GOOD AFTERNOON, EVERYONE.
I'M NINA SCHOR, DEPUTY DIRECTOR
FOR INTRAMURAL RESEARCH HERE AT
NIH.
IT IS MY PLEASURE AND MY HONOR
TO INTRODUCE THE SYMPOSIUM.
THIS IS DAY 4 OF OUR 2023
RESEARCH FESTIVAL.
AND THIS DAY ACTUALLY RECOGNIZES
THE WORK OF OUR SENIOR
INVESTIGATORS.
BOY, I CAN TELL YOU, YOU ARE IN
FOR A TREAT WITH THIS SYMPOSIUM
COMING UP.
THIS SYMPOSIUM WILL PRESENT AND
SHOWCASE FIVE INVESTIGATORS FROM
NIH WHO HAVE BEEN ELECTED IN THE
LAST TWO YEARS TO THE NATIONAL
ACADEMY OF THE SCIENCES.
SO, FIRST A FEW WORDS ABOUT THE
NATIONAL ACADEMIES.
THE NATIONAL ACADEMIES PROVIDE
RECOMMENDATIONS AND EXPERT
ADVICE ON ISSUES OF IMPORTANCE
TO SCIENCE, HEALTH, AND
ENGINEERING.
CURRENTLY, THERE ARE THREE
NATIONAL ACADEMIES.
NATIONAL ACADEMY OF SCIENCES,
THE NATIONAL ACADEMY OF
MEDICINE, AND THE NATIONAL
ACADEMY OF ENGINEERING.
AND NIH IS A PROLIFIC SOURCE OF
ELECTEES TO THESE ACADEMIES, AND
ELECTION TO ANY OF THESE
ACADEMIES IS WIDELY DEEMED TO BE
THE HIGHEST HONOR THAT THE
SCIENTIFIC COMMUNITY CAN BESTOW
UPON A SCIENTIST.
WE CURRENTLY HAVE MORE THAN 50
ACTIVE P.I.s IN THE NATIONAL
ACADEMY OF SCIENCES, MORE THAN
70 IN THE NATIONAL ACADEMY OF
MEDICINE, AND ONE IN THE
NATIONAL ACADEMY OF ENGINEERING.
SO, REALLY A FERTILE GROUND FOR
THE NURTURING OF THE SCIENCE AND
THE P.I.s THAT UPON WHOM THIS
HONOR IS BESTOWED.
SO, OUR FIRST SPEAKER TODAY, I
WILL INTRODUCE THE SPEAKERS
INDIVIDUALLY AS THEY COME UP TO
THE PODIUM OR ARE SEEN ON THE
SCREEN, IS DR. JULIE SEGRE.
AT THE OPENING OF THE FESTIVAL,
I TALKED ABOUT A VIRUS THAT
SHALL REMAIN UNNAMED, AND I'M
STILL NOT GOING TO -- VOLDEMORT
THE VIRUS, BUT IN ANY CASE, DR.
SEGRE IS COMING TO US VIRTUALLY
THIS AFTERNOON.
SO SHE IS A SENIOR INVESTIGATOR
AT NHGRI.
SHE IS AN INTERNATIONALLY KNOWN
EXPERT IN THE MICROBIOME AND
PARTICULARLY THE MICROBIOME OF
THE SKIN AND THE ROLES THAT
PLAYS IN MEDIATING THE
IMMUNOLOGIC EFFECTS OF THE SKIN
AND THE PROTECTION THAT THE SKIN
CONFERS UPON PEOPLE, AND THE
ROLE OF THAT MICROBIOME IN
DISEASE.
SHE APPLIES CUTTING EDGE GENOMIC
AND GENE SEQUENCING TECHNOLOGIES
TO BEST DEFINE THE MICROBIOME.
AND SO WITHOUT FURTHER ADO, DR.
SEGRE.
>> THANK YOU VERY MUCH, DR.
SCHOR.
IT IS A GREAT DISAPPOINTMENT TO
ME NOT TO BE WITH YOU ALL, AND I
HOPE I GET A RAIN CHECK SO THAT
I CAN MEET YOU.
BUT, YES, IT IS 3 1/2 YEARS
LATER, AND YET HERE WE ARE.
SO I WENT TO A -- THIS IS
IRONIC, A MICROBIAL PATHOGENESIS
MEETING, AND NOT SURPRISINGLY
TWO TO THREE DAYS LATER I CAME
HOME AND TESTED POSITIVE.
SO, UNFORTUNATELY, I'M STILL IN
MY PERIOD OF ISOLATION, BUT I'M
GLAD I'M FEELING BETTER ENOUGH
TO JOIN YOU TODAY.
REGRET NOT BEING WITH YOU ALL AT
THE RESEARCH FESTIVAL.
SO, I'M GOING TO KICK US OFF
TODAY AND IT'S REALLY A PLEASURE
TO TALK ABOUT MY WORK ON HUMAN
MICROBIOME AND AS DR. SCHOR
MENTIONED THE SKIN MICROBIOME IN
PARTICULAR.
AND THE TITLE IS FINDING OUR
FRIENDS AMONGST THE FOES.
AS MY WORK HAS BEEN ALSO AT THE
INTERSECTION OF ANAMICROBIAL
RETANS AND MICROBIOME.
PART OF MY WORK IS TO TALK WITH
THE IDEA IT MIGHT INSPIRE ANY OF
YOU AND IF YOU'RE INTERESTED IN
DEVELOPING MICROBIOME AS PART OF
YOUR OWN RESEARCH PROGRAM OR
QUESTIONS THAT YOU HAVE, I WOULD
WELCOME ANYONE TO SEND ME AN
E-MAIL NOW OR IN THE FUTURE.
THERE, OKAY.
SO, LET'S JUST GET STARTED WITH
THIS QUESTION OF WHAT DO I MEAN
WHEN I SAY THE HUMAN MICROBIOME.
BY THAT, I'M TALKING ABOUT THE
COLLECTION OF ALL MICROORGANISMS
AND THEIR DNA THAT LIVE IN
ASSOCIATION WITH THE HUMAN BODY.
SO I'M GOING TO GIVE A TALK
TODAY AND COVER BOTH BACTERIA,
FUNGI, AND VIRUSES.
AND REALLY ONE OF THE GOALS OF
THE HUMAN MICROBIOME PROJECT
THAT I WAS PROUD TO BE A PART OF
WAS TO MOVE THE DISCUSSION AWAY
FROM THINKING ABOUT ONLY THE
HUMAN MICROBIOME AND MICROBES
THAT COLONIZE US AS BAD OR EVIL
AND TO THINK AS MUCH ABOUT THE
BENEFICIAL ROLES THAT THESE
MICROBES PLAY ON OUR BODY.
TO THINK ABOUT THIS AS TO
REIMAGINE THAT WE THINK ABOUT
OURSELVES, HUMANS, AS THE SUPER
ORGANISMS, AND THAT WHEN I THINK
ABOUT MYSELF, IT'S THAT I AM A
COMBINATION OF THE HUMAN CELLS,
SO THESE 30 TRILLION HUMAN
CELLS, AS WELL AS AN EQUALLY
LARGE NUMBER OF BACTERIA AND
FUNGI AND PROBABLY EVEN MORE
VIRUSES.
BUT I CAME TO THIS WORK REALLY
STARTING AS A GRADUATE
OPPORTUNITY IN THE HUMAN GENOME
PROJECT WHERE WE WERE WORKING TO
DECIPHER THE HUMAN GENOME AND I
THOUGHT THE ORIGINAL GOAL WAS WE
WOULD STUDY THE HUMAN GENOME, 3
BILLION BASE PAIRS OF DNA, AND
ONCE REVEALED ABOUT THE 24,000
PROTEIN CODING GENES THAT WE
WOULD THEN HAVE THESE
BREAKTHROUGHS, AND I THINK THOSE
HAVE COME, EVEN MORE IS YET TO
COME.
WHEN WE THINK ABOUT HUMAN
DISEASE, A LOT OF IT IS STILL
ABOUT THIS HOST-MICROBIAL
INTERACTION AND AND MICROBES
THAT STIMULATE IMMUNE SYSTEM,
GENERATE RESPONSES OR CAUSE
INFECTION.
HERE IT'S A MORE COMPLEX GENETIC
QUESTION.
WHILE A BACTERIAL GENOME IS
SMALL, SO LIKE LET'S SAY 3
MILLION BASE PAIRS, RIGHT?
SO A THOUSAND TIMES SMALLER THAN
A HUMAN GENOME.
AND THE PROTEIN CODING OF THAT
IS TEN-FOLD LESS.
BUT NOW WHEN YOU THINK ABOUT THE
COMPLEXITY THAT THESE HUMAN
CELLS ALL HAVE MORE OR LESS THE
SAME CODING MATERIAL, BUT THE
BACTERIA AND FUNGI CAN HAVE
DIFFERENT GENOMES ACROSS STRAINS
AND SPECIES, YOU END UP WITH
AGGREGATE WHERE THE COMPLEXITY
OF THE GENOMES OF THE MICROBIOME
IS EQUAL OR EVEN LARGER THAN THE
HUMAN GENOME.
THERE'S A VAST SPACE TO EXPLORE
HERE.
I WANT TO MAKE SURE WE START
WITH THIS IDEA THAT THERE IS A
TREMENDOUS BENEFIT THAT MICROBES
PROVIDES, WHAT WE CALL SORT OF
ECOSYSTEM SERVICES.
SO, YOU KNOW, ONE OF THE GREAT
THINGS THE MICROBES DO THAT I'VE
HAD THE PLEASURE OF WORKING WITH
MY COLLEAGUE, DR. YASMINE
BELKADE, AN EXPERT, HOW MICROBES
TUNE THE IMMUNE SYSTEM, MAKE
SURE THE IMMUNE SYSTEM DOESN'T
FALL ASLEEP, ALSO TO RECOGNIZE
FRIENDS AND FOES.
THE SKIN MICROBES ALSO ARE GOING
TO SECRETE -- HELP BREAK DOWN
PRODUCTS THAT THE HUMAN CELLS
HAVE PRODUCED.
AND HELP US THEN MAINTAIN THIS
BARRIER.
YOU SEE THIS IN THE GUT WHERE
THEY WILL PROVIDE THIS BENEFIT
OF HELPING US TO DIGEST FOOD AND
NUTRITION.
BUT REALLY ALSO ONE OF THE MAJOR
THINGS THAT THE MICROBIOME DOES
IS TO PROVIDE COLONIZATION
RESISTANCE.
IT'S MUCH MORE ENERGY INTENSIVE
FOR SYSTEM TO MAINTAIN
STERILITY.
BUT YOU ARE CONSTANTLY BEING
EXPOSED ON YOUR OUTER SURFACES
TO PATHOGENS.
IF YOU HAVE MICROBES ALREADY
FILLING ALL THOSE NICHES, IT
MAKES IT HARDER FOR PATHOGEN TO
COME IN, AND TAKE UP RESIDENCE.
SO, THIS IS ONE OF ALSO THE
MAJOR BENEFITS OF HAVING
MICROBES COLONIZE YOU.
SO, MY GOAL AS SOMEONE IN THE
BEGINNING OF THE MICROBIOME
PROJECT HOW DO WE CHARACTERIZE A
MICROBIOME, AND IN EARLY WORK
THAT WE DID WITH DR. PATRICK
MURRAY, WE THOUGHT ABOUT LIKE
HOW ARE WE GOING TO CHARACTERIZE
ALL THESE MICROBES?
THE ORIGINAL WAYS HAD BEEN TO
PUT THEM ONTO PETRI DISHES.
AND THEN YOU WOULD GET THIS, YOU
KNOW, THESE CULTURED ISOLATES,
AND SOME OF THEM YOU WOULD SEE
YOU COULD CULTURE BETTER ON, YOU
KNOW, BLOOD AUGER OR BRAIN AND
HEART INFUSION, SO YOU KNEW YOU
WERE GETTING NOT THE FULL
MICROBIAL COMMUNITY, BUT ABOUT
HOW COMPLEX WITH MICROBIAL
CULTURING.
AT THIS POINT THE GENOME
PROJECT, WHEN I STARTED GRADUATE
SCHOOL IN 1990, WE'VE GONE
THROUGH MAJOR REVOLUTION IN THE
SCALE OF PROJECTS.
ON
ON THE LEFT THE CLASSIC IMAGE OF
READING THE GENOME IN BITS AND
PIECES BUT THAT MEANT AT THE
SAME TIME AS WE WERE
UNDERSTANDING THE COMPLEXITY OF
THE HUMAN GENOME, WE WERE HAVING
A REVOLUTION IN THE SEQUENCING
TECHNOLOGIES.
AND MOVING FROM THE ORIGINAL
SANGER SEQUENCING TO MUCH HIGHER
DENSITY SEQUENCING PLATFORMS.
ALONG WITH THAT CAME THE COMPUTE
POWER THAT WE HAVE AT THE NIH
THROUGH THE GREAT BENEFIT OF CIT
AND BIOWULF TO ANALYZE THESE
LARGE DATASETS.
SO, I THINK OF SEQUENCING AS A
NEW LENS TO OBSERVE BIOLOGY.
AND WHAT WE -- THE SAME WAYS
WE'RE CHARACTERIZING BACTERIA,
THINKING OF GRAM POSITIVE OR
GRAM NEGATIVE, UNDER MICROSCOPE
OR STAINS WE USE, NOW WE'RE ALSO
ADDING THIS OTHER FORM OF
CHARACTERIZATION WHERE WE'RE
SEQUENCING AND ABLE TO SEQUENCE
MICROBIAL COMMUNITIES AS WELL AS
INDIVIDUAL ISOLATES.
WE'RE ILLUMINATING THIS MUCH
GREATER SPACE BUT WE'RE THINKING
ABOUT USING THAT INFORMATION IN
THE SAME WAY SO I'M REALLY
THINKING ABOUT, YOU KNOW, HOW
THESE MICROBIAL COMMUNITIES COME
TOGETHER.
SORRY.
THE FIRST STUDY WE DID ON THE
SKIN MICROBIOME IN CONJUNCTION
WITH MY LONG-TERM CLINICAL
COLLABORATOR DR. HEIDI KONG AND
INSPIRED BY WORK WITH DR. MARIA
TURNER, WAS TO REALLY PUT FORTH
THIS IDEA THAT THE HUMAN BODY IS
AN ECOSYSTEM.
THIS WAS ONE OF OUR GREAT
STRENGTHS AS YOU THINK ABOUT
LIKE THE HUMAN MICROBIOME
PROJECT A LOT HAS REALLY BEEN
DRIVEN BY THE HUMAN GUT
MICROBIOME.
BUT THE SKIN HAS THIS REMARKABLE
ABILITY THAT WE HAVE ACCESS TO
THE TISSUE.
MORE THAN EVEN JUST HAVING
ACCESS, WE HAVE THESE
CLINICAL -- THE CLINICAL ACUMEN
OF DR. KONG, DR. TURNER, TO
THINK ABOUT HOW DERMATOLOGIC
DISORDERS MANIFEST AT SUCH
STEREOTYPIC LOCATIONS, AND SO
THE QUESTIONS THAT THEY BROUGHT
FORTH WERE THIS IDEA ABOUT IS
PSORIASIS ON THE OUTSIDE OF THE
ELBOW AND ECZEMA ON THE INSIDE
BECAUSE WE HAVE DIFFERENT
MICROBIAL COMMUNITIES AS WELL AS
HAVING DIFFERENT SKIN?
SO THIS WAS A STUDY THAT DOCTORS
KONG AND TURNER DESIGNED,
LOOKING AT 20 BODY SITES, AND
SURVEYED THEM BY LOOKING AT WHAT
WERE THE BACTERIAL COMMUNITIES.
THE OILY COMMUNITIES IN BLUE,
BACTERIAL ARE SMALL PIECHARTS,
YOU CAN SEE THE OILY SITES, LIKE
FOREHEAD OR GLOBELLAR, THE NOSE,
HAVE LIPID-LOVING BACTERIA,
WHEREAS THE MOIST SITES WILL
HAVE GREEN PROTEOBACTERIA,
STAPHYLOCOCCUS, DRY SITES ARE
SOME OF THE MOST DIVERSE
BACTERIAL COMMUNITIES.
AND IN FACT WHAT WE SAW WAS
THAT, YOU KNOW, THE BEND OF MY
RIGHT ELBOW IS MOST SIMILAR TO
THE BEND OF MY LEFT ELBOW, BUT
AFTER THAT, THE BEND OF MY LEFT
ELBOW IS THE MOST SIMILAR TO THE
BENDS OF YOUR LEFT ELBOW BECAUSE
MICROBIAL COMMUNITIES ARE
DEFINED BY THE ECOSYSTEM, BY THE
NUTRIENTS AVAILABLE, RATHER THAN
BY THE INDIVIDUAL.
AND SO, YOU KNOW, MY AXILLA IS A
SWEATY REGION SO THAT'S GOING TO
BE QUITE DIFFERENT THAN A MOIST
CREASE.
THESE KINDS OF IDEAS ABOUT WHAT
ARE THE COMMUNITIES WE DID THIS
THEN WITH THE BACTERIA, WITH THE
FUNGI, WITH THE VIRUSES, AND
THESE KINDS OF CHARACTERIZATIONS
THEN ENABLED US TO DO STUDY ON
CLINICAL POPULATIONS SEEN HERE
AT THE NIH, AND THIS WAS A
COHORT OF CHILDREN WITH ATOPIC
DERMATITIS, DR. KONG SEES.
AND IN THIS CASE, CHILDREN WERE
LOOKED AT, AT BASELINE, FLARE,
EPISODIC EXACERBATIONS, AND PUS
FLARE.
DR. KONG'S QUESTION WAS ABOUT WE
KNEW FROM EARLIER STUDY, THIS IS
JUST MOST RECENT STUDY, WE KNEW
FROM EARLIER STUDY AT THE FLARE
SOME KIDS HAD HIGH LEVELS OF
STAPH EPIDERMIS, STAPH AUREUS,
WAS IT MULTIPLE OR ONE STRANGE
AND HOW WERE THESE BACTERIAL
STRAINS DRIVING THIS DISEASE.
SO, IN THIS STUDY, SAMPLES WERE
PUT FORTH FOR SEQUENCING, AND
ALSO WE CULTURED INDIVIDUAL
ISOLATES SO WE COULD DO
STRAIN-LEVEL RESOLUTION.
WHAT WE SAW IF THERE IS A CHILD
WHO HAS STAFF EPIDERMIDIS IN
THEIR FLARE TIME POINT, SHOWING
JUST THE MICROBIAL COMMUNITY
THAT IS THE STAPHYLOCOCCUS, JUST
A SINGLE STRAIN OF STAPH AUREUS,
THAT WILL INCREASE IN THE FLARE,
BUT THEN WE SEE IF YOU MOVE TO
POST-FLARE TIME POINTS YOU'LL
SEE INCREASE IN STAPH EPI, AN
INTERESTING WAY THAT ALLOWS US
TO BUILD A MODEL THAT MAYBE THE
STAPH EPIIS HELPING RESOLVE THE
FLARE OF THE STAPH AUREUS.
IN THIS CASE ON THE RIGHT NOW
HERE WHERE I HAVE A CHILD WHO
HAS AT THE FLARE INCREASE IN A
LOT OF STAPH EPIDERMIDIS AS WELL
AS A LITTLE STAPH AUREUS, IN THE
FLARE TIME POINT WE'RE SEEING
INCREASE IN ALL OF THE STAPH
EPIDERMIDIS SUGGESTING THERE'S
SOMETHING ELSE IN TERMS OF
INITIATION AND POST-FLARE STILL
SEEING HIGH LEVELS OF STAPH
EPIDERMIDIS.
WE HAVE THESE STRAINS OF STAFF
EPI AND STAPH AUREUS WE'RE ABLE
TO USE IN ANIMAL MODELS AND
SHARE WITH THE COMMUNITY TO
START TO HAVE ANIMAL MODELS THAT
REALLY THEN PUT FORTH THE
LEGITIMATE SYSTEM OF HAVING THE
HOST WITH THE, YOU KNOW,
FILLEGRA MUTATION OR MOUSE MODEL
WITH THE MUTATION, USING THESE
STRAINS WE KNOW ARE INCREASING
IN A FLARE AND SHOWING TO DRIVE
DISEASE.
WE HOPE BY DOING THIS WE'LL BE
ABLE TO DEVELOP DIAGNOSTICS YOU
CAN IMAGINE IN BASELINE STATE
YOU CAN START TO MONITOR EITHER
THE MICROBIAL COMMUNITY OR
METABOLITES OF THE MICROBIAL
COMMUNITY AND PREDICT WHEN A KID
MIGHT TRANSITION FROM BASELINE
TO FLARE, AND INTERVENE EARLIER
THAN WHEN THE KID HAD ITCHED
THEMSELVES SO THEY ALREADY, YOU
KNOW, EXCORIATED THE SKIN.
IN THE SKIN MICROBIOME WE HAVE
THE GREAT PLEASURE OF BEING AT
THE NIH CLINICAL CENTER, AND
WORKING ON THESE FASCINATING
PATIENTS WITH INBORN ERRORS OF
IMMUNITY SO WE CAN BEGIN TO
UNDERSTAND WHAT IS THE
HOST-MICROBE INTERACTION.
WE THINK A LOT ABOUT HOW THE
MICROBES STIMULATE THE IMMUNE
SYSTEM, WE'RE ALSO FASCINATED TO
COME UP WITH THE CONCEPT OF
WHETHER THE MICROBES THEMSELVES,
YOU KNOW, ARE BEING SEEN BY THE
IMMUNE SYSTEM AND WHETHER THERE
IS SPECIFICITY TO THAT.
SO, THERE'S A LARGE COHORT OF
GENETICALLY DEFINED PRIMARY OR
IMMUNE -- PRIMARY IMMUNE
DEFICIENT PATIENTS,
FASCINATINGLY THEY HAVE -- YOU
KNOW, MANY PRESENT WITH ATOPIC
DERMATITIS WHICH GOT US
ORIGINALLY INTERESTED BUT WILL
ALSO HAVE OTHER ATOPES, AND WE
LOOKED ON THE LEFT AT DOCK8
DEFICIENT PATIENTS WITH ATOPY
BUT A LOT OF WARTS, AND GOT THE
FEELING THERE MIGHT BE
ADDITIONAL HPVs.
WHEN WE MINED GENOMIC DATA FROM
THE DOCK8 PATIENTS WE WERE ABLE
TO DEFINE 115 NEW HPV VARIANTS
INCLUDING ONE NO SPECIES, THESE
ARE THE 27 DOCK8 PATIENTS AND
YOU CAN SEE WHERE I LABELED THE
NOVEL SPECIES IN THE LIGHT BLUE
AND LIGHT GREEN YOU CAN SEE
THERE'S OFTEN MORE NOVEL SPECIES
BEING DETECTED THAN KNOWN
SPECIES OF HPV.
WE'VE EXPANDED THE REPERTOIRE.
AS WELL WORKING WITH DR.
NOTORANGELO HAVE LOOKED AT HIS
RAG DEFICIENT PATIENTS.
HERE AGAIN IT'S THIS INTERESTING
IDEA OF WHETHER THERE IS
INCREASED COLONIZATION IN
PATIENTS WITH IMMUNE
DEFICIENCIES.
WE SAW THIS IN THE DOCK8
PATIENTS.
WE SAW IN THE NARES THEY CARRIED
CORONAVIRUS, WE SAW THAT AGAIN
IN THE RAG-DEFICIENT PATIENTS
THAT WE IDENTIFIED RAG-DEFICIENT
PATIENT WITH NOVEL CORONAVIRUS
229E AS WELL AS SEEING
ADDITIONAL NOROVIRUS, INFLUENZA,
RHINOVIRUSES IN BOTH OF THESE
POPULATIONS.
WHAT'S REALLY INTERESTING ABOUT
THAT OF COURSE IS THAT WE'D LIKE
FOR PANDEMIC PREPAREDNESS HAVE A
BETTER SENSE OF WHAT ARE THE
VIRUSES THAT CAN COLONIZE HUMANS
AND I SEE THE NIH CLINICAL
CENTER'S PATIENTS WITH PRIMARY
IMMUNE DEFICIENCY AS A RICH
RESOURCE FOR UNDERSTANDING
VIRUSES THAT CAN COLONIZE HUMANS
AS WELL AS USING THIS
INFORMATION TO HELP TAILOR THEIR
CARE BECAUSE WE'RE OFTEN FINDING
VIRUSES THAT PERHAPS WE DIDN'T
EVEN KNOW TO TEST FOR.
ENOUGH ABOUT THE MICROBIUM
FRIENDS AND, YOU KNOW, LET ME
TALK ALSO A LITTLE ABOUT THE
FOES BECAUSE THIS HAS BEEN SOME
OF THE WORK THAT REALLY HAS BEEN
FOR ME PERFORMANCE FOREFRONT OF
HOW RESEARCH CAN BE EMBEDDED
INTO HOSPITAL LIKE THE NIH
CLINICAL CENTER, SO HERE IN THIS
TALK ABOUT THE NATIONAL
ACADEMIES, I WANT TO BRING BACK
TO A DECADE AGO, WHICH WAS
REALLY ONE OF THE SIGNATURES OF
MY CAREER AT NIH WHICH WAS THE
OPPORTUNITY TO WORK WITH DR.
TARA PALMORE, DAVID HENDERSON.
AND IN THIS CASE, IT WAS THE
FIRST PATIENT WHO WAS RECOGNIZED
AS COMING TO THE NIH CLINICAL
CENTER CARRYING A CARBAPENEM
RESISTANT KLEBSIELLA PNEUMONIA.
THE ENTEROBACTER ESIA LEAD THE
THREATS, NO ANTIBIOTICS LEFT TO
TREAT THESE BACTERIA.
IN THIS STORY WE HAD A PATIENT
COME INTO THE CLINICAL CENTER,
AND SHE WAS PUT IN ISOLATION AT
THAT POINT.
WE HAD A FAIR AMOUNT OF
EXPERIENCE AND PUT HER IN THE
LAST ROOM OF THE ICU.
AND A GOOD TWO MONTHS WENT BY,
AND THEN THERE WAS A SECOND,
THIRD -- OOPS.
SORRY.
SECOND, THIRD, FOURTH AND FIFTH
PATIENT WHO WERE IDENTIFIED AS
CARRYING KPC, KRE KPC.
THE TIMELINE IS WHEN PATIENTS
WERE IDENTIFIED AND THE ORANGE
ONES WERE IDENTIFIED AS CLINICAL
CULTURES, BLUE IDENTIFIED AS
SURVEILLANCE CULTURES.
THESE WERE VERY SICK PATIENTS IN
THE ICU.
AND SO I CALLED UP DR. HENDERSON
AND SAID, I'D LIKE TO SEQUENCE
THEM AND LET'S SEE IF WE CAN
FIGURE OUT ANYTHING.
WE TOOK PATIENT NUMBER 1 BECAUSE
WE THOUGHT THAT WAS GOING TO BE
THE OUTLIER, AND THOUGHT THAT'S
HOW WE WERE GOING TO UNDERSTAND
WHAT'S THE SIMILARITIES BETWEEN
2, 3, 4 AND 5 USING PATIENT 1 AS
THE REFERENCE.
THIS IS THE DECISION POINT.
OUR PRIMARY QUESTION WAS ABOUT
THE RELATEDNESS OF PATIENTS 2,
3, 4 AND 5, BUT WE ALSO HAD THIS
REALLY DEEP QUESTION ABOUT
PATIENT 1 AND WAS PATIENT 1
GOING TO MATCH 2, 3, 4 AND 5.
THE REASON THAT WAS IMPORTANT,
THE DECISION MATRIX DR. PALMORE
HAS TO MAKE, WHICH IS THE
QUESTION IF PATIENT 1 MATCHES 2,
3, AND 4 AND WE'RE ON THE LEFT,
THEN WE HAD NOSOCOMIAL, OR
HOSPITAL TRANSMISSION.
THAT WOULD MEAN THAT THE CONTACT
ISOLATION WAS INSUFFICIENT, THE
SCREENING OF PATIENTS WAS
INSUFFICIENT, AND THERE WAS A
NEED THEREFORE TO CHANGE THE
HOSPITAL PRACTICE.
ON THE OTHER HAND, IF PATIENT 1
DOESN'T MATCH 2, 3, 4 AND 5,
IT'S TWO SEPARATE INTRODUCTIONS,
AND THEN THAT MEANS THE CONTACT
ISOLATION IS SUFFICIENT,
SCREENING OF PATIENTS IS
SUFFICIENT, AND STAY THE COURSE.
THAT'S MORE ACTUALLY ON THE
RIGHT-HAND SIDE, THAT'S MORE
WHAT HAD HAPPENED TO THE NIH
CLINICAL CENTER DURING
ASENITOBACTER.
WE DIDN'T HAVE THE RESOLUTION,
MICRO LAB STILL RUNNING GELS,
ALL WOULD LOOK THE SAME.
BECAUSE 70% OF HEALTH CARE
ACQUIRED KLEBSIELLA PMEUMONIAE
IN THE U.S. ARE SEQUENCE TYPE
258 THAT LOOK THE SAME.
COULD GENOMIC DATA GIVE US MORE
INFORMATION TO RESOLVE THIS?
OKAY.
SO, PATIENT 1, I WANT TO TAKE A
BIT OF A DEEPER DIVE.
NOW, ON THE LEFT-HAND SIDE I'VE
DRAWN THESE DIAGRAMS.
THIS IS PATIENT NUMBER 1, AND
SHE HAD BEEN COLONIZED FOR SO
LONG THAT SHE ACTUALLY WAS
COLONIZED AT MULTIPLE SITES IN
HER BODY.
WE HAD ISOLATES, MULTIPLE
ISOLATES, FROM URINARY TRACT
OVER A WEEK OR TWO IN WHICH WE
KEPT CULTURES AND CLINICAL
MICROLAB KEPT CULTURING THEM.
WE ALSO HAD ISOLATES FROM THE
THROAT AND FROM THE GROIN AREA.
SO WE SEQUENCED ALL OF THESE
ISOLATES.
AND WHAT I'M SHOWING YOU IS THAT
ON THE VERY LEFT, THE PLAIN
CIRCLE, THAT'S THE URINE
ISOLATE.
WE'RE GOING TO CONSIDER THAT
ANCESTRAL OR REFERENCE STRAIN.
IN THE THROAT ISOLATE, THERE
SEEMS TO HAVE BEEN THREE SINGLE
NUCLEOSIDE CHANGES IN A 6
MEGABASE GENOME.
THAT WOULD HAVE HAPPENED NOT IN
CODING GENE OR ANYTHING BUT
SMALL ERRORS IN REPLICATION THAT
GET PASSED ON, IN A CLONAL
SPREAD.
THE GROIN SEEMS TO HAVE BEEN
SEPARATE, YOU KNOW, SEPARATE
COLONIZATION, YOU SEE THREE
DIFFERENT SNPs.
THE KEY, THESE SNPs ARE
IDENTIFIABLE TO THIS PATIENT.
SO THE MOMENT THAT WE ALL
REMEMBER IS SITTING THERE WITH
THE ORIGINAL SEQUENCE DATA, WE
GOT THE ISOLATES AND HAD THE
SEQUENCE DATA, AND WITHIN 24
HOURS WE HAD ANALYZED IT AND
WE'RE SITTING IN A CONFERENCE
ROOM WITH DR. PALMORE AND SAY,
WE HAVE A MATCH FROM PATIENT 1,
AND THE OTHER PATIENTS.
BUT THE PART WE DON'T UNDERSTAND
IS THAT WE THINK PATIENT 1
TRANSMITTED TO PATIENT 3,
BECAUSE THAT'S WHERE YOU SEE THE
SAME THREE SNPs IN PATIENT 3'S
THROAT ISOLATE AS IN PATIENT 1.
AND THEN WE BELIEVE PATIENT 3
TRANSMITTED TO PATIENT 2.
BECAUSE PATIENT 2 HAS AN
ADDITIONAL SNP WE DIDN'T SEE IN
EITHER PATIENT 3 OR 1.
AND DR. PALMORE SAYS, YEAH,
THAT'S ALSO WHAT I THINK
HAPPENED.
SO FOR THIS MOMENT WE HAD THE
GENOMIC DATA AND EPIDEMIOLOGIC
DATA LINING UP.
THE BOTTOM I SHOW PATIENT TRACE
DATA.
PATIENT 1 AND PATIENT 3 IN
TURQUOISE OVERLAPPED IN THE ICU.
AFTER PATIENT 1 HAD ALREADY LEFT
THE ICU, PATIENT 3 AND 2
OVERLAPPED IN THE ICU.
WHAT THIS WOULD SUGGEST IS THAT
THERE WAS A TRANSMISSION FROM
PATIENT 1 TO 3, THAT WENT
UNDETECTED FOR TWO MONTHS.
BUT WAS CARRIED BY THAT PATIENT
ENOUGH THAT THE PATIENT 3 COULD
GO ON AND TRANSMIT TO PATIENT 2.
I REALLY DO THINK PROBABLY THE
WORLD OF COVID HAS TAUGHT US
LIKE HOW THIS COULD HAPPEN BUT
I'M TELLING YOU THIS WAS MIND
BLOWING TO US AT THE TIME.
AND IT MEANT EXACTLY THAT.
WE HAD TO CHANGE THE -- ENHANCE
SURVEILLANCE OF ASYMPTOMATIC
PATIENTS, AND HAVE MORE CONTACT
ISOLATION.
THERE CONTINUED TO BE A CLUSTER
OF INFECTIONS AT THE NIH
CLINICAL CENTER, BUT WITH THIS
ENHANCED PRECAUTIONS THERE WAS A
HALT IN THE TRANSMISSIONS THAT
OCCURRED IN THE HOSPITAL.
WE WERE ABLE TO UNDERSTAND WHAT
WERE THE WEAKNESSES AND HOW WE
COULD CHANGE THE PRACTICE.
SO, A LOT OF WHAT WE DID WITH
GENOMIC DATA WAS THINK ABOUT THE
CONNECTIONS THAT WE COULD MAKE,
BUT WE ALSO WERE ABLE TO RULE
OUT CONNECTIONS.
SO ON THE LEFT OF THIS SLIDE,
I'VE DRAWN SORT OF WHAT WOULD
HAVE BEEN THE POSSIBLE
CONNECTIONS WHERE IF YOU HAD
LOOKED AT HOW ALL THE WAYS IN
WHICH THESE PATIENTS WERE ON THE
SAME BOARD OR ARE THE SAME
MEDICAL DEEM WHAT WERE
EPIDEMIOLOGIC LINKS BUT WHEN YOU
COMBINE EPIDEMIOLOGIC LINKS WITH
THE GENOMIC DATA, THEN YOU CAN
SEE WHAT ARE THE MOST LIKELY
ROUTES OF TRANSMISSION, AND THAT
MEANT THAT, FOR EXAMPLE, THERE
HAVE BEEN A NARRATIVE ABOUT TWO
PATIENTS WHO THERE WAS THE SAME
WOUND CART BEING USED ON THE TWO
SUBJECTS, BUT WHEN WE SAW THEM
IN THE GENOMIC DATA, ONE OF THEM
WAS LIKE PATIENT 9 AND THE OTHER
WAS PATIENT 16, YOU CAN SEE THEY
WERE ON TWO DIFFERENT PARTS OF
THE TRANSMISSION TREE, AND WE
DIDN'T NEED TO CONTINUE TO LOOK
FOR WHAT WERE THE LINKS.
SO, WITH THIS DATA WE WERE ABLE
TO STOP THE TRANSMISSION.
SO NOW I WANT TO TALK ABOUT THE
FINAL VIGNETTE WHICH IS REALLY
ABOUT NEW WORK THAT WE'VE BEEN
DOING ON CANDIDA AURIS COMBINING
SKIN MICROBIOME AND RESISTANCE,
WORKING WITH OUR COLLEAGUES AT
THE CDC.
SO CANDIDA AURIS IS FUNGAL
PATHOGEN, THAT IS AGAIN CDC IS
RATING THIS AS ONE OF ITS MOST
URGENT THREATS BECAUSE CANDIDA
AURIS ALSO HAS EVOLVED
RESISTANCE TO ALL KNOWN
ANTI-FUNGALS.
IF YOU THINK THERE'S A PROBLEM
WITH DEVELOPMENT OF ANTIBIOTICS,
ANTI-FUNGALS ARE HARDER BECAUSE
THEY ARE EUKARYOTES, HARD TO GET
IN THE SPACE TOXIC TO FUNGI AND
NOT TOXIC TO THE HUMAN.
CANDIDA AURIS IS AN ENIGMA.
IT WAS NOT KNOWN BEFORE 2009,
AND SINCE THEN FOUR CLADES OF
CANDIDA AURIS HAVE BEEN
RECOGNIZED, FOUR REGIONS OF THE
GLOBE, BUT THEY HAVE ALL NOW
COME TO THE UNITED STATES AND
ARE CAUSING OUTBREAKS IN NURSING
HOMES.
AND WHY ARE THESE CANDIDA AURIS
OUTBREAKS HAPPENING IN NURSING
HOMES AND HOW DOES IT CONNECT TO
THE SKIN MICROBIOME?
THE SKIN IS CONSTANTLY TURNING
OVER.
SO WE SHED THE ENTIRE LAYER OF
OUR SKIN EVERY TWO TO FOUR
WEEKS.
AND YOU'RE GOING TO SEE THAT AS
THOSE DUST BUNNIES AROUND YOUR
HOUSE.
SO, WE'RE SHEDDING FIVE TIMES
TEN TO THE EIGHTH HUMAN CELLS
PER DAY, BUT THAT MEANS WE'RE
SHEDDING, YOU KNOW, TEN TO THE
SEVENTH MICROBES PER DAY.
FOR THE MICROBES THAT CAN
PERSIST IN THE ENVIRONMENT, LIKE
ASENNICDOBACTER OR CANDIDA
AURIS, IT CAN PERSIST AS VIABLE,
THAT MAKES IT DIFFICULT TO
CONTROL THESE OUTBREAKS IN
CONGREGANT SETTINGS, I MEAN
NURSING HOMES.
WE'RE NOT SEEING THIS IN THE
HEALTHY POPULATIONS, BUT IN
THESE SICK OR OLDER PATIENTS
WE'RE SEEING CANDIDA AURIS
COLONIZE ONE RESIDENT IN THE
NURSING HOME, THEM BEING
BASICALLY RUNNING THROUGH THE
ENTIRE NURSING HOME, UNLESS
CASES ARE REALLY RECOGNIZED
EARLY ON AND ISOLATED.
SO, IN A STUDY THAT I'VE BEEN
VERY FORTUNATE TO CARRY OUT WITH
DR. MARY HAYDEN AND MY
COLLEAGUES AT THE CDC, WE SET UP
A STUDY TO LOOK AT RESIDENTS IN
NURSING HOMES TO TRAP
TRANSMISSION OF CANDIDA AURIS.
SOME OF THAT INITIAL WORK HAS
BEEN PUBLISHED, ABOUT HOW
DIFFICULT IT IS TO DETECT
CANDIDA AURIS BECAUSE IT CAN
COLONIZE ANY PART OF THE SKIN,
WHICH MAKES INFECTION CONTROL
VERY HARD.
BUT OUR LATEST WORK HAS BEEN
LOOKING AT THE AGGREGATE OF WHAT
IS COLONIZING THE SKIN.
SO WE WENT IN THIS LOOKING AT
THE SKIN AS WE WENT TO LOOK FOR
CANDIDA AURIS BUT I WANT TO TALK
ABOUT A SMALL TECHNOLOGY
REVOLUTION, THIS WILL BE THE
LAST PART, AND HOW THAT'S
CHANGING THINGS FOR US.
SO, I'M GOING IN AND LOOKING
AT -- I'M LOOKING FOR CANDIDA
AURIS.
WHEN WE WANT TO STUDY SOMEONE'S
SKIN MICROBIOME AND SEE, YOU
KNOW, WHAT STRAINS OF CANDIDA
AURIS, HOW MUCH THEY HAVE ON THE
SKIN WE SWAB THE SKIN AND WE
JUST PUT THAT SAMPLE -- WE CAN
CULTURE BUT WE WANT TO LOOK AT
THE STRAIN LEVEL VARIATION, WE
TAKE THAT SAMPLE, CLINICAL
SAMPLE, PUT IT STRAIGHT INTO THE
ILLUMINA SEQUENCER.
FROM THAT, WE CAN REASSEMBLE THE
CANDIDA AURIS GENOME.
THIS HAS REALLY BEEN ACTUALLY
THE REVOLUTION OF MY LAB, WHERE
FROM A COMPLEX SAMPLE OF THE
SKIN, I CAN THEN REASSEMBLE THE
ENTIRE GENOME OF CANDIDA AURIS
AND OTHER BACTERIAL, FUNGAL, AND
VIRAL SPECIES THAT ARE. THERE
HOW I THINK ABOUT THIS IS THAT I
THINK IT'S CLEAR TO EVERYONE HOW
YOU ALL SEQUENCE -- MANY OF YOU
SEQUENCE MICROBIAL GENOMES AND
KNOW YOU GET THESE READS AND
ASSEMBLE THEM AND GET A GENOME.
THAT'S LIKE IF I JUST GAVE
YOU -- I HAVE PUT ALL THE PIECES
FOR A PUZZLE INTO A BOX, AND YOU
USE THAT TO ASSEMBLE THE GENOME.
BUT WHAT I'M TRYING TO DESCRIBE
TO YOU HERE WITH SHOTGUN
METAGENOMICS TAKEN FROM THE SKIN
SAMPLE DIRECTLY I HAVE ONE BOX
WITH MULTIPLE PUZZLES IN IT,
RIGHT?
YOU'LL HAVE THE COMMENSAL
STAPHYLOCOCCUS AND CUDO BACTERIA
AND CANDIDA AURIS.
I PUT THESE TOGETHER IN ONE BOX
AND I SAY CAN YOU ASSEMBLE THEM
INTO GENOMES TO LOOK AT
TRANSMISSION NETWORKS.
THIS IS MORE EFFICIENT THAN
PREVIOUSLY WHERE I JUST LOOKED
AT KLEB PNEUMO, FROM ONE AT A
TIME.
WHEN YOU THINK IF I DID GIVE YOU
THESE PUZZLES, YOU WOULD SORT
THE PIECES INTO EITHER THE
COLORS YOU SAW OR THE SIZE OF
THE PUZZLE PIECES OR MAYBE SOME,
YOU KNOW, DIFFERENT MARKINGS
THAT SOME HAVE, SMALLER PIECES,
LARGER PIECES.
THAT'S EXACTLY WHAT WE'RE DOING
ALSO, LOOKING AT TETRA
NUCLEOSIDE, GC CONTENT, SORTING
INTO PUZZLES.
I WAS EXPECTING TO SORT OF LOOK
AT FRIENDS AND FOES AND CANDIDA
AURIS, AND INSTEAD WHEN WE
ASSEMBLED ALL OF THE GENOMES
WHAT WE FOUND WAS CANDIDA AURIS,
AND FROM THE SKIN OF THESE
RESIDENTS OF THE NURSING HOME
FOUND A CLONAL SPREAD OF CANDIDA
AURIS, IN ADDITION THE OTHER
GENOMES THAT CAME OUT OF THE
SKIN OF THESE PATIENTS IN THE
NURSING HOMES WAS E. COLI, STAPH
AUREUS, PSEUDOMONAS AERUGINOSA,
THE FULL ESCAPE OF ALL OF THE
BACTERIA WE CONSIDER AND ARE
MOST CONCERNED ABOUT WITH
MULTI-DRUG RESISTANCE.
WE ASSEMBLED FULL GENESOME FROM
THE SKIN OF THESE PATIENTS AND
ARE TRACKING, WE WERE TRYING TO
TRACK A CANDIDA AURIS OUTBREAK
BUT INSTEAD TRACKING
SIMULTANEOUSY SPREAD OF CANDIDA
AURIS, KLEBSIELLA PNEUMO, E.
COLI, AND STAPH AND PSEUDOMONAS,
HAVING THE SPREAD OF A LOT MORE
MULTIDRUG-RESISTANT ORGANISMS
THAN HAD BEEN PREVIOUSLY
APPRECIATED.
AND THIS BRINGS ME TO MY FINAL
POINT, WHICH IS, YOU KNOW, AS WE
THINK ABOUT ANTIBIOTIC
STEWARDSHIP IN HOSPITALS, AND SO
ON, ONE OF THE THINGS THAT COVID
HAS BROUGHT TO BEAR IS THAT
NURSING HOMES ARE PART OF OUR
HEALTH CARE ECOSYSTEMS, AND THEY
DON'T HAVE THE SAME LEVELS OF
INFECTION CONTROL BUT WE NEED TO
START THINKING ABOUT THEM AS
ALSO PLACES WHERE WE NEED
ENHANCED INFECTION CONTROL AND
CARE FOR THESE PATIENTS.
SO WITH THAT, I AM HAVING
TROUBLE NOW SOMEHOW -- OKAY.
MY OVERALL CONCLUSIONS, GENOMIC
SEQUENCING MAKES THE INVISIBLE
WORLD OF MICROBES VISIBLE AND
IDENTIFIABLE AND I HOPE WE'LL
REVOLUTIONIZE WHAT IT MEANS TO
BE HUMAN, HEALTHY, PROVIDE
INSIGHTS TO FIGHT MICROBIAL FOES
AND CULTIVATE MICROBIAL FRIENDS.
I'D LIKE TO ACKNOWLEDGE MY
COLLABORATORS.
IT'S BEEN THE PLEASURE AND HONOR
OF MY LIFE TO WORK AT NIH, AND
ALL OF THESE STUDIES WERE MADE
POSSIBLE BECAUSE OF THESE
COLLABORATIONS, IN PARTICULAR
THE LONG-TERM COLLABORATIONS
WITH DR. HEIDI KONG, TAR A
PALMORE, YASMINE BELKAID, NIH
AND CDC AND LARGER COMMUNITY,
ESPECIALLY SHOUT OUT TO MY LAB,
FROM 2011 AND 2013, AND REALLY
THE PERMANENT MEMBERS OF THE LAB
WHO ARE SHOWN IN BOTH OF THESE
PICTURES, GREAT MENTORS I'VE HAD
AT NHGRI AND NIH, AND MY FAMILY.
SO THANK YOU ALL.
AND I'M HAPPY TO TAKE QUESTIONS.
SORRY I COULDN'T JOIN YOU IN
PERSON.
[APPLAUSE]
>> WE'RE GOING TO MOVE ON, I
CONVEY GET WELL WISHES AND THANK
YOU FOR BEING PART OF THE
SYMPOSIUM.
NEXT SPEAKER IS DR. MICHAEL
LICHTEN, SCIENTIST EMERITUS AT
NCI.
HE'S AN EXPERT ON MEIOSIS, THE
PROCESS BY WHICH GAMETES ARE
FORMED, AND PARTICULARLY ON THE
PROCESS OF EXCHANGE OF CHROMATIN
DURING MEIOSIS, AND WHAT HAP%ES
WHEN THAT PROCESS GOES AWRY.
HE USES A YEAST MODEL TO LOOK AT
THESE RECOMBINATION EVENTS, AND
I'M GOING TO TURN THE PODIUM
OVER TO DR. LICHTEN.
>> THANK YOU, NINA.
YOU JUST BLEW 90% OF MY
INTRODUCTORY SLIDES.
[LAUGHTER]
OKAY.
OOPS, THE OTHER WAY.
HERE WE GO.
YOU WILL NOTICE A CERTAIN
REPETITIVE NATURE TO THE TITLES
OF THESE TALKS.
I DON'T KNOW HOW LONG IT'S GOING
TO EXTEND THROUGH THE SYMPOSIUM.
I'M INTERESTED IN MEIOSIS AND
RECOMBINATION, BUT IN PARTICULAR
IN THE REPAIR OF DNA BREAKS, AND
THE MECHANISTIC INSIGHTS WE CAN
GAIN.
SO, YOU KNOW EACH ONE OF US IN
EVERY ONE OF OUR CELLS, EVERY
ONE OF OUR CELLS THAT DIVIDES,
UNDERGOES DOZENS OF
DOUBLE-STRAND BREAKS, EVERY CELL
CYCLE.
AND THIS IS -- IN FACT, IF YOU
LOOK AT THE NUMBER THROUGHOUT
THE ANIMAL KINGDOM, IT'S WITHIN
A COUPLE OF FACTORS OF TWO OF
EACH OTHER.
SO THIS SEEMS TO BE PRETTY MUCH
UNIVERSAL AMONG EUKARYOTES.
INACCURATE REPAIR, CELLS ARE
DEPENDENT ON ACCURATE REPAIR,
INACCURATE REPAIR CAN LEAD TO
MUTATIONS AND CHROMOSOME
REARRANGEMENT, AND IN PARTICULAR
I LIKE TO SHOW THIS PICTURE
WHICH IS THE FACE OF THE ENEMY.
THIS IS A SPECTRAL YOUR CARE --
KARYOTYPE OF A BLADDER CELL BY
THOMAS REIDED A NCI.
I THINK THERE ARE OVER A DOZEN
CHROMOSOME REARRANGEMENTS IN
THIS SOLID TUMOR, AND EACH ONE
OF THOSE WAS A DOUBLE STRAND
BREAK THAT AT ONE TIME WAS
REPAIRED INCORRECTLY.
SO, THIS IS OF RELEVANCE FOR A
NUMBER OF THINGS BUT CERTAINLY
IN ETIOLOGY OF CANCER.
SO BREAKS CAN BE REPAIRED BY TWO
MECHANISMS, NON-HOMOLOGOUS
JOINING, BASICALLY JUST BASHING
AND LIGATION, AND THAT'S WHAT
GIVES YOU PROBLEMS LIKE THIS.
OR BY HOMOLOGOUS RECOMBINATION,
WHICH PRESERVES GENOME CONTENT.
BUT THIS IS -- DOUBLE-STRAND
BREAKS DON'T GO AWAY WITH THE
THOUGHT THAT DOUBLE-STRAND
BREAKS WITH JUST A CHALLENGE TO
THE CELL.
IN FACT, OUR CELLS AND CELLS OF
OTHER ORGANISMS MAKE WILLFUL USE
OF DOUBLE-STRAND BREAKS, WILLFUL
DAMAGING OF GENOMES TO CARRY OUT
VARIOUS FUNCTIONS.
ONE MIGHT WONDER WHY, BUT THE
FACT IS EVERY CELL THAT DIVIDES
IN OUR BODY IS REPAIRING 50
DOUBLE-STRAND BREAKS
EFFICIENTLY, SO WHAT'S A FEW
MORE?
SO, AMONG THOSE IS SWITCHING IN
FUNGI WITH ONE BREAK OR SO PER
CELL, EVERY MATING TYPE SWITCH,
GENERATION OF IMMUNOGLOBULIN
DEFICIENCY REPAIRED BY
HOMOLOGOUS RECOMBINATION IN VDJ
RECOMBINATION, CLASS SWITCH
RECOMBINATION, ET CETERA,
REPAIRING BY NON-HOMOLOGOUS END
JOINING BY A COUPLE BREAKS PER
CELL.
THEN YOU GET TO MEIOSIS, WHERE
THERE ARE ON THE ORDER -- THERE
ARE HUNDREDS OF HUNDREDS OF
BREAKS FORMED DURING THE PROCESS
OF MEIOSIS.
WHEN I WAS LOOKING FOR A MODEL
SYSTEM TO STUDY DNA DAMAGE
REPAIR, AND HOMOLOGOUS
RECOMBINATION, I FOCUSED ON
MEIOSIS
WHAT IS MEIOSIS?
MEIOSIS IS THE PROCESS, EXCUSE
ME.
MEIOSIS IS THE PROCESS BY WHICH
A DIPLOID GENOME IS DIVIDED INTO
FOUR HAPLOID GAMETES, SPERM --
EITHER SPERM OR THE EGG, AND IN
DOING SO IT GOES FIRST THROUGH A
ROUND OF REPLICATION, AND THEN
THE CELL DOES SOMETHING THAT --
EXCUSE ME, BEAR WITH ME FOR A
MINUTE, I'M GOING TO TRY AND
TURN ON THIS LASER POINTER.
THERE WE GO.
OKAY.
IT DOES SOMETHING THAT IT DOES
NO OTHER TIME IN THE LIFE CYCLE
OF THE ORGANISM.
RATHER THAN SEPARATE THE TWO
SISTER COPIES OF EACH
CHROMOSOME, IT GOES THROUGH AN
ELABORATE PROCESS OF TWO NUCLEAR
DIVISIONS, ENDING UP WITH FOUR
HAPLOID GAMETES WHICH INVOLVES
THE ASSOCIATION AND PAIRING OF
THE TWO COPIES OF THE SAME
CHROMOSOME, WHICH PREVIOUSLY
HAVE NO RELATIONSHIP TO EACH
OTHER, SO THEY HAVE TO BE
BROUGHT TOGETHER AND STABLY
PAIRED.
AND THEN CONNECTED BY
CHROMOSOME, BY CROSSOVERS THAT
LINK THE TWO HOMOLOGOUS
CHROMOSOMES AND PROVIDE THE
CONNECTION THAT ALLOWS THE
TENSION TO BE SENSED AT THE
FIRST DIVISION OF THE SPINDLE OF
THE FIRST DIVISION.
AND THEN THE SECOND DIVISION IS
A MORE MITOSIS-LIKE DIVISION.
SO, RECOMBINATION PLAYS A
CRITICAL ROLE IN BOTH THE EARLY
STEPS OF MEIOSIS, HOMOLOGS COME
TOGETHER AND ARE STABLY PAIRED,
AND IN COMING CROSSOVERS THAT AS
I SAID PROVIDE STABLE LICKS
BETWEEN HOMOLOGOUS CHROMOSOMES.
WHEN THIS GOES WRONG
CONSEQUENCES ARE DIRE.
MANY OF YOU HAVE RECOGNIZED THIS
CURVE, AMYPLOIDY WITH A FUNCTION
OF MATERNAL AGE.
I WANT TO POINT OUT ALL ACROSS
THIS CURVE, BUT IN PARTICULAR IN
THESE LATER STAGES WHERE
ANEUPLOIDY CAN BE HIGH THERE'S
CLOSE ASSOCIATION WITH
RECOMBINATION HAPPENING IN THE
WRONG PLACE.
IT'S IMPORTANT TO MAINTAIN -- TO
DO THIS PROBABLY IN ORDER TO
SEGREGATE CHROMOSOMES PROPERLY.
OKAY.
SO HOW DOES RECOMBINATION PLAY A
ROLE IN THIS?
AS I SAID, EARLY STEPS INVOLVE
MULTIPLE INTERACTION, MULTIPLE
TRANSIENT INTERACTIONS BETWEEN
HOMOLOGOUS CHROMOSOMES,
INITIATED BY PROGRAM
DOUBLE-STRAND STRAND BREAKS BY
SPO11 PROTEIN, HUNDREDS FORMED
PER GENOME.
NORMALLY MITOTIC CELLS SUCH
BREAKS WOULD BE REPAIRED BY
RECOMBINATION WITH SISTER
CHROMATID BUT IN THIS CASE USED
TO PAIR WITH HOMOLOGOUS, SO
SISTER RECOMBINATION IS
DISCOURAGED, RECOMBINATION WITH
HOMOLOGOUS CHROMOSOME IS
ENCOURAGED, SORTED INTO TWO
TYPES, MOST ARE SORTED OUT INTO
NON-CROSSOVERS, AND THEN A FEW
OF THEM ARE MATURED INTO
CROSSOVERS THAT, AGAIN, ARE
PROVIDING THE CONNECTIONS WITH
HOMOLOGS THAT ALLOW PROPER
SEGREGATION.
THAT ALLOW PROPER HOMOLOG
SEGREGATION.
SO, MY LAB'S BEEN INTERESTED IN
ALL OF THESE, MOLECULAR
MECHANISM OF ALL THESE STEPS
ALONG THIS PROCESS.
WE'VE STUDIED THE RULES THAT
DETERMINE WHERE SPO11 MAKES
DOUBLE-STRAND BREAKS, STUDY
MECHANISMS THAT DISCOURAGE
SISTER CHROMATID RECOMBINATION,
ENCOURAGE INTERHOMOLOG
RECOMBINATION BUT MOST OF THE
FOCUS HAS BEEN ON MECHANISMS
THAT DETERMINE WHETHER
RECOMBINATION EVENT IS GOING TO
FORM A CROSSOVER OR
NON-CROSSOVER, AND THE
REGULATORY MECHANISMS, THE
ACTIVITIES THAT REGULATE WHETHER
ONE STEP IS -- WHETHER ONE FATE
IS FOLLOWED OR THE OTHER.
SO, AND OUR WORK HAS BEEN DONE
EXCLUSIVELY IN BUDDING YEAST,
I'LL DISPENSE WITH THIS BRAG
SLIDE BUT JUST TO POINT OUT THAT
BECAUSE YEAST IS A SINGLE-CELLED
ORGANISM WE CAN GROW IT IN
LIQUID CULTURE, WE CAN INDUCE
MEIOSIS SYNCHRONOUSLY AND
EXTRACT DNA FROM CELLS AS THEY
ARE GOING THROUGH THE PROCESS OF
RECOMBINATION, AND THEREFORE BY
ANALYZING THE INTERMEDIATES AND
PRODUCTS AMONG THE DNA THAT WE
ISOLATE, WE CAN MONITOR
RECOMBINATION BASICALLY IN REAL
TIME.
AND SO WE CAN SEE THE
INTERMEDIATE STEPS, WHEN THE
INTERMEDIATE STEPS GO TO
PRODUCTS, ET CETERA.
UNLIKE MANY ORGANISMS
PARTICULARLY DURING OOGENESIS
RECOVER ALL FOUR HAPLOID
PRODUCTS AND AND TETRADS,
ANALYZE PARENTAL CONTRIBUTION IN
EVERY RECOMBINATION EVENT.
FINALLY, I WOULDN'T BE UP HERE
IF IT WASN'T TRUE MY MEIOSIS IS
SIMILAR TO MEIOSIS IN U.S. US,
DIRECTLY APPLICABLE TO OTHER
ORGANISMS.
THE PRINCIPLES WE'VE DISCOVERED
IN OUR STUDY OF THE MECHANISMS
OF MEIOTIC RECOMBINATION HAVE
OVER THE YEARS INFORMED
MECHANISMS OF REPAIR OF
DOUBLE-STRAND BREAKS AND
HOMOLOGOUS RECOMBINATION IN THE
MITOTIC CYCLE AS WELL.
OKAY.
I'M GOING TO TELL YOU ONE STORY
ABOUT MEIOTIC RECOMBINATION.
IT STARTS AROUND THE TURN OF THE
CENTURY WITH A POSTDOC WHO WAS
INTERESTED IN TRYING TO FIGURE
OUT THE RULES FOR NON-CROSSOVER
VERSUS CROSSOVER RECOMBINATION.
I WON'T SHOW -- I'LL SHOW A
LITTLE BIT OF DATA ENFORCING
THIS LATER ON, BUT WHAT HE FOUND
OUT IN FACT AFTER ONE HAD MADE
AN INITIAL STRAND INVASION
INTERMEDIA, NOT CROSSOVERS AND
CROSSOVERS WERE FORMED BY
DISTINCT, VERY DISTINCT AND
DIFFERENT BIOCHEMICAL
MECHANISMS.
AND HE GOT A HINT THAT THAT WAS
TRUE JUST BY LOOKING AT TIMING.
I'VE DRAWN A TIME LINE.
SO NON-CROSSOVERS APPEAR EARLY,
WELL BEFORE CROSSOVERS APPEAR,
AND WHEREAS CROSSOVERS DERIVE
FROM THIS INTERMEDIATE, DOUBLE
HOLLIDAY JUNCTION, AND THE
RESOLUTION OF THIS INTERMEDIATE,
AND ITS PRECURSOR CALLED SINGLE
END INVASION INTERMEDIATE, AND
THIS RESOLUTION IS DEPENDENT
UPON -- ACTIVATED BY IN YEAST
THE CDC5 POLO KINASE UNDER
CONTROL OF THE NDT80 TRANSCRIPT
FACTOR.
IF ONE MUTATED EITHER OF THESE
PROTEINS, THESE GENES, ONE
TERMINATED MEIOSIS WITH MATURE
NON-CROSSOVERS, AND DOUBLE
HOLLIDAY JUNCTIONS WITHOUT
PRODUCING COSTOVERS.
SO NON-CROSSOVERS WERE DERIVED
FROM DOUBLE HOLLIDAY JUNCTION
BUS NON-CROSSOVER FORMATION WAS
INDEPENDENT OF THAT.
AND WHAT THORSTON SUGGESTED
THESE WERE FORMED BY DISTINCT
BIOCHEMICAL MECHANISMS,
CROSSOVER FORMED BY CLEAVAGE OF
HOLLIDAY JUNCTIONS BUT
NON-CROSSOVERS FORMED BY
COMPLETELY DIFFERENT PROCESS BY
WHICH THIS EARLY STRAND INVASION
INTERMEDIATE WAS TAKEN APART BY
HELICASE AND AKNEELED --
A NEILSED, AFTER FILLING IN ONE
HAD A NON-CROSSOVER.
SINCE THEN, WE'VE LEARNED A LOT
MORE.
WE NOW KNOW THAT THIS PROCESS OF
CROSSOVER FORMATION FORMS IN
THE -- IS CARRIED OUT IN THE
CONTEXT OF AN ELABORATE PROTEIN
STRUCTURE THAT'S ASSEMBLED
BETWEEN HOMOLOGOUS CHROMOSOMES,
SYNAPTIC COMPLEX, IN PARTICULAR
BY ZM PROTEINS, THESE IN EFFECT
BIND TO AND PROTECT
RECOMBINATION INTERMEDIATES FROM
DISASSEMBLY AND NON-CROSSOVER
FORMATION, AND SET THEM UP FOR
THE RESOLUTION BY
MEIOSIS-SPECIFIC RESOLVEase TO
FORM CROSSOVERS.
THERE'S A THIRD PATHWAY OF
RECOMBINATION ACCOUNTING FOR A
MINOR FRACTION, 10% OF THE
PRODUCTS, WHICH OCCURS VIA
FORMATION OF INTERMEDIATE
SIMILAR TO THESE, ALTHOUGH THEY
HAVE MANY ALTERNATE STRUCTURES
AND THESE ARE, AGAIN, RESOLVED
UNDER CONTROL OF PROTEIN BUT A
SAID OF ENDONUCLEASES THAT ARE
NOT MEIOSIS SPECIFIC.
THESE ARE RESOLVED TO FORM
CROSSOVERS AND NON-CROSSOVERS.
SO THE QUESTION THAT AROSE THEN,
WELL, IF THERE ARE THESE
MULTIPLE PATHWAYS FOR
RECOMBINATION, WHAT'S REGULATING
THE BRANCH POINT, WHAT'S
DETERMINING WHICH PATHWAY WILL
BE FOLLOWED?
AND THE ANSWER TO THAT IS THIS
HELICASE, CONSERVED HELLICASE
COMPLEX THAT I'LL REFER TO AS
SDR.
WHAT I'LL TELL YOU IS NOW THE
LITTLE BIT OF EVIDENCE FOR THAT.
SO, STR IS THE HOMOLOG OF BTR, A
FAMILY, AND THESE GUYS ARE VERY
SIMILAR TO EACH OTHER BOTH IN
PRIMARY SEQUENCE AND STRUCTURE
BUT ALSO IN THE PHENOTYPES OF
MUTANTS THAT ONE SEES IN MUTANTS
LACKING THEM.
SO THEY BOTH SHOW PREMATURE
AGING, YES, YEAST GET OLD AND
AGE PREMATURELY.
THEY SHOW GENOME INSTABILITY.
AND MOST RELEVANT TO THIS TALK
INCREASED CROSSOVERS.
BOTH-- THIS IS AN ILLUSTRATION
FROM A VERY EARLY PIONEERING
PAPER SHOWING INCREASE THE
FREQUENCY OF SISTER CHROMATID
EXCHANGE IN THESE MUTANTS, EVERY
TIME YOU SEE A SWITCH FROM WHITE
OR GRAY TO BLACK IS A SISTER
CHROMATID RECOMBINATION.
OF COURSE, THESE PATIENTS ARE
ALSO HAVE PREDISPOSITION TO
CANCER.
YEAST DOESN'T GET CANCER, BUT
THE HOPE IS THAT BY
UNDERSTANDING WHAT'S GOING ON
HERE, WE CAN UNDERSTAND WHAT'S
GOING ON HERE.
OKAY.
OH, AND SO WHY IS THIS AN
ANTI-CROSSOVER, PREVENTING
CROSSOVERS?
THE HYPOTHESIS WAS THAT BTR OR
STR IS THE HELICASE THAT IS
INVOLVED IN NON-CROSSOVER
FORMATION, THAT IS IT'S TAKING
INTERMEDIATES THAT WOULD HAVE
MADE DOUBLE HOLLIDAY JUNCTIONS
AND THEN CROSSOVERS AND TAKEN
THEM APART AND TURNED THEM INTO
NON-CROSSOVERS.
THE IDEA WAS TO TEST THIS
HYPOTHESIS AND LOOK AT WHAT
HAPPENED DURING MEIOSIS IN SGS
1, TOP 3, RMI MUTANT.
THE PREDICTION WE MADE, THE
THREE DID VARIOUS ASPECTS,
PREDICTION WAS IF IT WAS TAKING
THINGS APART TO MAKE
NON-CROSSOVERS IN AN SGS1 MUTANT
SHOULD SEE A LOSS OF
NON-CROSSOVERS AND POSSIBLY
INCREASE IN THESE CROSSOVERS.
AND THAT TURNED OUT TO BE TRUE,
BUT ALSO NOT TO BE TRUE.
SO AS I ALLUDED TO EARLIER, WHEN
WE LOOKED AT -- WHEN ANAUD
LOOKED AT DNA LEVEL, IN
PARTICULAR UNDER CIRCUMSTANCES
WHERE HE BLOCKED FURTHER
PROGRESSION THROUGH THIS PROCESS
BY HAVING A -- BY CELLS THAT
FAILED TO EXPRESS CDC5 AND
TURNED ON CDC5, IN THE ABSENCE
OF CDC 5 HE SAW DOUBLE HOLLIDAY
JUNCTION INTERMEDIATES
ACCUMULATED, CROSSOVERS PRESENT
AT MUCH LOWER LEVEL, GRAPHS ARE
SHOWN HERE.
WHEN HE TURNED ON CDC5, HE SAW
RESOLUTION OF THE DOUBLE
HOLLIDAY JUNCTIONS, AND INCREASE
IN CROSSOVERS AS ONE WOULD
EXPECT IF DOUBLE HOLLIDAY
JUNCTIONS ARE RESOLVED TO FORM
CROSSOVERS BUT NO NET INCREASE
IN CROSSOVERS, AGAIN IN WILDTYPE
CELLS.
CONSISTENT WITH THE PICTURE THAT
THORSTON MADE THAT INDICATED
NON-CROSSOVERS WERE NOT FORMED
BY DOUBLE HOLLIDAY JUNCTION
INTERMEDIATE RESOLUTION.
HOWEVER, WHEN -- OH, I SHOULD
SAY THAT GENETICALLY IT WAS
SHOWN THAT THIS WAS THE MEIOTIC
RESOLVEASE THAT WAS DOING THIS.
HOWEVER, WHEN ARNAUD LOOKED AT
SGS1 MUTANTS HE FOUND A
DIFFERENT PICTURE.
IN THE ABSENCE OF CDC5
EXPRESSION WE DIDN'T GET
CROSSOVERS.
SAME AS IN WILDTYPE.
BUT WE ALSO DIDN'T GET
NON-CROSSOVERS.
AND WHEN YOU INDUCE CDC5 AND
INDUCED RESOLUTION EVER JOINT
MOLECULES, DOUBLE HOLLIDAY
JUNCTIONS, WE GOT BOTH
CROSSOVERS AND NON-CROSSOVERS.
SO, INSTEAD OF WIPING OUT
NON-CROSSOVER FORMATION
SPECIFICALLY, BOTH MEIOSIS --
OH, BOTH MEIOSIS-SPECIFIC
CROSSOVER FORMATION BUT ALSO
MEIOSIS-SPECIFIC NON-CROSSOVER
FORMATION HAD BEEN ABROGATED.
I SHOULD SAY THIS RESOLUTION WAS
NO LONGER DEPENDENT ON THE
MEIOTIC RESOLVEASE BUT WAS
DEPENDENT ON MITOTIC
RESOLVEASES.
THE LOSS OF SGS IS1 AND ALSO
TOP3 AND RMI1 RESULTED NOT ONLY
IN DEPLETION OF NON-CROSSOVER
PATHWAY BUT MEIOSIS-SPECIFIC
PATHWAY, A POPULATION OF THE
MINOR PATHWAY NOW ACCOUNTS FOR
ALL EVENTS WHEREBY JOINT
MOLECULES ARE FORMED AND THEY
ARE RESOLVED BY MITOTIC
RESOLVEASES.
SO THIS RAISED THE QUANDARY
BECAUSE AS I SAID WE THOUGHT OF
STR AS ENZYME THAT DISASSEMBLED
JOINT MOLECULES BUT DATA SAID IT
WAS MORE THAN FOR MAKING
NON-CROSSOVERS, THAT'S OKAY, BUT
ALSO IMPORTANT FOR THE MEIOTIC
CROSSOVER PATHWAY WHICH IS
BASICALLY ALL ABOUT PROTECTING
AND PRESERVING AND MAINTAINING
JOINT MOLECULES.
SO HOW COULD SOMETHING THAT
TAKES APART JOINT MOLECULES BE
INVOLVED IN A PATHWAY THAT'S
ABOUT PRESERVING THEM?
AND THE HYPOTHESIS THAT WE CAME
UP WITH WAS THAT IN FACT STR
WAS A CHAPERONE, YOU THINK ABOUT
PROMOTING A PROCESS LIKE FOLDING
PROTEIN BUT MOST CHAPERONES ARE
NOT THINGS THAT DRIVE FOLDING,
THEY ARE THINGS THAT UNFOLD
THINGS OR THAT TAKE THINGS
APART.
AND BY TAKING THINGS APART, WHAT
THEY DO IS THEY TAKE THINGS THAT
HAVE STARTED GOING THE WRONG
WAY, OKAY, AND RETURNED THEM TO
THE GROUND STATE, SO THEY CAN
TRY AGAIN.
AND THEY KEEP DOING THIS AND THE
FINAL STATE THAT THE PROPERLY
FOLDED STATE IS ONE A CHAPERONE
NO LONGER RECOGNIZED.
WE ARGUED, OKAY, THIS IS WHAT'S
HAPPENING HERE.
THESE INTERMEDIATES ARE
VULNERABLE TO STR AND GET TAKEN
APART AND THEY CAN BE RETURNED
BACK TO THE GROUND STATE, TO THE
DOUBLE STRAND BREAK STATE.
OKAY.
AND THIS WILL GO THROUGH
MULTIPLE CYCLES OF -- WE
HYPOTHESIZE MULTIPLE CYCLES OF
INVASION, DISASSEMBLY, NULL
UNTIL EITHER NON-CROSSOVERS
WERE -- YOU FORMED A
NON-CROSSOVER WHICH CAN'T BE
RECOGNIZED BY STR OR THESE
INTERMEDIATES WERE CAPTURED BY
THE ZMM PROTEINS OF THE
MEIOSIS-SPECIFIC STRUCTURE, THEN
ALLOWED TO MATURE, THEY WERE
ALSO PROTECTED.
OKAY.
SO ONE OF THE PREDICTIONS WAS
THAT IF WE WENT THROUGH MULTIPLE
CYCLES OF INVASION AND
DISASSEMBLY WE OUGHT TO SEE IT
AS TEMPLATE SWITCHING OR MOSAIC
HETERODUPLEX WITHIN THE PRODUCTS
OF RECOMBINATION.
BASICALLY I'VE DRAWN A
HYPOTHETICAL CASE WHERE THE BLUE
CHROMOSOME INVADES THE RED,
COPIES A LITTLE BIT OF RED, GETS
TAKEN APART, NOW IT HAS A LITTLE
BIT OF RED AGAIN.
HERE IT CAN NOW INVADE A BLUE
CHROMOSOME, COPY SOME BLUE, TAKE
APART AGAIN, ET CETERA, ET
CETERA.
AND IN THE END, WHAT ONE OBTAINS
ARE PRODUCTS OF RECOMBINATION
WHERE THERE ARE STRETCHES,
PATCHES OF RED DNA THAT ARE
INTERSPERSED WITH FULLY BLUE
DNA.
THE SAME IS TRUE OF CROSSOVERS.
AND SO THE PREDICTION WAS THAT
IF WE WENT THROUGH, IF THESE
MULTIPLE CYCLES OF INVASION AND
DISASSEMBLY WERE TRUE WE SHOULD
SEE EVIDENCE IN PRODUCTS OF
TEMPLATE SWITCHING.
A POSTDOC IN THE LAB SET OUT TO
DO THIS.
AND IN DOING SO, HE WENT BACK TO
THE VERY BEGINNING, TO TETRAD
DISSECTION AND ANALYSIS OF THE
DNA CONTENT OF THE SPORES THAT
ARE PRODUCTS OF MEIOSIS, SET UP
A MODEL, A RECOMBINATION --
LOCUS TO STUDY RECOMBINATION,
ABLE TO INSERT MULTIPLE
POLYMORPHISMS AT REGULARLY
SPACED INTERVALS, WHICH HAD A
SINGLE DOUBLE-STRAND BREAK IN IT
SO WE KNEW WHERE RECOMBINATION
INITIATED AND WE COULD THEN
FOLLOW AMONG THE PRODUCTS OF
RECOMBINATION WHETHER IT HAD A
STRAND FROM THIS PARENT OR
STRAND FROM THAT PARENT.
SIMPLY BY SEQUENCING DNA
EXTRACTED FROM COLONIES FROM
THESE SPORES.
SO THIS IS ACTUALLY A MASSIVE
UNDERTAKING.
IT INVOLVED DOING PACK BIOSIS
SEQUENCING, MULTIPLE MILLIONS OF
READS, AND THEN SORTING OUT
THOSE READS AND REASSEMBLING
THEM INTO THIS.
HE WOULD GET AN E-MAIL FROM THE
BIOWULF FOLKS BECAUSE THEY
COULDN'T FIGURE OUT WHAT COULD
BE MAKING SUCH HEAVY USE OF THE
CORES HE HAD REQUESTED.
BUT NEVERTHELESS, HE DID DO IT.
AND THE ANSWER WAS VERY CLEAR.
THAT BOTH THE NON-CROSSOVER AND
THE CROSSOVER PRODUCTS OF
MEIOTIC RECOMBINATION SHOWED
EVIDENCE, FREQUENT EVIDENCE --
EVIDENCE OF FREQUENT TEMPLATE
SWITCHING WHERE, YOU KNOW,
THINGS WENT FROM RED TO BLUE TO
RED TO BLUE STRAND, ET CETERA,
ET CETERA.
SO, THE ANSWER TO THE QUESTION
IS, YES, MEIOTIC RECOMBINATION
INVOLVES MULTIPLE CYCLES OF
ASSEMBLY AND DISASSEMBLY.
SO, THAT'S BASICALLY THE STORY I
WANTED TO TELL YOU TODAY.
JUST TO SUMMARIZE THEN, THAT
MEIOTIC RECOMBINATION AND BY
INFERENCE WE THINK REPAIR OF
DOUBLE-STRAND BREAKS IN MITOTIC
CELLS IS A REMARKABLY DYNAMIC
PROCESS, IT INVOLVES MULTIPLE
CYCLES OF INVASION AND
DISASSOCIATION, IT INVOLVES
FREQUENT TEMPLATE SWITCHING,
SWITCHING FROM ONE REPAIR
TEMPLATE TO THE OTHER.
AND THAT IS DRIVEN BY HELICASES.
WHAT I HAVEN'T TOLD YOU TODAY IS
THAT SIMILARLY, THE OTHER STEPS,
SECOND STEP OF RECOMBINATION,
FORMATION OF DOUBLE HOLLIDAY
JUNCTIONS IS SIMILARLY DYNAMIC,
THAT HE'S DOUBLE HOLLIDAY
JUNCTIONS FREQUENTLY GO MOVEMENT
ACROSS THE CHROMOSOME, THAT
BRANCH MIGRATION ADDS ANOTHER
LAYER OF DYNAMISM, IF THEY
BRANCH MIGRATE IN ONE DIRECTION
WILL BECOME DISASSEMBLED AND
RETURNED BACK TO THIS STATE.
SO, AGAIN, THERE'S POTENTIAL
REVERSAL OF BOTH THE EARLIER AND
THE LATER STEPS OF MEIOTIC
RECOMBINATION.
SO, WHAT'S THE POINT HERE?
WHY GO DO ALL THIS STUFF IF YOU
JUST ARE GOING TO BACK OUT
AGAIN?
AND I THINK THERE ARE TWO -- AT
LEAST TWO POTENTIAL REASONS FOR
THIS.
THE FIRST IS -- INVOLVES THE
FACT THAT HOMOLOG PAIRING, THE
PAIRING OF HOMOLOGOUS
CHROMOSOMES DURING MEIOSIS,
INVOLVES THE INITIATION AND
INVASION AT MULTIPLE SITES ALONG
THESE CHROMOSOMES, MULTIPLE
TRANSIENT EVENTS ARE USED TO
DRIVE THE PAIRING OF HOMOLOGOUS
CHROMOSOMES.
IF ONE EVENT HAPPENS IN A
REPEATED SEQUENCE, WHICH HAS
POTENTIAL TO RECOMBINE WITH A
REPEATED SEQUENCE ON THE WRONG
CHROMOSOME, YOU CAN -- YOU END
UP WITH ECTOPIC INTERACTION, AND
THAT WILL INTERFERE WITH PAIRING
AND INTERFERE WITH SUBSEQUENT
CHROMOSOME SEGREGATION, SO THE
ABILITY TO REVERSE INTERACTIONS
IS -- ALLOWS ONE TO THEN GO
ALONG THE PROPER PATHWAY OF
HOMOLOG PAIRING.
THE SECOND IS THAT HOMOLOG
PAIRING ITSELF BECAUSE IT'S
INVOLVING MULTIPLE INTERACTIONS
AT DIFFERENT PLACES, HAS THE
POTENTIAL FOR MAKING
ENTANGLEMENTS WHERE IN THIS CASE
TWO HOMOLOGS PAIRED HERE AND
HERE, HERE AND HERE, BUT THEY
ARE INTERDIGITATED AMONG EACH
OTHER, AND, AGAIN, THIS IS GOING
TO BE LETHAL FOR SUBSEQUENT
CHROMOSOME SEGREGATION AND SO
ONE WANTS TO BE ABLE TO BACK OUT
PERHAPS AT THIS LATER STAGE IN
ORDER TO, AGAIN, PAIR HOMOLOGS
PROPERLY.
AND THE LAST THING I WANT TO DO
IN THE MINUS 12 SECONDS THAT I
HAVE IS TO POINT OUT TO YOU
AGAIN AS I SAID EARLIER THAT
WHAT WE LEARN IN MEIOTIC -- IN
STUDY OF MEIOSIS IS ALMOST
CERTAINLY GOING TO BE APPLICABLE
IN THE STUDY OF DNA REPAIR UNDER
OTHER CIRCUMSTANCES.
IN FACT, REALLY WHAT MY ON
THETIC RECOMBINATION IS IS
MITOTIC RECOMBINATION BECAUSE A
BUNCH OF CHROMOSOME STRUCTURE.
THE PICTURE THAT WE'VE COME
ABOUT FOR MEIOTIC RECOMBINATION
IF ONE JUST TAKES OUT THE
MEIOTIC CHROMOSOME STRUCTURES
THERE'S A PRETTY ACCURATE
PICTURE OF WHAT'S HAPPENING IN
THE MY TO THE INCORPORATE
CELL -- MITOTIC CELL.
I'D LIKE TO THANK THE PEOPLE WHO
WORKED WITH ME OVER THE YEARS.
AND I WOULD BE REMISS IF I
DIDN'T END THIS TALK WITH THE
WAY WE END OUR PAPERS WITH AN
ACKNOWLEDGMENT TO THE INTRAMURAL
RESEARCH PROGRAM, CENTER FOR
CANCER RESEARCH, NATIONAL CANCER
INSTITUTE, IN THIS CASE IN
PARTICULAR TO THE FOLKS AT CCR
IN GENOMICS CORE AND SEQUENCING
FACILITY THAT MADE THIS POSSIBLE
AND THE FOLKS AT BIOWULF WHO
HELPED US CARRY OUT THE
ANALYSIS.
[APPLAUSE]
YEAH, I KNOW.
>> I'M VERY BAD AS A TIME
KEEPER.
>> IT WAS A MINUTE AND 12
SECONDS OVER.
>> THANK YOU FOR A WONDERFUL
TALK.
SPEAKING OF CONTINUITY, THEMES
AND SO FORTH, A LITTLE BIT MORE
ON DOUBLE-STRAND BREAKS AND YOUR
MEIOSIS TO MITOSIS I THINK WAS A
PERFECT SEGUE INTO THE NEXT
SPEAKER, DR. ANDRE NUSSENZWEIG
IS NIH DISTINGUISHED
INVESTIGATOR ALSO AT NCI CCR,
AND HE WORKS ON HOW CELLS REPAIR
DNA, PARTICULARLY DOUBLE-STRAND
BREAKS, BUT IN THE CONTEXT OF
ANOMALOUS REPAIR, IF YOU WILL,
BY TRANSLOCATION AND INSERTION
OF PIECES OF DNA FROM ONE
CHROMOSOME INTO THE NEXT, AND
THE ROLE OF THOSE -- THAT
PROCESS AND ITS REGULATION IN
CANCER BIOLOGY.
SO DR. NUSSENZWEIG.
[APPLAUSE]
>> THANK YOU, NINA.
I WILL TALK MORE ABOUT DNA
BREAKS BUT WANTED TO EXPAND A
LITTLE BIT ABOUT MYSELF BECAUSE
I HAVE A GOTTEN HERE IN A
ROUNDABOUT WAY.
MY TRAINING WAS ORIGINALLY IN
PHYSICS.
LET ME SEE IF I CAN GET THIS
POINTER TO WORK.
NOT SURE HOW.
WELL, SOMEONE CAN -- OH, THERE.
GOOD, GOOD.
MY TRAINING WAS IN PHYSICS.
I GOT A Ph.D. IN PHYSICS, THE
SAME DEPARTMENT AS MICHAEL
LICHTEN'S DADA PROFESSOR THERE
AT THE TIME.
I WENT ON AND DID A POSTDOC IN
PHYSICS AS WELL.
AND THEN, I STARTED TO DABBLE
INTO BIOLOGY, IN THE DEPARTMENT
OF MEDICAL PHYSICAL IT AT
SLOAN-KETTERING.
AL SINGER TOOK A RISK AND HIRED
ME IN EXPERIMENTAL IMMUNOLOGY
BRANCH.
AND THAT'S WHERE I LEARNED A LOT
ABOUT IMMUNOLOGY AND ALSO
STARTED WORKING ON DNA REPAIR.
AND I CONTINUED TO WORK ON DNA
REPAIR, AND I GUESS NCI LIKED
THE TOPIC OF DNA REPAIR, AND
THEY CREATED A BRANCH CALLED
LABORATORY OF GENOME INTEGRITY,
WHERE I CONTINUED TO WORK ON
THAT, BUT ALSO NOW FLIRTING WITH
NEUROBIOLOGY.
SO YOU MAY THINK THIS SOUNDS
LIKE THIS GUY IS A DILITANT,
DABBLING IN DIFFERENT AREAS, BUT
I WANT TO DEFEND MYSELF BY
SAYING THE STUDY OF DNA REPAIR
ALLOWS YOU OR GIVES YOU MAJOR
INSIGHT INTO DIFFERENT AREAS,
I'VE BENEFITED AND LEARNED FROM
THIS STUDY.
BECAUSE DNA DAMAGE OCCURS IN ALL
CELL TYPES, OCCURS IN MANY
DIFFERENT WAYS AS MICHAEL
LICHTEN POINTED OUT THROUGH
EXOGENOUS AGENTS LIKE CHEMICALS
IN THERAPEUTICS, AND WE'RE
INTERESTING IN ENDOGENOUS
SOURCES OF DAMAGE, REOXYGEN
SPECIES THAT MIGHT OCCUR IN
NORMAL METABOLISM BY ENZYMES,
NUCLEASES THAT PURPOSELY CUT THE
DNA AS MICHAEL MENTIONED, RAG
AID, SPO11 AND THIS DEMETHase,
TET ENZYMES.
SO IF YOU STUDY THIS YOU CAN
LEARN ABOUT IMMUNOLOGY BECAUSE
THIS IS IMPORTANT FOR DIVERSITY
OF THE IMMUNE SYSTEM, RAG AND
AID, THIS IS IMPORTANT FOR THE
DIVERSITY OF THE SPECIES, SPO 11
AND TET ENZYMES ARE IMPORTANT
FOR CELL IDENTITY.
OKAY.
AS MICHAEL POINTED OUT, THESE
ENDOGENOUS ENZYMES, NUCLEASES
ARE REALLY GOOD, REPAIR IS
REALLY GOOD BUT OCCASIONALLY YOU
HAVE MISTAKES AND THESE MISTAKES
HAVE CONSEQUENCES.
YOU GET MUTATIONS, CHROMOSOMAL
INSTABILITY, EPIGENETIC
ALTERATIONS.
AND THIS CAN LEAD TO TISSUE
DECLINE, AGING, AND OF COURSE
CANCER.
SO, YOU KNOW, BEING AT THE
CANCER INSTITUTE, THE FOCUS OF
MY LAB ANYWAYS IS ON BASIC
MECHANISMS OF CANCER, AND IT WAS
VERY INTERESTING THIS YEAR WHEN
THE NCI AND THE CANCER U.K.
ANNOUNCED THE CANCER GRAND
CHALLENGES, SO THESE WERE WHAT
THEY CONSIDERED TO BE NINE OF
THE TOUGHEST PROBLEMS, MOST
DIFFICULT PROBLEMS IN CANCER
RESEARCH, AND THESE NINE
PROBLEMS WERE BASICALLY A
CHALLENGE SO THAT YOU GET PEOPLE
FROM DIFFERENT AREAS TO
COLLABORATE, INTERACT, TRY TO
SOLVE THESE REALLY, REALLY TOUGH
PROBLEMS.
SO, WE STARTED WORKING ON THIS
PROBLEM, CHEMOTHERAPY INDUCED
NEUROTOXICITY.
WE DIDN'T JUST DECIDE TO WORK ON
IT.
LIKE EVERYTHING ELSE, YOU JUST
RUN INTO IT.
AND BUT WE BECAME VERY
INTERESTED IN THIS.
THIS IS -- YOU KNOW, REALLY A
CHALLENGING PROBLEM BECAUSE
CHEMOTHERAPY CONTINUES TO EXTEND
LIFESPAN OF PATIENTS, BUT THERE
IS FREQUENTLY PERSISTENT
NEUROLOGICAL SYSTEMS, SYMPTOMS,
AND COGNITIVE IMPAIRMENT WHICH
MAKES THE LIFE VERY, VERY
DIFFICULT.
AND THE CHALLENGE REALLY IS
BECAUSE THIS IS A SUPER
COMPLICATED PROBLEM.
THERE ARE MULTIPLE FACTORS THAT
CONTRIBUTE TO THIS COGNITIVE
IMPAIRMENT AFTER CHEMOTHERAPY,
INCLUDING AGE OF THE PATIENT,
GENDER, CANCER TYPE, DOSE,
CANCER REGIMEN, ET CETERA, ET
CETERA.
AND JUST TO GIVE AN EXAMPLE HOW
DIFFERENT CANCER REGIMENS CAUSE
NEUROLOGICAL CHANGES, WITH
TOXICITIES DEPENDING ON WHAT
COMPOUND, FROM HEADACHE TO
MENTAL STATE, MEMORY, COGNITIVE
ABILITY, EVEN SEIZURE ONE
PARTICULAR TYPE OF CHEMOTHERAPY,
ANTI-METABOLITES, ARE SHOWN HERE
IN RED, WE BECAME INTERESTED IN
THEM, AND THEY INTERRUPT NUCLEIC
ACID SYNTHESIS.
IN PARTICULAR, WE WERE
INTERESTED IN STUDYING THESE SO
CALLED SUICIDE NUCLEOSIDES WHICH
TERMINATE DNA SYNTHESIS AND
THESE APPARENTLY ARE THE LARGEST
CLASS OF ANTI-NEOPLASTIC AGENTS,
20% OF CANCER THERAPIES USE
THESE SUICIDE ANTI-METABOLITES.
LET ME GO BACK.
THESE METABOLITES, WHAT THEY
BASICALLY ARE -- THEY MAINTAIN
NUCLEOBASE, PRETTY MUCH, BUT
THEY HAVE A SMALL CHANGE IN THE
SUGAR MOIETY, AND A RESULT OF
THIS CHANGE IS THAT THERE'S --
WHEN YOU INCORPORATE THESE
ANTI-METABOLITES, THEY STOP
SYNTHESIS.
THIS KILLS RAPIDLY DIVIDING
CANCER CELLS AND ARREST DURING
REPLICATION OR THIS CAN GENERATE
DNA BREAKS AND MITOTIC CELL
DEATH.
WHAT ABOUT POST MITOTIC CELLS,
NEURONS?
HOW WOULD A SUICIDE
ANTI-METABOLITE IN NON-DIVIDING
CELLS HAVE THIS BAD EFFECT IN
POST MITOTIC NEURONS?
AND THEY DO.
THEY DEFINITELY DO.
AND THIS IS JUST ONE CLASSIC
EXAMPLE OF AN AGENT, THE
MAINSTAY WHICH HAS BEEN THE
MAINSTAY FOR 30 YEARS FOR AML
TREATMENT, AND KNOWN AT LEAST AT
HIGH DOSES INDUCES MAJOR
CEREBELLAR TOXICITY.
AND THERE'S ONE PARTICULAR CELL
TYPE ACTUALLY THAT IS PRIMARILY
AFFECTED IN TH CEREBELLUM, THAT
IS PURKINJE CELL FOR BASIC
MOVEMENT.
WE STARTED LOOKING AT
CYTARABINE, TREATED IPCs WITH
CYTARIBINE, IT IS VERY TOXICS.
WE LOOKED AT POST-MITOTIC
NEURONS DERIVED FROM THE SAME
iPSCs, TREATED WITH
CYTARIBINE, WE FOUND THAT THIS
WAS ALSO VERY TOXIC IN THE
NEURONS, NON-DIVIDING CELLS.
AND SO, YOU KNOW, THE CHALLENGE,
THE GRAND CHALLENGE HERE IS TO
UNDERSTAND AND PREVENT
CHEMOTHERAPY INDUCED
NEUROTOXICITY AND NEUROPATHY.
AND SO I'M GOING TO TELL YOU
TODAY ABOUT RECENT STUDY WHERE
WE'RE LOOKING AT THIS
CYTARIBINE, HOW IT TRIGGERS CELL
DEATH IN POST MITOTIC NEURONS.
WE STARTED WITH CRISPR
INTERFERENCE SCREEN IN NEURONS,
WE KNOW CYTARIBINE IS TOXIC,
WANTED TO SEE WHICH GENES IF WE
KNOCKED THEM OUT WOULD DECREASE
TOXICITY.
WE PERFORMED CRISPR INTERFERENCE
SCREEN IN COLLABORATION WITH
MICHAEL WARD AT NINDS ACTUALLY.
SO HE HAS THE iPSCs WITH
CRISPR INTERFERENCE MACHINERY WE
CAN INACTIVATE TRANSCRIPTION OF
VARIOUS GENES USING THE Cas9
FUSED TO KRAB DOMAIN, INFECT
WITH LIBRARY OF GUIDES AND
DIFFERENTIATE OUR iPSCs INTO
NEURONS, SIMPLY WITH SINGLE
TRANSCRIPTION WITH INDUCIBLE
TRANSCRIPTION FACTOR, WHEN YOU
GET NEURONS THAT HAVE ALL OF
THESE GUIDES THAT ARE INHIBITING
INDIVIDUAL TRANSCRIPTION, WE
TREAT OR NOT WITH CYTARIBINE AND
LOOK FOR GUIDES THAT PREVENT
CELL DEATH IN THESE NEURONS.
AND WE FOUND A LOT OF
INTERESTING HITS, THE CURVE IS
NOT SHOWING BUT THE TOP HITS ARE
SHOWING.
SOMEHOW THAT'S THE MAC-TO-PC
THING.
WE DIDN'T EXPECT IS THIS,
FACTORS INVOLVED IN METHYLATION,
THIS DNA METHYLASE WHICH WORKS
WITH THIS PARTNER, AND THIS IS
INVOLVES IN THE REVERSE PROCESS
OF DNA DEMETHYLATION.
AND WE ACTUALLY WENT BACK AND
CLONED INDIVIDUAL GUIDES FOR
TDG, TREATED WITH CYTARIBINE,
WHEN YOU LOSE THESE GENES YOU
GET RESISTANCE TO CYTARIBINE
PARTICULARLY WITH TDG.
HOW OR OTHER, THE ARA-C IS
BLOCKING THIS PROCESS OF
METHYLATION/DEMETHYLATION, AND
THE CHANGES OF METHYLATION OCCUR
AT REGULATORY ELEMENTS LIKE
ENHANCERS DURING
DIFFERENTIATION, AS WELL AS IN
RESPONSE TO STIMULI.
SO YOU IMAGINE THAT YOU HAVE --
THIS METHYLATION OCCURS AT CG
DINUCLEOTIDES, YOU HAVE
ENHANCER, CONVERTING TO NEURON,
THAT ENHANCER HAS TO OPEN UP,
THEN IT BECOMES DEMETHYLATED.
DMT1 IS RESPONSIBLE FOR
METHYLATION OF THE CYTOSINE AND
TET ENZYMES RESPONSIBLE TO
REVERSE THIS PROCESS, NOT JUST
GOING BACK TO UNMETHYLATED,
THERE'S A SERIES OF OXIDATION
STEPS WITH METHYL GROUP
OXIDIZED, TURNING TO
HYDROXYMETHYL CYTOSINE AND
OXIDIZED TO FORMAL FOLLOWED BY
CARBOXYL CYTOSINE, STILL NOT
ENOUGH.
THERE'S THIS ENZYME TDG THAT
REMOVES AND RECOGNIZES, EXCISES,
YOU HAVE TO REPAIR AND GET BACK
TO UNMETHYLATED CYTOSINE.
TO TEST HOW ARA-C MIGHT AFFECT
THIS PROCESS WE DEVELOPED HIGH
RESOLUTION METHODS TO DETECT
METHYLATION AND DEMETHYLATION.
SO, THIS IS WHAT I OUTLINED TO
YOU AT THE DNA LEVEL, TET
ENZYMES OXIDIZES METHYL GROUPS
TO CHANGE METHYLCYTOSINE
EVENTUALLY TO FORMAL CYTOSINE
AND CARBOXYL CYTOSINE.
THIS IS RECOGNIZED BY TDG WHICH
POPS IT OUT, EXCISES THE SPACE,
AND ENZYME AND NUCLEASE MAKES A
KNICK,
OR A MORE COMPLEX A LONGER PATCH
IS REPAIRED BY BASIC EXCISION
REPAIR NOT ONLY INVOLVING
CYTOSINE BUT A FEW MORE BASES
ARE SYNTHESIZED.
WE DEVELOPED WHO WAYS TO LOOK AT
THIS PROCESS.
ONE BY USING DIDEOXY CYTOSINE TO
BOX THIS SHORT PATCH BASE
EXCISION REPAIR AND THEN GOING
TO DETECT THIS BY THIS PROCESS
I'LL DESCRIBE, THIS THING WE
DEVELOPED.
THIS METHOD.
WE ALSO DETECTED LONG PATCH BY
INCORPORATING EDU, WE WOULD HOPE
GETS INCORPORATED SOMEWHERE
ALONG THE LINE AFTER THE
CYTOSINE IS INCORPORATED.
SO THIS IS A SEQUENCING APPROACH
THAT WE CALL
SYNTHESIS-ASSOCIATED REPAIR
SEQUENCING, WHERE WE SIMPLY TAKE
OUR POST MITOTIC NEURONS, ADD
EDU, ASK WHETHER THIS LONG PATCH
REPAIR IS HAPPENING, WE CAN
BIOATEN LATE AND SONICATE AND
PULL DOWN AND SEQUENCE AND IF
THERE ARE PEAKS, SPECIFIC SITES
IN THE GENOME, POST MITOTIC
CELLS UNDERGOING DNA REPAIR.
IF THERE ARE NO PEAKS IT'S
RANDOM OR DOESN'T HAPPEN.
I WILL GET BACK TO ARA-C.
WE DETECTED 50,000 HOT SPOTS OF
DNA REPAIR IN THE GENOME,
ABSOLUTELY NOT RANDOM BUT
ASSOCIATED WITH ENHANCERS, JUST
ADD EDU TO POST MITOTIC NEURONS
AND YOU SEE THESE SAR Seq
PEAKS CORRELATE WITH ENHANCERS.
AND THEY CO-LOCALIZE WITH THE
ENZYME, METHYL TRANSFERASE,
DIRECTLY BINDS TO THESE SITES,
NOT JUST ANY OPEN DNA, NOT
PROMOTERS OR GENE BODIES BUT
SPECIFICALLY NEURONAL ENHANCERS.
SO THIS IS A NICE METHOD,
SURPRISING RESULT BUT WE WANTED
TO KNOW EXACTLY WHERE WITHIN THE
ENHANCERS DO THESE ENDOGENOUS
DNA REPAIR INTERMEDIATES OCCUR.
AND SO WE USED A CHAIN
TERMINATOR, DIDEOXYCYTOSINE,
HAPPENING IN POST MITOTIC CELLS
AND USE THIS TO TERMINATE THE
CRANE -- THE CHAIN.
IF IT TERMINATES YOU HAVE A GAP,
WE TRY TO DETECT THE GAP USING
S1 NUCLEASE TO CUT THAT AND
WE'LL GENERATE NOW A
DOUBLE-STRAND BREAK WHICH WE
KNOW HOW TO DETECT.
SO BASICALLY PROTOCOL IS JUST TO
TAKE YOUR NEURONS, TREAT WITH
DIOXYCYTOSINE, EMBED THE CELLS
IN A PLUG AND THEN USE
RECOMBINANT, AND WITH END-Seq
ATTACHING HAIRPIN ADAPTER TO
LEFT AND RIGHT END OF THE BREAK
AND WE WOULD GET A READ ON THE
BOTTOM, CORRESPONDING TO LEFT
END, AND READS ON THE TOP
CORRESPONDING TO THE RIGHT END.
SO, HERE WE HAVE OUR SAR-Seq,
DNA REPAIR SYNTHESIS,
INCORPORATE EDU.
RESOLUTION OF ABOUT 500 BASE
PAIRS OR SO.
AND OUR END-Seq BY ITSELF
THERE'S NO DOUBLE-STRANDED
BREAKS.
TREAT WITH S1, SEE NO GAPS, BUT
WHEN WE USE OUR CHAIN TERMINATOR
TO HALT THIS PROCESS AND THEN S1
AND THEN END-Seq WE SEE PEAKS.
THESE PEAKS OVERLAP WITH
SAR-Seq, HIGH RESOLUTION,
SINGLE NUCLEOTIDE RESOLUTION,
CLUSTERS OF SINGLE-STRAND BREAKS
THAT OVERLAP WITH BLOBS OF
SAR-Seq PEAKS, WHEN WE LOOK
BACK WE SEE FIERY HIGH LEVEL OF
CG CONTENT.
CG METHYLATION, SO IT'S SMELLING
LIKE DNA DEMETHYLATION THAT WE
ARE DETECTING.
SO WE WANTED TO -- THIS IS
ENDOGENOUS SOURCE.
WE'RE NOT GOING ANYTHING TO THE
NEURONS.
AND SO WE WANTED TO KNOW IS THIS
REALLY DNA DEMETHYLATION?
WE STARTED EXAMINING THIS
GLYCOSYLASE, DEVELOPED DEGRON
SYSTEM TO ENACT RAPIDLY TDG WITH
DETAG SYSTEM AND FOUND THE
SINGLE STRAND BREAKS YOU SEE
WITHOUT DTAG WERE ELIMINATED
WHEN YOU ADD, THIS IS THE HEAT
MAP.
BOTTOM LINE IS THAT WE FOUND
WITHOUT DOING ANYTHING TO THE
NEURONS JUST ADDING EDU SITES OF
DNA SYNTHESIS ASSOCIATED WITH
ENHANCERS, NEURONAL ENHANCERS,
AND THESE ARE GENERATED BECAUSE
OF THIS ENZYME, TET, WHICH
BASICALLY CONVERTS THE
METHYLCYTOSINE TO UNMODIFIED
CYTOSINE BUT THROUGH A PROCESS
THAT INVOLVES DNA DAMAGE AND
WE'RE -- WE BASICALLY USED OUR
CHAIN TERMINATOR TO DETECT THAT
INTERMEDIATE.
OKAY?
AND SO, THIS IS IMPORTANT, IT
HAPPENS IN, WE SEE SHORT PATCH
BASE EXCISION REPAIR AND LONG
PATCH BASE EXCISION REPAIR, AND
THIS PROCESS IS IMPORTANT FOR
CELL IDENTITY BUT ALSO THIS DNA
SYNTHESIS COULD LEAD TO -- COULD
CAUSE MUTAGENESIS ACTUALLY.
AND CHRIS WALSH GROUP AND PETER
PARK PUBLISHED A PAPER PRETTY
RECENTLY IN WHICH THEY SEQUENCED
NEURONS FROM POSTMORTEM PATIENTS
AND THEY FOUND THAT HUMAN
NEURONS HAVE ACTUALLY ACCUMULATE
POINT MUTATIONS AT A RATE OF 20
MUTATIONS PER NEURON PER YEAR.
AND THEY ALSO ACCUMULATE
INSERTION-DELETIONS ABOUT THREE
PER NEURON PER YEAR.
AND THEY -- MUTATIONS WERE NOT
RANDOM AND THEY COMPARED SITES
OF MUTATIONS WITH OUR HOT SPOTS
AND FOUND THAT ENHANCER
ASSOCIATED HOT SPOTS WERE
ENRICHED FOR SOMATIC INDELS
FOUND IN THE PATIENT.
THIS MAY GO WRONG LEADING TO
INDELS.
BACK TO ARA-C, WHAT WOULD HAPPEN
IF YOU INTERRUPT THIS PROCESS OF
ACTIVE DEMETHYLATION WITH
CYTARIBINE.
IF THIS IS HAPPENING IN NEURONS
AND WE TREAT AND IT INTERRUPTS
THIS SYNTHESIS, WHAT IS THE CELL
GOING -- IS THE CELL GOING TO
DETECT IT AND RESPOND TO THAT
FORM OF INCOMPLETE DNA SYNTHESIS
OR DNA DAMAGE?
WE HAVE A VERY NICE WAY TO LOOK
AT DNA DAMAGE, THANKS TO BILL
BONNER, HERE AT NCI IN THE SAME
BUILDING 37 WHO DISCOVERED THIS
REMARKABLE HISTONE RARANT, H2AX,
WE USED HIS ANTIBODY AFTER
TREATING NEURONS WITH ARA-C, YOU
SEE THESE FOCI OF GAMMA H TO AX
INDICATING THERE'S A DNA DAMAGE
RESPONSE WHEN YOU TREAT NEURONS
WITH ARA-C.
IF YOU TREAT THE NEURONS WITH
ARA-A, ALSO VERY TOXIC TO CANCER
CELLS, THERE'S NO GAMMA-H2AX BUT
THERE'S A PROFOUND RESPONSE WITH
ARA-C.
DOES THIS DAMAGE RESPONSE
REQUIRE TET TDG? WE USED OUR
DTAG SYSTEM.
YOU GET THESE ROBUST GAM H2AX
FOCI, INACTIVATE, DOESN'T HAPPEN
BECAUSE YOU DON'T EXCISE THE
BASE, REINCORPORATE ACA-C,
DOESN'T GET THERE.
INDEED I SHOWED YOU THAT NEURONS
ARE VERY, VERY SENSITIVE TO
ARA-C, THEY DIE.
IF YOU INACTIVATE TDG THEY DON'T
DIE BECAUSE THEY DON'T
INCORPORATE THE ARA-C, THERE'S
NO CELLULAR DNA DAMAGE RESPONSE
SO CELLS DON'T DIE.
INTERESTING FACTS, MOST COMMON
CHEMOTHERAPY TO INDUCE
CEREBELLAR FUNCTION IS HIGH
DOSE, LOSS OF PURKINJE CELLS,
THEY HAVE AMONG THE HIGHEST
LEVELS OF TET PROTEINS, SO MAYBE
THIS PROCESS IS HAPPENING MORE
SPECIFICALLY IN PURKINJE CELLS
MAYBE CORRUPTED BY ARA-C.
WE STARTED TO INVESTIGATE THIS.
IN MICE.
SO WE TREAT MICE WITH ARA-C ON
DAILY BASIS FOR SEVEN DAYS, DO
BRAIN TISSUE SECTIONS, AND STAIN
FOR OUR GAMMA-H2AX.
AND THIS IS WHAT THE
CEREBELLUM -- WHAT A SECTION
LOOKS LIKE.
THESE ARE THE PURKINJE CELLS, IN
GREEN THE BIG CELLS WITH BIG
NUCLEOAND CYTOPLASM, STAINED BY
CAL BINDING, GAMMA-H2AX IS
CLEAN.
IF YOU SEAT FOR SEVEN DAYS YOU
SEE FOCI AND THEY REALLY ARE
VERY SPECIFICALLY FOUND IN THE
PURKINJE CELLS.
THAT INDICATES MAYBE TET TDG IS
JUST ACTIVE FOR SOME REASON IN
PURKINJE CELLS.
AND THIS IS AN ADULT MOUSE.
AND SO WE THEN HAVE MICE
DEFICIENT IN TDG, TREAT WITH
ARA-C OR MICE THAT ARE KIND
OF -- THESE ARE THE
TDG-DEFICIENT MICE, AND THESE
ARE THE WILDTYPE MICE.
YOU CAN SEE A LITTLE FOCI FOR
GAMMA-H2AX HERE IN THE PURKINJE
CELLS AND THEY ARE LARGELY
ABSENT IN THE TDG-DEFICIENT
CELLS THAT ARE TREATED WITH
ARA-C.
WE DID FLOW CYTOMETRY BY
ISOLATING PURKINJE CELLS AND
THEN WHEN YOU SEE WILDTYPE CELLS
TREATED WITH ARA-C YOU CAN SEE A
SHIFT, GAMMA-H2AX HAPPENING IN
THE PURKINJE CELLS.
IN THE TDG DEFICIENT WHEN YOU DO
THE SAME YOU DON'T SEE THIS
SHIFT SO THIS INCORPORATION AND
GAMMA-H2AX FORMATION IS
DEPENDENT ON TDG.
OUR HYPOTHESIS IS THAT THE ARA-C
NEUROTOXICITY IS CAUSED BY
INTERRUPTING A PROCESS THAT IS
ACTUALLY IMPORTANT FOR NEURONAL
CELL IDENTITY, NAMELY THIS
ACTIVE DNA DEMETHYLATION.
IT'S BEEN USED, CORNERSTONE OF
DRUGS FOR TREATMENT OF AML,
SPECIFICALLY WITH LOSS OF
PURKINJE CELLS, ONE SUGGESTION
IS WHAT ABOUT -- I DON'T KNOW
WHY PEOPLE DIDN'T USE THIS BUT
WHAT ABOUT USING ARA-A INSTEAD?
WE THINK THAT COULD KILL CANCER
CELLS.
BUT IT WOULD BE LESS TOXIC
BECAUSE IT WOULD AVOID
DOUBLE-STRAND BREAKS AT NEURONAL
ENHANCERS.
I DON'T KNOW WHAT THE TIMING IS
HERE.
>> YOU HAVE A COUPLE MINUTES.
>> I HAVE TWO MINUTES.
I WANT TO SHOW YOU CAN CHANGE A
CELL STATE AND ALSO INDUCE THE
SIMILAR PHENOMENON BY CHANGING
THE CELL STATE.
WE'RE GOING TO TURN
TRANSCRIPTIONAL CHANGES INTO
DOUBLE-STRANDED BREAKS.
SO HERE COMPLETELY DIFFERENT
SYSTEM.
WE TAKE BONE MARROW DERIVED
MACROPHAGES, GROW THEM, AND IN A
DISH, AND THEN WE TREAT THE
MACROPHAGES WITH LPS, WE'LL MAKE
A MAIN CHANGE IN THE
TRANSCRIPTIONAL RESPONSE.
AND YOU CAN SEE THAT IF YOU LOOK
AT FOUR DIFFERENT ENHANCERS AND
LOOK FOR MARKS OF ENHANCERS, YOU
CAN SEE LIKE H3K27 ACETYLATION,
WITHOUT LPS, YOU DON'T REALLY
SEE THAT MUCH BUT WITH LPS YOU
SEE ENHANCERS GET TURNED ON
AFTER LPS.
SO WE DID OUR LITTLE PDC OR
ARA-C SN 1 Seq, WITHOUT LPS WE
DON'T DETECT ANYTHING
WHATSOEVER.
BUT WITH LPS, WE CAN DETECT
THESE SINGLE-STRAND BREAKS
CONVERTED TO DOUBLE-STRAND
BREAKS, SO THESE ENHANCERS ARE
UNDERGOING ACTIVE DMEET LAYINGS
AND WE'RE CONVERTING THIS TO A
DOUBLE-STRAND BREAK SO WE CAN
DETECT IT.
AND WE CAN DO THIS ALSO JUST
WITH CELL BIOLOGY, WITH OUR
STAINING, WITH OUR BILL BONNER
GAMMA-H2AX, BONE MARROW DERIVED
MACROPHAGES AFTER ACA-C
TREATMENT, YOU DON'T SEE MUCH,
IF YOU HAVE LPS PLUS ARA-C YOU
GET NEW ENHANCERS UNDERGOING
ACTIVE DEMETHYLATION, TRIGGERED
A DNA DAMAGE RESPONSE, AND YOU
CAN SEE THESE SPOTS.
THESE LITTLE SPOTS ARE DEPENDENT
ON TDG BECAUSE IF YOU INACTIVATE
TDG YOU DON'T SEE THEM.
NOW WE CAN STUDY WHAT IS THE
ROLE OF ACTIVE DEMETHYLATION IN
A -- DURING TRANSCRIPTIONAL
RESPONSE.
WE STILL DON'T KNOW WHAT IT DOES
BUT WE KNOW THAT IT HAPPENS.
SO I JUST WANT TO END THERE.
AND THANK THE REALLY AMAZING
POSTDOCS THAT LED THIS PROJECT.
AND BEST HIRE I EVER MADE, ELSA
CALLEN IN MY LAB, GREAT
COLLABORATORS, AND OUR --
MICHAEL WARD WHO INTRODUCED ME
INTO THE SUBJECT OF NEURONAL
DAMAGE AND GOT ME INTERESTED IN
THIS.
I WANT TO THANK YOU.
[APPLAUSE]
>> APOLOGIES TO OUR ONLINE
VIEWERS, YOUR FIRST FIVE MINUTES
OF THE SLIDES WEREN'T SHOWING.
AND THEIR URGENT MESSAGING TOOK
CARE OF THAT AFTER FIVE MINUTES.
I COULDN'T REPLY TO ALL AT ONCE.
>> AGAIN, THANK YOU SO MUCH FOR
A WONDERFUL TALK.
I THINK ALL OF YOU ARE GETTING
THE SENSE OF THIS
INTERDISCIPLINARY NATURE OF WHAT
EVERYONE WHO HAS SPOKEN DOES AND
THE UNIQUE ENVIRONMENT OF THE
NIH THAT ALLOWS US TO REALLY
SEGUE BETWEEN FIELDS AND ORGAN
SYSTEMS.
NEXT SPEAKER IS NO EXCEPTION TO
THAT RULE.
DR. JOHN O'SHEA, SCIENTIFIC
DIRECTOR AND SENIOR INVESTIGATOR
AT NIAMS.
HE WORKS ON CD4 POSITIVE T CELLS
AND CYTOKINES THEY PRODUCE,
EXPERT IN MECHANISMS OF NOT ONLY
THE PRODUCTION OF THOSE
CYTOKINES BUT ALSO IN THE SIGNAL
TRANSDUCTION PATHWAYS THAT THEY
INITIATE AT RECEPTORS, AND HE
STUDIES THOSE PATHWAYS IN
EFFORTS TO DESIGN THOUGHTFULLY
THERAPEUTIC INTERVENTIONS WHEN
THOSE PATHWAYS GO AWRY.
SO, JOHN, THE PODIUM IS YOURS.
[APPLAUSE]
>> THANKS, NINA.
LET'S SEE.
MOUSE HERE.
I JUST RUINED MY JOKE THERE.
OKAY.
THANKS VERY MUCH FOR THIS
OPPORTUNITY.
WHAT I WANTED TO DO IN THE NEXT
FEW MINUTES IS TALK ABOUT THE
DISCOVERY OF JAKs AND JAK
INHIBITORS AND IT'S BEEN ABOUT
30-YEAR SORT OF BIRTHDAY PARTY,
LAST YEAR, FOR THE JAK/STAT
PATHWAY, AND I WANTED TO REFLECT
ON WHAT WE NEED TO DO FOR THE
NEXT 30 YEARS OR SO
I'LL TALK ABOUT THAT AND HOW
CYTOKINES MET THE GENOME, WHAT
THAT MEANS, COMMON AND RARE
DISEASES, SOME RARE DISEASES
MAYBE NOT SO RARE.
I'M PLEASED I'M IN THE NATIONAL
ACADEMY BUT WHAT IS THE REAL
HONOR IN MANY RESPECTS IS JUST
BEING HERE AT THE NIH.
THERE'S SO MANY GREAT PEOPLE AND
I JUST THINK WHEN I CAME HERE,
JUST BLEW MY MIND, ALL THE
AMAZING SCIENTISTS AROUND ME.
I THINK I INCLUDED MOST PEOPLE'S
NAMES HERE.
BUT IF I LEFT OFF YOUR NAME,
JUST SEND ME A NOTE AND NEXT
TIME I HAVE THE OPPORTUNITY TO
PUT UP THIS SLIDE I PROMISE I'LL
ADD THAT.
BUT IT DOES MAKE ME THINK ABOUT
THIS AMAZING ECOSYSTEM THAT WE
HAVE.
MICHAEL, I THINK FOR A LOT OF
TIME I WAS -- YOU KNOW, STARTING
OFF AS, YOU KNOW, I THINK YOU'RE
RESPONSIBLE FOR A LOT OF THIS
ECOSYSTEM.
NINA, I DON'T MEAN TO MAKE YOU
FEEL NERVOUS BY ANY MEANS BUT
IT'S REALLY -- YOU SAID THE SAME
THING, MICHAEL GETS CREDIT FOR
THIS?
YEAH.
JUST AN AMAZING PLACE.
AND REALLY IN MANY RESPECTS
THAT'S REALLY THE HONOR.
SO, HOW DID I GET STARTED IN
THIS BUSINESS?
I WAS A MEDICAL STUDENT, DOING
ROTATION IN INFECTIOUS DISEASE.
I SAW A PATIENT AT THE TIME THAT
HAD THE DISEASE THAT WE DON'T
CALL SO MUCH ANYMORE BUT ACUTE
RESPIRATORY DISTRESS SYNDROME
WAS NEW AT THE TIME.
THE ATTENDING TOLD ME THAT YOU
COULD TAKE THESE PATIENTS IN
SEPTIC SHOCK, GIVE THEM
ANTIBIOTICS, STERILE BLOOD
CULTURES BUT SOMETHING CAUSED
THIS INFLAMMATION, I DIDN'T KNOW
MUCH ABOUT THAT BUT HE THEN
STILL -- I CAN SEE THE POINTER,
MAYBE I'LL DO IT THIS WAY.
OKAY.
I'LL DO THAT.
WHAT HE HAD BEEN WORKING ON FOR
A VERY LONG TIME WERE THINGS
CALLED LEUKOCYTE ENDOGENOUS
PYROGENS.
I SAID CAN I JUST TAKE SOME TIME
AND WORK IN YOUR LAB?
I WORKED IN HIS LAB WHERE HE WAS
TRYING TO PURIFY WHAT ULTIMATELY
TURNED OUT TO BE INTERLEUKIN 1
AND TNF AND IL-6 AND OTHERS, HE
DIDN'T SUCCEED IN THAT BUT AS
YOU KNOW CHARLES WHO WAS HERE
AND MOVED ON AND EGAL GEARY DID
DISCOVER IL-1.
THE OTHER THING THAT HAPPENED
LATER WHEN I WAS A RESIDENT I
SAW A PATIENT WITH VASCULITIS.
THIS IS PETER GRAYSON'S IMAGE
HERE, BUT THAT'S -- WE OBVIOUSLY
DIDN'T HAVE THIS TECHNOLOGY BACK
WHEN I WAS SEEING THIS PATIENT.
BUT I THEN IMMEDIATELY WENT TO
THE LIBRARY AND THEN HAD TO READ
UP ON VASCULITIS AND I CAME
ACROSS THIS GUY HERE WHO YOU MAY
KNOW, TONY FAUCI, ALL OF US AT
THE TIME WOULD HAVE OUR WHITE
LAB COATS, SPECTRUM OF
VASCULITIS.
I CALLED TONY AND COLLEAGUES,
WHAT DO I DO WITH THIS PATIENT?
WHAT WAS REALLY NEW AT THE TIME
WAS TREATMENT OF AUTOIMMUNE
DISEASES WITH CANCER DRUGS,
CYCLOPHOSPHAMIDE, ET CETERA.
I GOT SO INTERESTED I WROTE TO
TONY, ANY CHANCE YOU COULD PICK
ME UP AND HAVE ME JOIN NIAID?
WHICH TURNED OUT TO BE THE CASE.
I CAME TO THE NIH, BUT TONY AT
THAT POINT MOVED ON TO ANOTHER
DISEASE WORKING ON HIV NOT SO
MUCH VASCULITIS.
I WAS MORE INTERESTED IN
CYTOKINE -- IN IMMUNE RECEPTORS,
AND STARTED OFF WITH COMPLEMENT
RECEPTORS BUT THEN WORKED WITH
BILL PAUL AND RICK CLAUSENER,
LARRY SAMUELSON TO WORK ON OTHER
RECEPTORS AS WELL, AND AGAIN
JUST AN AMAZING TIME AT THE NIH
TO SEE DISCOVERY AFTER STOVER.
AND SO WHEN I HAD STARTED MY OWN
LAB I STARTED THINKING ABOUT THE
ONE THING RICK TAUGHT ME ABOUT
WAS THAT TYROSINE KINASES SEEM
TO BE REALLY IMPORTANT FOR THE
PROXIMAL EVENTS IN SIGNAL
TRANSDUCTION BY LOTS OF THINGS,
FROM INSULIN TO GROWTH FACTORS,
ET CETERA.
WHAT WAS REVEALED WAS THAT
CYTOKINES ALSO INDUCED TYROSINE
PHOSPHORYLATION, BUT THE RACE
WAS ON, WHAT IS THE KINASE
THAT'S RESPONSIBLE FOR THIS.
AND SO WE DID WHAT A LOT OF
PEOPLE DID.
WE GENERATED LIBRARIES LOOKING
FOR KINASES IN MY CASE IN IMMUNE
CELLS.
AND REALLY THE KEY THING THAT
HAPPENED NEXT WAS THIS WORK BY
GEORGE STARK AND ALSO JIM
DARNELL.
JIM WAS A YELLOW BERET HERE AT
THE NIH AND HAD LOTS OF STORIES
ABOUT HIS TIME AT THE NIH.
BUT WHAT GEORGE DID WAS TO TAKE
CELLS AND MUTAGENIZE THEM AND
IDENTIFY CELLS THAT LACKED THEIR
SENSITIVITY TO INTERFERENCE AND
SANDRA PELEGRINEY REALIZED SHE
COULD COMPLEMENT CELLS WITH THIS
KINASE HERE WITH THIS AMBIGUOUS
NAME, TURNS OUT TO BE A JAK, A
KEY DISCOVERY.
JIM IS TAKING A DIVERSITY
APPROACH USING THESE ELEMENTS AS
BAIT TO DISCOVER STAT FAMILY OF
TRANSCRIPTION FACTORS.
SO, THIS IS AN AMAZING TOOL, ALL
THESE MUTAGENIZED CELLS, VERY
QUICKLY WE BEGAN TO SORT OF PUT
TOGETHER THE PATHWAYS OF
CYTOKINES, CYTOKINE RECEPTORS,
DIFFERENT JAKs, ET CETERA.
NOW TURNED OUT THAT THERE ARE 57
CYTOKINES THAT USE JAK/STAT
PATHWAY, PATHWAY IS CONSERVED
FROM INSECTS TO MAMMALS.
BUT THE KEY QUESTION REALLY WAS
WHAT DO WE KNOW ABOUT THE IN
VIVO CRITICALITY OF THIS
JAK/STAT PATHWAY.
WE MADE KNOCKOUT MICE, BERG AND
OTHERS, BUT THE FIRST JAK/STAT
KNOCKOUT CAME IN SLIGHTLY
DIFFERENT WAY, AND IT STARTS
WITH THIS COLLABORATION WITH
WARREN LEONARD.
JAK 3 IS HIGHLY EXPRESSED IN
IMMUNE CELLS, SUSPICIOUS IT
MIGHT BE INVOLVED IN SIGNALING
VIA THESE CYTOKINES HERE THAT
USE COMMON GAMMA CHAIN.
WHAT WARREN HAD SHOWN WAS THAT
MUTATIONS OF THE COMMON GAMMA
CHAIN SHOWN HERE UNDERLIE THE
DISEASE, BUBBLE BOY SYNDROME,
AND IF YOU HAD THESE MUTATIONS
YOU COULDN'T SIGNAL VIA
CYTOKINES.
SO WITH WARREN, WE SPECULATED
AND THEN PROVED THAT JAK 3
ASSOCIATES WITH COMMON GAMMA
CHAIN, IN THE SAME PAPER SAID,
WELL, WAIT A MINUTE, IF YOU HAVE
MUTATIONS OF GAMMA C MAYBE IF
YOU HAVE MUTATIONS OF JAK 3
YOU'LL GET A SIMILAR PHENOTYPE.
IN FACT GIGI NOTARANGELO ACTED
ON THAT AND LOOKED AT HIS COHORT
OF PATIENTS.
FABIO CANDOTTI, WHO CARED FOR
SOME OF THESE PATIENTS IN GIGI'S
COHORT WAS WORKING IN MY LAB AT
THAT TIME.
ANOTHER AMAZING PART ABOUT BEING
THEY NIH.
AND SO THEN WARREN COLLABORATED
WITH REBECCA BUCKLEY'S GROUP,
AND WHAT WE SHOWED WAS THAT
MUTATIONS OF JAK 3 UNDERLIE AND
FORM SCID THAT'S SIMILAR TO
X-LINK SEVERE COMBINED
IMMUNODEFICIENCY.
THIS IS THE FIRST KNOCK OUTIN
THE JAK/STAT PATHWAY, VERY
QUICKLY OTHER KNOCKOUT MICE
CONFIRMED THESE FINDINGS.
THE OTHER PART ABOUT THIS IS
THAT THE AMAZING PART ABOUT THIS
STORY WITH THE CYTOKINE
REVOLUTION WITH ALL THE CLONING
OF ALL THE MANY CYTOKINES THAT
WE HAVE IN THE GENOME PROBABLY
MORE THAN 200 OR SO THAT WAS
RAPIDLY CAPITALIZED UPON BY
MAKING A VARIETY OF MONOCLONAL
ANTIBODIES, THE NEXT LEVEL
BEYOND DRUGS LIKE METHOTREXATE
AND CYCLOPHOSPHAMIDE, ET CETERA.
WE HAD THE OPPORTUNITY TO REALLY
BEGIN TO SHUT OFF THE
INFLAMMATORY RESPONSES BY
TARGETING TNF, ET CETERA, ET
CETERA, ALL THE OTHER DIFFERENT
CYTOKINES SHOWN HERE BUT DESPITE
THAT, DESPITE THE IMPRESSIVE
RESULTS WITH BIOLOGICS, THERE
ARE PATIENTS WHO HAD INCOMPLETE
OR DIDN'T REALLY RESPOND TO
THESE DRUGS.
WHAT OTHER OPTIONS MIGHT WE
HAVE?
THE IDEA AT THE TIME WAS CAN WE
TARGET SIGNALING MOLECULES
MAINLY CAN WE TARGET JAKs?
IS WE MADE THIS BOLD CLAIM WITH
WARREN IN 1995.
WE SAID THIS WORK SUGGESTS WE
COULD TARGET JAKs, A NEW CLASS
OF IMMUNOSUPPRESSANT MOLECULES,
BUT AS YOU MIGHT IMAGINE, IT'S
ALWAYS EASY TO MAKE A PREDICTION
WHEN YOU KNOW THE ANSWER.
SO THIS IS IN 1995, BUT I WAS
FORTUNATE ENOUGH TO BE AT THIS
MEETING HERE IN LOVELY SAXTON
RIVERS, VERMONT, IN 1993.
I RAN INTO THIS GUY, PAUL
CHINGELLI, WE WORKED ON
COMPLEMENT RECEPTORS, SORT OF
COMPETITORS, HE WAS AT HARVARD,
I WAS WORKING WITH MIKE FRANK.
BUT IT WAS A FRIENDLY
COMPETITION.
BUT I SAID AT THE TIME WAIT A
MINUTE, PAUL, WHAT ARE YOU DOING
HERE AT THIS LYMPHOCYTE AND
ANTIBODIES MEETING?
I'M WORKING AT AT PFIZER, NOW,
AND PFIZER IS THINKING WE SHOULD
TARGET TYROSINE KINASE AS NEW
THERAPY FOR AUTOIMMUNE DISEASE.
HE'S RIGHT NEXT TO ART WEISS,
WHO JUST CLONED JAK 7, I THINK
HE WAS TRYING TO GET AWAY FROM
ME AND TALK TO ART.
OOPS.
I'M RUINING MY JOKES HERE.
[LAUGHTER]
IT'S OKAY.
YOU LAUGHED, THAT'S GOOD.
SO, YOU COULD ALSO SEE HERE ALL
SORTS OF OTHER NIH-ERS HERE,
MIKE LEOARDO AND ON AND ON.
WE HAD GONE INTO THE WOODS HERE,
WE HAD THIS COLLABORATION
CRADA, WHAT WOULD WE DID?
THAT'S A FORBES MAGAZINE WHETHER
THEY TALK ABOUT THE IDEA OF THIS
COLLABORATION BETWEEN PUBLIC AND
PRIVATE APPROACH WHICH, YOU
KNOW, LOTS TO SAY THERE BUT
MAYBE IN A DIFFERENT LECTURE.
IN ANY CASE, NOW THIS IS WHERE
WE ARE NOW.
THERE ARE 11 APPROVED
INDICATIONS FOR JAK INHIBITORS,
12 JAK INHIBITORS, MANY MORE ON
THE WAY, DRUGS APPROVED FOR
VARIOUS FORMS OF ARTHRITIS SHOWN
HERE.
ULCERATIVE COLITIS,
CORONAVIRUS'S DISEASE,
DERMATOLOGICAL DISEASES AND JAK
INHIBITORS USEFUL IN CYTOKINE
STORM ASSOCIATED WITH COVID-19.
WELL, IT'S INTERESTING IN THAT
REGARD THAT WITH THE CRADA WHEN
WE WERE WORKING WITH PFIZER, YOU
COULD IMAGINE THAT THERE'S SOME
RESTRICTIONS ON WE DO THESE
EXPERIMENTS AND I REMEMBER WE
SAID CAN WE USE TOFACINITIB?
I LIKED THE EXPERIMENT.
COLLABORATED WITH NCATS, HOME
BREW, WE SHOWED IN SEPTIC SHOCK
MODEL JAK INHIBITORS BLOCKED
THAT SEPTIC SHOCK PHENOTYPE,
USEFUL KNOWLEDGE DURING THE
PANDEMIC.
ELEVEN JAK INHIBITORS, NOT JUST
FOR PEOPLE BUT ALSO FOR DOGS.
THIS IS AN NIH DOG.
THIS IS THE JOKE I RUINED.
I'VE SHOWN THIS MANY TIMES.
WHEN I GO AROUND THE WORLD AND
SHOW PICTURES OF JAK INHIBITORS,
ET CETERA, ET CETERA, PEOPLE
HAVE A TENDENCY TO SHOW ME
PICTURES OF THEIR DOG SO IF
ANYBODY'S DOGS ARE ON A JAK
INHIBITORS, SEND ME A PICTURE.
I'LL PRESERVE, THESE ARE HIPAA
COMPLIANT DOGS BUT HAPPY TO SHOW
THESE PICTURES.
THIS IS WHERE WE WERE WITH
BIOLOGICS, TURN THE LIGHT ON,
TURN THE LIGHT OFF, YOU HAVE A
DISEASE ASSOCIATED WITH
CYTOKINES, BUT THEN JAK
INHIBITORS GAVE THE OPPORTUNITY
TO HAVE A RHEOSTAT, GETTING FROM
HOME DEPOT.
IT GIVES TWO OPTIONS TO BE
THINKING ABOUT HOW TO TREAT
THESE DISEASES.
BUT THE OTHER THING WE'RE
THINKING ABOUT WAS WHAT'S AHEAD
FOR THE NEXT 30 YEARS?
OBVIOUSLY WE HAVE A LOT TO DO
WITH UNDERSTANDING DISEASE
MECHANISM, MUCH BETTER, HAVE A
BETTER UNDERSTANDING OF
GENOMICS, PERSONALIZED MEDICINE,
CAPITALIZE ON GENE EXPRESSION.
THE OTHER THING THANKS TO CHRIS
GARCIA'S WORK, NOW JUST
RECENTLY, IN THE LAST YEAR OR
SO, WE FINALLY HAVE THE
STRUCTURE OF JAK SO THE FIRST
GENERATION OF JAK INHIBITORS
TARGETED THE CATALYTIC DOMAIN,
SO COMPETITIVE ANTAGONIST OF
ATP.
I REMEMBER WHEN PAUL WAS TELLING
ME ABOUT THIS, I WAS THINKING
ATP?
YOU'RE GOING TO TARGET ATP?
THAT'S NOT POSSIBLE.
IT IS POSSIBLE.
BUT IT'S ACTUALLY PROBABLY IF
YOU WANT TO GET MORE
SPECIFICITY, TARGETING GETTING
ALLOSTERIC INHIBITORS.
I WANTED TO GO BACK TO THINKING
ABOUT DISEASE MECHANISM AND THEN
PERSONALIZED MEDICINE, ET
CETERA, AND GENE EXPRESSION.
I'LL GIVE A FEW EXAMPLES OF
THAT.
SO, THIS IS THE CARTOON I SHOWED
JUST BEFORE, THINKING ABOUT WAY
BACK IN THE 20th CENTURY, THIS
IS HOW WE THOUGHT ABOUT
SIGNALING, PROMOTER CENTRIC,
THINKING ABOUT TRANSCRIPTION
FACTORS, BINDING THESE PORTIONS
OF THE GENOME BUT ONE THING THAT
WE FOUND IS THAT THE GENOME IS
ONLY 2% GENES AND 98% OF OTHER
STUFF, AND AT THE TIME WE DIDN'T
REALLY KNOW WHAT THIS STUFF WAS.
THAT GOES BEYOND PROMOTERS.
MUCH OF THE GENOME IS ACTIVE IN
CELL-SPECIFIC MANNER.
THERE'S THOUSANDS OF CELL
SPECIFIC ENHANCERS.
MUCH OF THE GENOME IS
TRANSCRIBED INCLUDING LONG
NON-CODING RNAs, AND MUCH OF
THE LINKS TO HUMAN DISEASE IS
NOT IN THE CODING PORTIONS OF
THE GENE BUT IN THE NON-CODING
PORTION OF THE GENOME, ET
CETERA.
THE OTHER PART WITH THESE ACTIVE
ENHANCERS WE HAVE BETTER
UNDERSTANDING OF THE LOOPING OF
THE GENOME, AGAIN CELL SPECIFIC
INACTIVATION DEPENDENT MATTERS,
I'LL COME TO THAT AS WELL.
ONE OF THE STRIKING FINDINGS,
THIS IS THE WORK FROM DAN
KASTNER AND MY BOSS LINDSEY
CRISWELL WE KNOW A LOT ABOUT OF
ROLE OF MHC AS A LINK IN
DISEASES, AUTOIMMUNE DISEASE,
BUT UP HERE IS STAT 4, STRIKING
TO US.
WE WORKED A LOT WITH STAT4 OVER
THE YEARS, DIDN'T EXPECT
NECESSARILY THAT IT WOULD BE
ASSOCIATED WITH RHEUMATOID
ARTHRITIS, LUPUS, ET CETERA.
THE PRESENCE OF THE STAT4 RISK
ALLELE, THIS VARIANT IS
ASSOCIATED WITH MORE SEVERE
DISEASE.
WE WERE THINKING IN TERMS OF
USING JAK INHIBITORS, MIGHT WE
TAKE THIS INTO CONSIDERATION,
PATIENTS WITH STAT4 RISK ALLELE
AND THOSE WHO DIDN'T.
I HAVE TO SAY THAT THIS WAS
BETTY DIAMOND'S IDEA.
AND WE HAD A CLINICAL TRIAL HERE
IN THE CLINICAL CENTER, VERY
SMALL TRIAL WITH 30-SOME-ODD
SUBJECTS.
AND I SAID AT THE TIME TO BETTY
THAT, YOU KNOW, WE'RE NEVER
GOING TO GET THE SENSITIVITY,
WE'RE NEVER GOING TO GET IT WITH
THAT FEW PATIENTS.
MAYBE IF WE HAD A THOUSAND
PATIENTS WE COULD SEE THAT.
THEY DIDN'T LISTEN TO ME IN
PLANNING THIS, THAT WAS REALLY
GOOD.
BECAUSE THEY DEVISED A PROTOCOL,
MARIANA INTERESTED IN THE ROLE
OF NEUTROPHILS IN LUPUS, ONE
THING THAT WAS CLEAR YOU COULD
SEE THAT THE EFFECT OF JAK
INHIBITORS AND LEVEL OF
NETosis WAS MORE PROFOUND WITH
STAT4 RISK ALLELE, BETTER
RESPONSE WITH JAK INHIBITORS.
SO GOING FURTHER ON THIS I GUESS
I WASN'T -- HAD LOOKED NOT JUST
FOR PATIENTS WITH LOSS OF
FUNCTION OF DIFFERENT STAT
MOLECULES AND LOSS OF FUNCTION
OF STATS, ET CETERA, ET CETERA,
BUT THIS DISEASE REALLY WAS
REALLY QUITE STRIKING TO ME.
AND REVEALS A COUPLE LESSONS I
THINK.
AND SO THIS IS A DISEASE CALLED
PAN SCLEROTIC MORPHEA, PATIENTS
HAVE THIS DEFORMING SKIN DISEASE
SHOWN HERE IN THIS PATIENT,
REALLY TRAGIC CHILD WHO HAS
ACTUALLY THREE AMPUTATION OF
LIMBS WITH INFLAMMATION IN THE
SKIN, REALLY AS YOU MIGHT
IMAGINE WAS REFRACTORY TO
VARIOUS THERAPIES.
AND WHAT HAD TURNED OUT, THIS
WAS A COLLABORATION OF RODERIC
AND KASTNER AND TOUROC,
UNIVERSITY OF PITTSBURGH, WHAT
THEY HAD WAS VARIANTS IN THE SH
2 DOMAIN, THE STRUCTURE OF
STATS, DNA COMING OUT AT YOU,
AND STAT MOLECULES BIND AROUND
THE DNA, IT'S THIS SH 2 DOMAIN,
THIS INTERACTION IS THE THING
THAT PROVIDES THE HINGE OVER
THESE STAT MOLECULES SHOWN HERE
AND THESE PATIENTS WHO HAVE GAIN
OF FUNCTION ALLELES HAVE
EXAGGERATED STAT ACTIVITY WHICH
I'LL SHOW IN A MOMENT AND SO
THAT THIS DISEASE HAS
EXAGGERATED RESPONSES TO
CYTOKINES THAT USE STAT4.
WE FOUND THAT JAK INHIBITORS ARE
EFFECTIVE IN THIS SETTING, THAT
THESE STAT4 VARIANT -- I'M GOING
TO GET INTO MORE DETAIL IN A
MOMENT BUT WE ASSOCIATED STAT4
WITH FUNCTIONING IN IMMUNE
CELLS, LYMPHOCYTES, ET CETERA,
NK CELLS.
IL-6 CAN SIGNAL THROUGH STAT4
AND ACTUALLY THEN GIVES A
FEEDBACK SO THERE'S MORE IL-6
PRODUCTION AND SO YOU HAVE
ABERRANT FUNCTION OF FIBROBLAST
AND INCREASE FIBROSIS AND
IMPAIRED WOUND HEALING.
THANKFULLY WITH THESE PATIENTS
WE NOW HAVE THE OPPORTUNITY TO
TREAT THESE PATIENTS WITH JAK
INHIBITORS.
TURNING TO ANOTHER PART OF THE
STORY, THIS IS WORK BY RACHEL
PHILLIPS IN THE LAB, STARTS WITH
CHRIS BYRON, THIS IS WHAT I WAS
REFERRING TO BEFORE WHEN WE
FIRST STUDIED JAK/STAT PATH WAY
HAD A TENDENCY TO TAKE
CYTOKINES, THIS USES STAT,
STAT1, IN THIS CASE, SHOWN HERE,
STAT1, STAT2, HOW INTERFERON
WORKS, IL-12, WORK WE DID SOME
TIME AGO WITH CHRIS BYRON IN MY
LAB, LINKED IL-12 TO STAT4.
SO WHEN WE WERE THINKING ABOUT
THE ACTION OF TYPE I
INTERFERONS, SIGNAL VIA STAT1
HOMODIMERS AND ISGF 3, BINDS A
DIFFERENT ELEMENT IN THE GENOME,
AND STAT4 ACTIVATES -- IS
ACTIVATED BY IL-12, THE PART
THAT DRIVES THIS ACTIVATION
PHENOTYPE, IN PARTICULAR INDUCES
INTERFERON GAMMA.
THAT'S THE KEY PART OF THE STORY
THAT I'LL EXPLAIN AS I GO ALONG
HERE.
SO WHAT WE FOUND AT THE TURN OF
THE CENTURY OR SO LONG BEFORE WE
REALLY HAD THE TECHNIQUES THEY
HAVE NOW IN TERMS OF SEQUENCING
AND THE LIKE, BUT WHAT CHRIS
BYRON HELPED US UNDERSTAND WAS
THAT IN LYMPHOCYTES THAT TYPE I
INTERFERONS ALSO ACTIVATE STAT4,
THAT MAKES A LOT MORE SENSE
BECAUSE LUPUS IS AN
INTERFERONOPATHY, AND THE
LINKAGE BETWEEN TYPE I
INTERFERONS AND STAT4 MAKES
SENSE WITH LINDSEY CRISWELL AND
DAN KASTNER'S WORK.
IN LYMPHOCYTES, IN THE BASAL
STATE, WHEN YOU FIRST HAVE AN
INSULT, INDUCTION OF
INTERFERENCE, TYPE I INTERFERONS
PREFERENTIALLY ACTIVATE STAT4
AND DRIVE ACTIVATION PHENOTYPE
IN INTERFERON GAM A LATER ON
STAT1 INCREASES AND THEN YOU
HAVE MORE PREDOMINANTLY THIS
ANTI-VIRAL RESPONSE.
OKAY.
SO WHAT MIGHT THAT MEAN?
SO, THERE ARE STAT1 LOSS OF
FUNCTION VARIANTS, STAT1 GAIN OF
FUNCTION VARIANTS.
AND YOU KNOW ALL THIS WORK FROM
STEVE HOLLAND AND COLLEAGUES
SHOWN HERE, I COULDN'T FIND A
PICTURE OF ALEX FREEMAN WITH HER
PARTNER OVER HERE.
BUT SOMEBODY SHOULD TAKE A
PICTURE.
CHARLIE, ARE YOU STILL HERE?
MAYBE HE'S NOT HERE.
>> WE'LL WORK ON IT.
>> OKAY.
IN ANY CASE, STEVE AND JOHN, ET
CETERA, IDENTIFIED PATIENTS WITH
STAT1 LOSS OF FUNCTION VARIANTS,
THESE PATIENTS HAVE BACTERIAL
AND VIRAL INFECTION BUT ALSO
IDENTIFIED PATIENTS WITH STAT1
GAIN OF FUNCTION VARIANT.
WHAT THAT RESULTS IN IS THAT
THEY HAVE IMPAIRED RESPONSES TO
FUNGAL INFECTIONS, CANDIDA SHOWN
HERE, BECAUSE INTERFERON GAMMA
AND EXAGGERATING INTERFERON
RESPONSES IMPAIRS THE TYPE 3
RESPONSE, IL-17 RESPONSES BUT
INDUCES LUPUS.
THE PARADOX HERE THOUGH IS THAT
PATIENTS WHO HAVE STAT1 GAIN OF
FUNCTIONS AND OTHER PATIENTS WHO
HAVE INTERFERONOPATHYS INCLUDING
CHILDREN WITH DOWN SYNDROME AND
THAT HAVE INTERFERONOPATHYS
BECAUSE THEY HAVE DUPLICATION OR
INCREASED EXPRESSION OF
INTERFERON RECEPTORS,
EXAGGERATED INTERFERON RESPONSE,
SHOULD HAVE IN PRINCIPLE
EXAGGERATED ANTI-VIRAL RESPONSE
BUT THAT'S NOT HOW IT WORKS.
THAT'S THE CASE FOR COVID-19.
SO WE WANTED TO UNDERSTAND THE
MECHANISM OF THIS.
AND SO WE MADE A MOUSE, SHOWN
HERE, WE PUT THE MUTANT ALLELE
IN SO THAT THE MICE WOULD BE
LIKE THESE PATIENTS THAT THEY
WOULD HAVE A ONE GAIN OF
FUNCTION ALLELE AND ONE NORMAL
ALLELE, AND THIS IS
COLLABORATION AGAIN WITH JOSH
MILNER AND STEVE AND LEE, WE
INFECT MICE WITH USUAL VARIANTS,
VIRUSES THAT WE USUALLY THINK
ABOUT BUT ALSO WITH SARS, I
MANAGED TO COVER THAT.
THE LEGEND HERE.
SUFFICE IT TO SAY THAT THE MICE
RECAPITULATE WHAT YOU SEE IN
PATIENTS, THAT THE MICE IN THIS
CASE THIS -- THESE MICE EXPRESS
THE MUTANT ALLELE IN ALL TISSUES
BUT WE OBVIOUSLY CAN DO THIS IN
A TISSUE-SPECIFIC MANNER AS
WELL.
AND WHAT YOU SEE IS INCREASE
SUSCEPTIBLABILITY TO VIRAL
INFECTION, THIS IS SARS-COV-2 AS
WELL.
SO WE WANTED TO UNDERSTAND THE
MECHANISM AND WHAT RACHEL FOUND,
IN THE INTEREST OF TIME I WON'T
SHOW ALL THE DATA, THAT THESE
MICE HAVE CYTOKINE STORM, AGAIN
AS WE HAVE BEEN HEARING OVER THE
PAST THREE YEARS OR SO, BUT ONE
OF THE SURPRISING THINGS IS THAT
THESE MICE DON'T MAKE INTERFERON
GAMMA.
I JUST TOLD YOU ACTUALLY THAT
THIS IS AN INTERFERONOPATHY, AND
YOU HAVE INCREASED RESPONSES TO
INTERFERONS, AND THERE'S A PAPER
THAT CAME OUT IN SCIENCE
TRANSLATIONAL MEDICINE BY
ANOTHER GROUP THAT MADE THESE
MICE AS WELL, AND THEY FOUND
THERE'S PATHOLOGY ASSOCIATED
WITH INTERFERON GAMMA.
IF THEY KNOCK OUT INTERFERON
GAMMA RECEPTOR THEY ACTUALLY
HAVE REDUCTION IN AUTOIMMUNE
DISEASE.
TURNS OUT WHAT HAPPENS IN THESE
MICE IS THAT YOU HAVE IMPAIRED
STAT4 ACTIVATION.
TYPE I INTERFERON IS
PREFERENTIALLY ACTIVATE STAT4.
IN THE MICE YOU HAVE REDUCED
STAT4 PHOSPHORYLATION.
MAY BE YOU CAN GIVE INTERFERON
GAMMA BACK?
THE PATHOLOGY IS DRIVEN BY
INTERFERON GAMMA.
I ALSO WAS THINKING IT'S GOING
TO BE REALLY HARD TO FIGURE OUT
WHAT'S THE DOSE OF INTERFERON
GAMMA, THE TIMING, ET CETERA, ET
CETERA.
BUT, AGAIN, RACHEL DIDN'T LISTEN
TO ME.
AND WHAT SHE DID WAS TO DO A
COUPLE THINGS HERE, AND WHAT SHE
DID WAS THIS IS THE NORMAL
RESPONSE TO INFECTION SHOWN
HERE.
AND HERE ARE THE MICE THAT HAVE
THE STAT1 GAIN OF FUNCTION
VARIANT.
AND THEY DID SOMETHING ELSE
CLEVER, HOW ABOUT IF WE BLOCK
INTERFERON GAMMA, DOES THAT GIVE
THE SAME PHENOTYPE?
AGAIN WE'VE DONE LOTS OF
DETAILS, LOTS OF SINGLE CELL
RNAseq, ET CETERA.
I'M GOING TO SPARE YOU THAT FOR
THE MOMENT.
AND WHAT YOU SEE WAS WHEN YOU
BLOCK INTERFERON GAMMA YOU SET
THE SAME PHENOTYPE.
IF I GIVE THE MICE INTERFERON
GAMMA EXOGENOUS YOU CAN RESOLVE
THIS CYTOKINE STORM.
SO, THIS IS PRETTY INTERESTING
IN A WAY THAT WHEN WE THINK OF
THE PATIENTS THAT WE'VE SEEN
WITH CYTOKINE STORM, IF SOME
PATIENTS, IN THE SETTING, IF
THEY -- EVEN IF THEY HAD BASAL
LEVELS OF INTERFERON GAMMA, YOU
KNOW, AT HIGHER LEVELS, THAN
NORMAL SUBJECTS, BUT THEY
DIDN'T -- WERE NOT ABLE TO SORT
OF GET THIS BOOST OF INTERFERON
GAMMA PRODUCTION YOU WOULD NEED
DURING THE INFECTIONS, THEN
ACTUALLY YOU COULD TREAT
PATIENTS WITH INTERFERON GAMMA,
THAT MIGHT BE RELATIVE FOR
PATIENTS WITH DOWN SYNDROME AND
INTERFERONOPATHYS, ET CETERA.
SIGNALING IS NOT JUST ABOUT THIS
ON/OFF SWITCH.
ONE PART, TO END QUICKLY, ONE
THING WE LEARNED IN TERMS OF
THINKING ABOUT CYTOKINES IS THE
COMPLEXITY OF CYTOKINE GENES
SHOWN HERE, SO HERE YOU HAVE
INTERFERON GAMMA, A TINY
INTERFERON GAMMA GENE, LITTERED
WITH ALL OF THESE ACTIVE
ENHANCERS, AND INCLUDES THINGS
LIKE LONG ENCODING RNAs,
INCLUDING INTERFERON GAMMA 1,
Th1 CELLS WITH ACTIVE
ENHANCERS, Th2 CELLS WHICH
DON'T, AND WHAT WE FOUND A FEW
YEARS AGO THAT IF YOU LOOKED AT
SUPERENHANCERS IN IMMUNE CELLS,
THEY OFTEN ENCOMPASS CYTOKINES,
CYTOKINE RECEPTORS, ANOTHER
STORY MORRISON PRESENTED THIS
TWO DAYS AGO BUT I'LL SPARE YOU
THAT AT THE MOMENT.
INTERFERE GAMMA AS 1 IS LINKED
TO IBD.
THIS IS ANOTHER EXAMPLE OF WHAT
I WAS SAYING BEFORE THAT THE
GENETIC LINK IS OUT HERE, NOT
ACTUALLY IN THE CODING REGION.
AND WHAT WE DID WAS WE MADE TWO
MODELS OF INTERFERON GAMMA AS 1
KNOCKOUT MICE AND SHOWED THAT
THIS GAVE REDUCED SUSCEPTIBLITY
OR INCREASED SUSCEPTIBILITY.
ONE THING, A COLLABORATION WITH
AHMED, HE FOUND IN HIS COHORT
VACCINATED WITH YELLOW FEVER,
LOOKING AT ANTIGEN-SPECIFIC
MEMORY T CELLS, HIGHER LEVELS OF
INTERFERON GAMMA AS1 EVEN WITH
NORMAL LEVELS OF INTERFERON
GAMMA.
FINALLY.
SO RETURNING TO WHAT I WAS
MENTIONING BEFORE ABOUT THE
LOOPING OF CHROMATIN, SO HERE WE
HAVE THE INTERFERON GAMMA GENE
SHOWN HERE.
HERE IS THE LONG NON-CODING RNA
THAT I MENTIONED.
THIS IS A CYTOKINE CLUSTER,
INTERFERON GAMMA IL- 22 AND IN
HUMANS IL-26.
HUMAN GENOME IS PLAID.
IS THIS PLAIDNESS IMPORTANT?
AND SO HUNG HU WHO WILL GIVE A
TALK OCTOBER 4, CAREFULLY
ASSESSED FOLDING OF CHROMATIN IN
THIS LOCUS, WHAT SHE IDENTIFIED
WAS CTCF BINDING ELEMENT, MINUS
70 REGION, USING CRISPR/Cas.
I'LL CUT TO THE CHASE.
WHAT WAS FOUND IF YOU ALTER
CHROMATIN STRUCTURE NK CELLS AND
MEMORY CD8 T CELLS, THAT'S FINE.
THEY CAN MAKE INTERFERON GAMMA.
WHAT HAPPENS IS THAT IN TH1
CELLS THAT START IN NAIVE STATE
AND POLARIZE TO Th1 CELLS
THAT'S WHERE THIS ELEMENT IS
CRITICAL.
ARCHITECTURE DOES MATTER IN THIS
SETTING.
AND SO THIS COMES BACK TO THIS
POINT THAT I THINK ONE OF THE
CHALLENGES FOR THE NEXT 30
YEARS, THAT WE HAVE BIOLOGICS,
THAT'S GREAT.
WE REALLY -- PATIENTS COME INTO
THE CLINIC, REALLY DO NOT COME
IN WHEELCHAIRS AS THEY DID WHEN
I WAS A RESIDENT, ET CETERA.
SO WE HAVE BIOLOGICS, CAN TREAT
THESE PATIENTS, AND WE ALSO HAVE
JAK INHIBITORS AS WELL.
I WAS THINKING ABOUT THIS, THIS
IS OUR BAND, FRANCIS COLLINS, ET
CETERA.
AND WHAT YOU MAY NOT THINK ABOUT
AS MUCH AS I THINK ABOUT WHEN
WE'RE PLAYING IS THAT SOMEBODY
HAS TO PUT ALL THIS STUFF
TOGETHER, YOU HAVE THESE MIXING
THAT'S GOING ON BEHIND THE
SCENE, SO THAT IT SOUNDS
BEARABLE TO YOU.
WHEN I'M OFF KEY OR PLAYING THE
WRONG SONG, ARRINGTON PRICE OR A
COLLEAGUE CAN TURN ME DOWN
QUICKLY.
I WAS THINKING THIS REMINDS ME
OF THE INTERFERON GAMMA GENE, WE
HAVE ALL THESE SWITCHES, JUST
SURROUNDING THIS REGION.
AND THE ISSUE OF COURSE IS DO WE
TAKE THAT INTO CONSIDERATION AS
WE'RE SORT OF THINKING ABOUT
TREATMENT?
YOU KNOW, WE HAVE BIOLOGICS, JAK
INHIBITORS, WHAT WE NEED TO
UNDERSTAND IS MORE SOPHISTICATED
BIOMARKERS IN PATIENTS,
CONSEQUENCE OF CYTOKINE
SIGNALING, ET CETERA.
AND REALLY UNDERSTAND AS I WAS
REFERRING TO BEFORE WITH THE
STAT 4 RISK ALLELE AND PATIENTS
HAVE VARIOUS GAIN OF FUNCTION
MUTATIONS TO UNDERSTAND THAT SO
WE CAN PICK THE RIGHT DRUGS AT
THE RIGHT DOSES.
REALLY ULTIMATELY I THINK WE
NEED TO UNDERSTAND THE
COMPLEXITY OF GENE REGULATION
THERAPEUTICALLY AND REALLY IN
MOST DISEASES EVERYTHING FROM
HYPERTENSION TO CANCER, IT'S
COMBINATORIAL THERAPY.
TYPICALLY YOU GO TO THE DOCTOR,
WHATEVER, SAY, NO, OKAY, THAT
DIDN'T WORK, LET ME GIVE YOU
THIS OTHER DRUG.
IT'S NOT RATIONALE, DOESN'T TAKE
INTO ACCOUNT ALL OF THE FACTORS
REALLY THAT REGULATE ALL THESE
KEY GENES.
I WOULD ARGUE THAT THIS IS JUST
THE BEGINNING OF OUR
UNDERSTANDING OF IMMUNE CELL
SIGNALING.
AND I THINK I MENTIONED PEOPLE'S
NAMES ALONG THE WAY.
AGAIN, JUST TREMENDOUSLY
THANKFUL JUST TO BE HERE AT THE
NIH.
SO THANKS.
[APPLAUSE]
>> DR. O'SHEA'S BAND IS PLAYING
TOMORROW AT THE CLOSE.
>> NOT ME.
>> WE GOT A SOUND PERSON.
YOU CAN ADD ME TO YOUR
ACKNOWLEDGMENT PAGE.
>> I HAVE TO MAKE A COMMENT.
I HAVE TO SAY FIRST OF ALL I'VE
ALWAYS THOUGHT IT IS INDICATIVE
OF INCREDIBLE HEALTHY EGO OF THE
SCIENTIFIC COMMUNITY THAT
BECAUSE WE DON'T UNDERSTAND 98%
OF THE GENOME WE JUST CALL IT
JUNK, CLEARLY NOT.
AND THE OTHER THING THAT
OCCURRED TO ME, IT'S ALWAYS BEEN
AMAZING TO ME, SINCE EARLY IN
MEDICAL SCHOOL, THAT THE SAME
PERSON THAT MAKES SUCH A ROBUST
IMMUNE RESPONSE THAT THEY
DESTROY THEIR GENES, THEIR
JOINTS, THEIR G.I. TRACT, THEIR
HEART, CAN'T MAKE AN IMMUNE
RESPONSE TO KILL OFF THE BUGS
THAT INFECT ALL OF US, I MEAN,
THAT IS -- THAT IN ITSELF SAYS
THIS IS A REGULATORY DISORDER,
AND NOT AN IMMUNE SINGLE
FUNCTION DISORDER.
ANYWAY, VERY INTERESTING.
I THINK YOU SEE THE THREAD IN
THIS THAT EVERY ANSWER TO A
QUESTION GENERATES 100 MORE
QUESTIONS.
AND THAT IS THE JOY OF WHAT WE
ALL DO.
SO OUR FINAL SPEAKER THIS
AFTERNOON IS DR. DEBORAH
MORRISON, SENIOR INVESTIGATOR AT
NCI CCR, AN EXPERT IN THE
FUNCTION OF THE RAS PROTEIN
KINASE PATHWAYS, AND SHE HAS
STUDIED BOTH THE NORMAL FUNCTION
OF THOSE PATHWAYS AND THE WAY
THOSE PATHWAYS AND THEIR
ABERRATIONS LEAD TO DISEASE.
AND, AGAIN, TO LOOK AT THE
BIOCHEMICAL AND STRUCTURAL
ASPECTS OF RAS AND THE PROTEINS
WITH WHICH IT INTERACTS TO TRY
AND DEVELOP NEW THERAPEUTIC
STRATEGIES.
DR. MORRISON.
[APPLAUSE]
>> IT'S GREAT TO BE HERE TODAY.
AND I WANT TO GIVE YOU -- TO
GIVE ME A CHANCE TO TELL YOU
ABOUT MY JOURNEY IN SCIENCE.
LET'S SEE.
I WANT TO GO FORWARD.
I NEED HELP.
JUST TO GET ME STARTED.
SO, ONE OF THE MAJOR FOCUSES OF
MY LAB HAS BEEN IN THE STUDY OF
SINGLE -- SIGNAL TRANSDUCTION
PATHWAY REGULATED BY RAF FAMILY
OF GTPaseS.
LET ME GO BACK.
CLEARLY I'VE NEVER USED THIS
POINTER BEFORE.
I WANT TO GO BACK.
BOTTOM?
NO, THIS IS GOING FORWARD.
>> THERE YOU GO.
>> ANYHOW, WE'RE INTERESTED IN
THE PATHWAY THAT'S REGULATED BY
THE RAF GTPase, AND THIS PLAY
AS VERY IMPORTANT ROLE IN
TRANSMISSION OF MANY CELLULAR
SIGNALS AND OFTEN SIGNAL IS
INITIATED BY EITHER GROWTH
FACTOR OR LIGAND BINDING TO
RECEPTOR TYROSINE KINASE ON THE
CELL SURFACE THAT LEADS GTP
LOADING, A CRITICAL FACTOR
COMPONENTS OF CASCADE, AND A
PROTEIN KINASE, THE RAF KINASES.
I AGAIN WORKING ON RAS AS A
FIRST-YEAR POSTDOC IN IT TOM
ROBERTS' LAB AT DANA-FARBER
CANCER INSTITUTE, 30 YEARS LATER
I'M STILL WORKING ON IT.
BE CAREFUL WHAT YOU CHOOSE AS A
POSTDOC TO STUDY.
SO, LET'S SEE.
IN ADDITION TO PLAYING AN
IMPORTANT ROLE IN NORMAL GROWTH
AND DEVELOPMENT, WE ALSO KNOW
THIS PATHWAY PLAYS A VERY
IMPORTANT ROLE IN HUMAN DISEASE
STATES AS WELL.
AND SOMATIC MUTATIONS IN VARIOUS
COMPONENTS OF THE PATHWAY ARE
KNOWN TO BE DRIVERS IN CERTAIN
CANCERS.
IN ADDITION, GERMLINE MUTATIONS
AND COMPONENTS OF THE PATHWAY
CAN ALSO BE CAUSATIVE FOR A
GROUP OF DEVELOPMENTAL
DISORDERS, SHOWN HERE, KNOWN
COLLECTIVELY AS RASOPATHYS.
BECAUSE OF THAT, FOR THE PAST 25
YEARS EFFORTS TO TARGET
COMPONENT PATHWAYS FOR
THERAPEUTIC INTERVENTION.
BUT I THINK ONE OF THE LESSONS
WE'VE LEARNED OVER TIME IS THAT
IT'S REALLY IMPORTANT FOR THIS
KIND OF DEVELOPMENT TO BE MOST
OFFICIAL AND TO WORK THE BEST
YOU NEED TO UNDERSTAND HOW
NORMAL SIGNALING THROUGH THE
PATHWAY TAKES PLACE.
AND IN -- WHAT HAS TURNED OUT IS
FOR THE RAF KINASES THEIR
REGULATION IS HIGHLY COMPLEX.
SO FOR THOSE WHO DON'T THINK
ABOUT THE RAFs ALL DAY EVERY
DAY, THEY ARE BASICALLY -- THEY
CONSIST OF C-TERMINAL CATALYTIC
DOMAIN, N-TERMINAL REGULATORY
DOMAIN, CONTAINS A RAF BINDING
DOMAIN.
WHICH TURNS OUT THERE ARE THREE
RAF FAMILY MEMBERS ARAF, BRAF, C
RAF, EVERY ONE CAN FUNCTION AS
EFFECTOR OF RAF, DIRECTLY BINDS
RAF, ALL CAN ALSO FUNCTION AS
INITIATING KINASE IN THE
CASCADE.
IF WE THINK ABOUT WHAT'S
DIFFERENT ABOUT THEM, WELL, IT
REALLY IS IN TERMS OF ENZYMATIC
ACTIVITY.
AND THE INTRINSIC CATALYTIC
ACTIVITY OF BRAF IS BY FAR THE
HIGHEST.
THEN C RAF, THEN ARAF.
WHAT DISTINGUISHES BRAF IS
DIFFERENTS WHERE THE ASTERISKS
ARE, DIFFERENCE IN SEQUENCES AT
THE BEGINNING OF THE KINASE
DOMAIN THAT DETERMINE ITS HIGH
LEVEL OF BASAL KINASE ACTIVITY.
SO, WORK FROM OUR LAB AND A
NUMBER OF LABS IN THE FIELD HAVE
KIND OF LED TO THIS MODEL OF HOW
THE RAFs ARE REGULATED.
BRAF AND CRAF ARE PRIMARY
DISEASE DRIVERS.
BRAF IS IMPORTANT IN CANCER,
c-RAF AND BRAF TOGETHER ARE
DRIVERS IN THE RAFOPATHYS.
THEY EXIST AS AUTOINHIBITTIVE
MONOMERS IN THE CYTOSOL AND IT'S
BOUND TO DIMER OF 14-3-3.
ONE HIGHLIGHT OF MY CAREER IN
THE LAST COUPLE YEARS HAS BEEN A
REALLY WONDERFUL COLLABORATION
WITH A YOUNG TENURE TRACK
INVESTIGATOR OF NCI, ABLE TO
SOLVE THE CryoEM STRUCTURE,
YOU CAN SEE THE 14-3-3 DIMER IS
A CRADLE, NIGH CASE ON TOP.
RBD WAS ALSO RESOLVED.
AND THE RBD REPRESENTS THE RAF
BINDING DOMAIN, A BREAKTHROUGH
WHEN WE LEARNED THEY WERE DIRECT
EFFECTORS OF RAF.
AND THEY CAN DIRECTLY INTERACT
RAS, FIRST IT RECRUITS THE RAF
KINASES TO THE PLASMA MEMBRANE,
PROMOTES THE RELEASE OF THE
AUTOINHIBITED STATE AND RESULTS
IN RAF DIMERIZATION WHICH WE
KNOW IS ESSENTIAL IN MOST
INSTANCES FOR RAF TO BECOME AN
ACTIVE KINASE.
AND WHAT I'D LIKE TO DO IN THE
REMAINING TIME IS TELL YOU A
LITTLE BIT ABOUT A RECENT
PROJECT THAT WE'VE DONE THAT I
THINK REALLY HIGHLIGHTS SOME OF
THE COLLABORATIONS THAT WE'VE
BEEN ABLE TO TAKE ADVANTAGE OF
HERE AT THE NIH.
AND ALSO, IT ALLOWED US TO
DOVETAIL IN AN EXCITING NEW
INITIATIVE, THAT'S ALSO GOING ON
AT NIH.
SO AS I SAID, BRAF IS ONE OF THE
RAF FAMILY MEMBERS THAT PLAYS A
ROLE IN HUMAN DISEASE STATE.
BOTH IN CANCER AND IN
RASOPATHYS.
WHAT'S INTERESTING IS THAT THE
CANCER ASSOCIATED BRAF MUTATIONS
HAVE BEEN FOUND TO OCCUR
PRIMARILY IN THE KINASE OR
CATALYTIC DOMAIN.
OFTEN STRONG GAIN OF FUNCTION.
AS A RESULT BECAUSE THEY ARE SO
ACTIVE, THEIR EMBRYONIC
(INDISCERNIBLE) IF YOU TRY TO
MAKE A MODEL.
RASOPATHY-ASSOCIATED MUTATIONS
TEND TO BE WEAKER GAIN OF
FUNCTION, OFTEN OBSERVED IN THE
N-TERMINAL REGULATORY DOMAIN, IN
PARTICULAR FOR RASOPATHYS, FOR
BRAF, CYSTEINE-RICH DOMAIN HAS
BEEN A UNIQUE HOT SPOT.
AND FOR THOSE MAYBE NOT SO
FAMILIAR WITH THE RASOPATHYS,
IT'S A DEVELOPMENTAL SYNDROME
AND THERE'S A GROUP OF THEM
LISTED HERE.
AND THEY ARE CAUSED BY GERMLINE
MUTATION IN MEMBERS OF THE RAS
OR RAS KINASE PATHWAY OCCUR IN 1
IN A THOUSAND TO 1 IN 2,000 LIVE
BIRTHS, FOR NOONAN 7% HAVE C RAF
MUTATION
70% IN CARDIO FASCIOCUTANEOUS.
THE RASOPATHYS CAN BE BROADLY
CHARACTERIZED BY DISTINCT
CRANIOFACIAL FEATURES,
CONGENITAL HEART DEFECTS,
PATIENTS ARE OFTEN SHORT
STATURE, THEY HAVE LEARNING
DISABILITIES, AND INCREASED RISK
OF CERTAIN CHILDHOOD CANCERS.
SO I SAID THAT FOR BRAF, AGAIN
WHICH 71% OF PATIENTS WITH CFC
HAVE BRAF MUTATION, CRD IS A
PARTICULAR HOT SPOT.
WE WANTED TO UNDERSTAND WHAT WAS
THE BASIS FOR THIS.
AND SO JUST TO LET YOU KNOW WHAT
A CYSTEINE-RICH DOMAIN IS, IT IS
A SMALL, 15 AMINO ACID DOMAIN IN
PROTEINS, COORDINATE ZINC IONS,
TWO TYPES FOUND.
TYPICAL AND ATYPICAL.
AND A CLASSICAL TYPICAL C1
DOMAIN IS CONTAINED IN PROTEIN
KINASE C, BINDS.
ATYPICAL ONES, ATYPICAL IS THAT
THERE'S A TRUNCATION IN LOOP 2,
WHICH DOESN'T ALLOW THEM TO
INTERACT, BUT THE ATYPICAL
CRDs ARE OFTEN FOUND IN
SIGNALING MOLECULES, CAN
INTERACT WITH BOTH LIPIDS AND
PROTEINS, AND ARE OFTEN INVOLVED
IN REGULATING ACTIVITY AND
LOCALIZATION.
WHAT WE KNEW ABOUT THE RFA CRD
BEFORE WE STARTED THIS PROJECT
WAS THAT THEY HAVE BEEN SHOWN
NOT TO BIND ESTER BUT INTERACT
WITH STERINE, AND RECENTLY
INTERACT CORRECTLY WITH RAS.
THESE FUNCTIONS OF CRD ARE
CRITICAL UNDER NORMAL SIGNALING
CONDITIONS.
BUT IT'S SURPRISING THAT THE CRD
PLAYS A ROLE IN RAF
AUTOINHIBITION AS WELL.
CRD IS IN MAGENTA.
IT FITS THERE AT THE CENTER OF
THE AUTOINHIBITED COMPLEX, MAKES
CONTEXT WITH KINASE DOMAIN AND
BOTH PROTOMERS OF THE DIMER
WHAT IS THE RELATIVE IMPORTANCE
AND HOW DID THEY INTEGRATE?
ONE IS A POSITIVE, ONE IS A
NEGATIVE, HELPING TO KEEP IT
EITHER QUIESCENT OR PROMOTING
ACTIVATION.
AND WE ALSO WANTED TO KNOW DOO
RASOPATHY MUTATIONS ALTER THESE
MUTATIONS.
SO FOR OUR MUTATIONS WE SELECTED
A PANEL OF THEM.
AND ACTUALLY SEVERAL OF THE
PANEL WERE IN A REGION THAT'S
KNOWN AS LOOP 1.
THAT'S ABOUT 11% OF THE
MUTATIONS CLUSTER IN AND AROUND
LOOP 1.
THOSE ARE HIGHLIGHTED IN ORANGE.
BUT THE ADDITIONAL ONES THAT
YOU'LL SEE ARE SCATTERED
THROUGHOUT THE DOMAIN.
WHAT THEY TEND TO DO IS ADD A
POSITIVE CHARGE TO THE DOMAIN.
IN PARTICULAR, THE Q2 57R
ACCOUNTS FOR 67% OF THE CRD
MUTATIONS IN THE RASOPATHY.
AND SO WHAT WE WANTED TO DO IS
ASK DO THEY ALTER THE FUNCTION.
CLEARLY I DON'T KNOW IF THIS
IS -- SO WE JUST WANTED TO LOOK
AT THE PANEL WITH THE DIFFERENT
ACTIVITIES THAT WE KNEW.
FIRST WAS PS BINDING.
SHOULD I BE POINTING AT
SOMETHING?
IT'S JUST NOT -- YEAH, MAYBE.
IF I CAN DO THAT MAYBE I HAVE
ADVANCE THIS DAY.
TO LOOK AT LIPID BINDING THIS
WAS A COLLABORATION WITH ANDY
STEVENS AT THE RAS INITIATIVE,
WE USED SURFACE PLASMA RESONANCE
BINDING.
AND SO WE COUPLED LIPO SENSOR TO
THE SURFACE, FLOWED OVER,
PURIFIED CRD, WILDTYPE OR
MUTANTS, WHAT YOU CAN SEE HERE
IS THAT IF WE HAD
PHOSPHO-SERRINE WOULD SEE
BINDING, AT THE CONTROL
LIPOSOMES WERE 100% CONTAINED NO
STEERENE WE SAW VERY LITTLE
BINDING.
WE BEGAN TO LOOK AT MUTANTS,
LOOP 1 VERY ALSO EFFECT ON PS
BINDING -- VERY LITTLE EFFECT ON
PS BINDING.
IF WE STEPPED BACK AND LOOK AT
STRUCTURE, THIS IS IN
ELECTROSTATIC SURFACE DEPICTION,
JUST THE CRD, WE'RE LOOKING AT
THE MEMBRANE BINDING VIEW,
THERE'S HYDROPHOBIC POCKET, WHAT
YOU SEE WITH THESE PUTTING IN
THESE BASIC RESIDUES, THEN
SURFACE BECOMES MORE POSITIVELY
CHARGED.
AND ALSO SHOWING SIDE VIEW, YOU
GET THESE BASIC PATCHES BUT THEY
ARE EXTENDING OUT.
SO WE THINK THIS ADDING THESE
POSITIVE CHARGES WOULD BE
CONDUCIVE FOR INTERACTIONS WITH
NEGATIVELY CHARGED PLASMA
MEMBRANE.
SO THAT MADE SENSE.
NOW WE WANTED TO LOOK AT EFFECTS
ON AUTOINHIBITION AND THOUGHT IT
WAS IMPORTANT TO DO IN THE CELL.
WE NEEDED TO KNOW HOW IT WAS
AFFECTING AUTOINHIBITION IN THE
CONTEXT OF EVERYTHING GOING ON
IN THE CELL.
RUSSELL SPENCER SMITH, A
POSTDOC, DEVELOPED A PROXIMITY
BASED ASSAY TO DO THIS.
IT'S A BIOLUMINESCENCE ENERGY
TRANSFER BASED ON RESULTS WE HAD
KNOWN FOR MANY YEARS IN THE
FIELD, IF YOU JUST EXPRESS THE
ISOLATE THE BRAF KINASE DOMAIN
IT CAN FORM CONSTITUATIVE DIMERS
AND SIGNAL TO MEK.
IF YOU EXPRESS THE ISOLATED
REGULATORY DOMAIN, IT COULD BIND
AND ACT TO BLOCK THE ACTIVITY OF
THE ISOLATED KINASE DOMAIN.
SO WE WONDERED IF WE TAGGED EACH
OF THOSE ISOLATED DOMAINS WITH A
TAG THAT IN THE RIGHT CONTEXT
COULD SERVE AS ENERGY DONOR AND
ENERGY ACCEPTOR, WOULD WE SEE A
SIGNAL THAT REFLECTED
AUTOINHIBITION?
SHOWN HERE AS WE DID INCREASING
AMOUNTS OF THE REGULATORY
DOMAIN, HOLDING THE KINASE
DOMAIN CONSTANT, YOU CAN SEE
WE'RE SEEING AN INCREASE IN BRET
SIGNAL THAT CORRELATES WITH
EXPRESSION OF SIGNALING TO THE
DOWNSTREAM SUBSTRATE MEK.
SO THIS WAS GREAT NEWS.
WE CAN DETECT AUTOINHIBITION.
WELL, NEXT IMPORTANT THING CAN
WE DETECT DISRUPTION OF
AUTOINHIBITION?
FOR THIS WE STARTED WITH ONE
THING WE THOUGHT WOULD BE AN
OBVIOUS CONTROL.
WHAT IF WE PUT IN AN ACTIVATED
RAF?
THAT SHOULD RECRUIT THE RBD AND
PULL THE REGULATORY DOMAIN AWAY
FROM THE KINASE DOMAIN.
THAT'S WHAT WE SAW.
ABOUT A 50% REDUCTION IN BRET
SIGNAL.
WE DID ANOTHER CONTROL,
INCORPORATING V600E ONE OF THE
MOST TRANSFORMING OR ONCOGENIC
CANCER-ASSOCIATED BRAF
MUTATIONS.
AND WHAT WE ALSO FOUND WAS, YES,
IT ALSO DISRUPTED
AUTOINHIBITION.
WHEN WE LOOKED AT MUTANTS IT WAS
A BIT SURPRISING, ALL THEM
DISRUPTED ON AUTOINHIBITION
EXCEPT ONE, THAT WAS THE E275K,
ABOUT THE TIME WE GOT THE
STRUCTURE OR FIRST CryoEM
STRUCTURES, AND WHEN WE STARTED
LOOKING AT THE STRUCTURES AND
WHERE THE CRD LAY, IT MADE ALL
THE SENSE IN THE WORLD BECAUSE
ALL OF THE POINT MUTATIONS THAT
DISRUPTED AUTOINHIBITION HAD --
WERE INTERACTING AND MAKING
CRITICAL INTERACTIONS THAT
HELPED HOLD THE AUTOINHIBITION
CONFIRMATION TOGETHER.
WHEN WE LOOKED AT E275 WHERE IT
LAY, IT'S LIKE OUT THERE WAVING
IN THE WIND.
AND SO IT'S NOT SURPRISING THAT
JUST CHANGING THAT RESIDUE IN
SIDE CHAINS WOULDN'T HAVE EFFECT
ON AUTOINHIBITION.
OKAY.
SO NOW WHAT ABOUT RAS BINDING?
SO, WE THEN WENT AND CONTINUED
TO THINKING IT WAS VERY
IMPORTANT TO LOOK AT THIS IN THE
CONTEXT OF THE CELL.
AND WE HAD ALSO DEVELOPED
ANOTHER -- THIS IS TRADITIONAL
BRET ASSAYS TAGGING VENOUS TO
KRAS OR THE LOOP, IF THEY ARE
ABLE TO BIND AN ACTIVATED KRAS
WE SHOULD SEE A SIGNAL.
WE ACTUALLY DID A TITRATION
CURVE AND WE INCORPORATED A
KNOWN MUTATION IN THE RVD THAT
COULD DISRUPT BINDING.
IN CONTEXT OF FULL LENGTH
PROTEIN, BINDING TO RAS, THIS IS
GOING TO BE SUBJECT TO EFFECTS
ON AUTOINHIBITION AS WELL AS TO
CHANGES IN MEMBRANE BINDING.
WHAT WE OBSERVED WAS THAT YOU
SEE LITTLE INCREASE IN RAS
BINDING IF AUTOINHIBITION IS NOT
RELIEVED.
SO FOR THE MUTANT, E2 75K WHERE
AUTOINHIBITION WAS A FINE
BUNCH.
THE OTHERS WHERE AUTOINHIBITION
IS RELEASED BINDING IS HIGHER
LEVEL, HIGHER AFFINITY AS WELL,
AT THE TOP OF THE LIST ARE THE
ONES THAT HAVE INCREASED
BINDING.
IF YOU CAN GET -- CAN OPEN AND
GET TO RAS, THEN THE ADVANTAGE
IS GIVEN TO YOU WITH BEING ABLE
TO HAVE A TIGHTER BINDING.
THIS IS A SUMMARY OF WHAT
MUTATIONS DO.
WE'VE KIND OF BEEN ABLE TO
CATEGORIZE HOW THEY AFFECTED
FUNCTIONS OF CRD BUT HOW DOES
THIS PLAY TOGETHER IN TERMS OF
THE BIOLOGICAL FUNCTION OF BRAF?
AND WE FIRST JUST DID A VERY
STANDARD, YOU KNOW, ASSAY IN
MAMMALIAN CELLS, LOOKED AT CELL
GROWTH, FOCUSED ASSAYS, AND
LOOKED AT SIGNALING DOWNSTREAM,
ABILITY TO ACTIVATE MEK BY
MEASURING p-MEK LEVELS, THEY
HAVE ELEVATED ACTIVITY ABOVE
WILD TYPE BUT WHAT'S NOTICEABLE
IS THAT THEY E275K THE GUY WHO
CAN ONLY -- HAS HIGHER BINDING
AFFINITY BUT DOESN'T RELIEVE
AUTOINHIBITION IS THE WEAKEST OF
ALL OF THE OTHER MUTANTS.
SO WE THOUGHT IT WAS IMPORTANT
ACTUALLY TO TRY TO GET INTO A
SYSTEM WHICH WOULD BE MORE
RELATABLE TO THE PATIENT.
WHAT PATIENTS WITH EXPERIENCING,
WORKING WITH NCI AND FREDERICK
AND BIGGER LABORATORY, TO
ESTABLISH SOME ASSAYS IN
ZEBRAFISH, MODEL SYSTEM OF
ZEBRAFISH, THAT WOULD
RECAPITULATE SOME THINGS SEEN IN
THE RASOPATHY PATIENTS.
SO, WE INJECT THE PROTEINS OR
RNA INTO THE ZEBRAFISH AT THE
ONE CELL STAIN, ONE DAY
POST-FERTILIZATION LOOK AT --
MEASURE EMBRYS, AXES, LOOKING
FOR DEFECTS IN CONVERGENT
EXTENSION MOVEMENTS REFLECTIVE
IN GETTING OBLONG EMBRYOS.
WE ALSO INJECTED AND LOOKED AT
THREE DAYS POST-FERTILIZATION,
WHERE YOU CAN ACTUALLY SEE HEART
EDEMA THAT MANY OF THE RASOPATHY
PATIENTS HAVE.
AND FINALLY WE ALSO LOOK AT
ANGLE OF THE CARTILAGE WHICH IS
A DIRECT MEASURE, INDICATIVE OF
GETTING INCREASED HEAD WIDTH AND
EYE SPACING, CHARACTERISTICS OF
THESE PATIENTS.
AND SO WE TOOK REPRESENTATIVE
FROM EACH OF OUR KIND OF CLASSES
OF THESE MUTANTS, AND WHAT YOU
CAN SEE HERE IS THAT MUTATIONS
THAT RELIEVE AUTOINHIBITION SHOW
GREATEST INCREASE IN CONVERGENT
EXTENSION MOVEMENTS SO THESE
CELLS ARE MOVING MORE THAN THEY
REALLY SHOULD DURING
GASTRILATION.
IF WE LOOK AT HEART EDEMA
MUTANTS THAT RELIEVE
AUTOINHIBITION INCREASE, CLEARLY
DIFFERENT, NOT AS SEVERE AS WITH
THE OTHER TWO.
WHEN WE LOOK AT ANGLE OF THE
CARTILAGE YOU CAN SEE FOR THE
E275K IT IS NO DIFFERENT THAN
WILD TYPE.
FOR THE OTHER TWO WITH RELIEF
AND AUTOINHIBITION YOU'RE SEEING
A WIDENING OF THAT ANGLE.
AND THIS WAS ACTUALLY EXCITING
TO US BECAUSE WE WENT AND
LOOKED.
THERE'S NOT A LOT OF PATIENT
DATA FOR THE RASOPATHY PATIENTS
BUT THERE'S SOME.
IN PARTICULAR, FOR THIS, E275K,
ESPECIALLY WHEN YOU'RE LOOKING
DOWN HERE WITH THE WIDE-SET
EYES, IT'S REFLECTIVE OF WHAT WE
SAW IN THE ZEBRAFISH EMBRYOS.
IT'S A LESS SEVERE DISEASE, IN
THIS ONE PATIENT, THERE'S ONE
OTHER PATIENT THAT HAS BEEN
REPORTED RECENTLY ALTHOUGH WE
DON'T HAVE MORE INFORMATION
ABOUT WHAT THEIR TRAITS ARE.
IT SEEMS ZEBRAFISH, WELL,
RESULTS IN THE ZEBRAFISH EMBRYOS
ARE CORRELATING WELL WITH
PHENOTYPES OBSERVED IN PATIENTS.
AND SO, YOU KNOW, WE THINK
ACTUALLY TAKING THIS I GUESS
BIOCHEMICAL CELL, BIOLOGICAL,
STRUCTURAL APPROACH, HAS BEEN
VERY INFORMATIVE AND STUDYING
THESE DISEASE-ASSOCIATE MUTANTS
CAN GIVE IMPORTANT REGULATORY
INFORMATION.
AND I THINK IT'S INTERESTING TO
THINK ABOUT THIS IN CANCER WHEN
YOU REALLY NEED TO BE HITTING
THE CELL WITH A SLEDGEHAMMER,
THOSE MUTATIONS ACTIVATE THE
CATALYTIC ACTIVITY, YES, THEY
ALSO RELIEVE AUTOINHIBITION, BUT
WHILE THEY ARE SUCH STRONG
DRIVERS IS BECAUSE OF ENHANCED
CATALYTIC ACTIVITY, WHERE WHEN
YOU HAVE TO SLIGHTLY UPREGULATE
THE PATHWAY, IN ORDER TO
MAINTAIN LIFE, THESE MUTATIONS
ARE ACTUALLY IMPACTING
MECHANISMS THAT ARE KNOWN TO
KEEP THE PROTEIN IN A QUIESCENT
STATE WHEN SIGNALING IS NOT
GOING ON.
WE'RE EXCITED TO BE PART OF A
NEW INITIATIVE CALLED THE NCI
ADVANCING RASOPATHY THERAPY,
ART, BRINGING TOGETHER PEOPLE
FROM THE CLINICAL GENETICS,
PEDIATRIC ONCOLOGY, NCI, NCI RAS
INITIATIVE TO IDENTIFY NEW
RASOPATHY MUTANTS SO LOOKING
FIRST AT PATIENT DATA AND SAYING
ARE THERE MUTATIONS IN SOME OF
THE KNOWN COMPONENTS THAT
CONTRIBUTE TO THE RASOPATHYS,
AND ANOTHER GOAL IS TO ANALYZE
THESE POTENTIAL NEW MUTANTS AND
DEVELOPMENT OF NEW THERAPIES.
I WANT TO SAY WE'VE ALREADY --
THAT'S WHAT WE'RE CURRENTLY
WORKING ON.
WE'VE STARTED WITH THIS ACTUALLY
LOOKING AT THE CRAF, ONE OF THE
OTHER MEMBERS OF THE RAS FAMILY
MEMBERS.
WE'VE ANALYZED THE PATIENT DATA,
AND WE FOUND ACTUALLY A COUPLE
POTENTIAL CLUSTERS, NOW IN THE
PROCESS OF ANALYZING THESE.
AGAIN, IT'S INTERESTING,
REINFORCES THE IDEA THAT BECAUSE
WHERE THESE CLUSTERS LIE ARE IN
RESIDUES WE KNOW ARE CRITICAL
FOR DOWNREGULATING PATHWAY UNDER
NORMAL GROWTH CONDITIONS.
WE THINK THE RASOPATHYS, YOU'RE
SUBVERTING SOMETHING THAT
NORMALLY KEEPS IT SUPPRESSED,
NOT AS, YOU KNOW, A CATALYTIC
ACTIVATION.
SO THERE'S REALLY A DIFFERENCE
THERE.
A COUPLE YEARS AGO WE CONDUCTED
A HIGH-THROUGHPUT SCREEN USING
OUR LIVE CELL ASSAYS, IN
COLLABORATION WITH MOLECULAR
TARGETS PROGRAM IN FREDERICK,
AND THEIR LARGE AND DIVERSE
LIBRARY OF NATURAL PRODUCTS.
WE'RE LOOKING FOR COMPOUNDS THAT
WOULD DISRUPT DIRECTLY OR
INDIRECTLY SOME OF THESE
CRITICAL PROTEIN INTERACTIONS
LIKE RAF, RAF BINDING, RAF
DIMERIZATION.
I WANT TO SHOW HOW WE'VE USED A
NATURAL PRODUCT INHIBITOR, AGAIN
SHOWING A MUTANT, AN N-RAS
MUTANT, REMEMBER RAS IS UPSTREAM
FROM RAF, WE KNOW SIGNAL
TRANSMISSION FROM A MUTANT RAF
DOES REQUIRE RAF DIMERIZATION.
AND YOU CAN SEE THAT EXPRESSION
OF THIS MUTANT IN THE ZEBRAFISH
EMBRYOS, YOU GET ELONGATED
EMBRYO.
AND IF WE TREAT THEM WITH THIS,
AS I CALL IT A RAF DIMER
DISRUPTER, WE CAN ACTUALLY
SUPPRESS THAT PHENOTYPE.
SO WE'RE STARTING TO SEE
SUPPRESSION.
IT'S TURNING OUT TO BE A VERY
RAPID WAY OF ASSESSING IF ANY OF
THESE NEW DRUGS OR NEW COMPOUNDS
MAY HAVE AN EFFECT ON SOME
RASOPATHY MUTANTS THAT ARE NOT
-- YOU KNOW, THEIR ACTIVITY
NEEDS TO BE DAMPENED DOWN.
IT DOESN'T HAVE TO BE COMPLETELY
TURNED OFF.
JUST DAMPENED DOWN, YOU KNOW, TO
HELP SOME OF THESE PATIENTS.
I'D LIKE TO THANK -- THERE ARE
THE PEOPLE IN THE LAB THAT
HELPED, COLLABORATORS, AND LIKE
EVERYONE ELSE, I THINK I HAVE
TO -- A DEBT OF GRATITUDE TO
THE INSTITUTE BECAUSE WHAT WE'VE
ACCOMPLISHED OVER THE YEARS IS
REALLY REFLECTIVE OF A GROUP
OF -- A TEAM OF COLLABORATORS
I'VE BEEN ABLE TO WORK WITH WHO
HAVE ALWAYS BEEN RECEPTIVE, YOU
KNOW, TO SAYING, SURE, COME TO
OUR LAB, YOU KNOW, WE'LL BE GLAD
TO HELP YOU DO THIS.
I'M REALLY GRATEFUL FOR HAVING
TO BE HERE, BASICALLY MY ENTIRE
CAREER AS AN INDEPENDENT
INVESTIGATOR.
SO THANK YOU.
[APPLAUSE]
>> THAT CONCLUDES THIS
PHENOMENAL SYMPOSIUM.
BEFORE I THANK THE SPEAKERS, I'M
GOING TO MAKE A SUGGESTION THAT
NEXT YEAR WE CALL THIS SYMPOSIUM
MYTH BUSTERS BECAUSE I THINK
WHAT WE'VE LEARNED, SKIN FLORA
CAN BE FRIEND AND FOE, WE'VE
LEARNED THAT YOU CAN BREAK DNA
IN THE COURSE OF MEIOSIS, AND
THEN USE THE MITOTIC APPARATUS
TO FIX IT.
WE'VE LEARNED THAT YOU CAN HAVE
A POST MITOTIC CELL, AND HAVE
DAMAGE DONE TO IT BY AN
ANTI-MITOTIC AGENT.
WE'VE LEARNED THAT THERE ARE
PATIENTS WHO HAVE ABERRATIONS OF
SORT OF GAIN OF FUNCTION
INFLAMMATORY ABERRATIONS OF
GAMMA INTERFERON.
AND YOU CAN GIVE THEM INTERFERON
TO MAKE THEM BETTER.
AND WE'VE LEARNED THAT YOU CAN
HAVE A PEPTIDE THAT INHIBITS THE
FUNCTION OF A SIGNAL
TRANSDUCTANT, AND IT CAN ALSO
ACTIVATE THAT SAME SIGNAL
TRANSDUCTANT AND SO I THINK
WE'VE LEARNED THAT THERE'S A LOT
MORE COMPLEXITY TO THE BIOLOGICS
SYSTEMS THAN WE EVER EXPECTED
AND I THANK ALL OF OUR
WONDERFUL, WONDERFUL SPEAKERS
FOR HELPING US UNDERSTAND THAT.
PLEASE JOIN ME IN CONGRATULATING
OUR SPEAKERS.
[APPLAUSE]