TRACO 2018 – Topoisomerases and Precision medicine

OUR FIRST SPEAKER IS THE CHIEF OF THE DEVELOPMENT CENTER THERAPEUTIC BRANCH. HE GOT HIS MD AND PH DEGREES FROM THE UNIVERSITY OF PARIS. HE HAS BEEN AT NIH SINCE 1981 AND HE’S CO-CHAIR OF THE DISCOVERY COMMITTEE FOR THE NIH EXPERIMENTAL THERAPEUTICS PROGRAM AND A MEMBER OF THE NCI DEVELOPMENTAL THERAPEUTICS PROGRAM AND HE’S IN CANCER RESEARCH AND HE RECEIVED NUMEROUS AWARDS SUCH AS THE PAUL EHRLICH AWARD. AND HIS TITLE TODAY IS, TOPOSOMBER ACES. >> THANK YOU. SO I DON’T THINK THIS IS WHAT IS GOING TO HAPPEN IF YOU. YOU’RE NOT JUST GOING TO RELAX AND WIND TOTALLY. THAT’S WHAT THEY DO. BECAUSE IN THE GENOME, YOU HAVE A LOT OF PROCESSING ENZYMES. THE GENOME NEEDS TO OPEN TO BE TRANSCRIBED AND BE REPLICATED, AND THIS IS DONE BY HELICASES. BUT WHEN YOU SEPARATE THE DNA STRANDS, AS A CONSEQUENCE, YOU ACTUALLY OVERTIGHTEN THE DNA HELIXES AND NOW THAT NEEDS TO BE RELAXED BY THE TOPOISOMERASES. THERE ARE A NUMBER OF REVIEWS THAT YOU COULD READ. THERE ARE OTHERS, OBVIOUSLY. YOU COULD GO BACK AND MANY OF THE SLIDES THAT I TOOK ARE FROM THESE REVIEWS, SO YOU COULD JUST GO BACK TO THE REVIEWS IF YOU WISH TO DO SO. THIS ONE IN 2010 WAS MOSTLY ON THE BIOLOGY, PROBABLY COULD BE UPDATED NOW BUT THE DRUGS REMAINED THE SAME. BOTH WERE CANCE CANS DISEASE. — CANCER DISEASE. THERE WAS AN UPDATE THREE YEARS LATER IN DRUGGING TOPOISOMERASES AND WHAT WE COULD DO BETTER AND WHAT WE COULD LEARN FROM THAT. AND THE MOST RECENT REVIEW WITH JOHN AND I, AND WE WORKED ON THIS FOR MANY, MANY YEARS, JOHN ON TOPO2 AND MYSELF ON TOPO1, AND HE IS A YOUNGER PERSON. BOTH ARE IN MY LAB AT THIS TIME. SO IF YOU WANTED TO TALK TOPOSOMBER ACES, YOU COULD COME TO BUILD 37. AND THERE IS ALSO A BOOK IN THE LIBRARY. SO I’M PROUD OF HAVING BEEN THE EDITOR OF ONE BOOK BUT HAVING DONE ONE, I WILL NOT DO A SECOND, BECAUSE IT’S A LOT OF WORK. [ LAUGHS ] BUT I WAS VERY PLEASED BECAUSE MY COLLEAGUES ALL SAID YES, INITIALLY. SO I GOT ALL MY CHAPTERS. FIVE YEARS FROM NOW WHEN I HAVE FORGOTTEN HOW PAINFUL IT WAS BUT IN THE END IT’S NICE TO HAVE SOMETHING SOMEWHERE WITH ALL THE PEOPLE AND ALL THE KNOWLEDGE. SO WHAT ARE THE PROBLEMS AND WHAT ARE THE ENZYMES? THE PROBLEM OF DNA IS DNA IS DOUBLE HELICAL. IT’S A DOUBLE STRAND. AND IT’S NOT AS FLEXIBLE AS ONE WOULD WISH. IT’S LIKE EVEN THAT CABLE, AND YOU CAN’T TURP IT AROUND. IT’S STIFF. SO WHAT HAPPENS IS IT MAKES SUPER CORD, LIKE YOUR BATTERY CHARMER OR YOUR iPHONE WIRE, YOU TAKE IT OUT OF THE BAG AND YOU WILL GET SUPER COILS AND GET THINGS LIKE THAT. SO ONE OF THE SOLUTIONS TO THESE SUPER COILS THAT YOU OVERWIND OR UNDERWIND THE DN A, ESPECIALLY WHEN IT’S OVERWOUND, AND IT’S NEGATIVE. WHEN IT’S UNDERWOUND, THE TOPOSOMBER ACES WILL ADJUST THAT AND ALL WILL DO IT. SO THERE ARE SIX. AND ALL OF THEM WILL DEAL WITH THAT RELAXATION PROBLEM, THAT IS PROBABLY WHY THE FIRST SLIDE IS APPROPRIATE. THEY ALL RELAX DNA. BUT SOME OF THE TOPODO MORE. AND TOPO2s, THERE ARE TWO, CAN DECAPINATE. SO WHEN WE HAVE RINGS THAT ARE INTERTWINED LIKE THIS, TOPO2 CAN OPEN ONE AND THEN DO THIS AND LET IT GO AND IT COULD DO THE OPPOSITE. SO IT’S CALLED CAT ONATION, FOR CIRCLE. AT THE END OF REPLICATION, DNA IS CAT ONIATED. SO IT NEEDS TO BE DECAT ON A LITTLED. TOPO2 IS ESSENTIAL FOR THAT. BUT IT MAKES KNOTS. SO WITHIN A SINGLE MOLECULE IT COULD BE KNOTTED AND THE TOPO2, BECAUSE IT WILL UNKNOT THE DNA AND COULD KNOT. SO IN ADDITION, TOPO2 CAN DO MORE. IT CAN DECAT ON 8, NOT, AND KNOT. AND THEN THERE IS TOPO3s THAT CAN RESOLVE THESE WHAT WE CALL JUNCTIONS WHICH HAPPEN DURING REPLICATION. YOU CAN HAVE A RECOMBINATION. AND THESE ARE AS A RESULT OF TOPO3 ALPHA. AND IN 2013, TOPO3 ALPHA WHICH WAS BEHIND IN THE FIELD WAS FIRST DESCRIBED IN 1990. NOT VERY MANY PEOPLE WORKED ON IT BUT I WILL DESCRIBE A LITTLE MORE LATER IN 2013. IT WAS FOUND TO BE AN RNA TOPOSOMBER ACE. SO IT IS NOT ONLY DEALING WITH DNA BUT ALSO RNA. TOPO3 BETA WILL DO THAT. SO I WILL FIRST INTRODUCE THE TOPOs. THEY ARE THROUGHOUT ALL THE ORGANISMS THAT HAVE DNA. I HAVE TOPOISOMERASES SO IF YOU TAKE EXAMPLE, THE HUMANS, SO IN HUMANS, THERE ARE SIX TOPOS AND THEY ARE DIVIDED IN THREE GROUPS AND THE NUMBERING SYSTEM IS BASED ON HISTORICAL LIKE THE NIH BUILDING ONE WAS THE FIRST BUILDING AND THEN BUILDING 50 WAS BUILT LATER. FIRST TOPO2 AND THEN TOPO3. BUT THEY ARE STILL GROUPED IN TWO MAIN GROUPS. THE TYPE I AND TYPE II. AND TO MAKE IT SIMPLE, TYPE I IS ONE STRAND OF DNA. TYPE II IS TWO STRANDS. SO IT MAKES IT QUITE EASY TO REMEMBER. TYPE I, ONE STRAND, AND TYPE II, TWO STRANDS. SO IN HUMANS, SO IN HUMANS, THERE ARE 4 TYPE IS AND THEN TWO TYPE IIS, TOPO12 AND BETA. IN ECOILY, THEY ARE TWO TYPE Is, TOPO1 AND TOPO3. SO ONE STRAND DNA AT A TIME AND YOU HAVE MORE TYPE IIs BECAUSE YOU HAVE GIRAISE AND YOU HAVE TOPO4. BUT IN E.COLI, THERE IS NO TOP 1B. SO WE DON’T UNDERSTAND AT THE END WHAT THERE IS FOR THE DIFFERENCE. WHAT IS INTERESTING FOR MEDICINE, AND IN ADDITION TO BIOLOGY, THESE ENZYMES ARE TARGETS OF VERY WIDELY AIDS AGENTS FOR CANCER AND INFECTIOUS DISEASE. TOP 2 IS TARGET OF ANTI-CANCER AGENT WIDELY USED AND TOPO4 IN BACTERIA IS THE TARGET OF BACTERIA. THESE ARE KWINNA LONES. THEY TARGET THE TOPO2s IN THE BACTER. >>> BUT NOT IN THE HOST SO THEY ARE SPECIFIC IN BACTERIAL DRUGS. SO IN HUMAN GOING BACK, YOU GET SIX ENZYMES DIVIDED IN THREE GROUPS. AND THE DIFFERENCE BETWEEN EACH OF THESE ENZYMES IS THE WAY THEY DEAL WITH DNA. SO I TOLD YOU TYPE I IS ONE STRAND OF DNA AT A TIME. TYPE II, TWO STRANDS. SO CLEAVAGE TO MAKE STAGGERED CUT, FOUR BASE PAIRS STAGGER, BUT ONE ON EACH STRAND IS HOMODIMER, AND THE ENZYME THEN CLEAVES THE DNA THAT WAY. THE INHIBITORS BLOCK THE ENZYME BY BINDING AT THE DNA INTERFACE. WE’LL COME BACK TO THAT. AND IN THE TYPE I, THERE ARE TWO TYPES DEPENDING ON WHERE THE TOPOCLEAVES THE DNA AND ATTACHES TO THE DNA. SO THE TYPE I A CLEAVES DNA BY ATTACHING TO THE 5-PRIME END. LIKE THE TOPO2, IT’S ATTACHED TO THE 5 PRIME END. BUT THE TOPO3B ATTACHED TO THE 3 PRIME END. AND SO THE A ENZYME ATTACHED TO THE 5 PRIME END, CATALYTIC TYROSINE, THE B ATTACHED TO THE 3 PRIME END. SO, GOING BACK, ANOTHER WAY TO LOOK AT THIS, SO FOR YOUR INFORMATION, THE CHROMOSOME LOCATION OF ALL OF THESE ENZYMES IN HUMANS. THE PROTEINS AND THEIR SIZE. SO THEY ARE FAIRLY LARGE PROTEINS. THE TOPO1 WORK IS MONOMER AND THE TOPO2 IS DIMER SO IT IS A VERY LARGE MACHINE. SO 340, ABOUT THE SIZE OF A NUKE ZOME, VERY LARGE. THESE ARE THEIR LOCATION. AND THESE ARE THE DRUGS. SO YOU SEE SOME OF THE ENZYME DO NOT HAVE DRUGS THAT ARE ALSO SHADED AND USEFUL FOR THERAPY. AND I THINK THERE IS STILL ROOM TO ACTUALLY MAKE GOOD DRUGS FOR THESE ENZYMES. NOW IF YOU THINK OF A HUMAN CELL, THE GENOME IS DIVIDED IN TWO PARTS, 95% OF YOUR GENOME OR MINE, IS IN THE NUCLEASE. BUT BEAR IN MIND THAT 5% AND IN THE HEART FOR EXAMPLE WHERE THERE ARE ALSO MITOCHONDRIA, PART OF YOUR GENOME IS IN YOUR MITOCHONDRIA AND THIS GENOME COMES FROM YOUR MOTHER ONLY. SO THE MOTHER AND FATHER GENOME IS IN YOUR GENOME BUT MITOCHONDRIA GENOME IS ONLY FROM YOUR MOTHER, ABOUT 5%. THESE GENOMES ARE TOPOISOMERASES, SO NUCLEAR, TOP 3B IS NUCLEAR AND TOP 13 IS ONLY MITOCHONDRIAL AND SOME OF THE OTHER OTHERS ARE SHARING BOTH COMPARTMENTS. BOTH IN NUCLEAR AND MITOCHONDRIAL GENOME AND THE SPECIFIC WITH THEIR ENZYME FOR THE TOPO2s ARE IN BOTH COMPARTMENT. SO THE WHOLE GENOME, NUCLEAR AND MITOCHONDRIAL GENOME ARE HANDLED BY THE NUCLEAR AND MITOCHONDRIAL GENOME BY THE TOPOISOMERASES AND IN AN IMBALANCE OF THESE TOPOISOMERASES IS THE STABILITY, AND THAT IS ONLY USED TO TREAT DISEASES AND ALSO WHEN IT IS NOT — SO I’LL DIVIDE IN THREE AND WE’LL TALK ABOUT THE TOP 1S AND THEN TOP 2S AND THEN TOP 3. SO TOP 1, WHICH IS CALLED TOP 1 AND TOP 1MT WHICH WE DISCOVERED AND WE DECIDED TO CALL IT TOP 1MT NOT TOP 1 PRIME OR WHATEVER úBECAUSE WE DIDN’T WANT TO
CHANGE THE NAME SO YOU WILL AGREE IT IS QUITE MARVELLISTIC. IT’S CALLED TOP 1MT. MITOCHONDRIAL. SO THE WAY TOP 01 WOULD WORK, SO I PUT THE TOP 02 HERE. THE TOP 01 WILL IN FACT MOST OF IT IS BOUND TO DNA AND PERIODICALLY IT WILL CLEAVE THE DNA, ONE OF THE STRANDS AND RELY GAND THE DNA. SO IT IS ALWAYS CLOSING THE DNA AND MOST OF THE TIME IT’S NOT PRODUCTIVE. AND THE REACTION TO DNA SUPER COIL, IT WILL ENABLE THE RELAXATION. SO THE CLEAVAGE REACTION WHICH IS DESCRIBED IN MORE DETAIL OPERATES BY THE ACTION OF A TYROSINERI SIDDUAL ENZYME WHICH WHEN IT IS BROUGHT IN PROXIMITY WILL GENERATE A 3 PRIME. THAT WILL GENERATE THE CLEAVAGE OF THE DNA AND WHEN THE DNA REALIGNS, THE 5 PRIME HYDROXYL WILL ELIMINATE THE TOPO. THAT’S WHY IT’S REVERSIBLE. THIS REACTION IS REMARKABLY EFFICIENT T WORKS WITHOUT ATP. DOESN’T REQUIRE THAT METAL. STILL WORKS AT ZERO DEGREES. SO THIS ENZYME IS REMARKABLY EFFECTIVE. AND IT IS TRAPPED BY THE — OTHER DRUGS THAT WE DESCRIBED THAT ARE SPECIFIC. AND THAT IS VERY DIFFERENT FROM THE TOPO2s WHICH I HAD SAID BOTH ARE LINKED TO THE 5 PRIME END AND STILL BY A TYROSINE BUT THIS REACTION USES ATP FOR TOPO2 AND REQUIRES MAGNESIUM. DOESN’T WORK AT ZERO DEGREES. AND ON EACH SIDE THERE ARE DIFFERENT DRUGS OF TOPO1 AND TOP 02. SO WHAT DOES REALLY TOPO1 DO? IT WAS DISCOVERED — AND IT WAS CALLED THE DNA UNWINDING BECAUSE THEY TOOK A TRACT FROM MURINE CELLS AND ADDED IT TO SUPER COIL PLASMID AND DISCOVERED THAT THE DNA WAS MAGICALLY RELAXED. VERY EFFECTIVELY WITHOUT MAGNESIUM, WITHOUT ATP. AND THIS WAS PURIFIED AT BEING TOPO1, THE DNA UNWINDING ENZYME. AND NOW IT’S WELL-KNOWN IT MAKES A NICK IN THE DNA AND DISSIPATING THE SUPER COIL, LETTING THE DNA ROTATE AROUND THE BREAK AND THEN RELY GATING THE DNA. AND TOPO1 IS ESSENTIALLY ESSENTIAL. BECAUSE AS THE DNA TRANSCRIBE OR REPLICATE OR ACCUMULATE SUPER COIL, YOU NEED TO RELAX THE SUPER COIL AND TOPO1 IS COUPLED WITH RNA AND DNA POLYMERASES. AND IN MOUSE, YOU CAN NOT KNOCK IT OUT AND IN HUMAN NOTHING EXCEPT RESIDUAL. BUT IN YEAST YOU CAN. YEAST CAN SURVIVE TOPO1 AND USES TOPO2 AND 3 BUT NOT HIGHER EUKARYOTES. SO IF YOU TAKE THE PARADIGM OF A TRANSCRIPTION MACHINE OR REPLICATION MACHINE THAT UNWINDS DNA, IN FRONT OF THE MOVING POLYMERASE THAT OPENS THE DNA, YOU GENERATE POSITIVE SUPER COIL, YOU TIGHTEN THE HELIX, AND AT SOME POINT IF YOU DON’T RELAX, YOU’RE GOING TO GET STUCK. SO THE TOPOWILL ENABLE THE DISSIPATION OF THE POSITIVE SUPER COIL ENABLING THE MOVEMENT OF THE RNA AND DNA TOPOISOMERASE. BEHIND, YOU GENERATE NEGATIVE SUPER COIL, DNAs OPENED UP AND THAT COULD CREATE ALTERNATIVE DNA STRUCTURES. SO IF YOU DO NOT RELAX DNA WITH TOPO, YOU CREATE A LOT OF – AUTO TRANSCRIPTION STALLS, REPLICATION STALLS, GENERATE R LOOPS AND GENERATES A LOT OF ALSOIVE DNA STRUCTURES. THE TOPOIS ALWAYS BALANCING THIS SUPER COIL. DNA SUPER COIL IN A SUPER COIL DOMAIN, THE TOPO, MAGICALLY RELAXES THAT AND THE POLYMERASES CAN MOVE ON. NOW THE WAY THE DRUGS WORK IS THEY INTERFERE WITH THE CLOSING REACTION AND THEN THE DNA BEING BROKEN OPEN, AND THAT GENERATES DNA DAMAGE. THIS IS NOT ONLY DUE TO DRUGS BUT ALSO TO DNA LESIONS. SO IN ADDITION TO DRUGS USED FOR CHEMOTHERAPY, THE TOPOISOMERASE COMPLEXES ARE ALSO VERY FREQUENTLY INDUCED, NAMELY NEURONS, FOR EXAMPLE, BY OXIDIZED BASE AND STRESS AND DNA LESIONS AND IF TOPOGETS ON DAMAGED DN A, IT GETS STUCK AND THAT IS EVEN WORSE. AND THAT IS WHY EVERY CELL HAS A DNA REPAIR MACHINERY FOR TOPO SIMMERAISE. A LITTLE BULLET POINT ABOUT DNA SUPER COILING, THE IDEA IS IN THE CONTEXT OF CHROMATIN WHERE THE ROTATION OF DNA IS CONSTRAINED, DNA SUPER COILING WHICH IS HOWEVER SUPER COILING AND UNDERTWISTING — REGULARLY GENERATED. IF YOU REMOVE A NUCLEOSOME, YOU IMMEDIATELY GENERATE 1 1/2 TURN OF NEGATIVE SUPER COIL. SO EACH TIME YOU MAKE A NUKESOME, YOU GET SUPER COILS BUT AS YOU TAKE NUCLEOSOMES AWAY, YOU PRODUCE THESE SUPER COILS. SO TOP 1 AND TOP 1MT REMOVES SUPER COILING BY UNTWISTING DNA WHEREAS AS TOP 02 ALPHA AND BETA REMOVE THE RIGHT WHEN THE STRANDS ARE BEING CROSSED. IT COULD PASS THEM ACROSS LIKE IF YOU’RE IN THE SHOWER YOU’RE THINK IS TWISTED. YOU HAVE TWO WAYS TO GO, YOU CAN JUST TURN IT, SO THAT IS WHAT TOP 01 WILL DO, OR JUST PASS IT AROUND, THAT’S WHAT TOP 02 WILL DO. SO THE BEST ADVICE IS DNA NEGATIVE SUPER COILS FACILITATE THE — WHICH IS GOOD WHEN YOU NEED TO TRIBE OR INITIATE REPLICATION AND GENERATE SINGLE STRAND FRAGMENT. NUCLEOSOMEAL FORMATION OR DISASSEMBLY ABSORB OR RELEASE SUPER COILS. THE POLYMERASE GENERATES POSITIVE SUPER COIL AND HAVE A NEGATIVE SUPER COIL BEHIND THEIR TRACK AND ACCESS POSITIVE SUPER COIL ARRESTS DNA TRACKING ENZYMES AND THE BLOCK TRANSCRIPTION ELONGATION AND INITIATION. THEY DESTABILIZE NUCLEOSOMES. NEGATIVE SUPER COILING COULD BE A GOOD THING IF YOU FAVOR DNA MELTING TO INITIATE REPLICATION AND TRANSCRIPTION, TO FORM D LOOPS AND TO GENERATE RECOMBINATION. AND NUKE SOMAL FORMATION. IF YOU HAVE TOO MUCH NEGATIVE SUPER COIL, THEN THAT FACILITATES THE ALTERNATIVE DNA STRUCTURES SUCH AS R LOOPS, G4 IN QUADRA PLEXES DISCOVERED HERE AT THE NIH, RIGHT-HANDED DNA, AND ALTERNATIVE STRUCTURE, WHICH THEN WILL ABSORB THE NEGATIVE SYRUP COILING AS THEY FORM AND THEY WILL ATTRACT REGULATORY — SUPER COILING. SO ALL ARE IN THE GENOME AND THE TOPOS ARE CROSS TALKING WITH ALL OF THIS. SO THE TWO TOPO1s THE NUCLEAR TOPO1 IS 765 AMINO ACID PROTEIN. WHEN WE DISCOVERED TOP 1MT, WE INITIALLY FOUND PARTS ON ANOTHER CHROMOSOME AND THEN SEQUENCED IT AND ENDED UP FINDING A MITOCHONDRIAL TARGETING SEQUENCE THAT CREATED THE NUCLEAR ACES SEQUENCE. SO ALL VERTEBRATES HAVE TWO TOPO1s. ON TWO DIFFERENT CHROMOSOMES. THE MITOCHONDRIA TOPO1. YEAST DOESN’T HAVE TWO. IT HAS ONE TOPO1, AND IN THE EVOLUTION PROCESS, DUPLICATION OF THE AN VEST RECALL TOPO1 HAPPENED IN THE CORE DAYS — ANCESTRAL. TOPO1B ALSO FOUND IN SOME BACTERIA AND IN SOME VIRUSES LIKE VACCINIA VIRUS, HAVE AN ABBREVIATED VIRUS OF TOPO. IT WORKS THE SAME JUST RELAXES DNA. SO WHAT ABOUT THE USE OF DRUGS THAT TARGET TOPO SIMMERAISE 1? THEY WERE DISCOVERED AT THE NIH, AT THE NCI AS EFFECTIVE ANTI-LOU KIMIC DRUG USING MURINE MODELS. THE DRUG WAS DEFINED AND IDENTIFIED FROM THE EXTRACT OF A CHINESE TREE — AND THAT’S WHY THE DRUG WAS — AS THE DRUG WAS DISCOVERED, DERIVATIVES THAT WERE WATER SOLUBLE WERE MAILED AND IN THE YEAR 2000, TWO WERE APPROVED IN THE UNITED STATES. TOPOT CAN WATER SOLUBLE DERIVATIVE AND HERE IN THE T CONDITION, WHICH IS A PRO DRUG WITH THIS WATER SOLUBLE GROUP, WHICH WITH ACTIVE INTERMEDIATE IS CALLED SN38, THIS IS METAB I WILL. TODAY IN SOUTH KOREA, THERE IS ANOTHER DERIVATIVE APPROVED, NOT IN THE UNITED STATES, IT’S CALLED BAITO AT THE CAN. ANOTHER WATER SOLUBLE DERIVATIVE. THE WAY THESE DRUGS WORK IS THE VERY SELECTIVELY HIGHLY SELECTIVELY BLOCK TOPO SIMMERAISE 1. AS TOPO RELEGATES ALL THE TIME IN THIS NICKING, CLOSING CYCLE, THE DRUG SNEAKS AT THE INTERFACE OF THE ENZYME AND THE DNA AND NATURE IS SELECTED THESE DRUGS BEAUTIFULLY TO JUST DO THIS. THEY BIND VERY SELECTIVELY AT THIS INTERFACE. AND WE DISCOVERED INITIALLY THIS NOTION THEY WERE VERY SELECTIVE BECAUSE THE BASIC PREFERENCE WAS HIGHLY SPECIFIC. IT WAS VERY, VERY BIASED FOR HAVING THYMINE AT MINUS 1 AND — AT PLUS 1. AND THE CRYSTAL STRUCTURE SHOWED THAT WHEN YOU HAVE A TOPOISOMERASE DNA COMPLEX WITH TOPO1 IN GRAY AND DNA IN GREEN, AND WHEN THE DRUG IS IN THE COMPLEX, YOU SEY IT IS BOUND EXACTLY HERE AT THE INTERFACE OF THE ENZYME AND THE DNA AND NATURE SELECTED THESE DRUGS TO ONLY DO THIS. IT HAS ALL THE CHEMICAL CHARACTERISTICS TO DO IT, ONLY ONE OF THE NATURAL TOPO SIMMERAISES IS ACTIVE. THE V NOT WHEN WE LOOKED AT THE R IT WAS NOT ACTIVE. SO IT IS HIGHLY-SPECIFIC FOR BLOCKING AND TRAPPING. SO THE NOTION OF TRAPPING AN ENZYME WAS BORN WITH THE TOPO SIMMER ACE. SO WHEN YOU HEARD THE TERM TRAPPING, IT WAS COINED HERE AND BECAUSE OF BIOLOGY AND THERAPEUTICS AND FOR POLYMERASE INHIBITOR, WE ARE ALL AWARE OF THE IMPORTANCE OF BRCA1 AND RECOMBINATION DEFICIENCY. IN FACT, WHEN I FIRST — WE WERE ALL WORKING IN CANCER AND WE FOUND VERY EARLY ON. IN 1988, TWO PAPER CAME, WHEN WE ARE STUDYING THIS, THIS IS NOT OUR WORK BUT IT WAS WORK DONE BY THESE GENETICISTS. INITIALLY, YEAST IS VERY RESISTANT TO THIS. AND YOU CAN HAVE SAID, IT’S VERY TRIVIAL BECAUSE CAMP TO THESEINS DON’T GET INTO YEAST T HAD ONE MUTANT WHICH IS HOMOLOGOUS FOR RAD 52 DEEFFICIENCY AND IT WAS VERY OBVIOUS THAT THIS BECAME VERY SENSITIVE. SO THE ARGUMENT THE DRUG WAS NOT GETTING IN WAS WRONG. YEASTS WAS ABLE TO REPAIR VERY EFFICIENTLY WHAT IT WAS DOING. AND THIS WAS NOT ONLY ONE LAB. THERE WAS A SECOND LAB BY JIM WONG THAT FOUND THE SAME. YOU CAN SEE HERE THIS IS A WILDTYPE RAD 52 POSITIVE YEAST, HIGHLY RESISTANT BUT AS SOON AS YOU KNOCKOUT RAD 52, IT BECAME VERY SENSITIVE. IN FACT, IF YOU GO BACK IN HISTORY, AND YOU HAVE TO GIVE A FAIR SHARE OF WHICH WAS THE FIRST DRUG THAT ACTIVITY WAS DETERMINED BY HOMOLOGOUS RECOMBINATION, IT IS THE CAMP TOW MICE IN. THEY SHOWED THIS IN HOMOLOGOUS RECOMBINATION DEFICIENT CELL. SO NOW WE CAN LOOK BACK AT ALL THIS IN THIS TIME. BUT IT HAS PRACTICAL IMPLICATION FOR MEDICINE TODAY. SO SOME YEARS AGO, WE DECIDED BECAUSE THIS WAS THE ONLY ONE TOP 01 INHIBITOR, WE DECIDED TO DESIGN AT THE NIH AND THE NCI AND LOOK FOR OTHER TOPO1 INHIBITORS THAT WERE NOT CAMP TOW THIS INS. THEY WERE EFFECTIVE AND TOP 01 WAS VALIDATED AND BECAUSE WE KNOW THAT DRUGS THAT HAVE THE SAME TARGET COULD HAVE VERY DIFFERENT CLINICAL APPLICATIONS. FOR EXAMPLE — MOLECULARLY YOU CAN NOT TELL THE DIFFERENCE BETWEEN THESE TWO. BUT IN A PATIENT, YOU WILL NOT GIVE — TO A CANCER PATIENT AND YOU WILL NOT GIVE AM BLAST IN TO SOMEBODY WITH GOUT. THESE DRUGS ARE VERY DIFFERENT APPLICATIONS. SIMILAR TARGET. THE ARGUMENT IF YOU MAKE ENOUGH TOP 01 INHIBITOR IT’S LIKELY TO HAVE DIFFERENT CLINICALPHARMACOLOGY. WE KNOW THERE WAS LIMITATIONS. IT PRODUCES VERY SEVERE INTESTINAL TOXICITY. BUT EVEN WORSE, THE CAMP TOW THESEINS ARE NOT STABLE BUT THEY ARE NATURAL PRODUCTS. IT IS VERY UNSTABLE TO P. AND AS SOON AS YOU PUT THEM IN SOLUTION, PH7 OR 7.4, IMMEDIATELY UNDERGO REOPENING FORMING CARBOXYLATE AND IN THE BLOOD THESE BIND SO SERUM ALBUMIN. SO IT’S A REAL PROBLEM. SO WE ALL KNEW THIS BUT THE DRUGS WENT ON AND THEY WERE APPROVED. AND THEN THE OTHER THING IS WHEN THEY BIND TO THE TOP 01 COMPLEX, THEY ARE NOT MAKING A COVALENT DETERMINATION. THEY ALWAYS COME ON AND OFF. IT’S VERY TRANSIENT THE WAY THEY WORK WITH THE TOP 01. SO, WE DECIDED TO DEVELOP OUR OWN TOPO1 INHIBITORS BASED ON THE ARGUMENT I HAVE GIVEN YOU. IF WE DEVELOP TOPO1 INHIBITOR, WE COULD ALWAYS THINK WE COULD TARGET THE PATIENT WHO ARE NOT RESPONDING TO THE PARP INHIBITORS WITH TOPO1 INHIBITORS THAT WOULD BE CAMP TOW THESE INS AND BEYOND. SO I TOLD YOU THAT THEY WERE THE ONLY CLASS AND I TOLD YOU THE LIMITATIONS. ALSO THE CAMPTOTHECIN HAS A SHORT PLASMA HALF-LIFE, HOURS. AND FOR PATIENTS AUTO NOT GREAT BECAUSE YOU WILL GET THE TOPOTARGETTING FOR A SHORT TIME. THEY HAVE BONE MARROW TOX AND SEVERE DIARRHEA. SO, THE SOLUTION TO THE PROBLEM CAME TO US. WE HAVE BEEN WORKING ON THIS CHEMICAL SERIES SINCE 1998. WE PUT IN THE PATENT AT THE TIME AND DEVELOPED THESE DRUGS WHICH WE CALL SI QUINIDINE. AND TODAY, WE SEE 20 YEARS LATER, WE HAVE THREE DRUGS IN THE CLINIC THAT ARE CALLED LMP400, LMP776, AND LMP744. SO THESE ARE IN THE CLINIC NOW IN BUILDING 10. AND THE LATEST STUDY WITH THIS DRUG WHICH WAS JUST PUBLISHED WAS TO COMPARE THE NEW DRUGS IN AN NCI-SPONSORED CLINICAL TRIAL NOT ONLY IN HUMANS BUT IN DOGS. SO WHAT WE DID IS WE TOOK DOGS WITH LYMPHOMA AND ALL THESE CLINICS HERE WERE COORDINATED THROUGHOUT THE UNITED STATES, AND THE DOGS WITH LYMPHOMA WERE TREATED. SO THIS IS WHAT THE NCI DOES. SO YOU COULD GO TO THE WEBSITE. SO THE OWNERS WOULD TAKE THEIR DOGS TO THE CLINIC THROUGHOUT THE UNITED STATES AND THEY WERE OFFERED THE OPPORTUNITY TO TREAT THESE DOGS FREE OF CHARGE WITH ANY OF THESE THREE DRUGS. AND OUR GOAL WAS TO DETERMINE THE MAXIMUM TOLERATED DOSE TO COMPARE THE ACTIVITIES, AND MAKE SURE WHICH ONE WAS THE BEST IN THESE REAL TUMORS AND DETERMINE THE PHARMACOKINETICS AND IT’S BETTER TO DO IN DOG TRIALS THAN HUMAN TRIALS, AND TARGET ENGAGEMENT WITH GAMMA H2X AND TOP 1 DOWNREGULATION. SO, THIS WAS NONE IN COLLABORATION HERE AT NCI AND JIM DOROSHOW, THE DIRECTOR FOR THE NCI. SO WE WORKED TOGETHER FOR A LONG TIME. AND THESE ARE THE CLASSICAL RESPONSE CURVES EACH DOG. THE DEEPER, THE MORE THE RESPONSE. MANY DOGS DO RESPOND TO THESE DRUGS WITH LYMPHOMA AND ONE DRUG WAS CLEARLY MUCH BETTER. LMP744. AND WHAT IS IRONIC IN A WAY, WE HAD SELECTED THIS DRUG FOR CLINICAL TRIAL BECAUSE IN MICE THESE TWO ARE BETTER AND THIS ONE IS NOT VERY GOOD BUT IN DOGS IT WAS VERY GOOD. SO, WHAT WE REALIZE IS THE TWO KWINNA DEAN AND TEE CAN HAVE ANTI-TUMOR ACTIVITY. SO WE DID THAT. BUT THE THIRD ONE WAS BETTER. THE DOSE LIMITING TOXICITY OF THESE DRUGS IS VERY ACCEPTABLE. THEY HAVE BON MARROW SUPPRESSION BUT NO DIARRHEA. FOR US THIS IS A WIN BECAUSE WE ARE LIMITED BY THIS. THESE DRUGS DID NOT PRODUCE DIARRHEA IN DOGS OR HUMANS OR MICE. THE CO-KINETTICS IS FAVORABLE. WE ARE TALKING MANY HOURS VERSUS JUST A COUPLE. THE ONE WE HAD NOT SELECTED SHOWS REMARKABILITY. WE WERE ABLE TO DO THIS BECAUSE OF THE DOGS AND THEN TARGET IS ENGAGED. AS A RESULT OF THIS, THIS LMP744 NOW IS BACK IN BUILDING 10. SO JIM DOROSHOW HAS PUT IT BACK IN BUILDING 10 AND WE ARE LOOKING AT THE PHASE I NOW TO SEE WHAT THE DRUG WILL BE DOING IN HUMANS. NOW IF YOU THINK OF CANCER THERAPY, GO BACK HISTORICALLY, THE BEST CANCER THERAPY AND TODAY REMAINS TRUE, IS THE TARGETED THERAPY. WHAT IS TARGETED THERAPY FOR CANCER? IT’S SURGERY. IF YOU CAN TAKE OUT THE TUMOR, THE CHANCES OF SUCCESS IS GREAT. THIS IS THE BEST TARGETED THERAPY. NEXT BEST THERAPY IS RADIO THERAPY. BUT WITH CHEMOTHERAPY YOU USUALLY DON’T DO TARGETED THERAPY. YOU DO IT BASED ON LIKE YOUR SIGNATURE SUCH AS BRCA. BUT YOU’RE STILL GETTING THE DRUG EVERYWHERE. SO THE BEST IDEA WOULD BE TO TARGET THE LIVER. AND WHAT IS HAPPENING NOW WITH THE TOPO SIMMERAISE INHIBITOR WITH TOPO1 IS SORT OF UNDER THE RADAR S BIG COMPANIES AND SMALLER COMPANIES REALIZED THIS AND TAKEN THE CAMP TOW THESE IN AS A WARHEAD AND ATTACH TO DELIVERY VECTORS THAT COULD BE MICROSOMES OR LIPOSOMES, ANTIBODIES, ANTIBODIES TARGETED TO DIFFERENT ELEMENTS. THIS DRUG FOR EXAMPLE IS DOING BEAUTIFULLY. A PATIENT WITH HEART 2 EXPRESSING TUMORS — MY HOPE IS THAT THESE DRUGS IN PHARMACOLOGY, IF WE COULD DELIVER TO THE TUMOR WITH THE RIGHT VECTOR CLEARLY WE WILL MAKE A DIFFERENCE. SO, I’LL GO TO THE TOP 02 NOW. SO THERE ARE TWO IN HUMANS AND VERTEBRATES. THERE IS DIVISION OF LABOR AND EACH GENE NEEDS TO BE EXPRESSED IN DIFFERENT CONTEXT. TOP 2 BETA IS THE MOST UBIQUITOUS. IT’S EXPRESSED IN BOTH REPLICATING AND DIFFERENTIATING CELLS AND IN EXPONENTIALLY GROWING CELLS. IT IS ABSOLUTELY CRITICAL FOR TRANSCRIPTION. WHEREAS TOP 2 ALPHA IS VERY CRITICAL, ESSENTIAL FOR REPLICATION. IT’S HIGHLY-EXPRESSED IN REPLICATING CELL AND CANCER CELLS, BREAST CANCER CELLS, BREAST CANCERS, TOP 2 ALPHA IS OFTEN AMPLIFIED AND IT HELPS CELLS TO DIVIDE. WHEREAS TOP 2 BETA IS THE HOUSEKEEPING TOPO2. THE TWO TOPOs IN HUMANS ARE VERY SIMILAR. THEY ARE ON DIFFERENT CHROMOSOMES BUT VERY SIMILAR. THEY DIFFER POSTALLY IN THE C TERMINIS, MORE VARIABLE. AND PROBABLY THEY FIND DIFFERENT FACTORS BASED ON THE C TERMINIS. THE TYROSINE IS IN THE MIDDLE AND THEY ARE SOMEWHAT SIMILAR TO BACTERIAL, EXCEPT IN BACTERIA, LIKE GIRAISE OR TOPO4, IT’S MADE IN TWO PARTS. SO YOU SEE IN BACTERIA, INSTEAD OF IN ONE CODING SEQUENCE, IT’S TWO DIFFERENT GENES, GIRAISE A AND B. AND THEN FOR TOPO4, YOU HAVE PAR E AND PAR C. SO HUMAN IS DIMERS. TOPO2 ALPHA DIMER, TOPO2 BETA DIMER. BUT IN BACTERIA, IT’S A TETRAMER TETRAMER. THE GIRAISE AB, AND SO ON. BUT THE BIOCHEMISTRY IS VERY CONSERVED. THESE ENZYMES ARE BEAUTIFUL IN A SENSE THEY HAVE TWO METAL IONS, THEY APPROACH DNA AND WHEN THEY APPROACH DNA, THE WAY THEY WORK IS THEY WORK ON CROSSOVER REGIONS MOSTLY. SO WHEN YOU HAVE TWO DNA HETEROPLEXES NEXT TO EACH OTHER, WHETHER IN THE SAME MOLECULE OR WHETHER IT IS JUST A CATAINATE IKE THIS, IT WILL DO THE SAME. YOU HAVE TWO STRANDS. ONE IS DUPLEX. ONE IS THE GATE AND ONE IS THE TRANSPORT. THIS IS THE CONVENTION. SO, THE TOPOWORKS LIKE THE NIH GATES. YOU CAN NOT ENTER THE NIH WITHOUT CLOSING THE GATE BEHIND YOU. YOU CAN NOT ENTER IN ONE GATE. SO THE DNA WILL ONLY GO THROUGH THE OTHER STRAND BY THE SAME PRINCIPLE. AND THE FIRST PRINCEEL, THE TOP GATE WILL OPEN — IT WILL LET ONE STRAND GO THROUGH. ONCE THIS IS IN THE SYSTEM, THEN THE STRAND, THE GATE STRAND WILL BE CLEAVED LETTING THE PASSAGE STRAND GO THROUGH AND THEN IT WILL GO THROUGH AND THEN WILL RELY GATE BEHIND AND THEN THE GATE WILL LET THAT GO AND EVERYTHING WILL CLOSE BACK WITH ATP. AND THEN IT IS VERY SIMPLE PROCESS WHICH IS TO TAKE ONE STRAND, OPEN, GET THROUGH, GO BACK. BUT IT NEVER GOES LIKE THIS. IT’S ALWAYS UNDER CONTROL IN THE TWO GATES. AND THESE VERY EFFICIENT REACTION DOES MIRACULOUS REACTION. IT CAN CAT ON 8 AND CAT ON 8, KNOT AND UNKNOT, RELAX SUPER COIL AND FOR GIRAISE YOU CAN ALSO GYRATE. SO IT IS A MAGNIFICENT MACHINE IN SOME WAYS AND MAGNIFICENT DIMER. WORKS IN MANY PROCESSES IN CELLS. SO IF YOU TAKE TRANSCRIPTION, FOR EXAMPLE, WHERE YOU HAVE HERE THE RNA POLYMERASE, YOU GENERATE POSITIVE SUPER COIL AHEAD AND THIS IS RELEASED BY TOPO1 OR TOPO2 ALPHA AND BETA. BEHIND GENERATE NEGATIVE SUPER COIL, THAT COULD BE RELIEVED BY TOPO1 OR TOP 03 BETA AND THEN TOPO2 IS ACTING AT THE ENHANCER REGION AND THEN YOU HAVE THE TOPO2 THE ENHANCER. SO ALL OF THESE TOPOs ARE TAKING CARE OF THE GROUPING OF CHROMATIN OF THE OPENING OF CHROMATIN AND MAKING SURE THAT THE POLYMERASES CAN DO THEIR JOB. BUT DURING REPLICATION, IT’S THE SAME PROBLEM. AS YOU OPEN THE DNA, YOU GENERATE POSITIVE SUPER COIL AHEAD. TOPO1 CAN BIC THAT UP BUT BEHIND YOU CAN GENERATE PRE KETAMINES AND THIS WILL BE REMOVED BY TOPO2. SO THEY HAVE A LOT OF INNER PLAY ARE REPLICATIONIN REPLICATIONING
AND TRANSCRIPTION THAT ARE ABSOLUTELY ESSENTIAL. NOW THE ANTI-CANCER DRUG FOR TOPO2 ARE LISTED HERE. ON THE TOP YOU HAVE ANTI-CANCER GROUP. SO THESE ARE THE STRUCTURES. YOU COULD APPRECIATE YOURSELF THAT CHEMICALLY THEY ARE QUITE DIFFERENT BUT THEY ALL HAVE IN COMMON THAT — SO THESE ARE THE ANTI-CANCER. [ READING ] ON THE BOTTOM, THESE ARE THE ANTI-BACTERIAL. THEY ARE SMALLER RING SYSTEMS AND THEY HAVE THESE GROUPS THAT HAS AN INTERACTION WITH THE METAL. AND ALL OF THESE ARE OF THE KWINNA LONES. SOME NAMES YOU WILL RECOGNIZE: [ READING ] THE INHIBITOR — ALL BIND WHEN THE DNA IS CLEAVED, SO THIS IS A TOPO2 DIMER AND THE DNA IS BARELY VISIBLE IN THE MIDDLE AND THE BREAKS WILL BE ON THAT PIECE OF DNA. THE DRUGS BIND LIKE THE TOPO1 IN THE CLEAVAGE SITE. AND THE REASON THE PLANAR MOIETY, WHICH IS SHOWN HERE, STACKS WITH THE BASE LIKE A SANDWICH WHEN THE DNA OPENS, THE BASE IS LIKE THIS AND THE DRUG FOLDS IN THE MIDDLE AND BY STACKING, IT JUST GETS STUCK, NON COVALENTLY, BUT GETS STUCK. SO THAT IS FOR EXAMPLE, FOR TOPO2. BUT IF YOU GIRAID OR TOP 04 FOR THE QUINAZOLINONES, IT’S THE SAME. SO A TETRAMIR, HAS THE SAME SHAPE WITH THE NIH GATE TYPE II GATES WITH THE HOLE HERE. THIS IS THE DNA THAT IS BEING BROKEN AND YOU SEE THE DRUG IS BOUND EXACTLY AT THE CLEAVAGE SITE. THAT CAN LOCK THE DOOR JUST BINDING THERE AND HERE IT IS. AND THIS IS CONTACT WITH THE METAL. SO SAME PRINCIPLE. THAT LED TO THE IDEA THAT THE TOPOISOMERASE INHIBITORS HAVE A COMMON MECHANISM OF ACTION WHICH IS REFERRED TO AS INTERFACIAL INHIBITOR BECAUSE THE DRUGS BIND AT THE INTERFACE OF THE DNA AND OF THE TOPOISOMERASE AND THIS LED TO THE NOTION OF INTEREFFICIENT INHIBITION OF MICROMOLECULAR INTERACTION. AND THAT WAS IN NATURE’S PARADIGM FOR MANY TOXINS. MANY TOXINS DO THAT. AND THE TARGET MICROMOLECULAR COMPLEXES. IN NATURE EVERYTHING MOVES. IF IF YOU PUT SOMETHING IN THE MIDDLE OF TWO THINGS THAT ARE MOVING IT WILL GET JAMMED, THESE MICROMOLECULAR COMPLEXES. SO IN THE CASE OF TOPO 1, TO SUMMARIZE, THE TRAPPINGS IS BECAUSE THE DRUG BINDS AT THE ENZYME DNA INTERFACE MAKING SPECIFIC FOR THE ENZYME, STACKING AGAINST HERE. THAT IS TRUE FOR THE CAMP TO THESE IN AND KWINNA 19. AND FOR TOPO2, IT TAKES PLACE AT THE DNA INTERFACE WITH THE HYDROGEN BOND WITH THE ENZYME. THE TOPO THREE. SO THIS IS A NEWCOMER. THERE IS NO DRUG ASSOCIATED HERE BUT THEY ARE VERY INTERESTING. AND ESPECIALLY FOR NOT ONLY FOR CANCER, BUT ALSO FOR NEUROSCIENCES. SO THE TOP 03 ALPHA IS PRIMARILY INVOLVED IN REPLICATION, SO IT’S A DNA TOPO SIMMERAISE. THE TOP 03 BETA IS A TRANSCRIPTION-ASSOCIATED ENZYME. IT’S BOTH A DNA AND AN RNA TOPO SIMMERAISE. FOR DNA PRIMARY WORKS IN R LOOPS MORE LIKELY AND D LOOPS AND FOR RNA, IT WORKS ON RNA KNOTS, RNA CAT ON 8. SO THE TWO ENZYMES 3A AND 3B, DO NOT WORK ALONE. THEY ARE THE EFFECTOR OF BIGGER MACHINES. SO IN THE CASE OF REPLICATION, IT IS ASSOCIATED WITH THE HELICASE AND RMI1. AND THE TOP 03 ALPHA WORKS ON DNA WHILE RMI1 AND BLACKBLOOM LOCATE THE TOP 03 WHEN IT NEEDS TO ACT. WHAT IS GOING ON IS WORK ON OUR LOOPS AND FRAGILE X PROTEINS WHICH ARE RNA BINDING PROTEINS. IN THAT CASE, THE TOP 03 BETA IS BROUGHT TO THE RNA THROUGH THE TDRD3FMRP COMPLEX AND THEN CAN DECATENATE OR WORK ON THE RNA AT THAT STAGE. SO THE TWO TOP 03 DIFFERENT PARTNERS THAT IS DIVIDE THEIR JOB IN THE CELLS WITH DNA AND RNA, REPLICATION AND TRANSCRIPTION. AND ESPECIALLY FOR THE TOP 03s, IT IS VERY CLEAR IF THE TOPO POLYMERASES ARE IN BALANCE, THAT LEADS TO GENERALLY INSTABILITY IN HUMAN DISEASE. SO AT LEAST WHY IS IT? BECAUSE THE TOPOISOMERASES THAT NORMALLY WORK SEAMLESSLY CAN GET STUCK, NOT BY DRUGS BUT IN THE COURSE OF NORMAL PHYSIOLOGY. SO IN THE CASE OF TOPO1, DURING REPLICATION, TOPO1 CLEAVAGE COMPLEX CAN BE CONVERTED TO A DOUBLE STRAND BREAK BY REPLICATION COLLISION. THAT CAN FORM, ESPECIALLY IF THE TOPO IS STUCK ON A SITE. THE TOPO2 EVENTUALLY, THE PH COMPLEX VECTOR IS LIKE THE NIH GATE. IF YOU REALLY PULL HARD, THE GATE WILL REALLY BE DISJOINTED AND THEN THE DNA MAKES A DOUBLE STRAND BREAK AND THAT WOULD LEAD TO RECOMBINATION. COLLISION WITH HELIXES AND POLYMERASES, IF THE COMPLEXES DON’T GO FAST ENOUGH, THE POLYMERASES WILL COLLIDE AND THAT WILL CREATE A PROBLEM AND IF YOU DON’T HAVE ENOUGH TOPO SIMMERAISE, YOU COULD BE LEFT WITH SUPER COIL, WITH KNOTS, OR WITH CAT ONAISE AND THAT WOULD BE VERY TOXIC. SO THIS IS AT LEAST THE — A LIST OF THE — IN ADDITION TO DRUG THAT WILL FORM THE TOPO SIMMERAISE COMPLEXES. SO WHEN YOU HAVE OXIDIZED LESION OF DNA, OR TOBACCO PRODUCTS, THIS WILL TRAP TOPO SIMMERAISES AND THAT WILL BE A PROBLEM FOR THE GENOME. IT WILL LEAD TO TOPO1 ENGAGEMENT ON DNA. NATURAL FOOD PRODUCTS DO THIS. GENETIC DEFECTS COULD BE UNREPAIRED AND TRANSCRIPTION ACTIVATION. SO FOR INSTANCE, IN THE CASE OF TOPOISOMERASE 1, IT RELAXES DNA VERY QUICKLY. THIS IS NORMALLY REVERSIBLE BUT IF THE DNA POLYMERASE CATCHES UP, NORMAL AT THE SHOULD NOT CATCH-UP, BUT IF THIS GOES TOO SLOW AND THE POLYMERASE GOES FASTER, IT WILL COLLIDE. THIS WILL GENERATE A DOUBLE STRAND END AND EVENTUALLY THE FORKS WILL REVERSE. SO THESE ARE THE CHICKEN FOOT. SO THIS LESION COULD BE VERY TOXIC. AND THE HUMAN DISEASES LINKED WITH TOPOISOMERASES ARE CLEAR NOW AND THEY ARE COMING UP. FOR TOPO1, THEY ARE NEUROLOGICAL DISEASES DUE TO LACK OF REMOVAL OF TOPO1 CLEAVAGE COMPLEXES WHEN THE DNA REPAIR ENZYME IS ONE OF THE EFFECTIVE. FOR TOPO2 BETA, CHROMOSOME TRANSLOCATION, THE SOURCE OF LEUKEMIA OR PROSTATE CANCER COULD HAVE THE TOPO2 B CLEAVAGE COMPLEXES NOT REVERSED ENOUGH. TOP 03, NEUROLOGICAL DISORDERS, SCHIZOPHRENIA, COGNITIVE IMPAIRMENT, THERE ARE PEOPLE IN FINLAND, A GROUP OF PATIENTS, OF PEOPLE WHO HAVE MENTAL RETARDATION SCHIZOPHRENIA AND IT WAS FOUND THEY HAVE A DELETION OF THE TOP 03 BETA GENE. THERE IS A RECENT PAPER OF FOCAL EPILEPSY IN PATIENTS IN TUE NEEDS WHERE IT WAS FOUND A LACK OF TOPO THREE BETA BECAUSE THE NEURONS JUST HAVE THIS PROBLEM, THEY DON’T HAVE RNA TOPOLOGY PROBLEM. AND THE REPAIR ENZYMES, VERY BRIEFLY WHAT THEY ARE, THEY GIVE RISE TO DISEASES IN HUMANS, ATAXIA AND NEUROPATHY, AND TDB2 INTELLECTUAL ABILITIES IN SEIZURE ATAXIA. BECAUSE IF THE TOPOs ARE NOT REMOVED, THAT WILL DAMAGE THE GENOME. SO HOW ARE THEY REMOVED? SO WHAT I TOLD YOU BEFORE IS THAT THE TOPO, WHEN THEY CLEAVE DNA, RELAX DNA, THEY GO THROUGH THIS COVALENCIY, WHICH IS NORMALLY VERY REVERSIBLE. SO ALL TOPOISOMERASES, CONSISTING OF A COVALENT BOND BETWEEN ONE END OF THE BREAK THEY MAKE IN DNA OR RNA, EITHER THE 3 PRIME OR 5 PRIME AND THE CATALYTIC RESIDUE AND IF THIS IS FROZEN IN THAT SITUATION, YOU HAVE A MASSIVE PROBLEM ON YOUR GENE. HERE I DREW THE TOPO1 CLEAVAGE INTERMEDIATE IN 3 PRIME END OF DNA. OR A TOPO2 CLEAVAGE LEAD TO THE 5 PRIME END OF DNA. NATURE DEVELOPED SPECIFIC ENZYMES TO HYDROLYZE THE BOND BETWEEN THE TOPOISOMERASE AND THE DNA. AND THESE ENZYMES, THE FIRST WAS DISCOVERED IN NIH IN BUILDING 35 BY HOWARD NASH. SO HOWARD CALLED THEM — WE DISCUSSED IT AT THE TIME, TIE ROW SEAL DNA PHOSPHODIE — IT’S A MOUTHFUL. BUT IT DESCRIBES WHAT IT DOES. WHAT THE ENZYME DOES IS CLEAVES THE TYROSINE OF THE TOPOISOMERASE AND WILL HYDROLYZE AND IT WILL LIBBER 8 TOPOISOMERASE AND REGENERATE THE DNA. — LIBBER 8. I SAID DO YOU WANT TO CALL IT 1? HE SAID, YOU NEVER KNOW. SO FIRST I’M HEARING WAS TDP1. BUT SOME YEARS LATER, THE SECOND TDP WAS DISCOVERED IN THE U.K. AND IT CLEAVES IN THE OPPOSITE DIRECTION. SO NOW THERE ARE TWO ENZYMES WHICH ARE ESSENTIAL BECAUSE IF THEY ARE DEFECTIVE, YOU GET NEUROLOGICAL DISEASE BECAUSE THE TOPOISOMERASE GETS THAT NEURON AND LACK OF THESE ENZYMES WILL LEAD TO THESE DISEASES. BUT AS EVERYTHING WHICH IS IMPORTANT, CELLS HAVE BACKUP AND THERE WAS A RECENT PAPER IN PNAS, DEMONSTRATING VERY CLEARLY THEY MR11 IS A BACKUP PATHWAY& FOR TOPO2. SO IF TDP2 DOESN’T DO THE JOB, THEN WHAT WOULD HAPPEN IS TDP2 WILL DO IT, THE MR11 WILL DO IT. AND THE SAME FOR 1. SO AT THE END OF THE DAY, FOR TOPO1 AND THE TOPO2 CLEAVAGE COMPLEXES, YOU HAVE VERY SPECIFIC SURGICAL INSTRUMENTS, SO TDP1 FOR TOP 1 STRICTLY EXCISE THE TOPO1 AT THE BOND. TDP AT THE TYROSINE BOND. BUT IN PARALLEL, CELLS WILL USE OTHER ENZYMES TO SHUT THE FLAP. AND THESE ARE THE ENDONUCLEASE. SO TDP1 TURNS OUT TO HAVE A LARGE RANGE OF FUNCTION WELL BEYOND TOPO1 BECAUSE IT IS A 3-PRIME CLEANSING ACTIVITY. AND TOPO2 IS ALSO INVOLVED IN SOME VIRUSES AS A VPG INDICATOR. SO THIS TURNS OUT TO HAVE ADDITIONAL FUNCTIONS. SO PUTTING IT BACK NOW IN THE CASE OF TOPO1, YOU HAVE THE TWO PATHWAYS THAT CAN ACT IN PARALLEL OR MAYBE WHERE THINGS ARE IN THE GENOME. TDP1 IS COUPLED WITH PARP AND THE — ARE AT THE NUCLEASE. WHAT IS INTERESTING, THIS CREATES ANOTHER POSSIBILITY BECAUSE IF YOU TAKE A CANCER AND THEN IF YOU WORK WITH A PARP-INHIBITOR, TDP1 WILL NOT WORK. CELLS ONLY DEPEND ON MR11 AND THAT IS A LETHAL SITUATION IF CANCER IS DEFICIENT IN MR11 OR DEFICIENT IN CC1, THEN THE PARP-INHIBITOR WILL SYNERGIZE TOPO1 INHIBITOR SELECTIVELY IN THE CANCER CELL TRYING TO CONVINCE THE CLINICIAN TO FIND THE TUMORS, AND THEY ARE NOT SO RARE, THAT ARE MR11 DEFICIENT OR ERCC1 DEFICIENT, TO DO A COMBINATION TOPO1 INHIBITOR WITH PARP-INHIBITOR AND THEN THAT SHOULD — THAT IS A PREDICTION. SO I’D LIKE TO SUMMARIZE WHAT I TOLD YOU. ALL THE CHEMISTRY WORK THAT LED TO THE CLINICAL DEVELOPMENT OF OUR OWN TOPO1 INHIBITOR IN THE CLINICAL CENTER WERE DONE IN COLLABORATIONS WITH MARK CURB MAN AT PURDUE UNIVERSITY. SO IT’S BEEN A VERY LONG-STANDING RELATIONSHIP. JIM DOROSHOW WITH THE DIRECTOR OF TRANSITION RESEARCH, IS WORKING TOGETHER FOR A LONG TIME. WITHOUT JIM I THINK THIS WOULD NOT HAVE GONE TO THE CLINICS. SO I’M REALLY GRATEFUL. AND ALL THESE DRUGS CAN HELP. THIS IS ALL TEAM WORK THROUGH THE NIH AND THE NCI. AND I HAVE TOLD YOU ABOUT HOWARD NASH, ABOUT TDPs. THIS HAS JUST BEEN A FANTASTIC WORK AND NOW THIS WORK NOW IN OUR BUILDING WE HAVE BECOME INTERESTED IF TOPO2 AND WHAT IT DOES TO THE GENOME. SO I’M HAPPY IF YOU HAVE QUESTIONS WHO ARE PRESENT, TO ANSWER SOME OF THEM. THANK YOU. [ APPLAUSE ] >> [ OFF MICROPHONE ] >> IT’S NOT SYNERGISTIC. THE TOPO2 INHIBITOR BLOCKS REPLICATION AND WHEN YOU BLOCK REPLICATION THE TOPO1 INHIBITORS ARE NOT VERY ACTIVE. BECAUSE THE WAY THE TOPO1 CANCER SELL IS MOSTLY REPLICATING, YOU BLOCK THE TOPO1 AND THE REPLICATION COLLIDES INTO THE BLOCKED TOPO1 COMPLEXES AND THAT IS HOW THEY ARE KILLED. IF YOU PUT THE TOPO2 INHIBITOR, AND IT IS NOT — SO IT WAS A GOOD TRIAL. IT DIDN’T WORK AND WE CAME UP WITH AN EXPLANATION. BUT GOOD COMBINATION FOR TOPO1 INHIBITORS. THE COMBINATION IS PARP INHIBITORS AND THEN WE HAVE THE RNA SO WE HAVE A CLINICAL TRIAL WITH TOPO T CAN WITH THE INHIBITOR AND THE RESPONSES ARE BETTER THAN WE EXPECTED.& SO I’M PRETTY PLEASED. BUT PARP INHIBITORS ARE VERY INTERESTING. BUT TOPO2 AND 1, NO. THANK YOU VERY MUCH. [ APPLAUSE ] CURT IS THE CHIEF OF THE LABORATORY OF USE OF HUMAN CARCINOGENESIS. GOT HIS MD FROM KANSAS UNIVERSITY SCHOOL OF MEDICINE. THEN HE DID AN INTERNAL MEDICINE TRAINING AT UCLA. AND HE HAS WON NUMEROUS AWARDS SUCH AS THE AACR PRINCESS AWARD AND HE IS EDITOR-IN-CHIEF OF THE JOURNAL CARCINOGENESIS AND TODAY HE IS GOING TO LECTURE US ON LUNG CANCER, PRECISION MEDICINE STRATEGY. KURT. >> GLAD TO BE HERE AND SEE SOME OF YOU. SO, LET’S GET RIGHT INTO IT. SO, PRECISION MEDICINE WAS COINED AND DEVELOPED BY FRANCIS COLLINS AND HAROLD VARMUS, ACTUALLY NOW A NUMBER OF YEARS AGO. AND HAS LED TO LOTS OF INTEREST AND FUNDING IN TERMS OF CANCER RESEARCH. THE KIND OF MEDICINE THAT I ORIGINALLY LEARNED WAS TRADITIONAL MEDICINE, WHICH INCLUDES TAKING A HISTORY, MEDICAL, LIFESTYLE AND FAMILY HISTORY AND SIGNS AND SYMPTOMS AND DOING STANDARD LABORATORY TEST. PREDICTION MEDICINE IS MULTI-LAYERED, IT’S INDIVIDUAL-CENTRIC AND INTERCONNECTED. AND IT UTILIZES VARIUOUS OMICS WHICH ARE LISTED HERE BUILT ON A FOUNDATION OF CLINICAL INFORMATION AND EPIDEMIOLOGICAL DATA. SO THIS IS A MORE EXTENSIVE PICTURE OR CARTOON OF WHAT IS GOING ON. SO, THE INFORMATION COMES WITH ALL OF THE OMICS, LEADS TO A KNOWLEDGE OF NETWORK AND A NEW TAXOMIC CLASSIFICATION OF INDIVIDUAL PATIENTS. AND THIS IS TO GUIDE AND IMPROVE CLINICAL MEDICINE. IT’S ALSO TO INFORM BIOMEDICAL RESEARCH AND THERE IS INTERCONNECTIVITY AND FORM MECHANISTIC STUDIES AND RICH INFORMATION COMMON. AND WE ADDED A FEW YEARS AGO NOW, A PREDICTION OF RESEARCH. THIS IS THE CAUSES OF PREMATURE DEATH IN 50 YEARS, TOBACCO AND SMOKING AND LUNG CANCER. IT’S NOT ONLY LUNG CANCER. IT’S MULTIPLE KINDS OF DISEASE STATES. AND PUT IT ALL TOGETHER, IT’S CLOSE TO 21 MILLION PREMATURE DEATHS FROM SMOKING TOBACCO. SOME OF YOU ARE MUCH TOO YOUNG TO SEE THE TELEVISION PROGRAM AND THE NEWS PROGRAM IN WHICH NINE CEOs OF TOBACCO INDUSTRY STOOD UP AND SAID THAT TOBACCO DOES NOT CAUSE DISEASE. THAT WAS DEFINITELY AN UNDERSTATEMENT. THIS IS A LITTLE BIT OLD. IN MALES, LUNG CANCER GOING DOWN IN INCIDENTS. IN FEMALE, IT LOOKED LIKE IT PLATEAUED OFF. IT’S STILL IN A PLATEAU IN THE RECENT 4-5 YEARS TOO. SO, IT’S A SERIOUS PROBLEM. IT’S DUE TO SMOKING. UNITED STATES HAS BEEN QUITE GOOD AND CANADA IN DECREASING THE AMOUNT OF SMOKING PER PERSON. IT USED TO BE LIKE IT IS IN THE SOVIET UNION AND SOME AREAS OF EUROPE IN WHICH THERE IS 35-60 PACKS A DAY. NOT A DAY, A WEEK. AND THAT’S CIGARETTES, ISN’T IT? OKAY. AND THAT IS DEATHS ASSOCIATED WITH THOSE. SO THERE HAS BEEN SOME PREVENTION THAT HAS BEEN ACCOMPLISHED IN SOME PARTS OF THE WORLD, INCLUDING IN THE UNITED STATES. BUT IT STILL IS THE MAJOR CAUSE OF CANCER DEATHS IN THE WORLD. AND THIS IS INCREASED IN MORTALITY UP TO ABOUT 1.5, ALMOST TWO MILLION NOW IN THE LATEST CENSUS, AND LUNG CANCER. AND WORLDWIDE IT’S LIVER CANCER, PRIMARILY IN ASIA AND THE OTHER CANCER TYPES. SO, A NUMBER OF YEARS AGO, IT WAS 1981, IT WAS MEETING IN GREECE 234 WHICH THE VERY FIRST REPORT OF SECOND-HAND SMOKE FOR ENVIRONMENTAL TOBACCO SMOKE CAUSING CANCER WAS PRESENTED BY A PROFESSOR. AND THIS IS A WATER CORAL THAT HE MADE. AND — COLOR. AND I WILL SAY THAT IT WAS AN AUDIENCE OF 400-500 PEOPLE, ALL FUGS SCIENTISTS TYPES, ALL STUDYING CANCER AND AT LEAST HALF OF THE GROUP DID NOT BELIEVE HIM. BUT HE WAS ABSOLUTELY RIGHT AND NOW THERE ARE 70, 80 STUDIES THAT HAVE SHOWN THAT EXPOSURE TO CIGARETTE SMOKE INCREASES YOUR RISK OF DEVELOPING LUNG CANCER. AND SINCE IT WAS IN GREECE, I LIKE SOME OF THIS FLOWERING LANGUAGE. OBVIOUSLY IN ENGLISH TRANSLATION, SOME MEN HAVE CONSTITUTIONS LIKE WOODED MOUNTAINS. MEN AND WOMEN. RUNNING WITH SPRINGS. OTHERS LIKE THOSE WITH POOR SOIL AND LITTLE WATER. STILL OTHERS LIKE LAND RICH IN PASTURES AND MASHES AND YET OTHERS THE BEAR, DRY EARTH OF THE PLAIN. I WAS SITTING AT THAT MEETING AND I THOUGHT, WELL, THAT WAS AN INTERESTING PRESENTATION. HOW COULD I MAY BE MAKE A CONTRIBUTION TO IT? WHAT I WANT TO DO IS ASK THE QUESTION WHETHER OR NOT EXPOSURE IN UTERO AND IN INFANTS BY THEIR PARENTS WHO WERE SMOKERS MIGHT CAUSE CANCER IN NEVER-SMOKERS. AND I HAD TO WAIT 30 YEARS OR MORE BECAUSE WOMEN DIDN’T START SMOKING UNTIL THE SECOND WORLD WAR. AND SO, IT TAKES 30, 40 YEARS, 50 YEARS, SOMETIMES TO GET LUNG CANCER. BUT ONE OF OUR FELLOWS, ACTUALLY TOOK THIS ON. WE PUBLISHED THIS ABOUT NINE YEARS AGO. I THOUGHT THIS WAS A CHILDHOOD EXPOSURE TO SECOND-HAND SMOKE IN GENETIC ALTERATIONS AND INNATE IMMUNITY LUNG CANCER RISK IN NEVER-SMOKER ADULTS. AND NEVER-SMOKERS ARE ABOUT 10-13% OF THE PEOPLE COMING INTO OUR CLINIC TODAY. THE CONCLUSION TO THAT STUDY WAS THAT PARENTAL SECOND-HAND SMOKE, EITHER PARENT OR BOTH, EXPOSURES IN CHILDHOOD ASSOCIATED WITH A DOZE DEPENDENT CREATES A LUNG CANCER RISK IN TWO DIFFERENT COHORTS. THIS IS ESPECIALLY TRUE IN THOSE INDIVIDUALS WHO HAD A HAPLOTYPE, WHICH IS INVOLVED IN I BELIEVE 8 IMMUNITY AND — INNATE — AND METABOLISM. AND THEY HAVE HYPERINNATE IMMUNE SYSTEM. THAT’S ABOUT ONE FOURTH. SO THERE IS 3-4 PEOPLE OR MORE IN THIS ROOM THAT PROBABLY HAVE THIS INHERITED CONDITION. AND THE OTHER INTERESTING THING, THAT IS VERY SURPRISING, IN THIS STUDY WITH THESE TWO COHORTS, THE NEVER-SMOKERS WERE GETTING CANCER, LUNG CANCER, IN THEIR 50s AND EARLY 60s INSTEAD OF LATE 60s AND 70s. SO, NOW LET’S GO TO THE EXPOSE EXPOSEOSOME. THIS WAS COINED BOY THE FORMER DIRECTOR OF THE ENVIRONMENTAL — INTERNATIONAL AGENCY FOR RESEARCH ON CANCER, CHRIS WILD. AND WE ARE ALL FAMILIAR WITH USING THE EXTERNAL ENVIRONMENT, INCLUDING TOW BAG BACKO SMOKE, INFECTIONS AND SO ON AND SO FORTH. HE ALSO INCLUDED THE INTERNAL ENVIRONMENT THAT WAS DIRECTLY RELATED TO THE EXTERNAL ENVIRONMENT SUCH AS OBESITY, CHRONIC INFLAMMATION. AND THESE TWO TOGETHER CAN BE USED FOR DEVELOPING CANCER BIOMARKERS OF RISK IN PROGNOSIS. THE CANCER GENOME OBVIOUSLY IS QUITE INTERESTING AND IMPORTANT, ESPECIALLY IN THE CANCER THERAPY, BUT IT ALSO — AND DRIVER GENES, BUT IT ALSO IS IMPORTANT IN GENOMICS SUSCEPTIBILITY. AND THE UNDERSTANDING OF CARCINOGENESIS ADDS TO THESE TO DEVELOP WAYS OF CANCER PREVENTION AND SCREENING STRATEGIES AND I’LL END UP WITH THAT IN A MOMENT. ALL RIGHT, GENOME. WELL, THE CANCER WHEN I WAS INITIALLY TAKING CARE OF CANCER PATIENTS, WAS SMALL CELL, SQUAMOUS CELL, ADENO. AND THEN IN 87, IT WAS DISCOVERED KRAS WAS DISCOVERED. AND IN 2004, A NUMBER OF eGFR MUTATIONS WERE DISCOVERED. AND THEN THERE WAS A NUMBER OF MUTATIONS THAT ARE TARGETABLE, MOST OF THEM, THAT WERE DISCOVERED LARGELY IN AROUND 2014. SO THE GENETIC LANDSCAPE OF LUNG CANCER IS FAIRLY WELL-KNOWN AT THIS POINT AND SOME OF THESE ARE TARGETABLE AND I’LL GET MORE INTO THAT LATER. THERE ARE A NUMBER OF CHEMICAL AGENTS THAT CAUSE CANCER, AND THIS GOES BACK ALL THE WAY BACK TO CHIMNEY SWEEPS AND VOTEAL CANCER WITH EXPOSURE TO SMOKE FROM WOOD AND CARCINOGENS. AND SECONDLY, DIETARY CARCINOGENS, SUCH AS ASPERGILLUS FLAVOR US, PRODUCED BY A FUNGUS, PRODUCES ALPHA TOXIN BY A FUNGUS THAT GROWS AND IS IMPORTANT FOR LIVER CANCER. AND THEN OF COURSE CIGARETTES. CIGARETTES IS A WITCH’S BREW OF 70 DIFFERENT KNOWN CHEMICAL CARCINOGENS NOW. BUT SOME OF THEM ARE BENZENE, FORMALDEHYDE, AND A NUMBER OF OTHERS. WELL, IN THE CHEMICAL CARCINOGEN SIS OF LUNG CANCER AND OTHER KINDS OF CANCERS, THE FIRST DEVELOPMENT IS IN THE 70s IN WHICH IT WAS CONCLUDED THAT MOST CARCINOGENS ACTUALLY ARE MUTAGENS. NOT ALL MUTAGENS ARE CARCINOGENS BUT MOST, NOT ALL, CHEMICAL CARCINOGENS ARE MUTAGENS AND THEY WERE RAPID TESTS THAT WERE DEVELOPED BY OTHERS TO IDENTIFY POTENTIAL CARCINOGENS THAT HAD IMPORTANT REGULATORY EFFECTS IN ALSO SMOKING. AND THEN THERE WERE A NUMBER OF VERY EXCELLENT REVIEWS, INCLUDING BY CHARLIE HEIDELBERGER WHO ALSO DISCOVERED 5FU, STILL USED IN OUR CLINIC, 50 YEARS LATER. AND THEN OUR GROUP WAS LOOKING AT MUTAGENESIS IN USING HUMAN TISSUES AND HUMAN CELLS. AND THEN THE NEXT ERA WAS IN THE 90s IN WHICH IT WAS DISCOVERED THAT p53 MUTATIONS WERE LINKED TO ENVIRONMENTAL CARCINOGENS. I WILL SAY THAT DAVID LANE GIVING A TALK ON THURSDAY, THE CODISCOVERER OF p53. SO IF YOU ARE REALLY INTERESTED IN p53, I’D RECOMMEND THAT YOU GO TO THAT TALK IN BUILDING 35. OUR GROUP AND OTHERS GROUP FOUND THERE WERE SPECIFIC MUTATIONS ASSOCIATED WITH ALPHA TOXIN EXPOSURE AND CARCINOMA. IN CHINA AND ALSO IN SOUTH AFRICA. AND THAT LED TO LOTS OF OTHER STUDIES LOOKING AT ALTER VIOLATE LIGHT AND VARIOUS OTHER CARCINOGENS WE ARE EXPOSED TO. — ULTRAVIOLET LIGHT. AND THIS IS A SUMMARY OF SOME OF THOSE CARCINOGENS THAT ARE TARGETING TISSUES, THAT CAUSE DIFFERENT KINDS OF MUTATION
MUTATION AND VARIOUS HOTSPOTS AND ALMOST ALL OF THESE ARE HOTSPOTS THAT HAVE GAIN-OF-FUNCTION. THE MOST COMMON MUTATION IN HUMAN CANCER STILL IS p53. AND OVARIAN CANCER IS ABOUT 95% N SQUAMOUS CELL CARCINOMA OF THE LUNG, IT’S ABOUT 70%. SO, THERE IS A LOT OF CONTINUED INTEREST IN p53 AND ITS MUTATIONS AND THEIR SIGNIFICANTS. AS I MENTIONED, SOME OF THE MUTATIONS ARE GAIN-OF-FUNCTION MUTATIONS. SO IT SWITCHES FROM JECKYL HIDE KIND OF SITUATION. IT SWITCHES FROM A TUMOR SUPPRESSOR TO AN ONCOGENE. THIS IS JUST A CARTOON SAYING THAT LOSS OF ONE ALLELE OF THE p53 GENE YOU LOSE SOME OF THE ONCOGENIC — EXCUSE ME, THE WILDTYPE TUMOR SUPPRESS AROR ACTIVITY. SOME OF THE MUTATIONS ARE NULL OR STILL HAVE SOME WILDTYPE ACTIVITY. LOSS OF THE ENTIRE GENE HAS — IS ASSOCIATED WITH INCREASED RISK OF CANCER AND VARIOUS MUTATIONS ARE VERY HIGH RISK AT DEVELOPING CANCER. p53 IS INVOLVED IN MANY DIFFERENT PROCESSES AS SHOWN IN THIS HALLMARK-LIKE SLIDE. GERMLINE MUTATIONS, THE LEAVE AR MEANIE SYNDROME. THESE KIDS START GETTING CANCER AT RELATIVELY EARLY AGE. SARCOMBS AS A VARIETY OF OTHER CANCERS. AND MUTATIONS CAN AFFECT ETIOLOGY. I JUST SHOWED YOU SOME SLIDES OF THAT. I’LL SHOW YOU SOME EVIDENCE THAT p53 MUTATIONS, AT LEAST CERTAIN TYPES WILL CAUSE DECREASE IN PROGNOSIS AND THEY ALSO CAUSE CHANGES IN TUMOR METABOLISM. YOU MUCOSA BARRIER AND I’LL SAY SOMETHING ABOUT THE MICROBIOME IS INTEGRITY IS DIMINISHED WITH p53 MUTATIONS. CHECKPOINT CELL CYCLE DNA REPAIR, APOPTOSIS, THAT IS INVOLVED WITH p53. CERTAIN microRNAs ARE UP REG LAIDED BY WILDTYPE p53, A TRANSCRIPTION FACTOR, BUT IS NOT BY MUTANTS. AND OF COURSE ALPHA TOXIN, WHICH I MENTIONED AND IN INFLAMMATION. NOW MOST RECENTLY, IS QUITE AN INTERESTING STUDY BY ALEXANDER AND MIKE STRAT EN, WHO ARE THE SANGER INSTITUTES, ALEXANDER IS NOW AT SAN DIEGO AT UCSD. AND THEY ARE LOOKING AT AND HAVE BEEN LOOKING AT TRYING NUCLEOTIDE SIGNATURES. AND THEY PUBLISHED IN 2013 OR SO, ABOUT 20-SOMETHING SIGNATURES NOW UP TO ABOUT 30. SOME ARE ASSOCIATED WITH AGING SUCH AS SIGNATURE 1 AND SIGNATURE 5. SOME ARE ASSOCIATED WITH DNA REPAIR PROCESSES AND INFLAMMATION SUCH AS AP BECK AND THE GREEN THINGS THAT ARE HERE. SOME ARE ASSOCIATED WITH OTHER KINDS OF GERM AND LINE MUTATIONS. SMOKING. THESE THREE GREAT EXAMPLES, THESE ARE DIFFERENT KINDS OF LUNG CANCER. AND IT GOES ON AND ON. AND SO IS THERE A LOT OF INTEREST IN REALLY DEFINING WHAT THESE VARIOUS OTHER MUTATIONS ARE OR SIGNATURES ARE, AND WHAT THEIR ROLE IN CANCER IS. AND THAT IS ONE OF THE THINGS THAT IS BEING CONSIDERED TO BE ADDED TO THE TCGA, WHICH IS THE COSMID MUTATION DATABASE. REQUIRES WHOLE GENOME SEQUENCING AND THAT IS REALLY EXPENSIVE. BUT OF COURSE PEOPLE WHO DO THOSE KINDS OF THINGS WOULD VERY MUCH LIKE THAT TO HAPPEN. IF YOU COMPARE SMOKERS VERSUS NON-SMOKERS, YOU SEE THE SIGNATURE 4, WHICH IS IN BLUE. THERE IS MORE MUTATIONS THAT ARE OCCURRING PER MEGABASE OF DNA THAN IN NEVER-SMOKERS. SO YOU MIGHT THINK, MAYBE THERE IS MORE NEO-ANTIGENS AVAILABLE IN SMOKERS THAN IN NON SMOKERS. THAT REMAINS TO BE PROVEN AND THAT THEY MIGHT BE MORE SUSCEPTIBLE TO IMMUNOTHERAPY, WHICH OF COURSE IS A MAJOR TOPIC AT THE MOMENT. THIS SIGNATURE LIKE THE p53 MUTATION CAUSED BY BENZ PIE REASON, IS FOUND IN SMOKERS AND FOUND IN MICE THAT HAVE HUMANIZED THE MICE WITH p53 AND YOU CAN TAKE HUMAN CELLS AND EXPOSE THEM TO BEN SPY REASON. THIS IS THE KIND OF EVIDENCE PEOPLE USE FOR LINKING PARTICULAR AMOUNT, CARCINOGEN, FOR EXAMPLE, OR A DRUG OR SOME ENVIRONMENTAL AGENT WITH THESE MUTATIONAL SIGNATURES. NOW I MENTIONED PROGNOSIS EARLY ON. AND THIS IS KAPLAN-MEIER PLOT. 100% SURVIVAL. 0% SURVIVAL. AND THIS IS TIME IN MONTHS SO THIS IS FIVE YEARS. SO THE RED LINE ARE CERTAIN HOTSPOT MUTATIONS THAT ARE GAIN-OF-FUNCTION, AND THEY ALSO ARE IN VARIOUS KINDS OF AS AS AND THEY ALSO ARE ASSOCIATED WITH POOR PROGNOSIS. SO, IT IS NOT BENIGN TO HAVE P53 MUTATION. IT CAN INCREASE YOUR CANCER RISK AND IT ALSO CAN CHANGE YOUR RESPONSE TO IMMUNOTHERAPY AND THAT HAS BEEN SHOWN NOW IN MELANOMA, AND PEOPLE ARE INVESTIGATING THAT IN OTHER CANCER TYPES. CHRONIC INFLAMMATION HAS BEEN RECOGNIZED SINCE 19 CENTURY, 18 CENTURY MAYBE AS BEING INVOLVED IN MANY DIFFERENT KINDS OF CANCER. IT CAN BE LARGELY INHERITED, MORE THAN ACQUIRED, AND HEMOCHROMETOSIS IS AN EXAMPLE OF THAT. OVERLOAD DISEASE, CROHN’S AND ULCERATIVE COLITIS, INFLAMMATORY BOWEL DISEASE AND PANCREATITIS, ASSOCIATED WITH PANCREATIC CANCER. AND CARTER’S FAMILY, THE PRESIDENT CARTER, MOST OF THE PEOPLE IN HIS FAMILY IN THE LAST GENERATION HAVE DIED OF PANCREATIC CANCER AND HAVE HAD FAMILIAR PANCREATITIS. HE DOES NOT. HE HAS METASTATIC MELANOMA RIGHT NOW TO HIS BRAIN. BUT IT’S UNDER THERAPY AND FOR OVER A YEAR NOW AND HE IS IN HIS LATE 90s NOW. SO HE IS A VERY FORTUNATE INDIVIDUAL. MORE FREQUENTLY, THE INFECTIONS ARE ACQUIRED MORE THAN INHERITED. THEY CAN BE VIRUSES. THEY CAN BE VARIOUS BACTERIA. THEY CAN BE PARASITIC CONDITIONS OR CAN BE CHEMICAL, PHYSICAL AND METABOLIC CONDITIONS SUCH AS ACID REFLUX AND ESOPHAGEAL CANCER, OBESITY, MULTIPLE TYPES OF CANCER AND OF COURSE SMOKING. SO THE GLOBAL IMPACT OF INFLAMMATION OR INFECTION IS ABOUT TWO MILLION — AND THIS IS BEING UPDATED AT THE MOMENT. TWO MILLION CANCERS PER YEAR ARE RELATED TO INFECTION AND OTHER CAUSES OF INFLAMMATION ARE ASSOCIATED WITH MANY MORE CANCERS PER YEAR. SUCH AS I SAID NOW SMOKING AND UP TO OVER 6 MILLION CANCERS, LUNG AND OTHER CANCER TYPES TOO. INFLAMMATION WE LOOKED AT A NUMBER OF DIFFERENT PRO-INFLAMMATORY CYTOKINES AND CONDITIONS THAT MIGHT HAVE AFFECT ON INCREASED RISK AND IN EUROPEAN AMERICANS, A, AND AFTER ROW AMERICANS, AA. SO THIS IS PROSPECTIVE STUDY THAT WE AND OTHER PEOPLE HAVE ANALYZED. THERE WERE VERY FEW AFRO-AMERICAN IN THAT PROSPECTIVE STUDY. BUT IN STUDIES OF POOR DIAGNOSIS, WE FOUND BOTH OF THESE ARE INCREASED IN EUROPEAN-AMERICANS AND AFRO-AMERICANS AND AFRO-AMERICANS ALSO HAVE INCREASE IN IL1 BETA, WHICH IS A PRO-INFLAMMATORY CYTOKINE IN IL10, WHICH IS AN ANTI-INFLAMMATORY CYTOKINE. IT’S A LITTLE MORE COMPLICATED WHEN YOU LOOK AT DIFFERENT RACIAL GROUPS. POOR SURVIVAL IS ASSOCIATED WITH THESE VARIOUS CYTOKINES AND TNF ALPHA, A HIGHLY REACTIVE PRO-INFLAMMATORY AMOUNT. TRANSCRIPTOME. ALL RIGHT. LET’S FIRST LOOK AT microRNA. SO MICRORNA IS QUITE INTERESTING. THESE ARE SMALL, NON-CODING RNAs THAT ARE EVOLUTIONARY CONSERVED AND REGULATE GENE EXPRESSION. I TRUST EVERYONE IN THIS ROOM IS STUDYING THEM OR HAS STUDIED THEMMER KNOWS ABOUT THEM. THEY WERE DISCOVERED BY VICTOR AM ROWS PRIMARILY IN YEAST IN 1994, I BELIEVE IT WAS. HE’LL PROBABLY WIN THE NOBEL PRIZE IN DUE TIME. AND THEDREADS OF GENES ARE REPRESSED BY EACH MICRORNA –DREADS — AND DESTABILIZING MESSAGE CURSE INTO A LESSER EXTENT INHIBITING TRANSLATION OF mRNA N2000, GARY RIPKIN, WHO I THINK IS GIVING A LECTURE HERE OR JUST GAVE A LECTURE HERE — IF YOU WANT TO HEAR MORE ABOUT THE HISTORY IN MODERN-DAY NON CODING RNAs, HE’S THE GUY TO LISTEN TO. AND THEN IT WAS RELATED TO CANCER IN 2004 BY KARL YOSHI, IN LYMPHOCYTIC LEUKEMIA AND THAT LED TO A LOT OF INTEREST IN microRNAs AS BIOMARKERS AND AS CAUSATIVE AGENTS OR CHANGES OF THAT THAT IS INVOLVED IF THE PROCESS OF CARCINOGENESIS AND A VARIETY OF OTHER DISEASES. SO AS YOU KNOW, microRNAs BIND TO MESSAGES. THEY CAN INHIBIT TRANSLATION OF GENES. BUT IF THERE IS A MISMATCH IN THE microRNA, THIS USUALLY WHEN IT BINDS TO THE MESSAGE, IT LEADS TO INSTABILITY OF THE MESSAGE, AND CLEAVAGE OF IT. SO TWO WAYS OF INHIBITING PROTEINS BEING MADE, EITHER BY REPRESSION OR BY DECREASING THE NUMBER OF mRNAs. BUT microRNAs CAN ALSO BIND TOO. OTHER PROTEINS ARE INVOLVED IN REGULATING DIFFERENTIATION AND EXPRESSION AND AS IT WAS ORIGINALLY SHOWED, THEY CAN BIND SUCH AS MI R21 AND 29 TO, RECEPTORS AND ACTIVATE RECEPTORS TOO AND INCREASE IN IL6 AND TNF ALPHA AND INFLAMMATION. WELL, THIS IS A PICTURE OF SUMO WHO IS BACK IN JAPAN, AN ASSOCIATE PROFESSOR AT THIS MOMENT IN TOKYO. AND THE HYPOTHESIS THIS WAS INVESTIGATE SAID THAT MICROARE. NA PROFILES WERE SIGNIFICANTLY DIFFERENT BETWEEN PRIMARY LUNG CANCERS AND CORRESPONDING TISSUES AMONG DIFFERENT HISTOLOGICAL TYPES. AND THAT PROVED TO BE THE CASE. AND WHAT HE FOUND IS THAT THERE WAS INCREASED MI R21, AND I’LL SAY MORE ABOUT THAT IN A MOMENT. AND A DECREASE IN LED7, WHICH WAS PREVIOUSLY FOUND BY OTHERS WHO I THINK WILL BE HERE IN JANUARY. THEY ARE AT HARVARD NOW. WE TEAMED UP WITH YOSHI AT THAT TIME AFTER WE HAD DONE THE LUNG CANCER STUFF AND ASKED, IN SIX MAJOR CANCERS, WHICH WERE THE microRNAs THAT WERE KNOWN AT THAT TIME? THERE WERE ONLY ABOUT 290. NOW THERE ARE MAYBE OVER 2000 microRNAs THAT HAVE BEEN DESCRIBED. WHICH OF THOSE MAJOR CANCER TYPES, WHICH OF THE MICRORNAs THAT WERE KNOWN AT THAT TIME WERE OVEREXPRESSED. MI R21, WAS FOUND TO BE INCREASED IN ALL OF THE MAJOR TYPES OF CANCER AND IT WAS SHOWN THAT IT WAS UPREGULATED IN 18 MAJOR CANCERS EVENTUALLY AND IT WAS A BIOMARKER AND SURVIVAL OF THESE. SO THERE HAS BEEN A LOT OF INTEREST IN MERE 21 AND AS WELL AS THE OTHERS BY US AND OTHERS. THIS IS A EXAMPLE OF A STUDY LOOKING AT MULTIPLE COHORTS AGAIN HERE IN THE UNITED STATES, IN NORWAY, IN JAPAN, BY OUR GROUP, AND THESE ARE KAPLAN-MEIER PLOTS. SO 100% SURVIVAL. 0% SURVIVAL. 5-YEAR HERE. ABOVE THE MEDIAN WHICH IS IN RED IS HERE, AND BELOW THE MEDIAN WHICH IS IN GREEN IS HERE. SO, IN EACH OF THESE COHORTS, THERE WAS AN ASSOCIATION WITH POOR SURVIVAL IF MERE 21 WAS OVEREXPRESSED. AND THESE ARE JUST SOME EXAMPLES OF OTHER KINDS OF CANCERS IN WHICH THE SAME KIND OF THING HAS BEEN FOUND. SO WHAT IS THE MECHANISTIC UNDERPINNING OF MERE 21 AND HUMAN CANCER? FIRST OF ALL, IT’S AMPLIFIED, GENE AMPLIFICATION AND IT HAS BEEN FOUND THAT IN THE 7th EDITION OF LUNG CANCER PATHOLOGY, THEY RECOGNIZED THAT THIS IS AN IMPORTANT CRITERIA IN THE PATHOLOGY. IT WAS DECREASED TRANSCRIPTIONAL SILENCING OF VARIOUS GENES WHICH IS SHOWN HERE. THEY ARE ASSOCIATED WITH CANCER. WE SHOWED THAT eGFR INCREASES MERE 21 OR KRAS INCREASES MERE 21 DURING THE SAME PATHWAY. SO EITHER ONE WILL LEAD TO AN INCREASE IN MERE 21. DAVID BALTIMORE AND HIS COLLEAGUES FOUND THAT VARIOUS INFLAMMATORY AGENTS, CYTOKINES, THROUGH STAT 3 WILL INCREASE MERE 21. AND I HAVE ALREADY MENTIONED THAT TOLL RECEPTORS THAT ARE INVOLVED IN INFLAMMATION AND HIGH PACKSIA CAN BE ACTIVATED BY THIS microRNA. SO IT’S NOT SURPRISING IT IS ALSO ASSOCIATED WITH MANY KINDS OF CANCERSES AND POOR PROGNOSIS. WE LOOKED AT PROTEIN CODING GENES ALSO IN AN UNBIASED WAY. AND WE FOUND THAT XVO1 WHICH IS EXPORTER OF SMALL MOLECULES IN BRC1 THAT YOU’RE ALL FAMILIAR WITH, INHERITED IN AN EFFECTIVE WAY, INCREASES RISK OF BREAST CANCER BUT THIS IS THE OVEREXPRESSION AND THAT HAS BEEN SHOWN TO ACTUALLY INCREASE MUTATION FREQUENCY AND HIP 1 ALPHA IS ALSO INVOLVED WITH VARIOUS KINDS OF CANCERS AND HYPOXIA AND ISCHEMIA IN TUMORS AND DECREASE IN TUMOR SUPPRESSOR GENE. THESE WERE ALL ASSOCIATED WITH POOR PROGNOSIS IN 12 INDEPENDENT COHORTS, THE FIRST 5 THAT WE LOOKED AT THESE WERE OUR STUDIES AND THESE ARE LOOKING AT DATA FROM OTHER STUDIES. AND THEN IT WAS CONFIRMED IN STAGE 1 AGAIN FOR PRIMARILY STAGE 1 LUNG CANCER ABOUT THAT SIZE LEGS THAN 3 CENTIMETERS, AND WITHOUT EVIDENCE OF PETAFT CEASE. AND THESE ARE — METASTASIS — AND THESE ARE OTHER STUDIES THAT WERE CONFIRMATORY OF WHAT WE FOUND. AS I SAID, WE ARE PARTICULARLY INTERESTED IN STAGE 1 OF LUNG CANCER BECAUSE THAT IS POTENTIALLY CURABLE. AND ONE B IS A LITTLE BIT LARGER. THAT IS 3 CENTIMETERS TO 5 CENTIMETERS IN SIZE BUT STILL LOW METASTASIS. BUT THIS IS JUST METANALYSIS SHOWING THAT RELAPSE-FREE SURVIVAL OF CANCER-SPECIFIC SURVIVAL, EVEN OVERALL SURVIVAL, THERE WAS A ASSOCIATION WITH THE EXPRESSION OF THESE GENES. WELL, I TALKED ABOUT mRNA AND THEN PROTEIN CODING. WHAT HAPPENS IF YOU MADE A COMBINATION OF THE TWO? AND SO THE HYPOTHESIS WAS COMBINATION OF PROTEIN CODING GENES SAFELY ALTERED IN LUNG CANCER AND ADENOCARCINOMA AND NON CODING MERE 21 IS A BETTER PROGNOSTIC CLASSIFIER THAN EITHER ALONE. AND THAT PROVED TO BE THE CASE IN A JAPANESE COHORT IN A U.S.-NORWAY COHORT THAT THE JAPANESE COHORT WAS LOOKING AT PRIMARILY CANCER-FREE OCCURRENCE AND THESE ARE STUDIES IN ADDITION WE ARE LOOKING AT PRIMARILY CANCER-SPECIFIC MORTALITY. BUT IN ANY CASE, HIGH LEVELS OF THESE GENES WHETHER IT WAS MICRORNA OR PROTEIN CODING, LED TO A DECREASE IN SURVIVAL. AND THE THE COMBINATION WITH MERE 21 WAS IN FACT EVEN BETTER THAN THESE PRIMARILY STAGE 1 LUNG CANCERS. SO, WHAT IS THE PRINCIPLE HERE? THE PRINCIPLE IS THAT IN THIS CASE, NON-CODING RNA AND CODING RNA, WHEN YOU COMBINE THEM, THEY ARE MECHANISTICALLY DIFFERENT FROM EACH OTHER. YOU END UP WITH LOW RISK OF BOTH NEGATIVE. HIGH RISK IF EITHER IS NEGATIVE OR VERY HIGH RISK IF BOTH ARE NEGATIVE. AND THE PRINCIPLE IS THAT THEY BOTH GIVE SOME DEGREE OF ACCURACY IN AGREEMENT. BUT THEY BOTH ALSO, EACH INDIVIDUAL ONE, ALSO LEADS TO MISCLASSIFICATION BUT MISS CLASSIFICATION OF DIFFERENT PATIENTS. MISCLASSIFICATION OF DIFFERENT PATIENTS. AND THIS FURTHER PROOF OF PRINCIPLE OF THIS HAS BEEN FOUND IN COLON CANCER, ESOPHAGEAL CANCER ESOPHAGEAL CANCER, LUNG CANCER, AND OF COURSE BREAST CANCER. ALL RIGHT, NOW EPIGENOME, WHAT IS EPIGENOME? EPIGENOME, ONE ASPECT IS DNA METHYLATION AND THIS IS ANNA ROBEELINGS WHO DID THESE STUDIES AT THAT TIME. AND THE HYPOTHESIS IS INTEGRATED BIOMARKER CLASSIFICATION OF STAGE 1 ADENOCARCINOMA BASED ON INDEPENDENT MESSAGE RNA MICRORNA AND DNA METHYLATION BIOMARKERS, WOULD FURTHER IMPROVE THE PROGNOSTIC CLASSIFIER. SO THREE INDEPENDENT MECHANISTIC AND STATISTICALLY BIOMARKERS TOGETHER WITH THAT GIVE YOU A BETTER RESULT? AND THE ANSWER WAS YES. BUT WE ALREADY KNEW THAT THE DNA METHYLATION OF LUNG CANCER, INCLUDING STAGE I, HAD SOME ASSOCIATION WITH LUNG CANCER SURVIVAL. AND THIS IS FROM A COLLEAGUE OF MINE, WHO IS IN BARCELONA. IN HIS STUDY OF LOOKING AT MULTIPLE SITES OF DNA METHYLATION, HE FOUND THAT THERE WERE FIVE DIFFERENT SITES IN WHICH DNA METHYLATION WAS ASSOCIATED WITH PROGNOSIS IN STAGE I OF LUNG CANCER. AND THIS IS VALIDATED IN THE SECOND COHORT. WHEN I LOOKED AT THESE RESULTS, I SAID, WELL, THAT’S VERY INTERESTING BUT MAYBE THESE ARE INTERDEPENDENT ON EACH OTHER. AND MAYBE ONLY ONE OF THESE IS SUFFICIENT TO GIVE YOU THE ACCURACY THAT YOU NEED. AND THAT PROVED TO BE THE CASE. SO MULTI-VARIANT ANALYSIS LED US TO LOOK ONLY AT HAWKS A9 AND WHEN WE DID AND COMBINED IT WITH THE PROTEIN CODING GENES AND THE MERE 21, THAT WE FOUND THAT THE COMBINATION OF ALL THREE MECHANISTICALLY-INDEPENDENT BIOMARKERS GAVE THE BEST PROGNOSTIC INDICATOR FOR PROGNOSIS IN STAGE 1A LUNG CANCER AND IN 1B LUNG CANCER. SO, THAT WAS PUTTING THREE THINGS TOGETHER AND NOW WE ARE PUTTING FOUR THINGS TOGETHER TO SEE IF WE CAN EVEN IMPROVE THE ACCURACY OF THE BIOMARKER. ALL RIGHT, MICROBIOME. AND I’LL JUST TOUCH UPON THIS. BUT IT’S A VERY EXCITING AND INTERESTING AREA OF RESEARCH, NOT ONLY IN LUNG CANCER BUT COLON CANCER AND A VARIETY OF OTHER CANCERS, LIVER CANCER, TIM WHO IS OVER IN BUILDING 10, AND PEOPLE AT HOPKINS PRIMARILY HAVE DONE THE COLON CANCER WORK, AND WE HAVE JUST DONE RECENTLY THE LUNG CANCER WORK. THE MICROBIOME WHICH WAS REALLY DEFINED BY THE NIH AS A DIRECTOR’S NIH DIRECTOR INITIATIVE, WENT ON FOR ABOUT EIGHT YEARS OR SO AND JULIE WHO IS HERE, WAS ONE OF THE LEADERS, AND THEY LOOKED AT DIFFERENT HABITATS WITHIN THE BODY, ON THE SURFACE OF THE BODY AND ON THE INTERIOR OF THE BODY. BUT ALL EXPOSED EITHER DIRECTLY OR INDIRECTLY TO THE ENVIRONMENT. SO THE MICROBIOME ISN’T JUST BACTERIA, IT’S ALSO FUNGI AND VIRUSES. AND IF YOU LOOK AT THE — YOU DON’T EVEN HAVE TO GO LOOK AT THE AXIS THERE, BUT IF YOU JUST LOOK AT THE DIFFERENT COLORS AND THE PERCENTAGES, THE DISTRIBUTION, YOU CAN SEE THAT THERE IS LOTS OF VARIATION FROM ONE HABITAT TO ANOTHER, FOR EXAMPLE FROM THE NOSE TO THE LUNG, TO THE SKIN, SO ON AND SO FORTH. SO, DIFFERENT PARTS OF THE BODY HAVE A DIFFERENT COMPOSITION OF BACTERIA AND VIRUSES AND FUNGI AND THEY ARE PART OF US AND ARE ABOUT 10 TIMES THE NUMBER OF CELLS WE HAVE IN OUR BODY. SO THEY ARE A LARGE PART OF OUR BODY. SO THERE ARE A NUMBER OF BACTERIA IN VIRUSES THAT ARE ASSOCIATED WITH THE CHANGES IN THE MICROBIOME. AND THIS RELATES PRIMARILY TO MECHANISMS ASSOCIATED WITH COLON CANCER OR STOMACH CANCER, H. PYLORI AND PEASO BACTERIA AND NUKE LOMA, WHICH CAN CAUSE CHANGES IN ATOUGH GEE, BETA CATREAT IN ACTIVATION, INHIBIT NK, JUST BE ON THE SURFACE OF THE CELLS. THE VIRUSES GENERALLY ARE INTRACELLULAR AND THEY CAN ACTIVATE ONCOGENES AND CAUSE INFLAMMATION. BUT THERE IS A BARRIER FOR EACH, LIKE I SAID, WITH COLON CANCER THAT HAS p53 MUTATIONS. BACTERIA GETS INTO THE MUCOSA AND THIS LEADS TO PRO-INFLAMMATORY SIGNAL THAT COULD BE EITHER ACUTE, WHICH IS VERY GOOD, BECAUSE YOU GET RID OF THE BACTERIA, OR CHRONIC, WHICH CAN BE BAD BECAUSE IT COULD CAUSE INFLAMMATORY BOWEL DISEASE, IT CAN CAUSE CHRONIC OBSTRUCTIVE PULMONARY DISEASE OR CAN BE INVOLVED IN CANCER. SO WHAT IS DYSBIOSIS? DYSBIOSIS IS A TERM IN WHICH THE MIXTURE OF DIFFERENT KINDS OF MICROBES IN A PARTICULAR HABITAT IS DISRUPTIVE. AND THIS OCCURS WITH ANTIBIOTICS, IT OCCURS WITH CANCER AND THE MOST DRAMATIC EXAMPLE IS SOMETHING CALLED C DIF, WHICH IS INVOLVED IN COLON DISEASE, AND PRIMARILY WITH CANCER THERAPY OR CHRONIC ANTIBIOTIC TREATMENT. AND SO IT IS PRIMARILY JUST ONE BACTERIUM THAT IS DRAWING IN SPITE OF ALL THE REST THAT ARE USUALLY LOST. SO, THIS CAN LEAD TO A FATAL DISEASE, AND THAT PARTICULAR BACTERIA IS USUALLY NOT VERY RESPONSIVE TO ANTIBIOTICS. BUT THE STUDY WE JUST PUBLISHED A COUPLE OF MONTHS AGO, WAS THE FIRST ONE IN LUNG CANCER. AND WE FOUND THAT CERTAIN TAXA, INCLUDING A C DIF, WERE HIGHER IN BOTH ADENOMA AND SQUAMOUS CARCINOMA ACTUALLY IN THE TUMORS AND IN THE TUMOR CELLS IN SMOKERS AND FORMER SMOKERS THAN NON SMOKERS AND THE GROUPS OF TAXA ASSOCIATED WITH SQUAMOUS CELL CARCINOMA WAS ENRICHED IN SMOKERS AND THAT THERE WAS A ASSOCIATION BETWEEN C DIFBORE AXE AND p53 MUTATION AND SQUAMOUS CELL CARCINOMA.& THIS IS AN UNBIASED ANALYSIS OF THE MICROBIOME AND THEN USING MACHINE LEARNING, A RANDOM FOR ANOTHER KINDS OF STRATEGIES. SO THIS IS THE STUDY THAT SHOWED THAT SQUAMOUS CELL CARS GNOME ARE ENRICHED TUMORS WITH p53 MUTATIONS WITH GENE INTERACTION OF LUNG CANCER TISSUE. AND YOU CAN READ THAT PAPER IF YOU ARE REALLY INTERESTED IN THE MICROBIOME. I WOULD SUGGEST THAT YOU SHOULD BE BECAUSE IF YOU’RE INTERESTED IN CANCER, YOU’RE GOING TO FIND THE MICROBIOME IS GOING TO BE INFLUENCING MANY ASPECTS OF THE KINDS OF STUDIES THAT YOU’LL BE DOING. LET’S END UP WITH METABOLOME. THAT’S PRODUCTION OF METABOLITES. AND THE HYPOTHESIS THAT MYA, NOW AT THE FDA AND HEAVY AT OHIO STATE, IN OUR GROUP, WAS UNBIASED METABOLOMICS AGAIN. DISCOVERED BIOMARKERS ASSOCIATED WITH RISK, DIAGNOSIS, PROGNOSIS, THERAPEUTIC OUTCOMES OF LUNG CANCER. AND IN FACT, THAT’S WHAT WE FOUND AT LEAST IN A COUPLE OF STUDIES NOW. AND ONE OF THE LIQUID BIOPSIES THAT WE HAVE BEEN STUDYING IS URINE. AND URINALYSIS. AND SO ARMSTRONG, THIS IS NOT FLOWERING LANGUAGE FROM A LIQUID WINDOW THROUGH WHICH PHYSICIANS FELT THEY COULD VIEW THE BODY’S INTERWORKERS,UNE LED TO THE BEGINNINGS OF LABORATORY MEDICINE — INNER WORK EGGS, URINE LED TO THE BEGINNINGS — AND YOU COULD GO BACK TO HI POCKRAITIES, TASTED URINE AND THE DIAGNOSIS OF DISEASE IN THEIR PATIENTS. NOW WHEN I WEPT TO MEDICAL SCHOOL, WHICH WAS ALSO A LONG TIME AGO, WE DIDN’T HAVE TO TASTE URINE YOU GO THERE WAS URINALYSIS. SO WE COULD TELL IF SOMEONE HAD A LOT OF GLUCOSE IN THEIR URINE WITHOUT ACTUALLY TASTING IT. USING THIS AS A LIQUID BIOPSY AND AGAIN UNBIASED AND THEN USING MACHINE LEARNING, WE CAME DOWN TO FOUR URINE AIRY AND METABOLITES THAT WOULD IMPROVE DIAGNOSTIC AND PROGNOSTIC PREDICTION OF NON-SMALL CELL LUNG CANCER. SO IT WAS ASSOCIATED WITH PROGNOSIS AND THESE ARE KAPLAN-MEIER PLOTS AGAIN. IF ALL FOUR WERE ELEVATED THE PATIENTS HAD A MUCH HIGHER PROGNOSIS. THIS ANALYSIS IS COMBINING THEM AGAIN. SO, IF ALL FOUR ARE ELEVATED, THEN THE AREA UNDER THE CURVE, WHICH IS A SIGNIFICANT VALUE FOR MAKING CLINICAL DECISIONS, WAS ABOUT, ALONG WITH THE MORE CLINICAL STUFF, WAS ABOUT .9, WHICH IS QUITE HIGH. THE QUESTION THAT CAME OR ONE OF THE QUESTIONS THAT CAME UP WAS, WHERE WAS THIS CRETE IN RIBOSIDE COMING FROM? WELL, WE ASKED THE QUESTION, INITIALLY WITH LUNG CANCER, BUT NOW WITH OTHER CANCERS, CAN COMES FROM THE TUMORS ITSELF AND FROM CELL LINES FROM THOSE TUMORS. SO IT’S MUCH HIGHER IN THE TUMOR THAN IN THE MATCHED NON-TUMOR AREA, AND CORRELATION BETWEEN TUMOR LEVELS AND THE URINE IS QUITE REMARKABLE IN TERMS OF THE R VALUE AND THE SIGNIFICANT. SO LET’S END UP WITH SCREENING WHICH I MENTIONED EARLIER. A LANDMARK SCREENING CAME IN 2011. PRIOR TO THAT, IT WAS PRIMARILY JUST CHEST X-RAY, WHICH PROVED NOT TO BE VERY GOOD AT IDENTIFYING SMALL CANCERS. BUT THEN LOW-DOSE TUMOR GRAPHIC SCREENING DEVELOPED AND IN THIS NEW ENGLAND JOURNAL PAPER, IT SHOWED THAT COMPARED TO X-RAYS, THERE WAS A 20% REDUCTION IN MORTALITY BY LOW-DOSE CT. THIS IS FAIRLY LARGE STUDY ALL SMOKERS, BETWEEN AGE 55 AND 70. SOME HAD X-RAYS AND SOME DIDN’T. THAT WAS THE GOOD NEWS. THE BAD NEWS, A HIGH POSITIVE RATE OF 96%. IT COULD DETECT THE SMALLER KINDS OF CANCERS INCLUDING STAGE 1A AND 1B. SO AGAIN 1A LUNG CANCER, NO EVIDENCE OF METASTASIS BUT PATHOLOGY. 3 CENTIMETERS OR LESS AND LUNG B5 CENTIMETERS OR LESS. BUT WITH MANY SCREENING TESTS, FALSE POSITIVES ARE A REAL PROBLEM WHETHER IT IS MAMMOGRAPHY OR VARIOUS KINDS OF BIOPSY TESTS. THERE IS HIGH FALSE-POSITIVE RATE ESPECIALLY USING LIQUID BIOPSIES OF VARIOUS KINDS. SO, IF YOU JUST PICTURE THIS AS 1000 INDIVIDUALS GOING TO A PLAY PLAY, AND THEY ALL RECEIVE THREE SCANS ON AN ANNUAL BASIS, EACH OF THESE BLACKEND AREAS WOULD REPRESENT SOMEONE WHO HAD LUNG CANCER. BUT ALL OF THESE ARE PEOPLE WHO DID NOT HAVE LUNG CANCER. AND THIS IS A INTERESTING PROBLEM FOR PHYSICIANS WHO ARE HELPING THEIR PATIENTS MAKE DECISIONS. AND INCLUDING YOU HAVE A SURGERY OR YOU DON’T HAVE SURGERY. YOU HAVE BIOPSY OR YOU DON’T HAVE BIOPSY. ABOUT 30% OF LUNG CANCERS THAT ARE VERY SMALL, YOU CAN’T REACH BY BIOPSY. SO, THERE IS A LOT OF OVERDIAGNOSIS ESPECIALLY IN PRIVATE HOSPITALS. SO IN SOME HOSPITALS, 50% OF THE PEOPLE GETTING SURGERY WITH THESE VERY SMALL NODULES, WHICH ARE CALLED INDETERMINANT NODULES, ARE ACTUALLY BENIGN. SO, AS SURGEONS SAY, CRACKING SOMEONE’S CHEST IS NOT A BENIGN PROCEDURE. SOMETIMES PEOPLE ACTUALLY DIE. AND IT CERTAINLY IS A LOT OF PAIN AND SUFFERING ASSOCIATED WITH IT. SO, ONE NEEDS TO IMPROVE LOW-DOSE CT SCREENING AND IT’S IMPORTANT FOR MANY REASONS, NOT ONLY BECAUSE THERE ARE A LOT OF PEOPLE WHO ARE ELIGIBLE FOR LOW-DOSE SCREENING, ALMOST 10 MILLION NOW, BUT IT’S PAID FOR BY THE U.S. GOVERNMENT. SO BOTH THE MEDICARE AND AT LEAST AT THIS POINT, AND THE AFFORDABLE CARE ACT, DEPENDING ON WHAT THE CONGRESS DOES AND WHAT THE ELECTION — I WOULD SUGGEST EVERYONE VOTES VERY SOON — SO IT’S PAID FOR. SO IT’S A VERY IMPORTANT SCREENING PROGRAM. WELL, WE PROPOSE THERE ARE A NUMBER OF WAYS OF EXAMINING USING LIQUID BIOPSIES TO IDENTIFY PEOPLE WHO ARE AT HIGH PRIORITY FOR SCREENING BY LOW-DOSE CT. AS I SAID, THERE ARE MANY MILLIONS OF PEOPLE WHO ARE ELIGIBLE BUT AT THIS POINT, THEIR CAPACITY TO SCREEN PEOPLE IS IN THE HUNDREDS OF THOUSANDS. SO THERE NEEDS TO BE SOME PRIORITIZATION IF POSSIBLE, AND THESE ARE THE ONES THAT WE PROPOSE BASED ON LABORATORY DATA. ONCE THE SCAN HAS BEEN DONE, THE QUESTION IS, IS IT A CANCER OR NON CANCER? AND I TALKED ABOUT IN DETERMINANT NODULES. AND THEN EARLY DIAGNOSTIC BIOMARKERS TO IMPROVE, IN COMBINATION OF LOW-DOSE CT, ARE ONE STRATEGY. AND THEN FINALLY, YOU REMOVE THE TUMOR AND STAGE I LUNG CANCER, PARTICULARLY STAGE I A, AT THE MOMENT, IS USUALLY NOT TREATED AFTER SURGERY. IT’S THOUGHT TO BE CURATIVE. BUT ABOUT 25% OF THE PEOPLE WHO HAVE STAGE I A LUNG CANCER HAVE A REOCCURRENCE. SO WE ARE QUITE INTERESTED IN IDENTIFYING THOSE PATIENTS WHO ARE CANDIDATES FOR ADJUVANT THERAPY, IMMUNOTHERAPY OR CHEMOTHERAPY USING A VARIETY PROGNOSTIC BIOMARKERS, NOT ONLY IN LIQUID BIOPSY BUT ALSO IN THE TUMORS THEMSELVES. SO THE OBJECTIVE IS TO USE PREDICTION MEDICINE STRATEGY TO IDENTIFY THOSE 25% OF STAGE 1 LUNG CANCERS WILL REOCCUR AND DIE OF THEIR DISEASE AND DECREASE THE FALLS POSITIVE RATE AND DECREASE FINANCIAL COSTS AND IMPROVE PATIENT CARE AND GUIDE MECHANISTIC STUDIES. SO, RIGHT NOW, STAGE I A IS, IF IT’S LOW-RISK, OBSERVE, WAIT ANOTHER SIX MONTHS AND SEE IF THAT INDETERMINANT NODULE, WHICH IS VERY SMALL, IF IT GROWS OR NOT F THEY ARE AT HIGH RISK, I RECOMMEND THAT IT BE REMOVED. SO, THAT IS THE OBJECTIVE OF SCREENING IN THE STRATEGY THAT WE AND OTHER PEOPLE ARE APPROACHING IT. AND THESE ARE MY COLLABORATORS, FELLOWS IN THE LABORATORY AND COLLEAGUES, AND THESE ARE COLLABORATORS OF PEOPLE THAT WE HAD THE OPPORTUNITY TO COLLABORATE WITH, TO LEARN FROM, AND TO HOPEFULLY DO SOME SYNERGISTIC RESEARCH. SO THAT IS MY STORY ABOUT LUNG CANCER. [ APPLAUSE ] >> [ OFF MICROPHONE ] >> THAT’S NOT ACTUALLY WELL-KNOWN. AND YOU CAN JUST IMAGINE THAT IT DEPENDS ON THE DIVISION AND THE CANCER CELLS. THE AMOUNT OF APOPTOSIS AND AMOUNT OF SINESENCE THAT OCCURS IN THE TUMORS OF VARIOUS KINDS. THAT WILL BE KNOWN WITH SOME OF THESE INDETERMINANT NODULES THAT PEOPLE FOLLOW FOR A LONGER PERIOD OF TIME. AND THEY DETERMINE THAT THEY ARE ACTUALLY A STAGE 1A BECAUSE IT INCREASES THEIR NODULE AND THEN THEY CAN SEE WHETHER OR NOT IT HAS PROGRESSED IN A VERY RAPID WAY OR NOT. BUT IT’S A VERY INTERESTING QUESTION. AND HAS THERAPEUTIC IMPLICATIONS IMPLICATIONS. >> [ OFF MICROPHONE ] >> SO THE QUESTION IS, IS THERE ANY OTHER KINDS OF IMAGING SUCH AS PET SCAN, AND THERE IS LOOKING AT GLUCOSE AND OTHER THINGS, AND THAT’S A VERY RAPID AREA OF INVESTIGATION AND PEOPLE ARE ASKING THEIR OWN QUESTIONS, CAN YOU ACTUALLY SCAN THE TUMORS FOR PD-1? AND IDENTIFY THOSE TUMORS THAT MIGHT BE MORE LIKELY BE CANCER AND THEN SECONDLY, THEY MIGHT BE MORE ABLE TO IMMUNOTHERAPY. SO THAT IS ANOTHER WHOLE FIELD OF RADIOLOGY THAT PEOPLE ARE — INVESTIGATING HERE AND ELSEWHERE ELSEWHERE. GOOD QUESTION. [ APPLAUSE ] THANK YOU.

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