT IS HIGHER
EXPRESSED.
AND ALK EXPRESSION IS ASSOCIATED
WITH LOWER IPPBL.
AND THIS OVER HERE AT THE A
BOTTOM I'M JUST SHOWING TRACKS
WHERE YOU CAN SEE THIS ALKATI
EXPRESSION IN SOME CELL LINES,
NOT OTHERS.
ALSO LOOKING A DATA --
EXPRESSION OF ALKATI IS
ASSOCIATED WITH NICHLT IPBL OR
MORE DELETERIOUS MUTATIONS.
SO PERHAPS THIS IS A LINK
BETWEEN NIPBL AND EXPRESSION OF
ALKATI.
ALL RIGHT THE NEXT THING PING'S
LAB DID WAS KNOCK DOWN AN NIPBL.
THERE IS TWO SRRNA WHICH IS
STRONGER ONE IS STRONGER.
SHE DID BOTH RNA SEEK AND K H
SEEK WHICH ALLOWS YOU TO MAP
DIFFERENTIAL USAGE OF START
SITES.
WHAT YOU CAN SEE IS THAT THERE
ARE WHEN YOU KNOCK DOWN NIPBL
THERE ARE MANY EVENTS, MANY
CHANGES IN EXPRESSION INCLUDING
ALKATI AND MANY ALTERNATIVE
TRANSCRIPTION SITES BY ALK, ALK4
AND OTHERS.
IN PARTICULAR THERE THIS KNOCK
DOWN INDUCES THIS ALKAT UING ON
JEN.
OK
AWN
.
>> IF YOU LOOK CLUSTER IN KRAN
DESCRIPTION SATURDAY S
START SITES.
IN ALK, IN NORMAL CIRCUMSTANCES
MOST OF THEM ARE ASSOCIATED WITH
PROMOTERS.
WHEN YOU KNOCK DOWN NIPBL YOU
GET AN EXPANSION SO NEW
INITIATION OF TRANSCRIPTION.
NEW TRANSCRIPTION START SITES AT
DISTAL INTERGENIC REGIONS.
WITH BOTH.
OKAY.
SO YOU ARE GETTING THIS GLOBAL
ALTERNATIVE PROMOTER USAGE WITH
KNOCKDOWN OF NIPBL.
OKAY.
A NEW OBSERVATION.
WHEN YOU START LOOKING AT SOME
OF THESE EVENTS, OKAY YOU BEING
WITH K H SEEK THIS ALKATI EVENT
AND INITIATION OF TRANSCRIPTION.
YOU CAN SEE RNA SEQ.
AND THERE IS AN TRANSCRIPT BUT
YOU CAN ALSO LOOK AT REPEAT
ELEMENTS.
AND YOU CAN SEE THAT THERE THIS
EVENT OVERLAPS WITH AN LTR
REPEAT.
OKAY.
AND IN FACT, WE SAW THAT IN
MULTIPLE CASES THAT YOU SAW
ASSOCIATION WITH ALTERNATIVE
TRANSCRIPTION INITIATION UNDER
NICHLT IPBL KNOCKDOWN WITH PRESS
ENOF AN LTR REPEAT.
OKAY.
LTRS OCCUPY 8 PERCENT OF THE
GENOME.
AND THEY HARBOR REGULATORY
ELEMENTS.
THEY HAD HARBOR TRANSCRIPTION
AND BINDING SITES.
IT IS KNOWN THAT SUBSET OF LTRS
ACTUALLY ACT AS PRIMARY
ALTERNATIVE PROMOTERS.
OKAY.
AND WHAT YOU CAN SEE HERE WHEN
WE LOOK AT ENELECTRONIC AND
DISTALLY, EVEN PROMOTER REGIONS
AND WE LOOK AT THE ALTERNATIVE
TRANSCRIPTION INITIATION SITES
YOU SEE AN ENRICHMENT OF SITES
THAT OVERLAP WITH LTR REPEATS.
EVEN IN THE PROMOTER CASE.
OKAY.
SO LTRS CARRY REGULATORY
ELEMENTS, ONE OF THEM THAT HAS
BEEN REPORTED BEFORE IS MITF
WHICH IS ALSO DISCUSSED IN
PREVIOUS TALKS.
AND INDEED WHEN YOU KNOCK DOWN
NIPBL YOU SEE INCREASED
OCCUPANCY OF MITF.
FOR EXAMPLE AT THE ALKTI LOCUS
AND OTHER SPOTS.
OKAY.
SO NEXT WE LOOKED AT OVERALL
CHROMATIN ORGANIZATION.
AND WHAT YOU CAN SEE PERHAPS, IS
THAT SOME OF THE STRONG TAD
STRUCT
STRUCTURES ARE BREAKING DOWN.
AND THIS IS PERHAPS WHAT YOU
WOULD EXPECT IF YOU REDUCED
EXPRESSION OF THE COHES
INLOADER.
SO QUANTIFY THIS WE USED A
METHOD CALLED ON TAD.
IT A HIERARCHICAL TAD.
HAS IF XU STATES A METHOD WHERE
IS CALLING TAD AND A SUBTAD AND
GIVING AN ANNOTATION.
SO THINK ELTON IS JUST SO A TAD
HAS NO SUBTADS FOR NESTED TADS
YOU HAVE LEVEL 1, LEVEL 2, LEVEL
3.
WHAT YOU CAN SEE UNDER LOSS OF
OR REDUCED NIPBL EXPRESSION
YOU'RE ALSO REDUCING THE LEVEL
OF HIESHG AROUND KEY OF TADS.
SO YOU HAVE SINGLETON AND AMONG
NESTED TADS THEY HAVE REDUCED
LEVELS.
OKAY.
AND THAT'S TRUE AT ALKATI AND
MANY OTHER LOCI.
SO LET'S LOOK AT THE HI C.
THIS IS NIPBL INSUFFICIENT CELLS
AND IN THE KNOCKDOWN.
WHAUK ASEE RIGHT AT THIS LOCUS
AND THESE ARE PART OF ON TAD
RESULTS, AND VISUALIZATION HERE,
ALKATI IS INSIDE THE SUBTAD.
AND UNDER LOSS OF EXPRESSION AS
OF NIPBL YOU HAVE DYSAGGREGATION
OF THE TAD AND A SUDDENLY ALKATI
IS NEAR THE BOUNDARY OF A TOP
LEVEL TAD.
IN GENERAL WHAT WE SEE, AND I I
DON'T SHOW THE QUANTIFICATION
HERE BUT JUST THE CARTOON.
YOU HAVE DYSAGGREGATION OF TADS.
THIS LOSS OF HIERARCHICAL
STRUCTURE.
AND NEW ALTERNATIVE
TRANSCRIPTION SITES THAT ARE
USED TEND TO BE NEAR THE
BOUNDARIES OF WHAT USED TO BE
SUBTADS AND ARE NOW TAD
BOUNDARIES.
OKAY.
FINAL POINTS.
WHAT WE CAN ALSO SEE IS THAT
THROUGH THIS LOSS OF
HIERARCHICAL TAD STRUCTURE
ALKATI WHICH IS AT THE BOUNDARY
OF WHAT WAS SUBTAD IS ALSO
GAINING INTERACTIONS WITH
UPSTREAM AND DOWN STREAM
ENHANCERS.
SO AGAIN, THIS IS FOR C.
IN BLACK IS THE CONTROL.
AND GREEN IS THE KNOCKDOWN.
AND YOU CAN SEE THE SPECIFIC
INCREASES.
SO AND HERE IS THE DATA WHERE
YOU LOOK AT EXPRESSION OF ALKRNA
AND YOU DO CRISPR.
A SPECIFIC ENHANCER.
ALK GAINS SHORT RANGE
INTERACTION ACTIONS WITH ACTIVE
ENHANCERS.
ALSO LOSES INTERACTIONS WITH UP
TRY METHYL DOMAIN.
THAT IS MAIN THING I WANTED TO
TELL YOU AND I'M ALMOST OUT OF
THE TIME.
THIS IS THE MODEL.
OKAY.
SO THAT IN LOSS OF NIPBL THIS
TYPE OF ASSOCIATED COHESIN OP
THEE.
YOU GET INITIATION TRANSCRIPTION
START SITES NEAR BOUNDARY
ELEMENTS.
THEY ARE ALSO ASSOCIATED WITH
LTR REPEATS.
YOU CAN ALSO THINK IN
PHYSIOLOGICAL CONTEXT MAYBE THIS
TAD STRUCTURE.
THIS HIERARCHICAL TAD STRUCTURE
IS PART OF GENOME ORGANIZATION
THAT PREVENTS EXPRESSION OF
LTRS.
OKAY.
I WILL JUST END BY THANK
EVERYONE WHO DID THE WORK.
ALISSA LONG IN THE LAB DID ALL
THE EXPERIMENTAL WORK.
THANK YOU TO ALL THAT DID WORK
ON THIS PROJECT.
AND I WILL JUST STOP SHARING AND
TAKE ANY QUESTIONS.
>> THANK YOU SO MUCH, CHRISTINA.
WE HAVE TIME FOR ONE QUESTION.
>> THANK YOU FOR YOUR TALK.
SO I WONDER WHAT ARE BIOLOGICAL
CONSEQUENCE OF NI PBL.
YOU MAY IMAGINE THIS IS GOING TO
INDID YOU GOS EXPRESSION OF NEW
ANTIGENS.
THAT IS HAPPENING, DO YOU HAVE A
RESPONSE?
>> SO THIS SOMETHING THAT,
ANOTHER PROJECTS SHE'S DEFINITE
LT IN IMMUNE RESPONSE.
WE WERE MAINLY LOOKING AT THAT
THE ALKATTI GENE AND OTHER
EVENTS.
BUT IT ALSO TRUE THAT THIS KIND
OF MORE PERMISSIVE TRANSCRIPTION
COULD HAVE COULD HAVE ADDITIONAL
CONSEQUENCES.
>> THANK YOU SO MUCH.
NEXT SPEAKER IS JASON LOCASALE
WHO WILL TALK TO US ABOUT
INTERFACE OF METABOLISM AND
EPIGEN
EPIGENETICS.
>> OKAY.
THANK YOU ORGANIZERS FOR PUTTING
TOGETHER WHAT HAS BEEN A REALLY
GREAT MEETING.
I CERTAINLY LEARNED A LOT.
I MEAN IT WAS ESPECIALLY NICE TO
SEE ALL THE HISTORY AND TO
REVISIT A LOT OF THAT AND LEARN
ABOUT IT.
AND SEE ALL OF THESE DIVERSE
AREAS OF EPIGENTICS COME IN AND
SEE ALL THE NEW DEVELOPMENTS.
IS IT REALLY A. REALLY GREAT
MEETING I'M REALLY LEARNING A
LOT.
ESPECIALLY HAVING BEEN FOCUSED
MAINLY IN THE METABOLISM FIELD
OVER THE YEARS.
OKAY.
SO THIS IS FIRST TIME I'M
TALKING TO MANY OF YOU.
I'LL JUST INTRODUCE OUR RESEARCH
AND THE PROGRAM OF DEVELOPING
OVER THE YEARS FIRST AND I'LL DO
THAT VERY BRIEFLY.
NEXT I'LL, YOU KNOW I WANT TO
KIND
KIND OF EMPHASIZE SOME OF THE
CONCEPTS RELATED TO THE STUDY OF
EPIGENTICS, PARTICULARLY AS IT
RELATES TO METABOLISM AND NOW
METABOLISM INTERSECTS, CONNECTS
SOMETIMES, INFLUENCES, YOU KNOW
THE ASPECTS OF ALL THE THINGS
WE'VE BEEN LEARNING ABOUT TODAY.
AND THEN, FINALLY, FOR THE
LATTER PART OF THE TALK, I'LL
TALK ABOUT AT DEFINING
METABOLISM AND A METABOLISMIC
NETWORK THAT WE'VE WORKING ON
AND I'LL TALK ABOUT HOW THAT IS
INFLUENCED BY DIET AND HOW THAT
COMES INTO THE CONCEPTS AROUND
EPIGENTICS.
>> OKAY.
SO OUR RESEARCH GENERALLY
SPEAKING IS IN YOU KNOW THREE
BASIC AREAS.
I MEAN WE'RE -- I ORIGINALLY
TRAINED IN CHEMISTRY AND
PHYSICS.
WHEN I WAS LOOKING FOR A POST
DOC I WANTED TO THINK ABOUT
AREAS OF STUDY WHERE I COULD
APPLY SOME OF THE SKILLS AND
SOME OF THE TECHNIQUES THAT I'VE
AND WAYS OF THINKING THAT
ACCUMULATED SINCE THAT TIME.
AND WANTED TO WORK IN AN AREA OF
BIOMEDICAL RESEARCH.
SO I EVENTUALLY DID A POST DOC A
CANCER HOSPITAL AT BETH IS RAY
HE WILL.
AND WILL YOU WAS A HIGHLY
ESTABLISHED IN GROWTH FACTORS
SIGNALLING FIELD.
HE REALLY MADE A LOT OF
CONTRIBUTIONS AND GETTING INTO
HOW SIGNAL PATHWAYS WERE
INTERACTING WITH METABOLISM.
IT WAS A PERFECT FIT.
IT ENABLED ME TO BRING IN A LOT
OF EXPERTISE THAT I HAD BOTH IN
CHEMISTRY AND ALSO IN PHYSICAL
SCIENCE.
SO MY RESEARCH OVER THE YEARS IS
FOCUSED A LOT ON METABOLISM, AND
A WAYS TO MEASURE METABOLISM.
COMPUTATIONAL APPROACH AND
ANALYZING STRUCTURE AND FUNCTION
OF METABOLISM.
THAT LEAD TO THINGS OF ABOUT
THINKING LIKE THERAPEUTIC AND
MORE RECENT DIET IN CANCER AND
ALSO WAYS THESE NETWORK OF
METABOLISM CONFER BIOLOGICAL
FUNCTION AND THAT ESSENTIALLY
GOT US THINKING TO THINKING
ABOUT EPIGENTICS AND I'LL
INTRODUCE THAT IN A BIT.
SO GENERALLY SPEAKING A STRONG
INTEREST IN TECHNOLOGY
DEVELOPMENT TO STUDY METABOLISM.
BOTH IN THE AREA OF ANALYTICAL
CHEMISTRY AND ALSO IN THE AREA
OF DEVELOPING MATHEMATICAL TO
TOOLS.
BOTH IN PHYSIC AND AI AND
MACHINE LEARNING.
THESE ARE JUST SOME OF THE KIND
OF EXAMPLES OF THINGS WE'VE
LOOKING AT OVER THE YEARS IN
THIS AREA.
LIKE ONE, WHETHER IT IS
DEVELOPING TRACING TOOLS AND
QUANTITATIVE ANALYSIS TO
UNDERSTAND THE STRUCTURE OF
METABOLISM.
THOUGHT ABOUT SINGLE CELL
ANALYSIS OF METABOLISM.
AND WAYS TO USE MACHINE LEARNING
TO UNDERSTAND NUTRITION AND DIET
AND HOW IT INTERACTS WITH
METABOLISM.
THESE ARE ALL THINGS THIS WE'VE
BEEN WORKING ON.
BUT THIS IS THE TOPIC OF WHAT I
WANTED TO TALK TO IN THE SPIRIT
OF THIS MEETING.
BASICALLY HOW METABOLISM
INTERACTS WITH ALL OF THE THINGS
WE'VE THINKING ABOUT AND ALL THE
CONCEPTS THAT HAVE BEEN
DEVELOPED AT THIS MEETING AND
OVER THE LAST 40 YEARS.
SO BASIC IDEA IS YOU'VE GOT
THESE METABOLIC NETWORKS THAT
ARE NODES WITHIN THESE METABOLIC
NED WORKS THAT INVOLVE SMALL
MOLECULES ACTIVE NODES, AND
THESE MOLECULES SERVE AS
CO-FACTORS, AS BINDING PARTNERS
A WELL SUBSTRATES AND PRODUCTS
OF ENZYMES THAT THAT WITHDREW
THINK ABOUT TO BE INVOLVED IN
CHROMA
CHROMATIN, DNA METHYLATION AND
THE GENERAL CONCEPT OF
EPIGENTICS.
AND THOSE ARE THE MOST COMMONLY
THOUGHT OF ALPHA, A SUBSTRATE
FOR METH LACES.
USED IN HI STONE.
ONE I'LL TALK ABOUT TODAY IS --
AND THAT IS OR SAND IS THE
METHYL DONOR THAT ARE USED IN
THE REACTION TIME SHOWN ON THE
RIGHT HERE.
THESE ARE JUST SOME EXAMPLES.
POTENTIALLY LINKED TO DIET TO
THINGS LIKE EPIGENTICS VIA
INTERACTIONS WITH CHROMATIN AND
AS WELL A DNA.
YOU CAN THINK OF A NUMBER OF
DIETARY FACTORS AS WELLS
ENVIRONMENTAL INFLUENCES THAT
INFLUENCE THESE MOLECULE THAT I
JUST TALKED ABOUT.
RANGING FROM HI POX SEE YA,
CALORIE RESTRICTION, EXERCISE,
TO MORE SPECIFIC THINGS LIKE
ALCOHOL INTAKE.
DIETARY FIBER, FAT INTAKE,
SOMETHING TALK A LOT MORE ABOUT
IN A BIT DEFINE LEARNING MORE
RECENTLY BEING A POTENTIALLY
MAJOR MODIFIER PS BASICALLY
THINKING THIS ONE WAY IN WHICH
THE ENVIRONMENT POTENTIALLY
INTERACTS WITH THE KIND OF
THINGS THAT WE'VE TALKING ABOUT
DURING THIS MEETINGS.
JUST DIFFERENT WAYS TO
CONCEPTUALIZE THIS.
THIS IS ONE KIND OF WAY OF
THINKING ABOUT IT IN TERMS OF
GENETICS.
AND I'LL TALK MORE ABOUT
EPIGENTICS IN A BIT.
BUT JUST THINKING ABOUT GENETICS
ONE WAY WE THINK ABOUT CANCER
AND CONTEXT OF GENETICS I KNOW
CANCER EPIGENTIC DISEASE BUT
THINKING ABOUT GENETICS FOR A
SECOND.
GENERALLY THINKING YOU THINK OF
CELLS REQUIRE MUTATION THAT COOP
NORMAL PHYSIOLOGY WHETHER THAT
WOUND HEALING OR INFLAMMATION OR
GROWTH FACTOR SIGNALLING.
BASICALLY CELLS ACQUIRE MUTATION
THAT IS ALLOW THEM TO COOP THESE
PATHWAYS.
THINKING ABOUT A CANCER
METABOLISM, GENERALLY SPEAKING
THE CRITICISM ESPECIALLY FROM
JET NETTIC IS ALWAYS RECEIVED AS
NOT MANY MUTATIONS IN METABOLIC
NETWORKS.
AND THE WAY I KIND OF GRAPPLED
WITH THIS AND TRIED TO THINK
ABOUT WHERE METABOLISM MIGHT BE
INVOLVED IN ACTUALLY DRIVING
DEVELOPMENT IS IT ACTUALLY VERY
CLOSELY CONNECTED TO YOU KNOW
CLASSES OF ENZYME THAT IS
COMMONLY MUTATED.
IN FACT, THESE ARE ONE WAY TO
THINK ABOUT METABOLISM IN THE
CONTEXT OF CANCER IS BASICALLY
IMPLODER, PART OF THE PROCESS OF
ACTUALLY MODIFYING THINGS LIKE
CHROMATIN AND DNA.
THE BIOCHEMICAL BASIS OF THIS IS
FAIRLY SIMPLE.
SO GENERALLY SPEAKING POST
TRANSLATIONAL MODIFICATIONS
THINGS LIKE PHOSPHORYLATION,
ASEAT LAGS, METHYLATION,
GENERALLY THEY HAVE AN ENZYME
AND SUBSTRATE AND CO-FACTORS.
THE MOST COMMONLY STUDIED ONE
ARGUABLY PROTEIN CAN I NIECES.
THOSE ARE ALL KINDS OF CELLULAR
PHYSIOLOGY.
BUT THESE KINASES USE METABOLIC
SUBSTRATE ATP.
FOR THE CASE OF KINASES THE
CONSTANT IS GENERALLY ON THE
MICRO MOLAR.
ATP IS ON AN ORDER OF MINI
MOLAR.
KINASES NEVER SEE SITUATIONS IN
CELLS WHERE THEY ARE LIMITED BY
ATP.
THAT'S IN CONTRAST TO THE ENZYME
IS LIKE HI STONES, ET CETERA
WHERE THOSE, THOSE HAVE A
SUBSTRATE KM OS N OUT ORDER OF
MICRO HAULER.
AND SUBSTRATE RANGES
CONCENTRATION RANGES ARE ALSO
MICRO.
SUGGESTING THAT CHANGES IN THE
CONCENTRATION OF THINGS LIKE SAM
OR ACTUALLY CHANGE THE RATE RATE
OF ENZYME.
THE ACTIVITY OF THE ENZYME.
AND WHAT I'LL SHOW LATER ON IN
THE TALK IN MANY CASES THEY CAN
BE JUST AS STRONG AS EPIGENETIC
MODIFYING FACTOR AS SOME OF
THESE ENZYMES I'M JUST LIMITING
THE SUBSTRATE AVAILABILITY.
THIS IS HIGHLIGHTED IN A REVIEW
THAT WE WROTE MANY YEARS AGO.
WHICH JUST BASIC COLLECTS A LOT
OF PRIMERS AND UNFORTUNATELY THE
GROUPS LIKE HAVE DONE A LOT OF
CHARACTERIZATION ON THE
ENTOMOLOGY AND DEFINING KINETIC
PARAMETER AND A LOT OF
INFORMATION ESPECIALLY AS IT
RELATES TO A DRUG DEVELOPMENT
AND COMPOUNDS.
THERASE LOT OF INFORMATION AND
THIS IS JUST AN EXAMPLE OF ALL
THE DIFFERENT KINETIC PARAMETER
AND HOW THEY ARE WITHIN THE
RANGE OF METABOLISM
CONCENTRATIONS.
IN TERMS OF THINKING ABOUT THIS
IN TERMS OF EPIGENTICS.
I REALLY WAS INSPIRED BY,
ESPECIALLY BY ANDY'S WORK OVER
THE YEARS.
AS HE TALKED ABOUT EARLIER, AND
BASICALLY BRINGING THE WADDING
TON LAND SKSCAPE INTO THEORY OFW
EPIGENTICS MIGHT WORK AND.
SOME POINT BUT, GENERALLY
SPEAKING, YOU KNOW THERE IS
STILL KIND OF DEVELOPING THIS,
BUT THERE TWO WAYS IN WHICH
METABOLISM MIGHT INTERACT WITH
EPIGENTICS.
ONE IS A POTENTIALLY ALLOWS
MOVEMENT ALONG THE LANDSCAPE.
IT IS EITHER AN ENVIRONMENTAL
DRIVER OR A CONTRIBUTES
SOMETHING TO THE PROCESS.
OR THE OTHER IS JUST THAT IT
ACTUALLY IS PART OF THE
LANDSCAPE JUST AS ENZYMES AND
OTHER ASPECTS OF EPIGENTICS ARE.
IN THAT CASE IT WOULD ACTUALLY
BE CHANGING THE ACTUAL STRUCTURE
OF THE LANDSCAPE.
BUT BOTH OF THESE KIND OF THINGS
MIGHT BE IN PLAY IN DIFFERENT
SITUATIONS.
AND THEN STILL A LOT OF WORK TO
DO IN TERMS OF CHARACTERIZING
THIS.
BUT THIS IS JUST BASICALLY A
FRAMEWORK OF THINKING ABOUT HOW
METABOLISM MIGHT BE INVOLVED IN
EPIGENTICS.
OKAY.
SO THE PATHWAY OR NETWORK WE'VE
WORKING ON OVER YEAR IS THIS ONE
CARBON METABOLISM.
IT BEGAN AS I WAS POST DOC IN
LOUIE'S LAB I WAS A WAR BIRD OR
ALTERED GLUCOSE METABOLISM AT
THAT TIME I MADE OBSERVATION
THAT CERTAIN CANCER CELLS WERE
DIVERTING THEIR GLUCOSE INTO
AMINO ACID SYNTHESIS.
VIA OXIDATION AND A TRANS AM
NATION REACTION AND INTERMEDIATE
ALT GLYCOLYSIS.
BUT GENERALLY SPEAKING THAT KIND
OF OPENED THE DOOR TO ME AND TO
MANY OTHERS AROUND THAT TIME IN
THINKING ABOUT WHAT THIS PATHWAY
ACTUALLY DOES AND HOW IT IS
CONNECTED TO CANCER.
CANCER BIOLOGY AND ONE WAY TO
THINK ABOUT THIS PATHWAY IT IS
BASICALLY AN INTEGRATION HUB OR
NUTRIENTS COME IN.
IT IS CALLED ONE CARBON
METABOLISM WITHOUT GETTING INTO
ALL BIOCHEMISTRY.
BUT THE MOVEMENT OF A CARBON
UNIT AROUND A COUPLE OF
DIFFERENT METABOLIC CYCLES.
AND FOR THE PURPOSES OF THIS
SYMPOSIUM, THAT CARBON UNIT IS
ALSO THE METHYL WOULD WORD THAT
A USE USED FOR HI STONES AND
DNA.
THERE ALSO A LOT OF OTHER
FUNCTIONS, RANGING FROM ANABOLIC
METABOLISM, REDUCTION
METABOLISM, ET CETERA.
IN STUDYING THIS WE WERE
BASICALLY PROFILING
CONCENTRATIONS OF, WE WERE USING
METABOLISM TO A PROFILE WILL
CONCENTRATION IRNLT MEDIATES IN
A PATHWAYS IN HEALTHY HUMAN
SUBJECTS.
AND WHAT WE FOUND WAS TMETHIONI
A MOST DYNAMIC.
I SHOULD MENTION THIS WILL
BECOME RELEVANT IN A MOMENT.
BUT THE TYPICAL TISSUE CULTURE
CONCENTRATION IS AROUND 100
MICRO MOLAR.
RANGE WE SAW IN HUMANS WAS ABOUT
THREE TO 35, THREE OF 35 MICRO
MOLAR.
IT TURNS OUT THAT THERE IS A
LONG STANDING HISTORY OF ME
THYM
ME
MEME
THIO NEEN.
IT HAS OFFICIALLY SUBSTANTIAL
EFFECTS ON HEALTH IN ANIMALS.
IT A CONCERTED FACTOR INVOLVED
IN AGING ALL THE WAY DOWN TO
YEAST.
DIET INDUCED AN OBESITY CAN OH
BELATED WITH ME THYMINE
RESTRICTION.
AND SOMETHING IT HAS A LOT OF
INTERESTING ANTI CANCER EFFECTS.
PARTICULARLY AS IT COUPLES TO
THERAPIES THAT ARE ALSO INVOLVED
IN ONE CARBON.
RADIATION WHICH AFFECTS
NUCLEOTIDE SYNTHESIS, ET CETERA.
SO THERE ARE A LOT OF
INTERESTING THINGS ABOUT DIETARY
ME TIE
ME THIO NEEN.
WHEN WE LOOK AT HUMAN FOOD.
I WAS BRIEFLY MENTIONING
EARLIER, IS THAT YOU KNOW, THERE
IS WIDE RANGES OF CONTENT IN
HUMAN FOOD.
YOU CAN WE CATEGORIZE IT IN THE
CONTEXT OF TYPICAL HUMAN DIETS
ON THE LEFT.
YOU CAN SEE WESTERN DIET YOU GO
FROM A WESTERN TO A, YOU KNOW TO
A VEGAN OR A JAPANESE KIND OF
SPACED DIET YOU CAN GET 3 FOLD,
FOR FOLD CHANGES IN CONTENT THAT
ONE EATS.
IF YOU LOOK ALONG DIFFERENT FOOD
GROUPS, GENERALLY SPEAKING ON
PER PROTEIN BASIS, THINGS LIKE
LEGUMES AND VEGETABLE BASED
PROTEIN ARE LOW IN ME THIO NEEN.
AND THINGS LIKE MUSCLE BASE
PROTEIN LIKE MEAT ARE HIGHEST IN
ME THIO NEEN.
WITH EGGS BEING THE HIGHEST.
SO YOU GET A VERY DYNAMIC RANGE
BASED ON FOOD.
AND WE WORK WITH NUTRITION
GROUPS TO SEE AND THERE'S BEEN
SOME CLINICAL TRIALS IN THE LAST
FEW YEARS SHOWING THAT DIETARY
CONTENT OF ME THIO NEEN CAN
INFLUENCE PLASMA IN A VERY
DYNAMIC ACUTE.
SO I MENTION ONE CARBON.
IT HAS LOT OF FUNCTIONS.
ONE I WILL TALK ABOUT AND FOR
THE REST OF THIS MEETING IS THIS
POTENTIAL INTERACTION WITH
EPIGENTICS.
SO THE KEY KIND OF FINDINGS THAT
WE MADE MADE OVER THE YEARS WAS
THIS BASIC CONCEPT THAT TYPICAL
TISSUE CULTURE CONCENTRATIONS OF
ME THIO NEEN IN TISSUE CULTURE
MEDIUM 100 MICRO MOLAR.
WE BRING IT DOWN TO THE LOWER
LIMIT OF WHAT HAS BEEN OBSERVED
IN HUMANS WHICH ABOUT THREE PRY
CHROMO LAR YOU CAN SEE DYNAMICS
HI STONE MODIFICATIONS.
THEY ARE ACUTE, REVERSIBLE AND A
SUFFICIENT TO ALTER DEPOSITION
OF MARKS ACROSS THE GENOME.
THERE IS A LOT OF WORK WE'VE
DONE ON THIS OVER THE YEARS.
I MEAN STILL A LOT OF OPEN
QUEST
QUESTIONS.
ONE IS JUST, YOU KNOW HOW THIS
POTENTIALLY INTERACTS WITH GENE
REGULATION, GENE EXPRESSION.
WHETHER THIS HAS ANYTHING TO DO
WITH EPIGENTIC WHICH INVOLVED
STABLE PHENO TYPES.
AND INHERITABLE.
AND A GENERALITY OR SPECIFICITY.
SOME OF THE ORIGINAL STUDIES WE
FOUND CERTAIN MARKS WERE VERY
DYNAMIC AND OTHER ONES WEREN'T.
I'LL TRY TO BRIEFLY TOUCH UPON A
COUPLE OF THESE QUESTIONS.
OKAY TO DO THIS WE HAVE DOING A
CLASSIC DIFFERENTIATION ASSAY
HERE.
THIS A MYO-GENESIS ASSAY.
I BELIEVE PETER WEATHERS
DISCUSSED THIS ONE PREVIOUSLY.
A
AND BASICALLY, LONG STORY SHORT
IS THAT THESE DATA ARE SHOWING
THAT ME THIO NEEN.
THERE IS A DEPENDENCE ON THE
DIFFERENTIATION IT IS DOSE
DEPENDENT.
AND BASICALLY, YOU KNOW, IT IS
REQU
REQUIRED.
DIFFERENTIATION, BUT NOT CELL
VIABILITY AND NOT PROLIFERATION
AT THE HIGHER DOSES IS REQUIRED,
REQUIRES ME THIO NEEN.
HI STONE, OR DYNAMICALLY
ARTICLED IN THESE THESE
SETTINGS.
WE BASICALLY PRO FILED A BUNCH
OF COMMONLY STUDY HIS STONE
MODIFICATIONS.
0 ON K 36, THAT WAS HIGHLY
DYNAMIC ONE IN THIS SETTING.
THAT IS AIDED BY SET D2.
IN THIS CASE YOU KNOCK OUT SET D
2 OR LOWER IT YOU GET SIMILAR
TYPES OF PHENOTYPE WITH RESPECT
TO DIFFERENTIATION AND YOU CAN
ACTUALLY RESCUE SOME
DIFFERENTIATION BY EXPRESSIONS
SECHLT D 2 ON TOP OF KNOCKDOWN
AND THAT WILL CHANGE WHETHER THE
CELLS RESPOND TO METHIONINE OR
NOT.
IF WE LOOK AT GENE ACTIVITY
ASSOCIATED WITH K 36, DURING THE
DIFFERENTIATION PROCESS AS WELL
AS IN RESPONSE TO METHIONINE.
WE HE ENRICHMENTS IN A MYO.
THAT IS IMPACTING GENES INVOLVED
IN THE IDENTITY OF THESE CELLS.
AND IF WE LOOK AT CHROMATIN
DYNAMICS, YOU KNOW, USING ATK,
DOING THAT EITHER WITH
METHIO
METHIONINE.
SET D TWOMENT 2 DO. KNOCK OUT .
IF EITHER METHIONINE RESTRICTED
CELLS OR WE KNOCK OUT THE ACTUAL
ENZYME THAT PUTS ON THE
METHYLATION GROUP.
OKAY.
SO THIS IS AT LEAST JUST SHOWING
THAT AT LEAST IN SOME BASIC
SIMPLE MODELS LIKE JUST A MYO-2
DIFFERENTIATION MODEL THAT THE
METHIONINE CAN ACTUALLY BE
INVOLVED IN THESE KINDS OF
EPIGENETIC PROCESS TO A SIMILAR
EXTENT IF NOT AT SAME EXTENT
THAT A KEY EPIGENETIC ENZYME IN
THIS CASE IS DOING.
OKAY.
AND SO NOW TO THINK ABOUT HOW
THIS MIGHT BE THOUGHT ABOUT IN
THE CONTEXT OF AN ORGANISM.
TO THAT WE'VE USING OUR CLASSIC
MODEL FOR DIFFERENTIATION AND
EPIGENETIC TRANSMISSION.
THE REASON WHY WE LIKE THIS
MODEL SO MUCH.
IT WAS A VERY PANDEMIC FRIENDLY
MODEL.
THE ENTIRETY OF THIS IS A CHEAP
MODEL.
THE ENTIRETY OF THESE DATA COST
ABOUT $200 TO COLLECT.
THIS IS QUITE IN CONTRAST TO THE
MICE AT LEAST I'M WORKING WITH.
BUT THE REASON WE WERE, BUT THE
KEY REASON WE WERE SO INTERESTED
IN THIS PERSPECTIVE OF
METABOLISM IS THAT YOU KNOW,
THAT THERE'S A HISTORY OF
CHEMICALLY DEFINED DIETS BEING
ABLE TO YOU KNOW SUPPORT THE
LIFE OF A FRUIT FLY.
A TYPICAL FRUIT FLY IT'S AGAR,
SOI, CORN MEAL, MOLASSES, YEAST
FOR PROTEIN.
BASICALLY YOU CAN COME UP WITH A
COCKTAIL FROM THE SIGMA CATALOG
AND DEFINE CHEMICAL CONSTITUENTS
BASICALLY RECREATE THE FRUIT FLY
DEVELOPMENT AND PROPAGATE ITS
LIFE AND ITS LIFE HISTORY.
AND SO WITH SELECTED DIET YOU
CAN DO VERY PRECISE CONTROLLABLE
FEATURES.
YOU HAVE VERY PREADVICE
CONTROLLABLE FEATURES ASSOCIATED
WITH THE DIET OF THE FRUIT FLY.
SO SOMETHING LIKE METHIONINE CAN
EASILY STUDIED.
THIS IS JUST TO SHOW THAT THAT
THESE CHEMICAL DIETS CAN
ACTUALLY, ACTUALLY REPRODUCE
FRUIT MY PHYSIOLOGY.
YOU CAN GET SIMILAR LIFE-SPANS
SIMILAR BODY WEIGHT AND A
DEVELOPMENT TIME AND FERTILITY,
ET CETERA.
BASICALLY YOU CAN CREATE ALL THE
PROPERTIES OF A FRUIT FLY WITH
THESE CHEMICAL DIETS.
AND SO WE WANTED TO LOOK AT THIS
IN CONTEXT OF SOME OF THE OLD
SCHOOL EPIGENETIC ASSAYS.
AND THIS IS AN OLD MODEL CALLED
POSITION EFFECT LOCUS.
LONG STORY SHORT.
THERE IS A GENE THAT SITS AT THE
BOUNDARY OF YOU KNOW HETEROAND
OPEN CHROMATIN.
AND DYNAMICS AND THIS GENE
DETERMINES EYE COLOR AND DYNAMIC
ASSOCIATE WITH THIS GENE IF THEY
ARE ALTERED POTENTIALLY BY WHAT
THE FRUIT FLY HAS CLASSICALLY
STUDIED FOR IS THE GENETICS,
THAT YOU KNOW IT TURNS OUT A LOT
OF THESE CHROMATIN MODIFYING
ENZYMES WERE CLONED IN THE
CONTEXT OF A GENES THAT GAVE
PHENOTYPES IN THIS KIND OF ASSAY
WHETHER THEY CHANGE THE COLOR OF
THE FLIY'S EYE.
JUST HIGHLIGHTING SOME DATA FROM
THAT.
IF YOU ACTUALLY PUT THE FLIES ON
METHIONINE, AND WE CAN DO TRANS
GENERATIONALLY,
INTERGENERATI
INTERGENERATIONALLY.
ET CETERA.
BUT THE BASIC IDEA IS THAT YOU
PUT THE FLY ON THE THIS DIET AND
YOU INTRODUCES A STABLE EYE
COLOR PHENOTYPE IN THIS MODEL.
OKAY.
SO JUST TO SUMMARIZE, I JUST
KIND OF HIGHLIGHTED OR
INTRODUCED A LOT OF ASPECTS OF
METABOLISM EPIGENETIC AND TALKED
ABOUT FINDING IN CELL STATE
TRANSITIONS AND WELL ORGANISMAL
PHENOTYPE AND PROPAGATION.
AND WITH THAT I JUST WANT TO
BRIEFLY THANK A FEW OF THE LAB
MEMBERS WHO WERE INSTRUMENTAL IN
THIS WORK.
MIKE REED A POST DOC ON THE
FRUIT FLY STUFF AND CONTINUED
AND MIKE IS STILL WORKING WITH
ME.
MIKE IS SENIOR SCIENTIST APPEAR.
AND A GRAD STUDENT AWAY
COMPUTATIONAL BIOLOGIES.
SHE HAS HER ON LAB NOW.
WITH THAT HAPPY TO TAKE ANY
QUESTIONS.
THANKS FOR YOUR TIME.
[ APPLAUSE ]
>> THE BINDING RESTRICTION ARE
SOME EXTENT OF THE METHIONINE
RESTRICTION IS, LARGELY IN THE
PROTEIN CONTENT OF THE DIET.
LIKE
THERE AROUND MANY OTHER GROUPS
HAVE, AS WELL.
IN THE CLINICAL NUTRITION FIELD
IS THAT GENERALLY SPEAKING
PLANTED BASED DIETS AND/OR LOW
IN METHIONINE.
MEAT-BASED DIETS OR FAIRLY
SUBSTANTIAL DYNAMICS HOW THAT
PROPAGATES TO EPIGENETICS THAT I
THINK WAS, I THINK AT THE VERY
LEAST IT IS INTERESTING.
IN SOME CASES, WE DON'T SEE A
LOT OF CHANGES.
BUT IN SPECIFIC LABORATORY
SETTING LIKE WHETHER CLASSIC
DIFFER REPUBLICAN SHAGS SELL
STRAIGHT.
LIKE MYOGENESIS OR FRUIT FLY
TRANSMITTING EYE COLOR.
WE'RE SEEING SOME PRETTY
INTERESTING EFFECTS.
EVENTUALLY FIGURING OUT HOW MUCH
THIS ACTUALLY PROPAGATES INTO
HUMANS IS, IT REMAINS TO BE
DETERMINED WHETHER YOU CAN
INTERFERE WITH THIS.
AND THERE SOME LITH ON
INTERRUPTING MINTERR
INTERRUPTING METHIONINE
METABOLISMS LIKE SOME PEDIATRIC
ONES.
ESPECIALLY THE K 27 FOR
INSTANCE.
AND THAT, THAT IS PRETTY
INTERESTING.
THERE IS ANOTHER ASPECT OF THIS
WHICH IS ACTUALLY SUPPLEMENT
LIKE YOU MENTIONED.
THERE THERE NUTRIENT SUPPLEMENTS
THAT CAN INTERFERE WITH A
METABOLISM AND METHIONINE
METABOLISM PROCESSING LIKE THE
ONE THAT WAS ON THE NEWS
RECENTLY.
I ACTUALLY HAD AN ENERGY DRINK
ABOUT AN HOUR AGO.
AND IT LACK OF COFFEE BREAKS.
SO THAT ENERGY DRINK ACTUALLY
HAS HIGH CONCENTRATION OF TORE
REASON IN IT.
IT DROWN STREAM OF METHIONINE
METABOLISM AND POTENTIALLY
DISRUPT THE PROCESS OF
METABOLISM.
HOW MUCH OF HAPPENING IN HUMANS
ON DAY-TO-DAY BASIS ON AN
INTERGENERATIONAL BASIS.
WHEN I USED TO TEACH UNDERGRAD,
AND LIKE I CAN REMEMBER I USED
TO TEACH A COUPLE LECTURES ON
EPIGENETIC AND UNDERGRADS AND
DUKE, I MENTIONED THAT.
AND THEY, YOU KNOW, WAS JUST
KIND OF AN INTERESTING CONCEPT
THAT LIKE WHAT YOUR PARENTS ARE
E
EATING, LIKE THAT COULD AFFECT
YOUR TRAITS.
AND LIKE IN EPIGENETICS
COMMUNITY THERE IS A HUNGER FA
M
FA MIN.
THERE AROUND THOUGHT TO BE SOME
SORT OF A TRANS MISSABLE
PHENOTYPE.
BUT WHETHER IT IS OBSERVED IN
DAY-TO-DAY, LIKE IF YOU HAVE, I
MEAN LIKELY LIKE PLANT BASED SRI
BEG
VEGAN IS MORE POPULAR.
A THERE IN PARTICULAR AREAS.
GENERALLY THOUGHT TO BE INVOLVED
IN THINGS LIKE HEALTH SPAN AND
ANTI CANCER, ET CETERA WHETHER
NEW UF THOSE EFFECTS ARE
OCCURRING THROUGH ANY OF THESE
PROCESSES DEFINITELY SOMETHING
YOU WANT TO WRITE ON.
>> OKAY.
>> THANK YOU SO MUCH.
WE'VE COME TO THE END OF THE
SESSION.
WE WILL HAVE ABOUT FIVE OR 10
MINUTES AND WE WILL RECONVENE AT
3:25.
ALSO BACK.
WE'LL HAVE YVONNE
FONDUFE-MITTENDORF TO TALK ABOUT
3-D CHROMATIN STRUCTURE AT THE
INTERSECTION OF TOXICOLOGY AND
DISEASE.
>> I'D LIKE TO THANK THE
ORGANIZERS FOR INVITING ME ABOUT
THE WORK IN OUR LAB.
AND THE WORK WE DO IN THE LAB IS
ON ARSENIC AND LOW-DOSE CHRONIC
EXPOSURE TO ARSENIC HAS BEEN
ASSOCIATED WITH SEVERAL DISEASES
INCLUDING DIABETES AND CANCER
AND YOU'RE WONDERING WHERE WE
GET ARSENIC FROM AND WE GET IT
FROM DRINKING CONTAMINATED WATER
AND FOOD SOURCES AND FOR THOSE
WHO LIVE IN THE AREA AND ARSENIC
SEEPS IN THE SOIL AND GETS IN
THE DRINKING WATER AND WE GET
EXPOSED THAT WAY.
MAKING IT MORE COMPLICATED THE
NEW YORK TIMES PUBLISHED EARLY
LAST YEAR THAT DUE TO CLIMATE
CHANGE WHEN THE WATER BED DRIES
UP THE DUST CAN EXPOSE PEOPLE.
YOU HAVE HIGH LEVELS OF ARSENIC
AND HAVING THE CONTAMINATION
THROUGH AIR AND DUST YOU COULD
HAVE EXPOSURE THAT WAY.
EXPOSURE CORRELATES TO HIGH
INCIDENCE OF CANCER.
ARSENIC DOES NOT DIRECTLY DNA
AND WE HYPOTHESIZE IT'S CHANGING
THE CHROMATIN STRUCTURE ORRIAN
DIRECTLY CHANGING THE EXPOSURE
LEADING TO GENE EXPRESSION.
-- INDIRECTLY CHANGING THE
EXPOSURE LEADING TO THE GENE
EXPRESSION.
WE DECIDED TO BUILD A MODEL
UNDERSTANDING CANCER IS A
PROCESS AND NOT ALL OR NOTHING.
SO WHAT WE DID WAS WE TREATED
CELLS WITH MICROMOLAR OF ARSENIC
AND WATCHED THE PROGRESSION AND
THE CELLS BECOME MORE MIGRATORY
AND AFTER SEVERAL WEEKS OF
TREATMENT THE CELLS BECOME LESS
MIGRATORY AND YOU CAN SEE THEM
FORMING COLONIES AND WHEN YOU IN
MICE AND WE SEE EPITHELIAL TO
WEEK 16 AND REVERTS TO
EPITHELIAL.
EMT BACK TO MET.
WE FOUND A TARGET FOR ARSENIC IS
CTC WHICH WE HEARD ABOUT AND
IT'S BEEN SHOWN PERSONS WOULD
HAVE THESE ARSENIC CAN INSERT
ITSELF INTO THE LOCATION IN
PLACE OF THE ZINC AND THAT WILL
NOT BE ABLE TO BIND AND THERE'S
11 ZINC FINGERS.
10 ARE TARGETED OF ARSENIC AND
THE 11th FINGER IS C3H1.
AND PROMOTERS THAT HAVE ONE
BINDING SITE WHICH IS A WEAK
BINDING SITE THAT PROMOTER IS
BOUND BY CTCF AND WE HAVE GENE
EXPRESSION.
HOWEVER, NOW IN ARSENIC
TRANSFORM CELLS OR EXPOSE CELLS
AT THE BINDING SO IING SITE IS R
BOUND AND WE THINK IT DOESN'T
LIKE TO BIND SO YOU NOW HAVE THE
11 ZINC FINGERS MODULATED BY
ARSENIC OR INHIBITED BY ARSENIC
AND CTCF IS NO LONGER ABLE TO
BIND AND WE'RE SEEING CHANGES IN
GENE EXPRESSION.
WE SEE A WEAK CTCF BINDING SITE
OR PROMOTER AND A STRONG CTCF
BINDING SITE AT THE ENHANCER.
WE THINK CTCF INTERACTION WOULD
BE CLOSE TO THE PROMOTER AND
DRIVE GENE EXPRESSION AND IF YOU
EXPOSE CELLS TO ARSENIC THE CTCF
SITE WE DON'T SEE BINDING BUT
SEE AN ENHANCE THE CTCF AT THE
ENHANCER SITE AND THINK BECAUSE
OF THAT IT'S LOOKING BACK OVER
TO THE PROMOTER TO DRIVE GENE
EXPRESSION.
IS IT TRUE ARSENIC IS TARGETING?
WE TRIED TO LABEL IT AND WHEN WE
PERFORMED GEL SHEET ASSAYS WE
SEE IT BINDS TO THE STRONG SITE
AND CAUSE A GEL SHIFT AND A WEAK
SITE CAUSES A LOT OF GEL SHIFT.
IF WE PLACE IT WITH THE ARSENIC
SEE THE REDUCTION OF CTCF
BINDING REEN TO THE STRONG
BINDING SITE BUT TO THE WEAK
SITE IT SEEMED TO HAVE ABOLISHED
CTCF BINDING.
WE MUTATED THE 11 ZINC FINGER TO
SEE IT AND SHOULDN'T SEE AFFECT
OF ARSENIC TO THE BINDING.
THAT'S WHAT WE SEE PERFORMING
THE SAME EXPERIMENT CTCF BINDS
NICELY.
THE STRONG CTCF BINDING SITE IN
THE ABSENCE AND PRESENCE OF
ARSENIC.
THE WEAK CTCF BINDING SITE IS A
BETTER BINDER IN THE ABSENCE OF
THE CTCF AND I SUMMARIZED THIS
IN THE TABLE WHERE WE SHOW WILD
TYPE CTCF REQUIRED 91 NANO MOLAR
FOR THE VALUES AND THE WEAK CTCF
BINDING SITE REQUIRES 110 AND IF
WE DALIZE WE GET THE BINDING AND
IT REQUIRES A LOT TO BIND TO THE
WEAK CTCF BINDING SITE.
WHEN WE PERFORM THAT IT DIDN'T
MATTER WHETHER YOU HAD ARSENIC
IN THERE OR NO ARSENIC.
WE SEE THE STRONG CTCF BINDING
SITE IS STILL BINDING TO THE
WEAK CTCF BINDING SITE IT SEEMS
IT MAKE IT A BETTER BINDER.
SO WE HEARD FROM TALKS THIS
MORNING WHAT DOES CTCF DO?
IT CREATES THE DOMAIN AND YOU
HAVE A PROMOTER TO DRIVE GENE
EXPRESSION AND BLOCKS THE
ENHANCER FROM ECTOPICALLY
ENHANCIE IING THIS FROM THE
PROMOTER AND IF YOU DISRUPT THIS
YOU CAN DISRUPT AND THIS COULD
DRIVE CARCINOGENESIS THAT WAY.
WE STARTED DOING HIGH SPEED DATA
AND ANALYZING THAT BUT I SHOW AN
EXAMPLE WHERE WE'RE LOOKING AT
INTERACTION BETWEEN THE A
COMPARTMENT AND AS WE GO THROUGH
THE MEDIATED TRANSFORMATION WE
START WITH THE INTERACTION AND
GO DOWN TO AN INTERACTION
STRENGTH ABOUT 3.
B AND B INTERACTION WE START
WITH 8 AND WE COME TO AN
INTERACTION STRENGTH OF ABOUT
4.3.
SUGGESTING POSSIBLY ARSENIC
DISRUPTING CTCF BINDING TO THE
WEAK CTCF BINDING SITE MAY BE
DISRUPTING THE INTERACTION
STRENGTH WE'RE GETTING.
AND WE'RE LOOKING THE AT NON
EXPOSED CELLS AT THE BASELINE.
THE WILD TYPE YOU SEE AS YOU
EXPOSE THE CELLS THROUGH THE
WEEKLY EXPOSURE OF ARSENIC.
WE SEE THE CELLS BECOME MORE
MIGRATORY AS EXPECTED DUE TO THE
TRANSFORMATION POTENTIAL.
HOWEVER, IF WE TAKE THE CELL
LINE OUT MUTATED TO C2H2 WE SEE
AN INHIBITION TO THE MIGRATION
POTENTIAL OF THOSE CELLS.
THE QUESTION IS ARE THERE OTHERS
TARGETED THAT MIGHT BE TARGETED
BY ARSENIC BECAUSE IF YOU'RE
SEEING ZINC IS THE ISSUE MORE
SHOULD BE TARGETED.
SO WHAT WE DID WAS AS WE EXPOSED
THE CELLS TO ARSENIC, WE NOW ADD
A TENFOLD AMOUNT CONCENTRATION
OF ZINC TO THE CELLS AND AGAIN
MEASURED THE MIGRATORY
POTENTIAL.
IF WE TREAT THE CELLS THROUGH
WEEK THREE AND ADD ZINC AS WE
CONTINUE THE CELLS WITH ARSENIC
WE SEE A DECREASE IN THE
MIGRATION POTENTIAL AND SEEMS TO
US THE EARLIER YOU DO THAT THE
BETTER FOR THE CELLS BECAUSE WE
SEE IF WE HAD TREATMENT AT WEEK
8 AS NOT AS DECREASED AS IF YOU
START EARLY SUGGESTING POSSIBLY
EARLIER TREATMENT WITH ZINC MAY
BE MORE BENEFICIAL FOR THE
CELLS.
WE THINK IT'S GIVEN A STRONGER
POTENTIAL COMPARED TO THE ZINC
AND THE ZINC IS STRONGER.
SO WHAT WE'RE DOING WITH THE
STORY IS WE THINK TOXICOLOGISTS
ALLOWED TO UNDERSTAND THE IMPACT
OF CTCF IN THE 3-D CHROMATIN
ARCHITECTURE BECAUSE PEOPLE HAVE
ARGUED IF YOU KNOCK DOWN CTCF
YOU SEE CHANGES BUT NOT MASSIVE
ENOUGH -- THEY'VE NOT SEEN
CHANGES IN GENE EXPRESSION
ASSOCIATED WITH THE MASSIVE
IMPACT OF CTCF BUT WE THINK IN
THE ONLY KNOCK DOWN AND MAY BE
ABLE TO EXPLAIN IT IN THE
EXPRESSION AND DOMAINS WE'RE
FIND.
WE'RE DOING EXPERIMENTS ON THE
C2H2 MUTANT TO SEE THE
DIFFERENCES IN THE GENE
EXPRESSION PATTERNS AND WHERE
IT'S BOUND AND WHERE YOU LOST
AND GAINED SITES OF CTCF BINDING
IN BOTH CELL TYPES.
AGAIN, BECAUSE OF THAT PEOPLE
HAVE ARGUED THAT POSSIBLY IT'S
BETTER TO HAVE A CELL TYPE
SPECIFIC 3-D GENOME REWIRER.
AND WE ALSO WENT OUT LOOKING FOR
THAT AND THE ONE WE THOUGHT WAS
INTERESTING TO US WHICH WE
THOUGHT WAS HIGHLY EXPRESSED IN
ARSENIC TRANSFORMED CELLS IS
HERE YOU START WITH YOUR
CHROMATIN AND IT PLAYS A ROLE
AND B COMPARTMENTS.
AND WHAT
B2 DOES IS BRINGS THE DNA TO
THE FAMILY TRAIT AND IT USES TO
BIND TO THE MATRIX AND ALSO HAS
THE DNA BINDING DOMAIN WHICH IT
USES TO BIND TO DNA AND YOU CAN
IMAGINE IT CAN BRING SEVERAL
REGIONS OF THE DNA TO THE MATRIX
THAT WAY AND BY BRINGING
CELLULAR REGIONS IT CAN BRING AN
ENHANCER AND BRING THE REWIRER.
AND WE HAVE A STORY WE THINK
CTCF MIGHT DIRECTLY BE
IMPLICATED IN CHROMATIN CHANGES
WHICH WOULD LEAD IT CHANGES IN
GENE EXPRESSION.
PEOPLE LOOK AT GENE EXPRESSION
AS TRANSCRIPT ABUNDANCE AND THE
ROLE OF A CIRCULAR RNA THAT
COMES THIS CAN GO BACK AND PLI A
ROLE IN ROW MATTIN MEDIATED
CHANGE CAN LEAD TO CANCER.
WHAT IS BACK SPLICING?
THIS IS TYPICALLY YOUR PRE mRNA
AND MATURE RNA LOOKS LIKE THIS
AND BACK SLICING YOU HAVE EXON 4
BACK SPLICING TO EXON 3 AND THE
ONLY WAY YOU CAN DIFFERENTIATE
THAT IS THIS BACKSPLICE JUNCTION
UNIQUE TO THIS mRNA.
PEOPLE HAVE LOOKED AT THIS FOR
YEARS AND ACTUALLY WOULD
DISCOUNT THEM AS JUNK RNA.
RECENT PAPERS HAVE SHOWN THEIR
FUNCTIONAL AND COULD PLAY IF
FOUND IN THE NUCLEUS CAN PLAYING
A ROLE IN REGULATING
TRANSCRIPTION AND COULD ACT AS
MICRO RNA SPONGES OR DEKOIZ --
DOUGH
DECOYS AND I'LL SHOW AN EXAMPLE.
LIKE EVERYTHING WE DO IN OUR LAB
WE START WITH PROFILING AND WHAT
ARE THE CIRCULAR RNA'S EXPRESSED
IN ARSENIC TRANSFORMED CELLS?
WHEN WE DID THAT WE SAW
DYSREGULATED CIRCULAR RNAs AND
THE QUESTION FOR US WAS THAT THE
ONLY TRANSCRIPT FROM THE GENE OR
WAS THE mRNA ALSO DYSREGULATED.
WE PERFORMED A NANO STRING
ANALYSIS AND TOOK THE TOP
CIRCULAR RNAs AND COMPARED THEIR
EXPRESSION TO THE EXPRESSION OF
THE LINEAR mRNA.
AND THE mRNA WAS UPREGULATED
AND THE CIRCULAR RNA FROM THIS
WAS ALSO DYSREGULATED.
AND THIS WAS EXCITING BECAUSE
RECENT PAPERS HAD SHOWN THE
EXPRESSION PLAYS A ROLE IN
INDEPENDENT GROWTH AND CELL
MIGRATION AND FOR IT TO BE
EXPRESSED AND MICRORNA THAT
NEEDS TO BE DOWN REGULATED TO BE
EXPRESSED AND LASTLY THERE'S A
PAPER THAT SHOWED RECENTLY
THERE'S A CIRCULAR RNA DIFFERENT
FROM THE ONE WE FOUND 8928 AND
IT SHOWED IT PROMOTES
PROGRESSION OF NON-SMALL LUNG
CANCER AND WE ASKED THE QUESTION
HOW IS THIS CIRCULAR RNA
EXPRESSED?
BECAUSE OF TRANSFORMATION OR
ARSENIC EXPOSURE?
WE PERFORMED ACUTE EXPOSURE OF
THE CELLS TO ARSENIC.
YOU SEE THE CONCENTRATION
INCREASED AND SAW THE EXPRESSION
OF THIS AS WELL AS THE CIRCULAR
RNA EXPRESSION.
WE THEN LOOKED AT IT IN A
TRANSFORMATION MODEL AND SEE THE
WEEKLY EXPOSURE OF ARSENIC AS IT
GETS EXPRESSED LIKEWISE THE
CIRCU
CIRCULAR RNA GETS EXPRESSED.
IS IT WE'RE MEASURING THE SAME
TRANSCRIPT OR MEASURING TWO
DIFFERENT TRANSCRIPTS.
I THINK WE'RE MEASURING TWO
TRANSCRIPTS AS SHOWN HERE.
IF WE KNOCK DOWN SAT B2 WE DON'T
SEE AN DECREASE IN THE CIRCULAR
RNA EXPRESSION BUT IF WE KNOCK
DOWN THE CIRCULAR RNA THE SAT B2
mRNA GOES DOWN.
SUGGESTING COULD IT BE THE
CIRCULAR RNA COMES IN TO PROTECT
AT LINEAR mRNA ?
WE THINK THAT'S TRUE BECAUSE IF
WE JUST OVER EXPRESS THE
CIRCULAR RNA BY ITSELF WE DON'T
SEE CHANGES IN SAT B2 BUT ONCE
WE EVERY EXPRESS WE SEE THE
CIRCULAR RNA COMING UP.
WE'VE SEWN DOWN OF THE CIRCULAR
RNA CAUSE CELL MIGRATION AND
PROLIFERATION AND LIKEWISE IF WE
EXPRESS SAT B2 IN NON-TREATED
CELLS WE SEE INCREASE IN CELL
PROLIFERATION AS WELL AS
INCREASE IN CELL MIGRATION.
WE WENT BACK AND ASKED THE
QUESTION WHAT ARE THE PATHWAYS
THAT ARE SIMILAR ONCOGENIC
PATHWAYS SIMILAR BETWEEN ARSENIC
TRANSFORMED CELLS AND SAT B2 AND
WE HAVE THE K RAS ONCOGENIC
PATHWAYS AND THIS WAS AMAZING TO
SEE BECAUSE IF YOU LOOK AT
ARSENIC TRANSFORMED CELLS AND
THIS IS ONCOGENIC PATHWAY WITH A
NUMBER OF GENES WE SEE FOR
ARSENIC EXPOSED CELLS WE SEE
WHEN THE GENES ARE EXPRESSED IN
ARSENIC TRANSFORMED CELLS AND
SEE SAT B2 CELLS EXPRESSED
CELLS.
HERE'S 600 WE SEE ARSENIC
TRANSFORMED CELLS AS WELL AS THE
SAT B2 CELLS AND THE K RAS
ONCOGENIC SIGNATURE GENES.
IF SOME OF THOSE GENES IN THE K
RAS PATHWAYS THAT WERE DOWN
REGULATED FOR ARSENIC
TRANSFORMED CELLS IN SAT B2
CELLS THEY WERE DOWN REGULATED
SUGGESTING IT'S POSSIBLE ARSENIC
IS TARGETING SAT B2 TO DRIVE THE
K RAS PATHWAY IN ARSENIC
TRANSFORMATION CELLS AND THE SAT
B2 CELLS WHERE YOU HAVE THE
PATHWAY UPREGULATED.
THOSE GENES WERE UPREGULATED AND
SAT B2 CELLS ALL REGULATED WITH
SIMILAR AND WHEN WE KNOCKED DOWN
SAT B2 OR THE CIRCULAR RNA WE
SEE A REVERSAL OF THE
UPREGULATION WENT DOWN AND FOR
THOSE AGAINST UP REGULATED WHEN
WE KNOCK DOWN SAT B2 OR CIRCULAR
RNA WE SEE THAT GOING BACK UP
SUGGESTING AGAIN THAT POSSIBLY
THIS IS THE PATHWAY THAT ARSENIC
IS TARGETING FOR DRIVING
CARCINOGENESIS AND THE CIRCULAR
RNA COMES UP AND MAYBE IT'S
PROTECTING SAT B2 TRANSSCRIPT
FOR DEGRADATION BY EXPUNGING THE
MICRORNA AND WE THINK IT TARGETS
SIMILAR K RAS PATHWAYS AS IN
ARSENIC TRANSFORMATION.
AND LASTLY WE SAW WHEN WE MAKE
THIS CIRCULAR RNA IT FORMS AN
ATG SITE SO COULD IT BE
TRANSLATED AND IF IT'S
TRANSLATED YOU IMAGINE IT WILL
FORM A PEPTIDE THAT LOOKS
DIFFERENT FROM SAT B2 BECAUSE
YOU WILL NOT HAVE THE DNA
BINDING DOMAIN.
INDEED WE'RE ABLE TO SHOW IT'S
TRANSLATED BECAUSE WE USE THIS
AND THE ONLY WAY IT'S EXPRESSED
OR TRANSLATED AND WE'RE ABLE TO
SEE THAT IS IF THE CIRCLE GOES
AROUND AND TRANSLATES THIS BUT
IF YOU TREAT THE CELLS TO TARGET
AND TREAT IT WITH A SUBSTRATE
YOU'LL BE ABLE TO READ IT WITH
LUMINESCENCE AND MAKE THE
PEPTIDE AND WE ALSO SHOW THAT
IT'S ACTUALLY A NUCLIDE
CO-LOCALIZED AND LASTLY WE'RE
ABLE TO SHOW THAT IT ASSOCIATES
WITH POLY SOMES AND WE DID
PROFILING AND mRNA ASSOCIATED
WITH HEAVY LIGHT AND VERY LITTLE
mRNA AND LITTLE ORGANOID FREE
AND THIS IS NOT TRANSLATED SEEN
WITH FREE.
IF WE THEN TREAT IT WITH EDT
WHICH SHOULD DISASSOCIATE AND
TRANSLATED.
SO THIS IS WHAT WE THINK IS
HAPPENING.
YOU HAVE SAT B2 WHICH IS THEN
MADE AND YOU CAN ACTUALLY OLI
OLIGOMERIZE AND IF IT INTERACTS
IT WOULD NOT BRING IT TO THE
MATRIX.
IN THAT CASE IT COULD ACT AS A
DOMINANT NEGATIVE SAT B2
FUNCTION AND WE HAVE PRELIMINARY
DATA THAT MAY BE TRUE AND IT
BINDS TO THE MATRIX ASSOCIATED
DOMAINS OF DNA AND BRING DOWN
ONE WHICH BINDS TO THE INNER
NUCLEAR MATRIX AND BRING OUT
HISTONES AND BRING DOWN SPLICING
FACTORS BUT SAT B2 BE PLAYING A
ROLE IN ALTERNATIVE SPLICING.
IT BRINGS UP ANOTHER AREA IN THE
LAB.
IF WE PULL DOWN WITH THE
CIRCULAR RNA WE LOSE WHAT BINDS
TO DNA AND LOSE SOME OF THE
MARKS AND WE RETAIN AND ARE ABLE
TO RETAIN DOWN 1 AND SEE SAT B2
COMING UP AGAIN SUGGESTING MOST
LIKELY THE CIRCULAR RNA COULD BE
BINDING AND WOULD BE ACTING AS A
DOMINANT NEGATIVE.
SO WE HAVE A POSSIBLE MODEL
WHERE WE HAVE SEEN THE CELLS ARE
EXPOSED TO ARSENIC THROUGH THE
PROCESS OF ARSENIC
TRANSFORMATION WEEKEND SEE THE
CIRCULAR RNA COME UP LIKEWISE
LINEAR mRNA AND THINK IT MY BE
DECOYING OR EXPUNGING THE
MICRO-RNA.
WHAT WE DON'T KNOW YET IS WHEN
DOES IT MOVE FROM NON CODING
FUNCTION TO A CODING FUNCTION.
WE HAVE A HYPOTHESIS THROUGH Y
JIO
-- MYOGENESIS AND CLEAVES SAT B2
AND THAT'S A SIGNAL TO DRIVE THE
CELLS TOWARDS MYOGENESIS.
WE HYPOTHESIZE MAYBE SOMETHING
LIKE THAT IS HAPPENS BECAUSE WE
SEE BANDS AND CLEAVED AND SEE
MORE OF SAT B2 AND LESS CIRCULAR
mRNA.
WE THINK THE CELL ONCE IT'S
DEGRADED IT WANTS TO MAKE SOME
FORM OF SAT B2 AND CONVERTS THAT
FROM A NON-CODING TO A CODING
FUNCTION.
IF THAT'S TRUE MAYBE WHAT'S
HAPPENING IS IT MIGHT CHANGE THE
3-D GENOME ARCHITECTURE THERE BE
DRIVING ONCOGENESIS THAT WAY.
AGAIN, I'M SO HAPPY I DO
TOXICOLOGY.
I WAS NEVER A TOXICOLOGIST BUT
ALLOWED US TO DISCOVER
REGULATORS AND THEIR FUNCTIONS I
WOULD NOT HAVE FOUND IF I WAS
DOING NORMAL GROWTH IN CELLS.
I'D LIKE TO THANK ALL THE
MEMBERS OF MY LABS, VIA FOR
THEIR SUPPORT AND COLLABORATORS
AND FUNDING.
THANK YOU.
>> MY BRAIN IS ON A FRIDAY
AFTERNOON SCHEDULE SO FORGIVE
ME.
I WANT TO MAKE SURE I'M
FOLLOWING ALL THE DIFFERENT WAYS
IT SEEMS LIKE SAT B2 IS
REGULATING ITSELF.
SO THE CIRCULAR RNA PLAY ACT AS
A SPONGE TO PROTECT THE FULL
LENGTH RNA AND ALSO BEING
TRANSLATE INTO A PEPTIDE AS ACTS
AS A DOMINANT NEGATIVE OF THE
PROTEIN.
IS THAT WHAT YOU SAID?
>> YES, BECAUSE IT DOESN'T HAVE
THE ABILITY TO BIND TO DNA AND
DIDN'T FUNCTION AS SAT B2 WOULD
FUNCTION.
NOT BRINGING THE DNA TO THE
MATRIX FOR TRANSCRIPTION.
WE'RE WORKING ON TESTING THAT.
>> THE DUAL LAYER OF REGULATION
OF THAT PROTEIN ITSELF MUST MEAN
IT'S VERY IMPORTANT.
>> EXACTLY.
WHAT WE'RE EXCITED ABOUT IS
MAYBE THIS IS THE SAME THING
HAPPENING IN MYOGENESIS BUT WE
WANT TO LOOK AT THE RNA WHICH
COULD BE TRANSLATED.
NOW WE'RE SEEING THIS HERE MAYBE
ACT THE SAME WAY IN OTHER AREAS
THAT NO ONE HAD LOOKED AT IT.
IT'S OPENED UP A LOT.
>> QUICK QUESTION FOR YOU.
IN THE ARSENIC WHEN YOU SEE ALL
THESE CHANGES AND DRIVING UP RAS
SIGNALLING YOU SEEING IT IN
TYPICAL WITHOUT MUTATIONS IN RAS
ARE THERE OTHER MUTATIONS.
>> THAT'S A GOOD QUESTION AND I
EXPECTED THAT.
WEN WHEN HE SAW THIS WE THOUGHT
MAYBE THERE'S A MUTATION BUT THE
FACT THAT WE'RE ABLE TO KNOCK
DOWN SAT B2 AND mRNA AND
REVERSE MAYBE IT'S NOT A
MUTATION AND THAT'S WHERE WE'RE
GOING FROM HERE.
>> THANK YOU.
>> OUR NEXT TALK IS FROM SEAN LI
FROM CEDAR-SINAI AND INTERESTED
IN THE EPIGENETIC SEX IN BLADDER
TUMORIGENESIS.
>> I'M VERY HONORED TO BE
INVITED.
THANK YOU AGAIN FOR THE
INVITATION AND HUMBLED.
TO SHARE THE STAGE WITH TRUE
PIONEERS.
THANK YOU ALL.
I'LL TALK ABOUT SEX BUT
E-GENETIC SEX.
EPIGENETIC SEX.
THIS IS KNOWN FOR AUTO IMMUNE
DISEASE MORE FEMALE DOMINANT
DISEASE FOR AUTO IMMUNE DISEASE.
WE'RE INTERESTED IN FEMALES FOUR
TO FIVE TIMES MORE.
TAKING ABOUT BLADDER AND IT
HAPPENS IN MAMMALS AND THEY ARE
WATER SEX IN TURTLES AND FROGS
AND THOSE ARE NOT EQUIVALENT IN
BLADDER BECAUSE THEY USE IT AS A
WATER REVERSE BECAUSE THEY CAN
COME BACK AND REABSORB WATER AND
WE HUMANS DON'T ABSORB WATER
FROM BLADDER.
WHAT'S INTERESTING ABOUT THE
BLADDER CELLS IT IS QUITE AN
EFFECTIVE BARRIER MUCH BETTER
BARRIER THAN THE BLOOD BRAIN
BARRIER.
IT'S MAKING SENSE BECAUSE THEY
WANT TO KEEP URINE AWAY FROM THE
TISSUE AND BLOOD.
IN ADDITION, BLADDER UROTHELIUM
CYCLE AND IT'S A VERY QUIESCENT
STRUCTURE.
IT'S AN EPITHELIAL STRUCTURE AND
UNLIKE GUT EPITHELIUM CONSTANTLY
AND BLADDER UROTHELIUM IS UNIQUE
IN A WAY THAT IT REGENERATES SO
UNDER A URINARY TRACT INFECTION
OR SUBJECT TO CHEMICAL INJURIES
IT REGENERATES VERY QUICKLY.
BECAUSE OF THIS RAPID
REGENERATIVE POTENTIAL OR
KINETICS BLADDER CANCER IS
RELATIVELY HIGH.
THIS IS PARTICULARLY TRUE FOR
MALE.
SO IT'S A NUMBER FOUR FOR MALES
AND THIS IS NOT THE CASE FOR
FEMALES.
IT'S QUITE SURPRISING.
WHAT'S INTERESTED IN TERMS OF
CLINICAL TRIAL PUBLISHED A FEW
YEARS BACK IN NEW ENGLAND
JOURNAL SO AS YOU CAN SEE HERE
THIS IS A TARGETED CHEMOTHERAPY
DRUG.
FOR MALES AND FOR BLADDER CANCER
A FEW FEMALES HAVE BLADDER
CANCER SO THERE'S OTHER
INDICATIONS IN TERMS OF
THERAPEUTIC RESPONSES INCLUDING
IMMUNOTHERAPY RESPONSES BETWEEN
MALE AND FEMALE.
SO WHAT'S ALSO INTERESTING ABOUT
THE GENDER DISPARITY IS YOU CAN
RECAPITULATE THIS IN RODENT
MODELS USING CARCINOGENESIS
MODEL WHICH WE USE IN THE MODEL
THIS FEEDS BBN IN THE WATER AND
MOUSE DRINK IT AND DEVELOP
BADDER CANCER AND MALES DEVELOP
AND DIE FROM BLADDER CANCER
SOONER THAN FEMALE.
YOU CAN ALSO RECAPITULATE THIS
USING GENETICALLY ENGINEERED
MOUSE MODELS AND IT'S NOT JUST A
BLADDER CANCER BUT MELANOMA YOU
CAN MODEL IN RODENTS.
IT SUGGESTED TO US IT'S NOT JUST
ENVIRONMENTAL.
ENVIRONMENTAL IS OBVIOUSLY
IMPORTANT AS YOU HEARD AND THE
GENETIC OF BIOLOGICAL BASIS FOR
GENDER DISPARITIES IN CANCER.
SO WE ARE INTERESTED IN THIS
QUESTION QUITE A FEW YEARS BACK
TRYING TO FIGURE OUT OR TRYING
TO UNDERSTAND WHY THERE'S A MALE
DOMINANCE IN CANCERS.
SO ONE OF THE FIRST THINGS
PEOPLE LOOKED IS DURING THE
EPIDEMIOLOGICAL STUDY DO SEE IF
IS THE REASON FOR SEX DIFFERENCE
AND THAT TURNS OUT TO BE NOT
TRUE.
SMOKING IS IMPORTANT BUT SMOKING
IS NOT THE REASON MALE HAS A
HIGHER INCIDENTS OF BLADDER
CANCER.
NEXT THING PEOPLE LOOKED IS SEX
HORMONES.
SO ANDROGEN IS A MAJOR DRIVER
AND CAN EXPOSE TO ANDROGEN AND
IT'S CLEARLY A MAJOR DRIVER AND
ESTROGEN DEPENDING ON THE
RECEPTORS I THINK WOULD BE
PROTECTIVE OR PROMOTING BLADDER
CANCER BUT THE EFFECT SIZE IS
SMALLER THAN ANDROGEN
SIGNALLING.
WE REPORTED LAST YEAR ANDROGEN
NOT ONLY FUNCTION IN UROTHELIUM
CELLS AND SIGNALLING THROUGH THE
IMMUNE MICRO ENVIRONMENT AND IT
INDUCES PREMATURE CD 8 T CELL
EXHAUSTION AND THIS WAS
PUBLISHED WITH CLOSE
COLLABORATION WITH MY COLLEAGUE
DR. LEE AT OSU.
ANOTHER REPORT ON SEX CHROM
CHROMOSOMES.
FOR PATIENTS WHICH LOST X
CHROMOSOME AND THEY HAVE
INCREASED BLADDER CANCER RISK
SUGGESTING THAT SEX HORMONE
IMPACT IS ALSO IMPORTANT
HOWEVER, THIS STUDY IS
CONFOUNDED WITH OTHER CLINICAL
OBSERVATIONS IN TERMS OF OTHER
COMORBIDITIES.
SO ONE CANNOT FOR CERTAIN FROM
OTHER COMORBIDITIES OR BECAUSE
OF SEX CHROMOSOME.
SO WE DECIDED I WANT TO MENTION
MY COLLEAGUES LY SOME MAY OR MAY
NOT KNOW THAT Y CHROMOSOME,
MALES HAVE Y CHROMOSOME AND IT'S
OFTEN LOST DURING AGING OR
ENVIRONMENTAL EXPOSURE AND ALSO
Y CHROMOSOME TRIGGERS ADAPTIVE
IMMUNITY DYSFUNCTIONING.
THAT'S NOT THE REASON MALE HAS
HIGHER RISK.
SO WE DECIDE TO LOOK AT THIS
QUIT A FEW YEARS BACK USING THE
MODEL.
THERE'S FOUR SEX TYPES RATHER
THAN TWO.
WE HAVE A NORMAL XX FEMALE, XX
CHROMOSOME OVARY, NORMAL OVARY
AND XX MALE.
XX SEX CHROMOSOME COMPLIMENT FOR
THE TESTES AND THIS IS LOSS OF
THE Y GONAD DEVELOPED TO IN THIS
CASE TRANSGENIC AND
OVEREXPRESSION OF XYL VIA
AUTOSOME AND THE GONAD DEVELOPED
TO TEST THIS INSTEAD OF OVARY.
AND CONVERSELY WE HAVE THE XLY
CHROMOSOME BUT LOSS OF XLY AND
THEY DEVELOP AN OAF OVARY
INSTEAD OF TESTES AND USING THE
MODEL ENABLES US TO SEGREGATE
THE EFFECT OF SEX CHROMOSOMES
VERSUS SEX HORMONE.
GONADO EFFECT VERSUS CHROMOSOMAL
ACT AND WE CAN LOOK AT THE
INTERSECT WHETHER THEY FUNCTION
TOGETHER OR NOT.
THIS IS A QUICK RESULT.
YOU CAN SEE XX IN THE MIDDLE IS
IS THE SURVIVAL CURVE.
XX CHROMOSOME WITH FEMALE
PHENOTYPE CAN DEVELOP AND DIE
FROM BLADDER CANCER MUCH MUCH
LATER AND IF YOU COMPARE XX
FEMALE VERSUS XX MALE THEY HAVE
THE SAME CHROMOSOMAL COMPLIMENT
BUT DIFFERENT GONADOCYTES AND
HAVE TESTES INCREASES THE CANCER
RISK AND ANDROGEN IS A RISK
FACTOR.
WHAT IS INTERESTING IF YOU
COMPARE XX FEMALE VERSUS XY
FEMALE THEY AN ALL HAVE ANDROGEN
AND OVARIES BUT THE ONLY
DIFFERENCE IS XX VERSUS XY.
IT INCREASES BLADDER CANCER
RISK.
WHAT IS INTERESTING IS IF YOU
LOOK IF THEY'RE INTERACTIVE AND
ANALYSIS AND HAVING XLY IS OVER
2.5 TIMES MORE HIGHER RISK.
AND HAVING TESTES IS 4.7 TIMES
MORE.
AND IF THERE'S A COMBINATION XLY
AND TESTES IT'S A 12.4 TIMES
MORE LIKELY TO DEVELOP BLADDER
CANCER IN THIS RODENT MODELS.
THIS CLEARLY SUGGESTS THERE'S
SOME INTERACTION BETWEEN
CHROMOSOMAL AND GONADAL AFFECT
AND WE'RE INTERESTED IN THE
INTERACTIVE MECHANISM.
ALL THIS INTERACTION LEADS TO AN
INCREASED CANCER RISK IF WE KNOW
THE MECHANISM, CAN WE TARGET IT
TO REDUCE CANCER RISK?
WE'RE INTERESTING TO LOOK AT THE
CHROME SOME FOR BLADDER CANCER
RISK AND HAVING A Y CHROMOSOME
IS EFFECTIVE AND LOOKING AT TWO
CHROMOSOME WHETHER IT'S BETTER
PROTECTED.
WE DID A QUICK RNA SEQ ANALYSIS
AND LOOKED AT THE TYPES AND
IDENTIFIED THE DIFFERENTIALLY
EXPRESSED AND KDM68 IS LINKED
AND ESCAPES IN ACTIVATION.
AND IT'S X CHROMOSOME LINK AND
MUTATED IN BLADDER CANCER ABOUT
24% IT'S INVOLVED IN REGULATING
POLY CHROME BECAUSE OF THE
METHYLATION AND PART OF THE
COMPLEX INVOLVED IN METH IN K4
MONOLATION.
AND ACTIVATION AND FUNGTION INTO
TWO MAJOR EPIGENETIC COMPLEXES
AND POTENT REGULATOR.
I'M GOING TO TALK BRIEFLY ABOUT
BLADDER CANCER MOLECULAR SUB
TYPE AND PATHOLOGY.
THE KEY IS BLADDER CANCER ARISE
PREDOMINANTLY FROM THE
UROTHELIUM STRUCTURE IN MOUSE IS
ONLY THREE LAYERS.
IN HUMAN MAYBE SEVEN TO NINE
LAYERS.
AND THEY CAN ENHANCE OR
COMPLIMENT EACH OTHER AND APPLY
SEX DIFFERENCES IN CANCER.
SO THAT LEADS TO WHAT WE WANT TO
DO IN THE FUTURE.
AND ESTABLISHED EPIGENETIC SEX
AND WHETHER THERE'S CONSENSUS
FROM TISSUE TO TISSUE, FROM
STAGE TO STAGE OR FROM SPECIES
TO SPECIES.
AND WE ARE ALSO VERY MUCH
INTERESTED TO KNOW THE POTENTIAL
IMPACT OF EPIGENETIC SEX,
HETEROGENEITY OR PLASTICITY AND
PLASTICITY MAYBE OF CANCERS.
AND WE WOULD LIKE TO KNOW
WHETHER IT'S USEFUL OR NOT OR
MAYBE IT'S USEFUL IN THE COMPLEX
OF EPIGENETIC SEX IN CANCER
RISK.
SO TO GIVE YOU A VERY BRIEF WE
DID LOTS OF EPIGENETIC HISTO
MODIFICATIONS FOR THE FIRST
ROUND.
YOU CAN SEE IF YOU PERFORM
ANALYSIS AND LOOK AT THE
ACETYLATION AND THIS IS A
POSITIVE CONTROL FOLLOWING X
CHROMATIN GENES AND YOU CAN SEE
HIGHER IN FEE FEMALES AND AS THE
NOT ALLELE SPECIFIC BECAUSE IT'S
A HIGHER SIGNAL.
IT'S AS EXPECTED AND YOU CAN SEE
THIS IS BINDING TO Y IN MALES
NOT IN FEMALES.
THIS IS OUR INTERNAL CONTROL
WHICH IS PERFECT.
WE SEE MALE PEAKS ARE HIGHER
CONSISTENTLY AND THE TWO PEAKS
APPEAR TO BE HIGHER IN MALES
THAN IN FEMALES.
SO WHAT WE WANT TO DO IS REALLY
TAKE AN DIAGNOSTIC APPROACH TO
LOOK NOT ONLY BASED ON THE GENE
JUST LOOK AT EPIGENETIC
MODIFICATION PATTERN.
CAN WE ESTABLISH EPIGENETIC
SITES HISTONE ASSCETYLATION AND
USING THAT CONSENSUS CAN WE
BUILD OUT THE IDEA OF EPIGENETIC
SEX.
WITH THEY WANT TO SAY SEX IS A
BIOLOGICAL VARIABLE IN CANCERS.
I THINK IT'S AN IMPORTANT
CONSIDERATION NOT ONLY WE HAVE
TO DO IT IN APPLICATIONS BUT WE
ARE DOING IT IN THE LAB AND IN
TALKING TO IAN WE HAVE TO USE
FOUR DIFFERENT SEX TYPES NOT
TWO.
WITH THAT I WANT TO THANK THE
PEOPLE IN THE LAB AND I'M
GRATEFUL FOR THE SUPPORT FROM
THE NIH AND NIDDK AND VERY MUCH
TO NCI.
>> WE HAVE TIME FOR A COUPLE
QUESTIONS.
>> INTERESTING TALK, SEAN.
I THINK ALSO VERY INTERESTING
POPULATION OF PATIENTS AT RISK
KIND OF EXCITED ABOUT
TRANSGENDER POPULATION.
ARE YOU AWARE IN HIGHER OR LOWER
RISK IN DEVELOPING BLADDER
CANCER IN THIS POPULATION YOU
PROBABLY HAVE A BETTER CHANCE TO
DECOUPLE THE EPIGENETIC AND
OTHER AFFECTS CAN BE MODULATED
AS WELL.
>> I THINK THAT THE JURY IS
STILL OUT IN TERMS OF
TRANSGENDER POPULATION.
WE TRY HARD TO GET IN TOUCH WITH
THIS POPULATION TO STUDY AND IN
TERMS OF WHAT IS ALREADY KNOWN
PROSTATE CANCER IS BETTER KNOWN
FOR TRANS GENDER FEMALES AND IS
LOWER AND HAVE INNER CANCER MY
COLLEAGUE PUBLISHED A PAPER IN
JAMA IN CERTAIN POPULATIONS AN
ANOTHER PUBLICATION THIS WAS
LAST YEAR OR EARLY THIS YEAR
TALKING ABOUT BREAST TISSUE AND
GENDER CONFIRMATION IN THERAPY.
WHEN MALES ARE EXPOSED TO
ESTROGEN OR FEMALES EXPOSED TO
ANDROGEN AND IT REALLY CHANGES
THE GENE EXPRESSION AND THIS IS
STILL YOUNG TO LOOK AT I THINK.
THAT'S A GREAT QUESTION.
>> OTHERS.
>> YOU TALKED ABOUT BLANDER
CANCER, WHAT OTHER SEX BIASES OF
TUMOR TYPES ARE THERE OR
INVOLVED MAYBE 6A AND ANYTHING
LIKE THIS YOU CAN TALK ABOUT?
>> FOR BLADDER CANCER, THE PLEK
SUB TYPE OR PATHOLOGICAL SUB
TYPE IT'S REALLY NOT CLEAR CUT.
THEY ARE INDICATIONS IT MAY BE
DITCH BUT IN THE DATA AND ONE
THING IS CLEAR OR SEMI CLEAR FOR
FEMALES IT APPEARS TO ACCELERATE
MUCH FASTER IN FEMALE DEVELOP
BLADDER CANCER IN TUMOR AND GET
POORER OUTCOME BUT THE QUESTION
IS IT WAS CONSIDERED TO BE FOR
LATER DIAGNOSIS RATHER THAN
BECAUSE FEMALES DON'T SEE
UROLOGIST AND FOR GUYS YOU GO TO
THE UROLOGIST AND GET A CHECK
UP.
WHEN YOU DO A SPECIFIC
COMPARISON IT APPEARS TO HAVE
FEMALES THAT HAVE A WORSE
OUTCOME.
OUR NEXT TALK IS BY TERESA
VINCENT FROM NEW YORK UNIVERSITY
mRNA POLYMERASE FUELLING TUMOR
CELL PLASTICITY.
>> THANK YOU FOR INVITING ME TO
THIS AMAZING DAY.
I FEEL VERY HONORED TO TALK IN
THIS SECTION.
I'LL SPEAK TO YOU A LITTLE BIT
ABOUT mRNA POLYMERASE 1 AND WA
WE BELIEVE IS AN UNDER STUDIED
AMONG THE THREE.
THERE'S A MEETING EVERY THIRD
YEAR WHICH BASICALLY STRUCTURE
LOOK AT POLYMER AS A 1 AND 3 AND
DECIDED TO MAKE A BREAKOUT
MEETING.
I HOPE I'LL GET YOU MORE EXCITED
ABOUT mRNA POLYMERASE 1 THAT
MAKES THE BIGGEST COMPONENT OF
THE RIBOSOME THE mRNA.
AND WE BELIEVE EVERYTHING mRNA
POLYMERASE FOR THE BIO GENESIS
OR RIBOSOME COULD BE A NEW
THERAPEUTIC TARGET FOR TREATING
CANCER.
MOSTLY I'VE BEEN STUDYING BREAST
AND I'M GRATEFUL TO THE NCI THAT
SUPPORT THE RESEARCH.
SO THIS IS ONE OF THE WORSE
BRAIN TUMORS THAT EXIST AND MOST
COMMON MALIGNANT BRAIN TUMOR.
80% OF CASE DBM AND POOR
SURVIVAL, 14 1/2 MONTHS AND THE
QUALITY OF LIFE IS POOR BECAUSE
OF THE INFILTRATIVE NATURE OF
THE TUMORS AND THOUGH IT
INCLUDES SURGERY AND RADIATION
HASN'T IMPROVED LONG-TERM
SURVIVAL WITH THESE TUMORS.
THERE'S A GREAT NEED TO LOOK AT
OTHER THERAPEUTICS AND HOW TO
TARGET THE TUMORS WHICH IS NOT
SOLELY FOCUSSED ON TUMOR
PROLIFERATION.
SO GBM SUB TYPES HAVE BEEN
STUDIED AND SIGNIFICANTLY
THERE'S BEEN GENERATED USING RNA
SEQ AND NEW SINGLE SEQUENCING.
PEOPLE HAVE COME UP WITH
CONSENSUS OF THREE DIFFERENT SUB
TYPES CAN WHICH IS SIGNIFIED BY
ONLY TWO EXPRESSION THE 1
MUTATION AND PROLIFERATIVE IN
THEIR NATURE.
THESE TUMORS ARE EASIER TO TREAT
OR EASIER BUT NOT AS RESISTANCE
AS THE MESSENCHYMAL.
AND THEY TRANSITION IN THE TUMOR
BETWEEN THE CELL TYPE.
THIS IS MEDIATED BY DIFFERENT
PROGRAMS THAT ARE DRIVEN BY THE
MICROENVIRONMENT THAT WAS BY THE
TREATMENT ITSELF.
BECAUSE RADIATION HAS BEEN SHOWN
TO INDUCE PLASTICITY AND INDUCE
A MER MIGRATORY PHENOTYPE.
I'LL TALK ABOUT THE MESENCHYMAL
POSITION FROM THE SUB TYPE.
HOW WE DECIDED TO DO THIS WAS TO
DO A VERY OVER ALL GENOMIC
STUDY.
SO WE BASICALLY HAD HUMAN PDX
LINES U1313 FROM THE HGG
DATABASE GENERATED WHERE I WAS
PREVIOUSLY AFFILIATED WITH THE
VSHS AND GROW THESE FOR 24 HOURS
WHICH IS KNOWN TO INDUCE A KIND
OF INVASIVE STEM LIKE PHENOTYPE.
AND WE DO RNA SEQUENCING AND
MASS SPECTOMETRY AND WANTED TO
LOOK AT THE DRIVERS AND THE CELL
STARTS TO BECOME MORE INVASIVE.
AND WE LOOKED AT THE MESENCHYMAL
GENE MADE IT COMFORTABLE AND
SAFE AND THE BETA CAN INDUCE
CHANGES SO IT'S IMPORTANT TO
LOOK AT THE TUMOR CELL
SPECIFICITY.
AND WHEN WE OVERLAID THE
PROTEOMICS AND 65% OF ALL THE
GENES DETECTED WERE NOT
SIGNIFICANTLY CHANGED WITH THE
mRNA.
AND THOSE WERE ACTUALLY INVOLVED
WITH ADHESION AND THERE WAS A
LOT OF CHANGES IN RNA PROCESSING
AND RIBOSOME GENESIS AND IF WE
DIDN'T LOOK AT THE PROTEOME WE
WOULD HAVE MISSED THOSE CHANGES.
SO THEN WHAT IS RIBOSOME RIBO
GENESIS AND THIS IS THIS
BEAUTIFUL MACHINE DISCOVERED BY
GEORGE PALADAY IN 1955 MADE IN
THE CELL AND IT'S BASICALLY
COMPOSED OF IF YOU WOULD BE
INSIDE A CELL YOU WOULD
ENCOUNTER RIBOSOME IN THE
NUCLEUS BUT THOSE WOULD BE
IMMATURE RIBOSOME AND GOING BACK
TO THE THREE POLYMERASES AND
WHAT I BELONGED TO POLYMERASE 1
AND 3 IS BASICALLY MAKING THE
RRNA.
AND ONE MAKES THE 47S.
AND 3 MAKES THE 5S.
YOU HAVE 2 HELPING OUT BUT WITH
mRNA TRANSLATION THEY'RE MAKING
THE SNOW RNAs AND IT'S HELPING
IN THE MODIFYING THE FIRST LONG
TRANS AND CUTTING IT AND
MODIFYING IT AND THE RIBOSOME
MAKES THE IMMATURE RIBOSOME AND
IT'S NOT UNTIL YOU HIT THE SI
CYTOPLASM AND IT RELEASES
CERTAIN FACTORS.
THE RIBOSOME IS COMPLEX BOTH HOW
IT'S MADE AND BEFORE IT'S ACTIVE
AND COMPETENT FOR CELLS.
SO HOW IS RIBOSOME MADE IN
CELLS?
IT'S BEEN SHOWN CLEARLY IN
PROLIFERATING CELLS ALL THREE
POLYMERASES NEED TO BE
COORDINATED DURING THE PROCESS.
SO THERE'S A TRANSCRIPTION
FACTOR AND POLYMERASE REGULATED
BECAUSE IT COORDINATES ALL THE
THREE POLYMERASES.
YOU HAVE ENOUGH mRNA AND GET
ENOUGH RIBOSOME EXPRESSED TO
MAKE THE RIBOSOME.
AND WHEN CELLS ARE CYCLED OFF
BECAUSE WHEN YOU ADD THE BETA TO
THE CELLS THEY'RE NO LONGER
PROLIFERATING.
THEY HAVE A CHANGE IN THEIR CELL
IDENTITY.
THIS IS WHAT WE DISCOVERED AND
TUMOR CELL PLASTICITY AND WHEN I
START MIDED MY LAB WE WERE ASKI
ABOUT THE CELL CYCLE ARRESTED IN
CELLS STARTING TO MIGRATE AND
STOP PROLIFERATING OR ARE THEY
MAKING RIBOSOMES?
STRANGELY ENOUGH IT HADN'T BEEN
STUDIED AND WHAT WE SEE HERE
THAT WE SAW THEY WERE MAKING
RIBOSO
RIBOSOMES WE SAW THIS IN
DIFFERENT STIMULI AND SAW IF
IT'S TRUE IT'S A RECREATION
DURING THE DEVELOPMENT.
WE DID THE EXACT SAME EXPERIMENT
IN MICE AND CHICKEN AND INDEBTED
EDU AND EU AND STAINED AND LOOK
FOR WHETHER THE CELLS CHANGED IN
THE CELL IDENTIFY MARK HERE AND
THOSE CELLS AND WE SEE OUT
CREATE THE TISSUES WE HAVE AN
ENRICHMENT AND THIS mRNA
SYNTHESIS IS NOT BEING DRIVEN BY
MIC ACTING IN THE PROLIFERATING
CELLS BUT DRIVEN BY SNAIL ONE OF
THE MOST IMPORTANT TRANSCRIPTION
FACTORS AND ALSO SHOWED WHEN WE
TURN ON SNAIL WE TURN ON mRNA
TRANSCRIPTION.
WE WERE THINKING CAN THIS BE A
GENERAL FEATURE OF PLASTICITY
PROGRAMS NOT JUST INDUCED BY TGF
BETA BUT LINKED AT THE CELLS
WHEN THEY CHANGE THEIR IDENTITY
THEY NEED TO MAKE OTHER TYPES OF
RIBOSOMES AND OTHER TYPES IN
ORDER TO CHANGE THE CELL
IDENTITY FROM THE PROLIFERATIVE
CELLS TO THE MIGRATORY CELLS.
WE LOOKED AT 47S THE FIRST
TRANSCRIPT AND COULD SEE AND WE
SEE AN INDUCTION AND WE HAVE
CLONES FROM ONE PATIENT AND FIND
CLONES OF PRONEURAL AND
MESENCHYMAL AND IT'S IMPORTANT
TO NOTICE HERE THAT MESENCHYMAL
CELLS DON'T ALWAYS PROLIFERATE.
THIS IS NOT JUST DUE TO THE
PROLIFERATION.
AND HOW IS THIS mRNA
TRANSCRIPTION REGULATED IN THE
CELL?
IT'S PRETTY COMPLICATED BECAUSE
mRNA IS DIFFERENTLY LOCATED IN
DIFFERENT CHROMOSOMES.
AND WE KNOW THEY'RE DIFFERENT
FROM DIFFERENT PEOPLE.
AND SOME OF US PERHAPS SITTING
UP THIS ROOM HAVE 500 COPIES AND
OTHERS CAN HAVE 1500 COPIES AND
IT'S THE SAME IF YOU COMPARE
DIFFERENT MICE STRAINS AND WE
DON'T KNOW WHY THAT IS THE CASE.
WHY WOULD SOME PEOPLE HAVE MORE
AND ONE OF THE PROBLEMS WITH
THIS RESEARCH AREA IS IS OUR DNA
GENES ARE REFERRED TO AS THE
DARK GENOME.
BECAUSE IT'S COMPLICATED AND
BASICALLY REPETITIVE GENES.
THEY'RE VERY SIMILAR AND WHEN
YOU DO THE BIO INFORM ATTIC
ANALYSIS IT'S MORE TO ASSEMBLE.
OF COURSE THERE'S HOT SPOTS FOR
CANCER IF THEY'RE REPETITIVE IN
SEQUENCING.
SO THE MAJORITY WILL WALK AROUND
HAD MOST ASSIGNED BY THE
COMPLEX.
THEN THEY WERE THINKING PERHAPS
IT COULD BE THE CASE THAT DURING
PNT THAT BECAUSE THERE HAS NOT
BEEN STUDIES HOW RNA SYNTHESIS
IS REGULATED SO WE THEN LOOKED
IS IT THAT HAVE YOU MORE OPEN
CHROMATIN IN THE CELLS.
THEY'RE OPENING MORE AND WHAT WE
COULD SEE IS WE HAVE ALMOST A 50
PATIENCE REDUCTION MEANING MORE
R DNA CHROMATIN AND MORE
RECRUITMENT AND SNAIL IS BEING
RECRUITED IN ORDER TO DRIVE THE
RNA TRANSCRIPTION.
WE WANTED TO DEMONSTRATE THE
NON-CYCLING CELLS ARE MAKE mRNA
IN RIBOSOMES BUT WE'RE FOLLOWING
UP WITH NCI IS TO DETERMINE WHY
ARE THERE AND THEY'RE INCREASING
THE SIZE BUT NOT DIVIDING.
WE STARTED THINKING ABOUT
RIBOSOME AND SINCE IT WAS
DISCOVERED IN 1975 FOR THE NOBEL
PRIZE IN FOR THE HYPE THE FACT
THE BEAUTIFUL CRYSTAL STRUCTURES
OF THE RIBOSOMES AND AN OLD
QUESTION WAS ALWAYS ARE THERE
DIFFERENT RIBOSOMES IN DIFFERENT
CELLS THAT ARE SPECIFICALLY
DESIGNATED TO TRANSLATE CERTAIN
mRNA s?
A FIELD THAT'S BEEN IN
DEVELOPMENT HAS BEEN STUDIED AND
VERY CONTROVERSIAL SOME PEOPLE
REALLY DON'T BELIEVE IN
HETEROGENEITY WITH RIBOSOMES
BECAUSE THEY'RE SAYING THEY'RE
TOO EVOLUTIONARY AND DOESN'T
MAKE SENSE BECAUSE OF THE
PRODUCTION MACHINERY TO MAKE
SPECIAL RIBOSOMES SENSE FROM A
METABOLIC POINT OF VIEW.
HOWEVER, WHAT'S SUGGEST THE IN
THE LITERATURE AFBD WHAT'S BEEN
DISCOVERED AND PROJECT IS
THEY'RE PAIR PARALOGS IN
DIFFERENT TISSUES AND DISEASES.
THERE'S MOST TRANSLATION
MODIFICATION AND THERE'S
PHOSPHORYLATION A COMMON
MARKETER FOR SIGNALLING AND 10
YEARS AGO IT WAS SHOWED NORD FOR
THIS TOCK ACTIVATED IN THE CELL
AND HAVE THE KINASE ACTIVATED IT
BINDS TO RIBOSOME.
FOR THEM TO BIND IT GETS
ACTIVATED.
AND WE DON'T NEED THAT MANY MORE
RIBOSOMES IN A CELL ONLY A FEW
THAT HAVE THE CAPACITY TO BIND
THE RIBOSOME TO PROPAGATE THE
SIGNAL IF RIBOSOMES ARE ALSO
SIGNALLING.
YOU ALSO HAVE mRNA VARIATION
BUT WE'RE VERY INTERESTED IN
THIS AREA WHICH IS CALLED RRNA
MODIFICATION.
AND RRNA MODIFICATION NEEDS TO
OCCUR WHILE THE mRNA IS BEING
TRANSCRIBED IT CAN'T COME AFTER
BECAUSE THE IMPORTANT SITES ARE
HIDDEN IN SIDE THE RIBOSOME.
AND IT OCCURS ON ALL MRNs AND
SHOWN TO PLAY A ROLE IN
EMBRYONIC DEVELOPMENT AND
PROGRAMMING.
IT'S ALSO BEEN SHOWN TO BE
IMPORTANT FOR MESENCHYMAL TYPES
AND THERE'S TWO AND THE MAJORITY
ARE RRNA AND WE USED THE HDC BIO
BANK AND LOOKED AT THE
EXPRESSION OF THE TWO AND ONLY
35 WAS -- 5 WAS CHANGED AND YOU
CAN FIND AP32.
IT'S INSIDE THE RIBOSOME IN THE
DECODING CENTER WHICH IS
IMPORTANT FOR mRNA TRANSLATION
WHERE IT COMES IN AND LEADS TO
DOWN mRNA.
WE FOUND THAT INTERESTING AND
BASICALLY LOOKED AT GBM AND SAW
IT WAS STRONGLY EXPRESSED AND
ALSO EXPRESSED [INDISCERNIBLE].
THEN WE WERE WONDERING WOULD
THIS BE THE SPECIFIC TRANSFERASE
IS COMING AND A MODIFYING THE
mRNA.
THAT'S WHY WE SEE MORE BEING
MADE.
AND WE DO SEE THE COMPLEX AND
ALMOST THE LOW ASSOCIATED
[INDISCERNIBLE] AND WHEN WE
LOOKED FOR THE SPECIFIC SELECT
OME ONE SITE WHERE WE'RE LOOKING
AT AND WE CAN SEE TWO DIFFERENT
CELL LINES WE CAN SEE AN UP
REGULATION AND HAVE MORE IN THE
MESENCHYMAL.
THEN WE WERE THINKING OKAY LET'S
SILENCE THIS AND LOOK WHAT
HAPPENS.
AS EXPECTED WHEN YOU SILENCE 5
YOU HAVE LESS OF THE SITE AND
WHEN WE DO THE PROTEOMIC
ANALYSIS WE ALMOST SEE A
COMPLETE STOP IN THE HALT SO
CELLS AND GENES UPREGULATED ARE
SILENCE AND ONE NUCLEOTIDE IS
HAVING THIS MASSIVE AFFECT ON
THE PROGRAM AND IT INDUCES
INVASION WHEN YOU SILENCE THIS.
THEN WE'RE THINKING THIS IS A
BEAUTIFUL SITUATION AND NICE
TARGET FOR SMALL MOLECULE
BECAUSE METTL3 HAS IT AND METTL5
THERE'S NO SMALL MOLECULE
INHIBITORS OUT THERE.
WE THOUGHT IF IT'S CODE
TRANSCRIPTIONALLY IF WE INHIBIT
THE mRNA TRANSCRIPTION WILL WE
INHIBIT SOME OF THE
MODIFICATION.
WE USED TWO DIFFERENT DRUGS AND
CX5461 WE DON'T UNDERSTAND HOW
TO GIVE PATIENTS AN RNA POLYMER
AS AN INHIBITOR WITH NO EFFECTS
AND THERE'S GREAT TOOLS AND
SINCE THEY'RE WELL TOLERATED IN
THE CLINIC WE STILL NOY KNOW TOO
LITTLE HOW'S IT'S REGULATED IN
DIFFERENT CELLULAR SPACE.
AND NPMR PASSES THE BLOOD BRAIN
BARRIER AND USED FOR GBM.
WE SAW THE DRUGS WORKED.
AND THEY REDUCED SYNTHESIS, BOTH
OF THEM AND POL 1 IS NO LONGER
RECRUITED AND LOST AMONG THE
TREME AND WE ALSO SEE SNAIL IS
NO LONGER RECRUTED MEANING WE
BELIEVE SNAIL IS ALSO DRIVING IN
THIS CONTEXT RRNA TRANSCRIPTION
AND COULD IT BE THAT BECAUSE
METTL5 NEEDS TO BE
CO-TRANSCRIPTIONALLY REGULATED
AND FOUND A NICHE HOW THE DRUGS
ARE WORKED AND LOOKED AT
RECRUITMENT OF METTL5 AND ADDING
THE DRUGS WE DON'T SEE AN
RECRUITMENT OF METTL5 AND SEE
SIMILAR TO WHEN WE ARE SILENCING
METTL5 WE REDUCE AND IN THIS
CASE THESE BOTH SILENCING OF
METTL5 AND REDUCING THE CAPACITY
OF THE CELLS.
WE LOOKED AT THE PHENOTYPE FOR
THE MECHANISM AND SAW THE REAR
ORGANIZATION OF THE CYTOSKELETON
WAS REDUCED AND UNDERSTAND FROM
AN UNBIASSED POINT OF VIEW HOW
THE DRUG ARE WORKING.
THESE ARE NOT INHIBITERS.
THEY HAD LITTLE AFFECT IN mRNA
TRANSCRIPTION.
AND THEY'VE DEVELOPED SMALL
MOLECULE INHIBITORS AND ITS WAS
NICE FOR US TO IS SEE THE
LITERATURE BECAUSE IT'S BEEN
PROVEN IT'S IMPORTANT.
IT HAS BEEN SHOWN FOR OTHER
CANCERS TOO OTHERWISE ALL THE
BEAUTIFUL DRUGS BEING DEVELOPED
AND WHAT WAS INTERESTING TO US
IT WAS PUBLISHED A TRANSLATIONAL
COMPONENT MEANING WE'RE STUCK IN
MAKING THE NEW MODIFIED mRNA
MICROSOMES AND WE DECIDE IT HAD
LOOK AT TRANSLATION.
SO WE JUST FIRST CONFIRMED THERE
WAS NO AFFECT ON mRNA
TRANSCRIPTION FROM THE DRUGS
HOWEVER, WE SAW BIG EFFECT AND
THEN WE DID POLY SOME ANALYSIS
AND WE BASICALLY ISOLATED THE
ACTIVELY TRANSLATING RIBOSOMES
AND THEN DID PCR AND IN LINE
WITH WHAT OUR HYPOTHESIS WAS WE
HAD MORE mRNA KINASE TRANSLATED
IN THE POLY SOME IN THE BETA AND
WHEN WE STOPPED THE RIBOSOMES
FROM BEING MADE WE'RE NO LONGER
TRANSLATING THE KINASE.
THEN COMING TO MY LAST SLIDE WE
WERE LOOKING IS THIS
THERAPEUTIC.
IT'S IN CLINICAL TRIAL AND SO
FAR IN ADVERSE EFFECT AND WOULD
LIKE TO DEVELOP THIS TOGETHER
WITH NCU PERHAPS FOR A FUTURE
CLINICAL TRIAL.
WE W
WE WERE LOOKING AT TWO ASSAY ONE
EXO -- IN VIVO AND EX VIVO AND
WE LOOKED AT MIGRATION.
WE SAW THE INHIBITORS REDUCED
THE MIGRATION OF BOTH THE GBM
CELLS.
THEN WE DID A MOUSE EXPERIMENT
WHERE WE BASICALLY DOSED THEM
TWICE A WEEK.
WE TRANSPLANTED THE F3030 CELLS
AND LET THEM GROW FOR TWO WEEKS
AND TREAT THEM FOR THREE WEEKS
AND THEN WE ISOLATED THE TUMORS
AND WE CHECKED AND WE ALSO REARE
REDUCING THE ONE SITE ON THE
RRNA AND WE SAW WHEN WE
SACRIFICED ANIMALS WE CAN SEE
THE CELLS ARE STARTING TO CREATE
A MORE ROUND SHAPE AND YOU CAN
SEE THESE SPIKY TUMORS.
THESE GBM TUMORS DON'T FORM THEY
BECOME HARD TO TREAT AND WE
COULD SEE LESS OF THE SPIKING IN
THE PHENOTYPE IN THE TREATED
TUMORS.
SO TODAY I HOPE I'VE BEEN ABLE
TO CONVINCE YOU THAT RRNA POLIO
-- POLYMERASE 1 IS IMPORTANT
AND THERE'S DIFFERENT COMPLEXES
FORMED IN DIFFERENT CELLAR STATE
WHETHER IT'S GBM OR BREAST
CANCER.
SO BASICALLY WHAT WE SUGGEST IS
THAT HAVE A BETA SIGNAL, OUR
COMPASS WILL BE OPENED OR CLOSED
DEPENDING ON THE SIGNAL.
DIFFERENT MODIFIERS CAN COME AND
MODIFY THE RRNA.
-- SORRY.
SO THE SUMMARY THEN IS BASICALLY
POL 1 IS AS IMPORTANT AN POL 2
BY RECRUITING DIFFERENT
TRANSCRIPTION FACTORS AN CAN
HAVE THE MODIFIERS THAT CAN
GENERATE THE SPECIFIC RIBOSOME
AND YOU HAVE POL 2 WORKING IN
CONJUNCTION WITH THIS AND THOSE
TWO TOGETHER WOULD THEN
BASICALLY FORM A SELECTIVE
TRANSLATION PROGRAM SUCH AS THIS
KINASE AND TRANSLATE THE mRNA s
TO PROMOTE TUMOR PLASTICITY AND
BELIEVE WE HAVE NEW DRUGS IF WE
CAN TARGET THESE PATHWAYS WE CAN
HAVE NEW THERAPEUTICS NOT
DEPENDENT ON DNA REPLICATION.
THANK YOU SO MUCH.
THIS IS MY LAB.
AND THANKS AGAIN.
>> A FEW MINUTES FOR QUESTIONS.
>> ANDY.
THIS IS INTERESTING.
I WANT TO ASK ABOUT PAPERS FROM
A WHILE AGO.
HE HAD THESE PAPERS ONE WAS IN
MOLECULAR CELL THAT I KNOW
ABOUT.
IT'S INTERESTING BECAUSE HE
TALKS ABOUT TRANS LOCATION OF
LAMIN TYPE PROTEINS BETWEEN THE
NUCLEAR MEMBRANE AT THE NUCLEUS.
IT DOESN'T HAVE A MEMBRANE OF
COURSE.
BUT THE VERY FIRST TIME ANYBODY
EVER LOOKED AT CTCF IN THE CELL
WAS AN IMMUNOLOCALIZATION
EXPERIMENT AT THE NUCLEUS AND
NOT JUST THAT BUT DATA IN THE
PAPER THAT SHOWED THE LOOPS
EITHER THE PROMOTER NEXT TO THIS
OR THAT ENHANCER.
THE QUESTION IS HAVE YOU THOUGHT
ABOUT HOW THE HIGH ORDER
STRUCTURE ISN'T JUST SOMETHING
OUT THERE ON THE NUCLEAR EDGE OR
WHATEVER OR EVEN IN THE INTERIOR
BUT COMES DOWN TO THIS REGION
WHICH IS THIS REALLY IMPORTANT
REGION INVOLVING
HETEROCHROMATINS SO THE
RIBOSOMAL GENES HAVE TO BE SHUT
DOWN EXCEPT FOR ONE AND WHAT ARE
YOUR THOUGHTS BECAUSE IT'S A
CONNECTION BETWEEN YOUR TALK AND
OTHERS.
>> HAVING ALL THE CHROMATIN IN
ONE PLACE AND HAVING THEM OPEN
VERSUS DOWN IS A BIG QUESTION
HOW THAT AFFECTS THE
TRANSCRIPTION IN GENERAL.
I ALSO THINK PERHAPS I
MISUNDERSTOOD THE QUESTION A
LITTLE BIT BUT IN GENERAL WE
THOUGHT OF mRNA POLYMERASE AS A
PASSIVE PLAYER.
IT'S HARD TO KNOW EXACTLY KNOW
HOW ALL THESE THINGS ARE
INTERLINKED BUT GIVEN THAT SO
MUCH ENERGY GOES -- 80% OF THE
CELLS ENERGY GOES TO MAKE
RIBOSOMES IS A FASCINATING THING
HOW THE CELL IS PARTICIPATING IN
THIS ENERGY AND ALSO WHEN IT
COMES TO THE SIZE OF THE NUCLEUS
WHICH HAS BEEN A CANCER
DIAGNOSTIC FOR A LONG TIME
PEOPLE HAVE LOOKED AT.
WE DON'T KNOW WHICH ONE OF
THESE RNA COPIES ARE OPEN OR
CLOSED IN DIFFERENT SETTINGS SO
WE STILL PROBE THE ONE RNA
SEQUENCE OVER AND OVER AGAIN FOR
OUR EXPERIMENTS.
YES, IF ANYONE WOULD LIKE TO
HELP I'M VERY OPEN FOR
COLLABORATION AND HELP.
>> LET ME ASK YOU A QUICK ONE, A
TRANSLATIONAL ONE.
ALL THESE INHIBITORS COULD COME
DOWN TO COMBINATIONS.
AND YOU HAVE THE LAST SUMMARY
SLIDE AND GOING THROUGH A SERIES
OF PGF BETA.
THANK YOU THOUGHT OF COMBINATOR
IAL STUDIES.
>> YES, WE'RE COMBINING WITH
RADIATION WE'RE COMBINING IT
WITH AND WE'RE TREATING THE
TUMORS.
[INDISCERNIBLE] AND WE'RE
CONTINUING THE TREATMENT.
WE HOPE FOR LONGER.
>> LAST IS CHIA LIN WEI.
TALKING ABOUT EXTRA CHROMOSOMAL
DNA TRANSCRIPTION.
>> IT WAS BELIEVED FOR THE
TRANSCRIPTION IS THROUGH A
HIGHLY ORGANIZED SUBNUCLEUS
STRUCTURE THROUGH THE ORGANELLE.
SO I THINK THERE'S 3-D
CONFIRMATION THAT ACTUALLY MOVE
THOSE RIBOSOMAL DNA LOCATION
INTO A VERY SPECIALIZED REGION
FOR TRANSCRIPTION.
I THINK BUT I THINK IN GBM THESE
ARE PROBABLY HIGHLY TRANSCRIBED
AS I UNDERSTOOD.
SO WHILE THIS IS BEING SET UP,
MY NAME IS CHIN LIN WEI.
SOME IN THE AUDIENCE I KNEW FROM
JACKSON LABORATORY OVER A MONTH
AGO.
AND I'M LEADING THE NORTHWEST
GENO CENTER.
TODAY TARGETS WORK BEING DONE IN
JAX BUT IAN CHARGED ME FOR THE
NEW FRONTIER.
I STARTED THE FIRST JUNIOR
FACULTY POSITION IN SINGAPORE IN
EARLY 2000s.
THE WORKED WITH ACB MEMBERS AND
THE FIRST SO-CALLED SENIOR
CORRESPONDING PAPER WE USING
SINGER SEQUENCE TO SEQUENCE IP
DNA AND SECOND WHEN ILUMINA GA
STARTED.
IT'S NOT REALLY MOVING.
AND WE ARE USING GA2 TO
SEQUENCING BY DNA ACROSS HUMAN
EMBRYONIC STEM CELL
DIFFERENTIATION AND OUR PAPER
GETS PUBLISHED A FEW MONTHS
LATER BUT NEVERTHELESS PAVED ME
TO THE SUBSEQUENT FOUNDATION OF
MY LAB WANTING TO STUDY -- SO MY
LAB IS STUDYING THE
REGULATION -- AND LOOKING AT HOW
GENETIC S AFFECT TRANSCRIPTION
AND IN FACT THE CHROMATIN
ORGANIZING STRUCTURE.
WE KNOW THE WHOLE DNA SEQUENCE
ADDING TOGETHER IS ABOUT TWO
METERS AND THE SIZE OF LeBRON
JAMES.
HOW IT GETS CODED INTO A 10
NUCLEI AND EXACTLY PRECISELY
EXECUTED TRANSCRIPTION
REGULATIONS AND HOW IT HAS TO BE
PERTURBED IN CANCER GENOME THAT
AFFECT THE TUMOR HETEROGENEITY
AND HOW TUMOR PROGRESSION IN THE
DRUG RESISTANCE.
AND KNOWING THAT EXPERIENCE OF
USING POWERFUL NEXT GENOMIC
TECHNOLOGIES -- AND FACILITATE
THAT UNDERSTANDING HAS BEEN THE
CENTRAL FOCUS OF MY LAB.
AND THE SEQUENCING IS SIMILAR TO
HI C.
YOU START WITH THE CROSS LINK
CHROMATINS TO SNAP FROZEN THE
ELEMENTS AND DIFFERENT FROM HI C
WE INTERROGATE THESE
INTERACTIONS WITH A FUNCTION AL
INTERACTION OF THE PROTEIN OF
INTEREST BY ANTIBODY.
ANG SIMILAR TO THE HIGH C OF THE
HEAT MAP IN THE MEASURE.
SECOND, BECAUSE WE USE CHROMATIN
PRECIPITATION WE CAN DETERMINE
PRECISE PROTEIN OR BINDING
LOCATION LIKE CHIP SEQ.
THIRDLY BECAUSE WE PULL DOWN THE
INTERACTING FRAGMENT USING THE
PROTEIN OF INTEREST WE CAN
GENERATE PRECISE RESOLUTION OF
LOOP INFORMATION INDICATING WHAT
POSITION THE TWO STILL
REGULATORY ELEMENTS COME
TOGETHER TO FORM INTERACTIONS.
SO THREE LEVELS OF INFORMATION
FROM DATA SETS AND BINDING
SITES, PROXIMITY AND
MEASUREMENTS AND THE GLOBAL GENE
I'M WIDE LEVEL AND SPECIFIC
INTERACTION REGIONS.
AND EXPAND UTILITY OF HI C TO
LOOK FOR NOT ONLY THE PROXIMITY
DISTAL REGULATOR INTERACTION AND
INFER STRUCTURE VARIATION.
FOR EXAMPLE THE THREE HERE TRANS
LOCATION, DELETIONS AND
APPLICATIONS.
SO THIS HAS BEEN DEMONSTRATED
THE UTILITY IN THE CANCER GENOME
INTERROGATION UNDERSTANDING THE
CHROMATIN INTERACTION AS WELL AS
THE PRIMARY STRUCTURE IN IMPACT
TRANSCRIPTION REGULATION.
SO AND WITH THE CANCER BIOLOGY
INVESTI
INVESTIGATOR IS IN GLIAL
BLASTOMA RESEARCH AND INTRODUCED
ME TO THE OBSERVATION HE MADE IN
THE GBM IS HIGH FREQUENCY OF THE
EXTRACHROMOSOMAL DNA FROM THE
FIELD IS A CHROMATINIZED
CIRCULAR DNA OFTEN FOUND IN THE
CANCER CELLS AND USUALLY KNOWN
TO CARRY HIGH COPY NUMBER FOR
FOCAL APPLICATION OF ONCOGENES
AND IT WAS BELIEVED TO BRING
HIGH COPY OF ONO GENE EXPRESSION
THUS GIVING THE CELLS A HIGH
LEVEL OF ONCOGENE EXPRESSION AN
AN ADVANTAGE THEREFORE GIVING
THE CANCER CELLS VERY HIGH
SO-CALLED ADVANTAGE IN TERM OF
GROWTH FITNESS.
IT WAS RECENTLY IN THE LAST FEW
YEARS IT WAS THE FREQUENCY OF EC
DNA FOUND BETWEEN THE METASTASIS
VERSUS THE PRIMARY TISSUE AND
BETWEEN THE AGGRESSIVE CANCER
TYPE VERSUS NON INDICATING THE
SIGNIFICANCE OF CC DNA IS VERY
IMPORTANT FOR US TO UNDERSTAND
THE TUMOR METASTASIS DRUG
RESISTANCE AN AGGRESSIVE TUMOR
PHENOTYPES.
AS A GENOME BIOLOGIST IT'S BEEN
VERY INTERESTING TO ME BECAUSE
EC DNA IS EXTRACHROMOSOMAL DNA
AND COULD ACTUALLY EVADE ALL THE
CHROMATIN FOLDING PRINCIPLE THE
TERRITORIES OF SO-CALLED DISTAL
REGULATION HAPPENS BETWEEN ON
THE CHROMOSOMES.
IT'S A QUESTION OF HOW IT'S A
CHROMATIN STATE.
DOES IT PERSIST WITH A UNIQUE
EPIGENETIC STATE AND CHROMATIN
GENERATION AND IMPORTANT FOR THE
FUNCTION BEYOND JUST THE VESICLE
OR VEHICLE CARRYING HIGH COPIES
OF ONCOGENES.
WE MEASURED THE GENOME WIDE
INTERACTIONS HERE SHOWING THAT
AND THERE'S HIGH CHROMATIN
INTERACTIONS AND SUGGESTING EC
DNA BEING SMALL, BEING CIRCULAR
AND FREELY MOVE AROUND THE
NUCLEUS MAKING A LOT OF
CHROMATIN CONTACT WITH THE
CHROMOSOMES.
AND THE FREQUENCY IS NORMALIZED
BY NUMBER AND SPIKING THE
CONTACT NOT BECAUSE THERE'S NOT
HIGH COPY NATIONAL BUT
SUGGESTING THE MOVING AROUND IN
THE NUCLEUS MAKING THE CONTACT.
NEXT, WE ASKED IF THAT CONTEXT
MEANING AND SPECIFIC REGIONS IS
IN CONTACT WITH A LOT OF
CHROMOSOMES AND THAT CARRIES
BROAD RANGE OF ESCALATION AND
IT'S BEING INDICATING ENHANCER
SIGNATURES AND THE HIGH LEVEL
K27 ACTIVITY AND BROAD SPAN OF
K27 ACTIVITY INDICATING A SUPER
ENHANCER TYPE OF SIGNATURE AS WE
HAVE SHOWN IS THE IMMUNOSTAINING
OF THE K27 STAINING AS THE STAIN
ON THE EXTRACHROMOSOMAL
SUGGESTING EXTRACHROMOSOMAL DNA
HARBOR A LOT OF ENHANCER
SIGNATURES.
AND THE ENHANCER SIGNATURE IT TO
MAKE SPECIFIC CONTACT WITH THE
CHROMOSOME ACTIVATING CHROMOSOME
INTERACTIONS.
ONE THING WE ARE TRYING TO PROVE
IS WHETHER THE SUPER ENHANCER AS
A HUB FOR THE TRANSCRIPTION HIGH
LEVEL TRANSCRIPTION FACTORIES
AND IT ALLOWED US TO MAKE THIS
AS A MOBILE ENHANCER.
SO ONE THING WE FIRST ASKED
WHETHER THIS TRANSCRIPTION
FACTORY CO-LOCALIZED WITH
TRANSCRIPTION ACTIVATE 1 AND
WHEN WE DID THE CHIP SEQ THE EC
CONTACT SHOWN AS A BLUE IS
HIGHLY OVERLAPPED AND SUPER
IMPOSED WITH THE BINDING AND THE
K27 SIGNATURES SUGGESTING THAT
THIS IS SUPER ENHANCER HUBS ARE
ACTUALLY CO-LOCALIZED WITH MET 1
AND NOT ONLY IN THE SPECIFIC
CELL LINE.
WE'RE SHOWING ACROSS MANY
DIFFERENT CELL LINE THIS
COLOCALIZATION IS CROSSING CELL
TYPE AND HAVING THE INTRINSIC
DOMAIN REGIONS IS KNOWN TO FORM
LIPIDS SEPARATED AND WHEN WE
LOOKED IS TO SEE WHETHER THIS IS
REALLY A PART OF LIQUID-LIQUID
CONDENSATE SO THE
EXTRACHROMOSOMAL STAINING
SHOWING THEY'RE HIGHLY
SIGNIFICANTLY CO-LOCALIZED AND
ALSO THE LIVE IMAGING STAINING
OF EC DNA SHOWS THE CONDENSATE
MORPHOLOGIES SUGGESTING THIS IS
REALLY A WAY OF EC DNA FUNCTION
TO FORM HIGHLY CLUSTERED
AGGREGATE IN THE PHASE SEPARATED
CONDENSATE FORMAT CARRYING
TRANS
TRANSACT -- TRANS ACTIVATOR AND
SEE IF THERE'S SPECIFICITY WHERE
THEY MAKE THE CHROMOSOME CAN BE
TACT.
WE USED A SINGLE MOLECULE,
CHROMATIN INTERACTION ANALYSIS
BY LEVERAGING 10X DROP LET
SYSTEM USING THE CHROMATIN
FORMALDEHYDE COMPLEXES, DILUTED
LOADING TO THE 10X AND THE
CHROMATIN FRAGMENTS CARRIED THE
SAME 10X BAR CODE SIGNATURE SO
THEY'RE SUGGESTING FORMING THE
SAME SINGLE MOLECULE COMPLEX.
WE CAN DECIPHER -- ECDNA
CHROMATIN INTERACTION REPERTOIRE
AND LOOKING HOW THE SIGNATURES
GENE OVEREXPRESSION AND THEIR
FUNCTION AND SHOWING THEY'RE
ENRICHED IN ONCOGENE
PROLIFERATION AND GENE INVOLVED
IN HIGH LEVEL CELL PROLIFERATION
ACTIVITY AND USE THE FISH TO
CONFIRM THE INTERACTION
REPERTOIRES BY SHOWING THE
CO-LOCALIZATION.
SO HERE WE HAVE VALIDATED
CONCEPT OF HIGH CHROMATIN
INTERACTIONS ON THE SUPER
ENHANCER SHOWING IN THE CONTEXT
FREQUENCY MAP SUPER ENHANCER
SHOWING IN BLACK AND THE MET 1
BINDING ARE SUPER IMPOSED AND
THE HIGH CONTACT FREQUENCY NOT
ON ONCOGENE BUT ACTUALLY ON THE
SUPER ENHANCER REGIONS.
AND THE NEXT IS IF THIS IS
INDEED A SEPARATED CONDENSATE OF
EC DNA USING TRANSACTING GENE
EXPRESSION IS ACTUALLY GOING
BEYOND JUST THE ONCOGENE
ACTIVATION AND A STRUCTURE BASED
WAY TO MODULATE CANCER CELL GENE
EXPRESSION.
THIS IS A STRUCTURE BASED
MECHANISM CAN IT BE EXPLOITED TO
USE AS A CANCER THERAPEUTIC
MEANING TO MODULATE CANCER CELL
PROLIFERATION.
USING A WELL KNOWN CHEMICAL THAT
CAN PERTURB THE CONDENSATE WE
WANT TO TEST THE IDEA CAN WE USE
THE STRUCTURE BASED PERTURBATION
TO PERTURB THE FUNCTION AND THIS
IS SHOWING THAT USING THE AGENT
TO PERTURB SEPARATIONS WE CAN
EASILY SEE THE EC DARN -- DNA
DIMINI
DIMINISHED AGGREGATION TREATED
IN THE CELL COLORECTAL AND IT
WORKED ACROSS MULTIPLE CANCER
TYPES AND MULTIPLE EC DNA
ONCOGENES AND HERE'S THE GMDDM
AND COULD BE A WAY TO MODULATE
SO-CALLED UNDRUGGABLE ONCOGENE
EXPRESSION AND WE ALSO
DEMONSTRATE THE AGGREGATION DOES
FOT CHANGE THE COPY NUMBER BUT
BY JUST PERTURB THEIR
AGGREGATION WE CAN MODULATE ON
COGENE EXPRESSION.
-- ONCOGENE EXPRESSION.
WE DEMONSTRATED BY USING THIS
PERTURBATION CONDENSATE WE CAN
CHANGE THE MET 1 BINDING,
PERTURB THE GENE EXPRESSION AND
SUPER ENHANCE ACTIVITIES AND
MODULATE THE EXPRESSION.
SO THIS IS DEMONSTRATING WHEN WE
LOOK AT THE LOOP INTERACTIONS
THE ECDM MEDIA LOOPS ARE
SELECTIVELY DOWNREGULATED IN
16HD SUGGESTING THE MECHANISM
MODULATED MAKES EXPRESSION AND
AGGREGATION THROUGH THE
INTERACTION AND CAN SHOW THAT
NEXT THE PERTURBATION AFFECT IS
GETTING MORE SENSITIVE IN THE
APOPTOSIS ASSAYS WITHOUT DOUBLE
MINUTE AND THESE ARE MORE
SENSITIVE TO THE TREAT AND
HIGHER APOPTOSIS SIGNIFICANTLY
AND THE STREAM DOES NOT HARM --
HARBOR DOUBLE MINUTE AND THIS
PHENOMENA CANNOT ONLY BE DEA
DEMONSTRATED IN ONE TYPE BUT GBM
AND PROSTATE AND COLORECTAL
DEMONSTRATING THE MECHANISM IS
NOT BASED ON SPECIFIC ONCOGENES
BUT A STRUCTURE BASED
PERTURBATION THAT AFFECT THE
FUNCTION.
OF COURSE YOU CAN ALWAYS ARGUE
THIS IS JUST A CORRELATION
BECAUSE IT'S NOT SPECIFIC AND
YOU MAY AFFECT OTHER THINGS THAT
YOU'RE NOT AWARE OF.
WE CARRY OUT CRISPR EPIGENETIC
EDITING OF THE SUPER ENHANCER
REGION WITH THE MOST
INTERACTIONS.
WE COMPARE THAT PERTURBATION AT
THE SUPER ENHANCER AND THE
PROMOTER SHOWING THAT BY
PERTURBED SUPER ENHANCER NOT
MAKING THE PBT1 PROMOTER HAS THE
MOST SIGNIFICANT AFFECT OF GENE
EXPRESSION SHOWING WE CAN BOTH
DOWN REGULATE MIK AND PBT1 AND
DEMONSTRATE EC DNA PRECIPITATED
AND THE EC HARBORING CELL HAS
LOST THE PROLIFERATION
ACTIVITIES.
HERE'S WHAT I REALLY WANTED
TO -- THE THINGS WE SEE IN A
CANCER CELL THE DNA HOW IS IT A
FIREBALL ACTIVATED WITH THE
CHROMOSOMAL INTERACTION AS THE
MOBILE ACTIVITIES.
IS THAT NEW?
AND LOOK AT THE RECEPTOR
REGULATED NOT INTRERCHLGED BY
ENHANCE R --
INTERCHANGED WORKING IN THE
EPISOME LOADING THE WHOLE CELLS
AND TRANS LOCATED AND IN CASES
IT SUGGESTS IT'S NOT JUST A
UNIQUE PHENOMENA CARRIED BY A
CANCER CELL IT HAS ENHANCED A
ACTIVATIONS CAN BE A MECHANISM
THAT PROVIDE CELL WITH
ADDITIONAL LEVEL OF
HETEROGENEITY OR IN ANDY'S WORD,
STOCHASTIC REGULATION THERE.
THIS IS ALSO FOR THE AUDIENCE
AND STARTING AS A GRADUATE
STUDENT LOOKING FOR ALL THE
REGULATORY REGIONS CLOSE TO
PROMOTER AND WHEN I STARTED TO
STUDY 3-D CHROMATIN INTERACTIONS
LOOKING AT REGIONS AT HUNDREDS
OF KBs AWAY FROM THE PROMOTER ON
THE SAME CHROMOSOMES AND LIKE
THE MOLECULE IN THE CANCER CELL
OR EVEN SOMATIC CELLS ALLOWS US
TO UNDERSTAND MAYBE THERE'S
ADDITIONAL LAYER OF
TRANSCRIPTION REGULATIONS THAT
LOOK AT THIS OUTSIDE THE
CHROMOSOME AND OFFER ADDITIONAL
LAYER OF PLASTICITY, AVOID AND
ALLOW THE CELL TO GENERATE MORE
HETEROGENEITY AND MORE DIVERSITY
THERE.
SINCE I'M THE LAST ONE TO TALK I
FEEL I'M OBLIGED TO LOOP BACK TO
THE FIRST TALK WE HEARD HOW THE
CHROMOSOMAL DNA VARIATION USE
THE MOBILE ENHANCER CONCEPT OF
CELLS INCLUDING CANCER CELLS AND
LOOKING AT EPIGENETIC
STOCHASTICITY AND LOOKING AT THE
HETEROGENEITY AND STRUCTURE
HETEROGENEITY AND GENE
EXPRESSION HETEROGENEITY AND
TOGETHER IT OFFERS SOME WAY TO
PROMOTE TUMOR EVOLUTION.
THAT'S THE CONCEPT I WANT TO
REALLY USE TO STIMULATE LOOKING
FORWARD IN THE NEXT I DON'T
KNOW, 10 YEARS, 20 YEARS
HOPEFULLY COME BACK HERE AGAIN
TO UNDERSTAND HOW THIS CANCER
EPIGENETICS EVOLVED USING THE
TECHNOLOGIES, GENOMIC
TECHNOLOGIES IN THE NEXT
GENERATION WE CAN LEARN.
I ALSO WANT TO THANK NCI BUT MY
OTHER GRANT IS NOT FUNDED BY NCI
SO I HOPE NCI WILL FUND MORE
EXTRACHROMOSOMAL RESEARCH IN THE
FUTURE.
THAT'S A PLEAD.
THANK YOU VERY MUCH.
SORRY ABOUT THE TECHNICAL
COORDINATION.
>> DON'T WORRY, I'VE BEEN THERE.
SO HAVE TIME FOR QUESTIONS
BEFORE WE HEAR CLOSING REMARKS
FROM THE AUDIENCE, PLEASE.
I'LL ASK YOU ONE.
I WAS TRYING TO GET A SENSE OF
BALANCE.
YOU HAVE A BIG DRIVEN TUMOR AND
HAVE THESE EXTRACHROMOSOMAL
ENHANCERS DRIVING MIC AND OTHER
CELLULAR STRUCTURES DRIVING MIK
WHAT'S THE PERCENTAGE OF HOW
MUCH DRIVE YOU GET THE
EXTRACHROMOSOMAL MATTERS THAN
OTHERS TO SPECIFICALLY BLOCK?
>> RIGHT.
IN OUR CRISPR CAS EXPERIMENTS
THIS IS VERY INTERESTING WHEN WE
BLOCK THE SUPER ENHANCER ON
EXTRACHROMOSOMAL DNA AND THE
EXPRESSION IS REDUCED
DRASTICALLY WE SEE ALL THE
ENHANCER SIGNATURES MOVING TO
THE CHROMOSOME.
THERE'S A MOBILITY OR DYNAMICS
AND WE SEE NUMBER OF GENE
ACTIVATIONS WHEN WE BLOCK THE
ENHANCER ON THE EXTRACHROMOSOMAL
DNA.
>> IS IT IN VARIOUS SWITCHES?
>> RIBOSOMAL DNA.
>> THAT CLOSES OUT AND CLOSING
REMARKS AND I WANT TO THANK ALL
THE SPEAKERS FOR THE SESSION AS
WELL.
>> SO THAT PRETTY MUCH WRAPS UP
I THINK A PRETTY INCREDIBLE DAY.
REFLECTION ON 40 YEARS ON CANCER
EPIGENETICS AND FANTASTIC TALKS.
WE WENT FULL CIRCLE FROM THE
HISTORY TO WHAT'S UP AND COMING.
I REALLY WANT TO EXTEND MY
PROFOUND GRATITUDE FOR EVERYONE
THAT CAME TODAY ESPECIALLY THOSE
THAT CAME IN PERSON.
I WANT TO THANK ALL THE SPEAKERS
WHO MADE THE TREK TO NIH AND FOR
THEIR GREAT PRESENTATIONS AND
AGAIN WE REALLY VALUE THE IN
PERSON PARTICIPATION AND EVEN
THOUGH IT MAY LOOK SPARSE AT
THIS TIME OF DAY, WE DID HAVE
SIGNIFICANT PARTICIPATION
THROUGH VIRTUAL PLATFORMS WITH
AT TIMES CLOSE TO 200 PEOPLE
WATCHING SO I'D BE ENCOURAGED
THERE'S A LOT OF INTEREST IN OUR
SYMPOSIUM TODAY AND WITH THAT
I'D ALSO LIKE TO THANK ALL THE
NIH STAFF THAT HELPED MAKE THIS
POSSIBLE AND OUR CONTRACTOR AND
THANKS ONCE AGAIN FOR COMING AND
HOPE YOU ALL HAVE A SAFE TRIP
HOME.