LET'S GET STARTED.
I AM SO DELIGHTED TODAY TO
INTRODUCE TODAY'S SPEAKER SIR
STEVEN JACKSON.
SO STEVE, STEVE IS THE
UNIVERSITY OF CAMBRIDGE
PROFESSOR OF QUICK BIOLOGY AND
SENIOR GROUP LEADER AT THE
CANCER UK INSTITUTE AT
CAMBRIDGE.
HIS RESEARCH IDENTIFIED KEY
PRINCIPLES BY WHICH CANCER CELLS
RESPOND TO AND REPAIR DNA DAMAGE
AND HOW THEY YIELD CANCER AND
OTHER AGE RELATED DISEASES.
IN ADDITION TO THE VARIOUS
COMPANIES THAT HE HAS FOUNDED TO
TAKE SOME OF HIS RESEARCH
FINDINGS TO THE CLINIC, STEVE IS
A FELLOW OF THE ROYAL SOCIETY,
UK ACADEMY OF MEDICAL SCIENCES,
THE EUROPEAN ACADEMY OF CANCER
SCIENCES AND [INDISCERNIBLE].
HE HAS RECEIVED NUMEROUS
NATIONAL AND INTERNATIONAL
PRIZES AND IN 2023 WAS AWARDED A
KNIGHT HOOD FOR HIS SERVICES TO
INNOVATION AND RESEARCH.
I AM VERY EXCITED TO HEAR WHAT
HE HAS TO SAY TODAY BUT BEFORE
WE START, I REMIND YOU ALL TO
PUT YOUR QUESTIONS IN THE CHAT
BOX AND WE WILL READ THEM OUT
AFTER HE COMPLETED HIS
PRESENTATION.
STEVE, IF YOU CAN START WHEN
YOU'RE READY?
>> GREAT.
CAN YOU HEAR ME?
>> WE CAN.
>> GREAT.
I WOULD LIKE TO START BY
THANKING KAREN, CHUN ZHANG, AND
COLLEAGUES FOR INVITING ME TO
THIS GROUP TODAY.
I WOULD LIKE TO THANK THE NIH
PER FUTURE--POTENTIALING THESE
SEMESTER NEARS ON, IT WAS
CRUCIAL DURING COVID TIMES BUT
IT STILL PLAYED AN AMAZING
IMPORTANT ROLE IN THE DNA REPAIR
FIELD AND IS IMPACTING WORLD
WIDE SO THANKS AGAIN FOR THAT.
SO I KNOW I WILL BE PREACHING TO
THE CONVERTED HERE BUT TO START
WITH, I'M JUST GOING TO GIVE A
BRIEF SUMMARY OF CELLULAR
RESPONSES TO DNA DAMAGE AND DNA
REPAIR.
POINT OUT IT'S MEDICAL
IMPORTANCE AND CLINICAL
POTENTIAL AND THEN I WILL ANY ON
TO TALK ABOUT SOME RECENT WORK
IN MY LAB WHICH PROVIDED NEW
INSIGHTS INTO THESE PATHWAYS,
AND ALSO PROVIDE FURTHER LINKS
TO HUMAN DISEASE.
SO WE'RE MORE AWARE OF THE FACT
THAT DNA IN OURSELVES AND EVERY
ORGANISMS' CELLS IS CONTINUALLY
BEING BOMBARDED BY A RANGE OF
[INDISCERNIBLE] AS WELL AS
EXOGENOUS DNA DAMAGING AGENTS
GIVING RISE TO A WIDE RANGE OF
DNA DAMAGE THAT NEEDS TO BE
DEALT WITH BY THE DNA REPAIR
SYSTEMS AND THE SYSTEMS.
APOLOGIES I HAVE A BIT OF A COLD
TODAY BUT HOPEFULLY MY VOICE
WILL HANG TOGETHER.
WHAT I WILL FOCUS ON TODAY IS A
BIT UNUSUAL FOR ME, I USUALLY
TALK ON DOUBLE STRAND BREAK
MECHANISMS BUT I WILL TALK TODAY
ABOUT DNA CROSS LINKS.
THESE ARE VERY, VERY IMPORTANT
LESIONS IN EMERGING ITS OF
BIOLOGICAL AND MEDICAL
IMPLICATIONS, OF COURSE DNA
CROSS LINK WILL BE A SEVERE
IMPEDIMENT TO PROCESS SUCH AS
TRANSCRIPTION AND REPLICATION
EMPLOY NOW WHEN WE THINK ABOUT
DNA REPAIR, WE CLEARLY WANT TO
KNOW HOW THE DNA REPAIR PROTEIN
COMPLEXES WORK BUT WE NEED TO
THINK ABOUT DNA REPAIR OR ALSO
THINK ABOUT DNA REPAIR IN THE
CONTEXT OF A BROADER DNA DAMAGE
RESPONSE AS IS OFTEN TERMED
WHICH LEADS US TO CONSIDER DNA
REPAIR AND THE CONTEXT BRINGS UP
CHROMATIN STRUCTURE, DNA
REPLICATION AND HOW IT'S
IMPACTED BY VARIOUS OTHER THINGS
INCLUDING NUCLEAR ARCHITECTURE
AND DNA METABOLISM AND OTHER
EVENTS.
WE ALSO NEED TO THINK ABOUT THE
SIGNALING PATHWAYS THAT ARE
TRIGGERED IN RESPONSE TO VARIOUS
FORMS OF DNA DAMAGE, AND HOW
THAT CAN LEAD TO INDUCTION OF
REPAIR FACTORS, TRANSCRIPTIONAL
PROGRAMS, CELL CYCLE, CONTROLS
INCLUDING CELL CYCLE CHECK
POINTS, AS WELL IN OTHER CASES
TRIGGERS APOPTOSIS OR
SENESCENCE.
AND WE ALSO NEED TO THINK ABOUT
HOW PROTEINS ARE INVOLVED IN
OTHER PROCESSES SUCH AS TELOMERE
MAINTENANCE UNDER
NORMAL/ABNORMAL CIRCUMSTANCES.
OVER THE YEARS WE LEARNED THAT
THESE RESPONSES ARE GOVERNED BY
THE ACTIONS OF A WIDE VARIETY OF
REVERSIBLE POST TRANSLATION
MODELS INCLUDE, UBIQUITINNATION,
POLYSATURATION, AND
HBB -ELATION, I THINK WHERE WE
STAND HOW THE DNA FIELD IS QUITE
A MATURE 1 AND WE KNOW NOW
THERE'S OVER 1000 HUMAN PROTEINS
WITH STRONG DNA DAMAGE RESPONSE
AND DNA REPAIR LINKS BUT MANY OF
THESE ARE STILL LARGELY
UNCHARACTERRIZED.
WE ALSO KNOW THAT DNA,
UNDOUBTEDLY OTHER DDR PROTEINS
AND REGULATORS TO BE IDENTIFIED
AND I THINK THE BIGGEST
CHALLENGE AND OPPORTUNITY FOR US
RIGHT NOW MOVING FORWARD IS TO
THINK ABOUT THIS IN TERMS OF THE
FUNCTIONAL INTERACTION LANDSCAPE
WHICH IS LARGELY UNEXPLORED AND
WHAT I MEAN BY THAT IS NOT
THINKING ABOUT INDIVIDUAL
COMOPPOSITE BEHAVIORIAL
PHENOTYPENTS OR PATHWAYS IN
ISOLATION BUT THINKING HOW THEY
CONNECT, REGULATE AND IMPACT ON
1 ANOTHER IN A BROADER RESPONSE,
AND WE NOW HAVE TOOLS AND
TECHNIQUES TO PICK THIS APART
AND I WILL BE HIGHLIGHTING SOME
OF THOSE IN MY TALK TODAY.
NOW WE KNOW THAT DNA DAMAGE AND
CELLULAR RESPONSE FOR DNA DAMAGE
UNDERLINE MANY DECS AND YOU WILL
BE AWARE OF JUST ABOUT ALL OF
THESE BUT I WANT TO HIGHLIGHT,
BUT THE OUTFIELD IS AMAZING IN
TERMS OF HUMAN BIOLOGY AND
DISEASE.
THESE FACTORS GOVERN HYPER
SENSITIVITY TO RADIATION AND
CHEMO THERAPIES USED IN CANCER.
THERE ARE DEFECTS IN THESE
PATHWAYS GIVE RISE TO
DEVELOPMENT DEFECTS,
DEFICIENCIES, NEURODEGENERATION,
PREMATURE AGING, INFERTILITY AND
DIABETES APPROXIMATE A RANGE OF
OTHER THINGS BUT MOST OF THESE
IS IN THE REALM OF CANCER WHERE
DNA DAMAGE AND THE DNA DAMAGE
RESPONSE AND REPAIR MECHANISMS
HAS HUGE RELEVANCE.
WE KNOW THAT DNA DAMAGE CAN GIVE
RISE TO MUTATIONS WHICH IS
CRUCIAL FOR CAR SIN O GENESIS,
AND WE KNOW THAT MANY DDR
DEFECTS INHERITED OR ACQUIRED
PROMOTE CANCER.
THE MAIN STAYS OF CANCER THERAPY
REMAIN IONINIZING RADIATION AND
MOST CHEMO TERRAPY THERAPEUTICS
WORK BY INFLECTING DNA DAMAGE.
OTHER AREAS WHERE DNA DAMAGE
IMPACT ON CANCER BUT OVER THE
LAST COUPLE DECADES WE LEARNED
THE DDR COMPARE MECHANISMS
CONTAIN VARIOUS ATTRACTED DRUG
TARGETS AND I IMAGINE, IN MY
VIEW, THE POSTER CHILD FOR THIS,
AT LEAST AT THIS TIME, ARE PARP
INHIBITORS INCLUDING PARP
INHIBITORS 1 AND 2, TAKEN BY THE
FIRST PATIENTS BY MY COMPANY
[INDISCERNIBLE] WHICH WAS
ACQUIRED BY AFT RAZENECCA AND
IT'S REACHING WELL OVER A
HUNDRED THOUSAND PATIENTS RIGHT
NOW TO RECEIVE THIS DRUG AND I
SHOULD ALSO POINT OUT THERE ARE
OTHER PARTNERSHIP --PARP
INHIBITORSS THAT ARE MAKING
INROADS.
THIS IS EXITING.
SOPHISTICATED WHAT IS
PARTICULARLY EXCITING IS HAS
PARP INHIBITORS BECOME MORE
ENTRENCH INDEED CANCER THERAPY
AND THEY'RE BEING BROADENED,
THEY'RE USED EARLIER AND EARLIER
AND IF WE LOOK AT THE SLIDE
GOING LEFT TO RIGHT, WE CAN SEE
THAT PARP INHIBITORS SUCH AS
[INDISCERNIBLE] ARE NOW MOVING
TOWARDS EVEN IN FIRST LINE
TREATMENT AND WHAT CLINICIANS
ARE SEEING IS THAT THE EARLIER
PARP INHIBITORS ARE USED WITH
THE RIGHT TYPES OF PATIENTS
THOSE WITH HRD, MORGS
DEFICIENCY, RECOMBINATION FOR
EXAMPLE, THE IMPACT ON THESE
PATIENT COHORT SYSTEM BECOMING
MORE EVER PRONOUNCED AND ON THE
RIGHT HAND SIDE, IT'S REALLY,
REALLY EXCITING DATA SHOWING
THAT THE [INDISCERNIBLE]
PROGRESSION FREE SURVIVAL IS
MUCH, MUCH BETTEROT TREATMENT
ARM AND OF COURSE YOU WILL NOTE
THAT THESE CURVES ARE FLAT
LINING OR STARTED TO FLAT LINE
TOWARDS THE END WHICH INDICATES
THAT MANY OF THESE PATIENTS ARE
BENEFITING FOR CONSIDERABLE
LINKS OF TIME AND INDEED THERE
MAY BE IN DUE COURSE, CERTAIN
INDIVIDUAL WHO IS COULD BE
CLAIMED AS CURES THROUGH THESE
KIND OF APPROACHES.
GOING ON FOR DNA REPAIR AND
ENZYMES IS NOW CAUGHT ON AND
THERE ARE A RAINCHL OF COMPANIES
AND OF COURSE ACADEMIC LABS IN
THIS SPACE IDENTIFYING SYNTHETIC
LETHAL AS SOMEWHAT OTHER
OPPORTUNITIES WHICH OFFER HOPE
IN THE NOTE TOO DISTANT FUTURE
FURTHER FDA AND E, INA APPROVED
MEDICINES FOR TREATING CANCER.
SO GOOD LUCK WITH ALL THOSE
PROGRAMS AND HOPEFULLY THERE
WILL BE POSITIVE NEWS IN VARIOUS
FRONTS OVER THE COMING YEARS.
BUT THAT'S IMPORTANT BUT AS I
MENTIONED A FEW MINUTES AGO,
MOST CANCERS ARE TREATED BY
SURGERY OR BY AND/OR RADIATION
AND CONVENTIONAL CHEMO THERAPIES
OR TRADITIONAL ANTICANCER CHEMO
THERAPIES AND WE DON'T ACTUALLY
REALLY UNDERSTAND HOW MANY OF
THESE TRADITIONAL CANCER CHEMO
THERAPY ACTUALLY WORK AND HOW
THEY SOMETIMES SUCCEED BUT OFTEN
FAIL.
NOW 1 OF THESE THERAPIES IS
BASED ON A COMPOUND CALLED
ATOPOCIDE WHICH IS SHOWN ON THE
LEFT HERE AND THIS IS USED IN A
RANGE OF OF DIFFERENT LIQUID AS
WELL AS SOLID CANCERS BUT THERE
ARE ISSUES WITH THESE
TREATMENTS, THERE'S A
2-3 PERCENT RISK OF DEVELOPING
SECONDARY LEUKEMIAS, SHOWING
CHROMOSOMAL TRANSWELLICATIONS BY
DNA REPAIR MECHANISMS BY NOT YET
FULLY UNDERSTOOD MECHANISMS.
AND OF COURSE CHALLENGES TO
MAXIMIZE EFFICACY, REDUCE SIDE
EFFECTS AND OVERCOME RESISTANCE
TO THESE KINDS OF DRUGS AND
MAYBE SIMILAR TO WHAT WE'RE
THINKING ABOUT IN THE CONTEXT OF
PARP INHIBITORS TO TAILOR
THERAPIES TO RIGHT CANCERS TO
THE RIGHT TIME TO THE RIGHT
PATIENTS.
SO WHAT DOES ATOPOCIDE DO?
IT BINDS TO AND TRAPS DNA
POLYISOMMER ACE 2, TOP 2 ALPHA
AND TOP 2 BETA, IT TRAPS THESE
IN A CLEAVAGE COMPLEX
INTERMEDIATE DURING ITS
ENZYMATIC CYCLE.
SO TOP 2 ENZYMES MODULATE DNA
TOPOLOGY DURING DNA REPLICATION,
RECOMBINATION, AND SISTER
CHROMATIN DECANTONNATION.
AND THE WAY TOP 2 ENZYMES WORK
IS TO GENERATE A DOUBLE STRAND
BREAK IN 1 DNA DOUBLE HELIX AND
THEN PASS THAT THROUGH ANOTHER
DNA DOUBLE HELIX, THEN RESEAL
THAT DOUBLE STRAND BREAK AND
THEREFORE OBVIOUSLY IN THIS
SCENARIO HERE CAN DISENTANGLE OR
WE MOVE TOPOLOGY FROM DNA
MOLECULES.
AND WHAT ATOPOCIDE DOES, IT
BINDS TO THIS REACTION ISHT
MEDIATE WHERE THERE'S A CLEAVAGE
COMPLEX, AND IN THIS CLEAVAGE
COMPLEX, TOP 2 IS ATTACHED TO A
LINKAGE TO THE 5 PRIME ENDS OF
THE DOUBLE STRAND BREAK.
SO WE HAVE A DOUBLE STRAND BREAK
IN THE DNA, BUT IT'S NOT NAKED,
IT'S AN ENZYME COUPLED COVALENT
INTERMEDIATE AND OF COURSE
TRAPPING THESE WITH ATOPOCIDE
POSES MAJOR PROBLEMS TO CELLS,
THIS IS CLEARLY GOING TO BE A
MAJOR BARRIER TO THINGS SUCH AS
REPLICATION OR TRANSCRIPTION.
SO TO DEAL WITH THESE KINDS OF
LESIONS AND PRESUMABLY NATURALLY
ARISING STORE CLEAVAGE COMPLEXES
WHICH PROBABLY TAKING PLACE ALL
THE TIME IN THE ABSENCE OF THE
ATOPOCIDE IN RESPONSE TO BASE
DAMAGE OR OTHER CHEMICALS, THERE
ARE A RANGE OF DNA REPAIR
SYSTEMS THAT DEAL WITH THESE,
NOW 1 OF THESE SYSTEMS SHOWN ON
THE LEFT HERE, IS MEDIATED, THE
TOP OF THE PATHWAY IS A PROTEIN
CALLED ZNF451, ALSO CALLED ZAT,
AND IT ATTACHING SUMO UNITS TO
TOP 2.
THIS THEN SERVES AS AN ATTRACT
ANT FOR TDP2.
TDP2, HAS SAME MOTIF, SAME
INTERACTION MOTIFS WHICH
RECOGNIZE THE SUMO RELATED TOP
2, TDB2 RECRUITMENT USES
ENZYMATIC ACTIVITY TO CLEAVE THE
FOSTER NURSED FOCUSED ON
TYROSOLLINKAGES, RELEASING THE
DOUBLE STRAND BREAK THAT CAN BE
REPAIRED BY JOINING MECHANISMS
OFTEN IN A GREAT FASHION.
THERE'S AN ALTERNATIVE TYPE OF
SET OF PATHWAYS THAT CAN ALSO
ARISE AT STABILIZED TOP 2
CLEAVAGE COMPLEXES AND AT THE
TOP OF THIS PATHWAY IS A SUMO
TARGETED UBIQUITIN LIGASE OR
STUBBLE CALLED RNF4, WHICH
ASSOCIATES WITH THE ASSIMILATED
TOP 2, MEDIATES UBIQUITINNATION
WHICH THEN LEADS TO PROTEOSOME
DEGRADATION
GRATES--GRATUEIGATION EMPLOY NOW
UNLIKE THE SITUATION WITH TDP2
WHICH CAN CLEANLY REMOVE THE
COVALENTLY ATTACHED TOP 2, RNF 4
DOES AND THE PATHWAY WILL NIBBLE
AWAY AND EAT AWAY AT THAT BUT IS
UNABLE TO REMOVE THE FINAL
PEPTIDE AND THEY MUST BE REMOVED
BEFORE DMA REPAIR TAKES PLACE
AND THOSE PEPTIDE BLOCKED DOUBLE
STRAND BREAKS ARE PROCESSED BY
TDP2, FOLLOWING INTO THE PATHWAY
IN THE MIDDLE OR THROUGH VARIOUS
NUCLEACES WHICH ALLOW MORE ARROW
PRONE PATHWAYS SUCH AS
NORMAL--SORRY ALTERNATIVE AND
JOINING POSSIBLY ENJOINING AS
WELL AS BY TDP2, FOLLOWING ON
INTO THE PATHWAY IN THE MIDDLE
ALL THROUGH VARIOUS NUCLEACES
WHICH ALLOW ERROR PRONE PATHWAYS
SUCH AS ALTERNATIVE END JOINING
[INDISCERNIBLE] END JOINING AS
WELL AS TDP2, FOLLOWING INTO THE
PATHWAY IN THE MIDDLE OR THROUGH
VARIOUS NUCLEACES WHICH ALLOW
MORE EROR PRONE PATHWAYS SUCH AS
[INDISCERNIBLE] ALTERNATIVE AND
JOINING POSSIBLY CANNED WHAT
JOINING AS WELL AS TDP2,
FOLLOWING INTO THE PATHWAY IN
THE MIDDLE OR THROUGH VARIOUS
NUCLEACES WHICH ALLOW MORE ERROR
PRONE PATHWAYS [INDISCERNIBLE]
ALTERNATIVE END JOINING AND
[INDISCERNIBLE] END JOINING AS
WELL AS TDP2 FOLLOWING IN THE
PATHWAY IN THE MITSD ALL THROUGH
VARIOUS NUCLEACES WHICH ALLOW
MORE ERROR PRONE PATHWAYS SUCH
AS NON[INDISCERNIBLE]
ALTERNATIVE END JOINING AND
POSSIBLY [INDISCERNIBLE] AS WELL
AS IN THE PATHWAY IN THE MIDDLE
ALL THROUGH VARIOUS NUCLEACES TO
ALLOW FOR ERROR PRONE PATHWAYS
--YOU CAN SEE THERE'S NF451 BUT
WE ALSO IDENTIFIED 2 FACTORS
THAT WE HIGHLIGHT IN RED, RAD 54
L2 EXPW ERCC6 L2 WHICH AT THAT
TIME WERE UNCHARACTERRIZED IN
TERMS OF THEIR DVR ROLES.
THESE ARE BOTH ATP ACE HELIX
CASE ENZYMES.
NOW FOLLOWING ENSUING WORK BY US
ESTABLISHED THAT RAV54 KNOCK OUT
CELLS WE CAN SPECIFICALLY MAKE
IN 1 RPA AND 1 UTOS BACKGROUNDS
WITH CRISPR CAS9 TECHNOLOGY, AND
WHAT WE FIND FOR THE KNOCK OUT
CELL SYSTEM THAT THEY'RE
SPECIFICALLY HYPER SENSITIVE TO
ATOPOCIDE.
SO KNOCK OUT CLONES FOR RAV54 L2
ARE MORE SENSITIVE THAN CONTROLS
TO ATOPOCIDE BUT WE DON'T SEE
THIS FOR A RANGE OF DNA OTHER
DAMAGINGLY AGENTS WE USED AND
THIS ACTUALLY CONTRASTED TO THE
SITUATION FOR ERCC 6 L2 WHICH WE
FOUND WERE HYPER SENSITIVE TO A
RAINCHL OF DNA DAMAGING AGENTS
AND HAVE BEEN NOW PUBLISHED BY
LABS OF [INDISCERNIBLE] AND
[INDISCERNIBLE] TO HAVE LINKAGES
AND MORE IN THE ENJOINING
PROCESSES.
WE CHOSE TO FOCUS ON RAD 54 L2
TO SEE HOW DNA INDUCED DAMAGE
COULD BE BROUGHT ABOUT AND A KEY
SET OF EXPERIMENTS THAT REALLY
SET US ON PATH TO UNDERSTAND
THANKSGIVING FACTOR WAS THROUGH
A COLLABORATION WITH
PETRA [INDISCERNIBLE] AND
[INDISCERNIBLE] IN GERMANY.
SO WE PROVIDED PETRA'S LAB WITH
CELL LINES EXPRESSING RFP ATTACK
EXPRESSING CELLS OR EXPRESSING
PROTEIN LINKED TO RAV54 L2.
WE THEN ISOLATED THE THESE
PROTEINS BY IMMUNO PRECIPITATION
AND THESE WERE THEN SUBJECT TO
MASS SPEC. PROTEOMICS BY PETRA'S
LAB.
NOW SATISFYING IF WE FIRST LOOK
AT THE DATA WE OBTAINED WITH NO
ATOPOCIDE WITH THE CELLS
UNTREATED WE FIND RAVPOOR L2
UNCONTROLLED, WE PULL IT DOWN,
WE SEE THAT, BUT YOU ALSO NOTE
THAT WE SEE SUMO 1, 2, 3, AND
THE TOP 2 IN HUMAN CELLS AND
ALPHA TOP 2 BETA AND ALPHA.
SO THIS DID SAY TO US THAT THIS
FACTOR GIVING THE SENSITIVITY IS
CONNECTED TO THE TYPES OF THINGS
THAT WE KNOW NEED TO OPERATE TO
MEDIATE TOP 2 CLEAVAGE COMPLEX
REPAIR.
THINGS BECOME EVEN MORE
INTERESTING WHEN WE LOOKA THE
ATOPOCIDE TREATED CELLS.
WE ALSO GET TOP 2 ALPHA AND TOP
2 BETA.
YOU WILL SEE NOW A VERY, VERY
STRONG INTERACTOR HERE IS AN
F451 WHICH IS NOT A STRONG HIT
IN THE UNTREATED CONDITION.
SO, THIS SUGGESTED TO US THAT
RAV54 L2 MUST BE CONNECTED TO
THE SUMO E3 LIGASE, AS AN F451,
WHICH HAS BEEN SHOWN A FEW
MOMENTS AGO TO SUMO ELATE TOP 2
ALPHA AND TOP 2 BETA.
SO, WHAT WAS RAV 54 L2 LOOK
LIKE, THIS IS A DEPICTION ON THE
LEFT-HAND SIDE, IT'S AN ATP ACE
HELIX CASE ENWRIEM AND IT ALSO
HAS A RANGE OF MOTIFS THAT ARE
PREDICTED TO IRPT ACT WITH SUMO.
THESE ARE CALLED SUMO
INTERACTING MOTIFS OR SIMS, SO
THIS ALLOWED US TO GO AHEAD AND
MAKE MUTATED VERSIONS OF RAV54
L2, .2 MUTATE INDEED THE
ANALYTIC SITE TO KILL THE
ACTIVITY OR MUTATE INDEED
MULTIPLE SIMS PRESUMABLY,
HOPEFULLY THEN TO PREVENT THE
ABILITY TO IRPT ACT WITH SUMO
RELATED PROTEINS.
SO THROUGH COMPLEMENTATION
TODAYS AS YOU WILL SEE IN THE
GRAPH BELOW, COMPARED TO
WILD-TYPE CELLS, SHOWN IN BLUE,
CELLS COMPLEMENTED--SORRY, CELLS
LACKING RAV54 L2 ARE HYPER
SENSITIVE TO ATOPOCIDE AND
IMPLEMENTATION OF THESE CELLS
WITH THE CATALYTIC DEAD OR SAME
MUTANT PROTEIN DOES NOT RESTORE
RESISTANCE.
BY CONTRAST, COMPLEMENTING THESE
CELLS WITH WILD-TYPE RAV54 L2
DOES.
AND IN FACT, WE CAN SEE THESE
CELLS ARE MORE RESISTANT THAN
THE WILD-TYPE CELLS.
SO THESE CELLS ARE INDICATING
THAT THE ATP-CASE, RAV54 L2 AS
WELL AS SIEWM OR INTERACTION
DOMAINS ARE IMPORTANT FOR ITS
ACTIVITY.
CONSISTENT WITH THIS, AND ALSO
CONSIST WENT THE PROTEOMICS
DATA, WE FIND THROUGH IP-IP
WESTERN BLOT STUDIES AS YOU CAN
SEE HERE THAT RAV 54 L2, AND IT
IRPT ACTING IN A CONSTITTATIVE
MANNER WITH ALPHA TOP 2 ALPHA
AND BETA, BUT IN RESPONSE TO
ATOPOCIDE TREATMENT, WHICH
GENERATES A MODIFIED FORM, TOP
2, THIS FACTOR BINDS TO THOSE
FORMS THE PROTEIN, HOWEVER, IF
WE LOOK AT THIS DELTA SAME
MUTANT SHOWN TO THE RIGHT HERE,
THAT'S INTERACT WITH THE TOP 2,
ALPHA AND TOP 2 BETA, BUT NOT
THE MODIFIED FORMS, EVEN THOUGH
THEY'RE EXISTING IN THIS
SCENARIO.
AND YOU WILL ALSO LIKE FROM THE
BOTTOM, ALSO CONSISTENT WITH THE
MASS SPEC DATA WE ALSO SEE AN
INTERACTION WITH ZEBF451 WHICH
IS REDUCED AND THIS IS LARGELY
AGGREGATED IN THE DELTA MUTANT.
SO WHILE WE'RE AT THAT STAGE, WE
CLEARLY CONNECTED IT INTO A
ATOPOCIDE SENSITIVITY AND
PRESUMABLY TOP 2 CLEAVAGE
COMPLEX PROCESSING AND/OR REPAIR
BUT WE WANT TO FIGURE OUT WHERE
IT MAPPED WITHIN THE NETWORK OF
PROTEINS ALREADY KNOWN.
SO THE APPROACH WE USED HERE AND
THIS IS AN APPROACH WE'RE USE
NOTHING MANY PROJECTS IN MY
GROUP IS TO CARRY OUT NOT 1
CRISPR KREENS BUT A RANGE OF
THEM ACROSS GENETIC BACKGROUNDS.
SO IF YOU THINK OF THIS SCENARIO
HERE WE MIGHT HAVE A WILD-TYPE
CELL BACKGROUND, CARRY OUT A
CRISPR SCREEN, FOR THE
PARTICULAR DRUG AND WE IESHES
DEBTIFY A SET OF GENES A-G.
OF COURSE IF WE CARRY OUT
ANOTHER SCREEN IN A CELL THAT'S
KNOCKED OUT FOR GENE A WE WOULD
NOT EXPECT TO SEE ANY FURTHER
SENSITIZATION FOR THE CRISPR
SCREEN, AND IN THIS SCENARIO
HEREY WE SEE THAT GENE C IS
NOTHING LIKE THAT DROPPING OUT
BECAUSE IT'S WORKING ON THE SAME
PATHWAY AS L ORA, WHERE B IS.
SO WE'RE ABLE TO DRAW UP
INTERACTION NETWORKS FOR THE
DRUG AT HAND BUT WE'RE STUDYING
AND THEREBY DRAW LINES OR
CONNECTIONS BETWEEN DIFFERENT
PATHWAYS THAT MIGHT BE BEING
USED IN DIFFERENT CONTEXT.
SO WHEN WE CARRY THIS OUT IN
THIS PARTICULAR EXPERIMENT HERE,
THIS WORK WAS CARRIED OUT BY
[INDISCERNIBLE] A POST DOC IN
THE GROUP WITH HELP FROM
[INDISCERNIBLE], A
BIOMATHEMATICIAN WHO JUST MOVED
DOWN THE ROAD FROM US, WHAT WE
SEE HERE ARE THE RESULTS FROM
PLOTS IN 2 DIFFERENT
BACKGROUNDS, WE HAVE THE
WILD-TYPE CRISPR ALONG THE
BOTTOM AND A IMK OUT IN THE
Y-AXIS AND ANYTHING IN THE LOWER
LEFT QUADRANT ARE DRIVING
SENSITIVITY.
VERY NICE CONTROL IN THIS
EXPERIMENT IS THAT THIS RAV54
L2, ITS, IT'S SERVING AS A GOOD
HIT IN THE WILD-TYPE BACKGROUND
BUT NOT DROPPING OUT IN THE
RAV54 L2 BACKGROUND BECAUSE IT'S
ALREADY KNOCKED OUT IN THAT BACK
IMROWND.
BUT WHILE WE'RE STRIKING IT,
EVERY OTHER FACTOR WE'RE SEEING
IN THIS SCREEN IS STILL LYING
CLOSE TO THE DIAGONAL.
AND THIS IS HIGHLIGHTING TO US
THAT RAV 54 L2 SEEMS TO BE WORKS
IN A DISTINCT MANNER FROM THE
BULK OF KNOWN RESPONDERS TO TOP
2 COMPLEX CANNED WHAT.
BUT 1 THING I NEED TO HIGHLIGHT
TO US IS I'VE LEARNED THAT THE
LIGHT WE USED FOR CRISPR
SCREENING THAT THE GAINS ARE NOT
VERY GOOD AND I WILL COME BACK
TO THAT IN A MOMENT AND I WILL
COME BACK TO THAT IN A MOMENT
BECAUSE THEY DO INDEED CONNECT.
SO VALIDATING THOSE CRISPR DATA
INDICATING THAT RAV54 L2 WORKS
IN A DISTINCT WAY TO OTHER
FACTORS WE'VE KNOWN ABOUT, IF
WHAT WE'RE SEEING ON THE LEFT
PLOT HERE, ARE SURVIVAL CURVES
WITH A RAINCHL OF DIFFERENT
KNOCK OUTS AND IF WE KNOCK OUT
BOGHT RAV 54, L2, MDP2, WHICH
RECORDS PLAYING KEY ROLE, IN
REMOVING THE CLEAVAGE COMPLEX,
WE GET MUCH MORE SENSITIVITY
THAN THE SINGLE MUTANTS ALONE
EMPLOY THE SENSITIVITY HERE IS A
LOW CONCENTRATIONS AND THAT
THESE CONCENTRATIONS ACTUALLY
NOT VERY MUCH SENSITIVE IN THE
KNOCK OUT ON HIS OWN, AND IT IS
WORKING AT A DISTINCT PATHWAY
FROM TDP 2, AND SIMILARLY, FROM
THE COMPETITION ASSAYS, WE LOOK
TO SEE IF RAV 54 L2 WAS WORKING
INSIDE OR OUTSIDE OR HR RELATED
PROCESSES HOMOLOGOUS COMBINATION
ASSOCIATED PROCESSES AND WE SEE
THAT IT'S ADD.
SO RAV54 L2 IS ALSO HAVING AN
IMPACT ON THOSE PROCESSES SO
WHAT IS IT DOING.
THROUGH SIMILAR PRPLTS -
-EXPERIMENTS THAT WHILE
KNOCKING OUT RAV54 L2,
SENSITIZING CELLS, AND THAT'S
SHOWN ON THE LEFT BUT I THINK
IT'S ACTUALLY EVEN MORE STRIKING
ON THE RIGHT, AND THIS IS
ACTUALLY PART OF THE STUDY THAT
IS AS YET UNPUBLISHED AND
SPEARHEADED BY [INDISCERNIBLE]
BONITEZ, IN MY GROUP AND SHE'S
CARRYING THIS OUT AND A RANGE OF
BACKGROUNDS A HERCULEAN EFFORT,
AND LOOKING AT CELLULAR
RESPONSES TO TOP 2 AND PROVIDING
A GENETIC INTERACTION MAP AND
THIS IS JUST 1 OF THOSE EXAMPLES
OF THESE KREENS OR 2 OF THEZ
KREENS AND WE'RE PLOTTING THEY
RESULTS OF A GENOME WIDE SCREEN
IN THE WILD-TYPE CELLS IN THE
ACCESS COMPARED TO NFR 451
BACKGROUND AND I HOPE YOU AGREE
WITH ME, IT'S A STRIKING FINDING
HERE THAT IT'S DROPPING OUT
QUITE STRONGLY IN THE WILD-TYPE
SETTING BUT BASICALLY IS SITTING
ON THE 0 LINE IN THAT 451.
SO IT'S WORKING DOWN STREAM OF
ZNF451, OR IT'S EPISTATIC TO
RAD54 L2 IN TERMS OF THIS
PHENOTYPE.
SO IN TERMS OF TRYING TO FIGURE
OUT PRESLICELY WHAT RAD54 L2 WAS
DOING WE WERE BENEFIT FRIDAY A
COLLABORATION WITH JOHN AND
KAREN NITRIC NITRIC OXIDE--KNIT
THE UNIVERSITY OF ILLINOIS ROCK
FORD.
AND JOHN'S LAB TOGETHER WITH US
CARRIED OUT A SET OF ASSAYS ON
OUR CELLS INCLUDING DUST ASSAYS
WHICH ARE WELL ESTABLISHED
ASSAYS LOOKING FOR BASICALLY DNA
PROTEIN CROSS LINKS THROUGH
WESTERN BLOT APPROACH AND THIS
ALLOWED US TO RECOGNIZE THAT
COMPARED TO CONTROL CELLS RAD54
L2 KNOCK OUT CELLS HAVE HIGHER
LEVELS OF SUMO RELATED PROTEINS
AS WELL AS TOP 2 PROTEINS ON
CHROMATIN, ON DNA,
CONVALENTINEDDENTLY ATTACHED TO
D NA CONTROLS.
THIS IS SOMEHOW SAYING THAT THE
PRESENCE OF RAD54 L2 IS
COUNTERING THESE COMPLEXES AND
THAT COULD BE VENTING THE
FORMATION OR PROMOTING THEIR
REPAIR.
DISTINGUISHING THOSE 2
POSSIBILITIES WE CARRIED OUT A
RANGE OF STUDIES SHOWING THEM ON
THE RIGHT, AND WHAT WE'RE
PLOTTING HERE ACROSS A RANGE OF
GENETIC BACKGROUNDS WITH OR
WITHOUT THE TOPOCIDE TREATMENT
OR TOPOCIDE TREATMENT AND
RECOVERY, ARE GAMMA H 2 X
2 FOCI,
AND THIS IS A DOUBLE STRAND
BREAK MARKER BUT THIS WILL ONLY
TAKE PLACE WHEN YOU GENERATE AN
ACCESSIBLE DOUBLE STRAND BREAK,
SO GAMMA H2 X IS NOT FORMED WHEN
YOU HAVE A COVALENTLY ATTACHED
TOP 2 CLEAVAGE COMPLEX, THIS
WILL BE FORMED WHEN THE TOP 2
GETS CLEAVED OFF, THE BREAK CAN
BE DIRECTLY RECOGNIZED BY
MEMBERS OF THE ATM, APR, AND THE
PK FAMILY AND LEAD TO
PHOSPHORYLATION OF H2 AX UNDER
THE MARKERS.
SO JUST TO BRIEFLY TAKE YOU
THROUGH THIS, IN A CONTROL
SETTING I'VE SHOWN HERE IN
PURPLE, WE CLEARLY GET GAMMA H2
X INDUCTION AFTER TREATMENT, THE
TOPOCIDE AND AS WE MOVE THROUGH
THE RECOVERY PERIOD, THESE ARE
LARGELY REPAIRED.
IN THE ABSENCE OF RAD54, AND
RAD54 L2, WE GET HIGH LEVELS OF
GAMMA RAY H2 X BUT THEY'RE
REPAIRED OVER SEBS QUEBT QUENT
PERIODS OF TIME ONCE WE REMOVE
THIS TOPOCIDE.
SO THIS IS SAYING TO US THAT
WITHOUT RAD54 L2, THESE BREAKS
CAN BE REPAIRED BUT SOMEHOW
THEY'RE A GREATER NUMBER OF
GAMMA H2 X FOCI BEING PRODUCED
IN THIS BACKGROUND COMPARED TO
WILD-TYPE, SO THAT MUST MEAN
THERE'S A GREATER AMOUNT OF TOP
2 DAMAGE AND PROCESSING TAKING
PLACE.
WE GO TO A DIFFERENT SITUATION
IN THE TDP 2 KNOCK OUT HERE
SHOWN IN TURQUOISE WHERE WE GET
THE HIGHER, SOMEWHAT HIGHER
LEVEL OF GAMMA H2 X FOCI BUT
THESE EXIST BECAUSE THEY'RE NOT
ABLE TO BE REPAIRED AND FINALLY
IN ORANGE WE GET THE GREATER
NUMBER OF DOUBLE STRAND BREAKS
AS TRIGGERING GRAMA RAY HTWORKS
X AND THEY'RE NOT COMPARED.
SO HOW CAN WE PUT THESE TOGETHER
IN A MODEL.
SO OUR DATA INDICATE THE RAD54
L2 ISHT ACT WITH THE TOP 2
ALFARIN AND TOP 2 BETA, AND
THEREBY THE SIMULATED VERSIONS
OF THIS PROTEIN AS IN ZNF451 AND
THAT SIMULATION, FUNNELS INTO
THE TDP 2 PATHWAY, FOR THE TOP 2
CLEAVAGE COMPACT REPAIR BUT ALSO
IMPACTS ON RAD54 L2 ACTIVITIES
AND OUR CURRENT MODEL IS THAT
RAD 54 L2 ACTIVITY HELPS TO
RESOLVE TOP 2 CLEAVAGE COMPLEXES
AND PREVENT DOUBLE STRAND BREAK
EXPOSURE.
WHAT WE MEAN BY THAT S&P THAT
TOGETHER WITH ZNF451, WE BELIEVE
THAT THE RAD54 L2, AND ITS
SOCKED D NA HELIX CASE
ACTIVITIES EITHER COUNTERACTS
THE INITIAL ASSOCIATIONS WITH
TOP 2 AND THE DNA OR WE RATHER
THINK, IT ACTUALLY CAN RECOGNIZE
THESE CLEAVAGE COMPLEXES AND
SOMEHOW ALLOW RNA, ALLOW TOP 2
TO BACK UP THAT INITIAL REACTION
AND BE RELEASED FROM THE DNA,
THEREFORE NOT GENERATING THE
DOUBLE STRAND BREAKS THAT NEED
TO BE REPAIRED THROUGH THE
CANNONICLE REPAIR PATHWAYS AND
THIS IS SOMETHING WE'RE NOW
FURTHER EXPLORING IN A RANGE OF
WAYS INCLUDING THROUGH BY
CHEMICAL STUDIES.
AND I WILL POINT OUT THAT OUR
WORK AND THE RELATED WORK FROM
CHEN AND COLLEAGUES HAS BEEN
PUBLISHED IN SCIENCE ADVANCES.
JUST TO ROUND OFF THIS FIRST OF
MY 2 PARTS OF MY TALK TODAY, I
WANT TO GO BACK TO THIS
OBSERVATION HERE THAT WHEN WE
PUT OR WHEN WE OVEREXPRESS RAD
54 IN WILD-TYPE CELLS WE GET
MORE RESISTANCE AND IN THAT
REGARD, IT'S INTERESTING THAT
THERE ARE DATA IN THE LITERATURE
INDICATING THAT THE EXPRESSION
OF RAD54 L2 IMPACTING ON
PROGRESSION FREE SURVIVAL IN
THIS PARTICULAR AML COHORT.
AND SO, THIS SUGGESTS THAT RAD54
L2 ACTIVITY MAY BE A MECHANISM
OF CANCER CELL RESISTANCE IN
CLINICAL SETTINGS, AND
HIGHLIGHTS RAD54 L2 AS A POETIC
TEBTIAL ANTICANCER DRUG TARGET.
ANOTHER POTENTIAL MEDICAL ANGLE,
WE WERE LED TO WAS THROUGH THE
REALIZATION THAT IT'S BEEN
ESTABLISHED BY KEITH AND
COLLEAGUES THAT TDP2 MUTATIONS
WHICH YOU WILL RECALL IS PLAYING
A KEY ROLE IN TOP 2 CLEAVAGE
COMPLEX REPAIR, IT INHERITED
MUTATIONS IN TDP2, CAUSE
NEUROLOGICAL DISEASE AND WE
THEREFORE WONDER WHETHER OR NOT
RAD54 L2 MUTATIONS MIGHT ALSO
CAUSE SIMILAR DISEASE.
NOW IT'S FAIRLY EARLY DAYSA THE
THIS STAGE BUT WE HAVE
COLLABORATED WITH PETER
[INDISCERNIBLE] AND IRENEA
[INDISCERNIBLE] IN THE
NETHERLANDS WHO HAVE BEEN
WORKING WITH A PATIENT
PRESENTING FAIRLY EARLY ON IN
DEVELOPMENT WITH SEIZURES,
DEVELOPMENTAL DELAY AND
INTELLECTUAL DISABILITY, WITH A
DE NOVO HETEROZYGOUS VARIANT IN
RAD54 L2.
AND A GRADUATE STUDENT IN MY LAB
CHAD [INDISCERNIBLE] HAS BEEN
ABLE TO MODEL THOSE IN HUMAN RP1
CELLS THROUGH CRISPR GENETIC
ENGINEERING AND BASICALLY SEEING
THAT THAT MUTATION DOES IMPACT
ON ATOPOCIDE SENSITIVITY.
SUGGESTING THAT THE INTELLECTUAL
AND OTHER PATIENT'S ISSUES MAY
BE DRIVEN BY THAT VARIANT.
OKAY, SO WHAT I'VE BEEN TALKING
ABOUT SO FAR IS ABOUT DNA
PROTEIN CROSS LINKS INVOLVING
TOP O ICE SOM RACE 2, 1 CAN FORM
THESE AND THAT'S A WHOLE OTHER
STORY ABOUT YOU WHAT I WILL TALK
ABOUT FOR THE REMAINDER OF MY
TALK NOW IS TO FOCUS ON OTHER
TYPES OF DNA PROTEIN CROSS LINKS
THAT ARE GENERATED BY THINGS
SUCH AS REACTIVE METABOLITES
SUCH AS FORMALDEHYDE AND AGENTS
SUCH AS 5 AZADECOXY SWREEN, ALSO
KNOWN AS DECIDE O BEAN A EABT
CANCER DRUG.
NOW WORK OVER THE YEARS HAVE
SHOWN THAT THESE OTHER CROSS
LINKS WHICH CAN ARISE WITH A
RANGE OF PROTEINS, HISTONES RNA
PROTEINS AND WHATEVER, THE KEY
PRINCIPLES OF REPAIR IN THIS
SCENARIO IS THAT THE DPCs NEED
TO BE DEBULKED, EATEN AWAY IF
YOU LIKE BY SPECIALIZED DTP
PROTEASE SUCH AS THE SPARTAN
ENZYME AND THE PROTEOZOME.
LIKE THE STORY I'VE JUST BEEN
TALKING WITH YOU, THIS DC REPAIR
IS ALSO WITH UBIQUITINNATION AND
SUMO-ELATION, AS WELL AS GLOBAL
GENOME REPAIR MECHANISMS.
ONE THING WE THOUGHT ABOUT A FEW
YEARS AGO NOW WAS WHETHER THERE
MIGHT BE A TRANSCRIPTION COUPLED
VERSION OF DMA CROSS LISTEN
REPAIR, THAT WILL MAKE INTUITIVE
SENSE, DNA CROSS LINKS DON'T
JUST INTERFERE WITH REPLICATION.
THEY ALSO INTERFERE WITH
TRANSCRIPTION.
SO, 1 COULD IMAGINE THAT SUCH A
MECHANISM WOULD HAVE ARISEN
DURING EVOLUTION.
SO NOW I'M GOING TO GO THROUGH A
UNPUBLISHED BUT SOON TO BE
PUBLISHED STORY PIONEERS BY
THESE INDIVIDUALS, 2 STORIES
ACTUALLY, BY THESE POST DOCS AND
AND A GRADUATE STUDENT.
THIS WORK IS ALSO BENEFITED FROM
A FANTASTIC COLLABORATION, 1 OF
SEVERAL COLLABORATIONS THAT
WE'VE GOT WITH JULIAN STINGLER'S
GROUP IN MUNICH AND THAT HAS
BENEFITED WITH OUR INTERACTIONS
WITH [INDISCERNIBLE] IN
[INDISCERNIBLE].
SO THE BRAIN WAVE THAT CHRIS
CARNIE A FEW WE'RES AGO WAS TO
REDUCE THE DPCs EXPERIMENTALLY
WITH 2 TYPES OF AGENTS AND THEN
COMPARE AND CONTRAST THE CRISPR
SCREEN OUTPUTS IMENERATED BY
THOSE 2 AGENTS.
SO THE AGENTS THAT THEY USE WERE
FORMALDEHYDE WHICH IS A POTENT
STATE AND THEN THE RAPID DPC
INDUCER.
IT'S CELL PSYCHE WILL BLIND, IT
OPERATES ACROSS THE CELL CYCLE.
IMPORTANTLY IT'S A NATURALLY
OCCURRING METABOLITE.
SO FORMALDEHYDE DNA DAMAGE AND A
REAL PHYSIOLOGICAL RELEVANCE.
INITIALLY IF YOU LIKE,
EXPERIMENTAL ISSUE WITH
FORMALDEHYDE IT CAN GIVE RISE TO
A RAGER OF OTHER ADDUCTS SO IF
YOU SEE A RESPONSE TO
FORMALDEHYDE IT'S DIFFICULT TO
DISENTANGLE WHICH LESIONS ARE
GENERATING THE PHENOTYPE EMPLOY
THE OTHER 18th THAT CHRIS
FOCUSED ON IS 5 AINTRRKS-2 PRIME
DEOXYICIDE O DEAN, OR 5
DECYTOBIEN, THIS COMPOUND SHOWN
HERE IS INTO DNA, AND
IMPORTANTLY, WHEN IT'S
INCORPORATED AT SITES OF DNA
METHYLATION, AT CPGs, WHEN THE
MAINTENANCE METHYLTRANSFERASE
COMES IN TO TRY AND BASICALLY
METHALATE THIS SITE TO MAKE IT
SYMMETRICAL, IT GETS STUCK, THE
CATALYTIC MECHANISM IS DISRUPTED
AND THAT LEADS TO DNMT1,
BECOMING A COVALENTLY ATTACHED
DPC IN THIS SCENARIO WHICH IS
OBVIOUSLY GOING TO GIVE PROBLEMS
TO THE CELL.
EXPERIMENTALLY YOU HAVE TO
REALIZE THIS IS ONLY GOING TO
TAKE PLACE IN S-PHASE AND IT
WILL ONLY REALLY GENERATE THOSE
TYPES OF LESIONS WHERE THERE'S A
CPG METHYLATION TAKING PLACE.
THIS IS USED IN THE CLINIC.
AND SO ANYTHING WE LEARN WITH
THIS AGENT POTENTIAL HAS
CLINICAL RELEVANCE.
SO WHAT I'M SHOWING HERE, IS THE
CRISPR SCREEN OUTPUTS, IN THIS
CASE IT'S NOT A CRISPR CAS 9
GENE, IT'S A CRISPR LIBRARY
SCREEN CARRIED OUT BY CHRIS
USING THE LIBRARY AND CELLS
INITIALLY GENERATED BY
APPROXIMATE [INDISCERNIBLE] IN
THE GROUP AND THIS IS IN K526
CELLS AND WE ARE LOOKING FOR
YEENS THAT WERE KNOCKED OUT GIVE
RISE TO SENSITIVITY TO
FORMALDEHYDE AND YOU CAN SEE
THERE'S A WHOLE RANGE OF THEM
THERE, WE KNOW THE KREEN WORKS
IMMEDIATELY BECAUSE SOME OF THE
STRONGEST SCREENING HITS ARE
THESE YEEPS AND THEIR PROTEINS
HERE ESD AND ADH5 AND
IMPORTANTLY THESE ARE INVOLVED
IN FORMALDEHYDE DETOXITION, SO
YOU KNOCK OUT WITH THESE, AND
YOU KNOCK OUT WITH MORE
PERRIST SENT FORM ALD HIGHS
WHICH WILL GIVE RISE TO MORE
DAMAGE AND MORE CELL KILLING.
SO THERE ARE MANY FACTORS HERE
AND THIS WASN'T ALL THAT
SURPRISING.
OTHER GROUPS INCLUDING
[INDISCERNIBLE]'S PAPER
HIGHLIGHTED HERE IN THE CELL
PAPER, HAVE MAPPED OUT CELLULAR
RESPONSES TO FORMALDEHYDE AND
SHOWN THE ARRANGE OF PROTEINS
INVOLVED IN NUCLEOTO THETIC
EXCISION REPAIR, ANEMIA PATHWAY
AND OTHER PATH WAGHTS IMPACT ON
THE--PATHWAYS IMPACT ON THE
REPAIR OF THOSE LESIONS BUT ALSO
IT'S KNOWN BY WORKING THROUGH
KPATEL AND COLLEAGUES THAT
TRANSCRIPTIONAL STRESS TRIGGERS
DNA RESPONSE IN MOUSE MODELS AND
THAT'S PROVIDED INSIGHTS INTO
POTENTIAL MECHANISMS OF HUMAN
DISORDERS, THAT I'LL TOUCH ON A
BIT LATER ON.
BUT I THINK THINGS GET MORE
INTERESTING WHEN WE FOCUS
SPECIFICALLY ON HITS IN OUR
SCREEN THAT RELATE TO A BATH WAY
THAT'S BEEN KNOWN FOR MANY
YEARS, IN FACT DECADES NOW,
TRANSCRIPTION COUPLED NUCLEOTIDE
ECCISION REPAIR.
AS YOU BE AWARE, NUCLEOTIDE
EXCISION REPAIR REMOVES UV
INDUCED LESIONS AND OTHER BULKY
DNAA ADDUCTS FROM DNA AND
BASICALLY ENDS UP THE SINGLE
STRANDED REGION AND WORKING ON
ITS REPAIR AND LIGATION, NOW
MUCH WORK OVER MANY YEARS AND IT
WORK MISS 2 DEFICIENT ARENAS AND
THERE'S TRANSCRIPTION COUPLED
REPAIR AND AND THE TOP OF THE
PATHWAY IS A CSB ENCODER BY THE
ARCC6 GENE AND RECOGNIZING A
STOLED RNETWORKA POLYMERASE 2,
ENCOULD YOU WANTERRING A
NUCLEOTIDE EXCISION OF TAR
TARGETS.
THIS RELEADS TO REACCUMULATE OF
ERCC8 GENE, AND WHICH AMONGST
OTHER THINGS LEAD TO
UBIQUITINNATION OF THE PUB UNIT
OF POLYMERASE AND OTHER FACTORS
INCLUDING UVSSA, THE DOWN STREAM
OF THE ECCISION REPAIR
TRANSCRIPTION COUPLED EXCISION
REPAIR ARE MEDIATED BY FACTORS
SUCH AS XBA, AND THE NUCLEACES,
AND XBF AND XPG.
AND AS YOU WILL NOTE FROM THE
LEFT-HAND SIDE, THIS RESPONSE TO
FORMALDEHYDE, ESSENTIALLY ALL OF
THOSE TCNER HITS SO THE LESION
IS GENERATED FOR FORMALDEHYDE
AND DRIVING TOCKSITY, LARGELY
BEING THE EFFECTS OF COUNTER
ACTED BY THAT SET OF FACTORS BUT
WE'RE NOT SEEING XPC, DROPPING
OUT IN THIS SCENARIO HERE, IT'S
INVOLVING GENOME REPAIR BUT NOT
TRANSCRIPTION COUPLED NER AND
THIS IS WHERE IT'S INDICATING
THAT THE TOXICITY GENERATED BY
FORMALDEHYDE IN THIS SCENARIO
HERE WILL BE LARGELY THROUGH THE
ACTIONS ON THE TRANSCRIPTIONALLY
ACTIVE GENES.
SO WHAT I'M PLOTTING ON THE
RIGHT HERE ARE THE RESULTS OF
CHRIS' CRISPR I-SCREEN WITH
CELLS TREATED WITH DECYTOBIN,
AND YOU WILL SEE A RAINCHL OF
POSITIVE CONTROLS.
THESE FACTORS SHOWN IN PURPLE.
SLC 29 A 1 ENCODES A TRANSPORTER
WHICH IS NECESSARY FOR
INCORPORATION OR OF THIS DRUG,
INTO CELLS, OBVIOUSLY IT'S NOT
GOING TO HAPPENOT IMPACT OF THE
CELL, IF IT CAN'T GET INTO THE
CELL AND DCK AND CMP K ARE
KINASES THAT MEDIATE
PHOSPHORYLATION OF THE CYTOBIN
INTO A TRANSPHOSPHATE VERSION
THAT, LOWS IT TO BE INCORPORATED
IN DNA AND MEDIATE ITS TOXICITY.
YOU CAN ALSO SEE THAT IN THIS
PLOT, I HIGHLIGHTED THE VARIOUS
NUCLEOTIDE EXCISION REPAIR YEENS
AND WITH STRIKING OBSERVATION IS
THAT WE CAN SEE CTSA AND CSB ARE
DROP OUTS IN THE--IN BOTH
SCENARIOS, WHEREAS--SORRY, I'VE
JUST BEEN INVITED TO THIS
SEMINAR.
--WHEREAS THE OTHER FABLGHTORS
THAT ARE GETTING YOU--FACTORS
THAT GETTING UP AS HITS IN THE
FORMALDEHYDE SCREENS ARE NOT.
SO KNOWLEDGING ABOUT THIS MORE,
WE DECIDED TO GO AHEAD AND
VALIDATE THESE SCREEN HITS AND
THESE DO VALIDATE SO YOU CAN SEE
ON THE LEFT-HAND SIDE HERE THAT
IF WE COMPARE TO WILD'RE
WILD-TYPE CELLS, CELLS KNOCKED
OUT FOR XPA OR CSB, ARE HYPER
SENSITIVE TO FORMALDEHYDE,
HOWEVER, WHILE CSB KNOCK OUT
CELLS ARE HYPER SENSITIVE TO 5
ACDC, THE EXPA KNOCK OUTS ARE
NOT.
WE GET SIMILAR RESULTS IN HAP1
AND ALSO IN HUMAN RBD1 CELLS.
SO THE DEFERENT YALE IMPACT THAT
WE'RE SEEING WITH DECYTOBIN AND
BASICALLY AT THE LEVEL OF THE
EARLY STAGES OF WHAT WE CALL
TCNER, INVOLVING TCAND CSA BUT
NOT THE DOWN STREAM COMPONENTS.
SO THROUGH STUDY IN
COMPLEMENTATION STUDIES IN THE
KNOCK OUT BACKGROUNDS WE'RE ABLE
TO SHOW THAT THIS FUNCTION
REQUIRES CSB ATP ACE ACTIVITY
AND WE SHOW THAT IF YOU LOSE THE
GLOBAL GENOME PATHWAYS THROUGH
KNOCKING OUT XPC, YOU SENSITIZE
CELLS TO UV LIGHT BUT NOT TO
FORMALDEHYDE OR RAISER OR THE
CYTOBIEN, AND EVEN IF YOU KNOCK
OUT XPA TOGETHER WITH XPC ARE,
YOU GET FORMALDEHYDE SENSITIVITY
BUT NOT DESIT BIEN SENSITIVITY.
FOR SPARTAN ON RF4, AND OR
EXPRESSION OF A PART AN DELTA
CTERMINAL TRUNK TERMINATED
CONSTRUCT WHICH IMPAIRS ITS
ACTIVITY WERE TO SHOW THAT CSB
IS COMPARING DPC COLERANCE
THROUGH A PATHWAY THAT ACTS IN
PARALLEL NOT IN THE SAME PATHWAY
BUT IN PARALLEL WITH ESTABLISHED
SPARTAN RF4 DEPENDENT
MECHANISMS.
SO WHAT WE THINK IS GOING ON
HERE IS THAT THE CROSS LINKS
AREN'T REQUIRING SEN ILLEGALSEN
CESS ENSEL OF CSA, AND CSB, AND
BUT NOT CONNECT WIDE NER.
NO IT'S SHOWN THROUGH CERTAIN
STUDIES THAT RNA POLYMERASES ARE
STOLE BY THE ACTIONS OF THE
DPCs AND IT'S NOT BEING
EXPORTS VIVO, BUT WHAT I'M
SHOWING YOU THROUGH THESE
STUDIES HERE WHICH WERE CARRIED
OUT LATER, I COMPARE IN THE LAB
ISSUE IS TO LOOK AT THE EFFECT
OF TRANSCRIPTIONAL CELLS AFTER
FORMALDEHYDE TREATMENT AND WE'RE
LOOKING AT WILD-TYPE CELLS AND
CSB KNOCK OUT CELLS.
SO BOTH SCENARIOS, IT WAS TRIEWT
WIDE FORMALDEHYDE AND LEADS TO
TRANSCRIPTIONAL SHUT OFF AS
MEASURED BY INCORPORATION OF EU,
SO THAT'S MASSIVE TRANSCRIPTION
BEING MEASURED HERE BUT IF WE
LOOK AT OVER TIME AFTER THE
FORMALDEHYDE IS KRAWN AND CELLS
RECOVER, WE SEE A GRADUAL
TRANSCRIPTION AND RECOVERY OF
TRANSCRIPTION IS MUCH MORE PRO
NOUNSED IN THE WILD-TYPE CELLS
THAN IN THE CSB KNOCK OUT CELLS,
SO THIS IS INDICATING THAT CSB
KNOCK OUT CELLS HAVE A PROBLEM
IN REACTIVATING TRANSCRIPTION
AFTER FORMALDEHYDE EXPOSURE AND
YOU MAY HAVE NOTED THIS THIS,A
PLIES BOTH TO PAN NUCLEAR
STAINING AS WELL AS NUCLEAR
STAINING SO THIS PRESUMABLY
MEANS THE IMPACT OF CSB IN THIS
REGARD IS NOT JUST RELATED TO
POL2 TRANSCRIPTION BUT ALSO POL1
TRANSCRIPTION.
AND WE GET SIMILAR RESULTS FOR
CSA KNOCK OUT CELLS BUT NOT FOR
XPA OR THE NER INCISION AND
NUCLEACES.
AGAIN SEPARATING THE DPC
ACTIVITIES OF THOSE COMPONENTS
FROM THOSE OTHER PROTEINS.
AS WAS SHOWN PREVIOUSLY FOR UV
DAMAGE FOR EXAMPLE, CSB AND CSA,
INTERACT WITH RNA POLYMERASE 2
AFTER FORMALDEHYDE TREATMENT AND
IN THE CASE OF CSA, ITS
ASSOCIATION BY CO IMMUNO
PRECIPITATION IS CSB DEPENDENT.
SO FINALLY, WHAT WAS SHARED WITH
YOU AS HOW WE USED AN
ESTABLISHED TECHNIQUE AND THIS
BROUGHT THIS INTO THE REALM OF
NEXT GEN SEQUENCING AND
APPROACHES AND BUT IT'S BEEN
KNOWN FOR TIME IF YOU SDS LY
CELLS AND CARRY OUT THIS, IT
WILL PRECIPITATE PROTEINS BUT
NOT DNA BUT IT WILL PRECIPITATE
DNA IF THAT DNA IS ATTACHED TO
PROTEINS.
SO WE THEREFORE REASONED IF WE
CARRY OUT THAT PROTOCOL ACTUALLY
SEVERAL ROUNDS OF THE PROTOCOL,
WE SHOULD SELECTIVELY RETRIEVE
REGIONS OF THE GENOME WHICH
CONTAIN DPCs, OF COURSE THOSE
REGION OF THE GENOME COULD BE
ANALYZED BY MASSIVELY
PARALLELLED NEXT GENERATION
SEQUENCING, SO WE'LL BE ABLE TO
IN A TBLOABAL WAY IF YOU LIKE,
LOOK AT REGIONS OF THE GENOME
THAT HAVE DPCs AND ACTUALLY
LOOK AT THEIR REPAIR OVER TIME.
BECAUSE WE'RE CARRYING OUT THESE
EXPERIMENTS WITH SYNCHRONIZED
RPE1 CELLS, THESE CELLS ARE IN
G1, SO WE'RE NOT GOING TO HAVE
TO THINK ABOUT THINGS SUCH AS
DNA REPLICATION, WHICH IS A WAY
TO IMPACT ON OTHER PATH WAYINGS
OF DPC REPAIR, AND WE CAN TREAT
THESE CELLS WITH FORMALDEHYDE
AND WASH IT OFF AND LOOK AT OVER
TIME THE REPAIR OF DNA DAMAGE.
SO WE'RE PLOTTING ON THIS--JUST
HERE, ARE BASICALLY THE RESULTS
OF 2 SUCH EXPERIMENTS.
ALONG THE BOTTOM, IS THE KNA
CROSS LINK COVERAGE IF YOU LIKE
EMPLOY THE GENOMIC LOCI WHICH
ARE PULLED DOWN THROUGH THE KCL
PRECIPITATION PROCEDURE AFTER
TREATING FORFORMALDEHYDE.
AND ON THE Y-AXIS, ANOTHER SET
OF DATA GENERATED AFTER THE
CELLS ARE INDUCED.
SO IF YOU THINK ABOUT AND CELL
THAT'S NOT REPAIRED SHOULD LIE
THERE, THE REGION OF THE GENOME
WHICH IS BEING REPAIRED SHOULD
BE LYING OFF THE DIAGONAL
TOWARDS THE BOTTOM, AND THE
STRIKING FINDING HERE IDENTIFIED
THE RANGE OF LOCI ACROSS THE
GENOME THAT WERE PREFERENTIALLY
REPAIRED AND IF WE MAP ON TO
THOSE DATA ESTABLISH RNA POL 2
TO OCCUPANCY DATA AND WE CAN SEE
THAT THOSE LOCI, TEBD TEND TO BE
LOCI THAT HAVE A HIGHER RNA POL
2 OCCUPANCY.
SO THIS IS A KIND OF APPROACH,
WE CAN ALSO LOOK AT WILD-TYPE OR
OTHER CSB BACKGROUNDS.
BUT I'M SHOWING THE WORK, THE
ANALYSIS WAS CARRIED OUT BY
[INDISCERNIBLE] IN THE GROUP, A
VERY EXPERIENCED AND VERY
VERSATILE EXPERIMENTAL BIOLOGIST
AS WELL AS A FANTASTIC
BIOINFORMATICIAN.
WE ARE COMPARING ALONG THE
BOTTOM, THE DAILY BASIS THEA
AFTER A 6 HOUR RECOVERY OF CSB
KNOCK OUT CELLS AND ON THE
Y-AXIS AFTER A 6 HOUR RECOVER
EVALUATION PROCESS WILD-TYPE
CELLS.
SO IN THIS SCENARIO HERE, ANY
GEANUS IN THE LOCUST PROMOTED BY
THE CSB WILL FALL BELOW THE
DIAGONAL HIGHLIGHTED HERE IN
BLUE AND THAT IDENTIFIED JUST
OVER A THOUSAND GENES THAT
WERE--WE BEING DEFINE AS THEIR
REPAIR BEING CSB DEPENDENT.
AND INTERESTING WHEN WE LOOK AT
THOSE GENES AND IN THIS CASE,
WHAT WE'VE DONE HERE IS TAKE
THOSE GENES AND BASICALLY PLOT
THEM IN A METAGENE PLOT, SO ALL
THE GENES ARE TOGETHER, WE GET
THESE--WE GET THESE CURVES HERE,
AND WHAT WE FIND IS THAT THE
IMPACT OF CSB, ON DPC REPAIR IS
MOSTLY WITHIN THE TRANSCRIBED
PORTION OF THE GENE, THE GENE
BODY.
SO THAT IS THE DIFFERENTIAL
BETWEEN THE WILD-TYPE SCENARIO,
HERE TO HERE, ORANGE TO RED, IS
LARGER THAN THE DIFFERENTIAL
HERE LIGHT BLUE TO DARK BLUE,
THOSE BEING IN THE PRESENCE OF
WILD-TYPE OR CSB KNOCK OUT
RESPECTIVELY, AND ANOTHER WAY OF
SHOWING THESE DAT IS LOOKING AT
THE DIFFERENTIAL BETWEEN
WILD-TYPE AND CSB KNOCK OUT ON
THE RIGHT HERE WHICH CLEARLY
INDICATING THAT THE IMPACT OF
CSB ON PROMOTING REPAIR IS
LARGELY CORRELATED WITH THE GENE
BODY.
THIS HAS BEEN A HIGHLIGHT OF
THIS BIG PEEK HERE AND IT'S
REFLECTING THE FACT THAT THE
TRANSCRIPTION START SITES TEND
TO BE LARGELY OPEN CHROMATIN
STRUCTURE, AND WE KNOW THROUGH
PROTEOMIC STUDIES THAT WE
CARRIED OUT IN CONJUNCTION WITH
THIS, THAT A LOT OF THAT, THOSE
DPCs ARE ACTUALLY
RNA-POLYMERASE 2, BEING CROSS
LINKED.
SO THE TAKE HOME MESSAGE HERE IS
THAT THERE ARE--THERE IS A SET
OF LOCI ACROSS THE GENOME THAT
ARE PREVENTIVE RENTIA WILY
REPAIRED IN TERMS OF DPCs,
THEY ARE CSB DEPENDENT AND THE
EFFECT IS LARGELY COMBOYNED TO
THE TRANSCRIBED PORTION OF THE
GENE.
SO TO SUMMARIZE, DPCs CAUSE
TRANSCRIPTION STRESS, WE THINK
THIS IS THE FIRST EVIDENT OF
TRANSCRIPTION COUPLED DECKER PC
REPAIR AND WE'VE SHOWN THAT CSB
PROMOTES DC REPAIR INDEPENDENTLY
OF CLASSIC TCNAR FACTORS.
THIS WORK HAS BEEN RECENTLY SENT
TO FOR PUBLICATION IN NATURE
CELL BIOLOGY AND HOPEFULLY WE
WILL BE ABLE TO READ IN THE NOT
TOO DISTANT FUTURE 2 WONDER AND
AND VERY COMPLIMENTARY STUDIES
BY THE LAG OF [INDISCERNIBLE]
AND [INDISCERNIBLE] WHICH WILL
BE APPEARING HOPEFULLY
BACK-TO-BACK TO BACK IN THE SAME
ISSUE.
FINALLY IN THE LAST 2 MINUTES, I
JUST WANT TO TRY TO RELATE THIS
BACK TO THE CLINICAL ARENA
AGAIN, AS YOU MAY BE AWARE,
THERE IS EXCISION REPAIR IN THE
SYNDROME BEING DESCRIBED OVER
THE YEARS, THERE'S BEEN THERE
KNOWN FOR MANY YEARS THAT
PATIENTS WITH MUTATIONS IN THE
LCC6--SORRY, ERCC6--ERCC8 WHICH
ENCODE CSBOR CSA RESPECTIVELY
AND WITH COCK CAIN'S SIN CHROME,
WHICH IS SEVERE DISORDER
PROGRESSIVE WEIGHT LOSS, MUSCLE
LOSS, NEURODEGENERATION AND
KIDNEY FAILURE AND THAT'S VERY
DIFFERENT PRACTICES PATIENTS
WITH GENES MUTATIONS AND IN THE
DOWN STREAM ASPECTS OF TCNER AND
THESE INCLUDE THE XPMUTATIONS
AND WHICH IS CHARACTERIZED BY
EXTREME UV SENSITIVITY, AND
INCREASE CANCER RISK.
SO CLEARLY THE IMPACTS OF CSA,
AND CSB IN NEUROLOGICAL ISSUE
TISSUES CANNOT BE THROUGH LIGHT
EXPOSURE SO THIS HAS TO BE
DRIVEN BY THINGS THAT DON'T
INCLUDE WHATY WE CALL CLASSICAL
SUBSTRATES.
[INDISCERNIBLE] GROUP INDUCES
TRANSCRIPTIONAL STRESS FROM
FORMALDEHYDE AND DRIVES PAIN
LIKE SYNDROMES PHENOTYPES IN
MICE.
HOWEVER THE RESPONSIBLE
FORMALDEHYDE INDUCED LESIONS
HAVE BEEN UNKNOWN, WHAT WE WOULD
LIKE TO PROPOSE IS THAT OUR
FINDING SUGGEST THAT EFFECTIVE
TRANSCRIPTION COUPLED DCP REPAIR
IS LARGELY CONTRIBUTING TO THE
UNIQUE NEUROLOGICAL FEATURES OF
CS.
FINALLY, WHAT'S MIXTURE AND I
WON'T DWELL ON THIS BUT NOW
WE'RE DELVING FURTHER INTO THIS
AND OTHER ARENAS BY CARRYING OUT
MORE CRISPR KREENS IN THE CSP
KNOCK OUT BACK GROUPEDS WHICH
ALLOWED US RECENTLY TO IERK
DENTIFY A NEW DPC FACTOR CALLED
TOPORS, WHICH WE THINK WILL WELL
IN PARALLEL WITH RNF4 AND THE
OTHER FACTORS I TALKED ABOUT
TODAY AND THIS WORK HAS BEEN
SPEARHEADED BY CHLOE
[INDISCERNIBLE] IN THE LAB WITH
ADVICE FROM CHRIS
[INDISCERNIBLE] AND OTHERS.
AND AGAIN WE'RE BENEFITING FROM
OUR FANTASTIC COLLABORATORS
INCLUDING JULIAN STINGLE,
[INDISCERNIBLE] AND OTHERS.
SO TO ROUND OFF THERE.
THIS IS A PICTURE OF MY LAB IN
SUNNY NORTH UP JUST AN HOUR AND
A HALF NORTH OF SUNNY CAMBRIDGE,
ALTHOUGH IT'S NOT SUNNY TODAY.
AFTER 30 YEARS PLUS IN THE
INSTITUTE MY LAB MOVED POT
CANCER RESEARCH UK CAMBRIDGE
INSTITUTE ON THE BIOMEDICAL
CAMPUS WHICH IS THE LARGEST AND
ARGUABLY THE BEST BIOMEDICAL
CAMPUS IN EUROPE, CERTAINLY THE
LARGEST AND IT'S A FANTASTIC
PLACE TO BE.
I WILL LEAVE IT THERE AND I WILL
TAKE QUESTIONS.
THANK YOU.
>> THANK YOU DR. JACKSON FOR THE
AMAZING TALK.
LET'S MOVE TO THE Q&A SESSION,
WE HAVE A FEW MINUTES, SO
AUDIENCE PLEASE TYPE IN YOUR
QUESTIONS IN THE CHAT BOX SO WE
CAN LOOK AT THAT.
WE HAVE A QUESTION RIGHT NOW
FROM [INDISCERNIBLE] ASKING WHAT
ARE THE STATUS OF RD54 L2 AND
ZNF451 IN TERMS OF THE MUTATION
COPY NUMBER?
ATOPOCIDE MEDIATED
[INDISCERNIBLE] CANCERS?
>> THAT'S A GREAT QUESTION.
THIS IS A NEW ARENA FOR US AND I
THINK THAT IS ANOTHER VERY
INTERESTING THING TO EXPLORE, WE
MANY IN THE FIELD I THINK ARE
NOW VERY WELL AWARE OF
MUTATIONAL SIGNATURES AND
MOWITATIONS BUT OF COURSE COPY
NUMBER CHANGES CAN DRIVE
PRONOUNCED PHENOTYPES AND I KNOW
THAT'S THE LAB AND OTHER LABS
ARE GETTING INTO BUT
UNFORTUNATELY I DON'T HAVE AN
ANSWER TO THAT QUESTION RIGHT
YOU.
>> KEN HAS A QUESTION, DO YOU
WANT TO ASK IT DIRECTLY?
>> THERE WE GO, WONDERFUL TALK.
REALLY SPECTACULAR.
I REALLY ENJOYED THE CHEMO
THERAPY ASPECTS OF IT AND THE
NEUROLOGIC XP GENETICS.
ONE OF THE ISSUES IN XP IS WHY
THE XBC PATIENTS DON'T HAVE
NEUROLOGIC DEGENERATION WHEREAS
THE XPA AND ALSO THOSE WITH XP,
OR XPV WHICH ARE HELIX CASES AND
THEY DIDN'T SHOW UP IN YOUR
SCREEN AT ALL.
SO EVERYONE'S BEEN LOOKING FOR
LESIONS THAT ARE NOT REPAIRED BY
NUCLEOTIDE EXCISION REPAIR AND
CYCLOPUREINS HAVE BEEN THE
PROMINENT 1S, YOU'RE SUGGESTING
THAT MAYBE OTHER 1S THAT HAS--DO
YOU KNOW OF ANYONE THAT'S BEEN
COOING THESE STUDIES THAT YOU'VE
BEEN SUGGESTING, LOOKING FOR
THESE LESIONS, AND THEN PATIENTS
WITH THESE NUCLEOTIDE EXCISION
REPAIR RELATED DECS THAT
PRESENCE ARE ABSENCE OR
NEUROLOGIC DEGENERATION.
>> THAT'S A GREAT QUESTION, WE
ALL KNOW WHAT TOOLS, WHAT TOOLS
WE CAN USE AT THE CELLULAR LEVEL
TO GENERATE SPECIFIC TYPES OF
LESIONS BUT I THINK WE'RE
ALREADY IN A POSITION TO COMPARE
OUR CRISPR STREAM DATA SETS FOR
EXAMPLE WITH THOSE OBVIOUSLY
ARISEN THROUGH OTHER TREATMENTS
SUCH AS UV AND WHATEVER.
OF COURSE, SOME MUTATIONS ARE
NOT ENTIRE LOSS OF FUNCTION
MUTATIONS THAT THEY COULD BE
SEPARATION OF FUNCTION MUTATIONS
IN MANY CASES, MULTIFUNCTIONAL
PROTEINS, AND TAKEN--THEY'S
WHOLE--A WHOLE--A WHOLE OTHER
ARENA THAT NEEDS TO BE EXPLORED
BUT I AGREE.
I THINK WE'RE TOUCHING ON
CERTAIN ASSPEBTS OF THE NUR O
DEGENERATION AND COCKAYNE'S
SYNDROME AND OTHER PATIENTS BUT
WE'RE NOT EXPLAINING OTHER
ASPECTS OF THESE DISORDERS WHICH
ARE DEALING WITH A WHOLE RANGE
OF OTHER LESIONS FROM ENDOGENOUS
TO EXOGENOUS CHEMICALS.
SO I'M RAMBLING HERE BECAUSE I
DON'T REALLY HAVE AN ANSWER BUT
IT'S A GREAT QUESTION.
>> WE ARE ACTUALLY PREPARING A
MANUSCRIPT, WE HAVE 18 XPA
PATIENT ANDS THEY RANGE FROM
SEVERE NEUROLOGIC DEGENERATION
TO PATIENTS [INDISCERNIBLE]
COLLEGE, DIFFERENT MUTATIONS IN
THE SAME GENE.
>> WOW, I GUESS SOME OF
THESE--ARE SOME OF THESE
POSSIBLY DOMINANT NEGATIVES OR
PARTIALLY FUNCTIONAL PROTEINS
THAT BASICALLY MIGHT GUM UP THE
WORK ANDS EVEN HAVE BROADER
EFFECTS RATHER THAN A KNOCK OUT
PERHAPS?
I LOOK FORWARD TO READING YOUR
PAPER.
>> THANK YOU.
>> DR., DO YOU WANT TO ASK A
QUESTION.
>> THANK YOU, STEVE, WE REALLY
ENJOYED IT, SOME OF US WOULD
ADVOCATE THAT CSV HAS A MAJOR
MITOCHONDRIA DISFUNCTION THAT
COULD BE A FACTOR IN THE SYSTEM,
I WOULD LIKE TO ASK YOU IF YOU
TRIED THE RESUSMGZ OF RNA
SYNTHESIS EXPERIMENTS THAT WERE
CLASSIC IN THE LITERATURE TO SEE
IF WHEN THERE IS A DELAYING
RESUSMGZ OF TRANGZ SCRIPGZ WITH
DAMAGE AFTER THESE AGENTS, THESE
CROSS LINKERS THAT WOULD
INDICATE THAT THE ACTIVE GENES
ARE LATER IN CSA AND B?
>> ABSOLUTELY, I THINK THE DATA
I SHARED WAS INDICATING THAT AT
THE CELLULAR LEVEL AT LEAST FOR
RNAPOL2, TRANSCRIPTION.
THE OOH RENA OF MITOCHONDRIA IS
NOT SOMETHING WE'VE NOT DELVED
INTO BUT IT IS VERY IMPORTANT
AND I WILL, ON THE BACK OF THIS
QUESTION, I WILL GO BACK IN MY
LAB AND SEE IF THERE AND
EXPERIMENTS WE COULD RUN TO
PROBE INTO THAT BUT IF YOU HAVE
ANY SPECIFIC QUESTIONS, WILL,
PLEASE DO E-MAIL US AND I WOULD
LIKE TO DISCUSS THAT.
>> GLADLY.
YEAH.
COMPREHEND.
>> WE HAVE A QUESTION FROM
[INDISCERNIBLE].
VERY INTERESTING TALK, THANK
YOU, I'M WONDERING WHAT'S
HAPPENING TO THE CELL SET AFTER
ATOPOCIDE TREATMENT.
THIS IS EITHER A WILD-TYPE OR
THE RD54 L2 KA, ARE THEY
PROLIFERATING OR SEN SENESCENC?
>> YES, IF YOU CARRY OUT THE
SCREEN, YOU MEASURE
PROLIFERATION EMPLOY SO THE
CELLS COULD BE DYING OR THEY
COULD JUST BE STOPPING
PROLIFERATING AND THEY WILL
STILL BEHAVE AS A KNOCK OUT AND
THE ONLY WAY YOU CAN REALLY
FIGURE OUT WHAT'S GOING ON IS
THEN TO GO IN AND HAVE A LOOK AT
THOSE THINGS IN A SPECIFIC WAY.
I THINK THE IDEA ISN'T CELL
DYING AND IF IT ISN'T WHERE'S IT
ANYTHING OFF.
THAT'S AN INTERESTING 1 AND
SOMETIMES IF YOU GET INTO THAT
IF YOU TAKE A LOOK AT THE
BACKGROUND AND LOOK AT FACTORS
THAT MAY GIVE YOU RESISTANCE
BECAUSE IN SOME CASES, THE THING
THAT'S ENDING UP KILLING THE
CELL MAYBE ISN'T THE DAMAGE BUT
MAYBE CHROMOSOMAL TRANSLOCATIONS
OR ABERRATIONS THAT ARE CAUSED
BY DNA REPAIR PROCESSES, SO
AGAIN IT'S A QUESTION ABOUT HOW
TO CLEAR OUT FROM ME BUT I THINK
IT'S A GOOD 1 AND I'M LOOKING
FORWARD TO DETAIL ABOUT WHAT'S
HAPPENING TO THESE CELLS THAT
DIE AND ALSO WHAT HAPPENS TO THE
CELL SURVIVE, WHAT SURVIVE
SYSTEM VERY IMPORTANT.
YOU KNOW WE'VE DONE THIS IN THE
PAST, 1 POISONS FOR EXAMPLE IN
THE PRESENCE OF ATM DEFICIENCY
AND WE FOUND THERE, THAT'S
ENDURING IS ACTUALLY A PATHWAY
THAT ENDS UP KILLING THE CELLS,
THAT CELLS WITHOUT ALL THIS END
JOIPING IN THE ATM BACKGROUND
ARE MORE RESISTANT TO TOP O ICE
ON SOM RACE, AS WELL AS PARP
INHIBITORS AND MAYBE A SIMILAR
THING MIGHT BE HAPPENING IN THIS
ARENA AS WELL.
I DON'T KNOW.
>> OKAY, WE HAVE A QUESTION FROM
WILLIE JAMES, COULD YOU EXPAND
YOUR COMMENTS ON THE
PESFISSILITY OF THE CRISPR
TARGETING SYSTEMYING AS MEASURED
BY NG S?
>> RIGHT.
SO, NEXT GENERATION SEQUENCING,
YOU KNOW, NOW PIONEERED BY
LUMENNA, DEVELOPED BY MY
COLLEAGUE NEXT DORDOOR TO ME AND
THE NEXT DOOR LAB IS THE LAB OF
[INDISCERNIBLE] WHO TOGETHER
WITH DAVID GENERATED THIS
TECHNIQUE.
IT'S MIND BOGGLING WHAT 1 CAN DO
NOW WITH THIS TECHNIQUE IF YOU
HAVE A BIO-INFORMATICS GROUP AND
TESTIFIES LOOK SIDE.
SO IT'S DEEP SEQUENCING.
OBVIOUSLY WHEN YOU CARRY OUT A
CRISPR SCREEN, I THINK THIS IS
THE QUESTION, YOU HAVE A WHOLE
POPULATION, WE PROBE THE LIBRARY
AT 1000 OR 5000 X AND WE HAVE
BIG COVERAGE AND THEN YOU JUST
BASICALLY PC R THAT POPULATION,
AND THEN BY DEEP SEQUENCING YOU
JUST SEE HOW MANY READS YOU GET
PER GUIDE AND COMPARE THEM TO 1
ANOTHER.
AND THAT IS BASICALLY TELLING
YOU THE RELATIVE DISTRIBUTIONS
OF THOSE GUIDES IN THE
POPULATION.
IT'S NOT ENTIRELY ACCURATE, AND
OF COURSE, THERE'S GOING TO BE
SOME DRIFT XTHERE'S GOING TO BE
IN STOI CASTIC SCATTER BUT THE
LEVEL OF DATA SETS ARE
SUFFICIENT, THAT THESE DATA ARE
INCREASINGLY TIGHT.
AND IT'S VERY--IT'S ALMOST NEVER
NOW THAT WE GET A DESCENT HIT IN
A CRISPR SCREEN THAT SCNT
VALIDATE.
THEY ALMOST ALL DO.
>> WE HAVE OUR LAST QUESTION
FROM JOHN [INDISCERNIBLE].
THERE'S ALREADY 54 L2 INFECTION
AFFECT THE TOPO2 ACTIVITY?
>> THAT'S GREAT QUESTION.
GREAT YOU ARE ON THE CALLMENT I
HOPE YOU WERE ATTENDING WHEN I
TALKED ABOUT OUR COLLABORATION
WITH YOU.
I THINK THATIA ANOTHER THING WE
SHOULD GO AHEAD AND LOOK AT IN
CELLS, WE HAVEN'T YET GONE ON TO
THAT BUT THAT'S ANOTHER THING.
AND MAYBE I WILL LOOK UP WITH
YOU TO SEE IF WE CAN ENGAGE WITH
YOU IN THOSE STUDIES AS WELL.
GREAT.
SO WITH TIME RESTRAIN, WE HAVE
TO WRAP UP OVER HERE, PLEASE
YOIN ME TO THANK DR. JACKSON
AGAIN FOR THE WONDERFUL TALK AND
WE ARE LOOKING FORWARD FOR YOUR
FUTURE WORKS IN THOSE GREAT
PUBLICATIONS.
>> THANKS AGAIN FOR THE
OPPORTUNITY TO TALK.
IT'S BEEN A PLEASURE AND IT'S
GREAT MEETING EVERYBODY AGAIN.
THANKS.
>> THANK YOU.
>> BYE-BYE.