>>IT'S VERY NICE TO SEE SOME
LEVEL OF IN PERSON CROWD.
I THINK I GAVE A TALK HERE A
COUPLE MONTHS AGO AND THIS IS
MUCH MORE PEOPLE HERE SO THANKS
FOR COMING.
IT GIVES ME GREAT PLEASURE TO
INTRODUCE LAZZERINI DENCHI AS
OUR CCR GRAND ROUND SPEAKER.
HE JOINED CCR IN 2018 AS A
STADTMAN INVESTIGATOR AND BECAME
SENIOR INVESTIGATOR IN 2021.
HE GOT TO A LIGHTNING START AND
HAS BEEN AWARDED THE NCI
RESEARCH HIGHLIGHT AWARD IN
2022.
AND THE NCI DIRECTOR'S AWARD FOR
BASIC BIOLOGICAL SCIENCES IN
2023.
CONGRATULATIONS TO EROS.
BEFORE COMING TO BETHESDA HE WAS
AN INVESTIGATOR AT THE SCRIPPS
INSTITUTE IN LA JOLLA AFTER IT
YEARS.
THE WORKED IN LAB SPIKED HIS
INTEREST IN TELOMERE BIOLOGY AND
CONTINUES IN THIS AREA TODAY.
THE STUDY OF TELOMERE REGULATION
SAY PRETTY CROWDED FIELD BECAUSE
IT HAS BROAD IMPLICATIONS FOR
AGING, SENESCENCE, CANCER AND
CLINICAL IMPLICATIONS BUT EROS
HAS DISCOVERED REPRESSED
ACTIVATION AT THE NATIONAL
CHROMOSOME ENDS, DISCOVERY OF
TELOMERE PROTEINS AND LENGTH AND
THE REQUIREMENT FOR TELOMERE
PROTEINS IN STEM CELLS VERSUS
DIFFERENTIATED CELLS.
HE WAS DOING THIS FANTASTIC WORK
AT SCRIPPS I FOLLOWED CLOSELY
AND AS SOON AS WE CONVINCED HIM
TO COME TO NIH AND WE WERE HAPPY
IT MAKE HIM AN OFFER AND HAPPY
HE JOINED.
AT NIH I'VE HAD OPPORTUNITY TO
SEE FIRST-HAND HOW HE APPROACHES
HIS -- HOW HE RUNS HIS LAB AND
APPROACHES HIS WORK.
TO GIVE AN EXAMPLE, HE RECENTLY
MADE AN UNEXPECTED FINDING ABOUT
TELOMERE END PROTECTION WHICH
WENT I THINK AGAINST THE DOGMA
THAT'S BEEN AROUND MORE THAN 25
YEARS WHICH I'M SURE HE'LL
MENTION.
AT THE SAME TIME HE FOUND OUT
ANOTHER COLLEAGUE OF HIS HAD
MADE A SIMILAR FINDING.
SO THEY WERE CONFIDENT THE
FINDINGS WERE TRUE.
THE ONLY PROBLEM WAS FIGURING
OUT THE MECHANISM.
THAT'S THE HARD PART.
SO EROS DECIDING HE PREFERRED TO
CRACK THE PROBLEM HIMSELF AND
THAT'S WHERE THE FUN WAS WHERE
HE DID ELEGANTLY AND I THINK
THEY CAME UP WITH SLIGHTLY
DIFFERENT ANSWERS AS WELL.
AND THIS IS PRECISELY WHAT I
FOUND TO BE HIS MODUS OPERANDI
AND STICKS TO HIS GUN AND ENJOYS
SOLVING SCIENTIFIC PROBLEMS
LEADING TO ELEGANT SOLUTIONS.
AT THE SAME TIME HE'S BEEN A
PLEASURE TO WORK WITH, A
TERRIFIC COLLABORATOR AND
ENCOURAGE YOU TO TALK TO HIM
AFTER HIS GRAND ROUNDS SO TALK
TO HIM.
THANKS.
>>THANK YOU.
THAT WAS NICE.
IT WAS A KIND INTRODUCTION.
IT'S BEEN A PLEASURE TO JOIN
NCI.
IT'S BEEN A PHENOMENAL TIME TO
BE HERE.
IT'S BEEN NICE TO WORK WITH
AND-DRE AND EVERYONE IN THE
BRANCH AND I'LL TALK ABOUT
TELOMERES AND TELL STORIES IN
WHAT WE'RE TRYING TO UNDERSTAND
WHEN TELOMERES AND CANCER.
MAYBE I WAS AMBITIOUS WITH THE
DEVELOPMENT PART.
YOU'LL TELL ME ABOUT IT.
FIRST OF ALL, WHAT ARE
TELOMERES?
THIS IS THE WAY EVERY MAMMAL
STUDIED SO FAR LOOKS LIKE AT THE
CHROMOSOMAL END.
EACH CHROMOSOME END AND I'LL TRY
TO DO THIS -- SO EVERY SINGLE
MAMMAL HAS THE SAME REPETITIVE
SEQUENCE AT EACH CHROMOSOME END
AND AT THE END THERE'S A STRETCH
OF SINGLE-STRANDED DNA.
AND THE ONLY DIFFERENCE BETWEEN
THE DIFFERENT SPECIES IN MAMMALS
IS THE LENGTHS.
IN HUMANS WE HAVE ROUGHLY AATC
AND MICE HAVE DIFFERENT
TELOMERES BUT THE OVER ALL
STRUCTURE IS THE SAME AND FOR
MAMMALS WITH A SLIGHTLY
DIFFERENT SEQUENCE FOLLOW THE
SAME T.
THE DIFFERENCE IS RELATED TO
WHAT TELOMERES DO.
THEY SOLVE TWO PROBLEMS
ASSOCIATED WITH LINEAR
CHROMOSOMES.
WHAT I FIND REMARKABLE IS THE
FACT THAT THESE PROBLEMS WERE
DISCOVERED BEFORE WE KNEW
ANYTHING ABOUT TELOMERES.
THE FIRST PROBLEM HAS TO DO WITH
THE SO-CALLED END REPLICATION
PROBLEM.
SO WHAT IS THAT?
THE END REPLICATION PROBLEM IS
THE FACT OUR DNA REPLICATION
WORKS.
WHEN YOU TRY TO DNA MACHINERY
TRIES TO COPY A LINEAR TEMPLATE,
THERE'S ALWAYS GOING TO BE A GAP
OF INFORMATION THAT IS LOST.
AND IT'S DUE TO THE FACT THAT IN
THE LIGAND STRENGTH NOW MATTER
HOW YOU PUT IT TO THE TIP OF THE
DNA THAT PORTION OF INFORMATION
WILL BE LOST.
SO WHEN A CELL DIVIDES A PORTION
WILL LOST AND IT WILL LEAD TO
CELL DEATH.
OF COURSE, WE DON'T DIE, WE'RE
STICKING AROUND AND THE REASON
IS WE HAVE WAYS TO DEAL WITH THE
PROBLEM.
FOR THE MOST PART THE MOST
FREQUENT WAY WE DEAL WITH IT IS
BY AN ENZYME CALLED TELEMERASE
WHICH ADDS NEW REPEAT USING ITS
OWN RNA AS A TEMPLATE AND WILL
ADD NEW SEQUENCES THEREFORE
COUNTER ACTING THE LOST
INFORMATION.
THIS IS THE HOW OUR CELLS DEAL
WITH THE PROBLEM.
AND THE SECOND PROBLEM IS MORE
PRESSING WHILE THE REPLICATION
PROBLEM IS ONLY A PROBLEM WHEN
CELLS DIVIDE THE SECOND PROBLEM
WITH TELOMERES WITH CELLS NOT
DIVID
DIVIDING.
OUR CELLS DE GAP IN THE DAMAGE
AND DAMAGE AND THIS WILL TRIGGER
DNA DAMAGE RESPONSE.
THE CELLS FIX IT AND IF THEY
CANNOT THEY WILL KILL THE CELL.
THIS IS GREAT FOR THE GENOME BUT
BECOMES A PROBLEM WHEN YOU DEAL
WITH CHROMOSOMAL ENDS IT'S A
BROKEN PIECE OF DNA IN THE
CHROMOSOME SO WE SHOULD BE
INDUCING DNA DAMAGE ALL THE TIME
IN EVERY CELL OF OUR BODY ALL
THE TIME.
WE DON'T DO IT AND IT'S BECAUSE
THERE'S A SET OF PROTEINS THAT
BIND TO CHROMOSOME ENDS AND THEY
SORT OF SHIELD OR HIDE THE
CHROMOSOME FROM THE DNA DAMAGE
ACTIVITY PREVENTING ACTIVATION.
THIS IS HOW YOU SOLVE THE END
PROTECTION PROBLEM.
I'LL SHOW AN EXAMPLE AND THIS IS
MY FAVORITE EXAMPLE BECAUSE IT
DEALS WITH MY FAVORITE PROTEIN
IN TELOMERES THIS PROTEIN CALLED
TR2.
IN THIS CASE WHAT I'M SHOWING IS
IN RED YOU HAVE CHROMOSOMES IN
GREEN YOU CAN HAVE THE TTAG
REPEAT AND TELOMERES AND THIS
DERIVES FROM A MOUSE MODEL WE
GENERATED THAT WAS GENERATED IN
THE LAB WHEN I WAS DOING MY
POSTDOC AND IN WHICH WE CAN
DELETE CONTINUALLY TR2.
IN THIS SITUATION TR2 IS
EXPRESSED, IT'S COMPLETELY FINE
BUT WE CAN DELETE TR2 AND I FELL
IN LOVE WITH THE PROTEIN AND
ASKED MYSELF HOW IT WORKS.
BY REMOVING THE PROTEIN AND
GIVING IT ONE SINGLE CELL CYCLE
EACH CHROMOSOME IS RECOGNIZED AS
A SITE OF DNA DAMAGE AND FUSED
TOGETHER TO REPAIR.
THIS IS THE BEST EXPLANATION AND
BEST EXAMPLE OF END PROTECTION.
THE FUNCTION THIS PROTEINS DO AT
TELOMERES.
ZOOMING OUT AND I WON'T GO INTO
ALL DETAILS AND THE WAY I SEE
ALL PROTEINS THAT BIND TO
CHROMOSOME ENDS THEY DEAL WITH
ALL THE DIFFERENT TYPES OF DNA
DAMAGE PRESENT IN A CELL.
YOU HAVE SPECIALIZED PROTEINS
THAT CAN SUPPRESS DIFFERENT
TYPES OF DNA DAMAGE.
THE RESULT IS OUR CHROMOSOME
ENDS ARE NOT STICKING.
THEY DON'T BECOME ENGAGED AB AND
THIS IS A FASCINATING PROBLEM
BUT THERE'S CLEAR IMPLICATION
FOR HUMAN HEALTH AND ESPECIALLY
FOR TUMORS.
THAT'S WHAT I'M GOING TO FOCUS
TODAY.
SO THE VERY FIRST PROBLEM I
INTRODUCED THE END REPLICATION
PROBLEM IS A STRONG IMPLICATION
FOR TUMOR DEVELOPMENT BECAUSE IT
REPRESENTS A MAJOR TUMOR
SUPPRESSIVE PATHWAY.
EACH CELL -- EACH CANCER IN
HUMANS NEEDS TO OVERCOME THE END
REPLICATION PROBLEM.
THE REASON IS HUMANS DON'T
EXPRESS THE TELOMERASE SO FOR A
TUMOR TO BE SUCCESSFUL AND GROW
NEEDS TO OVERCOME THE IMPULSE BY
TEL MERE SHORTENING.
THIS IS OUR OWN MARKER OF TUMOR
BIOLOGY.
THEN WAY THEY DO IS IT BY THE
MOST PART BY REACTIVATING
TELOMERASE AND THERE'S A
PROMOTION THAT ALLOWS FOR
REACTIVATION AND IT OCCURS IN
ROUGHLY 80% TO 90% OF HUMAN
TUMORS.
HOWEVER, THERE'S ANOTHER SEFT
TUMORS THAT DON'T EXPRESS
TELOMERES AND THESE INSTEAD USE
THE L PATHWAY WITH A TELOMERE
ELONGATION WE'LL GO IN THE
DETAILS TO AFTERWARDS AND
REPRESENT ROUGHLY 50% OF TUMORS.
WHAT IS INTERESTING IS NOT ALL
TUMORS ARE THE SAME WHEN IT
COMES TO TELOMERE MAINTENANCE
AND THE TUMORS OF THESE ORIGINS
INCLUDING SARCOMAS SEND TO HAVE
HIGH FREQUENCY OF RELIANCE ON
THIS PATHWAY WHICH IS A PATHWAY
WE DON'T UNDERSTAND VERY WELL.
OKAY.
SO THE SECOND PROBLEM THAT I
INTRODUCED THE END PROTECTION
ALSO AS IMPORTANCE DURING TUMOR
DEVELOPMENT AND THIS HAS TO DO
WITH GENOME INSTABILITY.
IT'S BEEN SHOWN IN MANY SYSTEMS
INCLUDING MOUSE MODELS AS WELL
AS IN CLINICAL STUDIES THAT WHEN
THERE IS -- WHEN TELOMERES
BECOME VERY SHORT THERE'S A
PHASE IN WHICH THEY CAN GROW
WITHOUT ANY INDUCED TELOMERE
ELONGATION.
IF THAT HAPPENS, TELOMERES ARE A
DRIVER FOR INSTABILITY AND ONE
NICE EXAMPLE IS PROBABLY ONE OF
THE MOST CATASTROPHIC GENE
REARRANGEMENT SEEN IN TUMORS AND
WHERE YOU SEE CANCER CELLS
TRIGGER IS BY END TO END
CHROMOSOMAL FUSIONS.
SO HOPEFULLY I CONVINCED WE ARE
STUDYING THIS HAS I MPLICATIONS
FOR CANCER BIOLOGY.
I'LL TELL YOU TWO STORIES OR TWO
ASPECTS OF OUR RESEARCH.
BY DOING SO HOPEFULLY I CAN SHOW
YOU WHAT WE DO AND HOPEFULLY
ENGAGE POTENTIAL COLLABORATION.
THE FIRST STORY HAS TO DO WITH
HOW IT BYPASSES TUMOR
SUPPRESSION.
WHAT I MEAN IS SO TELOMERE
SHORTEN AS A TOLD YOU AND
EVENTUALLY THEY BECOME SO SHORT
IT CAN INDUCE DNA DAMAGE
RESPONSE.
IF IT WAS PERFECT THERE WOULD BE
NO TUMORS.
UNFORTUNATELY WE KNOW THAT'S NOT
THE CASE.
THERE IS A QUESTION HERE, WHY
SOME CELLS CAN SURVIVE.
WHAT ARE MECHANISMS THAT ALLOW
CELLS TO SURVIVE AND ENGAGING
THE DIFFERENT PATHWAY.
THERE'S CLEARLY SOMETHING HERE
WE DON'T UNDERSTAND.
IT'S QUITE DIFFICULT TO STUDY IN
VIVO BECAUSE CLEARLY MOST OF THE
CELLS THE TUMOR CELLS THE
PRECANCEROUS CELLS DIE OFF SO WE
DON'T SEE THE RESULT OF THOSE
AND THE ONE THAT CANNOT HAS
TELOMERASE REACTIVATION AND WE
LOOK AT WHAT WE CAN RESPONSE THE
FUNCTION.
THE WAY WE DO IT INSTEAD OF
DRIVING THE SORTENING IS MESSING
WITH THE TELOMERES THAT BIND AND
MAYBE DIFFERENTIATION AND MAYBE
WE CAN LOOK AT THE WAY THE CELLS
RESPOND AND IT WAS MORE OF AN
IDEA AND WHEN I JOINED NCI I WAS
LUCKY TO RECRUIT MY FIRST
POSTDOC THAT TURNED OUT TO BE AN
AMAZING POSTDOC AND MARTA
DECIDED TO TAKE ON THE PROJECT
AND SOUGHT COLLABORATION WITH A
LAB THAT STARTED ROUGHLY THE
SAME TIME AND LOOKING AT THE
CELLS AND DIFFERENTIATION AND
LOOKED AT MICE CELLS SIMILAR TO
THE MODEL I SHOWED YOU BEFORE
WHEN YOU SAW THE CHROMOSOME
FUSIONS.
SO THE CELLS ARE PLURIPOTENT
STEM CELLS AND SO MARTA DERIVED
SOME DIFFERENTIATION FOR THE
SOMATIC CELLS WE CALLED
FIBROBLAST LIKE CELLS BECAUSE WE
DON'T KNOW WHAT THEY ARE BUT
THEY LOOK LIKE FIBROBLASTS AND
EXPRESS GENES EXPRESSED IN
FIBROBLASTS AND THE CELLS ARE
PROLIFERATING AND ISO GENIC TO
THE STEM CELL.
SO WE HAVE TWO POPULATION OF
CELLS GROWING AND THE DIFFERENCE
IS JUST DIFFERENTIATION STATE
AND SHE COULD SEE WHAT HAPPENS.
AND THE RESULT IS ONE OF THOSE
RESULTS THAT TO ME I'VE SEEN
MANY TIMES AND SPOKE ABOUT IT
MANY TIMES AND ALL THE TIMES I
SAY IT AND ALL THE TIME STILL
SHOCKED IT'S ALL BLACK AND
WHITE.
YOU HAVE THE EXPECTED PHENOTYPE
AND WE HAVE SOMATIC CELLS AND
END TO END CHROMOSOMAL FUSION
AND SEE THE SPAGHETTI LIKE
STRUCTURE ARE FUSED TOGETHER BY
THE END PATHWAY AND WHAT WE SEE
IN EVERY CELL.
WE DID IT IN HEPATOCYTES AND
EVERY CELL WE STARTED UP TO NOW
SHOWS THIS PHENOTYPE.
HOWEVER THE EMBRYONIC STEM CELLS
DO NOT SHOW -- THEY SHOW HARDLY
ANY TYPE OF FUSIONS.
MORE STRIKING, THEY KEEP
PROLIFERATING.
THEY PROLIFERATE A LITTLE BIT
LESS THAN THE COUNTERPART BUT
STILL PROLIFERATE.
SO THIS WAS TELLING US THERE WAS
SOMETHING UNIQUE ABOUT THESE
CELLS WE COULD DELETE TR2 BUT
SOMETHING WAS MODIFYING THE
PHENOTYPE.
THAT'S ONE OF THE THINGS WE
WERE LOOKING FOR.
THIS TRIGGERED A LOT OF WORK
FROM OUR PART TO TRY AND
UNDERSTAND WHAT IS THIS PATHWAY
AND WHAT CAN PREVENT FULL BLOWN
DNA DAMAGE RESPONSE OF
CHROMOSOMAL ENDS.
WE WERE NOT ALONE IN THIS.
SIMON BOLTON'S LAB CAME TO A
SIMILAR CONCLUSION AND PUBLISHED
A PAPER A COUPLE YEARS AGO.
AND I'M NOT GOING TO GO INTO ALL
THE DETAILS OF WHAT WE PUBLISHED
BUT LET ME TELL YOU WHEN WE
PUBLISHED A COUPLE IDEAS THE WAY
THIS COULD WORK SO ONE
HYPOTHESIS THAT THE TELOMERES
HAD A DAMPENED DNA DAMAGE
RESPONSE AND FOUND MOLECULES
THAT COULD TRIGGER THE RESPONSE
AND THE MOST STRIKING, EXCITING
IN MANY WAYS FOR ME WAS THE FACT
WE SAW INDUCTION OF A SET OF
GENES USUALLY EXPRESSED EARLY
DURING EMBRYOGENESIS AND SEEM TO
BE INVOLVED IN THE PROTECTION
PHENOTYPE AND AMONG THOSE WERE A
GENE WHICH I KNEW NOTHING ABOUT
AT THAT POINT BUT SEEMED
INTERESTING.
WE HAD SOME INKLING BUT NOT
COMPLETELY SOLID WHETHER THIS
WAS PROVIDING PROTECTION.
SO MARTA DECIDED THE ONLY WAY TO
KNOW IF THAT WAS INDEED THE CASE
WAS TO DELETE THIS AND THE
PROBLEM IS AT LEAST IN MICE
THERE ARE SIX GENES THAT ENCODE
ZSCAN4 AND THEY'RE COMPETITIVE
AND HAVE EXTRA GENES IN THE
MIDDLE SO WE CAN'T JUST DEPLETE
THE LOCUS.
WHEN WE SAW IT I THOUGHT THERE'S
NO WAY TO KNOCK IT OUT AND MARTA
IS VERY GOOD AND ALSO EXTREMELY
STUBBORN AND KNOCKED EVERY
ISOFORM OF ZSCAN4 AND THIS
DOESN'T DO JUSTICE TO THE AMOUNT
OF WORK SHE DID BECAUSE IT TOOK
A LONG TIME AND ONCE WE DID THIS
WE HAVE A CLEAN -- TWO ACTUALLY
CLEAN ZSCAN4 KNOCKOUTS SO THE
RESULT WAS CLEAR.
WHEN WE DELETE IT IN THE
PRESENCE OF ZSCAN 4 WE HAVE
LITTLE FUSIONS AND IN THE
ABSENCE OF THIS WE START TO GET
CHAINS OF CHROMOSOME FUSIONS.
SO MARTA IS NOW STUDYING THE
ZSCAN4 AND SHE IS NOW ON THE JOB
MARKET SO LIKELY FIGURE OUT WHAT
ZSCAN4 DOES IN HER OWN LAB WHICH
IS VERY EXCITING BUT FROM
ANOTHER POINT OF VIEW YOU CAN
SEE ON THE ONE HAND IT'S GOOD WE
CAN CONFIRM WHAT WE SUSPECTED SO
ZSCAN4 IS DOING SOMETHING BUT
NOT EVERYTHING BECAUSE WHAT I
SHOWED IN THE SOMATIC CELL YOU
GET THREE AND EVERY CHROMOSOME
IS FUSED TOGETHER AND THIS IS
STILL NOT A FULL BLOWN PHENOTYPE
THAT WE SEE HERE.
SO THERE MUST BE SOMETHING ELSE.
SO MARTA THEN JOINED FORCES WITH
ANOTHER POSTDOC IN THE LAB AND
THEY DECIDED TO SEE IF THERE WAS
ANYTHING WE WERE MISSING AND
DOING GENETIC SCREENS USING
CRISPR AND KNOCKOUT EVERY SINGLE
GENE AND THEN ASK IF THERE IS
ANY GENE THAT IS KILLING
SELECTIVELY ZSCAN4 KNOCKOUT
CELLS AND MAYBE THERE'S A
PROTECTIVE GENE IN THE GENOME
THAT IS PROTECTING TR2 KNOCKOUT
CELLS AND WE THOUGHT THAT GENE
WOULD DIE HOWEVER, IN TR2
PROFICIENT CELLS IT WOULD NOT
HAPPEN.
SO WE DID THIS AND I'M GOING
GIVE YOU JUST ONE IDEA OF THE
WAY WE SEE THIS.
SO USUALLY WHAT YOU DO IS YOU
GET A SCORE.
DEPENDING IF THE GENE IS
ESSENTIAL IT'S GOING TO BE THOSE
CELLS EXPRESSED IN THE RNA WOULD
BE LOST FROM THE POPULATION.
AND WE GIVE A BETA SCORE.
SO THE MORE NEGATIVE VALUE YOU
GET THE MORE ESSENTIAL A GENE IN
THESE SITUATIONS.
WHAT I'M SHOWING IS THE TOP
REPRESENTATION OF THE OLD GENOME
AND THE GENES THAT ARE DOWN HERE
SELECTIVELY LINKED HERE TO
KNOCKOUT CELLS.
THERE'S A COUPLE OF GENES IN
YELLOW.
I WON'T GO INTO GENES WE
VERIFIED PREVIOUSLY IN THE LAB.
ONE THING THAT WAS STRIKING FOR
US WE SAW A LOT OF GENES WITH
THE SAME NAME.
AND WHEN WE LOOKED FURTHER DOWN
THERE WAS ADDITIONAL GENE ALL
BELONGING TO THE SAME FAMILY.
THE FAMILY KNOWN AND IT WAS
SURPRISING FOR ME.
IT'S A PATHWAY THAT IS WELL
KNOWN TO BE SCANNING THE
TRANSCRIPTOME AND REMOVING ANY
GENE FOR INSTANCE.
BASICALLY A QUALITY CONTROL FOR
RNA.
AND SO WHY WOULD THE PATHWAY BE
LINKED WITH TR2 THAT WAS NOT
IMMEDIATELY APPARENT AND THERE
WAS SOME LITERATURE.
SOME OF THE GENES SEEM TO BE
CLOSE TO WHAT IS INVOLVED IN
TELOMERASE ACTIVITY.
WE THOUGHT MAYBE THERE'S
SOMETHING ELSE.
WE WANTED TO TEST IF THAT WAS
THE CASE.
WE WOULD DO THIS IN THE LAB AND
WE DELETE THE GENES AND ASK IF
THEY'RE VIABLE AND IF THEY'RE
NOT CAN WE DO ANYTHING TO SEE
WHETHER THEY PLAY A ROLE IN THE
CELLS.
IT WAS TOLD THE PATHWAY IS
ESSENTIAL IN SOMATIC CELLS IS
NOT ESSENTIAL IN THESE CELLS.
WE WERE NOT THE FIRST TO OBSERVE
THIS BUT CONFIRMED THIS.
WE DELETED A LOT OF THE NMD
FACTORS AND THEY CAN VERIFY THEY
DID KNOCKOUTS IN DIFFERENT WAYS
AND IMPORTANTLY THEN WE COULD
SHOW THAT THEY INDEED INVOLVED
IN THE PATHWAY.
WE DID THIS THANKS WITH A LOT OF
HELP FROM NIH.
AND HERE WE'RE GIVING YOU A
ROUGH OVERVIEW SO YOU TEND TO
HAVE A SPLICE ISOFORM.
FOR INSTANCE, YOU CAN HAVE CASES
IN WHICH THEY'RE RETAINED OR
CASES IN WHICH THEY'RE SKIPPED.
THOSE TYPE OF TRANSCRIPTS ARE
NORMALLY DEPLETED BY THE PATHWAY
BUT IN THE ABSENCE OF THESE
GENES THE EVENTS ARE ACCUMULATED
AND WE SEE A LOT OF THEM
CONFIRMING THE PROTEINS DO PLAY
A ROLE IN THE PATHWAY IN STEM
CELLS.
AND THE RESULT WAS REALLY
STRIKING.
SO HERE IS THE KNOCKOUT BY
THEMSELVES.
THE CHROMOSOMES ARE REALLY NICE
FROM WHAT WE CAN TELL.
THERE'S NO FUSIONS THEY LOOK
PRETTY HOWEVER, WHEN WE DELETE
TR2 IN THE CONTEXT WE CAN SEE
THE ENGAGEMENT OF END
CHROMESOMAL FUSION AND THIS IS
TRUE FOR THE MEMBERS OF THE
PATHWAY WE TEFFED TODAY.
-- TESTED TODAY.
THERE'S A SLIGHT VARIATION WE
STILL DON'T UNDERSTAND FROM IT'S
TRUE BIOLOGICAL OR NOT BUT MANY
HAVE HIGH LEVEL OF CHROMOSOMAL
FUSIONS.
AND TO PUT IT IN CONTEXT THIS IS
WHAT WE SAW WITH WHAT WE SEE IN
SOMATIC LEVELS AROUND 50% AND
HERE WE GET 20% OF FUSION.
WE ARE STILL NOT AT THE LEVEL OF
SOMATIC CELLS BUT IT'S A LOT OF
TELEFUSIONS.
WE WANT TO SEE IF THERE'S A SET
OF CELLS THAT CAN PROTECT
TELOMERES IN THE ABSENCE OF TR2.
THE GENES IN THE ABSENCE OF NMD
PATHWAY ARE EXPRESSED AT LOWER
LEVEL.
WE'RE GETTING THERE AND WHAT I
MEAN IS WE DID TRANSCRIPTIONAL
PROFILING AND FOUND AROUND 100
GENES THAT ARE CONSISTENTLY
AFFECTED IN NMD PATHWAY AND
THINK SOME OF THOSE ARE THE ONES
AND WE STILL HAVE TO DO A NUMBER
OF THINGS TO VERIFY THIS.
AND HOPEFULLY WE'LL HAVE AN
UPDATE SOON.
OKAY.
AND THIS CONCLUDES THE PART ON
THE TELOMERE PROTECTION AND
GOING BACK TO THE ORIGINAL
QUESTION DOES IT TRANSLATE TO
ANYTHING IMPORTANT POTENTIALLY
FOR CANCER AND I THINK SO.
WE LIKE TO THINK SO.
WE WERE LOOKING FOR FACTORS THAT
COULD MEDIATE THE RESPONSE TO
VERY SHORT TELOMERES.
INTERESTINGLY THERE'S A GENE NOT
EXPRESSED IN SOMATIC CELLS ONLY
EARLY DURING EMBRYOGENESIS AND
YOU HAVE SOME THAT DO EXPRESS
ZSCAN4 SO IT'S TEMPTING TO
SPECULATE AND MAYBE IN THOSE
CELLS THE INDUCTION COULD HAVE
HELPED SOMEHOW TELOMERE EROSION.
THIS IS SOMETHING MARTA IS
WISHING TO TEST IN HER OWN
RESEARCH AND NMD HAS BEEN SHOWN
TO BE POTENTIALLY IN SOME
SETTINGS FOR INSTANCE IN CELLS
WITH THE ABILITY THAT HAVE A LOT
OF INSTABILITY SO POTENTIALLY
THERE COULD BE A LINK WITH
TELOMERE DYSFUNCTION AS WELL AND
THIS IS SOMETHING WE NEED TO
TEST.
OKAY.
SO FOR THE LAST PART OF MY TALK
I'M GOING TO INSTEAD TELL YOU
SOMETHING ABOUT THE MECHANISM OF
TELOMERE ELONGATION.
IT'S AN AREA THAT IS DEF AND THE
IDEA IS WHEN CELLS UNDER GO
MASSIVE TELEMERE EROSION THEY
CAN ELONGATE AND ALLOW THEM TO
BE IMMORTAL.
HOW'S THIS WORK.
ROUGHLY 85% OF HUMAN CANCER IS
EXPRESSED IN TELOMERASE AND
THERE'S PROMOTER IN THE
TELOMERASE THAT ALLOWS
EXPRESSION BUT HOW'S IT
FUNCTION?
WHOEN YOU THINK ABOUT IT
TELOMERASE IS A TRICKY ENZYME TO
FUNCTION.
THE BRIAN LAB ESTIMATED ROUGHLY
10 TO 15 MOLECULES OF TELOMERASE
AND IN CANCER CELLS THAT
OVEREXPRESS TELOMERASE SO IT'S
AN ABUNDANT MOLECULE AND THERE'S
A LOT OF GENOME TO SCAN.
SO HOW'S IT FIND THE END TO ACT
IS UNCLEAR.
ONE INDICATION OF HOW IT COULD
WORK CAME OUT THANKS TO THE WORK
OF TOM CHECK WHICH IDENTIFIED AN
INTERACTION BETWEEN TELOMERASE
AND THE COMPLEX I TOLD YOU
BEFORE, AND THIS ALLOWS THEM TO
FIND TELOMERES BUT IT'S NOT
SUFFICIENT.
WHILE I'M DRAWING THIS IN A
SIMPLIFIED MANNER WITH THE END
OF THE CHROMOSOME ENDS WITH THIS
PROTEIN COMPLEX HERE, THE
REALITY IS A LOT DIFFERENT.
IN REALITY YOU CAN LOOK AT A
SINGLE CHROMOSOME AND THE
PROTEIN COMPLEX IS SPREAD
THROUGHOUT THE ENTIRETY OF THE
CHROMOSOME OR TELOMERE.
SO THIS INTERACTION CAN LOCALIZE
TELOMERASE TO THE TELOMERES BUT
ALSO POTENTIALLY A PROBLEM
BECAUSE THEN YOU'LL HAVE
UNPRODUCTIVE INTERACTION ALONG
THE TELOMERE.
HOW CAN TELOMERASE BE TREATED
HERE.
THIS IS ONE WAY OF SAYING WE
DON'T KNOW.
SO THERE'S SOMETHING ELSE WE'RE
MISSING.
SOMETHING THAT NEEDS TO BE FOUND
THAT CAN EXPLAIN HOW TELOMERASE
CAN BE AT THE END IN OUR
MODELLING.
SO THE NEWEST MEMBER OF THE LAB
JOINED A YEAR AND A HALF AGO AND
HE'S BEEN A PHENOMENAL IN THE
LAB IN LITTLE TIME MANAGED TO
GENERATE A LOT OF DATA AND
REALLY COOL THINGS TO STUDY
TELOMERASE ACTIVITY AND FIRST
IDENTIFIED ANY POTENTIAL
CANDIDATE THAT COULD INTERACT
WITH TELOMERASE IN TWO WAYS.
THE FIRST WAY WAS A PROTEOMICS
BASED APPROACHED AND USED
PROXIMITY BASED LIGATION AND
PROTEINS THAT COME IN CLOSE
PROXIMITY TO A BAIT AND USE THE
PROTEIN KNOWN TO INTERACT WITH
TELOMERASE AND THERE'S A MUTANT
THAT CANNOT ENACT AND COMPARING
THE TWO SITUATIONS HE'S HOPING
TO FIND FACTORS BOUND IN
PROXIMITY TO TELOMERASE AND
POTENTIALLY TO TELOMERASE AND
EXCLUDE THE ONE THAT INTERACTS
WITH MPP1.
THERE'S THE KNOWN COMPONENT OF
TELOMERASE AND HAS WHAT HAPPENS
WHEN HE DOES THIS DONE IN
COLLABORATION WITH THE
SPECTROMETRY CORPS.
YOU HAVE A LOT OF POTENTIAL HITS
THIS WAY AND IT CAN BE DIFFICULT
TO SEE EACH OF THEM IF YOU WANT
TO GO AFTER EACH OF THEM.
INSTEAD HE DID TWO EXTRA STEPS
TO CLEAN UP THE LIST.
THE FIRST WAS TO DO A CRISPR
SCREEN AND FIND ANYTHING IT THAT
IS ESSENTIAL FOR TELOMERASE
ACTIVITY AND HE HAS PARENTAL
CELL LINES WITH REALLY LONG
TELOMERES AND CREATED CELL LINES
THAT SURVIVE THANKS TO THE
TELOMERASE ACTIVITY WITH SHORT
TELOMERES.
THESE ARE THE LIMITATION THAT
ALLOWS THEM TO SURVIVE BUT JUST
BARELY HANGING ON THANKS TO
TELOMERASE ACTIVITY.
SO THEN THE TWO SITUATIONS
SHOULD BE GENETICALLY IDENTICAL
AND NOW WE CAN DO A CRISPR
SCREEN WHERE ASK WHO IS
RESPONSIBLE FOR THE VIABILITY OF
THE CELLS WITH SHORT TELOMERES.
AND WHEN HE DID THIS HE FOUND A
LOT OF GENES AND EXCITINGLY HE
FOUND TELOMERASE SUGGESTING
WE'RE HITTING THE PATHWAY WE
THINK.
AGAIN, THE TWO THINGS COMBINED
IS STILL HUNDREDS OF GENES
DIFFICULT TO ANALYZE ESPECIALLY
BECAUSE THE ONLY WAY WE REALLY
HAVE TO DATE TO MEASURE
TELOMERASE ACTIVITY IS TO DO
THESE TYPES OF EXPERIMENTS WHERE
YOU TAKE CELLS, DELETE WHATEVER
GENE YOU WANT TO AND CULTURE THE
CELL.
SO IN DOING THIS FOR 150 TARGET
GENES IS DEMANDING AND HE WAS
NOT GOING TO DO IT.
INSTEAD WE THOUGHT OF TRYING TO
DO A BETTER WAY TO TRY TO SEE IF
WE COULD DEVELOP A WAY TO SEE
TELOMERASE ACTIVITY IN A SINGLE
CELL CYCLE THAT WOULD ALLOW TO
US SCREEN ALL THE POTENTIAL
TARGETS IN A RELIABLE AND FAST
MAN
MANNER IDEALLY WE'D LIKE TO SEE
IT IN ACTION AND IT COMES WITH
ITS OWN TEMPLATE AND USE IT TO
ADD REPEATS AT THE END.
IF WE COULD JUST HAVE REPEATS WE
COULD DO IT BUT THEY'RE
IDENTICAL TO THE REST.
INSTEAD HE PROVIDED A MUTANT RNA
AND CHANGE IT AT SEQUENCE AND HE
CAN DETECT IT NICELY WITH A
PROBE I WORKED NICELY BUT WHEN
YOU INTRODUCE THE MUTATION IT
DOESN'T ALLOW ALL THE PROTECTIVE
PROTEINS TO BIND THEREFORE THE
CELLS DIE.
AS A RESULT IT DOESN'T REALLY
WORK BUT IN THESE CELLS WE CAN
GET RID OF THE PROTECTIVE
PROTEINS AND IT SEEMS TO WORK.
HE PUT THEM TOGETHER AND THOUGHT
MAYBE WE CAN OVEREXPRESS THE
MUTANT RNA IN THE CELLS AND SEE
IF THIS CAN BE USED YOU HAVE
CELLS THAT EXPRESS THE
TELOMERASE AND WILD TYPE AND
SOME WILL BE SYNTHESIZED AND YOU
CAN SEE WILD TYPE AS WELL AS
MUTANT ALMOST AT EVERY
CHROMOSOME.
I'M SAYING ALMOST BECAUSE SOME
ENDS ARE NOT LABELLED.
THAT MEANS TELOMERASE IN THE
GIVEN DIVISIONS ACT IN THIS
PARTICULAR TELOMERE.
THE CELLS ARE VIABLE AND GROW
AND SEEM TO BE COMPLETELY FINE.
THIS MEANS WE HAVE A POTENTIAL
ASSAY TO MEASURE TELOMERASE
ACTIVITY IN A SINGLE CELL CYCLE
OR AT LEAST IN THIS CYCLE.
BUT THERE'S A FEW CONTROLS WE
HAD TO DO.
THE FIRST IS IS THIS REALLY
TELOMERASE WE COULD TEST THIS
QUITE EASILY AND DELETED
TELOMERASE AND YOU CAN SEE IF
YOU EXPRESS THE MUTANT
TELOMERASE TEMPLATE IN WILD TYPE
YOU CAN SEE THE MUTANT TEMPLATE
AND THIS IS AN INTERFACE CELL
AND WORKS NICELY IN INTERFACE
CELLS BUT CANNOT GET COOPERATION
IN TELOMERASE KNOCKOUT TELLING
US YOU NEED A TO INCORPORATE
THIS REPEAT.
THAT'S GOOD.
COULD IT BE TELOMERASE ACTIVITY
IS DIFFERENT FROM EVERYTHING WE
KNEW AND THEREFORE IS NOT REALLY
RELEVANT FOR WHAT WE WANT TO
STUDY.
IN OTHER WORDS, CAN WE TEST
ANYTHING?
CAN WE DEPLETE ANYTHING TO HAVE
AN EFFECT ON TELOMERASE IN HUMAN
CANCER CELL LINES IS IMPORTANT.
FOR THIS WHAT WE DID WAS TO
DELETE TPP1 THE PROTEIN THAT
BINDS TO TELOMERES AND SHOWN TO
BE ESSENTIAL FOR TELOMERASE
ACTIVITY AND IT WORKS NICELY.
IF YOU DON'T HAVE TPP1 WE DON'T
HAVE INCORPORATION OF THE MUTANT
REPEAT AND IN THE WILD TYPE WE
SEE IT AND JUST A QUANTIFICATION
IF YOU DELETE A SECOND PROTEIN
BOUND TO TELOMERES IT SHOWS IT'S
NOT DELETING AS AN EFFECT AND
NOW WE'RE IN BUSINESS AND HE CAN
TAKE ALL THE CANDIDATE GENES AND
PUT THEM IN HIGH THROUGHPUT
FORMAT IN A SINGLE CELL CYCLE
WHETHER OR NOT THEY HAVE A ROLE
IN TELOMERE ELONGATION.
IT DOES THIS USING AN INDUCIBLE
SYSTEM CREATED INDUCED
EXPRESSION OF THE MUTANT
TELOMERASE AND LOOK DAYS AFTER
FOR INCORPORATION.
I THINK IT WILL A POWERFUL TOOL.
IT'S ONGOING.
I DON'T HAVE THE ANSWER BUT I
THINK IT WOULD BE INTERESTING
FOR A LOT OF YOU.
ANYBODY THAT IS INTERESTED IN
TELOMERASE ACTIVITY PLEASE COME
AND TALK TO ME BECAUSE IT CAN
SCREEN AND WE'RE EXCITED ABOUT
THIS.
HOPEFULLY WHEN WE COMPLETE THIS
WE'LL HAVE A BETTER IDEA HOW
THIS WORKS AND WHAT ARE FACTORS
THAT CAN RECRUIT THEM TO
TELOMERASE ENDS.
THE LAST THING I'LL TELL YOU
ABOUT IS ABOUT SOMETHING WE'VE
BEEN WORKING QUITE A LOT OF WITH
AND NOT TELL EVERYTHING AND THE
KEY IS UPREGULATED IN WAYS WE
DON'T UNDERSTAND IN ROUGHLY 50%
OF TUMORS.
GENETICALLY THERE MUST BE
SOMETHING THAT TRIGGERS THE
PATHWAY.
SO WE KNOW THAT GENETICALLY
USUALLY L POSITIVE CELLS HAVE
EXPRESSION AND IT'S A CHAPERONE
PROTEIN AND THE MUTATION ARE NOT
SUFFICIENT TO TRIGGER SO THERE'S
WE DON'T UNDERSTAND ABOUT THE
BIOLOGY.
BUT I THINK FROM A MORE
PRACTICAL POINT OF VIEW WOULD BE
COOL TO BE ABLE TO TARGET THIS
PATHWAY.
SO WE HAVE REALLY NO WAY OF
TARGETING THE PATHWAY SO IF
THERE'S A UNIQUE GENETIC
SENSIBILITY IN THESE KIND OF
CELLS WE DON'T KNOW AND ONE
REASON WE DON'T KNOW IS WE
REALLY STILL DON'T UNDERSTAND
FULLY THE PATHWAY OF TELOMERE
ELONGATION IN THE CELLS.
HERE COMES A Ph.D. STUDENT IN
THE LAB THAT JOINED ROUGHLY A
YEAR AND A HALF AGO AND ASKED
WHAT WE DO TO PERFORM GENOME
WIDE SCREENS TO SEE IF WE COULD
FIND ANY GENE THAT COULD KILL
POSITIVE CELLS BUT NOT NEGATIVE
CELLS.
THERE'S NOT MANY CELL LINES
AVAILABLE THAT ARE L POSITIVE
AND YOU HAVE A PROBLEM WITH THE
CONTROL AND WHAT YOU'LL COMPARE
IT TO.
USUALLY SO YOU COULD COMPARE IT
TO S CELLS THE SARCOMA CELL LINE
AND THESE ARE TWO COMPLETELY
DIFFERENT TUMORS.
YOU'RE MOST LIKELY GOING TO FIND
CELL TO CELL VARIATION RATHER
THAN ANYTHING TO YOUR PHENOTYPE
OF INTEREST PATH IN THIS CASE.
WE'VE BEEN THINKING WHAT WE CAN
USE AND MOUSE IS NOT A MODEL TO
USE BECAUSE MICE HAS LONG
TELOMERES THAT NEVER INDUCE ALT
AND WHEN I JOINED NCI MY
NEIGHBOR WSTUDIED DOGS AND
SARCOMA IN DOGS AND DOGS ARE A
BEAUTIFUL SYSTEM FOR CANCER
BIOLOGY AND FOR ALT IN OUR CASE.
THE REASON IS THEY DEVELOP ALSO
SARCOMA AND THERE'S A FRACTION
OF L POSITIVE CELLS.
IN ADDITION TO THIS, TELOMERES
IN DOGS ARE SIMILAR TO US.
ROUGHLY HAVE 10 KILO BASES AND
TELOMERASE SHUTS DOWN LIKE IN US
MAYBE THE DOG IS THE BEST SYSTEM
AND YOU DON'T HAVE TO HOUSE THE
DOGS.
PEOPLE CARE ABOUT DOGS SO THEY
HOUSE THEM THEMSELVES.
SO WE PAIRED WITH AIMY AND WHAT
WE DECIDED TO DO IS FIRST OF ALL
TRY TO SEE CAN WE DETECT ALT AND
POSITIVITY IN TUMOR CELLS OR
CANCER CELLS AND GIANNA TOOK
EVERYTHING AMY GAVE HER AND THIS
IS THE GOLDEN STANDARD FOR
DETECTING THE ROLE IN CYCLE PCR
TO SEE TELOMERIC DNA IN FREE
FLOATING CIRCLE FORM AND WE HAVE
A HUMAN SARCOMA CELL LINE WHICH
IS WHAT ALMOST EVERYBODY USES
WHEN THEY WORK WITH ALT AND WE
HAVE ER POSITIVE CELL LINES IN
HUMANS.
SO THE POSITIVE IS A DARK SIGNAL
FOR THE CELLS AND WE FOUND AND
GIANNA FOUND TUMOR SAMPLES THAT
ARE L POSITIVE IN THE DOGS AS
WELL AS CANCER CELL LINES
PRIMARY CANCER CELL LINE DERIVED
FROM DOGS FROM THE TUMORS THAT
ARE L POSITIVE AND A LOT OF L
NEGATIVE SAMPLES.
SO THE BEAUTY OF THIS IS AMY
ALSO COLLECTED A LOT OF
TRANSCRIPTIONAL DATA FROM THE
SAME CELLS.
SO WE HAVE A LOT OF INFORMATION
ABOUT THIS.
WE HAVE INFORMATION IN TUMORS AS
WELL AS IN THE CELL LINES.
SO NOW WE ARE IN THE PERFECT
POSITION TO ASK AND SORRY, THE
ADDITIONAL POSITIVE AND NICE
THING ABOUT DOGS IS GENETICALLY
THEY'RE CLOSE TO EACH OTHER THAN
WE ARE.
OKAY.
SO BASICALLY NOW WE ARE IN THE
PERFECT SITUATION OR SHE'S IN
THE PERFECT SITUATION TO PERFORM
THE SCREEN.
OF COURSE WE NEEDED A DOG,
GENOME WIDE LIBRARY AND WE WERE
IN THE PROCESS OF MAKING THIS
OURSELVES BUT WE WERE LUCKY TO
FIND A COLLABORATOR THAT
GENERATED THE LIBRARY AND GAVE
IT TO US AND NOW WE HAVE THE
LIBRARY.
SO NOW EVERYTHING IS SET TO
SCREEN THIS CANCER CELL LINES.
DOG CANCER CELL LINES AND FIND
ANYTHING THAT IS ESSENTIAL IN
POSITIVE BUT NOT L NEGATIVE
CELLS.
AND OF COURSE THE FINAL GOAL OF
THE PROJECT WOULD BE THEN TO
CROSS THIS WITH SCREENS IN HUMAN
CELLS AND WITH THE GENETIC DATA
OUT THERE WITH HUMAN CELLS AND
POTENTIALLY FIND COMMON TIPS.
SO WITH THIS I CONCLUDE JUST BY
AGAIN REMINDING YOU WHAT I TOLD
YOU.
I TOLD YOU HOW TELOMERES ARE
PROTECTIVE AND MAYBE IN CANCER
CELLS TO OVERCOME PROBLEMS WITH
TELOMERES.
I DIDN'T TELL YOU HOW IT WORKS
BUT WHAT WE'RE DOING TO TRY TO
UNDERSTAND THE MECHANISM OF
TELOMERE ELONGATION IN CANCER
CELLS.
IF YOU'RE INTERESTED IN THIS,
COME TALK TO ME AND I LOVE
TALKING ABOUT TELOMERES AND
ANYTHING ELSE.
AND I TRIED TO THANK EVERYBODY
IN THE LAB FOR THE DATA I
SHOWED.
I COULDN'T SHOW THE WORK OF SOME
BUT I WANT TO SHOW I HAVE THIS
AMAZING GROUP OF PEOPLE THAT
KEEPS WORKING IN A DIFFICULT
TIME AND IT'S BEEN A PLEASURE TO
WORK WITH THEM AND IN ADDITION
THERE'S A LOT OF PEOPLE THAT I
SHOULD THANK AND IT'S A
FANTASTIC PLACE IT DO SCIENCE.
THANK YOU.
>>UNFORTUNATELY WE CAN'T TAKE
QUESTIONS FROM THE PEOPLE THAT
ARE OUTSIDE BUT HAPPY TO TAKE
QUESTIONS.
>>THANK YOU.
THE DOG MODEL'S REALLY
ATTRACTIVE.
IT'S UNIQUE TO NCI SO GOOD
RESULT.
SINCE HAVE THE GENOMICS, I'M
CURIOUS WHETHER THE TUMORS ALT
BY THE PCR BASE WHETHER THEY
LOSE THE EXPRESSION.
I LOOKED AT THE GENES YOU HAD ON
YOUR LIST BUT THERE WAS NO CHART
AND I WOULD ASSUME THE TUMOR IS
TELOMERASE POSITIVE IT SHOULD
HAVE TERT POSITIVITY IN THE
EXPRESSION AND THE ALT SHOULD BE
TERT NEGATIVE HAVE YOU LOOKED AT
THE TERT IN THE TUMORS.
>>I'VE BEEN TOLD I HAVE TO
REPEAT THE QUESTION FOR THE
PEOPLE ONLINE.
SO THE QUESTION IS BASICALLY IN
THE ALT TUMORS -- SORRY, IN THE
DOG TUMORS IF WE COMPARE
EXPRESSION LEVEL DID WE FIND
TELOMERASE AND EXPRESSION IN THE
POSITIVE AND NEGATIVE.
IN THE CASE OF THE DOG, I DIDN'T
MINE THE DATA SO THE DATA I HAVE
IS WHAT A SHOWED YOU BUT YOU
SHOULD BE ABLE TO.
IF YOU GO TO DETMAP AND LOOK AT
THE -- TRY TO STRATIFY TUMORS
BASED ON THE TELOMERE LENGTH,
TELOMERASE SAY -- IS A GOOD WAY
TO DO IT AND IT'S NOT EXPRESSED
HIGHLY SO YOU'LL HAVE TUMORS
THAT CANNOT BE CHARACTERIZED.
YOU'LL FIND THE BOTTOM 1% AND
EVERYTHING IN THE MIDDLE WILL BE
QUESTIONABLE.
>>THE REASON I'M ASKING IS
BECAUSE I LOOKED AT THE CANCER
CELL LINES AND IT'S VERY CLEAR
THIS WAS BLACK AND WHITE.
WE CAN DISCUSS FURTHER TOGETHER
BUT IT WOULD BE NICE TO MINE THE
GENOMIC DATA FROM THE DOGS AND
SEE IF THAT CAN BE USED BECAUSE
IT COULD BE AN EASY WAY TO DO
RNA SEQ AND WHAT IS POSITIVE AND
NEGATIVE AND THEN WHAT YOU SAID
IS IT THERAPEUTIC AND SOMETHING
WE CAN DEAL WITH.
>>FOR SURE.
THAT'S A GOOD WAY.
IT'S NOT -- AGAIN, THERE'S A
GRAY AREA AND WHEN I DID DATA
MINING ON HUMAN CELLS AND I'M
NOT SURE HOW TO CHARACTERIZE.
THERE'S A LOT OF TUMORS IN THE
MIDDLE.
>>THANKS.
>>HI, EROS.
WHAT DO YOU THINK IS UNIQUE TO
THE EMBRYONIC CELLS THAT ALLOWS
THEM OR WHAT ADVANTAGE THEY HAVE
FOR NOT REGULATING THEIR
TELOMERES IN THE SAME WAY AND
ALSO DO YOU THINK THE SAME
MECHANISM IS AT PLAY IN HUMAN
EMBRYOS OR EMBRYONIC CELLS?
>>AGAIN, I'M REPEAT.
THE QUESTION IS ABOUT BASICALLY
IT'S WHAT IS UNIQUE ABOUT THE
CELLS AND WHY THEY DON'T SEEM TO
CARE SO MUCH ABOUT THIS
PROTECTIVE PROTEINS AND THE
SECOND PART OF THE QUESTION IS
WHETHER HUMAN CELLS BEHAVE IN
THE SAME WAY.
SO THE SECOND QUESTION IS ARE
REALLY EASY TO ANSWER AND THE
ANSWER IS I DON'T KNOW.
WE DIDN'T DO IT SO WORKING ON
CELLS HERE IT'S CHALLENGING.
SO WE STARTED A COLLABORATION
WITH A LAB IN WASH-U AND SO FAR
HE HASN'T BEEN ABLE TO GENERATE
TR2 KNOCKOUT CELLS BUT I CANNOT
KNOW IF THAT'S BECAUSE YOU CAN'T
OR -- SO THE FIRST QUESTION IS
THE MILLION-DOLLAR QUESTION,
LIKE WHY.
AND I DON'T KNOW.
THE TRUTH IS I DON'T KNOW.
IT COULD BE ONE POTENTIAL ISSUE
THERE IS THE CELLS, A, REALLY
SHORT SO THE CELLS DON'T STICK
AROUND VERY LONG.
WE ARTIFICIALLY BLOCK THEM IN
THIS SITUATION AND DURING EMBRYO
GENESIS WE HAVE THE BOOST OF
PROLIFERA
PROLIFERATION.
YOU MAY WANT TO FIND WAYS TO NOT
DEAL WITH THE CONVOLUTED WAY OF
PROTECTING TELOMERES FOR
INSTANCE IF YOU'RE THINKING OF
SECONDARY STRUCTURES AND THAT
COULD BE ONE EXPLANATION.
THE OTHER COULD BE INSTEAD OF
BEING A TELOMERE SPECIFIC THING
WE'RE SEEING, MAYBE WE'RE JUST
LOOKING AT SOMETHING MORE
GLOBAL.
FOR INSTANCE, THE CELLS ARE
KNOWN TO HAVE A WEIRD, LET'S
SAY, DNA DAMAGE RESPONSE.
IT COULD BE IN GENERAL THEY
DON'T RESPOND AS NICELY TO DNA
DAMAGED CELLS.
DOESN'T SEEM TO BE TOTALLY TRUE
FOR DOUBLE STRAND BREAKS AND
HAVE CANONICAL DNA RESPONSE BUT
THERE MAY BE THINGS WE DON'T
SEE.
>>BEAUTIFUL STORY.
THIS MAY BE A NAIVE QUESTION.
I WAS STRUCK BY THE PHENOTYPIC
DIFFERENCE BETWEEN THE CELLS WHY
YOU SEE LONG STRINGS OF INFUSION
AND YOU HAVE TWO OR THREE
CHROMOSOMES FUSING DO YOU THINK
IT'S A GAIN OF FUNCTION
PHENOTYPE OR LOSS OF FUNCTION
PHENOTYPE BECAUSE THE CRISPR
SCREEN SUGGESTS LOSS OF FUNCTION
GIVEN THE BIOLOGY BUT IS THAT A
REASON TO THINK THAT OR IS THERE
A POSSIBILITY THERE'S SOMETHING
NEW IN THE DIFFERENTIATED CELLS?
>>SO THE QUESTION HAS TO DO
WITH PRETTY MUCH THE AMOUNT OF
FUSIONS WE SEE.
SO IN THE SOMATIC CELLS WE SEE
LONG STRAINS OF SPAGHETTI LIKE
FUSIONS AND IN THE ESL CELLS WE
CAB GET FUSIONS WE GET FEW.
SO WHY IS THAT?
AND DON'T KNOW.
WE DON'T HAVE THE PROOF YET
HOWEVER, I THINK THERE'S A
COUPLE THINGS.
SO THE DIFFUSION ARE MAINLY
OCCURRING IN G1.
SO IT COULD BE IN THE EMBRYONIC
STEM CELLS WHEN YOU GET THE
FUSION FROM THE CRISPR SCREEN
THEY'RE STILL OCCURRING TUMORS
BUT THE G1 AND S CELLS ARE
RELATIVELY SHORT TO SOMATIC
CELLS.
YOU MAY NOT HAVE IF YOU HAVE
TIME.
IF YOU THINK ABOUT IT FROM A
PROBABILITY POINT OF VIEW YOU
HAVE TO FIND A PROTECTED
TELOMERE YOU HAVE TO FIND
ANOTHER AND AND ANOTHER ONE AND
ANOTHER ONE YOU MAY NEED MORE
TIME FOR THIS TO HAPPEN.
IT MAY BE A BORING EXPLANATION
BUT HOWEVER, IT COULD BE THERE'S
SOMETHING ELSE.
IDEALLY THAT'S WHY WE KEEP DOING
THE SCREENS.
IDEALLY I'D LIKE TO GET TO THE
POINT WHERE EVERYTHING FUSES AND
THEN I'D BE SATISFIED I FOUND
EVERYTHING.
>>THANK YOU.
>>I HAVE A QUICK QUESTION.
FIRST QUESTION OUT OF CURIOSITY
HAVE YOU LOOKED FOR THINGS THAT
ARE ESSENTIAL IN WILD TYPE CELLS
BUT STOP BEING ESSENTIAL IN
HUMAN CELLS PROTECTED.
>>THE QUESTION IS WHERE ARE WE
IN ADDITION TO LOOKING FOR
SYNTHETIC AND LOOKING FOR GENES
SYNTHETICALLY BOUND WITH THE
CELL TYPES.
AND IF YOU NOTICE BASICALLY THE
ESL CELLS WHEN WE DELETE EVEN IF
THEY SURVIVE AND DON'T GROW AS
WELL AS THE WILD TYPE.
ONE OF THE REASON WE DID THE
SECOND SCREEN WAS ALSO FINDING
THINGS THAT WOULD MAKE THE CELLS
GROW FASTER.
THAT COULD GIVE AN INDICATE OF
WHAT WAS HAPPENING.
AND WE DID FIND SOME GENES SO A
LOT OF CHROMATIN MODIFYING GENES
BUT DIDN'T FOLLOW-UP AND WE
DIDN'T VERIFY IT.
WE DON'T KNOW IF THAT'S THE CASE
BUT IT'S INTERESTING.
>>IT'S VERY INTERESTING.
AND SECOND QUICK QUESTION SO TO
FIND THE PROTEINS THAT HAVE
TELOMERE TO FIND THE ENDS OF
CHROMOSOMES YOU DID LIGATION
WITH TPP1 BUT WHY COULDN'T YOU
JUST USE TELOMERE BASED PROTEIN.
>>THE QUESTION IS FOR THE
PROTEOMICS WHY GO WITH TPP1
RATHER THAN TELOMERASE.
THE ANSWER IS TARGETING
TELOMERASE IS TRICKY.
YOU CAN AFFECT DYSFUNCTION.
THERE'S A LOT OF REPORTS ON THAT
SO WE DID NOT WANT TO TOUCH
TELOMERASE IF POSSIBLE.
>>OKAY.
>>SO THE OTHER THING WE
CONSIDERED DOING WAS INSTEAD
TARGETING THE RNA.
YOU CAN TAG THE RNA COMPONENT,
YOU KNOW AND THE FACTORS THERE
BUT TPP1 WAS THE EASIEST WAY TO
DO IT.
>>I SEE.
THANK YOU.
>>JUST WITH YOUR VERY COOL
ASSAY FOR MUTANT TELOMERASE
ADDITION, CAN YOU -- HAVE PEOPLE
STUDIED THE PROCESS THE CRISIS
THAT THEN LEADS TO RE-EXPRESSION
OF TELOMERASE AND BECAUSE THAT'S
WHAT HAPPENS RIGHT AFTER THE
PROCESS YOU GET RE-EXPRESSION
AND WONDERING ABOUT THE
FREQUENCY OF THAT IN VITRO.
>>I SEE -- SO IT'S A GOOD -- SO
BASICALLY WE -- WE DIDN'T DO IT.
YOU COULD DO IT.
SO ONE OF THE ISSUE WITH THAT IS
IN SOMATIC CELLS EXPRESSION OF
THAT SO WHENEVER THE MUTANT
WOULD BE EXPRESSED IT COULD
BECOME TOXIC.
SO YOU MIGHT BE SELECTING OUT
YOUR CELLS.
BUT IF YOU ONLY LOOK AT
SHORT-TERM IT COULD BE --
>>AND IN YOUR ESLs, DID YOU
LOOK FOR DEPLETION OF TRF
PROTEINS OR WOULD THAT TAKE TOO
LONG BECAUSE THAT'S YOUR
PREDICTION, RIGHT, YOU WOULDN'T
HAVE THE BINDING, YOU MENTIONED
THAT BUT PROBABLY SEE ROBUST
BINDING.
>>WE DIDN'T DO LIKE TO SEE IF
WE COULD FIND THE MUTANTS BUT
AGAIN WE DIDN'T INVENT THE
MUTANT RNA ASSAY.
SO THERE'S A LOT OF WORK DONE IN
VITRO AND SUPPOSEDLY NONE OF THE
PROTEINS BIND.
>>DOES IT DECREASE THE BINDING?
>>IT SHOULD DECREASE THE
BINDING.
WE DID IT IN THE MUTANT.
>>EVEN IN THE SHORT-TERM
BECAUSE TELOMERES ARE VERY LONG.
>>YOU WOULD DO A STAINING YOU
WOULD STAY AND YOU MAY HAVE 45KB
OF WILD TYPE AND A LITTLE BIT OF
DEN.
>>SO AS YOU SAID THE ALTERNATE
TELOMERE ASSAY HAS BEEN DONE BY
OTHERS IN OTHER TYPES OF CELLS.
I'M WONDERING IF YOU SEE ANY
CORRELATION BETWEEN THE -- IF
THERE'S ANY CORRELATION BETWEEN
THE LENGTH OF THE PRE-EXISTING
TELOMERES AND THOSE THAT ARE
CHOSEN TO HAVE NEW SEQUENCES
ADDED.
OF COURSE NOT EVERY SPOT WAS
LABELLED.
AND WHETHER THAT'S THE SAME IN
ESL CELLS AS IT IS IN OTHER CELL
LINES.
>>YES.
SO THE QUESTION HAS TO DO WITH
BASICALLY I'M GOING TO SLIGHTLY
REPHRASE IT.
CAN WE USE THE MUTANT TELOMERE
EASY TO STUDY THE ELONGATION AND
ACT ON THE SHORTEST TELOMERE
NOT.
SO THERE ARE INDICATIONS THAT'S
THE CASE.
IN OUR ASSAY WE DIDN'T DO THAT
YET.
SO AGAIN, THIS IS FAIRLY NEW.
BUT YOU CAN DEFINITELY DO IT AND
ONE THING -- ONE COOL THING WE
DID WAS TO USE ANDRE'S ASSAY TO
SEE WHETHER THE MUTANTS ARE AT
THE VERY END OR NOT OR HOW LONG
IT IS.
THINK OF FOR INSTANCE, MEASURING
THE LENGTH OF THE PROGRESSIVE
TELOMERASE.
THESE ARE THINGS WE'RE THINKING
OF DOING.
WE'RE IN THE PROCESS OF DOING IT
BUT THOSE ARE GOOD POINTS.
YEP.
>>SO I WANT TO THANK EROS AGAIN
FOR A BEAUTIFUL TALK AND THE
KIND OF TALK WHERE YOU WANT TO
INVITE HIM BACK.
IT'S PRETTY EXCITING.
YOU WANT TO KNOW WHAT THE
RESULTS ARE GOING TO BE IN A
YEAR, RIGHT.
I DON'T KNOW WHO ORGANIZES THIS
BUT CAN WE INVITE HIM BACK?
GREAT.
>>THANK YOU.
>>THANK YOU, THANK YOU.