TODAY WE HAVE YVES POMMIER AS OUR FIRST SPEAKER, M.D. AND PhD, University of Paris then he came to NIH, he's chief of developmental therapeutics branch, and he plays the role in the NCI development of therapeutics program, several other committees as well, and he serves as an editor in cancer research. HE ALSO HAS DONE SEVERAL GORDON CONFERENCES, RECEIVED AWARD FROM SOCIETY OF COMEE TALKING TODAY ABOUT TOPOISOMERASE. >> THANK YOU, TERRY. THANK YOU, EVERYBODY. THIS IS NOT RELATED TO ONE DISEASE BECAUSE WE'RE TALKING ABOUT ENZYME. WE'RE TALKING ABOUT UBIQUITIN ENZYMES, DNA TOPOISOMERASES ENZYMES. THERE ARE SEVERAL REVIEWS THAT YOU COULD G O BACK TO AND A NUMBER OF SLIDES ARE FROM THESE REVI EWS. SO, THESE ARE PROBABLY MORE OF A DRUG ORIE NTED ASPECT OF TOPOISOMERASES. AND THE FACT THEY ARE VERY IMPORTANT FOR M EDICINE BECAUSE THEY USE THE TOPOISOMERASE INHIBIT ORS ARE USED TO TREAT CANCER AND USED TO TREAT BAC TERIAL INFECTIONS, INCLUDING TUBERCULOSIS. AND I THINK THIS IS STILL AN ONGOING PROJE CT, STILL BEING DEVELOPED. MOST RECENT REVIEW WHICH I THINK NOW HAS A LARGE NUMBER OF CITATIONS, THE ONE WE WROTE WITH JOHN NITISS, UNIVERSITY OF CHICAGO, JOHN AND I WERE STARTED IN THE FIELD MANY YEARS AGO WHEN T HE FIELD WAS STARTED WITH THE DISCOVERIES OF THE FI RST TOPOISOMERASES, AND THEN SOON AFTER THE DI SCOVERY OF THE STRUCTURE OF DNA, IT WAS CLEAR THE HEL ICO RAISED THE PROBLEM, TOPOISOMERASES WERE DISCOVERE D. THERE'S A BOOK YOU CAN FIND IN THE LIBRARY , ON ALL THE TOPOISOMERASES AND CANCER, SO THERE AR E DIFFERENT ASPECTS OF THAT. SO THIS IS IN THE LIBRARY. I THINK IT'S ONLINE AS WELL. SO, WHY DO CELLS HAVE TOPOISOMERASES AND W HAT DO THEY DO? THE ISSUE WITH DNA IS BECAUSE IT'S HELICO, IT'S A RIGID POLYMER, AND DNA TENDS TO MAKE SUPER COIL, SO THE STRANDS TEND TO WRAP AROUND EACH OTHER , THAT'S WHAT WE CALL SUPERCOIL. AND WHEN THE DNA IS RELAXED, SHOULD TAKE A DOUBLE-STRANDED DNA, WHEN YOU OPEN IT UP, YOU GENERATE ON THE FLANK OF THE BUBBLE THAT Y OU'RE OPENING, SUPERCOILS, SO ON ONE SIDE YOU GE T POSITIVE SUPERCOIL, ON THE OTHER SIDE NEGATIVE SUPE RCOIL. THE TOPOISOMERASES DO THAT, ALL THE TOPOIS OMERASES DO THAT. TOPO 1, TOPO II, TOPO II BETA THEY DO IT E VERYWHERE. TOPO 3S, NEGATIVE DNA, THE OTHER IS WHEN D NA IS REPLICATED, IF YOU REPLICATE A CIRCLE, SUP ERCOIL, YOU END UP WITH THE TWO CIRCLES WILL BE IN TERTWINED, IN BACTERIA, VIRUSES, IN HUMAN CHROMATINS WHEN LOOPS DUPLICATE THEY TEND TO BE INTERTWINED, YOU NEED TO OPEN, LET THE OTHER STRAND GOES THROUGH, DECATINATION. THE ENZYME CAN PASS ONE DUPLEX THROUGH THE OTHER, UNKNOTTING, BUT COULD ALSO KNOT. IT'S NOT UNLIKELY GENOME IS KNOWN, IT HAS KNOTS, ON THE ROPE WHEN YOU GO SAILING THE KNOTS ARE USED TO MAKE PARTICULAR PULLS, MARKS, THAT'S PROBA BLY ALSO IN HUMAN CHROMATIN. ALSO OTHER STRUCTURES MAY EMERGE WHEN YOU HAVE WHAT WE CALL HOLLIDAY JUNCTIONS, TWO DUPLEXES A RE GETTING INTERTWINED DURING RECOMBINATION, TOPO3 AL PHA, AND R & D LOOPS, THIS COULD BE ALSO WITH TOPO3, SO TOPO3 BETA FOR EXAMPLE CAN DO THAT AND CAN ALSO UNKNOT& SINGLE-STRANDED RNA WHEN THE RNA MAKES KNO TS, THERE'S A VERY BROAD RANGE OF ACTIVITY OF THEY ENZYME. IF YOU TAKE A HUMAN CELL, THERE ARE SIX EN ZYMES, AND THE NOMENCLATURE IS GOOD TO KNOW BECAUSE T HE 1A ENZYME OF THE TOPO3 BETA, AND 1B, SO TYPE 1 ENZYME CLEAVES ONE STRAND, SO TOPO 1 AND TOPO 3 C LEAVE, AT THE TIME THE TYPE 2, DUPLEX OF DNA, SO TOP O 2 AND TOPO 2 BETA AND HUMANS HAVE SIX. E. COLI DON'T HAVE 1B, EMERGED FROM VIRUS THAT INFECTED EARLY CELL AND THEN WAS HIJACKED TO RELAX DNA. BUT E. COLI HAS ONE. TOPO 1 AND 3, GYRASE AND TOPO4. TOPO 1 AND 1MT, ANTI-CANCER TARGET OF WIDE LY USED DRUGS, TARGET CAMPOTOTHECINS. GYRASE AND TOPO-IV HAD TARGETS OF QUINOLON ES. AND THEY DON'T CROSS OVER. SO THE ANTIBACTERIALS DO NOT POISON THE HO ST ENZYME. BUT THE TOPO-II INHIBITORS TO NOT POISON T HE BACTERIA, SO THERE ARE DIFFERENT DRUGS. IF YOU THINK OF THE TOPO, I AND III, ODD N UMBERS HAD SINGLE STRAND BREAKING. IT WILL MAKE A KNICK, SINGLE STRAND BREAK, KNICK, AND THE NICK IS THEN USED TO RELAX DNA. THE DRUGS ARE SPECIFIC FOR EACH CLASS, BIN D, WHICH IS THE CLEAVAGE COMPLEX. SO THESE ARE CLEAVAGE COMPLEXES, THIS IS T HE NAME GIVEN. WE AND THERE IS NO INHIBITOR FOR TOPO 3, THER E ARE SIX ENZYMES, MOLECULAR SIDE, TOPO 2 IS HOMODIM ER, VERY LARGE PROTEIN, HOMODIMERS, TOPO 2 ALPHA AN D BETA. IF YOU TAKE A HUMAN CELL THERE ARE TWO KIN DS OF GENOMES, NUCLEAR DNA AND MITOCHONDRIAL DNA . DEPENDING ON THE CELLS, THE AMOUNT OF MITO CHONDRIA COULD BE UP TO 5% OF THE CELLULAR GENOME I S MITOCHONDRIAL DNA. THE TOPO 1 IS SPECIFIC FOR NUCLEAR TOP1MT IS SPECIFIC FOR THE MITOCHONDRIAL GENOME, THE RE'S A CLEAR DIVISION OF LABOR, TOP1MT. FOR TOP 2 ALPHA AND BETA, THEY GO IN BOTH. ALSO BOTH GENOMES HAVE THEIR OWN REPAIR EN ZYMES, I WILL DESCRIBE THAT AT THE END. LET'S TAKE THEM ONE AT THE TIME, START WIT H TOP 1 AND THE REPAIR. TOP 1, THERE ARE TWO NUCLEAR. WHEN WE DISCOVERED TOP1MT, IN 2000, WE DEC IDED NOT TO CHANGE THE NOMENCLATURE AND NOT TO CALL TOP1 ALPHA AND THE BETA, WE DECIDED TO CALL IT MT, MITOCHONDRIAL, BECAUSE IT WAS SPECIFIC FOR MITOCHONDRIA. SO AS I SAID, THE DIFFERENCES BETWEEN THE TOPO 1 AND TOPO 2 ARE MAJOR. THEY ARE BOTH TOPOISOMERASES BUT ARE DIFFE RENT. ONE A MONOMER, ONE A DIMER, TOPO 2 MAKES D OUBLE STRAND BREAK STAGGERED BY FOUR BASE PAIR. THE BREAKAGE OF THE DNA INCUMBENT THE ENZY ME USES TYROSINE. YOU SEE FOR TOPO 1, TARGET DNA BACKBONE, A ND FOR 2 IT'S ALSO TARGETING, BUT THE POLARITY IS D IFFERENT. IN THE TASTE OF TOPO 1 IT LINKS, SO THIS I S PROBABLY THE MOST CANONICAL MECHANISM FOR TOPOISOME RASE IN CLEAVAGE OF THE BACKBONE. IT CLEAVES THE BACKBONE, AND ENABLING ANOT HER STRAND TO PASS THROUGH THE BROKEN. 1 DOESN'T USE ATP, HAS ITS OWN INHIBITORS, TOPO 2 CONSUMES ATP, REQUIRES MAGNESIUM AND HAS ITS OWN SET OF INHIBITORS. TOPO 1, DNA UNTWISTING ENZYME, IF YOU WANT TO CALL IT THIS WAY, CRYSTAL STRUCTURE SHOWS THE E NZYME BINDS THE DNA AND MAKES A COVALENT BOND TO ONE OF THE ENDS OF THE DNA, AND THEN THE OTHER ST RAND ROTATES INSIDE THE ENZYME, TO CONTROL ROTA TION THAT DISSIPATES THE SUPERCOIL, REALIGNS AND THE N THE DNA RELIGATES. TOPO 1 IS ESSENTIAL FOR TRANSCRIPTION AND REPLICATION, DISSIPATES THE SUPERCOIL AND POTENTIALLY FOR DNA REPAIR. YET IN YEAST THE KNOCKOUT OF TOPO 1 IS VIA BLE, COMPENSATED BY TOPO 2 AND 3, BUT IN MOUSE CELLS, HUMAN CELLS, TOPO 1 IS ESSENTIAL. SO, WHAT I DESCRIBED AT THE BEGINNING, THI S IDEA WHEN DNA IS BEING TRANSCRIBED OR REPLICATE D, TRENDS BEING SEPARATED BY HELICASE, WHAT HAPPENS WHEN YOU HAVE A NUCLEAR MATRIX ATTACHMENT GENERATE SUPERCOIL ON ONE SIDE, NEGATIVE SUPERCOIL ON THE BAC K. FOR THE MOVEMENT YOU NEED TO DISSIPATE THE SUPERCOIL, OTHERWISE YOU CANNOT MOVE ALONG THE DNA DUPLEX. AND THIS DISSIPATION IS MOSTLY DONE BY TOP O 1, BINDING IN HELICO DOMAIN, ENABLE DISTRIBUT ING. FORMS CLEAVAGE COMPLEX NORMALLY AS WE SPEA K YOU'RE DOING IT ALL THE TIME, RELAXING THE DNA, N OTHING HAPPENS. CLEAVAGE COMPLEXES ARE INVISIBLE. EXCEPT WHEN THERE IS A DRUG LIKE TOPO 1 IN HIBITOR GIVEN TO A CANCER PATIENT, THE DNA IS DAMA GED, THEN RELIGATION IS NOT PROPER, CLEAVAGE COMPLEX ES CAN FORM. THIS IS IMPORTANT BECAUSE THIS IMAGE COMPL EX IN ADDITION TO DRUGS CAN BE INDUCED BY DNA LE SIONS, SOME FREQUENT, BASIC CARCINOGENIC ADDUCTS, THIS IS THE REASON SOME NEUROLOGICAL SYNDROMES WHE N THERE ARE DNA REPAIR DEFICIENT -- DNA REPAIR IS ALTERED, REPAIR IS ESSENTIAL. SO, THE TWO TOPO 1, NUCLEAR AND MITOCHONDR IA TOPO 1, DIFFERENT BY N-TERMINUS, UNCODED IN TWO CH ROMOSOMES, NUCLEAR TOP 1, SEQUENCE, TOP1MT WHICH EMER GED IN VERTEBRATES, THERE ARE TWO, AND IT HAS A MITOCHONDRIAL TARGETING SEQUENCE, VERY SHO RT BUT VERY EFFICIENT IN THE N-TERMINUS. IN THAT SENSE THE MITOCHONDRIA IN TOP 1 LO OKS LIKE THE VIRAL TOPO 1, IF YOU TAKE THE VACCINIA , IT'S A SHORT TRUNCATED VERSION OF THE HUMAN CELL. SO TOPO 1s ARE THEIR OWN SPECIFIC INHIBITO RS, DISCOVERED BY NCI, OUT OF THIS SCREEN OF P LAN EXTRACTS IN THE 1970s, BUT THE NCI WAS LOO KING FOR NEW CURES FOR CANCER, NEW DRUGS, NATURAL P RODUCTS, AND FOUND THIS VERY ACTIVE TOXIN FROM THIS PLANT, CAMPTOTHECIN, THE PRESENCE OF THE CHEMICAL IN THE PLANS. THIS CAMPOTHECIN IS VERY UNSTABLE. TODAY THERE ARE TWO USED CLINICAL AT THE C LINICAL CENTER EVERYWHERE IN THE WORLD, CAMPTOTHEC IN DERIVATIVES, MODIFIED TO MAKE THEM WATER S OLUBLE BECAUSE CAMPTOTHECIN IS WATER SOLUBLE. THE PRO DRUG, METABOLITE, OTHER CAMPTOTHEC IN IN OTHER PARTS OF THE WORLD. THEY DO THEY BLOCK TOPO 1? WE DISCOVERED THAT SOME YEARS AGO, TO DISC OVER A PARADIGM FOR DRUG MECHANISM OF ACTION. CAMPTOTHECIN LOOKS LIKE A BASE PAIR, WHEN WE LOOKED, THIS IS A TOPO CLEAVAGE COMPLEX WHICH GOES BACK AND FORTH, WHEN YOU PUT CAMPTOTHECIN, YOU GET A BREAK, BINDING AT INTERFACE HERE, BASED ON DNA AN ALYSIS AT THE TIME, TOOK TEN YEARS TO CONFIRM CAMPTO THECIN, THIS IS THE DERIVATIVE, IN GREEN IS DNA, T HIS IS THE BREAK, AND YOU CAN SEE THE DRUG BEAUTIFULL Y INSIDE THE CLEAVAGE COMPLEX. SO THIS IS WHAT WE CALL THE DRUG DOESN'T B IND ALONE, OR TO DNA ALONE BUT NATURE SELECTED THAT D RUG TO BIND ONLY IN THAT PARTICULAR POCKET. AND WHEN IT BINDS, THE TOPO IS SEQUESTERED INTO CLEAVAGE COMPLEX, CAN STILL EVENTUALLY GO BACK BUT ONCE THE DRUG IS IN IT IT WILL ACTUALLY PO ISON THIS. SO WE CALL THAT THE ENTER FACIAL INHIBITIO N BECAUSE THE DRUG BY THE INTERFACE OF TOPO 1 AND DN A. ANOTHER POINT THAT CAME VERY EARLY, VERY E ARLY IN YOUR LIFE, 1988 WHEN THE CAMPTOTHECINS WER E LOOKED AT BEFORE THEY WERE APPROVED IN 2000, IT W AS NOTED THAT IN YEAST THE CAMPTOTHECIN WERE INACTI VE. WHAT WAS FOUND INSTEAD OF SAYING THAT YEAS T COULD NOT TAKE UP CAMPTOTHECIN, IS THAT BY DOING MUTANTS OF RECOMBINATION, THEY WERE ALL SENSITIVE TO CAMPTOTHECIN. MOREOVER, IF YOU KNOCK OUT TOPO 1 IN THOSE , THEY BECOME RESISTANT AGAIN. THIS WAS CONFIRMED IN A PAPER IN 1988, AND CLEARLY SHOWING KEY PATHWAY FOR SURVIVAL TO CAMPTO THECIN, AND TO ME HISTORICALLY THIS SAYS CAMPTOTHE CIN WAS THE FIRST DRUG SHOWING LETHALITY IN HOMOLO GOUS RECOMBINATION CELL, FIRST DISCOVERED FOR T HE CAMPTOTHECIN, 1988, NOT 2012. NEW TOPO 1 INHIBITORS USED IN CLINICAL CEN TERS, BECAUSE CAMPTOTHECIN WERE EFFECTIVE, TOP 1 WAS VALIDATED, WE KNOW AMONG DRUG THOUGHTS IF YOU TAKE CHEMICAL CLASS THEY HAVE DIFFERENT ANTI-CA NCER ACTIVITY. FOR EXAMPLE, COLCHICINE IS USED TO TREAT G OUT, VINBLASTINE TO TREAT CANCER, CHEMICALLY TH EY LOOK DIFFERENT. THE IDEA MAYBE WE SHOULD LOOK FOR OTHER CH EMICALS, ALSO WE KNEW CAMPTOTHECIN HAD LIMITATIONS, SPECIAL TOXICITY, DRUG SUBSTRATE, CHEMICAL INSTABI LITY IS WHAT I TOLD YOU, IN THE BLOOD IN MINUTES C ONVERTED TO CAROBXIATE, BINDING TO CLEAVAGE COMPLEX , WE DEVELOPED TOP 1 INHIBITORS, AND DEVELOPMEN T NOW IS BEING BETTER UNDERSTOOD IN CONTEXT OF THE FACT THAT CAMPTOTHECIN IS COLLECTIVE FOR BRCA. DRUGS THAT HAVE GONE THROUGH THE CLINIC NO W WERE MADE AT PURDUE UNIVERSITY. WE STARTED THIS EFFORT IN 1998, AND IT TOO K MANY, MANY YEARS TO GET THE DRUG TO THE CLINIC B UT NOW THEY ARE IN THE CLINIC. ONE IS LMP400, AND LMP776 AND LMP744. THESE DRUGS RECENTLY ABOUT A YEAR AGO WE R ELEASED DATA WHERE WE TESTED DRUGS IN THE CONSORTI UM OF NCI. SO THESE ARE CLINICS AROUND THE UNITED STA TES, AND THE OWNER OF DOGS WHO HAVE CANCER CAN BRIN G THE PETS TO THE CLINIC AND THEN GET AN EXPERIMENTAL TREATMENT FOR NO COST. WE DECIDED TO LOOK AT THESE DRUGS IN DOGS WITH TUMORS. AND WE COMPARED THREE OF OUR INHIBITORS, A ND WE DID LYMPHOMA. THE IDEA WAS TO COMPARE ACTIVITY OF THE TH REE DRUGS TO DO PHARMACOKINETICS, EASIER IN DOGS THA N HUMANS, LOOK AT TARGET ENGAGEMENT, WITH JAMES DORO SHOW, AND AMY LEBLANC, RUNNING THE VETERINARY TRIALS , AND IT TURNED OUT SURVIVAL TUMOR RESPONSE PLOT IN ALL THE DOGS, EACH BAR IS A DOG, IF IT GOES UNDER IT MEANS YOU GET ACTIVITY. THE COMPOUND IN MOST OF THE DOGS, THIS ONE WAS THE BEST COMPOUND ACTUALLY IN THE TRIAL, WHICH IS INITIALLY NOT PUT IN THE CLINIC. SO, AT THIS STAGE WE FOUND THAT THE TWO CL INICAL COMPOUNDS WERE ACTIVE IN DOGS AND THEREFOR E THAT PUSHES FOR PHASE 2 CLINICAL TRIAL, AND THE THIRD COMPOUND WAS EVEN BETTER. THE DOSE, BONE MARROW IS EXPECTED FOR CHEM OTHERAPY, NO DIARRHEA, A BIG DEAL BECAUSE CAMPTOTHEC IN HAS THIS PROBLEM, AND LONG HALF-LIFE, SHOWS TU MOR RETENTION, SHOW TARGET ENGAGEMENT. SO, HAVING DONE THIS SO FAR WITH THE TOP 1 INHIBITOR THE NEXT QUESTION BECOMES WHEN YOU START F ACE 2 CLINICAL TRIAL YOU HAVE TO SELECT FOR PATI ENT SUSCEPTIBLE TO RESPONSE AND MEASURE SOMETH ING THAT WILL MAKE HYPOTHESIS. THE IDEA IS TO HAVE A MOLECULAR SIGNATURE. & WHO WILL RESPOND? SO I'VE TOLD YOU ALREADY HRD, BRCA PATIENT S SHOULD RESPOND, THE OTHER THING WE DISCOVERED IS DETRIMENTAL RESPONSE, SLFN11, GENE EXPRESS ION MARKER, WHICH IS IMPLEMENTED IN THE CLINI C. WE'RE DOING TARGETED DELIVERY TO MINIMIZE SIDE EFFECTS. THE NEXT PHASE DONE ALREADY FOR CAMPTOTHEC IN, THE PAYLOAD, COUPLED WITH ANTIBODIES. MOST PROMISING COMPOUND FROM JAPAN IS CAMP TOTHECIN DERIVATIVE WITH HER-2 ANTIBODY, RESPONSES ARE VERY, VERY GOOD. IT'S VERY LIKELY THE DRUG IS GOING TO BE F DA APPROVED BY THE END OF THE YEAR, OR VERY E ARLY NEXT YEAR. SO WE'RE PUTTING OUR OWN DRUGS ONTO THAT P ARTICULAR APPROACH. SO, TOP 2, ALPHA AND BETA, I CALL THEM TOP 2A AND TOP2B. THE DIVISION OF LABOR IS TOP2A IS PRIMARIL Y INVOLVED IN REPLICATION, REPLICATING DNA, RELAXING SUPERCOILS WHEN THEY ARISE WHEN DNA STRANDS DUPLEX CR OSS DURING REPLICATION. TOP2B IS MOSTLY INVOLVED IN TRANSCRIPTION, AND REGULATING PROMOTERS, AND PROMOTERS ENHANC ERS, SO THIS IS TRANSCRIPTION, NON-REPLICATION, SO THESE ARE THE SHAPE OF THE TWO ENZYMES, VERY SIMILAR . THEY ARE CATALYTIC POWER IN THE MIDDLE, AL PHA AND BETA ARE MOST VARIABLE IN THE C-TERMINUS. ALL THIS PART IS VERY COMMON. IF YOU COMPARE TO BACTERIA COUNTERPARTS, T HEY ALSO ARE SIMILAR, EXCEPT BACTERIA, HUMAN IS DIM ER 2 ALPHA, TIMER 2 BETA, IN BACTERIA YOU HAVE A TETRIMER, BUT MACHINERY IS THE SAME. WE KNOW ENZYMOLOGY VERY WELL. TOPO 2 TAKES TWO DUPLEXES, ONE DUPLEX OF D NA, ANOTHER DUPLEX OF DNA. GOES UNDER THE OTHER, NOT GOING THROUGH, T HIS IS GOING TO BREAK MY ARM, YOU'RE GOING TO PUT IT BACK, AND THEN MY OTHER ARM IS THERE. HOW DOES IT DO THIS? TWO DUPLEX, GATE AND TRANSPORT STRAND, ENZ YME WILL FIRST ENGAGE ONE OF THE DUPLEXES, PUT THE ONE ON THE TOP, CLOSE A LITTLE GATE, OPEN THAT OTHER STRAND, IT GOES THROUGH, ONCE IT'S THROUGH IT RELIGA TES AND RELEASES ATP. THIS IS THE GATE. TWO DOORS. EXACTLY WHAT YOU DO WITH TOPOISOMERASE. YOU STAIN THE MIDDLE OF TWO GATES AND GO T HROUGH THE NICK, THAT'S HOW IT RELAXES DNA. IT'S A GATED SYSTEM, AT LEAST TWO GATES, M AYBE THREE BECAUSE IN THE MIDDLE IT BREAKS. THE DNA NEVER LETS GO, DOUBLE STAND BREAK IS NEVER VISIBLE INSIDE THE TOPO 2, CAN DECATINATE AND CATINATE, CAN RELAX SUPERCOILING ON TWO ST RANDS, THE STRANDS PASS ON TOP OF EACH OTHER. IT CAN ALSO INDUCE NEGATIVE SUPER GYRASE. THIS DOUBLE STRAND DNA LOOKING AROUND AND COMING TOGETHER IS VERY REMINISCENT OF WHAT HAPPE NS WITH TRANSCRIPTION WHEN YOU HAVE THE ENHANCERS WITH PROMOTERS, AND DNA MAKES A BIG LOOP, AND W E NOW KNOW VERY WELL TOPO2B IS CLOSE TO THE REGIONING NOT ONLY RELAXING DNA BUT PROBABLY RIGHT AT THE PRO MOTER, SO THERE'S A LOT OF ACTIVITY ESPECIALLY IN NE URONS, AND TO KNOW HOW GENES ARE TURNED ON AND OFF AN D HOW TOPO2 BETA IS PART OF THIS PARTICULAR GYMN ASTIC. IN REPLICATION, TOPO 1 WILL RELAX SUPERCOI LS. IT DOESN'T NEED DNA, JUST TENSION, IT WOUL D JUST RELAX. TOPO 2 NEEDS TRANSFERENCE WITH THE OTHER. WHEN DNA IS REPLICATED, SOMETIMES YOU GET REPLICATION, CATINASE, AND YOU HAVE CONVER GING, YOU HAVE THIS, I TOLD YOU ALREADY DECATINATION . AS FAR AS DRUGS THAT TARGET TOPO 2, FOR TH E HUMAN THERE ARE A FLURRY OF DRUGS, VERY WIDELY U SED. THERE IS THE DOXORUBICIN, AND THERE ARE A NUMBER OF OTHER DRUGS, INHIBITORS ARE SHOWN HERE. FOR BACTERIA TOPO2 ALL THE ANTIBIOTICS YOU 'VE HEARD ABOUT, YOU KNOW, THE QUINOLONES SPECIFIC, THEY WORK IN THE SAME WAY. BY THIS I MEAN IT'S THE THE SAME INHIBITI ON. THIS IS A CRYSTAL STRUCTURE, HOMODIMER, DN A IS HERE INSIDE THE ENZYME, AND IF YOU LOOK FROM TH E TOP HERE YOU SEE THROUGH THE ENZYME YOU SEE THE DNA . AND THE WAY THE DRUG POISONS THE TOPO 2 IS BINDING AT THE INTERFACE OF THE TOPO2 AND THE DNA. SO AGAIN YOU'RE GETTING THE SAME PRINCIPLE OF AN INTERFACIAL INHIBITION. ANTIBACTERIAL IS THE SAME PRINCIPLE, THIS IS GYRASE, FOUR SUBUNITS, SAME KIND OF STRUCTURES, YO U CAN SEE VERY SIMILAR. LOOK FROM THE TOP, AND HERE IS A QUINOLONE . IT'S AGAIN BOUND AT THE CLEAVAGE SITE. WHAT DRUGS DO, THEY BIND AT CLEAVAGE SITE, THE LIGATION MAKES CLEAVAGE COMPLEXES. IT'S VERY PROMINENT MECHANISM THAT WAS I T HINK FIRST SEEN BY TOPOISOMERASES INHIBITORS FOR MANY MACROMOLECULES IN THE BODY, PROTEIN INTERA CTION COULD BY NATURAL PRODUCTS LIKE THAT. SO THESE ARE THE TYPE OF INTERFACIAL INHIB ITION, THE CAMPTOTHECIN, QUINOLONE IS AT THE CLEAVAGE SITE, AND THE DRUG SPECIFICITY IS DRIVEN BY SPECIFIC HYDROGN BONDS TO THE PEPTIDE CELL. FOR TOPO 2 AS CELL. SELECTIVITY FOR THESE DRUGS FOR THEIR PART ICULAR ENZYME IS NOT ONLY TO THE DNA STACKING INS IDE THE BREAK SITE BUT ALSO TO SPECIFIC INTERACTIO N, PROPERLY POSITIONED FOR EACH ROW AND FOR E ACH ENZYME. TOPO3, SO AGAIN WE GET TWO TOPO 3s, TOPO3 ALPHA UNCODED BY A, TOPO 3 ALPHA PRIMARILY INVOL VED IN REPLICATION, AND IT NEEDS TO HAVE SINGLE-S TRANDED REGION TO FUNCTION. SO THAT HAPPENS IN ANY CATINASE, PREVENTS RECOMBINATION, SO DEFICIENT TOPO 3A, COUPL ED WITH HELICASE, USUALLY HYPERRECOMEBINOGENIC, RE MOVES THE JUNCTION. TOPO 3B IS ONGOING WORK, RESULTS IN R LOOP S AND D LOOPS, THE ONLY ONE IN HUMAN CELL. THERE'S IS AN RNA TOPOISOMERASES, WIDELY K NOWN IN THE COMMUNITY BUT NOT TO THE WIDE COMMUNIT Y. THIS IS THE ONLY RNA USED FOR HUMAN CELLS, RODENT AND SO ON. SO, WHY WOULD YOU USE TOPO 2 VERSUS TOPO 3 DURING REPLICATION? IF YOU TAKE THE TOPO 2 PATHWAY, YOU CAN DE CATENINE ALL THESE TWISTED MOLECULES AND SEPARATE T HE TWO REPLICATED CIRCLES. BUT SOMETIMES YOU COULD GET AT THE END A S ORT OF -- SEE THE LOOP STRUCTURE, TOPO 2 DOESN'T REC OGNIZE THIS BECAUSE IT'S SINGLE-STRANDED. THAT'S WHERE YOU NEED TOPO 3 ALPHA, WHICH WORKS TOGETHER WITH BLM HELIC CASE, AND REMINISC ENT IN BACTERIA THE SAME SYSTEM, TOPO 1A, IN YEAS T, SO IN REPUTATION TOPO 3 ALPHA IS ALWAYS NEEDED. WHAT ABOUT TOPO 3 BETA, IT WAS FORGOTTEN F OR MANY YEARS, AND IN 2013 IT WOKE UP, WITH TWO PA PERS SHOWING THAT IN A GROUP OF PATIENTS IN FIN LAND, HIGH PREVALENCE OF NEUROLOGICAL DISEASE, WHEN T HEY SEQUENCED PATIENT, THE LOCUS, TOPO 3 BETA IS IMPORTANT FOR TRANSCRIPTION AND VERY IMPOR TANT AS AN RNA TOPO ISOMERASE, SO THIS WAS DONE IN BA LTIMORE AT THE AGING INSTITUTE AND BY LARGER GROUP OU TSIDE. AND THE POI NEAR -- AND STILL WORKING ON THIS. THEY ARE VALUABLE IN SPITE OF THE IMPORTAN CE OF THE ENZYME. SO, HOW DOES TOPO 3 WORK? THEY USUALLY WORK IN CONJUNCTION WITH HELI CASE, IN TOP 3B, SCAFFOLDING PROTEIN, RMI. THOSE WHO STUDY TRANSCRIPTION AND RNA META BOLISM, YOU KNOW ABOUT PROTEIN. IT'S MAKING A CONNECTION WITH NEUROLOGICAL DISEASE IN TOPO 3 BETA AS WE MOVE ALONG. TOPOISOMERASES GENOMIC INTEGRITY AND HUMAN DISEASES, OVEREXPRESSION OF OR LACK OF REPAIR LEADS TO GENOMIC INSTABILITY. IT'S BECOMING MORE AND MORE OBVIOUS THIS I S HAPPENING IN PATIENTS. WHEN I ADD CLEAVAGE COMPLEX ON DNA AND REP LICATION COLLIDES, YOU GENERATE DOUBLE STRANDS. THAT'S LETHAL. IF TOP 2CC IS SEVERED, IT'S AS IF YOU'RE T RYING TO BREAK THE DNA, YOU'LL NEED A BIG TRUCK TO DO THAT, NEEDS A LOT OF ENERGY, BUT TOPO 2 BREAKS M AY SEVER. IN SOME CASES YOU LEAVE BEHIND SUPERCOILS, OR KNOTS, OR CATENANESE, AND THIS IS DISASTER AT MIT OSIS. WE LISTED NUMBER OF CAUSES OF ALTERATION T HAT CREATE CLEAVAGE COMPLEXES AND LIST OF ENZYMES THA T DO THAT. FOR TOPO 1, YOU HAVE OXIDATIVE DAMAGE, TOP IS STUCK AND NOT REPAIRED, IF THERE IS TRANSCRIPTIO N IT WOULD COLLIDE, IT WILL FORM A HUGE COMPLEX, IF I T HAPPENS IN A REPLICATING CELL, THE REPLICATION COL LIDES, YOU MAKE DOUBLE STRAND AND SINGLE-STRANDED DOU BLE STRAND BREAK, AND THAT IS IRREVERSIBLE. THERE ARE DIFFERENT WAYS TO DEAL WITH THAT . WHEN THEY DON'T, YOU GET HUMAN DISEASE. FOR TOPO 1, LACK OF TOP1CC, BUT TOP2 BETA, CHROMOSOME TRANSLOCATION AS T2BCC, SECONDA RY LEUKEMIA, THOUGHT TO BE DUE TO TRANSLOCATI ON ARISING FROM COMPLEXES NOT REPAIRS. TOPO3B, NEUROLOGICAL DISORDERS. AND TWO REPAIR ENZYMES FOR CLEAVAGE COMPLE XES THAT LEADS TO ATAXIA AND NEUROPATHY. SO THE TOPO CLEAVAGE COMPLEXES AS I MENTIO NED GO THROUGH A COVALENT INTERMEDIATE, YOU SEE F OR THE TOPO 1, 1 AND MT, LINKAGE, FOR 2 AND 3 YOU GET A FIVE PRIME LINKAGE, NORMALLY REVERSIBLE, E LIMINATE THE PHOSPATE AND GET THREE PRIME, BUT IF I T GETS STUCK YOU GET CLEAVAGE COMPLEXES, WITH A C OVALENT TOPO CLEAVERRAGE COMPLEX, BREAK AND DNA PR EPPING CROSSLINK. HOW IS THIS REMOVED? THIS IS THE SCHEME OF THIS DRAWN IN DIFFER ENT WAYS. THIS IS TOPO 1 FOR INSTANCE, COVALENT CROS S-LINK FOR DNA BACKBONE, YOU CAN SEE COVALENT LINKAGE , LET'S ASSUME THIS IS NOT REVERSIBLE. WE KNOW CELL HAS TWO MAIN PATHWAYS, REMOVE LESIONS, ONE IS SURGICAL PATHWAY, DIESTERASE, PHOSPHO-DIESTERASE, TDP1 CAN EXCISE THEM. FOR TOPO2, THE POLARITY IS OPPOSITE, FIVE PRIME LINKAGE. TYROSINE DNA, ESTERASE, PDP2, AND THEN IT WILL RELEASE THE TOPO AND DNA REPAIR CAN TAKE P LACE. THERE'S BACKUP. BECAUSE THIS IS PROBABLY AT LEAST SO OFTEN THAT MANY WAYS TO DEAL WITH IT BEYOND THE PDPs, THEY USE NUCLEASE. AND THE NUCLEASE WILL CHOP THE DNA WITH TH E TOPO AND RELEASE THE THING AND DO A LITTLE GAP CLEA NING. SO, THAT'S TRUE FOR TOPO 1 AND TOPO 2, AND THESE NUCLEASES YOU'VE HEARD VERY PROMINENT LIKE MRE11, OR 81, AND WE KNOW IN BRCA DEFICIENT TUMORS M R 11 IS VERY DEFICIENT, SO THERE'S LACK OF REPAIR OFTY TOPO2 LESION, HYPOTHESIS MADE THAT IN BREAST CAN CER THE PREVALENCE OF BRCA DEFICIENCY IS BECAUSE B RCA IS NOT HELPING MR 11 TO REMOVE TOPO 2 CLEAVAGE CO MPLEX. THE IDEA OF PROMOTERS DRIVEN BY ESTROGEN, HE GET A LOT OF TOPO 2 CLEAVAGE COMPLEXES, MEMORY C ELLS DURING THE MENSTRUAL CYCLE OR LACTATION, I F THEY ARE NOT REMOVED AND BRCA NEEDS TO HELP TO BRIN G THE NUCLEASE TO THIS LESION, AND ONE OF THE ME CHANISMS PROPOSED ARE SELECTIVITY FOR OVARIAN AND B REAST CANCER IS BRCA CANNOT DRIVE THE REPAIR OF TOP 2. SCHEMATICALLY, YOU COULD THINK OF IT THIS WAY, TWO MAIN WAYS TO DEAL WITH THIS IS PHOSPHO-DIE STERASE, TDP1, ENZYMES ARE NOT EFFICIENT, THAT LEAD S TO NEUROLOGICAL SYNDROME, MOSTLY. IF NOT EFFICIENT, YOU ALSO GET (INDISCERNI BLE). SO TWO TDPS HAVE DIFFERENT ACTIVITY, DISCO VERED BECAUSE OF TOPOISOMERASES, BUT 1 HAS A GRE ATER RANGE OF DNA REPAIR FUNCTION, THIS IS ALSO NOT S O WELL ESTABLISHED, NOT SO WELL ACKNOWLEDGED, I W OULD SAY, IN THE DNA REPAIR FIELD BUT THIS REPAIR A LOT OF LESIONS BY ACTIVATING AGENT, BLOCKING LESI ON, THREE PRIME REPLICATING DNA, IT WORKS IN MITOCHO NDRIA, NUCLEASE, AS I'VE TOLD YOU TDP1 DEFICIENCY IS FOUND IN PATIENTS WHO HAVE NERVOUS SYSTEM STABIL ITY. IT IS ALSO INVOLVED PRESUMABLY DURING PROT EOVIRUS REPLICATION TO EXCITE THE TYROSINE OF THE END OF THE NUCLEIC ACIDS. SO NORMAL CELLS, TWO PATHWAYS TO REPAIR CO MPLEXES, COUPLED WITH PARP1, ALTERNATIVE PATHWAY IS XPF-ERCC1. IF ONE IS DEFICIENT, CELLS RELY ON THE SEC OND. THERE'S LETHALITY HERE, IF YOU BLOCK THE P ATHWAY WITH PARP INHIBITOR, THE CELLS BECOME EXCL USIVELY DEPENDENT ON THE MR 11 XPF PATHWAY, AND MA Y BE PROVIDING AN OPPORTUNITY TO GIVE PARP INHI BITORS WITH TOPO 1 INHIBITORS IN MRE11, AND MUTAT IONS ARE NOT RARE, THEY ARE FOUND IN MISMATCH REPAI R COLON CANCER, IT'S BEEN UNDERAPPRECIATED BUT IF WE TOOK THOSE PARTICULAR PATIENTS' TUMORS, MRE11 D EFICIENT, AND GAVE A TOPO1 INHIBITOR PLUS PARP INHIB ITOR MAYBE WE WOULD SEE RESPONSES WHERE WE DON'T SEE THEM NOW. SO I'D LIKE TO MAKE ACKNOWLEDGMENT, A LOT OF DRUG WORK IS DEVELOPING INDENOISOREQUESTSQUINOL INE. I'D BE HAPPY TO QUESTIONS OR IF YOU WANT T O SEND ME AN E-MAIL IT'S EASY TO FIND MY E-MAIL. >> THANK YOU. [APPLAUSE] ANY QUESTIONS? SO HOW FAST DO THE TOPOISOMERASESES UNWIND THE DNA? >> THE SPEED WE CALCULATED WITH NEWMAN IN THIS BUILDING, DOING SINGLE MOLECULE ANALYSIS, AND MY APPROXIMATION, ROTATION IS ABOUT 6,000 RPM . SO GIVES YOU -- >> PRETTY FAST. >> PRETTY FAST. THESE THINGS WORK IN A SECOND, A MILLISECO ND, WHEN WE DO BIOLOGICAL EXPERIMENTS IN HOURS AND MINUTES,& IT'S NOT AT THE SCALE OF WHAT CELLS DO. CELLS WORK IN THE MILLISECOND. AND THE PASSAGE, I DON'T KNOW QUICKLY PASS AGE, GOING THROUGH NIH, I THINK -- I DON'T KNOW THE S PEED ACTUALLY. >> THESE THINGS WORK VERY, VERY EFFICIENTL Y AND CONTINUOUSLY. YOUR CELLS, MY CELLS, WORKING ALL THE TIME . WHETHER WE TRANSCRIBE OR REPLICATE OR WHAT EVER WE DO. A VERY ESSENTIAL MACHINE. >> AND DO THEY CAUSE -- ALTER THE CELL CYC LE? DO CANCEL CELLS BUILD UP IN. >> USUALLY CANCER CELLS OVEREXPRESS THE TOPOISOMERASES BECAUSE THEY REPLICATE FAST , TRANSCRIBE A LOT, VERY COMMON OVEREXPRESSI ON OF TOPO1, IN BREAST CANCER AMPLIFICATION WITH HER-2 OF TOPO 2 ALPHA WHICH IS NEAR, AND THAT PROBA BLY ENABLES TO REPLICATE AND TRANSCRIBE QUICKL Y ONCE YOU PUT TOPO POISON, THEY ARE SENSITIVE TO POI SON, SO BECAUSE THEN YOU POISON THE ENZYME THAT TH E ENZYME IS USING, SO IT GOES BOTH WAYS. A LOT OF TOPO, SHOULD BE MORE SENSITIVE, T HAT'S THE CASE, USUALLY. SO, YEAH, CANCER CELLS TEND TO PUT A LOT O F THESE ENZYMES UP. >> I WAS WONDERING IF YOU CAN EXPAND ON TH E RELATIONSHIP BETWEEN THE BRCA MUTANT, BREA ST AND OVARIAN CANCERS, AND THE SYNTHETIC YEAST (INDISCERNIBLE) AND TOPOISOMERASES INHIBIT ORS, IN MY UNDERSTANDING AT LEAST RESISTANCE TO PARP INHIBITORS EMERGES QUICKLY, THEY ARE NOT AS GREAT IN THE CLINIC WHEN TREATING PATIENTS AS THEY ARE IN TISS UE CULTURE, WONDERING IF YOU SUGGEST WE CAN U SE THEM LIKE SEQUENTIALLY OR ONCE RESISTANCE EMERG ES OR IF IT'S LIKE -- BECAUSE I THINK ALSO ONE OF T HE MECHANISMS THROUGH RAB 51 LOSS, WHICH RESC UES THE WHOLE DEFECT, TOPOISOMERASES INHIBITORS WO ULD MAYBE NOT WORK AS WELL. >> YEAH, I THINK YOU'RE BRINGING A NUMBER OF QUESTIONS, SO CONNECTION WITH BRCA AND TOP O2 HAS BEEN WORKED ON LAST YEAR AND THE YEAR BEFO RE, IN KYOTO, (INDISCERNIBLE), AND THE CONCEPT AS I SAID IS THAT WHEN THE CELLS THROUGH ESTROGEN OR PR OSTATE CELLS THROUGH ANDROGEN, ANDROGEN RESPONSE, RESPONSIVE ELEMENT, THEY HAVE TO RETRIEVE TOPO 2 ON PROMOTER TO FACILITATE TRANSCRIPTION. NOW, THESE BREAKS OF THE PROMOTERS TEND TO BE PRETTY STABLE, AND TOPO CLEAVAGE COMPLEX IS THOUG HT TO (INDISCERNIBLE), AND IT'S A MYSTERY WHY TH EY ARE SO STICKY BECAUSE USUALLY TOP WILL GO AWAY, I N THOSE CASES THEY DON'T GO AWAY QUICKLY. THE IDEA IS CELLS USE THE NUCLEUS PATHWAY TO TAKE THEM OFF. THEY HAVE TO TAKE IT OFF. AND THE IDEA IS THE BRCA PROTEINS ARE RECR UITED TO FACILITATE RECRUITMENT OF THE NUCLEASE FOR THAT. SO IF YOU HAVE A BRCA DEFICIENCY, THEY ARE NOT REPAIRED. HORMONE DEPENDENT TUMORS, YOU GET THE MUTA TIONS, AND THEN YOU GET EVENTUALLY TRANSLOCATION AND ALL THE BAD THINGS. MAYBE THAT'S ONE EFFECT OF YOUR QUESTION. THE OTHER ASPECT OF THE PARP INHIBITOR, WH EN I FIRST LOOKED AT PARP INHIBITORS THE MOST STRIKIN G FINDING IS WHEN WE TOOK THE NCI 60 CELL LINE, ABOU T ONE HALF DO NOT RESPOND, EVEN TO 100 MICROMOLAR. SO WHAT IS IT? SOME RESPONDED AT 10 NANOMOLAR. SOME CELLS ARE BLACK AND WHITE, OTHERS RES POND AND NOT RESPOND. THE DETERMINANT OF RESPONSE WHEN WE LOOKED CAREFULLY WAS NOT THE BRCANESS TURNED OUT WHEN WE LO OKED AT UNRELATED, UNKNOWN, THIS IS ANOTHER STORY. IN THE CASE WHERE THE PARP INHIBITORS DON' T WORK, EVEN IF TUMOR IS BRCA DEFICIENT BECAUSE TH AT HAPPENS, BECAUSE AS YOU SAID, THE TOPO 1 I NHIBITORS MAY BE A WAY TO GO. THAT'S WHAT WE'RE PROPOSING. OR MAYBE YOU ADD TO PARP INHIBITOR THAT MA Y LIMIT THE RESPONSE. THAT'S WHAT WE'RE GOING TO BE TESTING WHET HER ADDING TOPO 1 TO PARP WILL LIMIT FRACTION OF PATI ENTS, FOR THE MOMENT IT'S MORE HYPOTHETICAL BUT CLEA RLY TOPO 1 INHIBITORS, THREE DRUGS, BRCA, HR DEPENDEN T, PARP INHIBITOR, YES. CISPLATIN, IN PART, BECAUSE PLATIN IS ALSO NER, WE ALL WANT TO SAY HR BUT MORE COMPLICATED, T HESE ARE ARE THREE CLASS BUT OTHERS DON'T DEPEND. NO HR, MANY DRUGS, THESE ARE THE THREE CLA SSES I KNOW, PARP INHIBITOR, PLATINUMS, TOPO 1 IN HIBITORS, BRCA DEFICIENT, HR DEFICIENT PATIENTS, FRO M THE PRE-CLINICAL MOLECULES THAT'S CLEAR. >> OKAY. IF THERE'S NO FURTHER QUESTIONS WE'LL TAKE 5 MINUTE BREAK SO CURT HARRIS, NEXT SPEAKER, CHIEF OF THE LABORATORY OF HUMAN CARCINOGENESIS, M.D. F ROM KANSAS UNIVERSITY, AND STUDIED INTERNAL MEDICINE AT UCLA, WASHINGTON VETERANS ADMINISTRATION HOSPITA L, AND NCI. AND THEN CAME TO NCI AND ADJUNCT PROFESSOR THE MEDICINE AT GEORGETOWN UNIVERSITY. HE'S WON SEVERAL AWARDS, INCLUDING THE ACR AWARD, AND HE'S CHIEF OF THE JOURNAL CARCINOGENES IS, AND HE'S GOING TO TALK TO US TODAY ABOUT LUNG CANCER, PRECISION MEDICINE STRATEGY. >> OKAY. ALL RIGHT, TERRY. SO, ALL OF YOU DIE-HARDS STILL HERE AFTER 5:00, ACTUALLY I FOUND MANY OF MY POSTDOCS ARE S TAYING AFTER 5:00 NOW. I DON'T KNOW WHY. THERE WAS A TIME I -- FORGET ABOUT THAT. LET'S TALK ABOUT SCIENCE, LUNG CANCER AND PRECISION MEDICINE SO WHAT IS PRECISION MEDICINE AND WHEN DID IT START? STARTED HERE AT THE NCI. AND HAROLD VARMUS AND FRANCIS COLLINS HAD AN INSTITUTE OF MEDICINE GROUP GET TOGETHER, AND COME UP WITH WHAT THEY CALLED PRECISION MEDICIN E. IT'S ESSENTIALLY AN UPDATE OF THE KIND OF MEDICINE THAT IS ONGOING AND WAS ONGOING. SO TRADITIONAL MEDICINE AT THAT TIME WAS T O -- THERE WE GO -- TO GET FAMILY HISTORY AND DO SIGN S AND SYMPTOMS, GET SOME LABORATORY TESTS, AND T HAT'S HOW IT WENT FOR MANY, MANY, MANY DECADES, IF N OT CENTURIES. SO WHAT'S PRECISION MEDICINE? IT'S MULTI-LAYERED. IT'S INDIVIDUAL CENTRIC, IT'S INTERCONNECT ED. AND ONE OF THE KEYS IS AN INFORMATION COMM ONS, WHICH HAS THESE OMICS, AND I'VE ADDED CLINICAL INFORMATION, EPIDEMIOLOGICAL DATA. AND THIS GETS PUT TOGETHER TO FORM A KNOWL EDGE NETWORK, AND A NEW MOLECULAR TAXONOMIC CLASSIFICATION OF INDIVIDUAL PATIENTS, TO GUIDE CLINICAL STUDIES, TO IMPROVE CLINICAL INVESTIGATIONS, TO PREVENT, EXCUSE ME, TO GUIDE PREVENTION RESEARCH, I ADDED THAT, THAT WA SN'T IN THE ORIGINAL, TO INFORM BIOMEDICAL RESEARC H. THESE ARE FEEDBACK LOOPS THAT ENRICH AND E XPAND THE INFORMATION COMMONS. SO, LUNG CANCER I THINK YOU HEARD ABOUT A SESSION ON LUNG CANCER, FAIRLY RECENTLY ACTUALLY. IT'S ONE OF THE THINGS WE STUDIED IN THE L ABORATORY OF HUMAN CARCINOGENESIS. THIS IS A -- AT THE TIME OF THE 50th ANNIV ERSARY OF THE SURGEON GENERAL REPORT, THEY DID A CALCULATION OF THE NUMBER OF CANCER-RELATE D DEATHS, AND DISEASE-RELATED DEATHS, AND CAME UP WI TH ABOUT, OH, OVER 20 MILLION PEOPLE DIED FROM TOBAC CO USE. AND OF COURSE THAT'S STILL ONGOING. IN THE UNITED STATES, THE INCIDENCE IS GOI NG DOWN, IN MALES, AND STILL PRETTY PLATEAUED IN FE MALES. LET'S SEE IF THIS WORKS ANY BETTER. YEAH, THAT WORKS BETTER. OKAY. THE DATA IN 2019, OR 2020, PROBABLY UPDATE OF THIS, AND THERE MAY BE A SLIGHT DECREASE IN FEMA LE LUNG CANCER BY THAT TIME. AND EVENTUALLY, MALE WILL COINCIDE WITH TH E FEMALE. SO, WHAT CAUSES LUNG CANCER? OBVIOUSLY TOBACCO SMOKE. WHY IS IT GOING DOWN IN THE UNITED STATES? THE UNITED STATES WE USED TO LOOK LIKE THI S. THAT'S 35 TO 60 SMOKE PACK YEARS, OR FREQU ENCY OF SMOKING I SHOULD SAY, STILL QUITE COMMON I N RUSSIA, ASIA, A NUMBER OF OTHER COUNTRIES. AND WE USED TO LOOK LIKE THIS, AND THAT'S WHY LUNG CANCER WAS SO -- HAS BEEN SO PREVALENT IN THE UNITED STATES. SO, THE VARIOUS PEOPLE IN LABORATORY OF HU MAN CARCINOGENESIS, THERE ARE FIVE P.I.s, INCL UDING MYSELF. WE FOCUS ON STUDYING THE MAJOR LETHAL TYPE S OF HUMAN CANCER. LUNG CANCER. SORRY ABOUT THAT. LUNG CANCER AND REID RYAN AND MYSELF. BREAST CANCER AND PROSTATE CANCER, STEPHAN , PANCREATIC CANCER, HUSSAIN. SO THAT'S -- AND WE'RE TALKING ABOUT, OH, PROBABLY CLOSER TO FIVE TO SIX MILLION DEATHS A YEA R FROM JUST THIS GROUP OF CANCERS. SO, A NUMBER OF YEARS AGO, ACTUALLY IN 198 1, I PARTICIPATED IN A MEETING IN GREECE. AND THIS IS CAPE SUNION, THIS IS A WATERCO LOR FROM PROFESSOR HIRAYAMA. PROFESSOR HIRAYAMA REPORTED THE VERY FIRST EVIDENCE OF PASSIVE SMOKING IN INDUCTION OF LUNG CA NCER. I WILL TELL YOU OF ABOUT, OH, FOUR OR FIVE HUNDRED PEOPLE AT THIS MEETING, AT LEAST HALF OF T HEM DIDN'T BELIEVE IT. BUT HE PROVED TO BE RIGHT. SO, THERE ARE PROBABLY OVER 100 PAPERS NOW IN THE LITERATURE SHOWING PASSIVE SMOKING, YOU GE T IN YOUR WORKPLACE, OR FROM YOUR SPOUSE, IS ASSOCIA TED WITH INCREASED RISK OF LUNG CANCER. AND I'M RATHER FOND OF THESE OLD KINDS OF QUOTES. THIS IS FROM HIPPOCRATES, SOME MEN/WOMEN H AVE CONSTITUTIONS LIKE WOODED MOUNTAINS RUNNIN G THE STREAMS, OTHERS LIKE THOSE WITH POOR SOIL AND LITTLE WATER, STILL OTHERS LIKE LAND, RICH IN PAS TURES AND MARSHES, OTHERS LIKE THE BARE, DRY EARTH O F THE PLAIN, PEOPLE DIFFER FROM ONE OTHER, EVEN OBVIOUS THOUSANDS OF YEARS AGO IN RISK OF DISEASE AND OTHER THINGS. SO I WAS SITTING AT THIS MEETING, I SAID, WELL, IS THERE ANY EVIDENCE OR COULD I FIND SOME EV IDENCE TO INVESTIGATE WHETHER OR NOT SMOKING OF YOUR PARENTS INCREASES YOUR RISK OF DEVELOPING LUNG CAN CER. SO THIS IS IN THE LATE '80s, OR EARLY '80s I SHOULD SAY. AND WOMEN DIDN'T START SMOKING IN THE UNIT ED STATES HEAVILY UNTIL SECOND WORLD WAR. AND THEN YOU DON'T GET CANCER UNTIL 50, 60 , 70 YEARS OF AGE. I FIGURED IT WOULD TAKE ABOUT 35 YEARS BEF ORE I COULD DO THAT STUDY. BUT IN FACT WE DID DO THE STUDY, OF 38 YEA RS LATER. AND THE HYPOTHESIS WAS THAT CHILDHOOD EXPO SURE TO SECONDHAND SMOKE AND GENETIC ALTERATIONS I N INNATE IMMUNITY INCREASED LUNG CANCER RISK IN NEV ER SMOKING ADULTS. SUSAN WAS INVOLVED WITH THIS. WE COLLABORATED, ONE OF THE COHORTS CAME F ROM THE MAYO CLINIC, ONE FROM OUR COHORT AT THE UN IVERSITY OF MARYLAND. AND WE IN FACT DID FIND THAT PARENTAL SECO NDHAND SMOKE EXPOSURE IN CHILDHOOD IS ASSOCIATED WITH DOSE DEPENDENT INCREASE IN LUNG CANCER RISK AMO NG NEVER SMOKERS, TWO DIFFERENT COHORTS. WHETHER IT WAS THE MOM OR THE DAD OR BOTH, MOM AND DAD BOTH IT WAS ADDITIVE. IT WAS EVEN SOME EVIDENCE THAT GRANDPARENT S TAKING CARE OF THE KIDS AT AN EARLY AGE INCREASED THEIR RISK. IT WAS ESPECIALLY TRUE IN MANNOSE BINDING LECTIN TYPE 2 HAPLOTYPE REFLECTIVE OF HIGH ACTIVE INNATE IMMUNE SYSTEM, AND SURPRISINGLY THESE CASE S DEVELOPED CANCER PRIMARILY IN THEIR 50s AN D 60s INSTEAD OF 60s AND 70s LIKE MOST OF US WOU LD. SO, THAT LED ME TO THINK ABOUT THE EXPOSES OME, SOMETHING THAT WAS DEFINED BY CHRIS WILD, WHO WAS DIRECTOR OF THE INTERNATIONAL AGENCY FOR R ESEARCH ON CANCER, AND IT INVOLVED NOT ONLY EXTERNAL ENVIRONMENT, TOBACCO SMOKE, INFECTION, DIE T, TOXINS, RADIATION, BUT ALSO THE INTERNAL ENVIRONME NT RELATED TO THAT. WE ADDED OBESITY, CHRONIC INFLAMMATION, LA TER ON THE GENOME CHANGES, AND EPIGENOME CHANGES, AND DEVELOPMENT OF CANCER BIOMARKERS OF RISK, DIAGNOSIS, FOR PREVENTION AND SCREENING, UNDERSTANDIN G CARCINOGENESIS FEEDS INTO THIS AND ALSO LE ADS TO CANCER THERAPY, AS I'M SURE ALL OF YOU KNO W. SO GENE OLD, WELL, HOW ABOUT LUNG CANCER I N THE GENOME? THE TRADITIONAL VIEW OF LUNG CANCER WHEN I WAS A RESIDENT AND MEDICAL ONCOLOGY FELLOW WAS S MALL CELL ADENO, AND SQUAMOUS, IN 1987 KRAS MUTATION WAS FOUNDS. P53 MUTATIONS WERE FOUND MUCH BEFORE THIS. FOLLOWING KRAS, EGFR MUTATIONS, AND FINALL Y IN 2014, THERE HAVE BEEN A COUPLE MORE THAT HAVE BE EN FOUND, PRIMARILY FUSIONS AND A FEW OTHER THINGS A SSOCIATED SUCH AS BRF WITH LUNG CANCER. SO, WHAT CAUSES LUNG CANCER? AND CAUSES CANCER IN MANY SITUATIONS? THERE WAS A PERIOD OF TIME WHICH PEOPLE WE RE STUDYING CHEMICAL CARCINOGENESIS, INCLUDIN G MYSELF. AND YOU COULD GO EPIDEMIOLOGICAL BACK INTO THE 18th CENTURY AND CHIMNEY SMOKE SWEEPERS DE VELOPED CANCER, ASPERGILLUS FLAVUS CAUSES FORMATIO N, LIVER CANCER, SMOKING, 66 DIFFERENT CHEMICAL CAR CINOGENS IN IT, SO IT'S A WITCH'S BREW OF CARCINOGE NS, ANDS THAT WHY IT'S SO CARCINOGENIC. THREE DECADES OR THREE ERAS LISTED HERE, E XAMPLES OF INITIAL SEMINAL ADVANCES IN EXPOSURE OF ENVIRONMENTAL CARCINOGENS, BEING MOLECULAR LY LINKED TO MUTAGENESIS AND TO EPIDEMIOLOGY FOR THA T POINT. AND SO BACK IN THE '70s, THEY WERE FINDING CARCINOGENS, CHEMICAL CARCINOGENS ARE MUTA GEN, SO THAT WAS ALL VERY EXCITING, BUT IT SORT OF TIED CARCINOGENS WITH DNA DAMAGE. AND THEN DISCOVERY OF P53 MUTATIONS LINKED ENVIRONMENTAL CARCINOGEN EXPOSURE. AND THE ADDITIONAL STUDIES DONE BY OUR GRO UP AND ABOUT THAT THAT I WON'T HAVE TIME TO TELL YOU ABOUT THAT. AND THERE'S ALSO REVIEW THAT WE WROTE FOR MULTIPLE KINDS OF CARCINOGENS THAT CAUSED P53 MUTAT IONS. THIS IS THE START OF MOLECULAR EPIDEMIOLOG Y, AND WHICH I AND OTHER PEOPLE CONTRIBUTED TO. THEN MORE RECENTLY, IN 2013, LUDMIL ALEXAN DER AND MIKE STRATTON PUBLISHED SEQUENCES, TRINUCL EOTIDE SEQUENCES INSTEAD OF LOOKING AT ONE GENE, LOOKING AT THE WHOLE GENOME. AND PUBLISHED IN "NATURE" AS ESSENTIALLY A LINK BETWEEN LOOKING AT A SINGLE GENE AND LOOKI NG AT THE ENTIRE GENOME. BEFORE THAT, IT WAS FOUND THAT AFLATOXIN, AS I MENTIONED, G TO T CONVERSIONS PARTICULARLY AT CODON, SMOKING AT VARIOUS HOT SPOTS, AGAIN G TO T WITH STRAND BIAS, AND STRAND BIAS IN THIS CASE MEANS ONE OF THE ALLELES, THE WILDTYPE ALLELE, SINCE THERE ARE TWO ALLELES, IS LOST. AND YOU HAVE MUTATION OF THE OTHER. ULTRAVIOLET LIGHT HAS A CHARACTERISTIC CC TO TT TRANSITION, WHICH IS NOT FOUND WITH ANY OT HER CARCINOGEN THAT'S KNOWN. ARISTOLOCHIC ACID CAUSES KIDNEY TUMORS, A TO T TRANSITION, THAT WAS SORT OF THE STATUS OF THINGS BACK IN THE EARLY '90s AND WENT FURTHER TH AN THAT. BUT P53 GOES BACK 40 YEARS NOW, WHEN IT WA S FIRST DISCOVERED. AND THEN FINDING MUTATIONS IN IT WAS DISCO VERED AFTER THAT. AND THERE'S SOME CANCER TYPES SUCH AS OVAR IAN CANCER P53 MUTATIONS ARE ALMOST OVER 90%. SOME ARE LOSS OF FUNCTION AS TUMOR SUPPRES SOR, AND OTHERS ARE GAIN OF FUNCTION. SO IT SWITCHES FROM A TUMOR SUPPRESSOR TO AN ONCOGENE, JEKYLL AND HYDE SWITCH, IF YOU W ANT TO THINK IT OF THAT WAY. NOT ALL MUTATIONS ARE EQUAL. SOME ARE PURELY LOSS OF MUTATIONS. SOME WITH LESS AND LESS WILDTYPE ACTIVITY SUCH AS THESE. OTHERS GAIN ONCOGENIC ACTIVITY, AND THESE ARE JUST EXAMPLES OF THAT. SO DEVELOPING THERAPIES AGAINST THE ONCOGE NIC FORMS OF P53 IS SOMETHING THAT THE FIELD HAS BEE N INVOLVED WITH FOR TEN YEARS NOW, AND PROGRESS ACTUA LLY IS BEING MADE, WE'RE LEARNING MORE ABOUT THAT IN MAY IN ISRAEL, WHEN THE NEXT P53 MEETING IS HELD. BUT P53 AND ITS FUNCTIONS AFFECT MULTIPLE LEVELS OF OMICS DATA, AND THIS IS JUST A NICE CARTOO N THAT WE'VE PUT TOGETHER, ANNA ROBLES AND MYSELF A FEW YEARS AGO. AND THERE'S -- WHY PEOPLE ARE STILL INTERE STED IN P53, IT JUST DOES LOTS OF THINGS. ALL RIGHT. NOW LET'S GO BACK TO COMPUTATIONAL ANALYSI S THAT LUDMIL AND MIKE STRATTON DID. THIS WAS THEIR INITIAL PAPER IN WHICH THEY DESCRIBED 21 DIFFERENT SIGNATURES, AND LOOKING AT MU LTIPLE CANCERS, INCLUDING LUNG CANCER, HEAD AND N ECK CANCER, AND IN THOSE SMOKING THERE WAS SPE CIFIC SIGNATURE THAT WAS ASSOCIATED WITH SMOKING , AND THOSE KINDS OF CANCERS. THERE ARE SIGNATURES THAT ARE ASSOCIATED W ITH AGE, THOUGH THIS REPLICATION OF CELLS, THAT'S A N INTERESTING ASPECT OF CANCER IN AGING. AND THESE ARE JUST SOME OTHER EXAMPLES. AND THIS HAS BEEN MARKEDLY EXPANDED SO THE RE ARE AT LEAST I THINK OVER 35 DIFFERENT SIGNATURES NOW THAT THEY CHARACTERIZE, AND THE THE AFLOTOXIN IS SIGNATURE 35, AS I RECALL. THERE WE GO. SO, ONE OF THE KINDS OF THINGS THAT THEY H AVE DONE IS COMPARE SMOKERS VERSUS NONSMOKERS, SO S IGNATURE 4, THAT'S THE BLUE LINES, MUCH MORE COMMON IN SMOKERS THAN IN NEVER SMOKERS, OR NONSMOKE RS. AND THAT'S -- THOSE ARE THE KINDS OF CORRE LATIONS THAT ARE -- THAT HAVE BEEN MADE, AND THEY ACTUALLY MAKE SENSE FROM KNOWING THE VARIOUS CARCIN OGENS THAT ARE INVOLVED. SO THIS IS AN EXAMPLE OF THE KINDS OF DATA THAT THEY SEE, C TO A TRANSITIONS THAT I MENTIONED, FOR EXAMPLE, WHICH IS SIGNATURE NUMBER 4. IF THEY TOOK HUMANIZED MICE, IN WHICH HUMA NIZED WITH HUMAN P53 AND EXPOSED THOSE MICE TO BENZOP YRENE, A PRIMARY CARCINOGEN IN TOBACCO SMOKE AND PO LLUTED AIR THEY GET THE SAME SIGNATURE OR TAKE HUMAN EMBRYONIC CELLS THEY GET THE SAME SIGNATURE WITH EXP OSURE, THE LINKS THEY ARE MAKING BETWEEN EXPOSURE TO VARIOUS CARCINOGENS AND MUTAGENS AND HUMAN CANCER. THIS SHOWS GAIN OF FUNCTION MUTATIONS PATI ENTS HAVE A SLOWER -- A POOR PROGNOSIS THAN THOSE WI LD TYPE VERSUS THOSE THAT ARE JUST LOSS OF MUTATIO N. AND IF YOU LOOKED AT CODON 249 IN LIVER CA NCER IT'S A GAIN OF FUNCTION BUT IT'S LIKE THIS. SO THAT ONE MUTATION IN LIVER CANCER IS HI GHLY DEADLY. IT'S CAUSED BY AFLOTOXIN. CHRONIC INFLAMMATION IS TIED TO HUMAN CANC ER, BACK TO AT LEAST THE 1800s. AND THESE ARE EXAMPLES OF PRIMARILY INHERI TED VERSUS ACQUIRED HUMAN CHROMATOSIS, APPARENT OVERL OAD, CROHN'S DISEASE, YOU CAN PUT OTHER DISEASE S, FAMILIAR PANCREATITIS, FOUND IN CERTAIN FA MILIES, SUCH AS CARTER'S FAMILY, HE'S ONE OF THE F EW MEMBERS OF THAT FAMILY, STILL ALIVE, DIDN'T GET PA NCREATIC CANCER. ALTHOUGH HE HAS METASTATIC MELANOMA NOW AT AGE 99 I THINK. MORE COMMON ARE ACQUIRED VERSUS INHERITED, VIRUS, BACTERIA. AND PARASITIC INFECTIONS. GLOBAL IMPACT IS TWO MILLION CANCERS PER Y EAR, CALCULATED OR RELATED TO INFECTION. OTHER CAUSES OF INFLAMMATION ARE ASSOCIATE D WITH MANY MORE CANCERS PER YEAR, FOR EXAMPLE IN ADDITION TO 66 CHEMICAL CARCINOGENS, SMOKING IS A V ERY POTENT INFLAMMATORY AGENT, AND IF YOU TOOK A DRAW OF CIGARETTES, IN YOUR EARLY AGE, HOPEFULLY N OT LATER, PROBABLY STARTED TO COUGH BECAUSE IT'S REA LLY TOXIC, AND IF IT WASN'T FOR NICOTINE, KIDS ARE BE ING HOOKED WITH NICOTINE WITH THESE CIGARETTES RIGHT NOW, NO ONE WOULD BE SMOKING. IN MY OPINION. VARIOUS AGENTS HAVE BEEN INVESTIGATED. WE FOUND IL-8 AND C-REACTIVE PROTEIN IS FOUND IN EUROPEAN-AMERICANS AND IN A STUDY THAT COLLECTED SAMPLES 20, 30, 40 YEARS AGO AT SHADY GROVE. WE FOUND THOSE WERE PREDICTORS OF DEVELOPING CANCER, ACTUALLY YEARS PRIOR TO DEVELOPING LUNG CANCER. AND AT THE TIME TIME CANCER WAS FOUND, IL-8 AND IL-6, VERY FEW AFRICAN-AMERICANS IN THAT INITIAL STUDY BUT AFRICAN-AMERICANS ALSO HAVE INCREASE IN IL-1 BETA AND IL-10, COMPARED TO EUROPEAN-AMERICANS SO THERE'S SOME HEALTH DISPARITY DIFFERENCES THAT WE AND REID RYAN AND OTHERS ARE INVESTIGATING. FOR SURVIVAL, SO THIS IS PRIMARILY AT STAGE 1 LUNG CANCER. STAGE 1 LUNG CANCER IS ABOUT THAT BIG. AND THERE'S NO METASTASES. OR NO DETECTABLE METASTASES. AND IF THE CASE OR YOUR PATIENT OR IF YOU'RE ON THE OUTSIDE YOUR CLIENT HAS ELEVATED THESE DIFFERENT CYTOKINES, OR MANNOSE BINDING LECTIN, WHICH I MENTIONED EARLIER, YOU HAVE INCREASE THE PROBABILITY OF DYING, OF RECURRENCE. NOW LOOK AT TRANSCRIPTOME. THIS WILL TAKE US BACK ABOUT TWO OR THREE DECADES NOW. VICTOR AND GARY DISCOVERED THERE WERE NON-CODING RNAs, THERE'S A NON-CODING RNA LECTURE AS PART OF THE SERIES, ISN'T THERE, TERRY? YOU KNOW ABOUT ALL OF THAT. AND THAT WAS QUITE INTERESTING AT THE TIME, AND CARLO CROCE DECIDED TO LOOK AT HUMAN CANCERS, AND HE FOUND THAT microRNAs ARE DIFFERENT, THEY EXPRESSED IN HUMAN CANCERS, AND THAT THEY CAN PREDICT RISK DIAGNOSIS OR PROGNOSIS, MOST STUDIES WERE DONE WITH ARTHUR. AS YOU LEARNED FROM NON-CODING RNA GROUP LESSONS, THE microRNAs CAN INHIBIT TRANSLATION, OR IF THERE'S SOME BIT OF A MISMATCH THEY CAN CAUSE MESSAGE TO CLEAVE. BUT THEY CAN ALSO BIND TO PROTEINS AND AFFECT PROTEIN FUNCTION. THEY CAN BIND TO RECEPTORS AND INCREASE IL-6 AND TNF ALPHA. THAT'S PRETTY INTERESTING IN TERMS OF INFLAMMATION. THIS IS NOZUMO WHEN HE WAS A YOUNG FELLOW IN THE LAB. AND WE FOUND microRNA PROFILES WERE SIGNIFICANTLY DIFFERENT BETWEEN PRIMARY LUNG CANCERS AND AMONG HISTOLOGICAL TYPES. AND MIR-21 AND THESE OTHER TWO WERE OVEREXPRESSED, LET-7 WAS UNDEREXPRESSEDDED, STAGE 1 LUNG CANCER, PRIMARILY INTERESTED IN STAGE 1 LUNG CANCER, WHY THEY RECUR. ANOTHER STUDY WE DID WITH CARLO, LOOKED AT SIX CANCER TYPES, AT THAT POINT THERE WAS I THINK 190, MAYBE 200, AND SO microRNAs THAT HAVE BEEN DISCOVERED, NOW OVER 2,000. BUT OF THOSE THAT WERE DISCOVERED AT THAT TIME, WE LOOKED AT THESE SIX CANCER TYPES, MIR-21 WAS OVEREXPRESSED IN ALL. WHEN WE LOOKED AT CANCERS IN THE FIELD UPREGULATED IN 18 MAJOR CANCERS, BIOMARKER OF POOR PROGNOSIS IN 14. SO WE FIGURED THAT WAS PROBABLY PRETTY INTERESTING AND IMPORTANT, OUR GROUP LOOKED AT THREE COHORTS, ONE IN MARYLAND, ONE NORWAY, ONE JAPANESE COHORT. THERE'S SOME DIFFERENCE HERE. WE USE CANCER SPECIFIC MORTALITY WHICH I MUCH PREFER, THE JAPANESE PREFER THE RELAPSE-FREE SURVIVAL. SO, THEIR DATA LOOKS BETTER THAN OUR DATA. AND THE REST OF THE WORLD. BUT NEVERTHELESS, IF MIR-21 IS HIGH, THAT'S THE RED, POOR PROGNOSIS, IN ALL THREE OF THESE COHORTS. AND THEN THE SAME IS TRUE IN LARGE NUMBER OF DIFFERENT KINDS OF COHORTS, SO HIGH LEVELS OF MIR-21 POOR PROGNOSIS, IN MULTIPLE TYPES OF CANCERS. WHAT'S THE MECHANISTIC UNDERPINNING OF THIS? WELL, ONE IS THAT MIR-21 IS AMPLIFIED IN THE GENOME, FAIRLY FREQUENTLY, ESPECIALLY IN LUNG CANCER. IT LEADS TO DECREASED TRANSCRIPTIONAL SILENCING. WE FOUND EGRF MUTATIONS YOU DON'T FIND KRAS MUTATIONS BUT YOU FIND INCREASE IN MIR-21, YOU FIND KRAS, DON'T FIND EGFR MUTATIONS, YOU FIND INCREASE IN MIR-21. SO THEY ARE IN THE SAME PATHWAY OR SIMILAR PATHWAY. DAVID BALTIMORE FOUND VARIOUS CYTOKINES INCREASE STAT3 IN ONE OF THE GENES STAT3 INCREASES IS MIR-21 AND DIFFERENT KINDS OF STRESS KINASE AND FREE RADICALS INCREASE MIR-21. HOW DOES IT WORK? INHIBITS A VARIETY OF DIFFERENT GENE EXPRESSION OF DIFFERENT GENES, AND MOST OF THOSE ARE ONES THAT ARE INVOLVED IN INHIBITING CANCER IN VARIOUS WAYS. OR IN INHIBITING MISMATCH REPAIR, SO YOU HAVE LESS MISMATCH WE PAIR WITH INCREASED EXPRESSION OF MIR-21. SO, THERE'S A LOT MORE DATA THAT'S BEEN COLLECTED SINCE I PREPARED THIS SLIDE A NUMBER OF YEARS AGO. SO, THEN WE WENT AND THERE WAS A TIME IN WHICH THERE WERE PEOPLE LOOKING AT EXPRESSION OF LARGE NUMBERS OF PROTEIN CODING GENES, WE TOOK A DIFFERENT APPROACH, DECIDED WE WANT TO LOOK AT MINIMUM NUMBER OF LARGE EXPRESSION OF CODING GENES, INCLUDING WE FOUND IN THESE STUDIES AND ANALYSIS, SOME OF THE WAYS INCREASE IN XPO 1, INVOLVED IN TRANSPORT OF microRNAs, BRCA 1 EXPRESSION HIGH LEVEL WHICH IS PREVIOUSLY SHOWN BY OTHERS TO CAUSE NEUROMIC INSTABILITY, HEP 1 ALPHA NOBEL PRIZE, THAT WAS WON THIS YEAR BY THREE DIFFERENT GROUPS. AND ALSO DECREASE DCL 1, TUMOR EXPRESSER GENE DISCOVERED HERE AT THE NCI. SO WE FOUND THIS FOUR-GENE CLASSIFIER IS QUITE REASONABLE IN IDENTIFYING AGAIN STAGE 1 ADENOCARCINOMA IN ORIGINAL FIVE COHORTS, AS YOU CAN SEE, SO 100% SURVIVAL, 0% SURVIVAL, AND THIS IS TIME IN YEARS. SO WHEN WE UPDATED THAT WITH SEVEN MORE COHORTS, STAGE 1 ADENOCARCINOMA OF THE LUNG, ESSENTIALLY THE SAME THING WAS FOUND. AND EVEN STAGE 1A, CHANGES IN THOSE THREE GENES, WAS ASSOCIATED WITH POOR PROGNOSIS, AND BOTH STAGE 1A AND STAGE 1B, THREE TO FIVE CENTIMETERS IN SIZE. AND THIS IS JUST LOOKING AT ALL THOSE DATA PUT TOGETHER. SO, WE DECIDED WE WANTED TO ASK COULD WE COMBINE PROTEIN-CODING GENES MECHANISTICALLY RELATED TO LUNG ADENOCARCINOMA AND MIR-21 TO FIND A BETTER CLASSIFIER THAN EITHER ALONE. THIS IS RELAPSE-FREE SURVIVAL VERSUS -- THIS IS A 4-GENE, MIR-21, 4-GENE, MIR-21, HIGH, POOR SURVIVAL, AND IF YOU PUT THE TWO TOGETHER, POOR SURVIVAL, AND ESPECIALLY IN PRIMARILY 1B VERSUS 1A. SO, HOW DID WE THINK ABOUT THAT AT THE TIME? AND THE -- WHAT WE CAME UP WITH WAS NON-CODING RNA AND CODING RNAs, PUT THEM TOGETHER, THEY ARE BOTH LOW, IF EITHER ONE IS HIGH, OR IF BOTH ARE HIGH, AGAIN THESE ARE SURVIVAL CURVES. BOTH CAN IDENTIFY PEOPLE WHO ARE AT RISK, BUT EACH MISCLASSIFIES SOME PATIENTS, BUT THEY MISCLASSIFIED OTHER PATIENTS. SO THE COMBINATION OF THE TWO IS BETTER THAN JUST ONE. WE FOUND THAT NOT ONLY IN LUNG CANCER BUT IN COLON CANCER, ESOPHAGEAL CANCER, BOTH ADENO, SQUAMOUS, LUNG CANCER WHICH YOU'VE SEEN, AND OTHER GROUP FOUND THAT TO BE IN BREAST SO THAT HYPOTHESIS ACTUALLY OR PRINCIPLE FOUND HAS BEEN MULTIPLE KIND OF CANCER CELLS. LET'S LOOK AT THE THIRD OMICS, THE EPIGENOME. AGAIN, WE'RE LOOKING AT LUNG CANCER. AND THIS IS DONE BY ANNA ROBLES, HYPOTHESIS WAS INTEGRATED BIOMARKER CLASSIFICATION STAGE 1 LUNG CANCER, ADENOCARCINOMA BASED ON INDEPENDENT MESSAGE, microRNA AND DEMETHYLATION MARKERS WILL IMPROVE THE CLASSIFIER. IT'S GOING FROM ONE TO TWO, NOW THREE. ONE OF OUR MANEL ESTELR STUDIED DNA MET LAYINGS, CAME UP WITH A SIGNATURE WITH ALL OF THESE MARKS, DNA METHYLATION MARKS, AND I LOOKED AT THAT AND I SAID, WELL, MAYBE ALL OF THOSE ARE DEPENDENT ON EACH OTHER, OR MAYBE THEY ARE INDEPENDENT OF EACH OTHER. WE ANALYZED THAT. WE FOUND THAT THEY ARE ALL DEPENDENT ON EACH OTHER. SO WE DECIDED, WELL, LET'S LOOK AT THE ONE THAT'S MOST HIGHLY EXPOSED, EXPRESSED, NOT EXPRESSED, FOUND, AND THAT'S HOXA 9. WE WENT FROM FIVE OR SIX DOWN TO ONE AGAIN. THE OBJECTIVE IS TO GET SMALLER AND SMALLER, THAT GIVES YOU BETTER AND BETTER RESULTS. WE PUT ALL THREE TOGETHER IN STAGE 1 LUNG CANCER. THESE ARE KAPLAN-MEIER PLOTS AGAIN, THIS IS A FIVE, THIS IS MONTHS, THIS IS FIVE-YEAR POINT. SO IF ALL THREE ARE POSITIVE, CLASSIFICATION OMICS ARE POSITIVE, VERY POOR PROGNOSIS. STAGE 1A IS A LITTLE BIT BETTER THAN STAGE 1B. SO, TAT WAS SOMETHING THAT IMPRESSED US, AND NCI HAS PATENTS ON ALL THIS STUFF. SO, NOW SOMETHING NEWER. THAT'S MICROBIOME. AND WAS THERE A LECTURE ON THE MICROBIOME? OKAY. SO, SHOULD BE FAMILIAR WITH THIS. AND MICROBIOME IS ABOUT TEN TIMES, 100 TIMES MORE BACTERIA AND FUNGI ON OUR BODY, IN OUR GUT AND ELSEWHERE, THAN THERE ARE HUMAN CELLS. AND THE MICROBIOME VARIES DEPENDING ON THE SURFACE THEY ARE ON, OR THE INTERIOR THEY ARE ON, AND SO JUDY SEGRAY AND OTHERS MADE IMPORTANT DISCOVERIES IN VARIATION, WE COLLABORATED WITH HER. IT WAS KNOWN A FEW YEARS AGO NOW THAT THERE WERE BACTERIA ASSOCIATED WITH INCREASED CANCER RISK, A PYLORI AND COLON CANCER, DYSBIOSIS, THAT MEANS A CHANGE IN THE FREQUENCY OF DIFFERENT CLASSES OF BACTERIA IN CANCER THERAPY. DOUG LOWY AND JOHN SCHNEIDER FOUND HUMAN PAPILLOMAVIRUS, VACCINE, RECENTLY PUBLISHED DATA ON ACIDOVORAX. ALL OF THESE HAVE DIFFERENT EFFECTS ON CELLS. EITHER ON THE SURFACE OF EPITHELIUM, FOR EXAMPLE, OR VIRUSES CAN CAUSE MUTATIONS, THEY CAN CAUSE PROBLEMS WITH THE MU COASTA BARRIER, ESPECIALLY CIGARETTES WILL CAUSE THIS, BACTERIA GET IN, AND CAUSE INFLAMMATION. AND SOME OF THE BACTERIA PRODUCE ACTUALLY PROTEINS WHICH ARE MUTAGENS, WHICH IS ANOTHER INTERESTING WAY OF CAUSING MUTATIONS IN HUMAN CANCERS, IN HUMAN CELLS, CAUSING CANCER. SO IN THE STUDY THAT WE RECENTLY PUBLISHED, I GUESS SOMETIME LAST YEAR, WE FOUND THERE WAS INTERACTION BETWEEN THE MICROBIOME AND SMOKING, P53 MUTATIONS, AND HUMAN LUNG CANCER, AND WE FOUND SEVERAL PACKS INCLUDING ACIDOVORAX WERE HIGHER IN SMOKERS VERSUS FORMER SMOKERS VERSUS NONSMOKERS, SO FORMER SMOKERS AND PEOPLE WHO WERE CURRENT SMOKERS ARE INCREASED FREQUENCY OF ACIDOVORAX. A GROUP OF FACTS ASSOCIATED WITH SQUAMOUS CELL CARCINOMA, AND AS I SAID ENRICHED IN SMOKERS. AND THAT THESE WERE ASSOCIATED WITH P53 MUTATIONS. SO, IT WAS P53 MUTATIONS ACTUALLY PLAYING A ROLE OR WAS IT A CONSEQUENCE OF THE FACT THAT THESE WERE SMOKERS? AND THAT'S AN INTERESTING KIND OF QUESTION. THIS CAME TO A CONCLUSION THAT THE SQUAMOUS CELL CARCINOMA ATTACKS IN PEOPLE WERE TP53 MUTATIONS, ESTABLISHING A MICROBIOME-GENE INTERACTION IN LUNG CANCER. NEXT QUESTION ARE BACTERIA PASSENGERS? OR ARE THEY PLAYING A ROLE IN THE DEVELOPMENT OF THE CANCER? WE'RE DOING A LARGE NUMBER OF ANIMAL STUDIES AND OTHER KINDS OF STUDIES RIGHT NOW, I'LL JUST SHOW YOU ONE SLIDE MAYBE. THAT GOT DELAYED. I GUESS IT DIDN'T GET TRANSFERRED, WHEN HE TRANSFERRED MY USB STICK. WE FOUND INDEED THAT ACIDOVORAX INCREASES THE SIZE OF THE TUMORS IN A MOUSE MODEL, KRAS AND P53 MUTATION AND THEY GOT TUMORS QUICKER THAN IF IT WAS ACIDOVORAX OR IF THEY WERE EXPOSED TO SHAM OR INTRANASAL OR LACTOBACILLUS, AN ANTI-INFLAMMATORY AGENT. SO, LOOKS LIKE IT'S RELATED TO INFLAMMATION, AND SO WE HAVE EXPERIMENTS NOW LOOKING WHETHER OR NOT INFLAMMATORY AGENTS EITHER CHEMICALS OR DRUGS OR LACTOBACILLUS WILL DECREASE FREQUENCY OF LUNG CANCER CAUSED BY ACIDOVORAX IN THE ANIMAL MODEL. SO LET'S -- WE'RE READY TO END UP, LOOKING AT THE METABOLOME, IN THE PREVIOUS SLIDE. AND HYPOTHESIS THAT THESE TWO POSTDOCS, MAJDA AND EWY, EWY HAS JUST TAKEN A HIGH LEVEL POSITION AT NCATS, AND MAJDA HAS A HIGH LEVEL POSITION AT FDA NOW. UNBIASED METABOLOMICS WILL DISCOVER BIOMARMERS ASSOCIATED WITH RISK, DIAGNOSIS, PROGNOSIS OF THERAPEUTIC OUTCOME OF LUNG CANCER. LET'S GO BACK TO PREVIOUS SLIDE, HIPPOCRATES AGAIN. SO HIPPOCRATES, IT'S SAID, TASTED URINE FOR DIAGNOSIS OF DISEASE IN HIS PATIENTS. NOW, I'M NOT AS OLD AS HIPPOCRATES, ALTHOUGH MY KIDS MIGHT THINK I AM. BUT I NEVER TASTED URINE, EXCEPT WHEN WE HAD SMALL KIDS. I WAS CHANGING DIAPERS. SO, BUT -- SO THAT WAS ONE OF THE THINGS THAT PHYSICIANS DID AT THIS STAGE. AND THEN A PHYSIOLOGIST, ARMSTRONG, IN EUROPE, THIS IS A PICTURE OF ALL THIS, WRITES FROM A LIQUID WINDOW THROUGH WHICH PHYSICIANS FELT THEY COULD VIEW THE BODY'S INNER WORKINGS, URINE BECAME THE BEGINNINGS OF LABORATORY MEDICINE. WE'RE NOT ALLOWED TO WRITE THIS KIND OF FLOWERY LANGUAGE BUT I THINK IT MAKES A POINT, AND THIS DUTCH PAINTING IS A GOOD EXAMPLE OF THAT. OKAY. SO WE DID BIASED SCREEN IN EUROPE, CHROMATOGRAPHY, MASS SPEC ANALYSIS, AND FOUND THERE WERE FOUR DIFFERENT METABOLITES, ONE CREATININE RIBOSIDE, ONE N-ACETYLNEUROAMINNIC ACID, CORTISOL SULFATE. PUT THEM TOGETHER, PROGNOSIS IS VERY POOR, THREE OUT OF THE FOUR ALSO POOR, AND GETS BETTER IF ONLY TWO, OR ONLY ONE. AND ALSO DIAGNOSIS, THESE ARE -- THIS IS A ROCK CURVE. SO THIS IS LOOKING AT SPECIFICITY AND SENSITIVITY, AND SO THE AREA IN THE CURVE HERE IS, OH, LET'S SEE IF I CAN READ IT HERE. IT'S ACTUALLY -- YEAH, IT'S DOWN THERE. IT'S ABOUT .9, WHICH IS ACTUALLY QUITE GOOD. AND PREDICTIVE VALUE, POSITIVE PREDICTIVE VALUE, NEGATIVE PREDICTIVE VALUE, PHYSICIANS INCLUDING MYSELF LOOK AT THE NEGATIVE PREDICTIVE VALUE BECAUSE YOU HAVE SOMEONE WHO HAS LOW DOSE CT SCANNING AND THERE'S A LESION THERE, THE NEGATIVE PREDICTIVE VALUE MIGHT ALLOW YOU TO SAY, WELL, WHY DON'T WE WAIT THREE MONTHS AND SEE IF THE LESION INCREASES OR NOT. BECAUSE MOST OF THEM ARE NOT CANCER. I'LL SHOW YOU THAT IN JUST A MOMENT. WE FOUND THE AMOUNT IN THE URINE WAS ASSOCIATED WITH THE AMOUNT IN THE TUMOR, AND THIS IS A CORRELATION CURVE, PRETTY GOOD. HIGH LEVELS IN THE URINE. IT'S ALSO THE CORRELATION HERE BETWEEN URINE AND TUMOR, ALSO HOLDS UP IF YOU LOOK AT SERUM, PLASMA. A CLINICAL TEST IS BEING MADE OF THIS, CLIA-BASED CLINICAL TEST FOR LICENSING AND HOPEFULLY PUT IN CLINICAL PRACTICE. LET'S GO TO LOW DOSE CT. THE TURNING POINT HERE WAS IN OLD DAYS THAT WASN'T SUCH A LONG TIME AGO CHEST X-RAY WAS THE SCREEN, YOU DIDN'T SEE ANYTHING UNTIL THEY GOT STAGE 2 OR 3 OR 4 CANCER. LOW DOSE CT DECREASED THE MORTALITY ABOUT 20%, IN THIS ORIGINAL STUDY, IN THE STUDY DONE IN ENGLAND IT WAS CLOSER TO 30%. AND WOMEN WITH BETTER PREDICTING CANCER, DECREASING MORTALITY IN WOMEN THAN MEN. SO THIS WAS A CASE-CONTROLLED STUDY, I SHOULD SAY, COMPARING X-RAY VERSUS LOW DOSE CT. AND THE FALSE POSITIVE RATE WAS PRETTY HIGH. I'LL MAKE A POINT OF THAT. BUT IT DID DETECT PRIMARILY EARLY STAGE, STAGE 1A AND 1B LUNG CANCER, IN WHICH THE CURE RATES ARE THE BEST WITH SURGERY. BUT THE HIGH FALSE POSITIVE RATE VERSUS THOSE THAT WERE DETECTED, YOU JUST LOOK AT THIS CARTOON OF -- I DON'T KNOW WHERE YOU GO FOR YOUR CONCERTS, BUT THIS IS THE STAGE, AND THIS IS THE BALCONY. THESE ARE THE SEATS. SO, LOW DOSE CT WILL DETECT 3 1/2 PEOPLE SCREENED IN THE WHOLE AUDIENCE, BUT FALSE POSITIVE RATE IS MUCH HIGHER. SO THAT'S A PROBLEM. AND THE PROBLEM IS THE FINANCIAL PROBLEM, IT'S A PROBLEM OF MORBIDITY. IT'S A PROBLEM OF ANXIETY. AND THIS ISN'T ONLY TRUE OF LUNG CANCER AND SCREENING, IT'S TRUE OF MAMMOGRAMS, A HIGH FREQUENCY OF FALSE POSITIVE MAMMOGRAMS, MY WIFE HAD FIVE NOW, ALL NEGATIVE, SHE REFUSES TO TAKE MORE BECAUSE GOING THROUGH BIOPSY, PSYCHOLOGY OF ALL THAT, SHE DECIDED THAT SHE HAD ENOUGH. THAT'S BETWEEN HER AND HER PHYSICIAN. SO LOW DOSE CT NOW IS FUNDED BY THE U.S. GOVERNMENT. SO THERE'S NO REASON THAT PEOPLE WHO FIT CRITERIA, AND THIS IS THE CURRENT CRITERIA, CAN'T GET LOW DOSE CT. BUT THERE'S ABOUT 10 MILLION OF THOSE FOLKS OUT THERE. AND THE CAPACITY FOR DOING LOW DOSE CT MAY BE 100,000 TO 200,000, IT'S INCREASING. BUT OBAMACARE, AFFORDABLE CARE, AND ALSO OTHER MEDICARE WILL PAY FOR IT, SO THERE IS A NEED TO IDENTIFY HIGH RISK INDIVIDUALS OUTSIDE CRITERIA, NEVER SPOK SMOKERS, 13% COMING IN ARE NEVER SMOKERS, FROM PASSIVE OR PARENTAL EXPOSURE OR IS IT JUST BAD LUCK? OR THEIR JOB? SO WE NEED CRITERIA TO IDENTIFY INDIVIDUALS, AND ALSO PRIORITIZE THESE INDIVIDUALS AND TO THOSE AT GREATEST RISK. SO THEY DON'T HAVE TO STAND IN LINE FOR SIX MONTHS OR A YEAR OR TWO OR THREE YEARS. AS I SAID, AT THE TIME IN 2010 THERE WERE ABOUT OVER 8 MILLION, THERE ARE OVER 10 MILLION NOW. SO I'M ABOUT READY TO END. SO WHAT'S OUR STRATEGY FOR EARLY STAGE LUNG CANCER, UNMET NEEDS? ONE OF COURSE IS IDENTIFY PEOPLE WHO ARE AT RISK, AND INCLUDING THOSE WHO ARE SMOKERS, OR NONSMOKERS, AND FOR PRIORITY SCREENING, AND LOOKING AT VARIOUS CYTOKINES, URINARY, TUMOR METABOLITES AS A QUICK ASSAY, AND ALSO NASAL EPITHELIUM IS BEING EXPLORED AND CLIA-BASED ASSAYS ARE IN PROGRESS. YOU FIND SOMEONE, DECIDE, WELL, THIS PERSON NEEDS A SCAN, THEN YOU NEED TO DETERMINE WHETHER OR NOT THE LESION IS A CANCER OR NOT. I WAS TELLING YOU EARLIER, MOST OF THEM ARE NOT CANCER. SO, YOU NEED EARLY DIAGNOSTIC MARKERS, AGAIN SERUM CYTOKINES, URINARY TUMOR METABOLITES, AND INDEED NASAL EPITHELIUM, AND OTHER BIOMARKERS ARE BEING DEVELOPED NOW. THIS IS A BIG AREA, LET'S SAY MORE ABOUT THAT. AND THEN THE NEXT QUESTION IS, WELL, IT'S CANCER, BUT YOU DO SURGERY ON THAT PERSON, AND YOU REMOVE THAT CANCER, DO THEY HAVE METASTASIS GOING ON? AND ABOUT 25, 30% WILL HAVE METASTASES, EVEN THOUGH YOU CAN'T SEE THEM. SO PATHOLOGIST MISSES THEM. THEY ARE MICROMETASTASES. SO IF YOU COULD IDENTIFY THOSE PEOPLE, MAYBE YOU COULD OFFER THEM ADJUVANT THERAPY. EITHER IMMUNOTHERAPY OR CHEMOTHERAPY. AND ONE RATIONALE MIGHT BE MICRO METASTASES MAY BE MORE SUSCEPTIBLE THAN A LESION THAT'S 4 OR 5 CENTIMETERS, WHAT KILLS THE PATIENT IS RECURRENCE OF ORIGINAL TUMOR SO THERE ARE SEVERAL WAYS PEOPLE ARE FOCUSING ON THIS. WHAT WAS I PREACHING ABOUT? STAGE 1 LUNG CANCER, CURATIVE SURGERY, 25% RECUR AND DIE, STAGE 1A OR 1B, SURGERY IN BOTH. SOME PEOPLE ARE LOW RISK, SOME ARE HIGH RISK, HOW DO YOU IDENTIFY THOSE PEOPLE AT HIGH RISK? AND PRECISION MEDICINE IS THE STRATEGY THAT PEOPLE ARE USING NOW, INCLUDING TO DECREASE FALSE POSITIVE RATE, DECREASE FINANCIAL COST, IMPROVE PATIENT CARE, AND GUIDE FURTHER MECHANISTIC STUDIES. AND THESE PEOPLE, YOU CAN OBSERVE TO SEE IF THEY CHANGE. THESE PEOPLE ADJUVANT THERAPY IS WHAT WE WOULD RECOMMEND. THESE ARE JUST SOME FELLOWS IN THE LABORATORY, SOME CURRENT PEOPLE, MANY COLLABORATORS, I THINK I'VE TRAINED OVER 130 POST DOCS. THEY ARE CHAIRMEN OF DEPARTMENTS, DEANS OF THIS, THAT AND THE OTHER, SO I'M VERY PROUD OF ALL OF THEM. WE GET TOGETHER ONCE IN A WHILE, THERE'S LIKE 60 OF THEM IN JAPAN, SO WE PARTY, YOU KNOW, SO I THANK THEM AND I THANK YOU FOR PUTTING UP WITH ME AFTER 5:00. [APPLAUSE] HAPPY TO ANSWER ANY QUESTIONS. YES? WHAT'S YOUR NAME? JOANNA? HI. SECOND FLOOR. >> [OFF MICROPHONE]. >> I HOPE IT'S NOT OVERHEATED LIKE OUR LAB IS. >> [OFF MICROPHONE]. >> GO AHEAD, WHAT'S YOUR QUESTION? >> (OFF MIC.). >> SO, YEAH. SO THAT'S REALLY A VERY INTERESTING QUESTION. SO PEOPLE HAVE BEEN THINKING ABOUT TUMOR MUTATIONAL BURDEN FOR VARIOUS REASONS, INCLUDING -- WELL, MAYBE MORE NEOANTIGEN, SO YOU'D HAVE YOUR IMMUNOTHERAPY MIGHT BE MORE SUCCESSFUL, IT'S CONTROVERSIAL. INITIAL RESULTS SUGGEST MAYBE THAT WAS GOING TO BE A CASE BUT THERE ARE LOTS OF RESULTS THAT DON'T FIND THAT TO BE THE CASE. AND TUMOR MUTATIONAL BURDEN VERSUS BIOMARKERS, THAT'S NOT BEEN WELL STUDIED YET. AND BUT THAT'S SOMETHING THAT CAN BE DONE, INCLUDING WITH PCGA DATA, OR WITH DATA THAT WE'VE COLLECTED, AND THAT'S ACTUALLY SOMETHING THAT WE'RE QUITE INTERESTED IN AND ITS RELATIONSHIP TO MICROBIOME, METABOLOME, SO THAT'S A SOPHISTICATED QUESTION AND A VERY GOOD ONE. THANK YOU. YEAH? >> [OFF MICROPHONE]. >> OKAY. SO, THERE MAY BE ANOTHER CONNECTION. SO, SOME BACTERIA ACTUALLY PRODUCE A PROTEIN THAT IS A MUTAGEN. AND SO THAT'S SOMETHING THAT WE'RE SEARCHING FOR, IN ADDITION TO ACIDOVORAX, PLASELLA IS ALSO ASSOCIATED WITH LUNG CANCER, THERE'S FIVE OR SIX SPECIES OF KLEBSIELLA IN ACIDOVORAX, THERE A 60 BASE PAIR SEQUENCE THAT USUALLY CAN TELL YOU MAYBE THIS PROTEIN IS THERE. AND SO THAT'S A BIOINFORMATIC KIND OF EXAMPLE, AND YOU HAVE TO ASK THE QUESTION, CAN YOU BIND THE PROTEIN AND THEN IS THERE A WAY OF INHIBITING THE PROTEIN, AND ONE OF OUR NEW COLLABORATORS FOUND A SIGNATURE FOR THAT PROTEIN IN ONE OF THESE SPECIES THAT LIKE THE SIGNATURES I WAS TALKING ABOUT WITH CHEMICAL CARCINOGENS, THEY WERE SIGNATURES ASSOCIATED WITH THESE PROTEINS, SMALL PROTEINS PRODUCED BY BACTERIA. AND SO THAT MIGHT BE ANOTHER CLUE TO WHO GETS CANCER AND WHO DOESN'T GET CANCER AND WHAT KIND OF CANCER AND SO ON AND SO FORTH. VERY GOOD QUESTION. TWO VERY GOOD QUESTIONS. WANT TO TRY A THIRD ONE? WANT TO GO HOME? OKAY. THANK YOU.