>>WELCOME TO THE
MONTHLY DNA REPAIR INTEREST
GROUP VIDEO CONFERENCES.
TODAY WE ARE VERY GLAD TO HAVE
DR. JO MORRIS JOIN US WHO IS
FROM UNIVERSITY OF BIRMINGHAM.
SHE STARTED IN INSTITUTE OF
CANCER AND GENOMIC SCIENCES AND
WORK AT KINGS COLLEGE IN LONDON
WITH ED SOLOMON.
BEFORE SETTING UP HER OWN GROUP
AT UNITED -- UNIVERSITY AT
BIRMINGHAM.
SHE WAS ONE OF THE FIRST
RESEARCHERS TO REPORT FOR
MODIFICATION IN DNA DAMAGE
RESPONSE AND INVESTIGATED
BRC1LIGASE, THE ASSUME MONTH
PROTEASE.
DR. MORRIS HAS INVESTIGATED HOW
BRC1 INTERACT WITH RD51 FOR
REPLICATION FOR PROTECTION.
SO WITHOUT FURTHER ADO WE WILL
TRANSFER TO DR. MORRIS.
FOR THE AUDIENCE IF YOU HAVE
ANY QUESTIONS, PLEASE TYPE IT
IN THE CHAT BOX.
WE WILL GET TO THE QUESTIONS BY
THE END OF THE TALK.
DR. MORRIS?
>>Jo Morris: CAN YOU HEAR ME?
>>YES.
>>Jo Morris: MY VIDEO CAMERA
HAS JUST GONE DOWN, I WILL USE
THE OTHER ONE, APOLOGIES FOR
THE AWKWARD ANGLE.
THANK YOU, ALL AND FOR INVITING
ME SOME TIME AGO.
>>WHAT I WOULD LIKE TO TALK TO
YOU TODAY ABOUT IS THIS
QUESTION HERE.
WHAT UNDER LIES SYNTHETIC
LETHAL RELATIONSHIP BETWEEN
POLYMERASE THETA TARGETING AND
BRCA 1/2 OR 53BP1 DEFICIENCY.
THE ANSWER IS IT DEPENDS.
WHICH DEFICIENCY AND HOW POL
THETA IS TARGETED.
MY APOLOGIES TO THOSE ON THE
LINE WHO UNDERSTAND POL THETA
BETTER THAN I, I APOLOGIZE.
AND THEN TALK TO LOSS COMPARED
TO ITS INHIBITION.
AND I WILL TAKE A LITTLE DETOUR
ALONG THE WAY TO TELL YOU ABOUT
MODEL WHICH IS BRCA C61G.
LET ME GET MY LASER POINTER
BACK.
WHERE IT FORMS MICRO FOLLOWED
BY LIGATION TO REPAIR DNA
BREAKS AFTER DISSECTION.
THERE'S PAPERS PARTICULARLY
THIS WEEK SUGGESTING A ROLE FOR
THAT IS IN MITOSI.
PRIOR TO THAT WE UNDERSTAND AS
BACK UP PATHWAY.
ONLY TO COMPLETE DNA REPAIR
WHEN OTHER ASPECTS OF MOLECULAR
COMBINATION WERE DISRUPTED.
WHAT HAS BECOME PARTICULARLY
INTERESTING IS THAT TARGETING
POL THETA SEEMS TO BE SYNTHETIC
LETHAL WITH A NUMBER OF
DIFFERENT SETTINGS.
THE MAIN INTERESTS HERE IS BRCA
1 AND 2 DEFICIENCY BUT NOT THE
ONLY DEFICIENCY SENSITIVE TO
POL THETA.
PAPERS HAVE SHOWN ANY NUMBER OF
REPAIR DEFICIENCIES DEPEND ON
POL THETA, ACTUALLY INCREASES
SENSITIZATION.
AND PERHAPS NOT SURPRISINGLY
THEREFORE A NUMBER OF COMPANIES
ARE INTERESTED IN GENERATING
INHIBITORS TO THE POLYMERASE
ACTIVITY AND SOME ARE SHOWN
HERE.
SO WE WANT TO UNDERSTAND A
LITTLE BIT MORE ABOUT THIS.
AT THE MOMENT, WHEN WE STARTED
THIS PROJECT WHAT WAS
UNDERSTOOD IS TARGETING POL
THETA WAS WITH MORE RECEPTION
AND REDUCE FILL IN OF NASCENT
SIPGLE STRANDED GAPS AND
INCREASE HR-INTERMEDIATES.
SUGGESTION IT IS SIGNIFICANT IN
BRCA 2 DEFICIENT.
WE ARE USING BRCA ZYGOTE RAT
CELLS.
THEY DIE IN REGENESIS AND IN
COMMON WITH OTHER MUTANT MODELS
IF WE COMBINE THEM WITH
DEFICIENT ANIMALS WE CAN RESCUE
THAT LETHALITY.
WE HAVE LIVE ANIMALS.
THAT ALLOWS US TO LOOK AT THE
BRCA DEFICIENCY ON THE BACK OF
BP1.
THEY HAVE RESISTANT.
FEATURES YOU NORMALLY CONSIDER
TO BE INDICATORS OF HR
COMPETENCY BUT INTRIGUINGLY
NEVERTHELESS SENSITIVE, THEY
ARE HR COMPETENT YET SENSITIVE,
SORT OF THE BASIS OF THE
BEGINNING OF THIS INVESTIGATION.
IF YOU TAKE A LOOK AT THESE
CELLS, RADIATE AND ASK WHERE
BRCA 1 IS, IT'S CLEAR THEY ARE
NOT ENTIRELY NORMAL, IN TERMS
OF THEIR BRCA -- THEY HAVE
ABOUT 30% WHAT YOU WOULD EXPECT.
20% OF WHAT YOU WOULD EXPECT.
THAT LED US TO WONDER DESPITE
THE HR COMPETENCY IF THEY ARE
NOT USING THE PATHWAY TO
SUPPORT THE HOMOLOGOUS -- WE
KNOW BRCA BARD1, THE LOADING OF
RAD51 CELLS DEFICIENCY FOR BRCA
1, 168 INDEPENDENTLY AND
ENABLES FOR RAD51 RECRUITMENT
AND ROLL FOR 52 FOR BOTH
IMPROVING -- SEPARATELY.
THESE ARE INVOLVED IN THESE
MUTANTS, LET'S SAY SLIGHTLY
COMPROMISED CELLS.
SO WE USED A NUMBER OF ASSAYS
BUT THE ONE I WILL SHOW YOU IS
CRISPR CAS9.
ALLOWS US TO MEASURE THE AMOUNT
OF HR.
AND PERFORM THESE ASSAYS IN
THESE CELLS AND WHERE WHETHER
THEY ARE INDEED DEPENDENT ON HR
SUPPORT MECHANISMS.
THEY REQUIRE THE MUTANTS, BRCA
1, IF WE DEPLETE WE SEE
INCREASE IN HR BUT ALSO
DEPENDENT ON 168 AND INDEED
BRCA 152.
IF HR IS SUPPORTED BY NON
CONICAL MEANS.
WE WONDERED IF THIS SUPPORT HAD
SOMETHING TO DO WITH THE POL
THETA SENSITIVITY.
AND THE PUNCHLINE IS IT DOES.
WE DEPLETED RF168 AND DIDN'T
SEE ANYTHING BUT SEE SOMETHING
DIFFERENT WITH THE 152.
WE TREAT THESE CELLS, WE SEE
REDUCTION IN VIABILITY.
BUT WHEN WE TREAT THEM WITH
BRCA152 INHIBITOR WHICH
DISRUPTS, WE SEE THE SAME
THING, THIS INTRIGUING BELL
CURVE WHERE THERE IS A SWEET
SPOT OF RAD 52 INHIBITOR.
RESCUE OF THESE CELLS FOR POL
THETA.
WE SEE THE SAME THING WHEN WE
TAKE HUMAN CELLS AND DEPLETE
THEM WITH BRCA 1.
SO WHAT'S GOING ON?
THE AMOUNTS OF RAD52 IS NOT
ENOUGH TO RESULT IN SENSITIVITY
BUT IF WE USE A SIGNIFICANT
AMOUNT MORE, WE DO SEE
SENSITIVITY.
THE AMOUNT WE ARE USING IS
QUITE LOW.
RAD52 ITSELF WE SEE IS
RECRUITED TO FO SI IN CELLS BUT
MORE FOR DOUBLE MUTANT --
WE KNOW THAT RAD52 INTERACTS
WITH, IS THERE SOMEONE ELSE WHO
CAN'T SEE THE SLIDES?
IS THERE ANYTHING YOU CAN DO
ABOUT THAT?
NO?
>> QUESTION FOR YOU.
I'M SEEING A BOX ITSELF.
IS THERE ANYTHING HOVERING OVER
YOUR SLIDES?
LIKE ANOTHER MENU OR?
>>Karen Usdin: JEROME IN
ADDITION TO THAT, IT SEEMS SOME
AREN'T ABLE TO SEE THE SLIDES
AT ALL.
>>Chun Zhang Yang: STOP
SHARING FOR ONE MINUTE AND THEN
START SHARING THE APPLICATION
AGAIN.
>>Jo Morris: HAVE I STOPPED
SHARING?
>>Chun Zhang Yang: NOT YET.
>>Jerome Battle: GO THROUGH
THE SAME PROCESS YOU WENT
THROUGH YESTERDAY.
HERE YOU ARE.
SHARE.
>>Jo Morris: IT SAYS IT'S
BLOCKED.
>>Karen Usdin: WE CAN SEE YOUR
SLIDES THOUGH THERE'S STILL
GRAY BOXES BLOCKING, THE TOP
AND BOTTOM, THE PERSON WHO
COULDN'T SEE IT PREVIOUSLY CAN
SEE IT NOW, SO AT LEAST THAT'S
CHANGED.
IT LOOKS LIKE SOMETIMES THE
TITLE OF YOUR SLIDE IS BLOCKED
OUT A BIT.
IF YOU CAN REPEAT THE TITLE OF
THE SLIDE AS YOU MOVE THROUGH
IT?
>>Jo Morris: OKAY, OKAY.
ALL RIGHT.
WHERE WERE WE?
>>Jerome Battle: GO AHEAD AND
PUT IT IN PRESENTATION MODE.
>>Jo Morris: THANK YOU.
THERE?
>>Karen Usdin: SOME OF US CAN
SEE IT.
WE HAVEN'T HEARD WHETHER THE
PEOPLE HAVING TROUBLE ARE STILL
HAVING TROUBLE.
THE TITLE IS BLOCKED.
IF YOU CAN READ OUT THE TITLE
OF EACH SLIDE.
>>Jo Morris: THE APPLICATION,
THAT'S YOU.
THE WEB THING.
>>Jerome Battle: OKAY, SO
CLICK ANYWHERE IN AN OPEN SPACE
SO THAT, IT SHOULD GO AWAY.
>>Jo Morris: NO, IT DOESN'T,
IT STAYS.
>>Karen Usdin: OKAY, SHOULD WE
JUST PROCEED.
IF YOU JUST READ THE TITLE OF
THE SLIDE IT SHOULD STILL BE
OKAY.
>>Jo Morris: I OF COURSE CAN'T
SEE THE TITLE, OKAY, WHERE WERE
WE?
I HAVE TOLD YOU THE DOUBLE
MUTANT CELLS ARE SENSITIVE AND
SEE INCREASE IN RAD52 WHEN WE
REMOVE POL THETA, WE CAN REMOVE
THE SENSITIVITY BY SLIGHTLY
DEPLETING AND DEPLETING RAD 52.
IT INTERACTS WITH SINGLE
STRANDED DNA IN CORPORATION
WITH SINGLE STRANDED BINDED
PROTEIN.
I WONDERED IF WE COULD PERFORM
THE SAME TRICK OF RESTORING
SURVIVAL BY SLIGHTLY REPEATING
RPA ITSELF.
WE TRIED THAT.
YOU CAN SEE LOW-LEVEL RPA
DEPLETION.
WHICH IS INDEED CAPABLE OF
REDUCING RAD 52 FOR THOSE
CELLS, DEPLETED FOR SIRPA,
SORRY, POL THETA.
WE SEE LESS RAD 52 IF WE
DEPLETE RPA AND SIMILARLY LOW
LEVEL CAN RESTORE SURVIVAL OF
CELLS EXPOSED TO POL THETA SIRPA
RAD 52 IS RESPONSIBLE FOR THE
LETHALITY OF CELLS EXPOSED TO
THE POL THETA SIRPA.
TO CHECK THIS MORE CAREFULLY WE
KNOW RPA IS CAPABLE OF CREATING
PROTEASES WE PRODUCED AND ONE
THAT LACKS THE BINDING REGION
WHICH DOES STOP WITH
INTERACTING WITH RPA.
SO IF WE DEPLETE RAD52 SLIGHTLY
WITH LOW CONCENTRATION of si
RPA.
IF WE EXPRESS SIRPA RESIST
ANESTHE ARE MORE SENSITIVE TO
THE POL THETA LOSS, IT IS THE
RAD 52 RESPONSIBLE FOR
SENSITIVITY OF POL THETA LOSS.
WHEREAS IF WE GIVE RAD 52
LACKING THE ABILITY TO INTERACT
WITH RPA WE DON'T SEE THAT.
SUGGESTING IT'S THE RAD 52 THAT
CONTRIBUTES TO THE SENSITIVITY
OF POL THETA LOSS.
IT WASN'T A SURPRISE TO US IT'S
RPA RAD 52 THAT APPEARS TO BE
RESPONSIBLE TO THE SENSITIVITY
OF THESE CELLS FOR THE REMOVAL
OF POL THETA.
WE HAVE KNOWN FOR A WHILE THAT
RESECTION IS ASSOCIATED WITH
SENSITIVITY TO POL THETA
TARGETING.
WE ASSUMED, AS IT TURNED OUT
INCORRECTLY, IT MIGHT BE THE
CASE OF RPAR52 -- WE TREATED
CELLS WITH MRE 11 EXO NUCLEASE
AS WE SAW LOW LEVEL, LOW
CONCENTRATION TREATMENT RESCUE
THE SURVIVAL OF THESE CELLS TO
POL THETA SIRPA.
BUT IT DID NOTHING, IT APPEARS
RAD 52 IS NOT DOWNSTREAM OF MRE
11, AT LEAST NOT WITH THESE
CONCENTRATIONS TO SUPPRESS MRE
11.
WHICH LED US TO THE POSSIBILITY
ACTUALLY MRE 11 IS DOWNSTREAM
OF RAD52 RPA SUGGESTED BY A
PAPER A FEW YEARS AGO FROM
LOPEZ'S GROUP.
WE DID AN ASSAY WE MEASURED THE
SINGLE STRANDED DNA EXPOSED TO
THE POL THETA TREATMENT AND HOW
MUCH WAS ASSOCIATED WITH MRE 11.
SO THIS IS THE ASSAY, WE LOOK
AT MRE 11 ASSOCIATED WITH THE
EXPOSED RDAU AFTER GIVEN FOR 24
HOURS AND ANTIBODY CAPABLE OF
PICKING IT UP IN SINGLE
STRANDED.
THERE'S MRE 11 ASSOCIATED WITH
THAT SINGLE STRANDED DNA.
INTRIGUINGLY WHEN WE GIVE LOW
LEVEL CONCENTRATION OF RAD 52
INHIBITOR THAT IS DECREASED
DRASTICALLY.
IT WOULD AMEER MRE 11 IS
DOWNSTREAM OF RAD 52 RPA,
SOMETHING LIKE THAT.
SO WHY IS THIS IMPORTANT?
WE HAVE THIS BIZARRE
RELATIONSHIP RPA, RAD 52 MRE11
IS IMPORTANT.
WHERE IS IT NEEDED.
AND WE SPENT LOTS OF TIME
TRYING TO FIGURE OUT.
A FEATURE THAT IS WORSENED BY
DECREASING POL THETA, NOT
WORSENED BY LOW CONCENTRATION
OF RAD 52 INHIBITION BUT
RESCUED WHEN WE COMBINED THE
TWO.
WE LOOKED AT SEVERAL FEATURES
REPORTED IN LITERATURE
IDENTIFIED TO BE IMPORTANT IN
POL THETA ACTIVITY ORAD 52.
THE SHORT STORY IS NONE OF
THESE FEATURES REALLY
CORRELATED WITH OUR CRITERIA.
NOT MADE WORSE BY POL THETA
LOSS OR NOT RESCUED BY THE
COMBINATION.
IT JUST DIDN'T CORRELATE WITH
OUR OBSERVATIONS.
WHICH LED US TO THINK ABOUT
SOMETHING QUITE DIFFERENT.
THIS COMES FROM LESSANDRO,
SUGGESTED THERE ARE PHASES
SUBSEQUENT TO NASCENT DNA
GENERATION.
ONE MUCH LATER IN G2.
TO LOOK AT THE G2M REPAIR, WHAT
HE DID ESSENTIALLY GIVE CELLS
NUCLEOTIDE ANALOG AS THEY ARE
GOING THROUGH S PHASE AND PUT
THEM IN NOCODAZOLE FOR A LONG
PERIOD OF TIME TO PUT THEM IN
G2M AND LAST FEW HOURS OF THAT
PROCESS GIVE THEM A SECOND
LABEL.
SO ANY SYNTHESIS OF THE DNA
THAT WAS GENERATED IN S-PHASE
COULD BE SEEN.
YOU GET THESE LITTLE DOTS
ARISING.
THE DENSITY OF THOSE PER
KILOBASE OF LABEL TELLS YOU HOW
MUCH YOU HAVE GOT LATER IN THE
CELSI KELL.
CELL CYCLE.
WHAT WE FOUND IS CELLS LACKING
BRCA 1 HAVE A DEFECT IN THE
ABILITY TO PUT THE FILL-IN
SOMETHING HE REPORTED.
HOWEVER THIS GETS WORSE WHEN WE
DEPLETE POL THETA AND
INTRIGUINGLY GETS BETTER IF WE
ALSO ADD LOW LEVEL RAD 52
INHIBITION.
SUGGESTING THIS IS THE POINT
WHICH THE RAD 52 IS CAUSING THE
ISSUE LATENT CELL CYCLE.
SO THAT DOES CORRELATE WITH OUR
SURVIVAL ASSAYS.
THE NOTION WE ARE COMING TO IS
POL THETA IS INHIBITING RAD 52
AND THAT IS DEPLETING
POLYMERASES LATER IN THE CELL
CYCLE.
WHICH IS THE -- POLYMERASES.
THE QUESTION IS WHERE IS THE
NEED FOR POL THETA RESCUE?
WHAT WE SEE IS THAT IN ADDITION
TO THESE G2N FILL-INOSINE THEY
SISES WE SEE INCREASE GAMMA RAY.
WE SEE CHROMOSOME BREAKS WHICH
ARE RESCUED BY THE INHIBITION
OF RAD 52 AND WE GET HUGE
NUMBERS OF MICRO NUCLEI RESCUED
BY INHIB BALDWIN COUNTY
COMMISSION INHIBITION OF RAD
52.
IS IT THE SAME WITH POL THETA
INHIBITION, THEY ARE LIKELY TO
BE USED IN THE CLINIC.
THE ANSWER TO THIS IS YES AND
NO.
SO YES, IF WE JUST TAKE 53DP1
CELLS TREAT WITH, WE SEE THE
SAME THINGS, SENSITIVE TO
INHIBITOR IS REDUCED,
SUPPRESSED BY RAD 52 INHIBITORS
OR SRNA AND SUPPRESSED BY
LOW-DOSE MRE 11.
THIS IS AN EXAMPLE, P31 BRCA 1
POSITIVE.
IF WE GIVE THEM THE INHIBITOR
ALONE, THEY ARE VERY SENSITIVE.
AND DECREASING VERY LOW LEVELS
OF RPA SIRNA WILL RESTORE THE
LEVELS OF THE CELLS.
AS YOU MIGHT ANTICIPATE THAT
RESCUE IS ALSO FOUND AT THE
LEVEL OF THE DNA SYNTHESIS, SO
WE REINHIBIT POL THETA IN THESE
CELLS WE SEE POOR DNA SYNTHESIS
WE CAN RESCUE BY INHIBITION OF
RAD 52 BUT IT'S DIFFERENT IF WE
LOOK AT BRCA 1 OR 53BP1.
IF WE INHIBIT IT DOESN'T WORK.
THIS IS ONE OF THE EXAMPLES,
NOTHING IMPROVES THE SURVIVAL
TO THE POL THETA INHIBITORS.
SO SOMETHING VERY DIFFERENT
GOING ON IN CELLS LACKING BRCA
1, THE SAME THING FOR BRCA 2
COMPARED TO CELLS LOOKING 53BP1.
ONE OF THE BIG DIFFERENCES IN
CELLS LIKE 53BP1 AND BRCA, IS
THESE DNA GAPS THAT OCCUR AFTER
TO REPRIMING WHEN ANTICIPATES
REPLICATION DIFFICULTY.
SO BP1 DEFICIENT CELLS WE DON'T
SEE GAPS GIVEN TWO LABELS AND
NUCLEASE.
THE BRCA BP1 CELLS WE SEE
APPEARANCE OF THESE GAPS.
LEADING US TO WONDER IF THIS IS
WHAT IS PERHAPS IMPORTANT.
HOWEVER, IF WE USE THE POL
THETA INHIBITOR IN CONTRAST TO
WHAT'S BEEN REPORTED BY SEVERAL
OTHER GROUPS WE DON'T SEE A
WORSENING OF THOSE GAPS THEY
DON'T GET BIGGER OR DIFFERENT
OR NOTHING REALLY CHANGES
LEADING US TO QUESTION WHETHER
THESE GAPS ARE IMPORTANT.
BUT I WILL COME BACK TO THAT.
MORE OVER, COMING BACK TO THE
DNA SYNTHESIS FILL-IN
EXPERIMENT, CELLS LACKING 53BP1.
CELLS ALSO LACK BRCA 1, DNA
SYNTHESIS IS NOT IMPROVED BY
THE INHIBITION OF RAD52.
THIS IS ZINT.
-- DIFFERENT.
IN THE BRCA CELLS YOU CANNOT
RESTORE BY INHIBITING RAD 52.
LEADING US TO WONDER IF IT'S
SOMETHING SPECIAL ABOUT THE
POLYMERASE ITSELF WHEN BRCA 1
OR 2 IS DEFICIENT.
THESE ALSO EXPRESS BP1.
OBVIOUS EXPERIMENT IS WHAT
HAPPENS IF WE DEPLETE POL THETA
AND GIVE THE POL THETA
INHIBITOR, AND PERHAPS WHAT YOU
MIGHT EXPECT HAPPENS.
JUST REMIND YOU THE INHIBITOR
REDUCES THE ABILITY TO PERFORM
THIS GAP TO FILL IN DNA
SYNTHESIS.
IT DOESN'T GET BETTER WE
COMBINE WITH INHIBITOR, IF IT
DOESN'T GET WORSE, WE COMBINE
WITH POL THETA DEPLETION.
IF WE DEPLETE POL THETA, WE
GIVE POL THETA INHIBITOR AND WE
GIVE THEM THE RAD 35 2
INHIBITOR, NOW WE CAN RESCUE.
SO I EXPLAINED THAT RIGHTLY,
LET ME DO IT ONE MORE TIME.
POL THETA INHIBITION, WE
RESTRICT THE ABILITY TO FILL
IN, IF WE DEPLETE POL THETA AND
GIVE INHIBITOR WE CAN RESCUE,
WHICH I THINK TELLS US THE POL
THETA DEFICIENT IS THE
DIFFERENCE.
WHAT IS SPECIAL ABOUT BRCA 1 OR
2 COUNTERING INHIBITING
POLYMERASE THAT IS DIFFERENT TO
THE SITUATION WITH 53BP1,
THERE'S A COUPLE WAYS TO ASK
THIS QUESTION.
FORTUNATELY BECAUSE WE ARE
USING THE 61C SYSTEM POOR FO
SI, WE CAN RESCUE BY OVER
EXPRESSING BARD 1 IT INTERACTS
A LITTLE BIT, NOT GREAT, BUT
RESCUE IT BY EVER EXPRESSING
BARD 1.
IF WE EXPRESS BARD 1 WE CAN
NEARLY RESTORE THE BRCA FO SI
TO NORMAL LEVELS.
AND INTERROGATE DIFFERENT
ASPECTS OF BARD 1.
I WON'T GO THROUGH ALL THE
CONTROLS BUT THE IMPORTANT BIT
IS THIS, MUTANT A, THIS RAD 51
BINDING REGION OF BARD 1 FAILS
TO SUPPRESS RAD 52
FOSI, OVER EXPRESSION OF WILD
TYPE BARD 1 IS CAPABLE WITH
CELLS TREATED WITH THE
INHIBITOR.
ANOTHER WAY WE CAN INTERROGATE
IT IS BY ASKING ABOUT THE
ABILITY TO BY PASS TO GO TO RAD
52'S ABILITY TO LOAD RAD 51.
THIS IS A COPY OF AN
EXPERIMENT, SHE TOOK RPA, TO
ONE OF THE REPEATS OF BRCA 2
AND SHE FOUND FUSING IT TO BRCA
4 OR 3 ONE OF THE LOADING
REPEATS WAS NECESSARY FOR BY
PASSING.
SO WE USED THAT TOGETHER WITH
THE SAME, TO THE EXTREME C
TERMINUS BRCA 2, INTERACTING
WITH RAD 51 OLIG.
WHEREAS THE EXTREME C TERMINUS
IS NOT.
AND THE BRC4 FUSION IS CAPABLE
OF SUPPRESSING RAD 52 FOSI, THE
POL THETA INHIBITOR.
AS YOU WOULD EXPECT, BOTH OF
THESE RESTORE THE ABILITY OF
THESE CELLS TO PERFORM THIS GAP
SYNTHESIS IN BOTH POL THETA
DEPLETED AND IMPORTANTLY, POL
THETA INHIBITED CELLS.
INTERESTINGLY, IT COMPLETELY
RESTORES THE ABILITY OF CELLS
TO NOT HAVE THESE S1 GAPS IN
NASCENT DNA AND AS YOU WOULD
EXPECT RESTORES RESISTANCE TO
THE INHIBITOR.
SO THOROUGHLY SPEAKING WHAT WE
HAVE ISIN' YIN TO THE YANG
DESCRIBED BY WOLFE NAYER AND
THE OTHER NAME ESCAPES ME.
BRCA 1 BRCA 2 -- JOINING IN
EXPOSING G2 PRIOR TO MITOSIS.
WE ARE FINDING POL THETA LIMIT
RAD 52 AND THIS IS BEHIND THE
TOXICITY OF POL THETA LOSS.
AND TO A DEGREE POL THETA
INHIBITION.
DEPENDING ON THE BACKGROUND.
AND JUST TO GO INTO THE DETAIL
OF WHAT I MEAN, SO WHAT WE ARE
SEEING IS IN BRCA COMPETENT P1
POSITIVE CELLS THERE'S NO GAPS,
THERE'S NO G2M GAPS EITHER.
WHEREAS IN 53BP1 CELLS THERE
ARE NO GAPS BUT THERE ARE G2M
GAPS.
WE HAVE NO IDEA.
BUT IN THE PRESENCE OF POL
THETA, IT'S REMOVING WHAT MIGHT
BE TOXIC RAD 52 ENGAGEMENT IN
THOSE GAPS ALLOWING GAP FILLING
THROUGH TRS POLYMERASES.
IN THE ABSENCE OF BRCA 1 OR 2
FOR MA MATTER WE ARE SEEING
GAPS IN NASCENT DNA AND GAPS IN
THE G2/M WHICH IN THE PRESENCE
OF POL THETA, REMOVING RAD 52
BUT ABSENCE OF POL THETA
APPEARS TO BE ENCOURAGING MRE
11 AND COUNTERACTING SOME OF
THE POLYMERASES.
SOMETHING DIFFERENT IS
HAPPENING WHEN WE HAVE
INHIBITOR TO POL THETA.
EITHER THE PRELIMINARIES IS
ENGAGED EARLY ON, PERHAPS TLS
OR LATER FOR THE G2/M GAPS AND
POLYMERASES ITSELF UNDER THESE
CONDITIONS WHICH IS TOXIC.
SO THE PEOPLE TO THANK ARE
[LISTING ON SLIDE]
THANK YOU FOR LISTENING AND
PUTTING UP WITH THE TREMENDOUS
TECHNICAL ISSUES.
I WILL DO MY BEST TO ANSWER
YOUR QUESTIONS.
>>Chun Zhang Yang: THANK YOU
FOR THE AMAZING TALKS.
FOR THE PARTICIPANTS, PLEASE
TYPE IN YOUR QUESTIONS IN THE
CHAT BOX SO THAT WE CAN
ELABORATE THOSE QUESTIONS.
SO FAR I DON'T SEE ANY
QUESTIONS.
>>Jo Morris: JUST TO FOLLOW, I
EXPECT GIVEN WHAT HAPPENED.
>>Karen Usdin: -- SAID THANK
YOU FOR THIS INTERESTING TALK,
BUT HAS NO QUESTION, JUST YET.
MARCELLA LATENCIA, I HOPE I'M
PRONOUNCING, SAYS GOOD WORK, DO
YOU PLAN TO ANALYZE SCE?
>>Jo Morris: DEPENDS ON
EXCHANGES.
WE HADN'T DONE, ACTUALLY.
NO, IT'S NOT ON OUR LIST OF
THINGS TO DO.
I WOULD BE INTERESTING TO KNOW
WHY YOU THINK THAT MIGHT BE A
GOOD IDEA.
>>Karen Usdin: PERHAPS WHILE
MARCELLUS IS THINKING OF THAT
WE CAN MOVE TO THE NEXT
QUESTION.
CHUN, DO YOU WANT TO TAKE IT?
MAYBE I WILL READ THE QUESTION
UP.
FROM KANAA, CELLS TO SIMPLE AL
KIN NATING AGENTS?
>>Jo Morris: WE HAVEN'T DONE
THAT.
I ASSUME OTHERS HAVE, I ASSUME
THE ANSWER IS YES.
BUT WE HAVEN'T DONE IT, BUT I
CAN'T DIRECTLY SAY MYSELF.
>>Karen Usdin: NICOLA, SHE
SAYS NICE TALK, JO.
IS THERE A PARALLEL WITH
RESISTANCE TO POP INHIBITORS TO
KNOCKOUT CELLS, TRAPPING
MECHANISM?
>>Jo Morris: I THINK THAT'S
RIGHT.
I THINK THERE HAS TO BE.
THE RAD52 INHIBITION ONLY WORKS
WHEN WE DEPLETE THE POLYMERASE
ITSELF AND INHIBITED CONTEXT,
YES, I THINK THERE MUST BE A
TRAPPING MECHANISM, VERY MUCH
SO, YES.
AND HELLO NICOLA.
>>Karen Usdin: NEXT QUESTION
IS FROM MITCH, I PRESUME MITCH
MCVEIGH.
FASCINATING.
APOLOGIES IF I MISSED THIS BUT
HAVE YOU CONSIDERED -- DOES
IT BEHAVE LIKE THE OTHER
INHIBITOR.
>>Jo Morris: WE HAVE TRIED IT.
AND WE ARE STRUGGLING WITH IT.
I DON'T KNOW IF THAT'S THE
EXPERIENCE FOR OTHER PEOPLE, WE
ARE A BIT CONCERNED IT'S OFF
TARGET EFFECTS.
I WOULDN'T WANT TO MAKE ANY
FIRM CONCLUSIONS.
WE ARE HOPING TO TEST THE
REPAIR INHIBITORS AS SOON AS
THE MTA IS SIGNED.
>>Chun Zhang Yang: I'M GOING
TO THE NEXT QUESTION.
CAN YOU ELABORATE ON THE DOSE
DEPENDENT ON THE RAD 52 KNOCK
DOWN.
>>Jo Morris: YEAH, HI PHILIP.
LOVELY TO HEAR FROM YOU.
IT'S EXTRAORDINARY, WE HAVE
THIS BELL CURVE.
TOO MUCH RAD52 INHIBITOR AND WE
HAVE AN HR DEFECT IN THESE
CELLS.
TOO LITTLE RAD 52 INHIBITOR,
ALL IN THE BACKGROUND OF POL
THETA, SORRY, WE HAVE THE
EFFECT OF POL THETA LOSS.
THERE SEEMS TO BE A SWEET SPOT
OF NOT TERRIBLY MUCH.
AND IT'S DIFFICULT WITH THE
INHIBITOR TO SAY EXACTLY WHAT
THAT IS, BUT WE CAN START TO
DELVE INTO IT WHEN WE USE THE
SIRNA, THE AMOUNT WE ARE LOSING
IS, YOU KNOW, 5-15%.
SO AT THAT VERY LOW LEVEL, AS
FAR AS WE KNOW WE HAVE
FUNCTIONAL RPA AND YET WE ARE
ABLE TO RESCUE THE SENSITIVITY
TO POL THETA LOSS.
I HOPE THAT HALFWAY ANSWERS
YOUR QUESTION.
GOOD.
>>Karen Usdin: AND THERE'S A
QUESTION FROM HAYA, GREAT TALK,
IN YOUR EXPERIENCE IS POL THETA
ACTING AS --
>>Jo Morris: THAT'S A LOVELY
QUESTION.
I DON'T KNOW.
I THINK IT'S TLS POLYMERASE,
THE DOUBLE STRAND BREAKS ARE
SUBSEQUENT TO THE ACTIVITY OF
THE POL THETA AND RAD52.
WE KNOW WE CAN RESCUE THE
BREAKS SO I'M GUESSING IT'S TLS
AT THE FORK.
AND PROBABLY I SHOULD HAVE SAID
THE BRC4 IS THE REGION OF
IMPLICATED STABILIZING WITH POL
ALPHA.
SUGGESTS IF YOU DOSE --
EXTRACT WITH THAT BRC4 YOU LOSE
THE GETTING THE REST OF THE
GAPS.
MY GUESS IT'S STEPPING IN AS
TLS, THAT'S BEFORE THE PROBLEMS
ARE ARISING, BUT IT'S A GUESS.
>>Chun Zhang Yang: I SEE A
QUESTION FROM SARA LE --
I WAS CURIOUS BECAUSE OF
CHROMATIZE HAPPENING IN HR,
WONDERING IF 53BP1 IN --
CELLS WOULD HAVE CHANGED THEM
AROUND.
JUST OUT OF CURIOSITY.
>>Jo Morris: CHANGE THE AMOUNT
OF WHAT?
>>Chun Zhang Yang: SHE DIDN'T
SAY EITHER.
>>Karen Usdin: I THINK RELATED
TO THE QUESTION OF SYSTEM
CHROMATID EXCHANGES.
>>Jo Morris: WOULD IT INCREASE
THE HR, YES IT DOES.
IF THAT'S THE QUESTION YOU ARE
ASKING.
IN RELATION TO WILD TYPE, OR
THE DOUBLE MUTANT HAS MORE HR.
>>Karen Usdin: OKAY, THERE'S A
QUESTION FROM MICHAEL.
GREAT TALK.
DO YOU THINK THIS ROLL OF RAD
52 IS DISTINCT FROM SUPPOSED
ROLE IN SINGLE STRANDED
KNEELING?
>>Jo Morris: YEAH, I DON'T
KNOW.
YEAH, THE THING IS -- NUMBER
OF ROLES OF RAD52 JUST GROWS BY
THE DAY, DOESN'T IT?
WE ARE WONDERING.
WE CAN'T DISTINGUISH IS WHETHER
IT'S ROLES IN RAD51 LOADING AND
HOMOLOGOUS MIGHT ALSO RELATE,
WE ARE PLANNING TO DRILL INTO
THAT QUESTION, I THINK THE A
TERRIFIC QUESTION.
WE DON'T KNOW.
IT ALSO WRAPS UP SINGLE
STRANDED DNA IN A
CONCENTRATION-DEPENDENT WAY.
LOVELY IN VITRO WORK SUGGESTS
TOO MUCH RAD 52 HOLD UP TOO
MUCH SINGLE STRANDED DNA IN A
WAY THAT MIGHT BE INCOMPATIBLE
WITH DOWNSTREAM REPAIR EVENTS.
SORRY, I DON'T KNOW IF I'M
EXPLAINING THAT VERY WELL, BUT
YEAH, WE DON'T KNOW IS THE
SHORT ANSWER.
>>Karen Usdin: THANK YOU.
ARE THERE ANY MORE QUESTIONS?
RIGHT NOW THE CHAT BOX IS
DEPLETED.
SO LAST-MINUTE QUESTIONS FROM
ANYBODY?
>>I JUST HAVE A QUICK QUESTION.
THE POL THETA INHIBITOR, HOW
SPECIFIC IS IT?
IT WAS ALREADY ADDRESSED IN THE
QUESTIONS.
BUT THE ONE YOU USED, HOW
SPECIFIC IS IT, ACTUALLY?
>>Jo Morris: WELL, FROM THE
PREVIOUS PAPERS IT LOOKS VERY
GOOD.
I THINK WHAT'S ENCOURAGING FROM
OUR PERSPECTIVE WE COMBINED IT
WITH POL THETA SIRNA AND SEE
BEAUTIFUL STASIS.
THEY ARE NO LONGER TERRIBLY
SPECIFIC.
BUT IT LOOKS PRETTY GOOD.
THERE'S A DERIVATIVE GOING INTO
CLINICAL TRIALS, SO I HOPE IT'S
GOOD.
>>Karen Usdin: OKAY.
ANY OTHER QUESTIONS?
NOT SEEING ANY QUESTIONS IN THE
CHAT.
I THINK UNFORTUNATELY CHUN HAS
DROPPED OFF THE CALL.
WE ARE HAVING INTERNET ISSUES
ON THE CALL ALL AROUND.
I THINK THIS HAS BEEN GOOD
DISCUSSION.
IF THERE ARE NO OTHER
QUESTIONS, I THINK JO, WE WILL
JUST THANK YOU SO MUCH FOR A
REALLY, REALLY INTERESTING TALK.
LOTS OF FOOD FOR THOUGHT.
WE WILL BE IN TOUCH.
WE WILL SEND YOU THE LIST OF
THE PARTICIPANTS AND QUESTIONS.
IF YOU WANT TO TOUCH BASE WITH
ANYBODY, YOU KNOW, THAT'S AN
OPPORTUNITY TO DO SO.
STEVE JACKSON, LAST MINUTE
INPUT SAYS GREAT TALK, JO.
CHUN IS BACK SO MAYBE HE CAN DO
THE SIGN-OFF FOR US.
>>Chun Zhang Yang: SORRY, JUST
LOGGED IN.
ALL QUESTIONS ARE DONE?
>>Karen Usdin: WE ARE NOT
GETTING ANY MORE QUESTIONS.
>>Chun Zhang Yang: OKAY, ALL
RIGHT.
SORRY ABOUT THAT.
I KEPT DROPPING OFF THE WEBEX
AS WELL.
I GUESS THERE'S OVERWHELMING
ISSUE.
REGARDLESS, THANK YOU, DR.
MORRIS FOR THE FANTASTIC TALK.
WE REALLY APPRECIATE YOU TO
PARTICIPATING AND GIVING US
SUCH AMAZING TALKS.
SO WE WILL KEEP IN CONTACT AND
GET BACK FOR ANOTHER INTEREST
GROUP, CONFERENCE.
THANK YOU SO MUCH, EVERYBODY.
>>Jo Morris: THANK YOU.
THANK YOU FOR THE QUESTIONS.
TAKE CARE, ALL.
>>Chun Zhang Yang: THANK YOU.
>>Karen Usdin: TAKE CARE,
EVERYONE.