>> WE HAVE A PROUD RANGE OF TIME
ZONES, SO GREETINGS TO YOU.
BEFORE I INTRODUCE OUR SPEAKER,
FOUR THINGS I WANT TO TELL YOU
ABOUT.
FIRST, I WANT TO THANK OUR
ADVISORY BOARD FOR ALL OF OUR
ADVICE AND GUIDANCE AND OUR A.V.
PEOPLE WHO MAKE THINGS WORK
SEAMLESSLY.
SECONDLY AND VERY IMPORTANTLY,
WE ARE COLLECTING NOMINATIONS
FOR STELLAR YOUNG INVESTIGATORS
TO SPEAK IN OUR ANNUAL YOUNG
INVESTIGATORS SHOWCASE THIS
YEAR.
THOSE OF YOU WHO WANT TO
NOMINATE SOMEBODY, PLEASE E-MAIL
US WITH YOUR NOMINATIONS WITH A
SHORT BIO WITH JUSTIFICATION,
THE SOONER THE BETTER.
THE REGISTRATION DEADLINE FOR AN
UPCOMING DNA STRUCTURES IN
BIOLOGY CONFERENCE THAT FOCUSES
ON UNUSUAL DNA STRUCTURES AND
THE EFFECTS OF TRANSCRIPTION,
REPLICATION AND REPAIR, THE
EARLY REGISTRATION DEADLINE IS
PAST, BUT YOU CAN STILL MAKE IT
IF YOU REGISTER SOON.
LATE REGISTRATION IS COMING UP
ON JULY 1.
AND THEN FINALLY, I WANT TO
MENTION THAT THE EARLY BIRD
REGISTRATION FOR THE 55th ANNUAL
EMGS MEETING ENDS ON JULY 15, SO
GET IN THERE WHILE YOU CAN.
NOW ON TO TODAY'S SEMINAR.
OUR SPEAKER TODAY IS DR. SHARON
BETH CANTOR.
DR. CANTOR HAS BEEN ON THE
FACULTY AT UMASS MEDICAL SCHOOL
SINCE 2003 WHERE SHE IS THE GLAD
SIS SMITH MARTIN CHAIR OF
ONCOLOGY AND PROFESSOR OF
MOLECULAR CELL AND CANCER
BIOLOGY.
SHE IS THE CO-DIRECTOR OF THE
PROGRAM GENOME INTEGRITY WHICH
SHE FOUNDED IN 2024.
DR. CANTOR RECEIVED HER PH.D. IN
BIOCHEMISTRY FROM TUFTS
UNIVERSITY AND COMPLETED
TRAINING AT DANA HARBOR CANCER
MEDICAL INSTITUTE AND HARVARD
MEDICAL SCHOOL.
SHE IS KNOWN FOR HER RESEARCH IN
BREAST AND OVARIAN CANCER GENES,
RESEARCH FOCUSED ON BROICA 1, BA
2, AND A GENE SHE IDENTIFIED AND
CLONED.
HER RESEARCH HAS CHALLENGED THE
CONVENTIONAL UNDERSTANDING OF
BRCANESS AND DNA REPAIR AND
TRANSLATION SYNTHESIS AS A
VULNERABILITY IN KEASHS.
HER SEMINAR IS DEFINING THE GAP
VULNERABILITY IN BRCA CANCER.
I JUST WANT TO REMIND EVERYONE
IN THE AUDIENCE TO PLEASE PUT
YOUR QUESTIONS IN THE CHAT AND
WE WILL, CHUN ZHANG AND I WILL
POST THE QUESTIONS TO SHARON
AFTER HER TALK.
SHARON, WITHOUT FUR ADO.
>> OKAY.
I THINK I GOT THAT.
ALL RIGHT.
HOPEFULLY YOU CAN SEE IT OKAY.
LET ME START SHARING.
IS IT SHARING?
>> NO.
NOT YET.
>> OH, THAT'S INTERESTING.
>> IT IS SHARING, BUT NOT
PRESENTING.
>> YES, THAT IS WHAT I MEAN.
SORRY.
YOU ARE NOT IN PRESENTER VIEW.
>> I'M JUST TRYING TO GET MY
CURSOR HERE.
OKAY.
ARE WE ALL SET?
IS THAT WORKING?
>> YES.
>> THANK YOU SO MUCH, CHUN
ZHANG, AND KAREN FOR THE
INVITATION TO PRESENT TODAY.
TO GET STARTED, I WILL JUST GIVE
YOU AN OVERVIEW OF WHAT I HOPE
TO COVER TODAY.
I WILL GO THROUGH THE
TRADITIONAL MOD OF BRCANESS AND
COMPARE TO THE GAP MODEL.
AND LOOK AT BOTH FOR THEIR
RELEVANCE TO CHEMO THERAPY
RESPONSE AND BETTER DEFINE THE
GAP VULNERABILITY.
SO WHEN WE THINK ABOUT
CHEMOTHERAPY, MECHANISMS OF
ACTION FOR THE LAST 30, 40 YEARS
TYPICALLY IT EVOKES A DOUBLE
STRANDED BREAK I'M SHOWING YOU
THAT LEADS TO THE SENSETIZATION
AND THE KILLING OF THE CANCER
CELLS.
THIS HAS BEEN WELL DESCRIBED
OVER THE YEARS.
THERE ARE SEVERAL REASONS, BUT
HER REDTY BREAST/OVARIAN GENES
WERE MISSING, HIGHLY SENSITIVE
TO CHEMOTHERAPY.
THE BEST KNOWN FUNCTIONS FROM
THESE PROTEINS FROM THEIR
ORIGINAL IDENTIFICATION FOR
FUNCTIONS IN REPAIRING DNA
DOUBLE STRAND BREAKS INVOLVED A
RAD 51 RECOMBINATION PROTEIN.
AND PROTECTING REPLICATION THAT
STALL AND REVERSE WITH RAD 51,
PROTECTING THEM FROM DEGRADATION
THAT WOULD BE EXPECTED TO LEAD
TO DOUBLE STEM BREAKS AND THE
FORMATION THAT HAS BEEN THOUGHT
TO BE THE FUNDAMENTAL NATURE OF
BRCANESS THAT I'M DEPICTING HERE
AS A CANCER CELL THAT CANNOT
HANDLE DOUBLE STRAND BREAKS
INDUCED BY CHEMOTHERAPY.
COMING FULL CIRCLE WITH THIS
MODEL, IT WAS SHOWN QUITE
CLEARLY THAT CHEMO RESISTANCE
WAS RESTORED HOOGEN COMBINATION
OR FOR PROTECTION.
WHY NOT CONSIDER THIS MODEL TO
BE SOLID?
WELL, IT HAS SEVERAL
EXPECTATIONS.
ONE OF THEM, OF COURSE, IS
CHEMOTHERAPY SHOULD GENERATE DNA
DOUBLE-STRANDED BREAKS.
WE LOOKED AT WHAT THE LITERATURE
REPORTED OVER THE YEARS AND WHAT
YOU'LL NOTE IS THAT EVEN WITH A
TOPSIDE AND IONIZING RADIATION,
THERE ARE FAR GREATER
SINGLE-STRANDED DNA THAN A
DOUBLE STRAND BREAK.
THE IDEA THAT THE DOUBLE STRAND
BREAK IS THE PREDOMINANT
DIFFICULT LESION HAS PERMEATED
THE FIELD INCLUDING THIS
RADIOLOGY TEXTBOOK WHO DISMISSED
THE SINGLE STRANDED DNA BREAK
NOT HAVING PROMINENCE.
COMING BACK TO EXPECTATIONS,
AGAIN, THE SOURCE OF THE DOUBLE
STRAND BREAK AGAIN IS A LITTLE
BIT MORE ELUSIVE.
AGAIN, THE SINGLE STRANDED DNA
BREAK BEING THE MORE COMMON
LESION.
IF YOU HAD THESE FUNCTIONS, IF
YOU DIDN'T HAVE THEM YOU SHOULD
BE SENSITIVE AND IF YOU DID
RESTORE THEM CONFIRM RESISTANCE.
THE GENES THAT REGULATE THESE
RESPONSES WERE NOT OVERLY
PREDICTIVE NOR IS THE HRD SCORE,
PERFECT BIOMARKER OF RESPONSE.
AND SO WE STARTED TO FOCUS ON
THIS OTHER FUNCTION OF THE BRCA
PATHWAY IN WHAT WE ARE CALLING
REPLICATION GAP SUPPRESSION OR
GAP SUPPRESSION.
AND SO HERE WE ARE NOT ALONE.
THIS AREA HAS REALLY EXPLODED.
MANY LABS HAVE CONTRIBUTED HERE
THROUGHOUT THE YEARS.
INCLUDING LOOKING AT THESE
PATHWAYS IN FROG EXTRACTS AND
REALLY ACROSS THE WHOLE SPECTRUM
OF THE PATHWAY THERE IS ROLES
FOR GAP SUPPRESSION.
SO WHAT REALLY CONVINCED US THIS
WAS AN IMPORTANT PATHWAY WAS A
TABLE LIKE THIS WHERE WE WOULD
LOOK AT A SERIES OF CANCER CELL
LINES WELL CHARACTERIZED IN THE
FIELD FOR HR -- WAS SUPPORTED.
WE LOOKED THAT FULLY ALIGNED
WITH RESISTANCE AND LOSS OF GAP
SUPPRESSION OR THE PRESENCE OF
GAPS FULLY ALIGNED WITH
CHEMOTHERAPY RESPONSE.
THIS OFFERS FOR CISPLATIN AND
THIS MODEL HELD UP.
EVEN IF PEOPLE DIDN'T LIKE THIS
MODEL, YOU HAVE TO AGREE GAPS
ARE HIGHLY PREDICTIVE OF
RESPONSE.
SO THIS TOGETHER LED US TO PUT
TOGETHER THIS NEW FRAMEWORK THAT
A HEALTHY CELL IS ABLE TO
WITHSTAND CHEMOTHERAPY BECAUSE
IT HAS THE ABILITY TO SUPPRESS
AND REPAIR REPLICATION GAPS
WHEREAS A BRCA WAS COMING FROM
INABILITY TO TAKE CARE OF THIS
AND CHEMORESISTANCE WAS RESTORED
GAP SUPPRESSION AND REPAIR.
OTHERS HAVE, OF COURSE, LOOKED
AT THIS AND HAD DIFFERENT
INTERPRETATIONS.
I WANT TO SHARE THAT WITH YOU
NOW IN A BRIEF SUMMARY.
FOR EXAMPLE, OTHERS HAVE
DETECTED GAPS FOLLOWING
CHEMOTHERAPY SUCH AS SHOWN HERE,
BUT THEY ARGUED IT IS THE SECOND
S FACE WHERE THESE DOUBLE STRAND
BREAKS FORM IS THE REAL KILLING
LESION.
WE ARE SAYING THIS IS THE
CRITICAL JUNCTION WHERE BRCA 1
PROTEINS ARE PROTECTING FROM
CHEMOTHERAPY.
WE WOULD AGREE THESE DRUGS MAKE
DOUBLE STRAND BREAKS.
THAT IS TRUE AND EASY TO SEE.
THEY ACTIVATE APOPTOSIS.
IF WE INHIBIT APOPTOSIS, YOU SEE
BREAKS IN KEERM THERAPY.
I SEE DOUBLE STRAND BREAKS
THEREFORE THAT IS THE
SENSITIZING LESION, SO I THINK
THERE IS MORE WORK TO DO TO
SOLVE THIS MYSTERY.
WE HEARD FROM ANOTHER GROUP THAT
BRCA'S PRIMARY ROLE IS IN HR.
SHE, OF COURSE, HAS PIONEERED
REPLICATION FWAPS THROUGHOUT HER
STUDIES.
SHE IS ABLE TO SEPARATE THIS
THROUGH A BRCA 2C2 TERMINATION
IN MOUSE OR IN HUMAN WITH A
SLIGHTLY DIFFERENT NUMB.
HERE USING THIS SORT OF STANDARD
DNA FIBER ASSAY, HER WILD TYPE
CELLS ARE NOT SHOWING ANY
CUTTING WHEN YOU ADD S1NUCLEAS.
WHEN SHE ADDS THE S1 IN THE BRCA
2 MUTANT, SHE IS SEEING S1
CUTTING SUGGESTING THIS MUTANT
ACROSS A SERIES OF CELL LINES
HAS REPLICATION GAPS.
SO THE ISSUE HERE IS WE ACTUALLY
DIFFER AND WE LOOKED AT THIS AS
WELL.
WE DID NOT PERFORM S1 ANALYSIS.
WE PERFORMED A DIFFERENT STUDY,
BUT WE DIDN'T SEE GAPS IN THIS
MUTANT.
SHE LOOKED HYDROXYREA.
THIS SHOWS DISPLAY RESISTANCE
AND HR PROFICIENCY AND FOR
PROTECTION DEFICIENCY, THE GAP
STILL REMAINS TO BE FULL YVETTED
-- FULLY VETTED.
THIS CAME OUT COVERING THESE
OUTSTANDING ISSUES IN THIS GAP
SPACE.
SO IF YOU ARE INTERESTED.
SHE HAS CONCLUDED THERE IS TOO
MUCH CLOSELY INTERTWINED
FUNCTIONS THAT WAS GOING TO BE
DIFFICULT TO TAKE THESE APART.
SO THIS IS, OF COURSE, WHERE WE
ARE TRYING TO WORK AND SETTLE
SOME ISSUES.
WE ARE TRYING TO DEVELOP A MORE
ROBUST AND UNIVERSALLY
ACCESSIBLE STANDARDIZED GAP
DETECTION ASSAY.
BECAUSE WE SEE UP WITH OF THE
ISSUES THAT PEEL APPLY DIFFERENT
AMOUNTS OF PARP INHIBITOR AND
DIFFERENT AMOUNTS OF TIME AND
HOW THEY PORTRAY THE DATA.
SO IT IS TRICKY TO COMPARE
ACROSS PAPERS.
WE ARE WORKING ON SOMETHING THAT
WE HOPE IS EASIER AND COMING
ONLINE IN THE FUTURE.
WE WILL EXAM THIS AND LOOK AT
OTHER HR MODELS AND SEPARATE
FUNCTION AND GET A SENSE OF
UNDERLYING THERAPY RESPONSE.
WE HOPE TO BETTER DEFINE THE
GAPS, WHERE THEY COME FROM AND
THEIR ULTIMATE FATE AND HOW THEY
KILL.
FOR THIS TALK I WANTED TO START
BY ASKING WHERE DO YOU, SO DO
GAPS EXPLAIN OTHER MODELS LINKED
TO RESTORED HR?
THIS IS A FAMOUS SPACE HERE.
WE HAVE LOTS OF PAPERS HAVE
DESCRIBED THIS ANTI-RESECTION
PATHWAYS PROPOSED TO DRIVE THE
SENSITIVITY TO DRUGS BECAUSE
THEY INTERFERE WITH DNA M
PROCESSING.
AND SO WE WONDER THEN, COULD WE
TEST THE ESSENTIALLITY OF HR FOR
RESISTANCE IN CELLS WHERE BRCA 1
IS MISSING AND TAKE OUT 50BP1.
WHAT IF WE DEPLETE C TIP-IN THIS
SCENARIO.
IF HR IS ESSENTIAL THE DOUBLE
KNOCKOUT CELL SHOULD BECOME
SENSITIVE.
HERE WE DEPLETED C TIP, WE SEE
IN OUR WILD TYPE AND DOUBLE
KNOCKOUT CELLS.
THE ABILITY OF RPA TO LOAD ON TO
AN END IS GREATLY CURTAILED BY
THE LOSS OF C TIP PRESENTING THE
SINGLE STRANDED DNA FOR RP
LOADING.
WE DISRUPTED RECEPTION.
IF YOU LOOK AT THIS, WE DID NOT
SEE A MASSIVE GAIN OR MUCH OF A
CHANGE WITH C TIP DEPLETION, WE
SAW A MILD ENHANCEMENT IN WILD
TYPE CELLS.
WE WENT FURTHER TO SAY WHAT IF
WE DISTRACT RAD 51 WITH SEVERAL
INHIBITORS.
HERE IT WAS STRIKING THE DOUBLE
KNOCKOUT CELLS WERE MORE
RESISTANT THAN THE WILD TYPE
CONTROL.
THIS DOUBLE KNOCKOUT CELL
REGAINED HR IS NOT HIGHLY
DEPENDENT ON THIS HR FOR ITS
BASIC FITNESS OR INHIBITOR
RESISTANCE.
WE CAN SUM THIS IN THE TABLE.
THESE CELLS DID NOT HAVE
PROTECTION AND WE CAN SEE GAPS
WERE STILL SUPPRESSED.
NOW, IF YOU WANT TO TAKE A BRCA
1 DEFICIENT CELL AND MAKE IT
SENSITIVE TO THERAPY, YOU CAN
GET RID OF LIGASE3.
HE WAS SURPRISED TO SEE HR
REMAINED INTACT, BUT HE COULD
SEE GAPS WERE PRESENT AGAIN.
SO, AGAIN, THIS SUGGESTS THAT
GAPS WERE CENTRAL TO THE THERAPY
RESPONSE.
AND WE WENT AHEAD AND JUST
CONFIRMED THAT.
IT IS QUITE STRIKING LIAGE3,
DEPLEGS WITH TWO DIFFERENT SR
RANNE REAGENTS CONFERS HIGH
SENSITIVE TO THESE CELLS PARPI
INHIBITOR.
WHAT IS THE SOURCE OF GAPS AND
HOW DOES BP1 CREATE THESE GAPS?
THOSE ARE TWO QUESTIONS WE ARE
LOOKING AT QUITE STRONGLY.
SO I WILL START WITH THE FIRST
ONE.
WHEN WE LEARNED THIS BEAUTIFUL
WORK THAT PARP HAS A CRITICAL
FUNCTION IN UNPRETESCHED ASSAYS
AND BASICALLY IN A BACKUP PATH
FILLS IN GAPS THAT THE
CANNONICAL PATHWAY COULDN'T TAKE
CARE OF.
YOU HAD TO KILL THIS PAR G
ENZYME WHICH WILL ACTSIVELY
REMOVE THESE.
YOU HAVE TO INHIBIT AND NOW YOU
CAN SEE THIS IN THE REPLICATION
ON A REGULAR S PHASE.
SO WE PONDERED WHETHER BRCA 1
WAS CONTRIBUTING TO LAGGING
STRAND BIOLOGY.
TO ADDRESS THAT WE SIMPLY ASKED
IN ABSENCE OF BRCA 1 WHAT
HAPPENS TO PARP ACTIVITY?
IT WENT UP.
IT WAS NOTHING LIKE GETTING RID
OF PHEN 1, WHERE YOU GET MASSIVE
PARP ACTIVITY, BUT IT WAS HIGHER
IN BRCA PATHWAY.
THERE COULD BE A ROLE FOR BRCA 1
IN BASIC BIOLOGY AND UNPRETERBE
AND PARP.
WE RECENTLY ASKED WHY THOSE WERE
DEFICIENT, NOT SENSITIVE TO PARP
INHIBITORS.
WE UNCOVERED IT IS CRITICAL TO
GET PARP ACTIVE IN THE S PHASE.
WHAT WE FOUND IN A SUMMARY FORM
HERE IN THE ABSENCE OF FENG J,
DANK CASE, PARP WAS GENERALLY
SEQUESTERED ON A G4 STRUCTURE
AND THEREFORE IT WAS BASICALLY
HELD IN INACTIVE POSITION.
SO YOU HAD LOW S PHASE PARP
ACTIVITY.
THIS WAS SIMILAR TO HAVING NO
PARP OR USING THE PARP
INHIBITOR.
ALL OF THESE SITUATIONS WE FOUND
TO BE TOXIC WITH BRCA DEFICIENCY
WE SAW ELEVATED PARP ACTIVITY.
THIS SUGGESTS IT WAS THIS COMMON
LESS OF S PHASE PARP ACTIVITY
THAT WAS DRIVING THE PARP
SYNTHETIC LETHALITY WITH BRCA
LOSS.
I WOULD SUGGEST TRAPPING HAS A
MORE MINOR ROLE BASED ON THESE
FINDINGS.
OKAY.
SO NOW COMING BACK TO HOW DOES
CREATING GAPS IN CELLS?
WE HAD LOOKED AT THE ACTIVATION
OF THIS PARP IN THE BRCA
DEFICIENT CELLS, WHICH WE COULD
SEE WAS RELATED TO 50BP1, IF YOU
GOT RID IT OF IN DOUBLE KNOCKOUT
CELLS, IT WENT DOUNT.
IF WE GOT RID OF THE 50BP1 WE
COULD SEE IT SOMEWHAT REDUCED
AND CRITICAL DOWNSTREAM TARGETS
OF THIS PARP ACTIVITY NOW SEEM
TO COME BACK INTO THE CHROMATIN.
THIS SUGGESTS WHEN YOU HAVE BOTH
PROTEINS MISSING THIS BACKUP
PATHWAY WAS WORKING BETTER IN AN
UNEREGULATED MANNER.
SO THIS FIT WELL WITH WHAT
YONKERS FOUND, LIGASE THREE AND
XRCC1 ARE TARGETS TO KNOCK OUT
WITH.
OTHERS FOUND FEN-1 INHIBITOR WAS
SENSITIZING TO BRCA CELLS.
YOU COULD RESCUE WITH LOSS OF
BP1 AND THE BACKUP PATHWAY WAS
HIGHLY COMPENSATING HERE.
MORE RECENTLY WE LOOKED AT
LIGASE1 AND RESCUE BY LOSS OF
53BP1.
IT WAS SHOWN BY SYLVIA
NORDAMER'S GROUP, THIS MAKES
SENSE TO US THROUGH THIS BACKUP
PATHWAY.
AND I ALSO WANT TO POINT OUT
THERE'S MANY EXAMPLES TO
SYNTHETICALLY VALUE LOSS OF PARP
WHICH ARE NON-RH GENES.
WE DON'T INVOKE DOUBLE STRAND
BREAK WHEN WE WANT TO WHEN WHEN
WE HAVE A BRCA GENE MUTATION.
LAGGING STRAND DEFECTS ARE
SENSITIZED TO PARP INHIBITOR AND
AGAIN, LEADING THE POSSIBILITY
THAT THE DOUBLE STRAND BREAK IS
NOT REALLY REQUIRED HERE.
OKAY.
BUT WHAT IS REQUIRED FOR THIS
MODEL TO HAVE ANY LEGS IS THAT
53BP1 HAS TO GET TO REPLICATION
FORKS AND IN CELLS THERE IS NO
DNA DAMAGE.
THIS GROUP HAD SHOWN THROUGH
PROXIMITY LIGATION ASSAY, THAT
50BP1 WAS RIGHT WITH NASCENT DNA
IN UNCHALLENGING CONDITIONS.
IT WAS RECENTLY SHOWN BY
WAGNER'S GROUP THAT 50BP1 IS ON
THE OKAZAKY FLAP.
IT SITS AS A BINARY BINDING
PROTEIN.
THAT FITS IT INTO THE THE FLAP.
SO WE WANTED TO SEE IF WE COULD
SEE A SIMILAR RELATIONSHIP.
SO WE PERFORMED THESE PROXIMITY
LIGATION ASSAYS AND WE COULD SEE
THAT AS COMPARED TO 50BP1
KNOCKOUT CELLS.
CLEARLY 50BP1 WAS SHOWING UP AT
THE REPLICATION FORK, PRESENT AT
THE FORK AND BEHIND THE FORK WE
COULD SEE THROUGH A 20-MINUTE
CHASE PERIOD.
WHAT IS INTERESTING IN THE
ABSENCE OF BRCA 1 THIS PRESENCE
WAS ENHANCED DRAMATICALLY, AS
YOU CAN SEE HERE, AND THOSE
IMAGES, AND QUANTIFIED AND THE
GREATEST ACCUMULATION WAS COMING
BEHIND THE REPLICATION FORK
POSSIBLY BECAUSE 50BP1 WAS
HANGING OUT ON LAGGING STRAND
FLAPS, NOT PROPERLY REMOVED.
BRCA 1 RESTRICTS 50BP1, IT COULD
BE RESTRICTING THROUGH ITS
ABILITY TO PROMOTE PROCESS AND
REPLICATION GAP SUPPRESSION.
IT IS CONFOUNDED BECAUSE WHILE
BPR1 LOSS WILL -- STILL LEADING
US TO NOT REALLY KNOW WHICH ONE
IS DRIVING THE RESISTANCE, EVEN
THOUGH WE CAN TARGET THIS AND
STILL SEE RESISTANCE.
ULTIMATELY, IT IS NOT GOING TO
BE FULLY CLEAR.
WHAT WE HAVE BEEN TRYING TO DO
IS FIND THINGS LIKE 53BP1
ELEVATED IN A BRCA DEFICIENT
SELL BUT PARTICIPATE IN NHR OR
REPLICATION GAP SUPPRESSION BUT
NOT BOTH.
SO CAN WE FIND THAT?
53BP1 IS ELEVATED IN A BRCA
DEFICIENT CELL.
WE ARE LOOKING FOR ANOTHER
PROTEIN THAT IS JUST LIKE 53BP1
THAT IS ENRICHED AND DIRECTING
THERAPY RESPONSE SIMILARLY.
HOW TO DO THAT?
HERE WE SET UP A COMPARISON
ACROSS ISOGENIC CELLS.
WE SUGGESTED JUST GRAP
EVERYTHING AND SEE WHAT WE CAN
FIND.
THIS WORK WAS RUN BY SYLVIA
AMIN, A GREAT GRADUATE STUDENT
ABOUT TO DEFEND HER PH.D. AND
THEN -- WHO HAS BEEN WORKING
WITH ME OVER 20 YEARS.
WHAT DID THEY FIND?
THEY FOUND PARP AND 53BP1 WERE
HIGHER IN THE DEFICIENT CELLS,
WHICH WE WOULD EXPECT.
WE FOUND HL THE F, THIS PROTHAT
IS INVOLVED ARE REVERSAL AND
SHOWN TO DRAW RESPONSE IN BRCA
DEFICIENT CELLS.
DEPLETION OF HL THE F REDUCED
FITNESS SOMEWHAT, BUT THE CELLS
THAT SURVIVED WERE MORE
RESISTANT TO A PARP INHIBITOR.
AND SO THAT WAS SATISFYING THAT
WE WERE FINDING THINGS IN THE
CHROMATIN THAT WERE CONTRIBUTING
TO BRCA RESPONSE IN A DEFICIENT
BACKGROUND.
WE ALSO FOUND THIS ENZYME HERE,
EEPD1, WHICH YOU HAD ALSO BEEN
FOUND BY YONKERS GROUP AS A GENE
WHOSE DEPLETION WOULD CONFER
RESTORED SENSETIZATION IN PARP
INHIBITOR IN DOUBLE KNOCKOUT
CELLS AND, AGAIN, WE COULD
CONFIRM THAT AS WELL.
WE THOUGHT THIS PROCEDURE HAD
THE MOBILITY TO FIND INTERESTING
FACTORS THAT WERE DRIVING
THERAPY RESPONSE.
LIKE 53BP1.
NOW, TURNING TO GOING A LITTLE
FURTHER, WE MOVED ON TO WORK
WITH JUST NOT DAN DEROACHER
CELLS, BUT THESE EMBRYONIC FIBER
CELLS WHICH HAS SIMILAR
GENOMICS.
WHAT WAS ENRICHED ACROSS CELL
LINES IN THE BRCA BACKGROUND
DEFICIENT OF 53BPR1.
WE SAW THE ONE THAT STOOD OUT
THE MOST WAS MDC1.
THIS HAS BEEN RECENTLY SHOWN BY
ANOTHER GROUP TO DIRECT THERAPY
RESPONSE IN BRCA DEFICIENT
CELLS.
SO THAT MADE IT QUITE
COMPELLING.
WE COULD SEE THE LEVELS DID NOT
CHANGE IN THE OVERALL EXTRACTS
IN THE CELLS UNLESS IT WAS
LEADED.
WHEN WE DID WESTERN BLOT
ANALYSIS, BRCA WAS UP.
WE COULD SEE THAT THROUGH A
CHOMATIN EXPERIMENTS WHERE IT
WAS HIGHER ONLY WITH A 53BP1 WAS
PRESENT IN THESE MAPS AS WELL
AND IN ANOTHER CELL LINE WE HAD
FROM KNEEL JOHNSON'S GROUP WHERE
THE RESISTANCES SHOW LOW 53BP1
LEVELS GO DOWN.
SIMILAR TO ANOTHER GROUP, WE
DEPREET, AND OUR FITNESS IS NO
REDUCED.
BRCA DEFICIENT CELLS ARE NOT
ONLY SENSITIVE TO PARP
INHIBITOR, BUT SENSITIVE TO WHEN
YOU INHIBIT TRANSLESION
SYNTHESIS.
WE WANTED TO CONFER RESISTANCE
TO THAT DRUG AS WELL.
SO QUICK STRIKINGLY WE COULD
RECAPITULATE THAT THE BRCA CELLS
ARE SENSITIVE, BUT THE DOUBLE
KNOCKOUT CELLS ARE HIGHLY MORE
RESISTANT THAN THE WILD TYPE AND
THE SINGLE KNOCK OUT.
MBC ONE DEFICIENT -- HL T F
DEFICIENCY DID NOT PROVIDE ANY
RESISTANCE HERE.
SO TOGETHER WE FOUND MDC1
SATISFIED REQUIREMENTS, IT
DIRECTS RESPONSE SIMILAR TO 50
3W BP1.
WILL MDC1 SEPARATE FOR
REPLICATION GAP SUH PRESENTATION
VERSUS HR.
TO BEGIN TO ADDRESS THAT WE
STARTED WITH LOOKING AT
REPLICATION GAPS BY THE DNA
FIBER ASSAY.
WE COULD SEE THE BRCA 1 KNOCKOUT
CELLS ARE CUTTING WITH THE
S1NUCLEASE, WE DEPLETE IT, THAT
CUTTING WAS SUBSTANTIALLY
REDUCED.
MOREOVER, WHEN WE WORKED WITH
OUR DOUBLE KNOCKOUT CELLS WHICH
IS 53BP1 DELETED WE COULD
RECAPITULATE WHAT WE PREVIOUSLY
SHONED.
A DOUBLE KNOCK OUT WITH MDC1 WE
COULD FIND A SIMILAR FINDING AS
WELL.
IT LOOKED LIKE MDC1 WAS DRIVING
GAP IN BRCA DEFICIENT CELLS.
WE FURTHER VALIDATED THIS
LOOKING AT RPA LEVELS IN
PARALATION CONTRIBUTED BY 53BP1.
WE COULD SEE IT WAS GOING DOWN
WE DEPLETION AND INHIBIT WITH
THE -- ENZYME.
IF YOU LOOK AT THE SINGLE STRAND
DNA IT WAS REDUCED BY MDC1
CONSISTENT MDC1 IS SUPPRESSING
GAPS IN BRCA DEFICIENT CELLS.
THEN JUST LIKE 53BP1 LOSS THIS
PARP INHIBITOR RESISTANCE WAS
DEPENDENT ON LIGASE3 AS WELL.
IS MDC1 AT THE REPA ZONE.
WE PERFORMED LIGATION ANALYSIS.
53BP1 IN THE WILD CELLS AND SO
THE MDC1.
WE SEE IN THE CHASE PERIOD.
NOW WHEN YOU LOOK AT THE B BRCA
1 CELLS, IT IS HIGHER SORT OF
MIRRORING THAT ACCUMULATION.
SO WE THEN BEGAN TO THINK, YOU
KNOW, HOW IS IT THIS REPIZOME
ENRICHMENT COULD PLAY OUT TO
CHROMATIN ENRICHMENT?
HOW ARE BOTH PROTEINS ENRICHED
IN THE CHROMATIN.
WE THOUGHT ABOUT THIS PHENOMENON
FROM SEVERAL YEARS AGO 53BP1
NUCLEAR BODIES.
THESE WERE IDENTIFIED TO BE
PRESENCE IN UNPERTURBED CELLS
AND PHENO COLON TREATMENT.
THESE NUCLEAR BODIES INCLUDED
BRCA 1 AND DEPEND ON -- IS BRCA
1 DEFICIENCY SIMILAR TO TREATING
CELLS WITH -- WILL IT CONTINUE
THROUGHOUT THE CELL CYCLE IN THE
CHROMAIN AND DEPEND ON H2AX.
THOSE ARE OUR QUESTIONS.
WE BEGAN TREATING CELLS WITH THE
APHIDICOLIN, MDC1 WAS ENRICHED.
AND COMPARABLE WITH 53BP1 CELLS.
IT IS ENRICHED IN THE CHROMATIN
THE BRCA DEFICIENT CELL.
IF YOU WANT TO KNOW WHETHER THAT
IS THROUGHOUT THE CELL CYCLE, WE
PERFORMED THIS CYTOMETRY THAT
ALLOWS US TO SEE IF WE EXTRACT
OUR CELLS THAT WE'RE JUST
LOOKING AT THE CHROMETIN, THE
MDC1 ENRICHMENT IS NOT IN A
PARTICULAR CELL CYCLE PHASE.
IT DOES SEEM TO GO THROUGH THE
CELL CYCLE.
WHAT ABOUT H2AX, IS THAT DEPEND
ON H2AX?
WE DEPLETED IT AND LOOKED AT THE
MDC1 ACCUMULATION AND SEE IT WAS
CLEARLY REDUCED.
IF YOU LOOK AT 53BP1 OR MDC1 OR
A2HX DEPLETED AND IT IS REPORTED
IN THE NUCLEAR BODIES.
IF YOU LOOK BY WESTERN BLOT IT
IS QUITE CLEAR.
THE DEPLETION IS REDUCING MDC1
AND REDUCING 50BP1CHROMATIN
ASSOCIATION.
AND SO WE NOW ASK IS H2AX IN THE
REPLIZOME?
THIS IS HAPPENING AT AND BEHIND
THE FORK CELL.
IN THE BRCA DEFICIENT CELL IT IS
ACCUMULATING BEHIND THE
REPLICATION FORK.
MDC1, 53BP1, H2AX ARE ENHANCED
BEHIND THE FORK WITH BRCA IS
MISSING.
IF YOU LOOK AT THE RESISTANCE OF
PARP INHIBITOR, WE SEE IT AS
WELL AS TELOS INHIBITOR.
I WILL POINT OUT THIS WAS
RECENTLY PUBLISHED.
SO FINALLY, IT COMES TO MDC1
RESTORE HOMOLGA CONFIRMATION?
WE WANT TO SEE SOMETHING
DIFFERENTIATE.
WE PERFORM THE STANDARD
EXPERIMENT AND ADD -- YOU CAN
SEE RAD51 FORMS, SHOWING THE
SITES OF DNA DAMAGE NOT IN BRCA
1 DEFICIENT CELLS, THAT HAS BEEN
KNOWN SINCE THE MID 90s.
THIS IS COMING BACK AS WE
LEARNED QUITE CLEARLY FROM
SEVERAL GROUPS WHEN YOU GET RID
OF 53BP1 AND WE CAN SEE THE RAD
FICATION IN THE RAD 51FOCI.
IF YOU LOOK AT THE DEPLETION AND
SCORE THE SAME RAD 51FOCI, WE DO
NOT SEE RESTORATION OF THE RAD
51NOCI.
WE THINK THIS IS ULTIMATELY THE
CORRECT OUTCOME HERE AND SO THIS
THEN LEAVES THE POSSIBILITY THAT
WE HAVE BRCA 1 AND MDC1
DEFICIENT CELLS RESTORING GAP
SUH PRESENTATION AND RESISTANCE
AND WE CAN CONTINUE SO FAR
WITHOUT BREAKING OUR MODEL,
AGAIN, STILL TO BE DETERMINED
WHAT IS GOING ON WITH THIS
MUTANT HERE AS TO WHETHER
THERE'S SOMETHING MORE TO TAKE
APART HERE.
THE INTERESTING TAKE HOME WORK
IS RAD 51 DON'T RESIDE WITH --
WE DO NOT SEE RAD 51 IS FOCI AND
WE DON'T SEE IN OTHER RESISTANT
CELL LINES.
RAD 51 FOCI IS BEING USED IN THE
CLINIC.
IT IS OBVIOUSLY, ONLY A PARTIAL
BIOMARKER FOR WHAT RESPONSE WILL
LOOK LIKE.
AND SO WHAT WE HAVE TURNED TO
THEN, OF COURSE, IS, YOU KNOW,
THE IDEA THAT FOR ONE, A
SNAPSHOT OF THE TUMOR YHROMATIN
IS CLINICALLY INFORMATIVE.
WE GRAB EVERYTHING IN THE
CHROMATIN AND YOU ARE NOT ADDING
ANY DNA DAMAGE.
IT SUPPORTS THE IDEA THAT THERE
IS ALREADY AN INHERIT GAP OR
OTHERWISE OTHER PROBLEM IN THE
BRCA DEFICIENT CELL FOR WHICH
PROTEINS ARE BEGINNING TO
ACCUMULATE AND TRYING TO DEAL
WITH.
MOREOVER, THE BIOMARKERS OF
RESPONSE COULD MAYBE BE MORE
LIKE THINGS THAT THE TUMOR HAS
HIGH GAPS.
WE CAN DEFINE GAP BIOMARKERS OF
THE DNA OR THAT THE PROTEINS
LIKE NBC1, GUAM-H1, WE THOUGHT
OF DOUBLE STRAND BREAK REPAIR
PROT
PROTEINS, MAYBE THESE ARE MORE
RELATED TO GAP INDUCTION.
BECAUSE AS WE FIND, THEY
ACTUALLY CAUSE THE GAPS IN THE
BRCA DEFICIENT CELL AND PERSIST,
WE THINK, IN THESE GAPS.
MOREOVER, CHEMOTHERAPY WILL WORK
IF WE ARE GOING TO MAXIMIZE GAPS
WITH A PARP INHIBITOR, AN ATR
INHIBITOR.
WE NEED TO SUPPRESS WAYS CANCER
ECELLS OVERCOME GAPS, EITHER BY
EXPRESSING MORE RPA THAT
PROTECTING THE GAP OR INHIBIT
TLS USEFUL FOR PREVENTING GAPS
IN THE FIRST PLACE AT THE
REPLICATION FORK OR FILL IN GAPS
POST REPUTABLY.
I WANT TO THANK MY TEAM.
MIN PENG HAS SPEARHEADED THIS
WORK AND WORKED CLOSELY WITH
SYLVIA ON THIS PROJECT.
WE HAVE A GREAT GROUP
CONTRIBUTING AS WELL.
I NOW KNOW WE ARE AT DANA FARBER
AS A POST DOC.
I WANT TO TURN ALSO TO A LITTLE
PLUG TO A MEETING THAT WE'RE
GOING TO HAVE IF YOU ARE
INTERESTED IN THIS AREA AND YOU
HAVE A STORY TO PRESENT OR JUST
WANT TO COME TO LISTEN, WE HAVE
A GREAT LINEUP.
SO PLEASE CHECK OUT THAT.
AND CONSIDER THAT IN YOUR
FUTURE.
ALSO AS MENTIONED EARLIER, BY
KAREN, WE STARTED A GENOME
INTEGRITY PROGRAM UMASS AND
INITIATE HIRING THIS FALL.
SO PLEASE CONSIDER THAT IF YOU
ARE LOOKING FOR A JOB IN THE
NEAR FUTURE.
AND SO FINALLY, I JUST WANT TO
LEAVE, YOU KNOW, WITH THIS SORT
OF IDEA THAT IF WE HAD COME TO
THE STUDY OF THE BRCA PROTEINS
AT THIS TIME, WE MIGHT HAVE SORT
OF STARTED WITH THINKING, OH,
THESE PROTEINS THEY REPAIR GAPS
AND THEY PREVENT GAPS AND AGAIN,
IS THIS THE FUNDAMENTAL ROLE FOR
THESE PROTEINS POTENTIALLY IN
THE REPLICATION FOR AN UNPER
UNPERTURBED CONDITIONS.
OTHER FUNCTIONS WERE
HIGHLIGHTED.
THEY ARE EASIER TO DESCRIBE IN
PHENOTYPES THAT REQUIRE
DIFFERENT ASSAYS THAT ARE JUST
COMING ONLINE NOW.
I GUESS I WILL STOP THERE AND
JUST WANT TO POINT OUT MY
ATTEMPTS TO TRY TO GET THE FIELD
TO THINK ABOUT THESE MODELS AND
WAS VERY HAPPY TO SEE THE --
INHIBITOR FIELD REIMAGINING
RATHER THAN A DOUBLE STRAND
BREAK BEING THE CAUSE OF
SYNTHETIC LETHALITY, IT WOULD BE
BRICK.
AND -- WHY INHIBTORS ARE
SENSITIVE TO BRCA DEFICIENT
CELLS.
SO I THINK I WILL STOP THERE AND
HAPPY TO TAKE ANY QUESTIONS.
THANK YOU.
>> CAN I JUST REMIND EVERYONE,
TO PLEASE PUT ANY QUESTIONS YOU
MIGHT HAVE FOR SHARON IN THE
CHAT.
THANK YOU, SHARON.
THAT WAS A REALLY, REALLY
INTERESTING TALK.
>> THANK YOU.
>> SO, YEAH, SO PLEASE TYPE IN
YOUR QUESTION IN THE CHAT BOX.
I SEE THERE IS A QUESTION FROM
PATTY OPRESKO, SHE MENTIONED A
GREAT TALK, SHARON.
DO YOU HAVE AN IDEA HOW LARGE
THESE GAPS ARE?
DO YOU KNOW IF TEMPLATE
SWITCHING HAS ANY ROLE IN
PROCESSING OR FILLING THESE GAPS
AND PARP INHIBITOR RESISTANCE?
>> OKAY.
YEAH.
PATTY, GREAT QUESTION.
WE DON'T HAVE ANY IDEA HOW BIG
THESE GAPS ARE.
WE ARE WORKING VERY HARD WITH
NANAPOR SEQUENCING TO TRY TO GET
A HANDLE ON THAT.
SO WE THINK THAT WILL BE REALLY
TELLING TO SEE HOW BIG THEY ARE
AND HOW MANY THEY ARE AND HOW
FAR APART THEY ARE.
SO WITH TEMPLATE SWITCHING, ITS
ROLE IN THE BRCA AND 53BP1
DEFICIENT SELLS WAS HIGHLIGHTED
BY WORK WHERE YOU COULD SEE A
MUTATION PATTERN THAT WAS MORE
CONSISTENT WITH A TEMPLATE
SWITCH PATTERN THAT CHANGED FROM
THE BRCA DEFICIENT CELL WITH
TRANSLESION SYNTHESIS MUTATION
PATTERN.
THERE IS EVIDENCE FOR TEMPLATE
SWITCH AND I THINK THE REAL
STRONG EVIDENCE FOR THE
MECHANISMS REGULATING THAT STILL
REMAIN.
YOU CAN IMAGINE YOU WOULD NEED
TO RESECT THE GAP TO GET IT A
CERTAIN SIZE FOR PROMOTING
TEMPLATE SWITCH.
UNDERSTANDING HOW THAT IS
REGULATED STILL IS OUTSTANDING.
>> JOHN HAS A QUESTION, ARE GAPS
HARD TO REPAIR WHEN LIGASE IS
NOT DEPLETED?
>> OKAY.
SO MEANING IF YOU HAVE THE
LIGASE, YOU SHOULD BE ABLE TO
REPAIR GAPS, CORRECT?
YES.
OKAY.
SO I THINK IT COMES DOWN TO
LAGGING STRAND SYNTHESIS IS
COORDINATED WHEN ONE OKAZAKY IS
COMING IN AND THE OTHER IS READY
TO BE PROCESSED AND THE LIGATION
WOULD BE READILY ACHIEVED.
IF YOU HAVE AN IMPEDIMENT WITH
PROCESSING OF THE FLAPS OR THE
FLAPS GET HIGHLY OVERLY
RESECTION, TOO MUCH OR TOO
LITTLE RESECTION COULD IMPACT
THE ABILITY TO PUT THE TWO FLAPS
TOGETHER AND SEAL THE DEAL.
SO, YES, CERTAINLY, YOU KNOW, IN
THE ABSENCE OF THE LIGASE YOU
HAVE A LOT OF PROBLEMS.
IF YOU HAVE A LIGASE AND NOT
RESECTING OR OVERSECTING
OKAZAKY, YOU ARE GOING TO HAVE
TROUBLE.
>> ELISE IS ASKING, ARE THERE
GENOMIC REGIONS MORE SENSITIVE
TO GAP FORMATION THAN OTHERS IS
>> YEAH.
THAT IS SUCH A GOOD QUESTION.
I WOULD THINK THE ANSWER WOULD
BE YES AND THAT IS WHAT WE HOPE
THE NANA4 SEQUENCING WILL SHOW.
FOR ONE, THE RELATIONSHIP
BETWEEN GAPS AND SECONDARY
STRUCTURE AND I HAD A SLIDE FOR
THAT IS HERE, THAT WE ARE
LEARNING FROM A MODEL FROM
ANOTHER GROUP, IF YOU HAVE A GAP
SUBSTRATE AND YOU HAVE THE
ABILITY TO FORM SECONDARY
STRUCTURES, WELL, THAT REALLY
INTERFERES WITH GAP REPAIR.
AND IF YOU CAN RAPIDLY RESECT
AND GET THESE SORT OF RAD 51
STRANDS BINDING THAT CAN
FACILITATE REPAIR.
GOING THE OTHER WAY, IF YOU HAVE
SECONDARY STRUCTURES, YOU KNOW,
THEY HAVE THE OPPORTUNITY TO
FORM IN A GAP OBVIOUSLY, THAT IS
GOING TO BE GAP REPAIR.
YOU WOULD EXPECT TO FIND
SECONDARY STRUCTURES IN REGIONS
OF UNREPAIRED GAPS.
SO THE ANSWER I GUESS IS YES.
THERE SHOULD BE A ROLE FOR
SECONDARY STRUCTURES INTERFERING
WITH GAP RESOLUTION.
>> SO HERE WE HAVE A QUESTION
FROM DAVID.
GREAT TALK.
CAN YOU DRIP HOW A T R AND THE
REPLICATION CHECKPOINTS GAP
FORMATION HOW DO YOU USE APR
INHIBITOR --
>> THE MECHANISM GAPS ARE
RESENTED FOR REPAIR AS OPPOSED
TO INTERFERING WITH A REPAIR, A
T R WILL ULTIMATELY REGULATE
THAT.
AND SO BECAUSE ATR HAS SO MANY
TARGETS IT WILL TAKE WORK TO
EXTRAPOLATE THE CRITICAL
PATHWAYS FOR THAT.
ATR INHIBITOR IS LEADING TO A
LOT OF GAPS.
THE MECHANISM BY WHICH THOSE
GAPS ARE FORMING WE DON'T QUITE
FULLY UNDERSTAND BEYOND THE MOST
OBVIOUS WHICH IS REPLICATION IS
NOT SLOWING PROPERLY AND NOT
BEING REGULATED BUT IN THE GAPS
ALREADY FORMED DOES IT HAVE A
ROLE?
I THINK THIS IS WHAT PEOPLE ARE
TAKING APART AND CERTAINLY
SEEING A ROLE FOR CHECKPOINT
SIGNALLING AND GAP PROCESSING.
>> OKAY.
THERE IS A COMMENT FROM YOUNG
KIM.
HOW IS IT ASSOCIATED WITH GAMMA
H2AX AND HOW MUCH WOULD YOU
EXPECT TO SEE AT UNPERTURBED
FORKS?
THE SECOND PART?
>> THROUGH GAMMA H2AX.
>> DOING THESE LIGATION ASSAYS
IN THE ABSENCE OR PRESENCE OF
ONE OR THE OTHER GENES IS
SOMETHING ONGOING.
IF YOU LOOK AT THE CHROMATIN IN
A CELL WHERE YOU DEPLETE H2AX,
BDC1 AND DROPS OUT.
IT IS THE SAME RESULT WITH
DOUBLE STRAND BREAKS AND NUCLEAR
BODIES.
I WOULD GUESS THE ABILITY FOR
MDC1 TO BE MAINTAINED IN THE
REPRIZOME IS HIGHLY DEPENDENT ON
H2AX.
IT IS PROPOSED TO GET TO THE
FLAP THROUGH ITS RANNE BINDING
DOMAIN.
IT COULD BE IT IS GETTING THERE
ON ITS OWN AND H2X MDC1 IS
COMING THROUGH, THE ORDER IS NOT
CLEARED, BUT IT IS PREDICTED FOR
MAINTENANCE.
TO ANSWER YOUR QUESTION, WE
HAVEN'T FULLY WORKED IT OUT.
HOW MANY MDC1 AS THE TOTAL?
I DON'T HAVE THE ANSWER.
>> HOW LARGE DO YOU THINK THESE
GAPS ARE FOR BRCA 1 DEFECTS?
>> OKAY.
SO IN ABSENCE OF BRCA 1, WE'VE
GOT GAPS.
WE DON'T KNOW HOW BIG THEY ARE.
PEOPLE WHO DO EM HAVE LOOKED AND
THEY CAN SEE THEM.
SO THAT MEANS THEY ARE BIGGER
THAN THE SIZE, YOU KNOW,
REQUIRED TO SEE THINGS.
I BELIEVE THAT, I DON'T KNOW, 50
TO 200 OR SOMETHING NUCLEOTIDES.
THE REASON THEY ARE SO TOXIC, IF
YOU RESECT THEM WITH OTHER
NUCLEASES YOU GET IN TROUBLE AND
NOT ABLE TO FILL THEM.
MY GUESS IS THEY ARE EXPANDING
AND SO HOW MUCH THEY ARE
EXPANDING AND WHAT IS THE
THRESHOLD THAT IS GOING TO
ULTIMATELY POTENTIALLY KILL
THESE CELLS REMAINS TO BE
DETERMINED.
>> OKAY.
WE HAVE ANOTHER QUESTION.
GREAT TALK.
THANK YOU.
DO YOU THINK PARP INHIBITOR
WOULD ASSERT THEIR EFFECTS OTHER
THAN --
>> SO.
YEAH, THIS IS A PAPER WE
PUBLISHED, NATURE COMMUNICATIONS
WHERE WE ASKED THE QUESTION IS
PARP INHIBITOR WORKING IN A BRCA
1 DIVISION BACKGROUND?
BECAUSE IT IS TRAPPING OR IS IT
THIS BASIC REPLICATION ACTIVITY
THAT IS THE THING THAT IS SO
IMPORTANT OR BOTH?
IS IT GETTING TRAPPED IN
REPLICATION GAPS FROM FAILURE OF
PARP ACTIVITY AND PARP ALSO
GETTING TRAPPED.
TOGETHER OUR DATA DON'T PROVIDE
ANY EVIDENCE FOR TRAPPING BEING
AS IMPORTANT AS THE PARP
ACTIVITY.
AGAIN, IF YOU REMOVE THE -- HELO
CASE WE SEE PARP ACTIVITY GOES
DRAMATICALLY DOWN.
PARP BECOMES SEQUESTERED.
IF YOU COMBINE WITH BRCA LOSS.
THAT IS SIMILAR TO KNOCKOUT
CELLS WHERE THERE IS NO
TRAPPING.
PARP KNOCKOUT IS HIGHLY TOXIC.
THAT WAS THE ORIGINAL
OBSERVATION IN 2005 NATURE
PAPERS.
LOSS OF PARP ALONE, NO HIB HIB
TOR, IS GOING TO BE TOXIC WITH
PARP BRCA.
WE KNOW PARP TRAPPING CAN BE
BAD, WHEN THE BRCA 1 DEFICIENT
BACKGROUND WE DON'T THINK THAT
TRAPPING IS A MAJOR CONTRIBUTOR
TO THE DEATH, BUT, AGAIN, WE
KNOW XRCC1 LOSS TRAPPING IS A
PROBLEM.
TO GET RID OF PARP IS BETTER.
TRAPPING CAN BE BAD, BUT IT IS
CONTEXT DEPENDENT.
THANK YOU.
>> I HAVE JUST ONE QUESTION
MYSELF.
>> OKAY.
>> SHARON, COULD YOU SPECULATE
ON WHETHER THE MDC IS SERVE AS A
BIOMARKER FOR THERAPY RESPONSE?
>> OH, YEAH.
YOU KNOW, WE ARE LOOKING AT THAT
NOW.
WE LOOKED ACROSS A SERIES OF
BREAST AND OVARIAN CANCER CELL
LINES.
WHEN BRCA 1 OR BRCA 2 WAS LOW,
MDC1, # 53BP1 TREND HIGHER.
SO THE QUESTION IS WHETHER THAT
WILL HOLD UP ACROSS TUMOR
SAMPLES.
WE ARE GOING IN THAT DIRECTION
NOW.
THAT IS WHERE WE ARE AT.
IT COULD PROVIDE A BIOMARKER FOR
GAPS AND, AGAIN, HOW MUCH SIGNAL
DO YOU NEED TO HAVE TO KNOW THEY
ARE GOING TO BE SENSITIVE IS THE
CRITICAL QUESTION.
WHAT IS THE LOAD THAT YOU NEED?
SO WE ARE HOPING WE CAN USE
THAT, COMPARE IT TO RAD 51 FOCI
AND SEE IF THIS IS EQUALLY OR
BETTER AS A MARKER.
>> THANK YOU.
>> YES.
>> WE HAVE AGES AGO, WE WERE
PUZZLED BY THE COMPARISON OF
SINGLE STRAND BREAKS AND DOUBLE
STRAND BREAKS AND WONDERED WHY
THEY WERE SO, IF SINGLE STRAND
BREAKS WERE NOT TOXIC, WHY WERE
CELLS DYING?
>> YEAH.
>> THE ANSWER IS EVENTUALLY YOU
LINE UP A SINGLE STRAND BREAK
OPPOSITE ANOTHER AND WOULD LEAD
TO A DOUBLE STRAND BREAK.
>> SURE.
>> IS THAT A CONCEPT STILL
VIABLE?
>> THIS IS VERY MUCH A CONCEPT.
SO PEOPLE GENERALLY THINK OF
SINGLE STRAND BREAKS OR PARP
COMPLEXES ARE COLLAPSING THE
FORK.
IT IS INEVITABLE A SINGLE STRAND
WOULD HIT A REPLICATION FORK AND
AT SOME CONFIRMATION WOULD LEAD
TO A DOUBLE STRAND BREAK.
IT COMES DOWN TO IS THAT HOW THE
CELLS DIE THROUGH THE DOUBLE
STRAND BREAK OR CAN A SINGLE
STRAND OF DNA BREAK EXPAND,
RIGHT, AND HAVE ITS OWN LETHAL
CONSEQUENCES OUTSIDE OF HAVING
TO HIT THE REPLICATION FORK.
SO THAT'S REALLY IMPORTANT
INTERESTING AREA THAT I THINK
NEEDS TO BE FIGURED OUT BECAUSE
OTHERWISE WE ARE GOING TO KEEP
INVOKING A DOUBLE STRAND BREAK,
WHICH MIGHT BE RIGHT, BUT IF IT
IS NOT RIGHT, MAYBE THERE ARE
OTHER THERAPIES WE CAN USE THAT
DON'T REQUIRE THAT SITUATION.
>> OKAY.
GO AHEAD.
THERE'S MORE QUESTIONS IN THE
CHAT.
>> WE HAVE A QUESTION.
GREAT TALK.
IT LOOKS LIKE A THE RX IS RICH
AT THE REPLICATING DNA ABSENCE
OF BRCA 1.
IS THERE ANY FUNCTIONAL FOR A
THE RX TO BE THERE?
THANKS?
>> SO THE PROTEIN IS A THE RX?
>> YES.
A THE RX.
ATRX.
AT THE REPLICATION FORK?
I DON'T THINK I HAVE A GOOD
ANSWER FOR THIS.
I DON'T THINK I KNOW TOO MUCH
ABOUT THAT.
SORRY.
YEAH.
>> OKAY.
THERE IS ANOTHER QUESTION FROM
JOHN.
ASKING DO COMMON SAS SAYS REVEAL
THESE GAP AND TELL YOU TIME FOR
REPAIR OR RESISTANCE?
>> YEAH.
I THINK, I MEAN, WE HAVE STAYED
AWAY FROM COMMON ASSAYS BECAUSE
THE EXPERTS I TALKED TO SAY THEY
DON'T FULLY ONLY REPORT ON
SINGLE STRANDED DNA BREAKS
VERSUS DOUBLE STRANDED DNA
BREAKS.
SO I THINK IF, YOU KNOW, THE
DRUG OF INTEREST WAS CLEARLY
ONLY MAKING ONE KIND OF LESION
YOU COULD USE IT TO ASK ABOUT
RESOLUTION AND WHATEVER.
BUT IF YOU DON'T KNOW AND YOU
ARE MIXING IN CHEMOTHERAPY, YOU
DON'T KNOW HOW IT WORKS AND
LOOKING AT THAT, YOU ARE CLEARLY
NOT ABLE TO DISCRIMINATE.
IT JUST ADDS COMPLEXITY TO
COMPLEXITY.
THAT IS WHY WE STAYED AWAY FROM
IT.
WE ARE TRYING OTHER APPROACHES
TO MEASURE GAPS IN DNA THAT
HOPEFULLY WILL BE ABLE TO
RESOLVE THESE ISSUES.
>> THERE ARE STUDIES DONE BY ONE
OF THE PIONEERS IN CELL CULTURE
AT THE CANCER INSTITUTE WAS
CATHERINE SANFORD.
AND SHE WAS DOING G2 CHROMOSOME
HYPERSENSITIVITY AT RADIATION.
AND SHE WOULD FIND SINGLE
STRAND -- LOOKING AT TERIATYPES
AND CHROMOSOME GAPS OR BREAKS
REPRESENTING SINGLE STRAND OR
DOUBLE STRAND DNA.
SHE WAS ABLE TO CHARACTERIZE
CELL LINES FROM CANCER PRONE
DISEASES TO NOT CANCER PRONE
DISEASES ON THIS LEVEL.
>> THAT IS INTERESTING.
>> SHE DID A SERIES OF STUDIES
ON THOSE.
VERY PRECISE USING THE RIGHT
GLASSWARE, THE RIGHT CULTURE
CONDITIONS, LIGHTS AND
EVERYTHING.
>> OH, THAT IS GREAT.
>> THERE IS A WHOLE SERIES OF
PAPERS ABOUT THAT AND THAT SEEMS
TO HAVE BEEN LOST TO HISTORY.
>> I'M GOING TO GO LOOK THAT UP.
HER NAME IS CATHERINE STANFORD?
>> SANFORD.
S-A-N-F-O-R-D.
>> WHY AREN'T LONG GAPS
ATTACKED --
>> IS THAT THE WORD, LONG?
>> YEAH.
LONG GAPS.
>> I'M NOT SAYING THEY ARE NOT.
THAT IS SOMETHING ONE CAN LOOK
AT.
HOW GAPS KILL IS NOT CLEAR,
RIGHT.
CERTAINLY THEY COULD CONVERT TO
DOUBLE STRAND BREAKS.
WE KNOW DOUBLE STRAND BREAKS ARE
VERY TOXIC AND COULD BE
ULTIMATELY HOW THEY KILL.
I'M JUST ARGUING THE
VULNERABILITY IS THE GAP THAT
BRCA 1 PROTECTS FROM.
THAT IS WHAT I'M REALLY ARGUING.
IF THEY ULTIMATELY CONVERT TO
DOUBLE STRAND BREAK AND THAT IS
HOW THE CELL DIES IS UNCLEAR.
OR WHETHER A GAP, YOU KNOW, WILL
TANGLE UP INTO A SECONDARY
STRUCTURE, WHICH, YOU KNOW, IS
GOING TO ITSELF BE BLOCKING
REPLICATION TRANSCRIPTION,
RECRUITING NUCLEASES LEADING TO
GENOMIC INSTABILITY AND
POTENTIALLY ELIMINATING CRITICAL
GENES.
THERE ARE SO MANY WAYS THIS
COULD UNFOLD AND THIS IS WHAT WE
ARE TRYING TO TAKE APART.
YOUR QUESTION IS A GOOD ONE.
IT IS POSSIBLE THAT THERE'S A
ROLE HERE THAT COULD FURTHER BE
INVOLVED IN THE MECHANISM.
ABSOLUTELY.
>> OKAY.
THERE IS ANOTHER QUESTION --
SORRY, I CANNOT PRONOUNCE RIGHT.
GREAT TALK.
DO YOU THINK LABELING LONG DNA
IS TO DETECT SS DNA FOCI?
>> SO NONDENATURING, WHEN YOU
LOAD UP THE CELL WITH AN ANALOG,
IF THERE IS A SPOT, IT WILL
RECOGNIZE IT.
I THINK IT IS VERY GOOD.
IT WORKS QUITE WELL.
AGAIN, YOU DON'T KNOW IN THE GAP
IS STARTING IN REPLICATION OR
NOT UNLESS YOU CAN MARK
REPLICATION.
BUT I THINK IT IS QUITE GOOD.
IT DOESN'T TELL YOU ANYTHING
ABOUT THE SIZE OF THE GAP,
RIGHT.
IT JUST SAYS YOU HAVE GAPS.
AGAIN, IT IS NOT GOING TO
DISCRIMINATE ABOUT NUMBERS AND
SIZE AND, BUT, YEAH, IF YOU WANT
TO KNOW IF YOUR DRUG OF ACTION
IS MAKING GAPS, YOU CAN DO THAT.
FOR SURE.
>> GREAT.
I DON'T SEE ANY ADDITIONAL
QUESTIONS.
>> OKAY.
GREAT.
THANK YOU.
>> PLEASE JOIN ME IN THANKING
DR. CANTOR FOR THE WONDERFUL
TALK.
AND THANK YOU SO MUCH FOR
TALKING.
WE'RE LOOKING FORWARD FOR MORE
FINDINGS FROM YOUR GROUP.
>> OKAY.
WELL, THANK YOU SO MUCH.
APPRECIATE YOU GUYS.
HAVE A GOOD DAY.
>> KEEP IN TOO MUCH.
BYE-BYE.
>> BYE.