THIS IS ALWAYS A LITTLE BITTER SWEET FOR ME BECAUSE I DO OF COURSE VERY MUCH VALUE MY MEMORIES OF DAVID DERSE BUT THIS REMINDS ME THAT HE LEFT US ALL TOO SOON. AND IN PARTICULARRING YOU ALL ALL--WELCOMING YOU ALL TO WHAT IS SIXTH DAVID DERSE MEMORIAL LECTURE, I WANT TO BEGIN WITH A PICTURE OF DAVE AND HIS COLLEAGUES. IT'S ENTIRELY FITTING THAT I DO THIS BECAUSE DAVE, DAVE, REALLY LOVED HIS LAB AND WAS DEDICATED TO HIS RESEARCH AND 1 OF THE THINGS THAT WAS PARTICULARLY POIGNANT ABOUT HIS UNTIMELY DEATH WAS THAT I THOUGHT HE WAS DOING REALLY SUPERB RESEARCH RIGHT UP UNTIL THE VERY END. BUT I ALSO, AS I ALWAYS DO WITH THESE OCCASIONS, WANT TO POINT OUT THAT AS IS TRUE FOR MANY OF US, WE'RE NOT ENTIRELY EMBEDDED IN THE LABORATORY 24/7 BUT WE HAVE OTHER INTERESTS AND OTHER DISTRACTIONS. ONE OF THE THINGS THAT I THOUGHT WAS ALWAYS PARTICULARLY COMPELLING AND WONDERFUL ABOUT THE RELATIONSHIP I HAD WITH DAVID DERSE IS THAT HE AND I SHARED A PASSION FOR WHAT MY WIFE CALLS HARASSING FISH. AND THIS IS A PICTURE OF DAVE WITH A SMILE AND A NICE LITTLE BROWN TROUT. THAT PICTURE WAS TAKEN ON TOWN CREEK, SO A PRETTY LITTLE CREEK MAYBE A HUNDRED MILES WEST OF HERE THAT MARYLAND PUTS FISH IN EVERY YEAR SO THAT PEOPLE LIKE DAVE AND I CAN GO AND HARASS. YOU CAN SEE FROM THE GRIN THAT ALTHOUGH HE LOVED HIS LAB PERHAPS MORE THAN ANYTHING, HE DID ENJOY BEING OUT OF IT. I THINK IT WASN'T JUST THE CATCHING OF FISH BUT IN FACT, FISHING IN A SENSE HAS A CONNECTION TO THE LARGER WORLD, TRYING TO UNDERSTAND HOW IT ALL WORKS, HOW FISH ARE IN, WHERE THEY MIGHT HIDE IN THE STREAM, HOW THEY MIGHT BE FOOLED OR CONVINCED. AND THIS IS A SIMILAR PICTURE OF DAVE BECAUSE HE'S JUST OUT IN THE WORLD SORT OF ENJOYING THE NATURE AND OUR PLACE IN IT. AND DAVE--1 OF THE THINGS THAT WAS REALLY WONDERFUL ABOUT DAVE AND I TREASURE HIM ABOUT HIM AND A FISHING WAS HE FIT INTO THE WORLD BEAUTIFULLY AND BY THAT I MEAN, THAT HE WAS--HE WAS 1 OF THE MOST RELAXED AND COMFORTABLE AND GENEROUS AND FRIENDLY PEOPLE I EVER MET. AND HE WAS ALWAYS A PLEASURE TO BE AROUND AND THAT GRIN THAT YOU SEE THERE, WAS HIS USUAL EXPRESSION UNLESS SOMETHING REALLY AWFUL HAD HAPPENED. SO I WANT TO TRY AND SEE IF I CAN CALL BACK FOR YOU THOSE THAT KNEW HIM, THE MEMORY OF THAT SMILE AND TRY TO IMPART TO THOSE OF YOU WHO DIDN'T KNOW HIM, THE FACT THAT IT IS POSSIBLE TO BE A REALLY FINE SCIENTIST AND A TRULY WONDERFUL HUMAN BEING AT THE SAME TIME. AND I WANT TO WELCOME OUR SPEAKER REUBEN HARRIS IN THE SPIRIT OF THE IDEA THAT DAVE WOULD HAVE LIKED VERY MUCH. HE LOVED SCIENCE DEEPLY AND HE WOULD HAVE BEEN ENTHRALLED TO HEAR THE LECTURE TODAY, AND SO, THE SADDEST PART IN SENSE IS THAT HE'S NOT HERE TO SHARE THE DAY WITH US WHICH IS WHAT I THINK I WOULD HAVE LIKED THE MOST. SO WITH THAT, I WILL ASK ERIC FREED TO INTRODUCE THE SPEAKER AND WE WILL BEGIN. >> THANKS A LOT FOR THAT STEVE. I THINK IT'S NO QUESTION AT ALL THAT WE MISS DAVE JUST AS MUCH TODAY AS WE DID WHEN WE HAD THE FIRST 1 OF THESE SEMINARS 5 YEARS AGO. SO ON BEHALF OF THE ENTIRE DRP, IT'S REALLY A PLEASURE AND AN HONOR TO WELCOME REUBEN HARRIS FOR THE SIXTH ANNUAL DERSE LECTURE AWARD. AS YOU KNOW RUBEN IS AN EXPERT ON APOBECS AND THIS WAS 1 OF THE MANY REASONS WHY WE SELECTED RUBEN TO GIVE THE LECTURE TODAY BECAUSE DAVE HIMSELF WAS QUITE INTERESTED AND FASCINATED REALLY IN ANTIVIRAL RESTRICTION MEDIATED BY THE ARKTS POETIC BECK PROTEINS AND THIS IS HIGHLIGHTED IN HIS PAPER FROM ABOUT 10 YEARS AGO FROM PNAS IN WHICH HE SHOWED THAT HTLB1 ESCAPES RESTRICTION VIA SEQUENCES AND NUCLEO-CAPS THAT BLOCK THAT INTO APOBECKS. AND THOSE HAVE SHARED RESEARCH INTEREST WITH DAVE SO FOR EXAMPLE, PAT GREEN AND JOE JEAN OUR FIRST 2 LECTURE AWARDEES WERE FOCUSED ON HTLV, AND STILL ARE, OTHER COLLEAGUES ON CELL TRANSFER AND LAST YEAR MIKE MALIN WHO IS A VERY PROMINENT APOBEC COLLABORATOR AND COLLABORATOR OF REUBEN'S. HE IS A NATIVE OF CANADA. HE DID UNDERGRADUATE STUDY AND Ph.D. RESEARCH AT THE UNIVERSITY OF ALBERTA, Ph.D. WORK WITH YOU AND SUSAN ROSEN BERG AND DURING THAT TIME MADE IMPORTANT CONTRIBUTIONS TO ADAPTIVE MUTATION AND A REALLY INTERESTING MUTATIONAL, BENEFICIAL MUTATIONAL PROCESS THAT OCCURS IN E.COLI AND BACTERIAL ORGANISMS AND HE FINISHED UP WITH SUSAN WHEN SHE MOVED TO BAYLOR A SHORT POST DOC ATL AND THEN IN 1998 WENT TO CAMBRIDGE TO DO POST DOCTORAL WORK WITH MICHAEL NEWBURGER, AND IT WAS AT THAT TIME THAT HE STARTED MAKING IMPORTANT CONTRIBUTIONS FOR UNDERSTANDING HOW DEAMNAISS AND AID IS ESSENTIAL FOR IMMUNE O GLIBBING O GENE CONVERSION AND THAT HE'S HOMOLOGUES CAN ACT AS DNA MUTATORS AND IN COLLABBUATION WITH MIKE MALIN SHOWING THAT APOBEC 3G WHICH MIKE DISCOVERED AS A RESTRICTION FACTOR IS A DEAMILLIO NAIS. AND THIS--DEAMNAIS AND THIS IS RESTRICTED BY APOBEC PROTEINS. IN 2003 RUBEN JOINED THE FACULTY AT UNIVERSITY OF MINNESOTA TWIN CITIES WHERE HE RAPIDLY CLIMBED THE LADDER. BECAME FULL PROCESSOR IN 2013 AND HOWARD HUGH'S MEDICAL INVESTIGATOR IN 2015. HE'S CONTINUED TO DO VERY IMPORTANT WORK ON THE STRUCTURE AND FUNCTION APOBEC PROTEINS AS THEY RELATE TO RETROVIRAL RESTRICTION. AND MORE RECENTLY IS ALSO BRANCHED OUT TO STUDY THE CONNECTION BETWEEN APOBECS AND CANCER. I THINK HE WILL TOUCH ON THIS TOPIC TODAY, AS MANY OF YOU KNOW, REUBEN IS A ATTENDEE AT NATIONAL AND INTERNATIONAL MEETINGS THESE ARE SOME PICTURES I TOOK A FEW YEARS AGO AT A CONFERENCE. AS WAS THE CASE WITH DAVE, REUBEN IS NOT LIVING IN THE LAB 247 AND TO SET THIS UP, I WANTED TO REMIND YOU THAT MINNESOTA IS OFTEN REFERRED TO AS THE LAND OF 10,000 LAKES. THIS IS A MAP FROM NORTHERN MINNESOTA SHOWING WHAT LOOKS LIKE MORE THAN LAND, SOME OF THESE LAKES HAVE AMAZING NAMES LIKE LAKE WINNIBISHAGOSH. [LAUGHTER] I'M NOT MAKING THIS UP. YOU CAN LOOK AT THIS LATER ON. NOT ONLY DOES MINNESOTA HAVE LAKES BUT IT'S VERY COLD. THIS IS A TEMPERATURE MAP FROM THE TYPICAL JANUARY DAY WHERE WE SEE IN THE WASHINGTON AREA, IT'S JUST AROUND FREEZING. THESE ARE TEMPERATURES OF COURSE IN FAHRENHEIT. MINNESOTA WHERE MANY OF US SPENT SOME TIME IT'S ABOUT 5 OR 10-DEGREES BELOW 0 AND THEN IN NORTHERN MINNESOTA IT WAS 36 BELOW 0, SO A FULL 60- DEGREES COLDER THAN WASHINGTON. SO NEXT TIME I HEAR ANYBODY COMPLAINING ABOUT HOW COLD IT IS HERE. KEEP THIS IN MIND. [LAUGHTER] >> [INDISCERNIBLE] >> ALMOST EXACTLY. AND THIS OF COURSE LEADS TO A LOT OF BAD JOKES. [LAUGHTER] SO THIS COMBINATION OF LATENT COLD WEATHER LEADS TO A LOT OF ACTIVITIES ABOUT FROZEN LAKES SO I THINK SOME OF THESE WOULD AGREE CATCHING MUSKEE THROUGH THE ICE, BUT OTHERS ARE MORE BIZARRE, FOR EXAMPLE, THE ICE AND JUMPING IN. THIS IS A POLEAR BEAR FESTIVAL IN MINNESOTA IN WHICH PEOPLE ARE LINED UP TO JUMP IN THIS HOLE IN THE ICE. I'M NOT QUITE SURE WHY PEOPLE DO THIS BUT IT'S TRUE OF A LOT OF THINGS. I'M NOT SURE WHAT THESE LADIES AND GENTLEMEN--I WOULD LOVE TO KNOW HOW LONG THEY POSED FOR THIS PHOTO. BUT I THINK THE MOST BIZARRE ACTIVITY--[LAUGHTER] --THAT INVOLVES FROZEN LAKES IS CURLING WHICH IS NOW AN OLYMPIC SPORT, HAS BEEN FOR SOME PERIOD OF TIME. >> NATIONAL TRIALS ARE RIGHT NOW. >> AND THIS IS THE ACTIVITY THAT RUBEN IS ACTUALLY VERY ACTIVE PARTICIPANT IN. I DON'T REALLY KNOW WHAT THE RULES OF CURLING ARE BUT CLEARLY IT'S A VERY INTENSE. AND REUBEN TAKES HIS ENTIRE LAB TO CURLING EVENTS. SO NEXT TIME YOUR P. I. ASK SAYS YOU TO DO SOMETHING UNUSUAL OUTSIDE OF THE LAB, BE GLAD YOU'RE NOT IN REUBEN'S LAB. AND FINALLY, WHEN SETTING UP THESE INTRODUCTIONS IT'S ALWAYS FUN TO GOOGLE THE SPEAKER'S NAME AND SEE WHAT COMES UP, SO WHEN YOU DO GOOGLE IMAGES ON REUBEN STRUCTURES YOU FIND OBVIOUS THINGS LIKE APOBEC STRUCTURES BUT YOU ALSO TURN UP OTHER THINGS LIKE I CAN'T QUITE EXPLAIN, BUT MAYBE LATER ON RUBEN WILL TELL US ABOUT THE CONNECTION BETWEEN REUBEN HARRIS AND THE IMAGES. SO--[LAUGHTER] --SO WITH THAT I WILL WELCOME REUBEN TO THE POOED IMRUM TO GIVE--PODIUM ON GIVE THE LECTURE ON HUMAN VIRUSES AND DISEASES AND PRESENT HIM WITH THE SIXTH ANNUAL DAVID DERSE AWARD, THERE WILL BE PHOTOS, MORE PHOTOS LATER ON, CONGRATULATIONS. >> [ APPLAUSE ] >> ALL RIGHT, THATIA A DIFFICULT INTRODUCTION TO FOLLOW. THANK YOU. YOU KNOW I AM HUMBLED TO BE HERE. DAVID WAS A FRIEND AND SCIENCE COLLEAGUE AND I WISH I HAD MORE TIME TO INTERACT WITH HIM. I AM GOING TO SHARE A FEW STORIES FROM THAT PERIOD INCLUDING, I WILL CHALLENGE YOU NOW TO REMEMBER WHERE THIS IS BUT DON'T BLURT IT OUT. DON'T BLURT IT OUT! AND HOPE TO ALSO CONVINCE YOU THAT MINNESOTA'S NOT NEARLY AS DESOLATE AS HE JUST DESCRIBED. IT CAN LOOK LIKE THIS FOR A GOOD FRACTION OF THE FALL. YEAH, AND HOPEFULLY HAVE SCIENCE TO SHARE WITH YOU AND QUESTIONS AS WE PROCEED. SO, THAT TITLEOT POSTER IS THE TRUE TITLE. --TITLE ON THE POSTER IS THE TRUE TITLE. I WAS THINKING OF ALTERNATIVE TITLES, 1 IS HERE, CANCER MUTA GENESIS, AND THE TITLE FOR DAVID IS BACK AND FORTH BETWEEN VIRUSES AND CANCER BECAUSE THIS IS A STORY THAT'S START WIDE MY INTEREST, MY CAREER INTEREST IN MUTE O GENESIS THAT TOOK ME INTO I VIROLOGY, AND TRANSFER OF ANY NORMAL CELL INTO A TUMOR. SO I'M ALSO GOING TO TRY TO USE THE TRACK PATH FOR COLLEAGUES WHO MAY BE WATCHING. THOUGH, I NEED TO SEE--ALL RIGHT, WE WILL SEE ABOUT THAT. MAYBE I'LL USE THE LASER. I THINK THIS WILL BE TRICKY. SO I WANTED TO START WITH THIS PAPER TOO BECAUSE IT WAS 2007. DAVID SHOWED NICELY, THE PUZZLE HERE WAS THAT APOBEC 3G RESTRICTINGS DEFICIENT HIV REALLY QUITE WELL IN A DOZE RESPONSIVE WAY. LIKE A SLEDGE HAMMER FALLING ON TOP OF THE VIRUS, BUT HTLB IS RELATIVELY RESISTANT BY APOBEC 3G AND THIS WAS IN LARGE PART DUE TO A FAILURE TO PACKAGE. SO SHOWING COMPARING SIDE BY SIDE APOBEC 3G INTO HIV, VERSUS HTLB, IT WAS CLEAR THAT THE NOT GETTING INTO THOSE PARTICLES AS WELL. AND HE WENT ON TO YOU KNOW AS JUST DESCRIBED, WORK ON A MECHANISM BY WHICH THAT VIRUS IS EXCLUDING THAT FROM THE PARTICLES AND THIS SORT OF CONTRIBUTES TO THE BROADER THEME OF HOW DO ANY AND ALL DNA BASED PARASITES AVOID THE APOBECS AND AVOID THE ENZYMES? AND THIS IS 1 MECHANISM BY WHICH THAT CAN HAPPEN. SO WE ALSO SHARED INTEREST, NOT ONLY IN APOBEC BUT ALSO IN A LITTLE BIT OF VIRAL AND HOST GENE NOMENCLATURE AND THIS WAS A COLLABORATIVE PAPER. IT'S THE ONLY COLLABORATIVE PAPER WHERE DAVID AND I ARE TOGETHER AS WELL AS SOME OF OUR OTHER COLLEAGUES IN THE FIELD AND IT WAS A NIGHT RALLYING POINT WHERE WE LEARNED THERE WERE A LOT OF DIFFERENT SPECIES AND WE DEPARTMENT HAVE CORRECT NUMBER CORRESPONDING HOM LOGS TO ALL THE DIFFERENT SPECIES SO WE NEEDED TO COME UP WITH A DIFFERENT NOMENCLATURE AND THAT WAS SHOWN HERE BY CALLING THEM INSTEAD OF BY APOBECS OR BY THE Z-DOMAIN OR THE Z1, 2, 3, BASED ON HALLMARK RESIDUES THAT DISTINGUISH THESE DIFFERENT DEAMNAIS DOMAINS. THIS I ACTUALLY SHOWED AT THE MEMORY MEMORY MEMORIAL SYMPOSIUM IN 2010 AND IT HIGHLIGHTS ANOTHER COMMON INTEREST OF DAVE AND I HAD MYSELF AND THAT IS AN INTEREST IN COMPARATIVE VIROLOGY, AND THESE LENTIVIRUSES, THESE DIFFERENT HOSTS OVER HERE AND THE MAIN POINT I WAS TRYING TO MAKE ON THIS PARTICULAR SLIDE IS THAT THERE'S ABSOLUTE CONSERVATION BETWEEN THE VIF PROTEIN ENCODED BY THE LENTIVIRUSES AND THEINATER CAPACITY TO NEUTRAL ICE THE Z3 APOBEC PROTEIN OF THE RESPECTIVE HOSTS. AND WHAT THAT TELLS US AS I TALK WITH TRAINEES OVER LUNCH IS THAT CONSUMMATION IS SYNONYMOUS TO IMPORTANCE AND YOU HAVE TO FIGURE OUT WHY IT'S CONSERVED AND THAL CASE THESE ARE CONSERVED BECAUSE THESE APOBECS ARE A THREAT TO THESE VIRUSES. SO THE APOBEC FAMILY, THIS IS THE CURRENT SCHEMATIC OF THE APOBEC FAMILY, A LOT OF PEOPLE IN THE CANCER WORLD ARE REFERRING TO APOBEC AS A SINGLE ENTITY, NOT A SINGLENTITY AND A MULTIGENE FAMILY. SO IT'S ROOTED WAY BACK IN THE EARLIEST VERTEBRATES, ALL OF THESE EARLY VERTEBRATES HAVE AID, ACTIVATION INDUCED DEAMNAIS AND ALL THESE ARE IMPORTANT FOR ANTIBODY DIVERSIFICATION. THATYA A LITTLE BIT OF THAT--THAT'S A LITTLE BIT OF THAT CONTRIBUTION WAS DONE WHILE I WAS A POST DOC IN MICHAEL NEWBERGER'S LAB. THIS GIVES RICE TO APOBEC 1, WHICH IS THE NAMESAKE OF THE FAMILY, APOBEC 1, IT'S A BIT OF A MISNOMER BUT IT'S A DESCRIPTIVE NAME THAT DOES TELL US WHAT APOBEC 1 DOES WHICH IS EDIT THAT PARTICULAR RNA AND OTHERS AND THEN REALLY INTERESTINGLY AT SOME POINT, PRIOR TO THE RADIATION OF PLACENTA MAMMALS, APOBEC 1 AND AID DUPLICATE AND DIVERGE TO THE APOBEC 3 LOCUST, WHICH IN ALL MAMMALS IS FOUND BETWEEN 2 CONSERVED GENES CBX6 AND 7 AND VARIES IN COPY NUMBER. SO THE ANCESTRAL MAMMAL HAD TO HAVE AN GREEN, ORANGE AND BLUE AND PRESENT DAY MAMMALS HAVE VARYING NUMBERS OF THESE DIFFERENT DOMAINS. ALL OF THEM HAVE A BLUE, AND ALL OF THEM HAVE VARYING COPY NUMBERS OF THE ORANGE AND GREEN AND SHOWN HERE FOR INSTANCE IS OUR FAVORITE MODEL ORGANISM, THE HOUSE MOUSE, THIS HAS A DOUBLE DOMAIN, GENE WHICH ENCODES A DOUBLE DOMAIN ENZYME OF THE Z2, Z3 TYPE AND HUMAN LOCUST IS COMPRISED OF APOBEC 3-H AND THESE ARE SINGLE DOMAINS LIKE APOBEC 3, A, C, H AND DOUBLE DOMAINS LIKE THE REST. SO THAT KIND OF GIVES YOU THE LAY OF THE LAND AS TO THE APOBEC FAMILY, I'M GOING TO GIVE AN INTRODUCTION WHICH IS GROUNDED IN OUR RESEARCH ON HIV 1 AND THEN SWITCH TO CANCER MUTE O GENESIS AND THEN COME BACK TO VIRUS RESTRICTION. SO IF IT SEEMS LIKE A BIT OF A SEE SAW, IT IS. OKAY? SO THIS IS OUR CURRENT WORKING MODEL FOR HIV RESTRICTION BY APOBEC 3 ENZYMES AND WORK FROM MY LAB AND RENE'S AND DAVID'S AND OTHERS THAT HAVE COMBINED TO SHOW THAT 4 APOBECS HAVE THE CAPACITY TO PACKAGE INSIDE PARTICLES, TROJAN HORSE TYPE MECHANISM AND HIDE INSIDE UNTIL THE TARGET CELL IS FOUND AND THEN DURING REVERSE TRANSCRIPTION, WHICH SHOULD BE WITHIN THIS PERMEABLE CAP SID CORE, BUT THAT'S HARD TO DETECT SO IT'S DEPICTED FREELYY HERE, DURING TRANSCRIPTION, THE VIRAL SET O SEENS AND THEN THESE TEMPLATE THE INCORPORATION OF THE ADO 19S IN THE RESULT OF THE PHENOMENON TO THE HYPER IN YOUITATION. --MUTATION. THESE 4 CAN DO IT AND OF COURSE THE LENTIVIRUS IS THRIVED BECAUSE THEY ALL ENCODE A PROTEIN CALLED VIF, WHICH FOR YEARS WAS CALLED A VIRUS--VIRAL ACCESSORY PROTEIN. IT'S A FUNDAMENTAL VIRAL PROTEIN THAT THESE CAN'T DO WITHOUT. AND THIS RECRUITS CBS BETA AND OTHER CELLULAR PROTEINS TO FORM AN E3 UBIQUITYIN COMPLEX THAT DEGRADES THE APOBECS, SO THAT'S HOW THE VIRUS CAN THRIVE IN THIS HIGH MUTATIONAL ENVIRONMENT. SO A LOT OF OUR WORK OVER THE YEAR SYSTEM BEING DIRECTED TO TOWARD UNDERSTANDING THE APOBEC MUTATION SIGNATURE IN THE VIRUS AND BY THAT I MEAN, IF YOU LOOK CAREFULLY AT PATIENT-DERIVED SEQUENCES 1 CAN FIND 2 DISTINCT PATTERNS. ONE PATTERN IS G-A MUTATIONINGS MUTATIONS SO I WILL START UP HERE, SO THE BUSINESS IS ON THE CD NA STRAND WHERE THOSE ARE DEAMINATING CDU IN A TC CONTEXT OR A CC CONTEXT. OT VIRAL PLUS STRAND THAT IS THEN CONVERTED INTO AN A. G. THAT'S 1 OF THE DISCIPLINARY NUCLEOTIDE SIGNATURES AND THE OTHER 1 IS THE GA IS CONVERTED INTO A PLUS STRAND AA BECAUSE THE U, ONCE IN THERE BASE PAIRS LIKE A T, SO WHICH APOBECS ARE RESPONSIBLE FOR THE SIGNATURE? I GAVE YOU THE PUNCH LINE BUT ROAD TO GETTING TO THAT POSITION WAS ACTUALLY QUITE LONG AND ARDUOUS. SO TO SHARE A COUPLE OF THOSE EXPERIMENTS BY MEANS OF INTRODUCTION, APOBEC 3G IS THE ONLY APOBEC THAT DEAMINATES PREFERENTIALLY IN A CC CONTEXT. SO IT WAS THE OBVIOUS CULPRIT FOR MAKING THE GG-A G PATTERN. SO WE DELETED THIS. CRISPR IS ON THE BLOCK, AND BEFORE THAT WE HAD HOMOLOGOUS ASSOCIATION AND BEFORE THAT ADENO VIRUS, SOPHISTICATEDY WE GO OUT WITH A SELECTIVE MARKER AND TAKE OUT APOBEC 3G, REPEAT THE PROCESSOT OTHER CHROMOSOME AND GENERATE AN OLD CELL LINE THAT LACKS APOBEC 3G, AND THEN ASK WHAT THE RESULTING HYPER MUTATION SIGNATURE IS, 1 CAN SEE THAT IT GZ--GOES FROM BEING A GG FOCUSED AND GA FOCUSED SIGNATURES NATURE TO BEING AN EXCLUSIVELY GA FOCUSED SIGNATURE. SO AS PREDICTED FROM OVER EXPRESSION EXPERIMENTS WE COULD TAKE AWAY THE GG COMPONENT OF THE HYPER MUTATION PROGRAM. THE OTHER PART WAS HARDER TO DO BECAUSE IN THAT VERY SAME T-CELL SYSTEM THAT EXPRESSES MULTIPLE APOBECS ENDOGENOUSLY, WE FOUND THAT EITHER DELETING APOBEC 3 F OR KNOCKING DOWN APOBEC 3D DIDN'T CHANGE VERY MUCH THE OVERALL BALANCE BETWEEN GG AND GA MUTATION BUT WHEN WE KNOCK DOWN APOBEC 3D AND IN THE ABSENCE OF APOBEC 3 F, WE COULD TAKE AWAY THE COMPONENT. SO WHEN YOU PUT 2 AND 2 TOGETHER, THAT REALLY IMPLICATED OR DEMONSTRATE THAD APOBEC 3G AND D ARE CONTRIBUTING TO THIS MUTATION PROGRAM IN HIV. SO, THE MODEL THOUGH, YOU WILL RECALL, DEPICTS THAT PERSNIKETY 1 APOBEC 3 H THAT DAVE AND I TALKED ABOUT FROM TIME TO TIME WHICH IS THE HUMAN Z3 SINGLE DOMAIN FACTOR AND THIS GUY IS ALSO INVOLVED AND IT'S KIND OF--THE OPOETIC BECKS, EVEN THOUGH THEY'RE VARIABLE IN COPY NUMBER BETWEEN SPECIES BUT THEY'RE ALSO VARIABLE WITHIN A SPECIES SO THAT WILL BE 1 OF THE THEMES OF TODAY'S LECTURE AS WELL. SO SOME OF THAT VARIATION IS SHOWN HERE AND THIS WAS ORIGE NAWILLLY REPORTED BY MICHAEL EMMER MAN'S GROUP WHERE APOBEC 3 H HAS A NUMBER OF DIFFERENT SNPs OR NATURALLY OCCURRING POLYMORPHISMS AND THESE SNPs ARE SINGLE NUCLEOTIDE POLYMORPHISMS ARE IN BLOCKS CALLED HAPLO TYPE BROCKS, AND SOME OF THESE BLOCKS LIKE HAPLOTYPE 2 ENCODE A PROTEIN. I WILL SHOW YOU THAT IN A SECOND. HAPLOTYPES 1, 3, 4 ENCODE A PROTEIN AND WE KNOW WHY BECAUSE THERE'S A DELETION IN THERE OF A DISPARAGING AND ALPHA HELIX, SO THE ONLY 2 YOU NEED TO CARE ABOUT IS THIS 1, POSITION 15, THERE'S APARRAGING OR DELETION AND AT 105 THERE'S GLYSEEN OR ARE--ADMINISTRATIVE GENERATEDDINE. SO WE HYPOTHESIZED THAT THIS WOULD CONTRIBUTE TO THEOVERALL MUTATION AND PROCESS. BUT TO TEST THAT HYPOTHESIS, WAS KIND OF TOUGH. SO WE CAN ALL OVER EXPRESS AND DAMAGE A VIRUS AND EVEN SUPPRESS A VIRUS REPLICATION COMPLETELY BUT IT'S HARDER TO INTERROGATE SORT OF LIKE THE LITTLE BROTHER APOBEC AND THE PRESENCE OF MULTIPLE OTHER REALLY DOMINANT APOBECS IN AN ENDOGENOUS SYSTEM, SO TO DO THAT, WE TOOKAT VANNEDDAGE OF VIRUSES MOLECULAR PROBES IF YOU LIKE, SO WE HAVE LAB STRAINS LIKE HIV 1 NL43, 3 B, LAI, THEY ALL HAVE SLIGHTLY DIFFERENT VIFS AND THEY'RE NOT ALTOGETHER PERFECTLY FUNCTIONAL AGAINST 3 H BUT BETWEEN THE EMMER MAN AND SIMON LABS WE WERE ABLE TO DO AMINO ACIDS THAT WERE IMPORTANT FOR NEUTRALIZING IT. SO WE COULD MAKE A HYPER VIF, SOMETHING THAT BINDS AND GRADES APOBECS REALLY WELL AND WE COULD MAICA VARIANT THAT--MAKE A VARIANT THAT DOESN'T RECOGNIZE 3 H BUT RECOGNIZES ALL THE LITTLER APOBECS. SO WE COULD USE THESE AS PROBES TO ASK WHETHER THE ENDOGENOUS APOBEC 3 H IS IMPORTANT FOR RESTRICTION AND MUTATION, SO CONTROLS ARE SHOWN HERE. SO ESSENTIALLY ALL THESE VIRUSES GROW ON A T-CELL LINE THAT HAS NO APOBECS IF YOU HAVE APOBEC 3D, YOU THEY ALL GROW PERFECTLY WELL AND IN AN INDISTINGUISHABLE, IF THESE CELLS EXPRESS, LET'S LOOK OVER HERE FIRST AT HIGH LEVELS OF APOBEC 3 H, ONLY THE HYPER VIRUS GROWS WELL ON THOSE CELLS EXPRESSING HIGH LEVELS OF H, AND IT GROWS WITH THE LOWER EXPRESSING MEDIATE LEVELS SO WE CAN USE THESE VIRUSES AS PROBES THEN THAT GO INTO PRIME AREY CELLS AND ASK IF THE 3 H IS ALSO CONTRIBUTING TO RESTRICTION. AND HYPER MUTATION, SO FOR TRAINEES, I CALL THIS THE MONEY EXPERIMENT, EVERY PAPER ALWAYS HAS 1 KIND OF IMPORTANT EXPERIMENT THAT IS ABOVE THE OTHERS. AND THIS IS IT WHERE WE USE THESE VIRUSES TO REPLICATE AND INTERROGATE THE FUNCTION OF THE ENDOGENOUS APOBECS IN T-CELLS WITH DONORS FROM DIFFERENT HAPLOTYPES. SO HERE'S HAPLOTYPE 1 WHICH IS AN UNSTABLE HAPLOTYPE, ALL THOSE REPLICATE JUST FINE BUT IN A PERSON BHO HAS A HAPLOTYPE 2 OR 3 H PROTEIN, ONLY THE HYPER VIRUS REPLICATES WELL, THE LAB OF THE INTERMEDIATE AND THE HYPOVIF IS RESTRICTED BY THOSE CELLS. SO THE WESTERN BLOTS ARE VERY INTERESTING AND 1 CAN LOOK ESSENTIALLY AT WHAT'S HAPPENING TO THE ENDOGENOUS APOBEC 3 H IN THE INFECTED CELLS. SO IF 1 LOOKS OVER HERE IN THE CELL LIAISON SAITS, AT APOBEC 3 H. IF THERE'S A HYPOVIF IN THE SYSTEM, THERE'S A LOT OF APOBEC 3 H AND IT'S INDUCED OVER THE TIME OF INFECTION, SO EARLY IN THE INFECTION IT'S LITTLE AND QUITE A LOT. IN THE HYPER INFECTION, THERE'S A LOT LESS APOBEC 3 H BECAUSE IT'S INTEGRATED BY THAT VIP, AND WHEN YOU LOOK AT THAT, THE DIFFERENCE IS MORE AND STRONGER BECAUSE AGAIN, HYPER VIF IS ABLE TO TAKE DOWN ENDOGENOUS A3 H, WHEREAS A30 BECK VIF CANNOT. SO WHEN YOU ASK WHAT'S THE RESULT IN G-A MUTATIONAL SPECTRUMOT VIRAL GENOME, YOU CAN SEE FROM THESE CELLS THAT EXPRESS THE STABLE APOBEC 3 H, THE HYPER VIF ACCUMULATES MUTATION IN THE GA MUTATION CONTEXT WHEREAS IN CELLS NOT EXPRESSING APOBEC 3 H ORE UNSTABLE FORM OF APOBEC 3 H, THERE'S NO DIFFERENCE IN THE MUTATIONAL PATTERN IN THE RESULT AND VIRUSES. SO THIS SAID, THAT IN ADDITION TO APOBEC 3G AND F AND D, NOW WE HAVE APOBEC 3 H PLAYING A ROLE IN HIV RESTRICTION AND MUTE O GENESIS. SO THIS IS THE CURRENT REPERTOIRE OF ENZYMES THAT'S RESPONSIBLE FOR THIS. AND SO THERE'S ONGOING WORK IN MANY LABS TRYING TO FIGURE OUT HOW WE CAN USE THIS INTRINSIC œREALLY QUITE POWERFUL AGAINST THE VIRUS TO EITHER EXTINGUISH THE VIRUS BY HYPER MUTATION MECHANISM OR PERHAPS FOR INSTANCE INHIBIT VIF FUNCTION TO ALLOW THIS PROGRAM TO HYPER MUTATE THE VIRUS. SO THERE'S A THERAPEUTIC ASPECT TO THIS THAT HOPEFULLY OVER THE NEXT SOME YEARS WILL COME TO FRUITION. SO THIS PICTURE I KNOW JOHN KNOWS THE RESULT OR THE ANSWER, IT'S DOWN HERE, THIS IS GRAVES MOUNTAIN LODGE IN VIRGINIA. HOW MANY OF HAVE YOU BEEN THERE? SO I FIRST MET DAVID AT THE FAMOUS DRUG RESISTANT PROGRAM MEETING WHEN IT WAS HELD IN SHAN TILLY VIRGINIA IN 2004 WHICH WAS A VERY NICE MEETING LOTS OF GREAT PEOPLE FROM HERE AND THE SMALL MEETING AND REALLY NICE INTRODUCTION TO THE FIELD OF RETROVIRUS RESEARCH THIS MEETING WAS ORGANIZED BY HUNG FANG AT THE PATHOGENESIS AREA AT THIS LITTLE RESORT LODGE CALLED GREG'S MOUNTAIN RESORT LODGE. SO AT THIS MEETING [INDISCERNIBLE] SPOKE AND I SPOKE IN THE SAME SESSION AND DAVID WAS ATTENDING THIS MEETING AND THIS WAS 2 MONTHS BEFORE HE PASSED AWAY AND ACTUALLY I HAD A FOND RECOLLECTION. WE STAYED IN THESE SORT OF CABINS UP ON THE HILL. BETWEEN OR AFTER, I CAN'T REMEMBER THE DETAILS WE WERE SHARING A BEER UP AT 1 OF THESE LODGES, TALKING ABOUT SCIENCE AND LIFE AND ENJOYING THIS BEAUTIFUL ENVIRONMENT. AND THAT'S WHERE I SHARED SOME OF OUR EARLIEST STUDIES ON APOBECS IN CANCER MUTE O GONE SIS WITH DAVID. SO THIS IS WHERE I SWITCH GEARS ALSO AND I'M HAPPY TO TAKE QUESTIONS THROUGHOUT FOR--IF ANYONE WANTS TO TALK ABOUT VIRUSES OR CANCER. PLEASE INTERRUPT, I DON'T MIND AT ALL. SO MAYBE THERE'S EMERGING--SO IN THE LAST FEW YEARS MY LAB AND OTHERS HAVE BEEN UNCOVERING AN IMPORTANT ROLE FOR APOBEC ROLES IN ENZYMES IN CANCER MUTE O GENESIS. SO IN HINDSIGHT IT'S NO SURPRISE. THESE ARE THERE TO DESTROY VIRUSES, A LOT OF VIRUSES APPEAR THAT ARE USING THE APOBECS TO THEIR OWN ADVANTAGE TO FACILITATE THEIR OWN EVOLUTION, THE BEST EXAMPLES ARE APPLIED LOMA AND POLYOMA VIRUSES BUT 1 COULD ARGUE THAT FOR HIV. BUT IT'S NOT A SURPRISE BUT THEY'RE ALSO--THE TUMOR CELLS ARE AWLTS TAKING ADVANTAGE OF APOBECS TO FACILITATE TUMOR EVOLUTION, SO THIS FUNDAMENTAL DNA CYTOSCENE DEAMINATION MECHANISM WHICH IS THE HALLMARK OF ALL APOBEC ENZYMES IS NOT ONLY LEVERAGED FOR THE BENEFICIAL SIDE AND INNATE IMMUNE RESPONSE BUT ALSO IN THE DETRIMENTAL SIDE TO MUTATION PROGRAMS. SO I WANTED TO SHARE WORK ON OUR CANCER MUTE O GENESIS AND THEN MAKE A SWING BACK TO RETROII RETROVIRROLOGY. SO THIS IS GOING BACK TO PIE POST DOC. SO ERIC POINTED OUT MY PAPER, AT THE TIME WE DISCOVERED THE APOBECS WE DIDN'T HAVE REAL GOOD BIOCHEMICAL TOOLS SO WE EVERYEXPRESSED THE MINIE.COLI, IF YOU PUT IT ON HAY PLATE CONTAIN--A PLATE CONTAINING A DRUG, THEY WILL GROW UP TO FORM COLONIES IN THE PRESENCE OF THE DRUG. SO THEY'RE DRUG RESISTANT. IF YOU ADD AN AID OR APOBEC IN THE SYSTEM, YOU WILL INCREASE FREQUENCY OF DRUG RESISTANCE MUTATION IF YOU TAKE THE PATHWAY AWAY, YOU WILL INCREASE MUTATION FREQUENCY AND WHEN YOU PUT THEM TOGETHER, HAVE YOU SINNER GESTIC MUTATION. THIS TOLD US IMMEDIATELY THAT THEY'RE ALL WORKING WITH THE DEAMINATION MECHANISM AND THAT THE CELLS NORMAL CELL EXCISION SYSTEM AND REPAIR SYSTEM IS COUNTERACTING A ROT OF THAT DAMAGE AND THAT--LOT OF THAT TISSUE CULTURE MEDIA AND THAT SUGGESTED A ROLE IN CANCER MUTOGENESIS. BUT THE HARD PART OF PICKING OF THE NONAPOBEC FAMILY MEMBERRINGS OF THE CYTOSEEN DEAMNAIS IN THE 50 + CANCER TYPES WHERE DO YOU START THE BATTLE AND TESTING THIS HYPOTHESIS? SO WE CHOSE BREAST CANCER, I WILL TELL YOU WHY SHORTLY BUT IT'S TO TEST THE HIGHWAY POGHT SIS IN THE--HYPOTHESIS TO TEST OTHER SOURCES OF MUTATION IT CANCER TOO. SO 1 CAN REGARD A TUMOR AS THE END POINT OF SOMETHING THAT STARTED OUT AS A VERY PRISTINE GENETIC PROGRAM PASSED FROM OUR PARENTS TO US THAT OVER TIME ACCUMULATED DAMAGE AND MUTATION. SOME OF THOSE SOURCES OF DAMAGE ARE LIKE INTERNAL SOURCES, LIKE DNA REPLICATION ERRORS ANDOXIDATIVE DAMAGE. SPONTANEOUS HYDROLYTIC DEAMINATION OF METHYL CYTOSWREEN IS THE MOST ABUNDANT MUTATION IN CANCER AND THERE'S EXTERNAL SOURCES, MANY OF WHICH CAN CONTROL AND WE CHOOSE MATT HOLT TO LIKE UV LIGHT. WE ALL LOVE TO GO TO THE BEACH AND CATCH A FEW RAYS IN THE SUMMER TIME, LIKE THAT SARSEEN PICTURE YOU SHOWED OF ME WEARING MY ORANGE CRUSH T-SHIRT OF ALL THINGS AND YOU KNOW TOBACCO CARCINOGENS AND OTHER EXTERNAL SOURCES THAT CAN BE CONTROLLED. WE HYPOTHESIZED THAT THE APOBECS HAVE A MUTATIONAL ROLE IN THIS PROCESS. SO THE PUNCH IS AT LEAST 1 APOBEC, 3 B IS A MAJOR PART OF CANCER MUTE O GENESIS IN AND AMONG OF THEST ALL OF THESE AND CONTRIBUTING TO LARGE FRACTIONS OF THESE MUTATIONS TO THESE CANCER TYPES AND BREAST CANCER IS 1 OF THE MAIN MODELS WE CHOSE TO STUDY MAINLY BECAUSE THERE'S EXPERTISE IN MINNESOTA. THEREYA A NUMBER OF MODEL SYSTEMS IN CELL LINES AND IN ADDITION TO THAT, 1 CAN GET PATIENT DERIVED TISSUES WITH ESSENTIALLY TUMOR TISSUE AND MATCH NORMAL TISSUE. SO THERE'S SO MUCH VARIATION IN APOBECS FROM PERSON TO PERSON, WE WANTED TO HAVE NORMAL FROM THE SAME PERSON, SAME PEOPLE FROM THOSE INITIAL STUDIES AND WE'RE ABLE TO SHOW UPREGULATION OF APOBEC 3 B AND OVERALL CANCER MUTE O--MUTOGENESIS PROGRAM. SO 1 OF THE MAIN PIECES OF DATA AND SEGUES OFF OF OUR HIV 1 STUDIES IS IF THE SIGNATURE FITS, IT MUST HAVE BE THE ENZYME THAT MADE THAT PARTICULAR MUTATION SIGNATURE. SO, WE GOT A LITTLE BETTER OVER THE YEARS AT PURIFYING APOBEC ENZYME EXPWS SHOW THAT RECOMBINANT APOBEC 3 B, PREFERS CYTO69S IN A TCAG CONTEXT. REALLY SIMILAR TO WHAT SOME OF THOSE OTHER APOBECS AF-D, H, DO, AND THAT CONTEXT IS REMARKABLY SIMILAR TO THE ACTUAL DISTRIBUTION OF CYTOSEEN MUTATIONS IN LOOKING OVER HERE. THIS IS A HUNDRED TCGA, THE CANCER GENOMEAT LOS EXPOEMS AND OVER HERE THIS IS A HUNDRED TRIPLE NEGATIVE BREAST CANCER AX OHMS AND THE CYTOSEEN MUTATION PROFILE IS VERY, VERY SIMILAR, THIS IS BIOCHEMISTRY BELOW SHOWING IT REALLY DOES PREFER TO DEAMINATE CYTOSIGNS IN THESE MOTIFS, TCG, AND TCA, BUT THIS IS REALLY IN CONTRAST TO THE ACTUAL DISTRIBUTION OF CYTOSEEN IN OUR GENOME. SO HERE IS, IF YOU TAKE THE ENTIRE HUMAN EXOME AND YOU DISTILL IT DOWN TO WEB LOGO FORMAT, CYTOSEENS ARE PRECEDED EQUALLY BY THE 4 DNA DATABASES AND FOLLOWED IN A BIAS WAY BY ATOR C. THAT'S A VERY DIFFERENT PATTERN THAN THIS AND THIS IS A VERY DIFFERENT PATTERN THAN OBSERVED IN OTHER CANCER TYPES SO IN MANY LIVER CANCERS THERE'S CLEARLY NO APOBEC 3 B COMPONENT. AND IN MOLE ANOMA WHERE WE KNOW THE SOURCE OF CYTOSEEN MUTATION IS THE MOST ABUNDANT SOURCE OF CYTOSEEN MUTATION IS CROSS LINKING OF ADJACENT BASES AND THE DNA REPAIR SYNTHESIS THAT OCCURS OPPOSITE THOSE LESIONS INSERTS OFTEN FOLLOWS AN A-RULE INSERTING AD19 AND THAT ALWAYS RESULTS IN C-C MUTATIONS BUT THAT'S DIFFERENT CONTEXT. SO WE CAN LOOK AT THE ACTUAL PATTERNS IN CANCER AND INFER WHO CAUSED THE DAMAGE AND IN THIS CASE, APOBEC CAUSED THE DAMAGE. ANOTHER IMPORTANT PIECE OF DATA, I KEEP LOOKING FOR WAYS TO SUMMARIZE THESE DATA IN WAYS THAT HOPEFULLY STRIKE HOME AND HIGHLIGHT THE IMPORTANCE OF THIS MUTATION PROCESS IN CANCER. AND RECENTLY, WE CAME UPON THIS WAY WHICH IS A SIMPLE PIE GRAPH USING--I HATE TO SAY IT BUT MICROSOFT PELLET COLORS. BUT THIS IS TAKING THE ENTIRE CANCER GENOME ATLAS WORTH OF BREAST CANCER MUTATION DATA AND COMPRESSING IT INTO WHICH MUTATIONAL SIGNATURE IS PRESENT. SO APOBEC MUTE O GENESIS IS PART OF THAT PRIMARY MUTATION MODE. BRACA 1 AND 2 ARE HOMOLOGOUS HOMOLOGOUSDEFEESHESS ARE IN THE AGING PROCESS, THE SPONTANEOUS PROLIFERATION OF METH8 HOURS C-T AND THERE ARE OTHER PROCESSES CONTRIBUTING. BUT THEN WHEN YOU LOOK AT METASTASIS WHICH ARE A SPECIAL SUBSET OF THOSE THAT GROW A LOT OF THOSE PRIMARY TUMORS AND ARE ULTIMATELY WHAT ARE THE--THIS IS THE MAJOR CLINICAL CHALLENGE, NOT THE LOCAL FOCUS TUMORS NOW THE APOBEC MUTATIONAL PROCESS BECOMES BY FAR THE MOST DOMINANT PROCESS AND OTHER PROCESSES LIKE AGING BECOME COMPRESSED AND THAT'S BECAUSE THE TUMOR IS OPERATING ON ITS OWN TIME, NOT THE BIOLOGICAL AGE OF THE PATIENT'S TIME. SO THAT ARGUED STRONGLY THAT APOBEC IS CONTRIBUTING TO THIS OVERALL TRANSFORMATION PROCESS AND ONGOING TUMOR EVOLUTION LIKE I WOULD ARGUE CONTRIBUTING TO VIRUS EVOLUTION. SO A LOT OF PEOPLE--A LOT OF THE CRITICS OF THIS WORK HAVE SAID, WELL, YOU KNOW THE APOBEC MUTATIONS ARE THERE BUT ARE THEY ACTUALLY CAUSING ANY OF THESE PHENOTYPES WE SEE IN CANCER. SO THERE ARE A NUMBER OF DRIVER MUTATIONS IN YOU LIKE THAT 1 CAN LOOK AT IN DIFFERENT CANCER TYPES AND THEN 1 CAN ASK WHAT CAUSED THESE PARTICULAR DRIVERS AND IN THIS CASE, WE'RE LOOKING AT PI3 KINASE WHICH IS A DOMINANTLY ACTIVE KINASE IN MANY DIFFERENT BREAST CANCERS. THERE WERE 2 WAYS TO FLIP THE SWITCH FOR THIS KINASE, THERE'S DOMINANT ACTIVATING SIGHT OUT HERE AT THE 1047 POSITION WHICH IS MUTATE INDEED MANY DIFFERENT CANCER TYPES SO THIS IS 140 OUT OF A THOUSAND OR SO BREAST CANCERS BUT WHEN YOU ADD THESE 2 UP, THERE'S ALSO 140 INDEPENDENTLY OCCURRING ACTIVATING MUTATIONS THAT ARE OCCUR IN APOBEC HOT SPOTS. SO SHOWN DOWN HERE ON THE BOTTOM STRAND IS TCA. AND ON THE TOP STRAND, SO WHEN THAT TRCA IS MUTATED TO TTA, THAT RESULTS IN A GLUTAMATE TO LISINE SUBSTITUTION AND THAT IS AN APOBEC HOT SPOT. AND 1 CAN ARGUE THAT THESE MUTATIONS ARE ONLY OCCURRING IN TUMORS IN WHICH APOBEC 3 B IS OVEREXPRESSED. ALSO, IN FAVOR OF A CONTROL IN CONTRIBUTING TO TUMOR EVOLUTION, WE HAD THE GOOD FORTUNE OF COLLABORATING WITH JOHN MARTIN'S GROUP IN THE NETHERLANDS AND THERE THEY HAVE A FANTASTIC HEALTH SYSTEM WHERE PATIENTS HAVE TO OPT OUT TO NOT HAVE THEIR SAMPLES CONSIDERED FOR RESEARCH AND THIS IS A REALLY IMPORTANT THING. I SEE FRANK GRINNING BUT IT IS SUPER IMPORTANT. SO THOUGH THEY HAVE ARCHIVED OVER THE YEARS MANY NITROGEN FLASH FROZEN AND PARAFFIN EMBEDDED SAMPLES AND WE WERE ABLE TO GO BACK AND ASK WAARE THE APOBEC LEVELS IN THE PRIMARY TUMORS AND DO THEY CORRELATE WITH EVENTUAL THERAPEUTIC OUTCOME AND THAL CASE IT'S ESTROUGH ATOM GEN RECEPTOR PROZ POIF BREAST CANCER WHICH IS THE MOST COMMON TYPE. AND THIS COHORT WAS PROCURED AND ANALYZED IN THE 90S OR EARLY 2000S WHEN 1 OF THE ONLY THERAPEUTIC OPTIONS IN ARK DITION TO SURGICAL RESECTION WAS TAMOXIFEN WHICH BLOCKS THE FUNCTION SO HERE WE'RE LOOKING AT MONTHS AFTER RECURRENCE OF WOMEN WHOSE POSITIVE BREAST CANCERS OCCURRED AND WERE GIIVE TO MONOTHERAPY BECAUSE THEY KNEW THEN THAT WAS IT AN E. R. POSITIVE BREAST CANCER AND WE'RE CORRELATING WITH THE APOBEC EXPRESSION LEVELS IN THE ORIGINAL TUMORS TUMORS AND 1 CAN SEE THAT THOSE ORIGINAL TUMORS SO YEARS EARLIER IN MANY CASES HAD LOW LEVELS OF APOBEC B3 TO THE BEST AND THOSE WHO TUMORS HAD THE I HIGHEST LEVELS IN THOSE ORIGINAL TUMORS BY A LONG WAY. SO THIS IS--WE LIKE TO PLOT THIS IN CORTILES BECAUSE THIS IS A RARE EXAMPLE IN THE CLINICAL DATA SET WHERE WE ALMOST HAVE A DOSE RESPONSE A LOT LIKE WE CAN EASILY ACHIEVE IN THE LAB WITH CELL BASIS. SO TAKEN TOGETHER, THIS STRONGLY IMPLICATES THE APOBEC MUTATION PROCESS OF ONGOING TUMOR EVOLUTION INCLAUDING METASTASIS AND RECURRENCE AND WE WANTED TO BE ABLE TO MODEL THIS IN THE LABORATORY, SO, THERE ARE A NUMBER OF HUMAN BREAST CANCER CELL LINES THAT ARE ESTROUGH ATOM GEN RECEPTOR EXPRESSING AND DEPENDENT ON ESTROUGH ATOM GEN FOR GROWTH AND WE CAN TAKE THOSE CELL LINES AND PLACE THEM SUBCUTANEOUSLY WITHIN THE MEMORY OF THE ANIMAL AND THEY'LL FORM LARGE TUMOR MASSES OVERTIME IN AN ESTROUGH ATOM GEN DEPENDENT WAY AND ASK IF IT'S IMPORTANT FOR THE MUTATION PROCESS IN THESE CELLS BY ONCE THE TUMORS REACH A CERTAIN VOLUME WE CAN APPLY THE TAMOXIFEN MONOTHERAPY IN THE TO SYSTEM AND ASK WHAT HAPPENS OVER TIME? AND IN MANY CASES WE CAN SUPPRESS TUMOR GROWTH BUT UNFORTUNATELY, A LOT LIKE THE CLINICAL SCENARIO, RESISTANCE DEVELOPS FREQUENTLY AND BY THE END OF THIS YEAR-LONG EXPERIMENT, ALMOST ALL ANIMALS IN THE APOBEC 3 B HIGH GROUP HAVE DEVELOPED RESISTANCE TO TAMOXIFEN. BUT IF WE XOK DOWN APOETIC DECK 3 B PRIOR TO AND GRAFTING THESE CELLS IN ANIMAL AND FOLLOW IT ALONG IN PARALLEL 1 CAN SEE THAT WE IMPROVE THE RESPONSIVENESS OF THESE TUMORS TO TAMOXIFEN AND DECREASE THE PROBABILITY OF THESE TUMORS BECOMES RESISTANT TO THE DRUG. SO WE CAN TURN DOWN TUMOR EVOLUTION BY KNOCKING DOWN APOBEC 3 B. >> IS THIS KNOCK DOWN THE THIS. >> NO, NO, SO THERE'S LOTS OF CONTROLS WE KNOCK DOWN. --THAT'S THE ONLY THING WE'RE EFFECTING AS WE KNOW IN THE CELLS. BUT TO SORT OF SPEAK TO THAT QUESTION, ANOTHER WAY WE CAN ALSO OVEREXPRESS APOBEC 3 B IN THE SYSTEM, AND WE CAN SPEED UP THE RATE OF TAMOXIFEN RESISTANCE AND THAT'S SHOWING COMPARING THE ORANGE TO NOW THE RED OVER EXPRESSING APOBEC 3 B OVER EXPRESSING CELLS OVER HERE AND THAT ACCELERATED TUMOR IS DEPENDENTOT CATALYTIC ACTIVITY OF THE ENZYME. SET OF EXPERIMENTS WHERE WE'RE ABLE TO HOPEFULLY MODEL THE RESISTANCE TO TAMOXIFEN OF AN E. R. POSITIVE BREAST CANCER CELL LINE. SO ALL OF THIS, I APOLOGIZE BECAUSE THERE'S A LOT OF DATA I'M NOT ABLE TO SHOW IN THE INTEREST OF TIME AND MY BURNING DESIRE TO GET BACK TO SOME VIROLOGY AS WELL, BUT THIS LEADS TO A MODEL WHERE THERE'S A VERY INTERESTING CROSS ROADS BETWEEN VIRUSES AND CANCER. AND APOBECS AT THE HEART OF THIS MODEL. SO NORMAL EPITHELIAL CELLS AND AT POINT THERE HAS TO BE A TUMOR INITTIAITING EVENT AND THIS IS THE CASE FOR ANY CANCER, SOME CASES THAT MIGHT BE INHERITED AND OTHER CASES IT MAY BE ACQUIRED. IN SOME OF THESE CANCER TYPES I DIDN'T SHOW THE DATA BUT SOME OF THE CANCERS WITH THE HIGHEST APOBEC MUTATION LEVELS AND APOBEC EXPRESSION LEVELS ARE CERVICAL CANCERS AND IN THAT CASE, WE KNOW THAT APPLIED LOMA VIRUS IS THAT INITIATED EVENT AND THAT UPREGULATES APOBEC 3 B. IN ANOTHER DNA VIRUS THAT UPREGULATES APOBEC 3 B ARE THE POLY-OMA I HAVE--VIRUSES, ALL OF THESE UPREGULATE APOBEC 3 B. WE KNOW FROM OTHER WORK THAT ACTIVATING SIGNAL TRANSDUCTION PATHWAYS LIKE THE NONCONNICCAL PATHWAY, LEAD TO UPREGULATION AND AT THE END OF THE DAY, HIGHER LEVELS OF MUTATION IN THOSE CELLS ARE REALLY THE FUEL FOR ONGOING TUMOR EVOLUTION AND ALLOW THAT TUMOR TO SAMPLE MORE GENETIC SPACE AND ULTIMATELY OUTGROW IT'S CONSTRAINTS. NOT UNLIKE VIRUSES IN THE CONTEXT OF A SINGLE DRUG& SELECTION. SO ALL STORIES ALWAYS HAVE A NEXT STEP AND NEXT STEP AND THE TRAINEES AND I WERE TALKING ABOUT THIS. IT'S NEVER DONE. THAT'S 1 OF THE NEAT THINGS ABOUT SCIENCE IS THE GOALPOSTER CONTINUALLY MOVING AND THAT'S PART OF WHAT MOTIVATES US. AND IN THIS STORY A COUPLE YEARS - AGO, WE WERE THROWING A GIG CURVE BALL AND THAT CAME IN THIS FORM BECAUSE THERE ARE NATURAL VARIATIONS IN THE APOBEC LOCUST AND IT'S LIKE OUR HIV STORY WHERE WE HAD TO PEEL BACK THE ONION TO GET AT THE APOBECS THAT WERE INVOLVED IN HIV MUTE O GENESIS. IT WAS NOT CLEAR AT THE OUTSET. SO THE NATURALLY OCCURRING VARIANTS ALL THROUGHOUT THE WORLD AND THROUGHOUT THIS ROOM AND 1 OF THE BIGGEST IS A FULL DELETION OF THE APOBEC 3 B GENE. IT'S 29.5 KB, IT TAKES OUT THE WHOLE GENE AND AXONS AND FUSES THE REGION OF APOBEC 3A TO THE TRANSLATED REGION OF APOBEC 3 B. SO IT'S GENETICISTS WE WANT TO ASK, IS APOBEC 3 B REALLY REQUIRED FOR ALL THE THINGS I WAS JUST TELLING YOU? THIS IS DEPICTING WHERE THESE VARIANTS ARE IN THE WORLD. A3 B DELETION IS PREDOMINANTLY OCCURRING AT HIGHER ALLELE FREQUENCIES IN SOUTHEAST ASIA. THE KEY EXPERIMENT, 1 OF THE MONEY EXPERIMENTS WHERE WE THEN TOOK ALL THE BREAST CANCERS AND SORTED THEM FOR APOBEC 3 B POSITIVE. SO THIS IS 2 ALLELES OF WILD TYPE A3 B,IE TO APOBEC 3 B AND THIS IS ANOTHER WAY OF PREDICTING CYTOSEEN MUTATION PATTERN WHERE WE'RE LOOKING AT 16 OF THE TRI NUCLEOTIDECONTEXT AND I CIRCLED THE CYTOSEEN MUTATIONS THAT ARE THE POETIC BECK SIGNATURES, AND THESE TUMORS WITH 35,000 MUTATIONS AND HERE WITH A COUPLE THOUSAND MUTATIONS THEY'RE STILL OBVIOUSLY A VERY STRONG MUTATIONAL SIGNATURE WHICH IS PREDOMINANTLY A TCA AND TCT AND SOME TCC, AND TCG. SO THAT'S DEPICTED OVER HERE AGAIN AND WE'RE LIKE, OKAY, THERE'S NO APOBEC 3 B IN THE SYSTEM SO WHO IS THE OTHER APOBEC CONTRIBUTING TO THIS ADDITIONAL APOBEC MUTATION SIGNATURE? SO THEN WE COME BACK TO THE VIRUS. SO THIS APOBEC CAN BE A BIT PERSNIKETY OF GETTING TO THE BOTTOM OF THE STORY AND THE HIV MUTOGENESIS AND CANCER MUTOGENESIS, SO AGAIN WE'RE LOOKING AT DIFFERENT SNPs. SO I WANT TO CALL OUT HOP LOW TYPE 1. --HAPLOTYPE 1. SO WE THOUGHT THIS WAS AN UNSTABLE PROTEIN IN CONTRAST TO HAPLOTYPE 2 AND THESE ARE THE MOST COMMON ALLELES IN THE WORLD SO WE'LL DEAL WITH THESE NOW, AND THESE ENCODE NONFUNCTIONAL PROTEIN. SO THAT SHOWN HERE ON A WESTERN BLOT WHERE A HAPLOTYPE 1 IS WEEKLY EXPRESSED, HAPLOTYPE 2 STRONGLY AND 3 AND 4 MAKE NO BAND ON THIS. SO--AT THE MRNA LEVELS THEY'RE ALL EXPRESSED ABOUT THE SAME LEVELS AND THESE ARE ACTUALLY DATA FROM PRIMARY CD4 POSITIVE T-CELLS. AND THIS IS AN EXPERIMENT FROM THE ZANG LAB AT MICHIGAN STATE, SHOWING KIND OF WHAT I JUST TOLD YOU ABOUT IN A VIRUS CONTEXT WHERE HAPLOTYPES 2, 5, AND 7 CAN EXPRESS IN CELLS, PACKAGE INTO VIRUS PARTICLES AND RESTRICT VIRUS INFECTIVITY WHEREAS HAPLOTYPES 1, 3, 4 AND OTHERS CAN'T. SO WE THOUGHT THIS WAS A REAL DICHOTOMY, KIND OF LIKE APORKS DECK 3 B WHERE YOU HAVE 1 FUNCTIONAL GENE, 1 FUNCTIONAL PROTEIN AND NONFUNCTIONAL 1S. SOPHISTICATEDY WE WENT IN WITH THE HYPOTHESIS THAT WOULD BE STABLE HAPLOTYPE 2 THAT WOULD BE RESPONSIBLE FOR THIS ADDITIONAL MUTATION SIGNATURE IN CANCER. BUT THE MONEY EXPERIMENT RIGHT HERE SAYS OTHERWISE. SO NOW IF YOU TAKE THE WORLD WIDE DATABASE OF BREAST CANCERS, SEPARATE THEM INTO--I ALREADY DID SEPARATE THEM INTO APOBEC 3 B AND THOSE THAT DON'T AND THEN FURTHER SEPARATE INTO APOBEC 3 H HAPLOTYPES. SO, GOING AROUND THE HORN STARTING RIGHT HERE, SO THIS PARTICULAR SET OF INDIVIDUALS, THEIR CANCERS CLEARLY HAVE AN APOBEC MUTATION SIGNATURE, THEY HAVE HAPLOTYPES 1 AND 2, HERE HAPLOTYPE 1 AND 1. SIGNATURE HERE 1 AND 3. APOBEC SIGNATURES NATURE, WHEN YOU HAVE HAPLOTYPE 1, YOU DON'T HAVE A MUTATION SIGNATURE. ERGO, IT WASN'T HAPLOTYPE 2 STABLE VERSION TAKEN--THEY RESTRICTS HIV THAT WE THOUGHT THAT WOULD BE--THAT WE HYPOTHESIZED WOULD BE MUTATING THE TUMOR GENOME. IT'S ACTUALLY HAPLOTYPE 1. SO, THIS IS HELD UP IN OTHER INDEPENDENT DATA SETS. HAPLOTYPE 1, WHAT'S THE MECHANISM? SO HAPLOTYPE 1 IF YOU GUYS REMEMBER, AT POSITION 105 IS DIFFERENT FROM HAPLOTYPE 2 AND THE STABLE HAPLOTYPE HAS AN R-GENNINE AND THIS 1 HAS A GLYCINE THERE AND IT'S ENOUGH TO TAKE THE 2 ENZYME WHICH IS NORMALLY PREDOMINANTLY CYTOPLACENTAS MIC AND CAUSE--CYTOPLASMIC AND CAUSE A CELL WIDE CONCENTRATION IN THE NUCLEOUS PHENOTYPE AND THEN WE OF COURSE DID CONTROLS. APOBEC 3 B AND STRONG AND NUCLEAR, APOBEC 3G IS STRONG AND CYTOPLASMIC. SO OUR WORKING MODEL FOR THIS IS THAT THE HAPLOTYPE 1 ENZYME CAN DIFFUSE THROUGH THE CELL AND DAMAGE GENOMIC DNA AND BECAUSE IT'S WEEKLY EXPRESSED, IT'S DOING IT AT A SMOLDERING LEVEL, SO IT'S OVERALL POTENCY IS MUCH LOWER THANNA POETIC BECK 3 B BUT OVERTIME, IN THE CASE OF TUMOR EVOLUTION IN HUGH MANS THAT COULD BE YEARS, APOBEC 3 H HAS TIME TO MAKE AN IMPACT AND WE CAN ONLY SO THAT IN THE ABSENCE OF APOBEC 3 B, SO THE ANALOGY I LIKE A LOT WHICH IS A3 B IS LIKE A RAGING MUTATOR AND H-HAPLOTYPE 1 IS LIKE A SMOLDERING MUTATOR. SO TO CAP THIS OFF. SO THERE ARE NO--THAT'S WHY WE CAN STILL HAVE A VERY LARGE CLINICAL PHENOTEEP WITH THE APOBEC 3 B WITHOUT THE COMPLICATIONS FROM WHOSE APOBEC 3 H HAPLOTYPE OR WHICH HAPLOTYPE THE PATIENT HAS. SO WE'RE CONTINUING TO WORK ON THIS AND UNDERSTAND HOW THESE 2 ENZYMES CONTRIBUTE TO CANCER MUIT, OGENESIS OVERALL SO IN THE FINAL PART OF THIS I WANT TO SHARE REALLY RECENT DATA BECAUSE SOME OF THESE GUYS KNOW I CAN'T TALK WITHOUT TALKING ABOUT NEW STUFF. SO THIS STUFF'S PRETTY NEW. THIS A POST DOC IN MY GROUP, NADINE WHO'S BEEN WORKING ON THE STRUCTURE FUNCTION OF THE APOBEC ENZYMES FOR THE LAST 5 + YEARS IN MY GROUP. AND THESE IS FOCUSED ON--HAS FOCUSED ON UNDERSTANDING INTERACTION BETWEEN VIRKS IF IN THE POETIC POETIC BECKS AND TO DO THAT, YOU HAVE TO HAVE BOTH OF THESE COMPONENTS WELL BEHAVED BIOCHEMICALLY OR YOU CAN FORGET IT BOTH OF THEM ARE TOUGH. SO RECENTLY SHE WAS ABLE TO MAKE PROGRESS WITH APOBEC 3 H. SO, 1 OF THE ANNOYING THINGS ABOUT THE APOBECS, IS THAT THERE ARE--THEY'RE INTERFERED AND THIS IS AN OLDER OBSERVATION, IN THE LITERATURE IS THAT RNA BLOCKS THEIR DAMNAIS ACTIVITY. AND WE THOUGHT, AND THAT'S DEPICTED HERE WHERE, YOU CAN'T REEVEN PURIFY APOBEC 3 H UNLESS YOU REET WITH RNAs OR YOU GET A LOW YIELD AND PRETTY INSOLUBLE STUFF COMING UNDER THE CELLS AND IN E.COLI, AS AS HUMAN CELL LIAISON SAIT, YOU GET ALMOST NO ACTIVITY FROM APOBEC 3 H FOR SEVERAL OTHER APOBECs UNLESS YOU TREAT WITH RNA, SO WITH RNA ACTIVITY, WITHOUT IT NO ACTIVITY. SO WE THOUGHT THIS WAS BECAUSE--LET SEEEE WHAT--LET'S SEE WHRA I--WHAT I PUT NEXT. THIS IS THE COORDINATING SIGHT. WE THOUGHT IT WAS BINDING WE THOUGHT IT WAS BECAUSE THE RNA AND DNA WOULD BE COMPETING ON THE SAME SITE. SO THAT MAKES SENSE. IF IT'S NOT BUNDLED UP WITH RNA CAN CAN'T ACCESS DNA. SO A PICTURE OF THAT IS SHOWN HERE AND THIS IS BEAUTIFULLY PUT ON THE COVER OF THAT POSTER, WHOEVER DID THAT, KUDOS REALLY NICE. BUT THIS IS ANOTHER DEPICTION OF APOBEC 3 A AND BINDING SINGLE TRAND--STRAND DNA, YOU CAN SEE THE FOSTER NURSED FOCUSED ON DISCIPLINARY ESTER BACKBONE BEAUTIFULLY POSITIONED BETWEEN LOOPS AND THE CYTOSEEN ANYWAYS SHOVED INTO THE ACTIVE SITE OF THE ENZYME AND THE MINUS 1 T I'VE BEEN TALKING ABOUT FOR THE LAST 30 MINUTES POGDZED BETWEEN THESE 2 LOOPS. SO THIS IS ACTUALLY EXPLAINS THE MINUS 1 T SPECIFICITY WITH THE OTHER 3 B, AND THE PREFERRING APOBECS BUT THIS DNA BINDING MECHANISM, SO GOING BACK HERE, WE THOUGHT, RNA MAY BE BINDING IN A SIMILAR WAY, BUT NADINE NOTICED IS THAT IF YOU TURN THIS PROTEIN A LITTLE BIT, THERE'S A PREDICTED VERY STRONG POSITIVELY CHARGED PATCH IN ANOTHER PART OF THE PROTEIN THAT MAY ALSO BE CONTRIBUTING TO NUCLEOTIDES CLITIC ACID BINDING. SO THERE ARE LOTS OF RESIDUINGS THROUGHOUT THESE LOOPS, AT THE END OF THE DAY, THE APOBECS ARE CENTRAL GLOBULAR ENZYMES SO THE DEVIL'S IN THE DETAIL WHICH IS IN THE LOOPS AND THE CREVICES OF THE PROTEIN. SO WE MADE A BUNCH OF MUTANTS AND WE PREDICTED IN RNA AND DNA ARE BINDING THE EXACT SAME WAY, WE CAN DISRUPT RNABINDING BUT WE COULDN'T DO IT WITHOUT DISRUPTING DNA BINDING BUT IF THEY'RE BINDING DIFFERENT SURFACES, WE MIGHT BE ABLE TO SEPARATE THESE ACTIVITIES SO WE MADE A WHOLE BUNCH OF MUTANTS AND THESE ARE UNDERGRADUATE AND ROTATING GRADUATE STUDENT WHO CONTRIBUTED TO THIS PROJECT. THEY'RE HAPPY TO DOING THESE DRUG RESISTANCE ASSAYS, USING E.COLI AND NICE COLORFUL PLATES. THAT'S KATE SHOWING HERE WITH RICK, SHOWING THE PLATES WITH THE ORIGINAL EXPERIMENTS I DID COUNTING LOTS AND LOTS OF COLONIES TO FIGURE OUT THESE MUTATING AND THERE'S A TON OF DATA BUT THIS IS MUTATION FREQUENCY ON THE Y-AXIS AND LOTS OF MUTANTS ON THE XKS-AXIS SO TO ORIENTALLY. WILD TYPE APOBEC 3 H WILL MUTATE 10 FOLD ABOVE THE BACKGROUND. ONE OBSERVES IF WE PUT NONE IN THE CELLS OR CATALYTIC MUTANT OR APOBEC 3 H AND THAT'S 10 FOLD, SO THAT'S INTERESTING AND THAT'S A CLUSTER OF MUTANTS OUT HERE THAT ARE NOW HYPER MUTATORS, SO THESE ARE MUTATING E.COLI UP TO A HUNDRED FOLD, 10 TO A HUNDRED FOLD ABOVE WILD TYPE 3 H LEVELS AND A HUNDRED OR MORE FOLD ABOVE BACKGROUND LEVELS SO WE CALL THESE HYPER MUTANTS. THEY ARE CLUSTERING IN LOOP 7 AND ALPHA 6 HELIX WHICH IS THE PATCH 2 REGION. SO NOT ONLY DID WE NOT DESTROY D NA DEAMNAIS ACTIVITY IN THIS REGION BUT WE CREATE HYPER IN YOUITATORS, OKAY? --MUTATTORS, OKAY? WHAT WAS MORE INTERESTING IS WHEN WE TAKE ADVANTAGE, THERE'S MORE DATA BUT WE TAKE ADVANTAGE OF THE FACT THAT THESE PROTEIN RS TAKEN BUT WE DISRUPT THE BINDING DOMAIN THAT THEY'LL SHOW& ACTIVITY IN THE ABSENCE OF RNAs AND JUST TO POINT OUT WILD TYPE 3 H AND NO ACTIVITY IN THE ABSENCE OF RNAs SO THIS IS LOOKING AT SUBSTRATE CONVERSION TO PRODUCT BUT IF YOU LOOK DOWN HERE FOR INSTANCE, THESE R-GENERATEDDINES 176 FOR INSTANCE, THESE MUSEUM STANTS SHOW ACTIVITY EVEN IN THE ABSENCE OF RNA TREATMENT AND WHEN WE TREAT THESE LIAISON SAITS WITH RNAs WE CAN OF COURSE FREE UP THE DNA DEAMNAIS ACTIVITY OF WILD TYPE 3 H AND MANY OTHER MUTANTS SO WE CREATED DNA HYPER IN YOUITATORS BY DISRUPTING THE INTERACTION DOMAIN. WHAT WAS EVEN MORE INTERESTING NOW, IF YOU TAKE THE MUTANTS AND PROFILE THEM BIOCHEMICALLY, SO WE CAN PURIFY THEM. I WON'T GO INTO THE PURIFICATION SCHEME BUT THE WILD TYPE ON THE EXCLUSION COLUMNS AS A DIMER AND THE HYPER MUTANT PROTILES AS A MONOMER, SO IT'S AS IF THIS RNA IS PROVIDING A INTERACTION SURFACE FOR APOBEC 3 H. AND OVER HERE, WE'RE SHOWING YOU THAT IT'S RNA, THAT'S IN THIS PREPARATION AND CORRELATED WITH DIMERIZATION, BECAUSE IF WE DO AN OLD SCHOOL KIND OF EXPERIMENT WHERE WE TAKE THE ENZYMES THAT WE PURIFY, TREAT IT WITH PROTEASE TO CUT IT UP AND THEN EITHER TREAT IT WITH DNAs OR RNAs AND ASK WHAT NUCLEAIC ACIDS ARE LEFT OVER VERY CLEARLY THE TREATMENT WITH RNAs DECREASES WITH THE DNAs DOESN'T. SO RNA BINDING REALLY STRONGLY. WHAT WAS INTERESTING IS USUALLY WHEN WE MAKE A BUNCH OF MUTANTS WE DO IT WITH THE AIM OF DEVELOP--OF PURIFYING MONOMERIC ENZYMES SO MONEY MERS OFTEN BEHAVE BETTER AND THEY'RE MORE AMENABLE TO STRUCTURAL STUDIES SO IN THIS CASE, THE MONITOR MERWASN'T VERY WELL BEHAVED BUT THE WILD TYPE WAS WELL BEHAVED. THAT NADINE COULD EXPRESS AS A CHERRY TYPE CLEAR OR CHERRYISH-COLORED SOLUTION AND MAKES LOVELY CRYSTALS AND REFRACTED RESULTS IN A STRUCTURE OF 2 APOBEC 3 H MONITOR MERS, REALLY--MO MERS POSITIONED BY THE RNA BETWEEN THEM. SO THESE 2 MONOMERS ARE CRADLING THE RNA WHICH IS CONSISTENT WITH THE RNA DNAs TREATMENT EXPERIMENT AND WITH THE FACT WE COULD NEVER EVER GET ACTIVITY WITH THE PROTEIN UNLESS WE TREAT WITH RNAs AND ALSO WITH THE FACT THAT WHEN WE POLLUTE ALL OF THOSE MUTANTS I JUST DESCRIBED THAT TURNED INTEREST A HYPER DNA YOU HADITATOR,--MUTATOR THEY ALL MAP TO THE DNA BINDING SURFACE. SO THIS IS ANOTHER DEPICTION OF THAT SO YOU SEE THE VERY BASIC POSITIVE CHARGE PATCH 2 THAT'S IMPORTANT FOR THIS RNA INTERACTION, LOTS OF RESIDUES ARE INVOLVED AND LIKE I SAID ALL OF THOSE ARE THE 1S THAT WHEN WE MUTATE THEM MAKE IT INTO A DNA HYPER MUTATOR. YOU CAN KIND OF SEE IN THIS VERSION HERE, CAN YOU SEE HOW THE 2 MONOMERS ARE MORE OR LESS CRADLING THE RNA. I SHOULD TELL YOU TOO, TO GET THIS STRUCTURE AND PURIFY THIS PROTEIN, SO WHEN YOU SEE THE ENDS OF THE RNA HERE, YOU HAVE TO IMAGINE THAT FROM THE E.COLI, THEY'RE OF COURSE LONGER STRUCTURED RNAs THAT ARE BOUND BY APOBEC 3 H AND RNAs A-TREATMENT TRIMS THEM DOWN TO THE LENGTH SHOWN IN THE CRYSTAL STRUCTURE. SO 1 CAN--SO THE ACTIVE SITE IS ACTUALLY HERE AND HERE IN THE MONOMERS AND SO 1 CAN IMAGINE MOW DNA COULD ACCESS THAT TREND RNAs TREND PROTEIN AND ACTIVE SITE. SO IT IS DEFINITELY DUPLEX RNA, BUT IT'S RNA, THERE'S 7 DIFFERENT RNA BASE PAIRS WE CAN'T MAKE OUT THE XABLGHT IDENTITIES OF THE BASIS BECAUSE IT'S A MONTAGE OF DUPLEX RNA WE PULLED FROM E.COLI SO LOTS OF DIFFERENT RNA SEQUENCES ALTHOUGH WE VERY VERY INTERESTED IN WHAT RNAs ARE PHYSIOLOGICALLY BOUND BY THIS ENZYME IN NORMAL HUMAN CELLS. BUT TO COME BACK TO THE MECHANISM, AND THE CANCER MUTE O GENESIS, SO WHAT'S REALLY INTERESTING ABOUT THIS RNA BINDING DOMAIN, IS IT'S REQUIRED FOR PROPER SUBCELLULAR LOCALIDESSATION AND FOR HIV RESTRICTION. AND I WILL TELL YOU, SO RNA, IN OTHER WORDS IS A FUNDAMENTAL PART OF THE REGULATORY ACTIVITIES OF THIS PARTICULAR APOBEC. SO SHOWING UP HERE, AND SHOWING YOU BEFORE, WILD TYPE IS PREDOMINANTLY HYPER PLASMIC AND TO PICK A MUTANT LIKE THIS 1 OUT HERE, THE 175-176 TO DOUBLE GLUTAMATE, THIS IS MOSTLY CELL WIDE IN 293 AND MOSTLY NUCLEAR AND HELIOS POSITIVE EXCITATORY CELLS THERE'S QUANTIFICATION BELIFE ON EARTH BUT UPSHOT IS WHEN YOU DISRUPT THIS AND TURN IT INTO A DNA HYPER MUTATOR, YOU SIMULTANEOUSLY DISRUPT THE CYTOPLASMIC RETENTION MECHANISM. SO THAT COULD BE RELEVANT TO CANCER MUTTEE GENESIS BECAUSE 1 COULD IMAGINE HOW THAT SAME R-GENINE TO GLUE MARIOUS SEEN CHANGE BETWEEN HAPLOTYPE 2 AND 1 ENABLES IT TO GO INTO THE NUCLEAR COMPARTMENT. ALL OF THESE ARE ABLE TO ACCESS THE NUCLEAR COMPARTMENT AND PRESUMABLY CONTRIBUTE TO GENOMIC DNA DAMAGE. NOW BACK TO HIV. EVERYBODY STILL WITH ME. WE'RE ALMOST DONE. REALLY. SO, THE WILD TYPE APOBEC 3 H AND SO THIS IS UNTAGGED APOBEC 3 H EXPRESSION IN 293 CELLS, RESTRICTING VIF DEFICIENT HIV 33 WHICH IS A MODEL CITIZEN LEAK DLOAN AND THIS IS REALLY RESTRICTED IN A DOSE RESPONSIVE WAY SO THE MORE YOU EXPRESS THE MORE PACK KNOWLEDGES AND THE MORE RESTRICTION YOU GET, THE CATALYTIC MIEWITANT RESTRICTS PRETTY GOOD AND OTHERS REPORTED THIS IN THE LITERATURE BEFORE, ALMOST AS GOAs WILD TYPE BUT NOT QUITE SUGGESTING THAT A GOOD PART OF HIV RESTRICTIONS BY APOBEC 3 H IS DEAMNAIS INDEPENDENT. SO WHAT WE'RE CONTRASTING THESE WITH IS WHAT HAPPENS WITH THESE RNA BINDING DEFICIENT DNA HYPER MUTATORS. SO THESE GUYS, EXPRESS TO VARYING DEGREES IN CELLS BUT THEY'RE REALLY DEFICIENT IN PACKAGES INTO PARTICLES. SO THIS IS CONSISTENT WITH EARLIER LITERATURE SHOWING THAT THE APOBEC 3G AND H AND INTERACTION WITH GAG WHICH IS NECESSARY FOR VIRAL PROTEIN OR POETIC APOBEC PACKAGES INTO VIRUS PARTICLES IS MEDIATED BY RNA AND IN THIS CASE, THAT IS ABSOLUTELY ESSENTIAL FOR THE PROTEIN TO GET INTO VIRUS PARTICLES AND RESTRICT. SO, THIS IS NOT MY WORK, BUT THIS PUTS IT INTO A BIGGER CONTEXT, SO THIS IS--THE SHAPE STUDY WHICH REALLY TRIED TO MAP THE STRUCTURAL ELEMENTS IN THE HIV GENOME AND THIS IS 1 RENDERING OF ALL OF THE STRUCTURAL ELEMENTS IN PART OF THE HIV GENOME. IT'S A MASSIVELY STRUCTURED RNA. SO IT'S NOT HARD TO IMAGINE HOW APOBEC 3 H MIGHT ACTUALLY EXPLOIT SOME OF THESE STRUBLGHTURES FOR PREFERENTIAL PACKAGES INTO THE PARTICLES AND A REALLY NEAT STUDY LAST YEAR FROM PAUL'S LAB USED CLIP SEQ TO ASSOCIATE APOBEC 3 H F, AND G WITH DIFFERENT RNA CELLS IN THE VIRUS AND IT LINKED APOBEC 3 H TO STRUCTURED REGIONS OF THE RNA GENOME. AT THE TIME THEY DIDN'T KNOW IT BOUND RNA QUITE LIKE I JUST SHOWED YOU BUT IT FITS NICELY TOGETHER. SO WHAT I'VE SHOWN NUCLEOTIDES CONCLUSION REGUARDING APOBEC 3 H IS THAT IT CONTRIBUTES BOTH TO CANCER MUTE O GENESIS AND HIV INFECTION, IT'S DOING 3 THINGS, REGULATING DEAMNAS ACTIVITY WHICH IS BENEFICIAL FOR THE CELL THIS THE INDIVIDUAL. IT'S KEEPING IT IN THE CYTOCELL WHICH IS BENEFICIAL FOR THE CELL OF THE INDIVIDUAL BUT IT'S POSITIVELY REQUIRED THE RNA BINDING POSITIVELY REQUIRED FOR THE ANTIVIRAL ACTIVITY AND PACKAGING INTO PARTICLES AND ULTIMATELY FOR VIRUS RESTRICTIONS AND BASED ON THE DEAMNAIS DEFICIENT MUTANT I WOULD SUGGEST THAT THAT COULD BE A DIRECT BLOCK TO REVERSE TRANSCRIPTION. AND THAT IS ALSO BEING REPORTED IN THE LITERATURE, I'M BLANKING THIS EXACT SECOND BY THE GROUPS ABOUT I THINK IT WAS JUDITH LEVINs GROUP, BUT ACTS AS A BLOCK TO REVERSE TRANSCRIPT ACE. SO THIS X-RAY STRUCTURE DEMONSTRATES NOVEL DUPLEX FOR THE APOBEC DIMERIZATION. AND FINALLY, LAST BUT NOT LEAST, THANK YOU GUYS AGAIN FOR THIS PRIVILEGE OF PRESENTING HERE TODAY. AND I'D LIKE TO JUST BRIEFLY ACKNOWLEDGE A COUPLE MEMBERS IN NIGH GROUP WHO CONTRIBUTED TO THE LATTER STORY. SOPHISTICATED NADINE WAS THE POST DOC THAT GOT THE STRUCTURE. SHE WAS ASSISTED BY A FEW LAB MEMBERRINGS FOR VIROLOGY, TALENTED UNDERGRADS WHO TIRELESSLY COUNTED--NOT TIRE TIRELESSLY COUNTED THEE RESISTANT COLONIES AND DAN FOR LOTS OF LOCALIZATION AND THE COLLABORATORS I MENTIONED DURING THE TALK AND THE HHR, AND NIH FOR THEIR SUPPORT ASK YOU GUYS FOR YOUR ATTENTION TODAY. THANK YOU. [ APPLAUSE ] I THINK WE HAVE TIME FOR QUESTIONS. >> LET'S HAVE AN APOBEC 1 FIRST. >> ACTUALLY HAD A QUESTION ABOUT THE CANCER CELL. [LAUGHTER] >> I WAS WONDERING WHETHER YOU EXCLUDED APOBECS FROM CONTRIBUTING CANCER AND FOCUS ON A3 H I WAS WONDERING LIKE OTHERS IA OR F OR KREBS CYCLE? >> SO IT'S HARD--I CAN NEVER SAY NEVER. SO IN THAT PARTICULAR STUDY, THE GENETIC SAYS ACTUALLY CONTROL FOR THAT. IN THOSE CANCERS HAVE THE APOBEC 3, LACK A3, BUT HAVE H1, THEY ALSO HAVE THE OTHER PARKS POETIC BECKS, SO THE ARGUMENT IS THAT NONE OF THE OTHER APOBECS ARE CONTRIBUTING BUT THE APOBEC 3 H AND CO SEGGREGATING MOST OF THE TIME WITH ITSELF AND THE APOBEC 3G BUT IT'S ALMOST WITH THE APOBECS IN THE LOCUST. WE USE THE LINKAGE ARGUMENT TO SUGGEST IT'S NOT SOMETHING LINKED BUT ACTUALLY HAPLOTYPE 1 THAT'S DOING IT. AND MEN WITH HIGHER YOU PURIFY--WHEN YOU PURIFY EQUAL AMOUNTS THEY'RE EQUAL POTENT ENZYMES SO THE FACT THAT WE DON'T SEE IT RESTRICTING HIV AND PROBABLY BECAUSE IT'S EXPRESSED AT A LOWER LEVEL AND IT'S DEDEFECTIVE IN PACKAGES SO WE THOUGHT WAS AN DEFECTIVE ENZYME SO WHEN YOU LOOK AT IT, IT'S AN ACTIVE DNA DEAMNAIS, AND NEVER SAY NEVER BUT I WILL PUT A LOOT OF MONEY ON IT. STEVE? >> SO I WILL ASK A QUESTION. SO WHEN YOU HAVE B-DELETION, IT WENT BY BIKE, YOU GO IT LOOKED TO ME LIKE YOU HAD AN A-B FUSION? >> YEAH. >> HAVE YOU LOOKED AT THE PROPERTY OF THAT CREATURE AND SEE IT'S A B-DELETION, BUT IT'S REALLY SOMETHING--THERE'S SOMETHING ELSE IN THERE, RIGHT? >> ABSOLUTELY. SO, OTHER LABS HAVE HAD A REALLY CLOSE LOOK. SO THE ACTUAL PROTEIN THAT'S PRODUCED IS A3A. IS THERE'S NO AMINEE ACID CHANGE WHATSOEVER. ONLY LITTLE CHANGE IS IN THE 3 PRIME UTR. SO NOW INSTEAD OF A3, IS 3 PRIME UTURKS R, IT HAS THE UTR FROM A3 B. SO WANE HOBSON'S LAB, LEADING THE CHARGE AND A COUPLE OF OTHERS HAVE SUGGESTED THAT THAT LEADS TO INCREASED STABILITY OF THE A3A TRANSCRIPT AND MAYBE INCREASE PRODUCTION OF A3A, WHICH WOULD BE DAMAGING TO THE GENOME POTENTIALLY, WE'VE ARGUED AGAINST THAT FOR THAT LINKAGE ARGUMENT I JUST MENTIONED TO [INDISCERNIBLE] AND ALSO IN ALMOST ALL OF THOSE SOLID TUMOR TYPES WE ALMOST NEVER SEE A3A EXPRESSED AT THE MRNA LEVEL. SO IT IS THERE IT'S A LITTLE BIT GOING A LONG WAY AND I DON'T THINK THAT IT IS. SO I DON'T THINK IT'S CONTRIBUTING TO THE CANCER MUTE O GENESIS PROGRAM HERE. IN OTHER STUDIES WE REALLY LINKED IT TO MYELOID LINEAGE CELLS IT'S STRONGLY INTERFERON INDUCED, YOU KNOW IT'S AN ASH MAZING ENZYME. IT'S 1 OF MY FAVORITE APOBECS BECAUSE IT IS A GOOD DEAMNAIS, BUT BECAUSE OF THAT IT'S DEVELOPMENTALLY CONSTRAINED. IT'S NOT A FACTOR HERE BUT WE'RE NOT DONE BATTLING BECAUSE IT'S HARD TO SAY NEVER. RIGHT? NOT YET. BUT THE CLINICAL DATA SPEAK TOW THAT ALSO. VERY STRONGLY. AND THEN IF IT WERE A ANOTHER APOBEC, WE WOULD NEVER HAVE THESE MASSIVE CORRELATIONS WITH A3 B AND POOR CLINICAL OUTCOMES. RIGHT? >> SO IN THE CONTEXT, IF A3 H IS THE MAJOR PLAYER AND THERE ARE PEOPLE WHO DON'T SEEM AT LEAST IN THESE ASSAYS TO HAVE THESE, DOES THAT SHOW UP, IS THAT A BIG ENOUGH EFFECT TO SHOW CLINICALLY? >> SO WITH B--SO, THE QUESTION IS THERE ARE PEOPLE WITHOUT B-- >> [INDISCERNIBLE] >> OKAY, AND THERE ARE PEOPLE WITHOUT H. >> YEAH. >> ASK THERE ARE SOME THAT LACK BOTH. >> YEAH. >> THE CLINICAL STUDIES WITH COHORTS LIKE THAT HAVEN'T BEEN DONE, I COULD PUT IT UP AGAIN BUT THE WORLD WIDE DISTRIBUTION OF APOBEC 3 B AND H IS--IS NOT FAVORABLE FOR THAT SORT OF STUDY. SO THE B-LEAD HIGHER FREQUENCY IN SOUTHEAST ASIA, 1 COULD DO THE STUDY IN SOUTHEAST ASIA, IT'S HARD TO DO WITH A NORTH AMERICAN OR EUROPEAN COHORT BECAUSE THERE AREN'T ENOUGH B-DELETES TO GET TO H BUT I SUGGESTED THIS TO MORE THAN 1 GROUP OVER THERE AND I HOPE IT'S GETTING DONE BECAUSE I THINK IT WILL COME OUT. AND 1 THING I THINK MAYBE SOMEBODY'S THINKING ABOUT NOW BUT THE 1 TECHNOLOGY TRANSFER WE--1 THING WE DON'T HAVE IN THESE STUDIES IS WE DON'T HAVE THE END MUTATIONAL SIGNATURE OR PATTERN. WE DON'T HAVE THE TIMELINE. SO SOME OF THESE SIGNATURES COULD HAVE ACCUMULATED OVER A VERY LONG PERIOD OF TIME. AND IF YOU REMEMBER--LET ME GO BACK TO THAT 1 ACTUALLY. LET ME GO BACK. SO YOU WILL SEE THE TIME SO,--IN THIS PARTICULAR--SO THIS IS THE STUDY WE'RE TALKING ABOUT WHERE WE SEPARATED THE DIFFERENT GENOTYPES SO, 1 COULD SEE THESE MUTATIONS AT ROUGHLY EQUAL PROPORTIONS IN EACH OF THESE APOBEC GROUPS, SO THIS IS THE--THESE ARE ALL IN CPG MOTIFS, SO THIS IS THE SPONTANEOUS DEAMINATION CYTOSEEN CORLACE THAT IS WITH THE PATIENT. THIS HAPPENS OVERTIME AND THE H-SIGNATURE WAS SO STRONG IN THE ABSENCE OF B COULD HAVE ACCUMULATED OVER A MUCH LONGER PERIOD. ITE? AND THAT'S THE EARLIER 1 HERE. SO ... IT REALLY NEEDS TO BE DONE. IT'S KILLING ME THAT IT HASN'T HAPPENED ALREADY. BUT I CAN'T INFLUENCE STUFF IN SOUTHEAST ASIA VERY EASILY. SURE. >> YOU THINK YOU WOULD BE ABLE TO INDUCE [INDISCERNIBLE] APOBEC INDUCES EXPRESSION PRODUCE TUMOR CELLS WITH CERTAIN MUTATIONS? CAN YOU PRODUCE THIS [INDISCERNIBLE] CELLS? >> SO CAN YOU USE APOBEC TO PRODUCE TUMOR CELLS? >> YES. >> SO WE'RE TRYING TO MODEL THIS IN MICE. SO THERE'S QUITE A FEW CLINICAL STUDIES NOW SHOWING THAT THIS APOBEC 3 B DELETION DOES NOT CORKORALATE WITH TUMOR INS--CORRELATE WITH TUMOR INCIDENCE. SO THE DEVELOPMENT OF THE INITIAL PRIMARY TUMOR IS NOT AT ALL INFLUENCERRED BY THE PRESENCE OR ABSENCE OF APOBEC 3 B. IT'S WHAT HAPPENS DURING TUMOR EVOLUTION THAT I THINK CAN BE REALLY INFLUENCED BY APOBECS. SO WE'RE WORKING ON DEVELOPING A MODEL FOR TUMOR EVOLUTION AND TUMOR HETEROGENEOUS ROW HETEROGENEITY. AND IT WON'T BE AN APOBEC-ONLY MODEL, IT WILL BE APOBEC OVER THE TOP OF OTHER DRIVERS TO LOOK AT HETEROGENERATED AITY IN A MOUSE MODEL. >> ANY DATA AT OWL PROGNOSIS OR ANYTHING? >> SO JOWN ASKED IF THERE ARE ANY DATA ON PROGNOSIS? >> AND SO, THERE ARE THOSE DATA THAT WE SHOWED HERE, WE ALSO--WE AND OTHERS HAVE PUBLISHED DATA ON UNTREATED BREAST CANCER, ESSENTIALLY BREAST TUMOR PATIENTS THAT ONLY RECEIVED SURGICAL RESECTION AS THE PRIMARY CARE AND IN THOSE CASES APOBEC 3 B CORRELATE WITH POOR OUTCOMES. SO YOU CAN USE A3 B IN THE TUMORS AS PROGNOSTIC MARKER FOR ULTIMATE OUTCOME. SIMILAR DAT WAHAVE SHOWN UP FOR LUNG CANCER AND 1 OTHER TUMOR, I'M FORGETTING RIGHT NOW, A MYELOID TYPE AND I THINK STUDIES ARE UNDER WAY TO LOOK AT OTHER TUMOR TYPES IN OVERALL OUTCOMES. WE'LL JUST HAVE TO WAIT AND SEE, AND I KNOW THERE ARE ALSO STUDIES ONGOING TO LOOK AT RESISTANCE TO DRUGS, NOT JUST TAMOXIFEN WHICH ISN'T SO MUCH A STANDARD OF CARE ANYMORE BUT SOME OF THEM MORE FRONT LINE TARGETED THERAPIES AND I THINK WE WILL SEE IN MANY OF THESE CANCERS THAT OUR APOBEC CANCERS THAT IT'S CONTRIBUTING TO THE OVERALL RESISTANCE MECHANISM. THERE'S NO REASON TO SUSPECT IT'S NOT, RIGHT? IF IT'S PART OF THE FUEL? YES? >> WHAT IF SOMEONE PUT [INDISCERNIBLE] PROTEIN [INDISCERNIBLE] WOULD IT HELP? >> SO WE'VE SUGGESTED THAT MUCH TOO, AND ACTUALLY THE SIV--SO HOW DO YOU STOP APOBEC IN THE TUMOR? OKAY, IS THE QUESTION AND 1 POTENTIAL ANSWER IS YOU COULD USE VIF, SO SIV MACK 239 VIF IS A GOOD INHIBITOR OF APOBEC 3 B, SO WE PUBLISHED A LITTLE BIT ON THAT THAT 1 COULD POTENTIALLY USE THAT, BUT THEN YOU FALL INTO THE--THE BIG PROBLEM OF HOW DO YOU DELIVER IT AND HOW DO YOU PREVENT THAT FROM BEING RECOGNIZE AS A FOREIGN ANTIGEN. SO, IT COULD BE USEFUL BUT YOU THEN HAVE A DELIVERY PROBLEM AND AN E3. AND ALSO FOR VIF TO FUNCTION IT NEEDS CBF BETA AND THAT'S LARGELY CONFINED TO THE HEMATOPOIETIC COMPARTMENT INCLUDING T-CELLS AND I DON'T THINK, I DON'T KNOW BUT I DON'T KNOW IF IT'S ALL SOLID TUMOR CELLS. SO YOU NEED TO MAKE THE LIGASE. SO VIF'S A TOUGH 1 BUT WHY NOT USE SOMETHING THAT MOTHER NATURE'S INVENTED AGAINST APOBEC. SURE. SURE. >> ANY INTEREST IN EXPLORING HEALTH DISPARITIES?& BECAUSE IN AFRICAN AMERICANS IN THE UNITED STATES, FOR INSTANCE PROSTATE CANCER HAS SIMILAR INSTANCE? [INDISCERNIBLE] TWICE THE MORTALITY COMPARED TO WHITE MEN BECAUSE IT BECOMES METASTATIC SO I'M CURIOUS IF YOU'RE THINKING OF LOOKING AT THAT BECAUSE OF THE BREAST CANCER. >> WE ARE REALLY INTERESTED IN THAT AND ABSOLUTELY. SO THERE ARE SNPs AND THERE ARE VARIATIONS IN THE LOCUST THAT COULD ULTIMATELY EFFECT HOW MUCH THE APOBEC MUTATIONAL PROGRAM IS IPT IMPACTING CANCERS IN DIFFERENT PEOPLE. >> YEAH, WE'RE TOTALLY INTERESTED, IT'S JUST WE HAVEN'T QUITE GOT THAT FAR YET. >> YEAH, SO I'M SURE THERE'S 2--THAT'S OKAY. >> YOU SHOW THE ASSOCIATION OF APOBEC EXPRESSION AND THE MUTATION [INDISCERNIBLE] IN CANCER BUT WE ALREADY HAVE PROVEN EVIDENCE THAT'S CLOSE RELATIONSHIP AND [INDISCERNIBLE] DIRECTION. >> SO I THINK THE BEST CAUSE AND EFFECT DATA SO FAR IS WHAT I SHOW YOU. SO IF YOU TAKE AWAY, ALBEIT THERE IS A LAW OF DIMINISHING NUMBERS HERE BUT IF YOU TAKE AWAY B AND HAPLOTYPE 1 THERE'S NO LONGER AN APOBEC SIGNATURE. I SHOW YOU BREAST CANCER DATA BUT IF YOU LOOK ACROSS TCGOR ICGC, OR BIG DATA SETS THERE'S NO EXAMPLE OF A TUMOR WITH AN APOBEC SIGNATURE THAT DOESN'T HAVE 1 OR THE OTHER. SO THAT'S PRETTY COMPELLING CAUSE AND EFFECT DATA. AND THE OTHER CAUSE AND EFFECT ALTHOUGH NOT--WE NEED TO RECREATE IT IN A MODEL SYSTEM BUT THE PI3 KINASE ACTIVATION IS TOO STRONG TO BE NOT CAUSE AND EFFECT. SO YOU REALLY SEE THAT FOR INSTANCE CERTAINLY--CERTAINLY SPECIALLY IN HPV DRIVEN CERVICAL CANCERS WHERE APOBEC 3 B IS UPREGULATED BY THE VIRUS, THAT IS THE DOMINANT KINASE MUTATING FASHION SO I THINK YOU HAVE A VERY STRONG--SO I SHOULD ALSO SAY, SO NO 1 PIECE OF DATA I'VE SHOWN YOU IS A SMOKING GUN, RIGHT? NONE OF THEM. I WISH. SOME OF THE GENETIC STUDIES ARE PRETTY DEFINITIVE BUT IT'S ALTOGETHER A COMPELLING ARGUMENT THAT APOBEC IS CONTRIBUTING TO THIS AND CONTRIBUTING TO TUMOR EVOLUTION. >> FRANK? >> IF IT CONTINUE WITH HPV, IT'S SO COMPELLING FOR CERVICAL, THERE ARE PLENTY OF ORAL LESIONS, SOME ARE HPV ASSOCIATED AND SOME AREN'T, AND DO YOU GET THE SAME LEVEL OF APOBEC INDUCED CHANGES IN THOSE CANCERS? >> SO IF YOU SEPARATE THEM OUT, THOSE THAT HAVE HPV INDUCED ORAL CANCERS HEAD AND NECK CANCERS THOSE WITH VIRUS IN APOBEC, HAVE HIGHER MODES THAN THOSE WITHOUT. BUT SOME OF THOSE LIKE BREAST CANCER IS NOT AT LEAST DIRECTLY A VIRUS DRIVEN CANCER. SOME OF THOSE LESIONS WITHOUT VIRUS ALSO HAVE HIGH APOBEC. SO WE'VE SEPARATED ALL THAT OUT IN THE CORRELATIONS ARE VERY, VERY STRONG ALSO. >> SO IF YOU LOOK AT JUST THE HPVs AGAIN, VIRALLY INDUCED 1S VERSUS NONVIRAL, THE NONVIRAL TEND TO BE MORE DIFFICULT TO TREAT, TEND TO BE MORE METASTATIC SO WHAT'S UP WITH THAT. >> FRANK'S ASKING A GOOD QUESTION, WHY ARE THE NONVIRAL CANCERS HARD TO TREAT THAN THE VIRAL APOBECS. ONE REASON IS I DIDN'T GET INTO BECAUSE IT'S AN ENORMOUS PROBLEM, IS A LOT OF THESE CANCERS THAT HAVE HIGHER MUTATIONAL LOADS CAN BE CONSTRAINED BETTER BY THE IMMUNE RESPONSE SO IN THESE CASES, THE APOBEC CANCERS IN MANY CASES ARE COMING SOON ON IMMUNOTHERAPYS SO LIKE MELANOMA, LIKE YOU KNOW HEAVILY LUNG CANCERS WITH A LOT OF SMOKING LESIONS, THOSE APOBEC CANCERS ARE BETTER CANDIDATES FOR IMMUNOTHERAPY. SO IN THIS CASE, IF YOU CAN PROVOKE A GOOD IMMUNE CONTROL MECHANISM, THE APOBEC CANCERS WILL FAIR BETTER THAN THE NONAPOBEC CANCERS AND I THINK THAT MAY BE THE CASE IN LUNG BUT FOR BREAST, I DON'T KNOW. I THINK IT DEPEND OTION THE MICRO ENVIRONMENT SO WE WILL SEE DEMENDENTS ON THE LESION SO THAT'S WHY IT BECOMES ENORMOUSLY COMPLEX, BUT I'LL ALSO BET HEAVILY ON THE BETTER IMMUNE RESPONSES WITH APOBEC. >> BEFORE YOU GO-- >> [INDISCERNIBLE]. >> YOU DIDN'T SAY IT BUT ARE YOU ATTRIBUTING THAT EFFECT TO NEOIMAGE FORMATION? >> NO, ABSOLUTELY. SO FOR INSTANCE IN PI3 CAIN ACE, THAT IS A JUICY BRAND NEW NEOEPITAUPE AND IT'S EASY TO IMAGINE HOW A POINT MUTATION MECHANISM LIKE THIS CAN MAKE NEW EPITAUPES THAT THE IMMUNE SYSTEM CAN FOCUS ON. YEAH. IT'S JUST, WE HAVE TO SOME OF THESE IDEAS. RIGHT? YEAH? >> JUST TO FINISH UP ON THAT. VARIANT OF BREAST CANCER, INFLAMMATORY BREAST CANCER, RELATIVELY INFREQUENT BUT MUCH MORE--HAS MUCH MORE MUTANT ASPECT FOR HAPPEN LO GENESIS, HAVE YOU LOOKED AT THOSE? >> NO. SO 1 THING WE WANT TO DO MOVING FORWARD IS BE ABLE TO SEE THE PROTEIN IN THE TISSUE AND COLLABORATE WITH PATHOLOGISTS TO REALLY GET A HANDLE ON THINGS THAT ARE CORRELATING WITH THE APOBEC MUTATION PROGRAM INCLUDING BET ARER IMMUNE RESPONSES AND INFLAMMATION. AND IF YOU HAVE TO BE PRIED AWAY FROM THE TUMORS ITSELF, HOW ABOUT OTHER INFLAMMATORY CIRCUMSTANCES? [LAUGHTER] SO RHEUMATOID LOGIC DISEASES. >> NO IT'S DEFINITELY WORK LOOKING AT. YOU HAVE TO PICK YOUR BATTLES AND YOU PROBABLY SAW THIS THAT WE APPROACH THE CANCER MUTATION PROGRAM FROM A COMPLETELY UNBIASED CRITERIA. SO OUR SOLE SELECTION WAS NORMAL AND NEGATIVE, AND I DIDN'T CARE IF IT WAS E. R. HR. TWO, ONLY CRITERIA WAS NORMAL PAIRED TUMOR. SO WE'RE STARTING FROM FUNDAMENTALS AND WE'RE BUILDING UP AND I THINK IT WILL GET REALLY COMPLEX. ESPECIALLY WHEN YOU FACTOR IN THE IMMUNE RESPONSE, FROM FUNDAMENTALS THERE'S NO REASON TO EXPECT THAT THE APOBECS ARE NOT CONTRIBUTING, I OFTEN HAVE THE SLIDE, BUT I DIDN'T PUT IT IN, JOHN, SORRY. BUT YOU VIEW IT AS A BALANCE. IT'S A DOUBLE EDGED SWORD. HELPING TUMOR EVOLUTION IF IT GOES TOO FAR ANDNY O ANTIGENS THEN THAT WILL BACK FIRE IN FAVOR OF THE HOST AND AGAINST THE TUMOR. YEAH, SAME HAS THE VIRUS. >> ALL RIGHT. >> LET'S THANK REUBEN FOR A FANTASTIC TALK. [ APPLAUSE ] WE HAVE ALL KINDS OF OTHER GOODIES OUTSIDE. SO PLEASE.