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OUR FIRST SPEAKER TODAY IS SONIA JAKOWLEW SHE GOT HER PH.D. AT RUTGERS UNIVERSITY, THEN SHE DID A POSTDOCTORAL FELLOWSHIP IN FRANCE, SUBSEQUENTLY SHE CAME TO NCI IN THE 80s AND SHE STARTED WORKING WITH MICHAEL SPORN AND ANITA ROBERTS ON TGF-BETA AND SHE IS GOING TO TALK TO US ABOUT THAT TODAY CURRENTLY SHE IS IN THE CANCER TRAINING BRANCH AND THE TITLE OF HER TALK IS TRANSFORMING GROWTH FACTOR BITA IN LUNG TUMORIGENESIS SONIA >> THANK YOU IT’S GOOD TO BE HERE TODAY I’LL BE TALKING TO YOU ABOUT TRANSFORMING GROWTH FACTOR BETA IN LUNG TUMORIGENESIS AND I WILL JUST TOUCH BRIEFLY ON THE LUNG CANCER DISEASE AND LEAVE THE HEAVY LIFTING FOR LUNG CANCER DISEASE TO THE SPEAKER COMING IN A FEW WEEKS TO THE CLASS SO LIKE MOST PAST YEARS, LUNG CANCER WAS DETERMINED TO BE THE MOST COMMON CANCER DEATHS AMONG BOTH MEN AND WOMEN IN THIS COUNTRY LAST YEAR THERE WERE OVER 221,000 NEW CASES OF LUNG CANCER THAT WERE DIAGNOSED IN THIS COUNTRY LAST YEAR AMONG BOTH MEN AND WOMEN, AND OVER 158,000 DEATHS DUE TO THIS DISEASE SO IT’S A SIGNIFICANT DISEASE BUT BECAUSE LUNG CANCER IS PRIMARILY CAUSED BY TOBACCO SMOKING, AND THE INCIDENTS OF SMOKING HAS DECREASED IN THIS COUNTRY, THANKFULLY, MOST CASES OF LUNG CANCER NOW OCCURS IN FORMER EXSMOKERS HOWEVER, THERE ARE MANY, MANY PATIENTS WHO HAVE LUNG CANCER WHO HAVE NEVER SMOKED SO THERE IS LUNG CANCER WHICH INCLUDES EXPOSURE TO RADONS, EXPOSURE TO COAL, AND COAL DUST AND THINGS THAT — ENVIRONMENTAL CONDITIONS THAT PRECIPITATE INFLAMMATION IN THE LUNGS SO, FIVE-YEAR SURVIVAL RATE FOR LUNG CANCER IS STILL NO LESS THAN 15% SO A LOT OF WORK HAS YET TO BE DONE ON THIS DISEASE NOW, I STARTED ON TRANSFORMING GROWTH FACTOR BETA OR TGF-BETA AND BEGAN IN THE EARLY 80s AND TGF-BETA HAS BEEN SHOWN TO BE A MULTI-FUNCTIONAL REGULATOR OF CELL GROWTH IT IS A RATHER POTENT INHIBITOR OF THE GROWTH OF MOST NORMAL EPITHELIAL CELLS AND WIDESPREAD EXPRESSION TGF-BETA HAS BEEN SHOWN TO PLAY A PIVOTAL ROLE IN MAINTAINING EPITHELIAL HOMEOSTASIS AND HAS COME TO BE ASSOCIATED WITH VARIOUS TYPES OF CANCER, INCLUDING LUNG CANCER SO, AND FINALLY, TGF-BETA HAS BEEN SHOWN TO HAVE WHAT WE CALL CONTRACT-DEPENDENT, INHIBITION OR STIMULATION OF CELL PROLIFERATION AND NEOPLASTIC TRANSFORMATION, DEPENDING ON THE ENVIRONMENTAL CONDITIONS SO TGF BAIT AT IS AN ATTRACTIVE CANDIDATE FOR NEW THERAPEUTICS INTERVENTIONS IN CANCER NOW TO UNDERSTAND TGF BAIT AYOU HAVE TO GO BACK TO THE BEGINNING AND THIS HAS ITS ROOTS IN ANOTHER GROWTH FACTOR SO IT WAS IDENTIFIED IN THE EARLY 80s AS BEING A POLYPEPTIDE THAT WAS SECRETED BY [ INDISCERNIBLE ] AND STIMULATED AND PERFORMED

COLONIES THIS WAS USED AS THE ASSAY FOR THE GROWTH FACTOR BY JOE AND GEORGE AT THE NCI IN THE 70s AND SHORTLY AFTER THE GROWTH FACTOR WAS IDENTIFIED, IT WAS DETERMINED THAT THERE WERE ACTUALLY TWO DIFFERENT PROTEINS THAT MADE UP THIS GROWTH FACTOR AND THEY WERE CALLED — ISOLATED FROM THE SARCOMA VIRUS TRANSFORM CELLS THAT THEY USED TO FIGHT SARCOMA GROWTH FACTORS AND ONE CLASS WAS SHOWN TO COMPETE WITH EPIDERMAL GROWTH FACTOR, EGF RECEPTOR BINDING AND THIS CLASS WAS PDF ALPHA AND ANOTHER – TGF ALPHA AND ANOTHER CLASS WAS HOPE IS NOT TO BE ABLE TO HAVE TGF BINDING BUT DID FORM COLONIES IN THE PRESENCE OF EGF AND THIS WAS CALLED TGF-BETA SO SARCOMA GROWTH FACTORS REALLY IS A GROWTH FACTOR THAT IS REALLY COMPOSED OF TWO DIFFERENT PROTEINS THAT FORTUNATELY OR UNFORTUNATELY, CARRY THE NAME OF TGF THEY ARE VERY DIFFERENT PROTEINS AND DO VERY DIFFERENT THINGS SO THIS WAS REPORTED BY ANITA ROBERTS AND MICHAEL SPORN IN THE EARLY ANXIETY SO FOLLOWING THIS — IN THE EARLY 80s SO FOLLOWING THIS THE LAB HASTENED TO PURIFY DFG BETA FROM ROVARIETY OF DIFFERENT SOURCES, INCLUDING HUMAN PLATELETS, HUMAN A ASENTA AS WELL AS BOVINE KIDNEY NOW TO DEMONSTRATE THE PURIFICATION FROM BOVINE KIDNEY, IDNEYALLY OBTAINED FROM THE FLORIDA HEALTH AND EXTRACTED FOR SEVERAL HOURS WITH ETHANOL AND THEN SENTRI FUSED TO GET RID OF THE WASTE AND THEN PRECIPITATED NVERNIGHT WITH ABOUT 50 LITERS 0 ETHER ETHANOL IN THE COLD ROOM NEXT MORNING IT WAS THEN DISSOLVED IN ONE MOLOR ACID AND THEN APPLIED TO 80 LITERS PE LITER WAS THEN SELECTED AND WHAT AW WHAT A ONE LITER IS AND THESE WERE THEN REDISSOLVED 100 GRAMS OFURIFICATION TISSUE THEY WOULD GET — MICOGRAMS OF TGF-BETA THIS IS THE ENORMITY OF THE COLUMNS NEEDED TO PURIFY TGF-BETA AND ON THE RIGHT, LOWER WORK, THIS IS 55-GALLON BARREL TO GIVE YOU SOME IDEA OF SIZE NOW THE ASSAY FOR TGF-BETA INCLUDED THE ROTH OF NORMAL RED KIDNEY CELLS AND RK CELLS SO TYPICALLY, TO PERFORM THIS ASSAY, A PLATE WAS MADE AND THEN A MIXTURE OF MEDIA SERUM AND RK CELLS, EGF AND SAMPLE, WHICH MAY OR NAY NOT HAVE CONTAINED TGF-BETA WAS APPLIED AND THEN ALLOWED TO INCUBATE AT 37 DEGREES FOR A WEEK AND THEN STAINED AND THE COLONIES WERE THEN COUNTED ON THE PLATES USING AN IMAGE ANALYSIS SYSTEM, WHICH IS SHOWN AT THE BOTTOM OF THE SLIDE HERE THAT WAS STATE-OF-THE-ART AT THE TIME SO, IF NO TGF BAIT IS PRESENT IN THE SAMPLE AND NO COLONIES WOULD BE COUNTED IF TGF-BETA IS PRESENT IN THE SAMPLE, THOUGH, COLONIES WOULD APPEAR OVER THE ONE WEEK, AND IF THEY WERE A SPECIFIC SIZE OR LARGER, THEY WOULD BE COUNTED SO, AFTER THE FINAL HIGH-PRESSURE CHROMATOGRAPHY PURIFICATION, SAMPLES WERE THEN

PUT IN A GEL AND RAN AND PURIFIED TGF-BETA, MIGRATED AS A 25,000 MOLECULAR WEIGHT PROTEIN ON SINGLE BAND ON THE GEL SO, DOCTORS MICHAEL PORN AND ANITA ROBERTS ARE CREDITED WITH THE DISCOVERY OF TGF-BETA AT THE NCI AND I HAD THE GOOD FORTUNE TO WORK WITH BOTH OF THESE INVESTIGATORS FOR SIX YEARS AND IT WAS ONE OF THE HAPPIEST AND INFORMATIVE AND EXCITING TIMES OF MY LIFE, MY RESEARCH LIFE AND THEN FORTUNATELY, WE LOST UNFORTUNATELY WE LOST ANITA FROM GASTRIC CANCER WHEN SHE PASSED ON, SHE HAD THE ENORMITY OF BEING THE SECOND-MOST HIGHLY-PUBLISHED FEMALE INVESTIGATOR IN THE WORLD AND MICHAEL S PORN, HAS SINCE LEFT THE NIH AND SHE ENDOWED PROFESSOR OF MEDICINE AT DARTMOUTH COLLEGE AT NEW HAMPSHIRE AND HE IS AT THE RIPE YOUNG AGE OF 84 STILL GOING AND STUDYING TGF-BETA LET’S HEAR IT FOR THE YOUNGSTERS SO FOLLOWING THE IDENTIFICATION AND PURIFICATION OF TGF BAIT ATHE SEQUENCE WAS DETERMINED AND ACTUALLY IT WAS FOUND THAT TGF-BETA WAS OR IS INITIALLY FORMED AS WHAT IS CALLED A PREPRO MOLECULE OF 391 AMINA ACIDS IT INCLUDES THE SEQUENCE AT THE END TERMINIS, A MIDDLE PORTION, WHICH WAS THEN CALLED THE LATENCY ASSOCIATED PEPTIDE OR LAP FOR SHORT, AND THEN TGF-BETA SEQUENCE AT THE SAME TERMINIS OF 112 AMINO ACIDS NOW THE AMINO ACID SEQUENCE HAS BEEN DETERMINED AND I GIVE YOU A PRETTY DOLLAR PICTURE DENOTING THE 9 CHARACTERISTICS SISSTEINS THAT HIGHLIGHT THIS CLASS OF PROTEINS AND I’LL TALK ABOUT THAT A LITTLE LATER SO, IT CONTAINS A SINGLE PEPTIDE, THEREFORE THIS IS A SECRETED PROTEIN NOW THE CRYSTAL STRUCTURE OF TGF-BETA WAS DETERMINED IN THE EARLY 90s THIS WILL HAPPENS TO BE A REPRESENTATIVE — REPRESENTATION OF TGF-BETA TWO BECAUSE FOR UNKNOWN REASONS, TGF-BETA TWO IS MUCH EASIER TO CRYSTALLIZE THAN TGF-BETA ONE SO TGF-BETA 2 AND 1, OCCURS AS A DIMER OF TWO IDENTICAL MONOMERIC CHAINS HELD TO GO BY A BOND WITH A HYDROPHOBIC POCKET, THAT OTHER PROTEINS CAN INTERACT WITH NOW, IT GF BETA IS A CONIKEAL MEMBER WHAT HAVE IS CALLED THE SUPER FAMILY THIS SUPER FAMILY CONTAINS, IN ADDITION TO THE TGF-BETA ISOFORMS, THE BMPs, GDF, GROWTH DIFFERENTIATION FACTOR, AS WELL AS ACT EVAPORATES AND INHIBITTANTS NOW TO SUMMARIZE WHAT I HAVE BEEN TALKING ABOUT TGF A 25,000 MOLECULAR DISULFIDE-BONDED HOMODIMER THERE ARE THREE HIGHLY HOMOLOGOUS ISOFORMS IDENTIFIED IN HUMANS AND THESIS ARE CALLED TGF-BETA, 1, 2 AND 3 THEY ARE SEPARATE PROTEINS BUT THEY ARE HOMOLOGOUS AND IN ADDITION, TGF-BETA 4 HAS BEEN IDENTIFIED IN BIRDS TGF-BETA 5 HAS BEEN IDENTIFIED IN AM PHIBIANS AND TGF-BETA 4 AND 5 HAVE MOST CLOSELY HOMOLOGY TO TGF-BETA ONE THE PRINCIPLE SOURCES IS TGF-BETA IN THE HUMAN ARE PLATELETS, BONE AND SPLEEN MOST CELLS HAVE BEEN SHOWN TO EXPRESS TGF-BETA AS WELL AS ITS RECEPTORS AND TGF BAIT IS – USUALLY SECRETED IN A LATENT, IN ACTIVE FORM WHICH MUST BE ACTIVATED BEFORE TGF-BETA CAN ACT ON EXPRESSION OR DO ANY OF ITS ACTIVITIES

AND WILL THE SUPER FAMILIES ARE TGF-BETA ENCOMPASSES NOT ONLY THE TGF-BETAS BUT ACTIVINS AND BMPs AND GDFs MEMBERS OF THE TGF SUPER FAMILY CONTROL CENTRAL CONTROL MODULES FOR A NUMBER OF BIOLOGICAL PROCESSES, INCLUDING DEVELOPMENT IMMUNE SYSTEM REPRODUCTION, ANGIOGENESIS, AGING, TISSUE REPAIR, METABOLIC REGULATION AND HOMOSTAYSIS SO IT EXPANDS A WIDE RANGE OF ACTIVITIES TGF-BETA CAN INHIBIT PROLIFERATION OF CELLS AND REGULATE ISOTOSIS AND STIMULATE ACCUMULATION OF EXTRACELLULAR MATRIX AND PROMOTE CHEMOTAXIS OF CERTAIN CELLS SO ANYTHING ELSE TGF-BETA CAN’T DO? THAT WAS THE QUESTION IN THE EARLY 80s SO, THE PATHWAY HAS BEEN DEVELOPED OVER THE YEARS AND THIS WE WILL CALL THE DEPENDENT PATHWAY BECAUSE OF THE INVOLVEMENT OF THE PROTEINS IN THIS PATHWAY SO THIS BEGINS BY THE LIGAND SHOWN IN BLUE AT THE TOP AND IT CAN BE TGF-BETA ITSELF AS WELL AS ACTIVIN INHIBINS AND BMPs, BY THE TYPE — LOST MY MOUSE SORRY THE TGF-BETA LIGAND BINDS FIRST TO THE TYPE II RECEPTOR, WHICH IS PHOSPHORYLATED ONCE THIS BINDS, IT THEN FORMS A COMPLEX AND RECRUITS ANOTHER MOLECULE CALLED THE TGF-BETA TYPE I RECEPTOR TO FORM A COMPLEX THE TYPE II TGF-BETA RECEPTOR THEN TRANSFERS TYPE I RECEPTOR AND THAT ENABLES IT TO THEN BE TRANSDUCED TO OTHER PROTEINS CALLED THE SMAD PROTEIN DEPENDING ON THE LIGAND IS, THE SMADS ARE SPECIFIC TO THE LIGANDS TGF-BETA ACT VINCE THEY ARE SMAD 2 AND 3 FOR THE BMPs, WE HAVE SMAD 1 AND SMAD 5 AND SMAD 8 SO THESE CARRIES THE SIGNAL OR TRANSPHOSPHORYLATED AND THEN DELIVERS THE SIGNAT TO ANOTHER SMAD CALLED SMAD 4, WHICH IS CALLED A — SMAD SO THIS SMAD IS UNABLE TO TRANSDUCE A SIGNAL INTO THE NUCLEUS WHERE IT CAN THEN PARTICIPATE AND AFFECT TRANSCRIPTION SO THE WHOLE PROCESS CAN BE CIRCUMVENTED BY WHAT ARE CALLED IN HIM TORY SMADS SMAD 6 AND SMAD 7 WOULD CIRCUMVENT THE WHOLE PROCESS SO THERE ARE REGULATORY SMADS THAT MAKE THE TGF-BETA PATHWAY GO FORWARD AND THERE ARE INHIBITORY SMADS THAT STOP THE PATHWAY ITSELF SO, IN ADDITION TO THE LABORATORY, CLINICALLY, TGF-BETA HAS BEEN STUDIED IN THE CLINIC AS WELL IT’S BEEN SHOWN THAT TGF BAIT AT IS A TYPE OF TUMOR SUPPRESSOR FOR EXAMPLE, GERMLINE MUTATIONS HAVE BEEN SHOWN TO OCCUR IN VARIOUS COMPONENTS OF THE TGF-BETA PATHWAY TO CAUSE FAMILIAL PREDISPOSITION TO CANCER AND THIS HAS BEEN SHOWN QUITE NICELY WITH SMAD 4 IN JUVENILE POLYFOCUS SYNDROME THE SECOND EXAMPLE IS TGF-BETA PATHWAY COMPONENT MAYBE SOMATICALLY MUTATED OR DELETED IN SOME HUMAN CANCERS NOW THE TYPE II TGF-BETA RECEPTOR HAS BEEN SHOWN TO BE MUTATED IN HUMAN, COLORECTAL CANCER WHILE SMAD 4 HAS BEEN SHOWN TO BE DELETED IN MANY CASES OF PANCREATIC CANCER

THIRDLY, REDUCED EXPRESSION OF TGF-BETA SIGNALING PATHWAY COMPONENTS ARE OVEREXPRESSION OF THE INHIBITORS HAVE BEEN SHOWN TO BE ASSOCIATED WITH CANCER DISEASE PROGRESSION AND FOR THIS IT IS DEMONSTRATED AS A TYPE I AND TYPE II TGF-BETA RECEPTORS HAVE BEEN OR CAN BE ORE EXPRESSED WHILE SMAD 7 CAN BE AS AN INHIBITOR CAN ALSO BE OVEREXPRESSED IN SEVERAL TYPES OF CANCER NOW, IN ADDITION TO BEING A TUMOR SUPPRESSOR, CLINICALLY, TGF-BETA HAS BEEN ALSO SHOWN TO BE A POSSIBLE TUMOR PROMOTOR IN THE CLINIC SO, ELEVATED LEVELS OF TGF BAIT AT 1 HAS BEEN SHOWN TO BE OCCURRING IN MANY ADVANCED TYPE OF HUMAN TUMORS AND TO CORRELATE WITH METASTASES AND OFTEN WITH CORE PROGRESSION AND I HIGHLIGHTED A NUMBER OF TISSUES IN WHICH WE SEE ELEVATED LEVELS OF TGF-BETA 1 IN TUMORS, INCLUDING THE LUNG AND SHOWN ON THE RIGHT IS A PATHETIC ADENOCARCINOMA THAT HAS BEEN HISTORICALLY STAINED FOR TGF-BETA 1 WITH A VERY SPECIFIC ANTIBODY AND TGF-BETA SEEMS TO SIT AT THE INTERFACE BETWEEN THE TUMOR CHROMATIN AND MICROENVIRONMENT — PA REVEREND MA — WHAT IS GOING ON? WHAT IS THE ROLE OF TGF-BETA IN CARCINOGENESIS? IS IT A HERO LIKE WE ALL THAT THE IN IN THE BEGINNING? OR HAS IT TURNED INTO A PART-TIME VILLAIN? WELL, THE ANSWER IS PROBABLY BOTH TGF-BETA IS A PROXIMAL EFFECTOR OF THE MALIGNANT PHENOTYPE IT’S ALSO A POTENT GROWTH INHIBITOR AND TUMOR SUPPRESSOR, ESPECIALLY EARLY ON IN THE PROCESS AND IT IS ALSO A PRO METASTATIC FACTOR NOW HOW DOES THIS OCCUR? WELL, OVER THE YEARS, A UNIFYING HYPOTHESES HAS BEEN DEVELOPED WHEREBY TGF-BETA CAN SWITCH FROM BEING A TUMOR SUPPRESSOR TO PRO-ONCOGENIC FACTOR AS CANCER PROGRESSES SO WHEN WE START IN THE NORMAL EPITHELIUM, THE NORMAL EPITHELIUM AND NORMAL CELLS, TUMOR SUPPRESSOR ACTIVITIES OF TGF-BETA DOMINATES OVER ANY PRO-ONCOGENIC ACTIVITIES BUT, AS CHANGE IS OCCUR IN THE GENETIC AND EPIGENETIC CONTEXT, THE LEVEL OF TGF-BETA RESPONSIVENESS GOES DOWN AND AS TUMORIGENESIS OR CARCINOGENESIS PROGRESSES, THE EXPRESSION AND/OR ACTIVATION OF TGF-BETA INCREASES SO WHEN WE HAVE A INVASIVE METASTATIC CANCER, THEN THE PRO-ONCOGENIC ACTIVITIES OF TGF-BETA PREDOMINATE OVER THE TUMOR SUPPRESSOR ACTIVITIES SO WE HAVE ESSENTIALLY A TUMOR SUPPRESSOR SWITCHING AND TURNING INTO A PRO-ONCOGENIC MOLECULE NOW, IN ADDITION TO THE SMAD PATHWAY, THERE ARE A NUMBER OF TGF — WHAT IS CALLED TGF-BETA SMAD-INDEPENDENT PATHWAY THAT IS ENTER INTO THE PICTURE AND THIS INCLUDES A NUMBER OF PATHWAYS SUCH AS THE MAP KINASE PATHWAY, THE REST AND THE PP2A PATH WAY WE WERE PARTICULARLY INTERESTED IN THE RAS MAP KINASE PATHWAY BECAUSE THE KRAS PROTOONCOGENE IS VERY IMPORTANT IN CANCER MUTATION IN AT LEAST 25-50% OF HUMAN LUNG TUMORS MUTATION OF EVEN ONE ALLELE OF KRAS CAN INCREASE APPEARANCE OF LUNG LESION EARLY ON AND IT’S BEEN DEMONSTRATEED

THERE IS CROSS TALK THAT OCCURS BETWEEN THE SMAD-DEPENDENT PATHWAY AND THE RAS/MEK SIGNALING PATHWAY IN SEVERAL TYPES OF CELL TYPES AND ACTIVATION OF THE RAS PATHWAY CAN MODULATE TGF-BETA SIGNALING THROUGH THE SMADS ED AND LASTLY, IN-VITRO STUDIES IN CELL LINES HAVE SHOWN THAT TGF-BETA DOMINATES OVER THE MITOGENIC AFFECTS OF RAS BUT WHEN RAS IS ACTIVATED, THIS CAN OVERRIDE THE ANTI-PROLIFERATIVE AFFECT OF TGF-BETA AND TURN IT MORE INTO A PRO-ONCOGENIC FACTOR AND IS THIS A PROBLEM SO, THIS SLIDE REPRESENTS AT LEAST FOUR DIFFERENT WAYS THAT THE TUMOR SUPPRESSOR CAN TURN INTO A TUMOR PROMOTOR HOW DOES THIS OCCUR? WELL, WE HAVE THE NORMAL CONDITION WHERE THE — WHERE ENORMOUS CELLS WERE TGF-BETA INTERACT WITH THE TYPE I AND TYPE II RECEPTORS THROUGH THE SMAD PATHWAY, AND OTHER PATHWAYS, AND TO RESULT IN TUMOR SUPPRESSION BUT IF SOMETHING OCCURS AND THE — SAY FOR INSTANCE THE DECREASE IN THE AMOUNT OR LEVEL OR EXPRESSION OF THE TYPE II RECEPTOR OR THERE MAY BE HYPERACTIVATION OF THE SMAD OF MAP KINASE PATHWAY WITH ACTIVATES RAS OR THERE MIGHT BE DECREASED SMAD LEVELS OR ACTIVITY, OR THERE MAY BE OTHER THINGS THAT MAYBE HAPPENING IN THE TUMOR SUPPRESSOR ARM OF THIS PATHWAY THEN YOU HAVE A TUMOR PROMOTOR SITUATION IN LIEU OF A TUMOR SUPPRESSOR SITUATION SO WE HAVE BEEN KIND OF INTERESTED IN THE HYPERACTIVATED MAP KINASE PATHWAY IN OUR STUDIES SO, THE BROAD GOAL OF MY LABORATORY WAS TO DETERMINE THE ROLE OF TGF-BETA IN THE DEVELOPMENT AND MA LIGNANT TRANSFORMATION OF LUNG EPITHELIAL CELLS AND THIS WORK WAS DONE IN THE EPITHELIAL CARCINOGENESIS SECTION OF NCI SO, WE HAVE THREE OBJECTIVES THE FIRST IS TO EXAMINE THE EFFECT OF TGF-BETA 1 DELETION IN KRAS MUTATION ALONE AND THEN IN COMBINATION ON LUNG TUMOR INCIDENCE AND PATHOLOGY THEN TO DETERMINE THE EARLY EVENT THAT MIGHT OCCUR IN THE DEVELOPMENT OF LUNG LESION AND HOW THEY PROGRESS AND THEN TO IDENTIFY ANY POTENTIAL SIGNAL TRANSDUCTION PATHWAY CHANGES THAT MAY OCCUR WITH INCREASES LEVELS OF TUMORIGENESIS SO TO DO THIS WORK, WE EMPLOYED FOUR MOUSE MODELS THE AJ, THE C57BL6TGF BAIT HETEROZYGOUS MODEL AND THE: [ READING ] WE GENERATED IN COLLABORATION WITH MIT SO, WE HAD TWO QUESTIONS DOES THE LUNG TUMORIGENESIS AFFECT THE TGF-BETA SIGNALING PATHWAY? AND THE RECIPROCAL QUESTION? DOES IT AFFECT LUNG TUMORIGENESIS? SO, WE TRIED THE AJ MOUSE MODEL BECAUSE THIS MOUSE IS VERY SUSCEPTIBLE TO CHEMICALLY-INDUCED LUNG TUMORS THE TUMORS DEVELOP IN A TIME-DEPENDENT FASHION THEY PROGRESS FROM HYPERPLASIA TO ADENOMA AND THEN CARCINOMA VERY SIMILARLY TO WHAT OCCURS IN HUMANS AND THE CARCINOMAS THAT DEVELOP ARE HISTORICALLY SIMILAR TO THE HUMAN LUNG ADENOCARCINOMAS, WHICH ARE THE CHIEF CARCINOMAS THAT APPEAR IN HUMAN LUNG CANCER AND THE SAME MOLECULAR MUTATIONS OCCUR IN BOTH HUMAN AND MOUSE LUNG TUMORS, SUCH AS OVER-EXPRESSION OF KRAS AND LOSS OF p53 EXPRESSION SO, WE USE ETHEL CARBAMATE AS OUR LUNG-SPECIFIC CARCINOGEN,

AND ETHEL CARBAMATE CAN GO THROUGH TWO PATHWAYS WHERE THERE IS A DETOX PATHWAY THAT RESULTS IN JUST HARMLESS ETHANOL ALSO A BIOACTIVATION PATHWAY THAT PROCEEDS TO ASSIST E1 IN THE LUNG AND THIS RESULTS IN VINYL CARBAMATE AND VINYL CARBAMATE APOXIDE WHICH ARE THEN ABLE TO BIND TO MACRO MOLECULES AND CAUSE NASTY AFFECTS SO, FOR PRODUCTION OF TUMORS IN THESE MICE, WE INJECTED ETHEL CARBAMATE IN 2-MONTH-OLD MICE AND THEN SACRIFICED THEM AT MONTHLY INTERVALS UP TO A YEAR WITH 20 MICE PER SACRIFICE FOR A STATISTICAL REASON SHOWN ON THIS SLIDE IS IMMUNOHYSTERICAL CHEMICAL STAINING OF TUMORS AT VARIOUS STAGES OF TUMORIGENESIS AND STAINED FOR SPECIFIC ANTIBODIES TO TGF-BETA 1 AND THE TYPE I AND TYPE II RESEPTEMBERRORS BEING AND YOU WILL SEE IN THE LEFT HAND AND RIGHT HAND MIDDLE PANEL THIS BROWN STAINING FOR TGF-BETA ONE AND THE TYPE I RECEPTOR THROUGHOUT THESE TUMORS HOWEVER, ON THE RIGHT FOR THE TYPE II RECEPTOR, STAINING FOR THIS PROTEIN IS ADMONISHED IN THE TWO MONTH TOMBORS AND ABOUT 4 MONTHS AND CATER MONTHS IT’S VIRTUALLY GONE SO WE SEE DECREASED EXPRESSION OF THE TYPE II RECEPTOR PROTEIN IN TUMORS WITH ADVANCING TUMORIGENESIS THIS IS SHOWN MORE CLEARLYO THIS SLIDE IF YOU’LL DIRECT YOUR ATTENTION TO THE LEFT SIDE OF THE SCREEN SPECIFICALLY PANEL D&D, THE RED ARROW SHOWS DISTINCT EXPRESSION OF THE BROWN STAINING FOR THE TYPE I AND TYPE II RECEPTOR VIRTUALLY IDENTICAL LEVELS IN TUMORS BUT IF YOU LOOK AT THE TUMORS IN COMPARISON, THERE IS INTENSE STAINING FOR THE TIME 1 RECEPTOR WHILE THERE IS VERY DIMINISHED STAINING FOR THE TYPE II RECEPTOR THIS IS ALSO WE LOOKED AT THE EXPRESSION OF THE mRNAs FOR THESE PROTEINS AS WELL AND USING FIVE CELL LINES, MOUSE LINE CELL LINES, WE SHOW EXPRESSION OF THE TYPE I AT VARIOUS LEVELS IN THESE CELL LINES WHILE THE EXPRESSION OF THE TYPE II RECEPTOR MESSAGE IS VIRTUALLY GONE IN THESE 2 CELL LINES AND I HIGHLIGHTED THE 5th, PCC4 BECAUSE THESE WERE DERIVED FROM A ETHEL CARBAMATE INDUCED MOUSE LUNG TUMORS THIS MIRRORS WHAT WE ARE SEEING IN THE AJ MOUSE SO WE ARE JUST SHOWING DECREASED LEVELS OF PROTEIN IN mRNA FOR TYPE II RECEPTOR WITH ADVANCING TUMORIGENESIS IN THESE MICE WE ALSO LOOKED AT LUNG TUMORS FROM BP INDUCED TUMORS WE SAW THE SAME THING IF YOU’LL DIRECT YOUR VISION TO THE RIGHT SIDE OF THE SCREEN, WE SEE DECREASED LEVELS OF EXPRESSION OF THE PROTEIN FOR THE TYPE II RECEPTOR AND DECREASED EXPRESSION OF THE MRNA FOR TYPE II RECEPTOR IN COMPARISON TO THE TYPE I RECEPTOR AND TGF-BETA LIGAND ITSELF WHEN WE STAIN FOR THE PROTEINS THEY ARE ALMOST IDENTICAL SO OUR MOUSE MODEL REFLECTS THERE IS WITH DECREASED EXPRESSION OF THE TYPE II RECEPTOR WE SEE CORRELATION OF LUNG TUMORS IN OUR MOUSE MODEL

SYSTEM OUR NEXT QUESTION WAS, DOES THE DELETION OF TGF-BETA WONG AFFECT LUNG TUMORIGENESIS? AND FOR THIS, WE SHIFTED TO THE BLACK TGF-BETA 1 MOUSE BECAUSE THE TGF-BETA KNOCKOUT MOUSE WAS ENGINEERED AND GENERATED IN THE EARLY 90s AND IT IS BORN IT THRIVES, BUT THREE WEEKS OF AGE, IT STARTS TO GET VERY SICK AND SUCCUMBS TO A GENERAL SYNDROME SO THIS MOUSE WON’T BE GOOD FOR CARCINOGENESIS STUDIES BUT THE KNOCKOUT MOUSE, THE TGF-BETA 1 HETEROZYGOUS LITTER MATE IS BORN, THRIVES AND SURVIVES T DOESN’T SUCCUMB TO A WASTING SYNDROME SO IN COLLABORATION, WE DID A STUDY AND LOOKED ON BECAUSE SHE WAS INTERESTED IN LIVER TUMORIGENESIS, WE DOSED THESE OR CHALLENGED MICE WITH DIMETHYL NITROSE MEAN, WHICH IS SPECIFIC FOR LIVER TUMORS AND WE SHOWED THAT THERE WAS AN INCREASE LEVEL OF TUMORIGENESIS IN THE LIVER OF HETEROZYGOUS MICE SHOWN IN RED COMPARED TO THE WILDTYPE MICE NOW UNEXPECTEDLY IF YOU FOCUS YOUR ATTENTION ON THE RIGHT SIDE, WE SAW EVEN GREATER NUMBER OF LUNG TUMORS FORMING IN THESE MICE SO THESE MICE HAD INCREASED TUMOR INCIDENTS OF LUNG TUMORS IN THEIR LUNGS SO THIS WAS SURPRISING SO IN ORDER TO BE ABLE TO LOOK AT IT IN A BETTER MOUSE MODEL, WE CROSSED WILDTYPE TO DERIVE TWO DIFFERENT PHENOTYPES, TGF BAIT AT HETEROZYGOUS MICE AND THE WILDTYPE LITTER MATE OUR PLAN WAS THEN TO TREAT THEM AS CARCINOGEN, ETHEL CARBAMATE AND LOOK AT THE LUNG TUMOR WE ISOLATED LUNGS AND STAINED THEM FOR TGF-BETA 1 ANTIBODY AND AS WELL AS LOOKED AT IN SITU HYBRIDIZATION FOR TGF-BETA 1 LIGAND AS WELL IF YOU LOOK AT THE TOP MANUAL, THE MIDDLE PANEL SHOWS DECREASED EXPRESSION OF TGF-BETA 1 PROTEIN AND MESSAGE IN HETEROZYGOUS MICE COMPARED TO WILDTYPE MICE AS WAS EXPECTED WE PERFORMED NORTHERN BLOTTING ON THESE MICE LUNGS AS WELL AS COMPETITIVE RTPCR AND THE SHOWED REDUCED EXPRESSION OF THE TGF-BETA 1 LIGAND AND HEIGHT ROWZYGOUS MICE COMPARED TO WILDTYPE SO WE HAD THE RIGHT MICE WE WENT ALONG AS WE HAD DONE WITH THE AJ MICE THIS TIME INJECTING WITH ETHEL CARBAMATE AS BEFORE IN 2-MONTH-OLD MICE WITH HETEROZYG US AND WILDTYPE MICE AND SACRIFICED GROUPS OF 20 MICE OVER A 12 MONTH PERIOD AND WHEN WE DID THIS, WE EXTRACTED AND LOOKED AT THE MICROSCOPEICALLY HYPERPLASIA ADENOMA AND CARCINOMA AND IN PANEL A, YOU SEE THAT IN PANEL B, YOU’LL SEE THAT IN THE TGF BAIT 1 TET ROWZYGOUS MICE DRAWN IN RED, HERE IS DEFINITE OCCURRING BY ONE MONTH WHEREAS IT TAKES 2-4 MONTHS BEFORE WE SEE ADENOMAS AND HYPERPLASIA OCCURRING IN THE WILDTYPE MICE

NOW THE STRIKING THING IS WE SEE CARCINOMAS APPEARING BY 4 MONTHS IN THE TET ROWZYGOUS MICE BUT TAKES ALMOST ONE YEAR AND 12 MONTHS BEFORE WE SEE ANY CARCINOMA STARTING TO APPEAR IN THE WILDTYPE LITTER MATES IN THESE MICE, WE SEE INCREASED TUMOR INCIDENTS BUT A DECREASE TUMOR LATENCY IN THE TGF-BETA ONE HETEROZYGOUS MICE COMPARED TO WILDTYPE MICE THIS IS WITH BORN ALLELE TGF-BETA SO WE THEN PROCEEDED TO STAIN FOR TYPE II RECEPTOR AND UNLIKE THE ORIGINAL MICE, WE SIGH REDUCED STAINING FOR TYPE II RECEPTOR PROTEIN AND CARCINOMAS BUT NOT IN THE ADENOMAS OR HYPERPLASIA SHOWN IN THE RIGHT PANEL SO IT SEEMS LIKE THE LEVEL OF OR TYPE II RECEPTOR IS MAINTAINED IN EARLY TUMORIGENESIS BUT IT IS OVERTAKEN WITH INCREASING TUMORIGENESIS SO WE ALSO LOOKED AT THE RELATIVE LEVEL OF THE TYPE II RECEPTOR mRNAs WITH INCREASING TUMORIGENESIS IN THESE MICE AND SEEMS LIKE DECREASING LEVELS OF THE TYPE II RECEPTOR mRNA WITH INCREASING LUNG TUMORIGENESIS FROM HYPERPLASIA TO ADENOMA AND THEN TO CARCINOMA SO OUR NEXT QUESTION IS DELETION OF TGF-BETA ONE AND MUTATION OF KRAS COMBINED AFFECT LUNG TUMORIGENESIS? AND FOR THIS, WE GENERATED THE TGF-BETA ONE HETEROZYGOUS KRAS LIKE R. MOUSE NOW I WON’T BORE YOU WITH THE DETAILS ESSENTIALLY WE CROSSED MICE AND C7 BLACK 6 STRAIN BECAUSE WE HAD TO GO BACK TO THE ORIGINAL BECAUSE WE HAD DEFINITE PROBLEM WITH NUMBER OF OFFSPRING GENERATESSED FROM JUST THE AJ MICE WITH — ACTIVATABLE MICE AND THIS IS HAVING WE GENERATED FOUR DIFFERENT PHENOTYPES, THE DOUBLE MUTANT TGF-BETA 1 WILDTYPE WHICH WE CALLED THE KRAS SINGLE MUTANT TGF-BETA 1K KRAS WILDTYPE WHICH WE CALLED THE SINGLE MUTANT AND THE LAST WAS THE WILDTYPE COMBINES SO WE LET THESE MICE GROW AND AT 6 MONTHS LOOKED — STARTED LOOKING AT THESE 4 MONTHS BUT THESE ARE 6 MONTH LUNGS AND IN A AND B, YOU SEE THESE PEARLY WHITE TUMORS THAT ARE FORMING ON THE EXTERIOR OF THE LUNG AND THESE ARE VERY EVIDENT IN THE DOUBLE MUTANT SHOWN IN PANEL A AND THE KRAS SINGLE MUTANT SHOWN IN PANEL B WHEREAS IN PANEL C AND PANEL D, THE TGF-BETA SINGLE MUTANT AND THE WILDTYPE DO NOT REALLY SHOW ANY TUMORS ON THE LUNG, JUST AN OCCASIONAL ONE VERY INFREQUENTLY WE LOOK AT THE AFFECT OF TGF-BETA ONE DELETION ON MOUSE SURVIVAL AND PARTICULARLY ON MORTALITY AND LIKE TO DIRECT YOUR FOCUS ON A PLAT FOR A&B A IS A DOUBLE MUTANT AND SIGNIFICANT DECREASE IN MORTALITY, AND LIFESPAN, AND B, WHICH IS THE KRAS SINGLE MUTANT IS ALSO AFFECTED BUT LESS SO COMPARED TO THE TGF-BETA 1 HETEROZYGOUS SINGLE MUTANT SHOWN IN C LIFESPANS IN THE DOUBLE

MUTANT AND SINGLE KRAS MUTANT MICE WE LOOKED AT PATHOLOGY OF LUNG LESIONS AND SHOWED INCREASE HYPERPLASIA AND ADENOMA IN THE LATE KRAS SINGLE MUTANT SHOWN IN GREEN BIIT SEEMS IN THE SINGLE MUTANT A PROTECTION UP TO A CERTAIN POINT BUT IN THE DOUBLE MUTANT THAT PROTECTION IS GONE AFTER CERTAIN TIME WE STAINED FOR TGF-BETA IN LUNG TUMORS AND SHOWN BY THE PINK ARROW, YOU SEE DECREASED EXPRESSION OF TGF-BETA ONE IN THE TYPE II RECEPTOR IN THE DOUBLE MUTANT TUMORS AND THEN DOUBLE MUTANT ON THE RIGHT WE SEE DEFINITE EXPEDITED REDUCTION OF TYPE II RECEPTOR AND DEFINITE INCREASE IN PRODUCTION OF SMAD 3 OCCURRING BY TWO MONTHS COMPARED TO 3 AND 4 MONTHS IN THE WILDTYPE SO WE PERFORMED A NUMBER OF SIMILAR WESTERN BLOTS IN ADDITION TO THE SMAD 3 WE SEE REDUCED EXPRESSION OF THE SMAD 4 AND SMAD 7 PROTEIN AS WELL AS INCREASED EXPEDITED EXPRESSION PRODUCTION OF KRAS AND RAV 1 WE ALSO LOOKED AT THE RAF 1 WHICH IS INTEGRAL IN THE KRAS PATHWAY AS WELL WE PERFORMED OR LOOKED AT APOPTOSIS INDEX IN THE SINGLE MUTANT AND DOUBLE MUTANT AND SHOWN IN RED WE SEE DEFINITE REDUCED APOPTOSIS IN THE DOUBLE MUTANT ADENOMA COMPARED TO THE SINGLE KRAS MUTANT SO, OUR MOUSE MODEL SHOWS AND SEEM TO CORRELATE THE DECREASED EXPRESSION AND PRODUCTION OF THE TYPE II RECEPTOR CORRELATES WITH INCREASED LUNG TUMOR PROMOTION, ACTIVATED KRAS MAP KINASE SEEMS TO CORRELATE NICELY WITH INCREASED LUNG TUMOR AND COMPROMISED APOPTOSIS CORRELATES WITH INCREASED LUNG TUMOR PROMOTION AND I DIDN’T SHOW HERE WHAT DECREASED SMAD 4 ALSO SHOWS CORRELATION WITH INCREASED LUNG TUMOR PROMOTION IN OUR MOUSE MODEL SO I’D LIKE TO ACKNOWLEDGE THE PEOPLE INVOLVED WITH THIS STUDY AND I’D LIKE TO THANK TYLER FOR THE KRAS DISPOSABLE MOUSE THAT ENABLED THIS STUDY SO I’D LIKE TO TAKE ANY QUESTIONS YOU MIGHT HAVE AT THIS TIME [ APPLAUSE ] [ OFF MIC ] >> PEOPLE ARE WORKING ON IT THE PROBLEM IT’S VERY TIME CONSUMING TO PURIFY AND VERY EXPENSIVE ABOUT 10 YEARS AGO, THERE WAS A GREAT PUSH ON TO TRY TO GET BETA IN WOUND HEALING AND THERE WERE CLINICAL TRIALS THAT WERE STARTED TO DO THIS BUT THE PROBLEM IS GETTING ENOUGH

FOR LONG TERM STUDY AND THAT IS A PROBLEM HERE AND THAT IS THE PROBLEM WITH MANY OF THESE GROWTH FACTOR STUDIES SO UNFORTUNATELY, IT’S STILL IN THE PIPELINE >> WHAT IS THE BEST TARGET? RECEPTOR, LIGAND OR SMAD >> TAKE YOUR POISON IT DEPENDS ENTIRELY ON THE SITUATION IN SOME SITUATIONS, IT IS THE LIGAND AND REMEMBER ALTHOUGH I CALLED IT TGF-BETA ALL THE TIME, WE ARE TALKING ABOUT THE TGF-BETA SUPER FAMILY SO THERE IS A LOT OF INVOLVEMENT OF MANY DIFFERENT MOLECULES HERE SO WHAT MAY WORK IN ONE TYPE OF CANCER OR DISEASE MAY NOT WORK IN ANOTHER TYPE OF DISEASE SO IT’S KIND OF A LOT TO KIND OF FUSS OVER IN THE BASKET OF GROWTH FACTORS YVES POMMIER RECEIVED NIH MERIT AWARD FOR LOUISIDATION OF SUMMERAISE AS A TARGET FOR ANTI-CANCER DRUG HE IS ALSO EDITOR ON CANCER RESEARCH, ONE OF THE DISTINGUISHED JOURNALS HE RECEIVED A PAUL EHRLICH AWARD AND PUBLISHED HUNDREDS OF MANUSCRIPTS AND WILL TALK TO US ABOUT DNA TOWNSHIP OTHERWISE SUMMERAISES AND POISONING AND ANTIBACTERIAL DRUGS TOPOSOMBER ACES SO WHAT I DID AT GIPPING OF THE THIS IS NOT A FIELD THAT WILL SHRINK ANY TIME SOON IT GOT A LOT OF — AND DNA COULD BE LOOKED UNDER DIFFERENT CONFIGURATIONS WHEN IT WAS UPGRADEIENT AND FAST — AND THAT WAS CALLED FORM ONE PLASMID DNA AND SOMETHING SLOW THAT WAS CALLED FORM TWO AND THEN WHEN BROMIDE CAME ABOUT FOR YOU IT IS GIVEN BUT IN THOSE DAYS IT WAS NOT A GIVEN AND GELS WERE DEVELOPED IT WAS REALIZED THAT FORM 1 WAS A SUPER TWISTED DNA WHICH THEN WAS CALLED SUPER COIL DNA WHEREAS THE FORM 2 WAS RELAXED, FULLY RELAXED DNA AND THEREFORE THERE WAS A NEED TO UNDERSTAND WHAT THE PROCESSES WERE TO GO FROM ONE TO THE NEXT FORM AND IN THE FIRST TOPOISOMBER ACE WAS ISOLATED FROM BACTERIA AND VERY SOON AFTER FROM MURINE TISSUE SO TO KEEP TRACK, I PUT SOME RETCHESES THAT ARE THE ONES THAT I WILL USE DURING THE PRESENTATION THAT IS WHY THEY ARE MINE BECAUSE THEY ARE REFERRED TO THIS SLIDE MY INTEREST HAS BEEN LONGSTANDING INTEREST IS TO TAKE ADVANTAGE OF THE TOPOICESOMER ACES FOR THERAPEUTIC PURPOSES AND DRUGGING THEM AND THIS IS PRETTY MUCH UPDATED AND MORE RECENTLY A TOOK ANOTHER CHALLENGE WHICH WAS TO WRITE A REVIEW WHICH IS JUST COME OUT SO IT’S NOT EVEN — THERE ARE NO PAGES YET BUT THAT WAS UNIVERSITY OF CHICAGO AND JOHNNY IS MORE EXPERT ON TOPO2 AND ON YEAST AND SO TOGETHER, WE WROTE THAT REVIEW, WHICH COVERS NOT THE BIOLOGY NOT SO MUCH THE DRUGS REALLY BECAUSE THIS WAS COVERED, BUT THE EMERGING NEW RICS MERGING ROLE OF TOPOISOMERASE IN TRANSCRIPTION, REPLICATION AND NOW VERY MUCH IN THE MIND OF ALL OF US IN GENOMICS STABILITY I’LL USE SOME OF THE SLIDES

IF YOU WANTED TO GO DEEPER, IS THERE A BOOK IN THE LIBRARY WHICH I PAINFULLY EDITED NOW BUT TWO 3 YEARS AGO I SWEAR I’M NEVER GOING TO EDIT ANOTHER BOOK BECAUSE IT STAY LOT OF WORK AND I’M NOT SURE MANY PEOPLE READ THE BOOK IT’S GREAT HAVE A BOOK BUT NOWADAYS EVERYBODY WANTS SOMETHING ON THE FLY BUT I LOVE BOOKS AND THIS IS ONE NICE TO HAVE IF YOU WANT TO LOOK AT IT THE CHAPTERS ARE ALL DIFFERENT ASPECTS OF TOPOISOMERASES IF YOU LIKE THAT SO LET’S GET INTO THE TOPIC SO NOT COUNTING SPO11, WHICH IS IN GERMLINE CELL, WHICH IS ESSENTIAL TO TO GENERATE DIVERSITY OF WHO WE ARE WHICH IS A TOPOISOMERASE PROTEIN SO NOT COUNTING THIS IN ANY OF YOUR CELLS, ESPECIALLY REPLICATING CELL, THERE ARE 3 TYPES OF ISOMBER ACES AND 6 TOPOISOMERASE GENES IN THE HUMAN AND THERE ARE THREE GROUPS THERE IS THE TYPE — WE CALL THEM 1, 2, 3 AND IT STAYS SIMPLE BECAUSE IN EACH GROUP THERE ARE TWO ENZYMES AND TWO GENES WHERE IT GETS A LITTLE MORE COMPLICATED IS THE FACT IF YOU LOOK AT THE TYPES, THERE ARE TWO TYPES TYPE I AND TYPE II, AND TYPES DEFINES THE FACT THAT THE ENZYME CLEAVES ONE STRAND AT THE TIME SO IT’S TYPE I, AND WHEN THEY CLEAVE TWO STRANDS AT A TIME, IT’S TYPE II NOT SO HARD TO REMEMBER AND THEN YOU GOT AN A HERE BECAUSE HUMANS WHEN THEY HAVE TYPE II A BUT IF YOU GO TO RK BACTERIA, THEY HAVE A LOT MORE TOPOISOMERASES THAN WE DO HAVE A DIFFERENT KIND IN HUMAN, THERE ARE ONLY TWO WAYS BUT HUMAN HAVE ONE A AND ONE B AND ONE A WAS NAMED BECAUSE IT WAS THE FIRST TYPE OF TOPOISOMERASE DISCOVERED IN E.COLI ANYWAY, SO TYPE I, TOPO1, IN THE TOPO1 ENZYME, I’LL GO BACK LATER ON THE DETAILS OF THIS THE ENZYME THIS IS DUPLEX DNA SO ENZYME CLEAVED THE BACKBONE AND THAT IS A WAY TO RELAX THE DNA OR DO WHATEVER NEEDS TO BE DONE TO CHANGE STRUCTURE OF DNA IN TOPO1, THERE ARE TWO ENZYME, TOP 1 AND TOP 1MT TWO DIFFERENT GENES AND CHROMOSOMES IN THE TOPO2, SO TOPO2 CLEAVED TWO STRANDS SO INSTEAD OF ONE BREAK ON ONE STRAND, YOU GET A BREAK ON BOTH STRANDS BUT EACH SIDE IS CLEAVED BY ONE HOMODIMER SO IT’S A HOMODIMER IN ALL CASES FOR THE TOPO2 AND THERE ARE TWO TYPES OF TOPO2 TOPO2 ALPHA AND TOPO2 BETA WE WILL COME BACK TO THAT THESE PROTEINS ARE RATHER BIG A SINGLE MONOMEROF TOPO2 IS 170 KILODALTON AS A DIMER IT’S TWICE THAT SO BICKER THAN A NUCLEOSOME AND THEN A TOPO3 CLEAVES ONLY ONE STRAND AS 2 GENES AND 2 ENZYMES, 3 ALPHA AND BETA WE WILL COME BACK AT THE END BECAUSE THIS IS THE EXPANDING FIELD IN THE TOPOISOMERACE ESPECIALLY THE LAST ONE IT HAS BEEN WORKED ON FOR MANY YEARS BUT IT’S COMING UP AS BEING VERY CRITICAL THE TRANSACTION FOR CHANGING DNA STRUCTURE GO THROUGH THE NIX OR BREAKS IN THE DNA, WHICH ARE CALLED CLEAVAGE COMPLEXES AND THE ENZYME MAKES THE BREAK BUT ALSO LIGATES BACK THE BREAK SO THIS NORMALLY VERY TRANSIENT AND THEN THERE ARE DRUGS THAT POISON THE BREAKS FOR TOPO1 AND TOPO2 THERE ARE DRUGS WE COME BACK TO THAT SO IF YOU LOOK AT THE BIGGER PICTURE NOW, IF YOU COMPARED HUMANS VERSUS E.COLI, SO IN HUMAN YOU GET THE TYPE I, WHICH IS TOP 3 ALPHA AND BETA, THESE ARE THEIR SIZE IN KILODALTON THE LINKAGE TO THE DNA WHICH IS COVALENT AND I’LL COME BACK TO THAT WHEN THEY DO THEIR TRANSACTION HOW MANY TIMES PAY PASS ONE STRAND AROUND THE OTHER? ONE TIME AT THE OTHER FOR THE TOPOTYPE I A AND TYPE I B, ABOUT SAME SIZE YOU SEE THE LINKAGE HERE, THE 3 PRIME END, THIS IS A VERY DIFFERENT FROM THE 1A AND 2A AND ONLY PASSES ONE STRAND AT A TIME AND THEN THE TWO A. YOU GET THE TWO ALPHA AND BETA LINKAGE 5 PRIME E.COLI ON THE OTHER HAND IS SIMPLER IN A WAY T DOESN’T HAVE 1B ONLY 1A AND IT HAS A TOPO1 IN

E.COLI AND TOPO3 E.COLI THAT’S WHERE IT GETS CONFUSING IT’S HISTORICAL ON THE TYPE II SIDE, ON THE OTHERP OTHER HAND, E.COLI IS MORE SOPHISTICATED IT GETS TWO ENZYMES AND TOPO4 EACH ENZYME IS MADE OF TWO GENES AND IT’S A TETRAMER SO ABSOLUTELY MORE COMPLEX BUT THE MECHANICS IS VERY MUCH THE SAME IN TERMS OF MEDICINE AND IMPACT ON THERAPEUTICS, YOU SEE ALL THESE ENZYMES, THE TOPO1 IS AN ANTI-CANCER TARGET OF DRUGS THAT ARE PRESCRIBED EVERY DAY THE 2S HAVE BEEN USED AS A TARGET BEFORE WE KNEW THAT DOXORUBICIN WAS TARGETING TOPO2 BUT THERE ARE VERY PROMISCUOUS DRUG IN CANCER TREATMENT DOCKSY RUBE SIN IS PRIMARILY USED AND TOPOSAID IS WIDELY USED AND USED IN CANCER AND IMMUNE DISORDERS AND A BIG CHURCHING OF TARGETING TOPOIS ON THE BACKSIDE GIANT RAYS AND TOPO4 ARE THE TARGET OF KWINN OHLONES IT’S A HUGE IMPACT IN BUO MEDICINE INCLUDING TB THOSE TARGET ONLY THE BACTERIAL AND THEY DO NOT TOUCH THE HOST TOPOISOMERASE GIVING THEM SELECT ACTIVITY THE DRUGS THAT TARGET THE HOST DO NOT TARGET THE BACTERIAL TOPO SO INSIGHT OF VERY SIMILAR STRUCTURES, THE DRUGS ARE VERY SELECTIVE SO DIFFERENCES BETWEEN TOP 1 AND TOP 2, YOU HAVE REGROUPING BECAUSE THEY HAVE THE SAME MECHANICS SO TOP 1S WORK AS MONOMERAND TOP 2S WORK ADD DIMERS OR TETRAMERS WHEN THEY MAKE THEIR TRANSACTION IN DNA, THE TOP 1 CLEAVES ONE STRAND AT A TIME AND LINKS THE TOW 3 PRIME END SO DRAWN HERE AS IT USES TYROSINE TO ATTACK DNA AND MAKES A COVALENT LINKAGE AND THEN A NICK IN THE DNA THIS IS THE OH AND THEN THIS CAN COME BACK AFTER THE DNA HAS BEEN RELAXED IF YOU HAVE A DRUG THAT GETS POISONED BY THE — [ INDISCERNIBLE ] AND OTHER DRUG ON THE TOPO2 SIDE, SO DIMER, LINKAGE TO THE 5 PRIME END AND 4 BASE PAIR STAGGER IS ALWAYS A 4-BASE PAIR STAGGER NOT LIKE A BLUNT DOUBLE STRAND BREAK LINKAGE TO THE 5 PRIME END, 3 PRIME HYDROXYL THE DRUGS ARE SPECIFIC TO TOPOTWO AND DOXORUBICIN AND TOPO4 KWINN LONES AND THE DIFFERENCES IS THAT TOPOONE IS A VERY ECONOMICAL ENZYME AND DOESN’T BURN OR NEED ATP TOPO2 NEEDS IT T REQUIRES MAGNESIUM TOPO1 WORKS EVEN ON ICE SO IT IS VERY EFFECTIVE TOPO2 DIFFERENT WORK AT ZERO DEGREES AND DRUGS ARE SPECIFIC FOR EACH TYPE OF ENZYME SO A GOOD DIVISION IN TERMS OF THE BIOCHEMISTRY AND THE PHARMACOLOGY FOR THESE TWO TYPES OF ENZYME IF YOU COMPARE THE MOST CHROMOSOME LOCATION SHOWING YOU THEY ARE INDEPENDENT IN THAT WAY AND THEIR NAME AND THEIR SIZE AND NOW PAY ATTENTION TO THIS BECAUSE OUR GENOME IS NOT JUST OUR NUCLEASE OUR GENOME IS THE MITOCHONDRIA AS WELL WHICH IS CALLED CHROMOSOME M AND CHROMOSOME M YOUR MOMMY CHROMOSOME, IS VERY ABUNDANT IN THE CELL AND TOPOISOMER IS — IN CASE OF TOPO1, THERE IS A SPECIFIC NUCLEAR ENZYME AND THEN MITOCHONDRIAL ENZYME, FOR TOP 2A AND B, THEY SERVE BOTH COMPARTMENTS AND CAN GO TO BOTH NUCLEUS AND MITOCHONDRIA AND THEY DO THEIR DEED ON THE CHROMOSOMES WHETHER IN THE

MITOCHONDRIA TOP THREE IS IN NUKE LAWS AND CYTOPLASM THE DRUGS YOU CAN SEE THAT SOME OF THE ENZYMES ARE STILL NOT BEING TARGETED AND WE DON’T HAVE ANY DRUGS FOR THE 3S AND THEN THE MECHANISM YOU UNDERSTAND BETTER AT THE END OF THE LECTURE BUT TOPO1 SWIVELS THE DNA AND MAKES ROW TAIN WHERE AT TOPO2 MAKE A STRAND PASSAGE WITH DOUBLE-STRANDED DNA AND THE TOPO3S MAKE A STRAND PASSAGE WITH SINGLE STRANDS AND THIS IS THE POLARITY, LINKAGE OF ENZYME AND THESE ARE THE REACTION LATER OW CAN GO BACK TO THIS ONE WHO HAS GONE THROUGH THE LECTURE SO THE WAY THE ENZYMES, WHAT THEY HAVE IN COMMON, IS THE WAY THEY BREAK DNA THEY ALL USE A TYROSINE AS THE NUCLEO FILE TO TAG THE PHOSPHOBACKBONE AND DEPENDING ON THE ENZYME IT HAS A DIFFERENT POLARITY OR GEOMETRY IN THE CASE OF TOPO1, THE GEOMETRY WILL LINK IT TO THE 3 PRIME END AND IN THE CASE OF TOPOTWO AND TOPO3, THE LINKAGE WILL BE THROUGH THE 5 PRIME END AND THIS SETS TOPO1 APART FROM ALL OTHER TOPOICE OMER ACES AND BRINGS IT TO THE FAMILY OF THE CRE RECOMINATE AND IT’S BELIEVED THAT THE TOP 1B EVOLVE FROM A RECOMBINASE USED FOR RELAXING — BUT MOST BASIC TOPOISOMERASES ARE THESE, THE 5 PRIME LINKAGES SO THIS IS GOING TO TALK ABOUT MORE DETAIL ABOUT TOP 1 AS I SAID THERE ARE TWO TOP 1 THERE IS NUCLEAR, WHICH IS THE ONE WE MOST COMMUNITY IS REFERRING TO WHEN WE SAY TOP 1 YOU HAVE TO UNDERSTAND THIS IS NUCLEAR TOP 1 AND WHEN WE DISCOVER TOP 1MT, WE MADE THE DECISION AT THE TIME NOT TO CHANGE — THIS REMAINED TOP 1 BUT JUST TO ADD M TO THAT ONE ONE TO SAY THIS IS MITOCHONDRIAL THIS DOESN’T GO THROUGH NUCLEUS THIS IS FOR CHROMOSOME M AND THIS IS FOR THE REST OF THE GENOME THE RELAXATION OF DNA BY TOPOISOMERASE WAS DISCOVERED EARLY ON WHEN RUBBING IN THE BROMIDE IF YOU TAKE THE DNA, USUAL AT THE RUNS FAST AND WHEN YOU PUT A DROP OF NUCLEAR EXTRACT YOU CAN DO IT IN YOUR LAB ON THIS DNA, IT WILL BE MIRACULOUS, YOU SEE YOUR DNA BECOMES RELAXED AND THE ENZYME DOING THAT WAS CALLED THE DNA TWISTING ENZYME BECAUSE THE DNA AND OVER TWISTED HERE SEE THE HELICAL STRUCTURE BUT IT’S OVERTWISTED AND THE TOPOISOMBER AS 1B WITHOUT ATP AND EVEN ON ICE, WILL RELAX IN DNA AND CONVERT IT TO A FULL CIRCLE NO BREAK FULL CIRCLE AND THE MANAGE SICK IN THE CLEAVAGE COMPLEX WHEN TYPO ISOMBER ACE SURROUNDS THE DNA, CLEAVES ONE STRAND AND THEN CONTROLS ROTATION OF BROKEN STRAND ANDCATHCULATE IN THIS BUILDING THE SPEED IS QUITE IMPRESSIVE 6000RPM SO IT IS ESSENTIAL AS IMAGINE FOR TRANSCRIPTION AND REPLICATION WHY? BECAUSE WHEN YOU TAKE A PIECE OF DNA, THE BINDING TO MOLECULAR MATRIX AND NEED TO TRIBE AND REPLICATE AND YOU WILL UNDERSTAND THIS IF YOU TAKE A ROPE AND THEN I HAVE ANOTHER ONE AND THEN ONE OF YOU IS GOING IN THE MIDDLE TO START TO SEPARATE THE STRANDS OF THE ROPE ON EACH SIDE YOU’LL GET SORT OF OVER WINDING AND UNDER WINDING ON EACH SIDE SO ONE SIDE YOU GET SUPER COALING AND ON THE NEGATIVE SIDE SUPER COALING IF YOU CAN NOT RELIEF THE TWO POSITIVE SUPER COALING, NO WAY YOU CAN GO BACK TO THIS BECAUSE IT WILL BE KNOTTED AND SO YOU NEED SOMETHING TO TAKE AWAY THE SUPER COIL THAT’S WHAT TOPO2 WILL DO WITHOUT MUCH EFFORT AND THIS IS ACHILLES HEEL OF TOP 1 NORMALLY IT’S VERY TRANSIENT BUT IF IT GETS STUCK THESE COULD BE EXTREMELY DANGEROUS AND THAT’S WHAT WE TAKE ADVANTAGE OF WAS ANTI-CANCER DRUG AND WE REVIEW

LATER THAT DNA DAMAGE CAN DO THAT IT CAN TRAP THE TOPO ISOMBER ACE 1 AND COMPLEX IS FORMED AS A WAY TO DESTROY CHROMATIN SO A FEW WORDS ABOUT SUPER COILING ALSO IN REVIEW IN THE CONTEXT OF CHROMATIN WHERE THE ROTATION OF DNA CAN STRAIN, DNA SUPER COILING UNDER TWISTING, IMAGINE OVER TWIST OR UNDER TWIST IF YOU GO IN A SENSE OF EXLICKS YOU OVER TWIST, YOU TIGHTEN LIKE YOU SQUEEZE THE WATER OUT OF SOMETHING AND THEN THE OTHER WAY YOU’LL JUST UNTWIST SO, ON THE RIGHT IS THE WHENEVER YOU HAVE MADE SO MUCH TWIST THAT THE STRANDS FLIP ON TOP OF THE OTHER SO TOP 1 AND MT REMOVES SUPER COILING BY DNA UP TWISTING THEY SWIVEL DID THE NA WHEREAS TOP 2 ALPHA AND BETA REMOVES — THERE A WORK AT THE CROSSOVER POINT AND YOU SEE WHY LATER AND SOME BASIC CONCERNING SUPER COILING SUPER COOLING TIGHTEN DNA AND NEGATIVE SUPER COILING OPENS HELIX AND THAT IS VERY IMPORTANT BECAUSE FOR ANYTHING TO GET INTO THE DNA YOU FIRST NEED TO OPEN IT A LITTLE BIT AND NEGATIVE SUPER COILING IS A FACT OF LIFE ABSORB AND RELEASES SUPER COILING SO YOU CONSTRAIN SUPER COILING WHEN YOU TAKE A NUCLEO SEEM AWAY YOU GET NEGATIVE SUPER COIL AND POLYMERASE GENERATE NEGATIVE POSITIVE SUPER COILING AHEAD AND TIME TO ACCUMULATE THE SUPER COILS AND NEGATIVE SUPER COILING BEHIND TOO MUCH POSITIVE SUPER COILING THAT WILL ARISE YOUR TRACKING ENZYME AND SUPPRESS TRANSCRIPTION, STABILIZE NUCLEOSOME NEGATIVE SUPER COILING FACILITATE MELTING OF THE STRAND AND THAT IS NECESSARY TO INITIATE REPLICATION AND TRANSCRIPTION IF YOU HAVE TOO MUCH NEGATIVE SUPER COILING, DNA CAN FLIP AND MAKE AN R LOOP, IT CAN MAKE A QUADROPLEX OR MAKE CDNA OR AT A VERY STRANGE STRUCTURE THAT ARE NOTED ALWAYS DESIRABLE Y SO NEEDS TO BE REGULATED SO THESE OTHER TWO TOP 01s, THEY ARE VERY SIMILAR IN THEIR BIOCHEMISTRY THE ONLY DIFFERENCE IN THE END TERMINIS IT HAS A VERY SHORT TARGETING SEQUENCE AND TOP 1 AS NUCLEAR LOCATION SIGNAL AND THE THING THAT TALKS TO PROTEINS, IT NEEDS TO BE DRIVEN TO WHERE IT HAS TO WORK IT CAN’T WORK EVERYWHERE IT WILL BE EXTREMELY DAMAGING IF IT WERE TO LET FREE THESE ARE PRIMITIVE IN — TOPO1B SUCH AS VACCINIA VIRUS AS OWN TOPO1B SO TOPO1S ARE SELECTIVELY TARGETED BY A CLASS OF DRUG ROUTINELY USED IN CANCER TREATMENT SO TOPOT CAN USED FOR OVARIAN CANCER AND SMALL CELL LUNG CANCER AND SECOND LINE, WHICH IS ALSO USED IN SOME PEDIATRIC CANCERS AND THEN BROADLY USED FOR COLON CANCER AND IN ASIA AND JAPAN AND OTHER INDICATIONS SUCH AS LUNG CANCER AS WELL SO BOTH OF THEM IN PEDIATRIC CANCER AND IN ASIA ALSO DRUGS AND THIS IS A NATURAL ON PRODUCT WHICH IS DERIVED FROM A TREE DISCOVERED AT THE NCI BY THE NCI AND THEN THE DRUG COMPANY JUST EVOLVED THIS TODAY SO THIS DRUG HAS BEEN APPROVED FOR 10 YEARS NOW SO THEY ARE NOT MOVING SO MUCH STILL VERY USEFUL THEY ARE NEW TOPO1 INHIBITORS BEING DEVELOPED 3 OF THEM ARE IN CLINICAL TRIAL HERE AT THE NCI WE MAKE THOSE DRUGS AND THEY ARE REFERRED TO NP744 AND 76 AND 400 AND IT JUST FINISHED PHASE 1 WE HAD ACTIVITY IN ONE PATIENT AND THOSE LIMITING TOXICITY WITH BONE MARROW NOBODY DIED THE QUESTION IS PHASE II OR NO PHASE II 766 IS FINISHING PHASE I NOW AND 744 WE BELIEVE IS ABOUT TO START PHASE I HERE SO THESE DRUGS, THE WAY THEY WORK IS QUITE REMARKABLE SO THIS IS A STRUCTURE AND TOP 01

REACTION TOPO1 FIRST DNA BY MAKING COVALENT LINKAGE 3 PRIME END NORMALLY DNA REALLIANCES AND THIS REVERSES VERY READILY THIS IS THEN IN THE BREAK SITE AND NO THE ANYTIME BRAKE SITE THAT’S HOW WE DISCOVERED OR HYPOTHESIZED THIS MECHANISM THEY WERE THE BRAKE SITES THAT TURNED INTO HAVE A 5 MINUTE MINUS ONE AND ONE PLUS ONE SO SUB SET OF THE TOPOONE CLEAVAGE SITE AR TRAPPED AS THE DRUG BINDS IN HERE SO WE PROPOSE THIS IN 1990 SOMETHING AND IT TOOK ABOUT 10 YEARS TO REALLY CONFIRM THIS THIS IS THE CRYSTAL STRUCTURE SO IN THE CRYSTAL STRUCTURE, THE DNA IS GREEN, THE BREAK IS HERE, THE DRUG IS THERE AND YOU CAN SEE EXACTLY LIKE HERE SO WHAT HAPPENS THE DRUG PREVENTS THE RELYINGATION OR LIGATION AND THEREFORE THE TOPOISOMERASE TRAPPED IT’S NOT COVALENT BUT IT’S TRAPPED FOR LONG ENOUGH THAT THE TOPOCAN’T GET AWAY AND THEN THAT LEADED TO COLLISION POLYMERASES AND DISASTER AND THEREFORE CANCER CELLS DIE THE NATURAL PRODUCT IT WAS FOR LONG TIME MYSTERRUOUS WIDE PLANT WHICH PRODUCED THE OTHER TREES AND WHY ARE THEY NOT KILLED BY THE TOX THANE THEY MAKE? AND INITIALLY WE THOUGHT IT WAS BECAUSE THE TOXIN WAS ONLY MADE IN THE BARK BUT THAT IS INCORRECT IT’S ALSO IN THE LEAVES AND WHAT WAS FOUND IS IN PRODUCING PLANTS, ENCODE A MUTATION ON THEIR TOPOISOMERASE 1 GENE, A SINGLE-POINT MUTATION AND THIS WAS REPORTED IN 2008 BY JAPANESE GROUP AND THE JAPANESE GROUP FOUND IN THE PLANT A MUTATION THAT WE HAD GENERATED OR SELECTED IN A HUMAN LEUKEMIA CELL LINE VERY RESISTANT WE HAD PUBLISHED THIS SOME YEARS EARLIER THAT TELLS YOU WHAT YOU DEFINE BY TARGETED THERAPY IS THAT IF YOU MUTATE THE TARGET AS ONE RESIDUE IT BECOMES IMMUNE TO THE DRUG THEREFORE THEY ARE ABSOLUTELY TARGETED AND THAT IS EACH MORE CLEAR IN THE YEAST BECAUSE YEAST YOU CAN DELETE TOPO1 AND YEAST CAN SURVIVE AT THIS STAGE, NOBODY HAS FOUND THAT HE COULD TARGET ANYTHING AND THE SELECT ACTIVITY IS DRIVEN BY THE FACT THEY JUST BIND INTO THE POCKET HERE INTO THE CLEAVAGE COMPLEX OF THE TOPOISOMBER ACE 1 AND THAT IS RESISTANT RESIDUE THERE NEXT TO THE DRUG BINDS AND INTERFERE WITH THE DRUG BINDING AND RENDERS ENZYME EFFICIENT AND DRUG RESISTANT WE HAD KNOWN FOR A LONG TIME THEY HAD LIMITATION YOU CAN CURE A MOUSE THERE WAS A PAPER IN SCIENCE IN THE OLD DAYS SHOWING YOU CAN REALLY CURE A MOUSE BUT YOU CAN NOT CURE A HUMAN BECAUSE YOU CAN NEVER GIVE ENOUGH DRUG BECAUSE THEY CAN’T PUT THIS IN CHEMICALLY UNSTABLE AND THE CHEMICAL STABILITY IS DUE TO THE FACT THAT THEY HAVE ALPHA HYDROXY — AND AS SOON AS — THESE AGENTS SO OTHER REASON TO

PUT THIS IN LIMITATION IS BECAUSE YOU DON’T WANT THE DRUG TO GO AWAY SO QUICKLY SO THAT IS WHY THESE DRUGS WERE DEVELOPED, THE ONE I MENTIONED BEFORE SO THESE ARE THE ADD NOWS THAT WE DEVELOPED AT THE NCI AND WHICH ARE IN PHASE I CLINICAL TRIAL THEY ARE HIGHLY POTEDDENT AND PURIFIED ENZYME AND ALL THE TEST HIGHLY SPECIFIC THEY ARE CHEMICALLY STABLE BECAUSE AS YOU CAN SEE FROM THE STRUCTURE, THIS IS ALPHA HYDROXY, AND OTHER DRUG RESISTANT SO THIS IS CLEAR AND ATTRACT A DIFFERENT SITE AND MORE STABLE SO THAT IS WHY THE CLINICAL TRIALS ARE GOING ON THERE IS ANOTHER CHANGE IN THE PARADIGM OF CLASSICAL CHEMOTHERAPY ONCE YOU FOUND A WARHEAD, WHAT NOW WE CAN REALLY START THINKING AND DOING IS TO DO TARGETED DELIVERY AND I’M A FIRM BELIEVE THER IS A NICE WAY TO GO YOU CAN TAKE A VERY TOXIC DRUG BUT NOT LETHAL AND MAKE SURE YOU CONCENTRATE IN THE TUMOR AND THIS HAS BEEN WORKED ON BY DRUG COMPANY YOU CAN SEE HERE 1, 2, 3, 4, 5 5 EXAMPLES OF ONGOING DEVELOPMENT OF WAR HED ASSOCIATED LIPOSOME TO PEG OR TO ANTIBODIES TO TARGET THESE DRUGS SELECTIVELY TO THE TUMOR AND MAYBE THEN YOU CAN DO WHAT WE — SO IN MICE, IF YOU INCREASE 10 TIME THE AMOUNT IN THE TUMOR, YOU PROBABLY GOING TO GET THE CURE WE SEE IN THE MICE SO I THINK A LOT OF CRIN CALL TRIALS NEED TO BE DONE TO LOOK AT THIS BECAUSE THEY MAY BE VERY HOPEFUL AND ONE WAS FDA APPROVED FOR PANCREATIC CANCER SO LET’S MOVE TO TOPO2 THERE ARE TWO THE ALPHA AND MAYBE BETTER NOW TO CALL THEM TUCKED AWAY BUT TIME WILL TELL OR TOPO2 BETA B THIS IS VERY SCHEMATIC BUT EASY FOR YOU TO REMEMBER TOP 2A IS REPLICATION ENZYME AND TOP 2B IS TRANSCRIPTION ENZYME ONE REASON TO KNOW THIS OR TO THINK THAT WAY IS THAT TOP TWO A. IS HIGHLY EXPRESSED IN REPLICATING AND CANCER CELLS AND IT’S NOT EXPRESSED IN NON-REPLICATING CELLS SO IF YOU TAKE YOUR HEART OR YOUR BRAIN, THERE IS NO TOPO2 WAY BUT IS THERE A LOT OF TOP 2BA IS DRIVING REPLICATION AND B IS THE HOUSEKEEPING TRANSCRIPTION ASSOCIATED ENZYME EXPRESSED IN REPLICATING AND DIFFERENTIATED CELL BETWEEN NEURONS AND MUSELS AND THIS YOU HAVE TOP 2B AND IT GOES BOTH TO THE NUCLEUS AND MIGHT ROW QUANDARYIA SO POLYPEPTIDES SHOWN HERE, THIS IS HUMAN TOP 2A AND TOP 2B AND WITH DOMAIN, THE CLEAVAGE RELYINGATION DOMAIN IS HERE, VERY SIMILAR YOU CAN SEE BUT THE CENTER IS HIGHLY CONSERVED IT’S HIGHLY CONSERVED WITH THE GYRATE AND TOPO4 — BINDING WITH A BUNCH OF RESIDUE AND THIS IS VERY CONSERVED WHEN YOU GO FROM HUMAN TO E.COLI STOW THAT IS THE WAY THEY WORK AND THEY WORK WELL THAT WAY SO WHAT DO THEY DO? HOW DO THEY DO THE TRICK? SO ONE WAY TO SEAL THE TRICK IS LIKE THIS YOU TAKE COMPLEXES OF DNA AND ONE IS THE GATE AND THE OTHER IS FOR T FOR TRANSPORT SO ONE IS A GATE AND THE OTHER GOES THROUGH YOU HAVE TO THINK OF THE NIH GATES BECAUSE IT WORKS THE SAME IT’S A TWO DOOR CAN’T GO ONE DOOR

SO FIRST GATE IS YOU’RE COMING TO THE ENTRANCE AND THIS IS CLOSED YOU GET INSIDE FIRST DOOR OPENS AND YOU GET IN THEN YOU’RE IN THE MIDDLE AND THEN YOU GOING THROUGH THIS OPENING ONCE YOU’RE IN IT, IT CLOSES BEHIND YOU AND THEN OPENS ON THE OTHER SIDE AND THEN IT CLOSES BEHIND YOU AND YOU HAVE GONE NOW YOU’RE INSIDE THE NIH SO THIS IS A TWO-GATE MECHANISM AND BURPS ATP AS IT DOES THAT WITH THIS MACHINE YOU CAN TAKE TWO CIRCLES, IF YOU HAVE A KNOT ON DNA, IT CAN UNKNOT IT BECAUSE IT WILL PASS THE STRANDS THROUGH THE OTHER IT CAN KNOT IT AND IT COULD RELAX BECAUSE YOU CAN IMAGINE THAT IF YOU RELAX SUPER COILING AND IT WILL DO THAT PRETTY WELL AND IT WILL RELAX POSITIVE AND NEGATIVE AND THE ENZYME THAT GENERATES NEGATIVE IS GIRAISE THE ONLY ENZYME, THE OTHER TOPORELAX THE SUPER COILING DO NOT GENERATE NEGATIVE 3 SO HOW DO THEY FUNCTION? HOW DO THEY INTEGRATE THE FUNCTION WITH THE BIOLOGY OF THE CELL? SO LET’S ASSUME YOU HAVE TRANSCRIPTION UNITED WHICH BARRIERS AND DNA HERE AND THE POLYMERASE IS THERE AND YOU HAVE THE RNA HERE SO AS THE DNA OPENS AND AS WE SAID, YOU HAVE TO GENERATE FIRST NEGATIVE SUPER COILING HERE NEGATIVE SUPER COILING WITH THE INITIATION OF POLYMERASE AND THE RNA WILL GET GOING AND THE POLYMERASE WILL MOVE BUT AS THE POLYMERASE MOVES AND I’M GOING TOWARDS — SUPER COILING ACCUMULATES TOPO1 WILL TAKE IT AWAY IN REGIONS WHERE NO NUCLEOSOMES, JUST USING THE SWIVELLING OF THE STRAND SO 2 AND 1 WILL TAKE AWAY THE POSITIVE SUPER COILS AHEAD OF THE FORK TOO MUCH NEGATIVE SUPER COIL WILLING GENERATE R LOOPS AND THIS WILL BE DONE BY TOPO1 AND 3 BETA WE COME BACK TO THAT THE PROMOTORS TO GET ACTIVATED RECRUIT TOPO2 BETA AND TOPO2 BETA IN THE PROMOTOR APPEARS TO MAKE TRANSIENT BREAKS NOT SO TRANSIENT THAT ENABLE INITIATION OF TRANSCRIPTION AND THAT IS WHERE TOPO2 BETA IS COMING BACK INTO FOCUS FOR NEURONS AND FOR OTHER SYNDROMES IN NEURONS OR AGAIN MUSCLE CELL, TAKE AWAY THE TWO ALPHA IT WILL BE SUFFICIENT AND WILL DO IT IF IT’S A CANCER CELL IT WILL USE BOTH TOPO2 ALPHA AND BETA TO DO THE TRICK IF YOU THINK OF REPLICATION AND ROLES THAT ARE VERY SPECIFIC IF YOU TAKE THE REPLICATION AND FIRST THOUGHT WAS THE INITIATION, AGAIN THE INITIATION OF REPLICATION NECESSITATED MELTING TO INITIATE THESE FRAGMENTS OF DNA AND WHEN YOU DO THAT, WILL YOU GENERATE NEGATIVE SUPER COILING BUT YOU NEED THAT NEGATIVE SUPER COILING THEREFORE IF YOU HAVE TOO MUCH TOPO1, YOU CAN NOT INITIATE REPLICATION AS YOU INITIATE THIS, GENERATE SUPER COALING IN THE FLACK AND YOU HAVE TO DISSIPATE THE SUPER COILING FOR THE FOLKS TO MOVE SO AFTER INITIATION WHEN THE HELICASE IS MOVING ALONG, WHEN YOU GENERATE THE SUPER COILING, IN FRONT OF THE REPLICATION FORT, THE TRANSCRIPTION TOPO1 WILL TAKE AWAY THE SUPER COILING AND NUCLEOSOME-FREE REGION AND TOPO2 WILL TAKE AWAY ALPHA IN THAT CASE MOSTLY IN THE CROSSOVER AROUND THE NUCLEOSOME VERY EFFICIENTLY

SO BEHIND THE REPLICATION FORK, YOU WILL GENERATE NEGATIVE SUPER COILING THAT COULD BE DISSIPATED BY TOPO1, TOP 2 ALPHA AND TOP 3 ALPHA AND THEN YOU CAN FORM — WHEN THE FORCE CONVERGING, YOU HAVE EXSAYS OF POSITIVE SUPER COILING AND IF YOU WOULD NOT TAKE AWAY THE SUPER COILING, YOU COULD NEVER GO ALL THE WAY SO THE TOPOICER MERWILL TAKE AWAY THE SUPER COALING AND TOPO1 AND 2 WHEN ALL IS FINISHED AND EVERYTHING HAS BEEN REPLICATED, YOU END UP WITH TOPO2 BY CLEAVING TWO STRAND AT A TIME WILL THEN DECAT ON 8 AND IN YEAST, IF YOU TAKE A TEMP SENSITIVE MUTE APT, IT WILL DIE BECAUSE IT CANNOT DECATENATE THE CHROMOSOME THE DRUGS FOR TOPO2, THERE ARE LOTS OF THEM TOPOAND TOX I RUBE SIN AND THEN YOU COULD SEE ALL THESE DRUG LOOKS SIMILAR AND DIFFERENT IN COMMON THEY HAVE THIS AROMATIC POLYCYCLIC THING AND SORT OF SIDE CHAIN BUT THEY REALLY LOOK DIFFERENT AND NOW IF YOU TAKE THE ANTIBIOTICS VERY SPECIFIC TO GIRAISE, AND TOPO4 BUT NOT THE HUMAN ENZYME, YOU CAN SEE THEY LOOK DIFFERENT AND THESE ARE ALL THE KWINN OHLONE THAT IS YOU HAVE ENCOUNTERED THESE ARE SPECIFIC FOR BACTERIAL ENZYME AND THOSE ARE SPECIFIC FOR CANCERS BUT NOT THE BACTERIA WHAT IS IN COMMON? WHAT IS IN COMMON IS INTERESTING IF YOU TAKE FOR EXAMPLE HERE TOPOSITE, WHAT IS CLEAR IS THE DRUG WORKING EXACTLY LIKE THE TOPO1 INHIBITOR THE STRUCTURE OF THE TOPOISOMER IS TO CLEAVAGE COMPLEX AND A TOPOSITE REVEAL THAT THE DRUG IN THE CLEAVAGE SITE LIKE THE BINDING NEW CLEAVE ANNUAL SITE OF THE TOPO1 CLEAVAGE COMPLEX AND ONE DRUG IN EACH OF THE TWO CLEAVAGE SITES YOU CAN SEE VERY INTRICATE GEOMETRY AND THE DRUG, THE PLANTERRING HERE TENDS TO CIRCULATE BETWEEN THE BASE PARIS AND THE SIDE CHAINS TEND TO REACH TO CONFER SELECT ACTIVITY SO HIGHLY SPECIFIC FOR ALPHA AND BETA THEY DON’T TOUCH TOPO1 AND IT IS BECAUSE THEY HAVE ALL THE INTRICATE INTERACTIONS AND THE ANTIBACTERIAL IS THE EXACTLY SAME PRINCIPLE IF YOU TAKE THE STRUCTURE RADIO – THIS IS A DIMER AND TOP 04 VERY SIMILAR MACHINE AND THE DRUG AGAIN IN THE CLEAVE ANNUAL SITE WITH THIS AGAINST THE BASE AND THEN HYDROGEN BOMBS TO SPECIFIC RESIDUES TO THE BACTERIAL TOPOISOMER AND THAT LED TO ONE CONSENT WHICH IS THAT THESE DRUGS MADE US REALIZE THAT NATURE DEVELOP A PARADIGM TO POISON THESE DNA ENZYMES TO BIND THE DRUGS AT THE INTERFACE OF THE ENZYME AND DNA SO THAT CAME OUT AND THIS IS WHAT WAS REFERRED TO AS THE INHIBITION PRINCIPLE VERY PROWL BECAUSE YOU CAN TARGET MICROMOLECULAR COMPLEXES YOU USE INTERFACES AS THESE THINGS MOVE AROUND AND THEN JAM THEM SO THAT’S WHAT — IF YOU LOOK AT THEM IN COMPARISON, YOU HAVE A SINGLE STRAND BREAK AND YOU STACK AGAINST THE BREAK WITH THE — AND TH EN HAVE HYDROGEN BOMB AND THEN TOPO2, YOU STACK AND HAVE DIFFERENT HYDROGEN BOMB SPECIFIC FOR THE PARTICULAR ENZYME THESE DRUGS DO NOT WORK AT PICO MOLAR THIS DRUG WORKS NOT VERY HIGH AFFINITY BUT HIGHLY SPECIFIC THEY WORK AT MICROMOLAR SO NATURE DOESN’T NEED TO REALLY GET PICO MOLAR WHEN IT HAS SUCH SELECT ACTIVITY SO TOPO3

WE CAN BE CONFIDENT IS THERE A CLEAR DIVISION OF LABOR TOP 3 IS REPLICATION AND IT’S A DNA TYPO ISOMBER ACE AND WILL WORK ON SINGLE STRAPPEDDED REGION BUT IT NEEDS TO HAVE DUPLEX DNA TO ENGAGE NOT ENGAGE ON SINGLE STRANDED DNA IT RESOLVES SOMETHING WHERE ONLY ONE IF I COULD SPLIT MY ARM, ONLY HALFWAY IS GOING THROUGH, NOT MY WHOLE THING AND THEN YOU CAN CUT ONE STRAND AND IT WILL RESULT AND PREVENTS RECOMBINATION BECAUSE AT THE OTHER REPLICATION — TOP 3 BETA IS THE HOTTEST NOW THE ONE WHERE I THINK WE LEARN THE MOST IT IS HIGHLY INVOLVED IN TRANSCRIPTION IT’S A DNA TYPO ISOMBER ACE RESOLVING R LOOPS AND ALSO RNA TOPOISOMER I DIDN’T TALK TOO MUCH ABOUT THAT SHOW HOW DOES IT WORK? IF YOU TAKE A REPLICATING CIRCLE WHEN YOU FINISH REPLICATION YOU HAVE THIS KATE ONIATED REGION WHICH CAN BE RESOLVED IF YOU HAVE TOPO2 BY JUST CUTTING ACROSS TWO STRANDS AT A TIME AND END UP WITH THE PRODUCT IF YOU DON’T DO THIS PERFECTLY YOU WILL END UP WITH SINGLE STRANDED REGION AND THESIS ARE HIGHLY RECOMGENIC SO IF IT DOESN’T DO THE JOB FULLY YOU NEED DO THE JOB BY TOPO3A AND IT WORKS WITH BLOOM SYNDROME PROTEIN OR WITH VERNER WITH THE — SO THIS IS TOP 3A AND THEN THE NEWCOMER IS THE 3 BETA AND OF AND THAT CAME TWO YEARS AGO, 3 YEARS AGO WHERE IT WAS FOUND FOR ONE NOBODY TALKED TOO MUCH ABOUT 3 BETA BUT TWO PAPERS CAME OUT IN NATURE AND NEUROSCIENCE AND THIS LIFTED THE WHOLE THING AND THERE IS A REGION OF PEOPLE WHO HAVE VERY HIGH FREQUENCY OF SCHIZOPHRENIA AND MENTAL RETARDATION A LOT OF THESE PEOPLE THERE AND WHEN THEY LOOKED AT THEIR GENOME AND LOOKED FOR WHAT WAS WRONG WITH THESE FAMILIES, THEY FOUND THERE WAS A DELETION IN THE TOPO3 BETA LOCUS AND THAT WAS A SURPRISE WHY WOULD TOPOTHREE BETA SO IMPORTANT FOR THAT? AND THEN AT THE SAME TIME, THE INSTITUTE OF AGING IN BALTIMORE WORKING ON TOPO3 BETA FORMED THAT IT WAS RNA TOPOISOMERASE SO THE IDEA IS THAT RNA IS SUSCEPTIBLE TO MAKE KNOTS AND TO BE UNTANGLED ESPECIALLY DURING TRANSLATION AND TOPO3 BETA IS IN THE CYTOPLASM AND ESSENTIAL TO RESOLVE RNA PROBLEMS AND IN NEURONS WHERE YOU HAVE VERY LONG GENES, THAT TENDS TO HAPPEN OR IF YOU HAVE R LOOPS TOPO3 BETA WILL TEP TO DISSOLVE THEM AND THERE ARE FACILITATE TRANSCRIPTION BUT IF YOU DON’T HAVE TRANSCRIPTION IN THE NEURON, THAT SHOWS IMMEDIATELY BECAUSE OF HIGH LEVEL OF SOPHISTICATION SO YOU END UP BEING SCHIZOPHRENIC OR MENTAL RETARDED IF SOME OF YOUR NEURONS DON’T FUNCTION RIGHT SO THE DIVISION OF LABOR BETWEEN THE TWO TOPO3s IS THAT THEY WORK OR DRIVEN TO THEIR SIGHT OF ACTION BY A DRIVER IN THE CASE OF TOPO3 ALPHA, IT IS RMI1 ALSO DISCOVERED BY IN BALTIMORE AND RMI1, TOGETHER WITH BLOOM AND GAUGE TOPO3 ALPHA IN REPLICATION IN TERMEDIATES AND RESOLVE THE JUNCTIONS AND THEREFORE IT IS CRITICAL IN TRANSCRIPTION, THE PARTNER IS TDR3 RNA BINDING PROTEIN AND THEN ENGAGES TOPO3 BETA ON THE RNA AND ON R LOOPS WITH THE FRAGILE X COMPLEX AS WELL SO THIS IS IT EMERGING AS BEING VERY IMPORTANT AND THAT TOPO3 BETA IS MUCH MORE IMPORTANT THAT WE HAD ANTICIPATED BEFORE AND THAT LEADS ME TO WHAT I WANT TO FINISH WITH IS THE TOPOISOMERASE AND GENOMIC

INTEGRITY AND HUMAN DISEASES WHICH IS SOMETHING THAT IS REALLY EMERGING NOW SO NOT ONLY DRUGS BUT DNA ALTERATION AND PHYSICAL LONG CALL PROCESSES LEAD TO THE PERSISTENCE OF COMPLEXES AND FOR ONE ENZYME, TOP 1 AND 2, WE HAVE MANY EXAMPLES WHERE PERSISTENCE LEAD TO GENOMIC DAMAGE SO I’M NOT GOING TO SAY ANYTHING ABOUT THE ANTI-CANCER DRUG BECAUSE WE HAVE COVERED IT AND WE US TO THERAPEUTICALLY BUT IF THE DNA IS VERY COMMON, THAT POISONS TOPO1 AND 2 AND BOTH IN THE NUCLEUS AND IN THE MITOCHONDRIA YOU HAVE 86 SITES SAME THING TOPO1 GETS SLUGGISH AND WILL GET COLD TOBACCO PRODUCTS EN USE AND TRAP THE COMPLEXES BOTH TOPO1 AND 2 NUCLEOTIDES ARE COMING BACK AND I’LL SHOW YOU SOME EXAMPLES WHY WOULD IT BE SO BAD? LET’S SAY YOU HAVE A TOPO1 CLEAVAGE COMPLEX WHICH NORMAL IS VERY REVERSIBLE AND NEEDS TO GO ON AND OFF AND VERY QUICKLY SO NORMALLY IT’S COUPLED WITH REPLICATION AND REPLICATION FOLLOWS BUT IF THIS IS TALK THE REPLICATION CAN COLLIDE AND THAT WILL GENERATE WHAT WE CALL REPLICATION RUNOFF T JUST COLLIDED AND NOW TOPO1 CANNOT LIE GADE ANYWHERE AND THAT LEADS TO GENOMIC DAMAGE AND I’LL TELL YOU AT THE END, HOW THIS IS RESOLVED, OR, ANOTHER TRICK IS THAT THEY COLLIDE BUT IT CAN REVERSE WE COULD TAKE ONE OR THIS ONE AND THIS IS A TOPO1 CLEAVE ANNUAL SITE WHICH ARE VERY FREQUENT AND IN THE GENOME MANY, MANY DNA BOUND LET’S ASSUME YOU HAVE A NICK TOPO1 IS OBLIVIOUS TO THE PRESENCE T WILL STILL CLEAVE HERE IF YOU HAVE QUICKED HERE AND TOPO1 MAKING HERE, THEN THIS MAKES DOUBLE STRAND BREAK AND THE DOUBLE STRAND BREAK AND DNA COMES APART SO WHAT HAVE YOU DONE IS SINGLE TOPOISOMERHAS CONVERTED INTO A DOUBLE STRAND BREAK AND ANOTHER THING LIKE THIS YOU IF HAVE A NICK ON THE SAME STRAND, TOPO1 CLEAVES UP STREAM AND THAT COULD FLY-OFF BECAUSE IT’S NOT HELD BY TOPO1 NOW SOUBED LOOK THERE AND THE MOST RECENT, STILL A REALLY ONGOING WORK IN OUR LAB IS THE INTERESTS THAT WE THINK OF THE GENOME BEING ALL DNA AND BUT WHAT IS COMING SUPTHERE IS A LOT OF RNA THAT GETS INCORPORATED INTO THE GENOME ON PURPOSE OR BY ACCIDENT AND THIS IS VERY FREQUENT IT’S BEEN ESTIMATED IN YEAST FOR INSTANCE THEY COULD BE 1-1,000 AND IN YEAST THIS RIBONUCLEOTIDE IS REMOVED BY RNAH2 IS ESSENTIAL SO IF YOU HAVE PATIENT YOU WILL GET A LOT OF THIS HOW BAD IS THIS? PRETTY BAD BECAUSE YOU HAVE THE POLYMERASE THAT GETS STUCK AND WHAT IS WORSE IS IF TOPO1 COMES AND HERE BEFORE RNA2, IT WILL IMMEDIATELY CONVERT THIS INTO A NICK AND TOPO1 BINDS TO THE END HERE OF THE DNA AND BECAUSE IT IS A RIBOWITH A TWO PRIME HYDROXYL, THEN ATTACKS THE FIRST AGAIN AND THEN LIBERATES THE TOPO1 BUT GENERATES A TWO PRIME, 3 PRIME CYCLIC WITH A NICK SO WHAT TOPO1 DOES IS ACTS AS RIBONUCLEASE AND THAT COULD BE PRETTY BAD AND BEEN SHOWN IN YEAST NOW THAT IF THAT IS GENERATED A SECOND TOPO1 SITE COULD BE FORMED RIGHT UP STREAM THAT IS WHAT I SHOWED YOU RIGHT BEFORE AND WHEN THIS FORMS, THIS COULD THEN DISASSOCIATE AND TOPO1 WILL RELY GATE ACROSS AND THIS WILL FORM TWO-BASE DELETION AND IN

REPEATED SEQUENCES, YOU END UP WITH THIS DELETION COULD BE REPAIRED BUT THAT’S A PROBLEM THE OTHER ONE WHICH I ALLUDED TO IS IF YOU HAVE MADE A NICK ON ONE STRAND, AND RELEASED TOPO TOPOCOULD JUST JUMP ACROSS AND GO TO THE OPPOSITE STRAND AND IF IT FINDS A SITE THERE IT WILL CONVERT INTO A DOUBLE STRAND BREAK SO A RIBOIN DNA CAN GENERATE A DOUBLE STRAND BREAK JUST WITH TOPO1 AND WE COULD DO THAT BIOCHEMICALLY SO WHAT ARE THE HUMAN DISEASES LINKED WITH TOPOISOMER ACES SO FAR? THE LIST MIGHT BE EXPANDING IN THE CASE OF TOPO1, WE ARE LOOKING AT NEUROLOGICAL DISEASES DUE TO THE LACK OF REMOVAL OF TOPO1 CLEAVAGE COMPLEXES, AND I’LL GIVE YOU A LITTLE KEY AT THE END OF WHY THIS IS TOPO2B, WHEN TOPO2 CLEAVES DNA, IT NEEDS RETORELY GATE IN THE PROMOTOR IF IT DOESN’T RELY GATE QUICKLY, ANOTHER PIECE OF DNA CAN DO A TRANSLOCATION AND THE CAUSAL IMPLICATION OF TOPO2 BETA IS NOW PRETTY WELL ESTABLISHED AS IA SOURCE OF LEUKEMIA, SECONDARY LEUKEMIA AND PRIMARY, AND IN TRANSLOCATION SITE IN PROSTAT CANCER WHEN THE ANDROGEN RESPONSIVE GENES A TOPO2 BATA SALE IN THE PROMOTOR AND KNACKED LEAD TO RECOMBINATION IN THE CASE OF 3 BETA, AS INDICATED EARLIER, THE LACK OF 3 BETA LEADS TO NEURODEVELOPMENTAL DISORDERS SCHIZOPHRENIA AND COGNITIVE IMPAIRMENT AND THEN, IF YOU HAVE A LACK OF REPAIR OF THESE CLEAVE ANNUAL COMPLEXES, YOU END UP WITH THIS TYPE OF DISEASE NEUROLOGICAL DISEASE IN THE CASE OF THAT ENZYME AND WILL — [ READING ] IF YOU CAN NOT REMOVE THE TOPOCLEAVE ANNUAL COMPLEXES, I’LL SHOW YOU HOW THEY DO IT, SO THE WAY THE TOPOISOMER IS REPAIRED AND BECAUSE OF THE DANGER, ALL SPECIES GOING ALL WAIT THROUGH YEAST HAVE BATTERY OF EP ZYMES TO REMOVE TOPOCOMPLEXES AND SO WHEN THEY ARE EFFICIENT, THAT’ST THAT’S WHEN YOU END UP WITH DISEASES HOW DO THEY DO IT? YOU CAN LOOK AT THE REVIEW OR FIND A LITTLE SCHEME THAT IS SAYING, SO IF YOU HAVE A TOPOISOMER, IT IS LINKED TO THE 3 PRIME END OF KNA HERE AND THIS IS THE DNA THERE IS A SPECIFIC ENZYME LINKED TO THE 3 PRIME END AND DNA THE ENZYME WILL CLEAVE IN THE MIDDLE HERE AND IT REMOVES TOPOONE AT THE SITE OF LINKAGE AND REGENERATES 3 PRIME PHOSPHATE IN THE CASE OF TOPO2, A SPECIFIC ENZYME IN HUMAN AND VERTEBRATES AND YEAST SAME ENZYME BUT IN HUMANS YOU GET A TDP TWO ENZYME SPECIAL AND SEE THE POE LATERY IS THAT THE TOPOTWO IS LINKED TO THE 5 PRIME END SO TDP2 WILL AGAIN REMOVES THE TOPO2 AND REGENERATE THE 5 PRIME FIRST FATE CELL HAVE A BACKUP AND ALTERNATIVE PATHWAY TO CHOP THE PIECE OF THE DNA A LITTLE BIT OF STREAM OF THE TOPOISOMBER ACE LINKAGE BY NIKE LACES AND THESE NUCLEASES ARE VERY COMMONLY KNOWN LIKE CTIP THAT COU3 KNOWN LIKE CTIP THAT COULD CLEAVE THESE AND THEN GAP REPAIR SO THE NCI DIRECTOR FOR TRANSITION RESEARCH IS DOING

CLINICAL TRIALS WITH HIM AND THEN THE DEVELOPMENTAL THERAPEUTICS PROGRAM HERE WERE VERY CONNECTED TO THEM SO IF YOU HAVE ANY QUESTION, NOW I AM HAPPY TO TAKE THEM [ APPLAUSE ] [ OFF MIC ] >> IT IS HARD TO TELL WHERE THE HETEROZYGOTES HAVE MINOR FORMS OF THAT AND IT’S VERY NEW IT WAS FORMED IN A PATIENT THAT HAS SYNTHETIC DISABILITY SEIZURES AND FOUND TO START TO SCRATCH THE TIP OF THE ICEBERG I THINK YOU ARE ALL BURNED-OUT? YOU CAN LOOK AT SLIDES AND SEND 01:51:08.328,00:00:00.000 ME E-MAIL IF YOU HAVE ANY

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