GOOD AFTERNOON. LAST WEEK YOU SHOULD HAVE ALL RECEIVE AND E-MAIL FROM ME AND IF YOU'RE INTERESTED IN SIGNING UP TO VISIT THE CORES, EITHER THE TUMOR BOARDS OR THE LABS PLEASE RETURN E-MAIL AND INDICATE YOUR CHOICES BY THE END OF THE WEEK.SO TODAY WE HAVE THE GREAT PLEASURE OF HAVING YVES POMMIER AS THE SPEAKER.HE RECEIVED HIS MD AND Ph.D. DEGREE FROM THE UNIVERSITY OF PARIS.HE'S BEEN AT THE NIH SINCE 1981.HE'S, UH, CHIEF OF THE LABORATORY OF MOLECULAR PHARMACOLOGY IN BUILDING 10.HE'S ALSO A COCHAIR OF THE DRUG DISCOVERY COMMITTEE OF THE NCI EXPERIMENTAL THERAPEUTICS PROGRAM.HIS TOPIC IS DNA TOPOISOMERASE AND THEIR POISONING BY ANTICANCER AND ANTIBACTERIAL DRUGS.YVES. >> GOOD AFTERNOON.SO THIS TITLE IS BROADER THAN WHAT I'M GOING TO BE DISCUSSING.WE'LL NOT BE DISCUSSING THE ANTEBACTERIAL DRUGS BUT THIS IS A REVIEW THAT CAME OUT JUST LAST YEAR AND PROBABLY BRINGS YOU UP TO DATE.THIS REVIEW IS A CHALLENGE AND THE ONLY REASON I ACCEPTED IT AT THE TIME WAS THAT BECAUSE IN ONE REVIEW I HAD TO DEAL WITH THE VERY LARGE NUMBER OF DRUGS THAT ARE USED IN MEDICINE THAT INCLUDES NOT ONLY ANTICANCER AGENTS BUT ALSO ANTEBACTERIAL AGENTS AND THAT TARGET A VERY LARGE FAMILY OF ENZYMES.SO IT WAS A HUGE CHALLENGE BUT I THINK THERE WAS NO SUCH THING AS A SINGLE REVIEW THAT WOULD TRY TO PUT EVERYTHING IN ONE PLACE.TODAY, WE'RE NOT GOING TO BE TALKING ABOUT THE ANTEBACTERIAL ASPECT.I'LL TOUCH UPON IT JUST SO YOU UNDERSTAND WHY IT IS CONNECTED. SO THE FIRST ISSUE IS THAT WHAT ARE DNA TOPOISOMERASES, AND I WILL GO OVER SOME GENERAL SLIDES BECAUSE -- AND I THINK IT'S IMPORTANT YOU GET AN APPRECIATION OF THE GENERAL MECHANISMS AND THE RELEVANCE OF THESE ENZYMES AND OF THE DRUGS THAT ARE BEING USED TO TARGET THESE ENZYME AND TREAT PATIENTS. UM, AND THEN I'LL GIVE YOU MAYBE AT THE END A MORE SPECIFIC INSIGHTS.SO THESE ENZYME CALLED DNA TOPOISOMERASE DRIVED THEIR NAME FROM THE TOPOLOGY AND THEY CHANGED THE TOPOLOGY OF DNA.AS YOU WELL-UNDERSTAND, DNA'S A VERY LARGE POLYMER, ABOUT TWO METER LONG IN SINGLE CELL WHICH IS HIGHLY PACKAGED, HIGHLY ORGANIZED YET ACCESSIBLE FOR TRANSCRIPTION AND REPLICATION, TRANSCRIPTION MANY TIMES DURING THE CELL CYCLE AND REPLICATION ONCE, BUT ONCE AND FAITHFULLY.TO DO THIS FAITHFULLY, YOU NEED TO UNRAVEL THESE POLYMER IN A [INDISCERNIBLE] MANNER WITHOUT SCREWING DOWN THE REPUTATION AND [INDISCERNIBLE] MACHINERY.HENCE -- ALL CELLS IN FACT IN THREE DOMAINS OF LIFE WHERE YOU TAKE ARCHIA, YOU BACTERIA OR YOU CARRIER HAVE TOPOISOMERASE.ALL CELLS THAT HAVE DNA HAVE TOPOISOMERASES.AND IN HUMANS, YOU HAVE TWO MAIN FAMILIES THAT ARE EITHER TYPE ONE OR THE TYPE TWO TOPOISOMERASES, AND THEY ALL USE THE SAME MECHANISM.THEY WILL USE A TYRO-SEME TO ATTACK THE DNA BACKBONE AND THERE BY RETRIEVE THE DNA.IF YOU WERE TO THINK OF THESE ENZYMES WITH REGARD TO WATERING HOSE, THAT WOULD BE THE MAGIC TOOL BOX.IN THE SAME ENZYME YOU BOTH HAVE THE KNIFE TOR SCISSORS YOU TR HAVE THE GLUE.SO THEY WILL CUT THE DNA, CHANGE IN ANY WAY IT NEEDS TO BE CHANGE AND THEY WILL GLUE IT BACK.ONE ENZYME WILL DO BOTH.SO IT'S SORT OF VERY AMAZING MACHINES, AND THE [INDISCERNIBLE], WHICH IS SHOWN HERE WILL ATTACK THE BACKBONE WITH A DIFFERENT POLARITY DEPENDING WHETHER IT'S A TOPO ONE OR T PORTION O TWO.IN THE CASE OF TOPO ONE THE CASE IS SUCH THAT IT WILL LINK ITSELF TO THIS FREE [INDISCERNIBLE] OF THE DNA [INDISCERNIBLE] LEAVING THE DNA OPEN AND THEN IT WILL REVERSE THAT REACTION ONCE THE DNA HAS BEEN CHANGED IN WHICH EVER WAY IT NEEDS TO BE, AND FOR THE TOPO TWO THE LINKAGE IS A FIVE PRIME END, AS WITH THE TOPO 3.THERE ARE THREE MAIN NAMES THAT ARE IN HUMAN CELLS.THE TOPO ONE IS SORT OF A LONER.THE ONLY ENZYME THAT [INDISCERNIBLE] AND IN THAT SENSE A TOPO ONE BELONGS TO A FAMILY OF [INDISCERNIBLE].IN FACT THE TOPO ONE IS MORE DERIVED FROM THESE [INDISCERNIBLE] WHEREAS THE TOPO THREE AND TWO ARE OF THE CLASS KAL FORM OF THE OTHER TOPOISOMERASES THAT YOU FIND IN BACTERIA MUCH MORE COMMONLY THAT LEAVE TO THE FIVE PRIME MAN OF THE DNA.SO THERE ARE OTHER DIFFERENCES BETWEEN THIS THREE MAIN CLASSES THAT ARE SHOWN HERE.SO ON THE LEFT YOU THE TOPO 1, IN THE MIDDLE, TOPO 2 AND IN THE RIGHT TOPO 3. FIRST WAS TOPO 1 BY [INDISCERNIBLE] AND THEN THE TWO WAS THEN DISCOVERED LATER ON BY GENE WONG AND THEN THE TOPO THREE MORE RECENTLY.THE WAY THIS IS TLAUN DRAWN IS TO SHOW THE [INDISCERNIBLE] BY WHICH THE DNA IS BEING PROCESSED.THIS ARE CLEAVAGE COMPLEXES.THESE ARE [INDISCERNIBLE] BY WHICH THE [INDISCERNIBLE].IN THE CASE OF TOPO 1, YOU COULD SEE AS I SAID BEFORE, THIS IS THE ONLY ENZYME THAT LINKS TO THE THREE PRIME MAN, AND IF YOU LOOK AT THE TOPO 2 AND 3 THEIR LINKAGE IS THROUGH THE SIZE PRIME MAN.OTHER DIFFERENCE; YOU COULD SEE IN THE MIDDLE THAT TOPO 2 WORKS AS DIMER.THERE ARE TWO BOWLS AND TOPO 2 WORKS IN A CONCERTED FASHION, IN HOMO DIMER WORKS TO CLEAVE DNA IN A DOUBLE STRAND BREAK WITH A FOUR BASE PAIR STAGGER.IS A [INDISCERNIBLE] SPACES FOR THE CLEAVAGE OF DNA WITH A FIVE PRIME OVERHANG.THAT FORMS A GATE INTO THE ONE OF THE DUPLEX TREND IS THROUGH WHICH WILL GO ANOTHER DUPLEX AND I WILL SHOW YOU THE DETAIL THOFS MECHANICS LATER.THEN WHEN THE ENZYME IS DONE THE ENZYMES WILL DENE GAIT.TOPO 1 AND 3 DO NOT NEED ATP, TOPO TWO NEED ATP.THE DRUGS THAT TARGET EACH OF THESE ARE VERY SPECIFIC AND VERY DIFFERENT.INITIALLY DERIVED FROM NATURAL PRODUCTS.IN THE CASE OF TOPO ONE, IN THE [INDISCERNIBLE].IN THE CASE OF TOPO TWO, [INDISCERNIBLE].AND YOU ALREADY HAVE SENSE THESE ARE DRUGS USED IN THE CLINIC TO TREAT CANCER PATIENTS.THERE ARE NO TOPO THREE INHIBITORS TO DATE, AND TOPO THREE BEING THE E VIF WENT RENT IN TOM SOME WAYS TO THE TOPO ONE IN BACTERIA, THERE ARE ALSO NO TOPO 3 INHIBITORS USED IN ANTIBIOTICS IN BACTERIA TODAY.SO THESE THE THREE FAMILY; THE TYPE ONE, THE TYPE TWOE, THE TYPE THREE.THE TYPE THREES IN FACT CALLED A TYPE 1 A BASED ON THE FACT THAT IT CLEAVES THE DNA BY MAKING A SINGLE STRAND BREAK TO LINK IT A SINGLE STRAND BREAK.SO.I DON'T KNOW WHAT ELSE THAT IS.IGNORE I GUESS. SO TYPE ONE A, TYPE TWO A, THIS IS THE MORE FORMAL CLASSIFICATION OF THESE ENZYMES. SO THERE ARE SIX GENES IN HUMAN CELLS.THIS HAS BEEN SHIFTED AROUND BECAUSE OF THE FILE.THERE ARE TWO TOPO ONE B, WHICH ARE TOPO ONE AND TOPO ONE MITOCHONDRIAL.YOU AND VI SIX GENES.THEY AROSE BY DUPLICATION SUCH AS THESE AND TWO TOPO THREE GENES THAT SHOULD BE HERE, THESE ARE THE TOPO THREE ALPHA, TOPO THREE BETA.GIVE YOU PERSPECTIVE OF THE SIZE OF THESE MACHINES.THESE ARE MOLECULAR MACHINES THAT ARE FAIRLY LARGE BUT EXTREMELY EFFICIENT.IN THE CASE OF THE HUMAN, THESE THE SIX ENZYMES GROUPED IN THE TYPE ONE A FOR THE TOPO THREE, YOU HAVE THE MOLECULAR SIZE.THEY'RE ALL IN ABOUT THE HUNDRED [INDISCERNIBLE] AT LEAST.THE TOPO TWO ARE LARGER.THEY ARE ABOUT 170 [INDISCERNIBLE] FOR THE MONOMER NAKING THE DIMER 350; LARGE MACHINE IS GOING TO BIND DNA.MUCH BIGGER THAN THE [INDISCERNIBLE].T TOPO TWO DAY BAY TA 180 TWICE AND THEY USUALLY DON'T INTERMIX. T 180 TWICE AND THEY USUALLY DON'T INTERMIX. A 180 TWICE AND THEY USUALLY DON'T INTERMIX. TOPO THREE [INDISCERNIBLE] ABOUT THE SAME BUT WORKS AS MONER.MONOMER, MONOMER, MONER DIMERS.THIS IS THE LINKAGE THAT I DESCRIBED BEFORE.THE ONLY ONE LEADING TO THE [INDISCERNIBLE] IN THE TOPO ONE.THE TOPO ONE LINKING TO FIVE PRIME END IS TOPO THREE AND TOPO TWO.E. COLI IS A MUCH SIMPLER ORGANIZATION.IT DOES NOT CONTAIN THE TOPO ONE B, WHICH IS THE ONE TARGETED BY CANCER [INDISCERNIBLE].IT DOESN'T ONLY TOPO ONE A AND TWO FORM, WHICH IS CALLED TOPO ONE, TOPO THREE, AND MORE IMPORTANTLY FOR MEDICAL USE, E. COLI OR OTHER BACTERIA HAVE THE TYPE TWO A AND THERE ARE TWO FORMS, THE JIE RACE AND TOPO FOUR, BOTH TARGETED BY ANTI[INDISCERNIBLE]. SO LET'S GO BACK, IF YOU WISH, TO THE [INDISCERNIBLE] MECHANISM TO PUT IT BACK TOGETHER.SO IF YOU THINK OF THE TWO MAYBE CLASSES FOR THERAPEUTICS IN HUMANS IT'S TOPO ONE AND TOPO TWO.TOPO ONE, AS I SAID ARE TWO GENES; THE NUCLEAR TOPO ONE WHICH IS DEVOTED TO THE NUCLEAR GENOME AND THE MITOCHONDRIAL TOPO ONE DEVOTED TO THE MIE TOE COUNTRY Y'ALL [INDISCERNIBLE].THESE ENZYME WILL BIND THE DNA, FORM A CLEAVAGE COMPLEX BY LINKING TO THE STRAIT PRIME MAN.SWIVEL THE DNA AROUND AND [INDISCERNIBLE] THE DNA WHEN IT IS BEING DERETAXED.THE POISONS FOR THE TOPO ONE THE [INDISCERNIBLE] WHICH [INDISCERNIBLE] THE TOPO ONE CLEAVAGE COMPLEX IN A [INDISCERNIBLE] CIVIL MANNER SHIFT THE [INDISCERNIBLE] TOWARDS THE [INDISCERNIBLE].THE INTERMEDIATE IS THE COVALENT LINKAGE OF THE TOPO ONE THROUGH THE [INDISCERNIBLE] WITH ITS [INDISCERNIBLE].WHICH IS REVERSIBLE [INDISCERNIBLE] TOPO ONE GOES AWAY.IN THE CASE OF TOPO TWO, HOMO DIMERS IN THE CASE OF HUMAN, TOPO TWO ALPHA HOMO DIMERS.IN THE CASE OF BACTERIA, YOU HAVE [INDISCERNIBLE] JIE RACE AND TOPO FOUR.THE DRUGS THAT INTERFERE WITH THESE MOSTLY IN CANCER THERAPY AND IN ANTIBACTERIAL THERAPY TRAP THE POE TOE CLEAVAGE COMPLEX.LY SHOW YOU CRYSTAL STRUCTURES LATERER AND THEN POISON THESE ENZYMES BY [INDISCERNIBLE] THE CLEAVAGE COMPLEX.THE [INDISCERNIBLE] WITH THE ENZYME LINKED AT THE THREE PRIME MAN OF THE DNA AND LEAVING A FIVE PRIME [INDISCERNIBLE] HAND.IT WILL EVENTUALLY REVERSE, BUT IT JUST SLOWS DOWN THE RADIATION ONCE THE DRUG IS IN THE SYSTEM. SO LET'S FIRST START WITH THE TOPO ONE.I'LL GO INTO TOPO ONE AT FIRST, THEN GO BACK TO TOPO TWO AND END UP A LITTLE BIT ON TOPO ONE.SO THE TWO TOPO ONES, WHAT ARE THEY?TWO GENES THAT ARE ENCODED IN THE NUCLEUS.ONE WHICH IS DEVOTED TO THE MIE TOE COUNTRY Y'ALL AND ONE TO HUMAN GENOME.DEPENDING ON THE CELL, YOU COULD HAVE # 9% OF THE GENOME IS IN THE NUCLEUS OR SOME CELLS YOU COULD HAVE MAYBE 80%, MEANING 20% OF THE DNA IS ACTUALLY IN THE MIE TOE COUNTRY Y'ALL DNA.THAT'S WHY THE CELLS, ACTUALLY THEY HAVE TWO ENZYMES.ONE WHICH IS SPECIFICALLY TARGETED TO MIE OCHONDRIA WHICH IS THE UPPER UNMADE IN THE NUCLEUS, AND THE OTHER ONE, WHICH STAYS IN THE NUCLEUS AND DOES NOT WORK IN THE MIE TOE COUNTRY COUNTRY MIE TOECHONDRIA.THE SGROOEN MIE TOCHONDRIAL TOPO ONE WAS THE [INDISCERNIBLE].THE TWO PROTEINS, THE TWO POLYPEPTIDES ARE RATHER SIMILAR.MOSTLY IN THE CATALYTIC DOMAIN.YOU COULD SEE E THE SIMILAR IS I TTIES UP TO 90% IN THE CATALYTIC DOMAIN OR YOU COULD SEE THE CATALYTIC [INDISCERNIBLE] IN HERE.THEY'RE MOST DWER GENT IN THE INTERIM NOUS DOMAIN THAT'S OF USE.THE PAYWAY THE TOPO ONE WORK AND THIS WILL GO INTO MORE DETAIL LATER.THESE WERE INITIALLY CALLED THE DNA UNTWISTING ENZYME.WHEN DNA WAS VISUALIZED BY [INDISCERNIBLE] BROMIDE STAINING, UH, IT WAS IMMEDIATELY REALIZED THAT YOU COULD ACTUALLY SEE THE UNWINDING OF DNA.BEFORE THAT THE DNA WAS SEPARATE BY SEDIMENTATION.[INDISCERNIBLE] PRO MIED MADE IT EASY TO DETECT THE CONVERSION OF [INDISCERNIBLE].IN FACT, WHEN INITIALLY WAS DISCOVERED THAT DNA WHEN YOU EXTRACT FROM BACTERIA FROM VIRUSES USUALLY SUPERCOIL, BUT NEGATIVELY SUPER CORD AND IF YOU TAKE THAT NEGATIVELY SUPER CORD AND MIX IT WITH A DROM DROP OF TOPO ONE TDNA WILL BE IMMEDIATELY RELAXED EVEN AT ZERO DEGREES.THE ENZYME WAS [INDISCERNIBLE] UNTWISTING ENZYME.AND THE UNTWISTING ACTIVITY IS LINKED TO THE FACT THAT THE ENZYME AS SOON AS IT SEES A CROSSOVER OR A SUPER CORD IN THE DNA WILL VERY QUICKLY BIND TO IT, THEN CLEAVE ONE OF THE STRANDS, LET THE DNA SWIVEL AROUND AND [INDISCERNIBLE].THE DNA WILL GO QUICKLY FROM SUPERCOILED TO RELAX BECAUSE THE ENZYME WILL LINK TO ONE END, LET THE OTHER END ROTATE AROUND INTO THE CLEAVAGE COMPLEX AND ACTUALLY WE'RE MEASURING THIS WITH [INDISCERNIBLE] HERE IN THIS BUILDING AND THIS STAGE WITH SINGLE MOLECULE.WE COULD STUDY EXACTLY HOW THIS IS BEING RELAXED AN THEN THE ENZYME RELY GAITS WHEN THE [INDISCERNIBLE] AND THEN GOES AWAY.THAT'S TRUE FOR MIE TOE COUNTRY Y'ALL AND THE OTHER DNA.THE DRUGS USED IN THE CLINIC TO POISON AND TARGET TOPO ONE ARE QUITE MANY.THE TOPO ONE-TARGETED DRUGS ARE SHOWN IN DIFFERENT PANELS HERE.PANEL A, OTHER DRUG THAT ARE CLINICALLY APPROVED, AND THEY'RE IN THE UNITED STATES.TWO D-FDA-APPROVED COMPOUNDS.T CAN IS APPROVED FOR COLON CANCER BUT ALSO QUITE EFFECTIVE IN LUNG CANCER AND SOME [INDISCERNIBLE] TUMORS. IN ASIAN, IN KOREA, [INDISCERNIBLE] HAS ALSO BEEN APPROVED.IT'S NOT USED IN THE UNITED STATES.ALL THESE DRUGS ARE DERIVATIVES OF [INDISCERNIBLE] AND THEY WERE MADE TO MAKE WATER SOLUBLE PRODRUG OR WATER SOLUBLE DRUGS OF [INDISCERNIBLE] WHICH IS A NATURAL PRODUCT. AT THIS STAGE -- AND I WILL GO LATER ON THIS -- THERE ARE SOME OTHER NON-[INDISCERNIBLE] THERE ARE IN CLINICAL DEVELOPMENT AND TWO OF THEME ARE IN CLINICAL TRIALS HERE AT THE IN BUILDING 10, AND THEY ARE CULL CALLED THE [INDISCERNIBLE].THESE TWO DERIVATIVES IN CLINICAL TRIAL IN BUILDING TEN.THIS OTHER ONE IS IN CLINICAL TRIAL IN MANY PLACES IN THE UNITED STATES AND THIS WAS INITIALLY DEVELOPED BY THE UNIVERSITY OF NEW JERSEY AND IT'S BEEN LICENSED TO ENZYME AND NOW TO [INDISCERNIBLE]. LET'S LOOK AT THE TOPO TWOS NOW.SO THE TOPO TWO FAMILY, THERE ARE TWO GENES AND TWO ENZYME IN ANY CELL.THESE GENES ARE ABSOLUTELY ESSENTIAL.TOPO TWO ALPHA AND BETA ARE MADE OF THE LARGER PROTEINS OR ABOUT 170, 180 KILO DAK TON KILN KILO MER.THE [INDISCERNIBLE] IS NOT IN THE END LIKE TOPO ONE, IT'S IN THE MIDDLE SO THERE'S CLEAVAGE REDONATION HERE.THEY'RE VERY SIMILAR.YOU COULD SEE THE SIMILARITY OR IDENTITY OF THESE PROTEINS, AND THE MAIN DIE VER JENS -- UP TO 90% SIMILARITY, WHICH EXPLAINS WHY THE TOPO TWO INHIBITORS USED IN THE CLINIC TARGET BOTH ENZYME THAT THE STAGE, BOTH ALPHA AND BETA.THEY ARE SOMEWHAT SIMILAR TO THE BACTERIAL AUTO LOGS WHICH IS EITHER DNA JIE RACE IN BACTERIA OR TOPO FOUR IN BACTERIA.THE DIFFERENCE IN BACTERIA IS BACTERIA MAKE THOSE ENZYME OUT OF TWO GENES.IT'S TWO PIECES.IN HUMANS, EACH OF THEM IS MADE AS ONE POLYPEPTIDE YET WORKING AS A HOMO DIMER BUT ONE POLYPEPTIDE.SO IN BACTERIA, THE SYSTEM IS A LOT MORE COMPLEX, MADE OF TETRAMER.HUMANS ARE MADE OF DIMERS AND BACTERIA HAVE THE TWO FORMS, OKAY.SO WHAT IS THE DIVISION OF LABOR?WHY DO HUMAN CELLS NEED AND USE TWO TOPO TWO?THE DIFFERENCE IS BETWEEN THE TWO ENZYME.TOPO TWO ALPHA IS ASSOCIATED VERY TIGHTLY WITH DNA REPLICATION.TOPO TWO DAY BAY TA IS MORE ASSOCIATED WITH TRANSCRIPTION, AND IT MAY BE VERY SIMPLISTIC WAY TO THINK OF IT IS TOPO TWO ALPHA IS A REPLICATION-DEPENDENT UNRAVELLING ENZYME.TOPO TWO BETA IS MORE A TRANSCRIPTION-RELATED UNRAVELLING ENZYME.THIS IS FAR TOO SIMPLICITY BUT AN EASY WAY TO REMEMBER.SO MANY CELLS THAT DO NOT DIVIDE INCLUDING NEURONS HAVE A LARGE AE MOUNT OF TOPO TWO DAY BAY TA BUT ALMOST NO TOPO TWO ALPHA, WHERE AS A VERY RAPIDLY DIVIDING CELL, LEUKEMIA CELLS FOR INSTANCE HAVE A LOT OF TOPO TWO ALPHA OR CANCER CELL AND THEY STILL HAVE TOPO TWO BETA BUT MAKE A LOT OF TOPO TWO ALPHA.IN BRS CANCER THERE IS A LOT OF [INDISCERNIBLE] GENE AND YOU FIND A LOT OF TOPO TWO ALPHA ALONG WITH THIS GENE.TOPO TWO ALPHA CAN BE SEEN AS A [INDISCERNIBLE] UNRAVELLING ENZYME.THIS IS MORE NUTS AND BOLT, I DON'T WANT TO GO THROUGH THIS.FOR THOSE WHO CAN READ THE REVIEW THAT I HAD IN THE FIRST SLIDE YOU CAN UNDERSTAND THE CATALYTIC MECHANISM.THESE ARE VERY BEAUTIFUL MACHINES THAT UTILIZE MAGNESIUM TO SET THEIR STRUCTURES UP AND BIND DNA.DOESN'T MATTER FOR THE PURPOSE OF THIS PICTURE.SO WHAT DO TOPO TWOS DO THAT TOPO ONE DO NOT DO?WHAT YOU HAVE TO KEEP IN MIND IS TOPO TWO MAKES A DOUBLE STRAND BREAK OR A TOPO ONE MAKE SINGLE STRAND BREAK.WHY DO YOU NEED TO MAKE A DOUBLE STRAND BREAK?WELL, IT'S VERY EASY. THINK OF THIS; IF YOU HAVE TWO DNA CIRCLES THAT ARE [INDISCERNIBLE], THE ONLY WAY YOU COULD SEPARATE THOSE CIRCLE IS BY MAKING A DOUBLE STRAND BREAK.OKAY.YOU NEED TO OPEN UP ONE, LET GO THROUGH.THAT'S WHAT WE CALL DECAT NATION.THIS IS THE MOST OF USE WAY CHI BIWHICH TOPO TWO CAN DO ITS JOB.WHY IS THIS RELEVANT?BECAUSE ANY TYPE YOU DIVIDE ANY CIRCLE, WHICH IS SUPERCOIL WHICH HAD IS CALLED A [INDISCERNIBLE] STRIP.IF YOU KIT CUT NIT THE MIDDLE AND IT HAS ONE SUPER TURN, YOU'LL END UP WITH ONE INTERLINK.IF YOU HAVE MORE THAN ONE, THE NUMBER OF INTERNS WILL BE EQUAL TO THE NUMBER OF CIRCLE TURNS.IF YOU TRY TO DEVELOP A SUPER COIL CIRCLE OR SUPER COIL DNA DOMAIN, YOU'LL END UP WITH A LOT OF LINKAGE POINTS.THEY NEED TO BE RESOLVED, AND AT THE END OF REPLICATION, TO SEPARATE THE CHROMOSOME, YOU ABSOLUTELY NEED TOPO TWO ALPHA.ABSOLUTE NEED.IF YOU KNOCK IT OUT IN YEAST OR ANYWHERE, THE CHROMOSOME CANNOT SEGREGATE.THEY ARE INTERTWINED.SO OTHER THAN THIS INTERTWINING WORKS, IF YOU TAKE TWO DNA SEGMENTS, WHICH WE COMMONLY REFER IN OUR JARGON AS THE T AND G FOR GATES AND THE TRANSPORTED.SO THE ONE WHICH HAS A GATE AND THE OTHER ONE THAT WILL GO THROUGH THE TRANSPORT SEGMENT, THE TOPO TWO WILL FIRST BIND TO THE GATE SEGMENT AS THIS, AND I WILL SHOW YOU CRYSTAL STRUCTURE SHORTLY.IN THE PRESENCE OF ATP, THEN THE TOPO TWO WILL CHANGE ITS STRUCTURE AND CAPTURE THE TRANSPORTED SEGMENT AT THE TOP OF THE ENZYME.IN THE NEXT STEP, THE ENZYME WILL OPEN THE INSIDE AND CLEAVE THE G-STRAND, YOU COULD SEE THE DARK GREEN NOW BECOMES DISBELIEVED.WHAT YOU'RE DOING NOW IS ENABLING THE T SEGMENT TO GO THROUGH.IT GOES THROUGH THE ENZYME THEN THE ENZYME WILL RELY LI GAIT THIS SEGMENT HERE BUT NOW THE TRANSPORTED SEGMENT IS THE LOWER PART OF THE ENZYME HOMO DIMER AND IN THE END BY DIE DROEIZING ATP, YOU COULD FREE THE T SEGMENT AND THEN REGENERATE THE SEGMENT YOU HAVE GONE BACK TO WHERE YOU WERE.BUT THE DIFFERENCE IS YOU HAVE MADE THIS.YOU HAVE OPENED THAT, LET IT GO THROUGH THE ENZYME, LET IT GO ALL THE WAY, RELY GAIT FREE.YOU COULD ASSUME ATP AND YOU HAVE DONE IT.AMAZINGLY, THIS REACTION IS VERY FAST.MUCH FASTER THAN YOU WOULD THINK.YET IT DOES ONE THING AT A TIME.SO THE ENZYME WILL TAKE THIS AND I'LL JUST GO LIKE THIS AND BURN ATP EACH TIME.THAT'S WHAT TOPO TWO DOES.THIS REACTION VERY HAS THE ABILITY TO DECAT NATE CIRCLES.COULD GO THE OTHER WAY.COULD TAKE TWO CIRCLES, CAT NATE THEM.YOU COULD GO BACK AND FORTH DEPENDING ON THE ENERGIES AND HOW THE DN SASHGS BEING PUSHED.IT HAS ALSO THE ABILITY TO UNKNOT DNA.IF YOU HOSE HAS NOT WHAT TOPO WILL DO, IT JUST CHIEF, PATCH, GLUE UNTIL IT'S TOTALLY UNKNOTTED.IT WILL UNKNOT.IT CAN ALSO RELAX SUPERCURLING.IF YOU HAVE SUPERCURL DNA IT CAN RELAX VERY EFFICIENTLY BUT IT WILL DO THIS BY JUST GETTING THE REGION SUPER COIL, TWO STRANDS ONE DUPLEX AT A TIME BURNING ATP.NP THE CASE OF JIE RACE, WHICH IS THE BACTERIAL ENZYME, IT CAN EVEN GENERATE SUPERCORDING. WHEN THE [INDISCERNIBLE] SHE WORKED ON THE MECHANISM OF JIE RACE, IT WAS CALLED THAT BECAUSE IT GYRATES DN MAKING SUPERCOOL.THIS IS THE REACTION THAT GYRATE DOES, IT MAKES A DNA PASSES THROUGH AND GETS DNA WRAPPED AROUND THE ENZYME.ALL THESE INCREDIBLE REACTIONS ARE MADE BY THE TOPO TWO.SO THE TOPO TWO HAS LOT IN VERY, VERY CAPABLE ENZYME DOES THESE.HOW DO THE DRUGS WORK?SO THE ANTICANCER DRUGS AND THE ANTEBACTERIA DRUGS BLOCK THE RELY GAGS REACTION.SEVERAL STEPS YOU COULD SEE HERE.THE [INDISCERNIBLE] ANTIBIOTICS, SOME OF THE ANTICANCER AGENTS, OTHER ANTICANCER AGENTS HERE AND I WILL SHOW YOU A CRYSTAL STRUCTURE SHORTLY.SO WHAT ABOUT TOPO THREE?WELL, SORT OF THE LONER.IT DOESN'T HAVE ANY MEDICINAL APPLICATION AT THIS STAGE, YET THIS IS AN ESSENTIAL ENZYME AND WHY IS BECAUSE PROBABLY BECAUSE TOPO THREE AT LEAST TOPO THREE ALPHA IS LINKED TO REPLICATION.ANOTHER WAY DNA IS REPLICATED AFTER YOU [INDISCERNIBLE] HERE IS IF YOU ASSUME THAT THIS CIRCLE WHICH IS SUPERCOILED GETS REPLICATED FROM THE TOP HERE TO THIS REACTION, IF YOU TRY TO REP SATE IT, YOU'RE GOING BUILD THESE INTERTWINED SEGMENTS, BUT THIS IS SA A SINGLE STRAND THAT STAGE AND THIS CAN BE RESULT BY TOPO TWO OR IF TOPO ONE CAN GETS IN IT'LL RESULT IN AND IT WILL [INDISCERNIBLE].SO THE VIEW IS THAT THE TOPO THREE WHICH FORMS A [INDISCERNIBLE] DIMER WITH [INDISCERNIBLE] -- THESE ARE INVOLVED IN HUMAN DISEASE SUCH A BLUE HETICASE ACTUALLY DEACTIVATING NEWLY [INDISCERNIBLE] CIRCLES.BLUE SYNDROME PATIENTS HAVE A VERY MARKED PREDISPOSITION TO CANCER AND THERE ARE VERY SHORT PEOPLE.THEY HAVE PROBLEMS IN REPLICATING THEIR DNA.TOPO THREES ARE VERY IMPORTANT, YET THEY HAVE NOT BEEN TARGETED FOR MEDICINAL PURPOSE AT THIS STAGE. SO THAT'S AS MUCH AS I WILL SAY ABOUT THE TOPO THREE. SO THE TOPO TWO TARGETED DRUGS, WHAT ARE THEY? THEY ARE VERY LARGE FAMILY, AND FOR THOSE WHO WORK IN THE CLINIC AND TREATING CANCER PATIENTS, YOU WILL RECOGNIZE MANY DRUGS THAT YOU USE AND YOU KNOW TOPO TWO TARGETED BUT ARE USED EVERY DAY.THIS INCLUDES THE ATOPO SIDE DRUG WHICH IS USED IN LARGE TUMOR MOST SUCCESSFULLY TESTICULAR CANCER WHICH CURES IT WITH PLATINUM COMPOUNDS.THIS IS THE ATOP POE SIDE OF [INDISCERNIBLE].THERE IS A DERIVATIVE TO [INDISCERNIBLE] USED IN SOME LEUKEMIA.THEN YOU WILL RECOGNIZE, HERE, [INDISCERNIBLE] USED BREAST CANCER PATIENT USED TO TREAT SOME [INDISCERNIBLE] DISEASES.VERY LARGE NUMBER OF CANCERS ARE TREATED BY ATROE PSYCH LEANS.SO THAT WILL BE THE MORE, YOU KNOW, THE OLD [INDISCERNIBLE] SOME MORE RECENT DERIVATIVES.YOU CAN SEE [INDISCERNIBLE].MORE RECENT DRUGS THAT MAY BE COMING, AND THERE ARE SOME IN THE PIKE.IN FACT T PIPELINE OF TOPO TWO DRUGS IS STILL GOING.I WAS REVIEWING RECENTLY A PAPER ON THE -- I ALSO WAS UNDER THE IMPRESSION THAT TOPO TWO FIELD WHERE THE PIPELINE WAS GETTING DRY.IN FACT, IT'S NOT DRY.THERE ARE TWO OR THREE DRUGS IN CLINICAL TRIALS TARGETED TOPO TWO.YET WE HAVE A LOT OF TOWERS IN KINASE TARGETING DRUGS, BUT WE ALSO STILL HAVE TOPO TWO TARGETED DRUGS IN CLINICAL PIPELINE.ONE BIG CHUNK WHICH AGAIN I SAID I WOULD NOT SAY ANYTHING ABOUT ARE THE ANTI[INDISCERNIBLE] TARGETED DRUGS.YOU CAN SEE HERE [INDISCERNIBLE]ACID [INDISCERNIBLE], ALL THESE DRUGS THEY ARE VERY, VERY COMMON TO USE TO TREAT ANTIBACTERIA DISEASES ARE TARGETING THE BACTERIA TOPO TWO, BUT YET THE TWO TOPOS ARE SO DIFFERENT BETWEEN BACTERIA AND VERTEBRAE THAT THE BACTERIA DRUGS ARE SPECIFIC FOR THE BACTERIUM ENZYME AND DO NOT AFFECT THE [INDISCERNIBLE] HOST ENZYME.ON THE OTHER HAND, THE ANTICANCER TOPO TWO DO NOT TARGET HAVE MUCH OF THE BACTERIA ENZYME EITHER.SO WE HAVE A VERY GOOD DIVISION OF LABOR HERE. SO I'LL SPEND A LITTLE MORE TIME ON TOPO ONE, AND THEN THAT WILL LEAD ME TO SOME OF MECCA NIS TICK AND HOW THESE DRUGS WORK.SO TOPO ONE, SO I GO BACKWARDS IF YOU WISH TDNA-UNTWISTING ENZYME.MORE DETAILS OF HOW AND WHY IT WORKS.BECAUSE WE HAVE CRYSTAL STRUCTURE OF ALL THESE THINGS, WE HAVE EVEN SINGLE MOLECULE ANALYSIS TO LOOK AT THE RELAXATION.LET'S GO BACK.YOU TAKE A PIECE OF DNA, A VIRAL PIECE OF DNA AND IT IS NEGATIVELY SUPERCOIL AND ONE REASON IS NORMALLY THESE DNAs ARE WRAPPED AROUND PROTEINS IN [INDISCERNIBLE].IT WOULD BE THE HIS TOEN CORE.[INDISCERNIBLE] IF YOU PUT A DROP OF TOPO ONE TDNA WILL GO RIGHT AWAY HERE.SO AND THE MECHANISM [INDISCERNIBLE] AND THE DNA ROTATES INSIDE THE ENZYME AND THEN THE DNA GETS FULLY RELY GAITED.WHY THIS IS IMPORTANT? IS BECAUSE FOR TRANSCRIPTION AND REPLICATION TDNA BECOMES VERY QUICKLY SUPERCOILED IN THE VICINITY OF DNA REPLICATION AND TRANSCRIPTION MACHINELY RI. KNOCKING OUT TOPO ONE IS LOOET UNTIL MICE.IT IS LETHAL IN FLIES.IT'S ONLY IN YEAST WHERE THE KNOCKOUT IS VALUABLE.THERE ARE SOME ABNORMALITIES IN THE GENOME.THE GENOME IS [INDISCERNIBLE] AND STABLE IN THE [INDISCERNIBLE] YEAST.MOST NOTABLY IN RIBOSOMAL DNA --SO TOPO ONE IS VERY IMPORTANT TO MAINTAIN GENOMIC STABILITY IN JOOE YEAST AND ABSOLUTELY ESSENTIAL IS MORE COMPLEX ORGANISMS.SO I HAVE ALREADY DESCRIBE TO YOU AND TALKED ABOUT THE [INDISCERNIBLE].THESE WERE DISCOVERED BY THE NCI IN THE 60s.THE NCI HAD A VERY LARGE PROGRAM OF SCREENING OF NATURAL PRODUCTS THAT WOULD HAVE BENEFICIAL ACTIVITY AGAINST CANCER.THAT'S HOW THE DRUG WAS DISCOVERED OUT OF THE BARK OF THE CHINESE TREE [INDISCERNIBLE], WHICH LEAD TO THE NAME [INDISCERNIBLE].AND THIS IS STILL THE CASE, THE DRUG IS PRODUCED OUT OF NATURAL PRECURSOR OF [INDISCERNIBLE] THREE OR OTHER TREES FROM INDIA OR FROM CHINA.THESE THE TWO [INDISCERNIBLE] DERIVATIVES USED IN [INDISCERNIBLE] AND [INDISCERNIBLE].AND WHAT YOU HAVE TO REALIZE AND WHAT WAS REALIZED VERY EARLY ON WZ BEAUTIFUL AS THESE DRUGS ARE IS THE CHEMICAL STRUCTURE BEAR THESE ALPHA HYDROXY AK TONE HERE AND THIS IS VERY UNSTABLE. AT LEAST [INDISCERNIBLE] -- IN THE CLINIC, WHAT REALLY HAPPENS IS WHEN THESE DERIVATIVES OF [INDISCERNIBLE] ARE GIVEN, VERY QUICKLY IN THE BLOOD THE RING OPEN AND GIVE A MAJORITY OF THE DRUG IS INACTIVE [INDISCERNIBLE]WHICH IS NOT HIGHLY DESIRABLE. NOW, HOW DO THESE REALLY WORK AT THE MOLECULAR DETAIL?WE KNOW QUITE A GOOD DEAL.SO I WOULD POSE TO YOU THAT [INDISCERNIBLE] AND [INDISCERNIBLE] TOPO TWO, [INDISCERNIBLE] FOR TOPO ONE ARE TARGETED AGENTS.THEY'RE HIGHLY-TARGETED, BUT THE TARGET [INDISCERNIBLE].SO YOU COULD CALL THEM TOPO ONE OR TOPO TWO-TARGETED AGENTS.HOW DID WE GET TO THAT CONFUSION?AS I INDICATED TO YOU TOPO ONE CLEAVES AND RELY GAIT DNA.TOPO ONE SEND TO RELY GAIT DNA VERY FAST.MAKE SURE DNA GETS RELY GAITED BEFORE IT GOES AWAY.IN THE PRESENCE OF DRN TURN, WHAT WE REALIZE AND WE AND OTHERS IN THE MID 80s, IS THAT THE CLEAVAGE COMPLEX WAS SHIFTED TO WHERE A CHIEFED INTERMEDIATE.YET, IT WAS REVERSIBLE, AND WHEN WE LOOKED CAREFULLY AT THE BASE SEQUENCES THAT [INDISCERNIBLE] THE CLEAVAGE SIDE, WE FOUND THAT THE TOPO ONE CLEAVAGE SIDES THAT WERE TRAPPED BY [INDISCERNIBLE] PRETTY MUCH ALL THE TIME [INDISCERNIBLE] PLUS ONE POSITION AND THIGH MIEN OF MINUS ONE.WE PROPOSED THAT THE REASON THIS WAS IS THE DRUGS WERE BECAUSE THEY LOOK LIKE A BASE PAIR, YOU COULD SEE THE STRUCTURE, THEY LOOK LIKE A FUSED BASE PAIR.WE SAID THE DRUGS WERE [INDISCERNIBLE] IN THE CLIFF TOPO ONE COMPLEX AND THEREBY PREVENTING THE RELY GAGS.SO THEY WERE LIKE A REJ IN THIS OPEN GATE IN THE DNA.THEY WOULD FALL IN AND THEY WOULD SLOW DOWN THE RELY GAGS.IT TOOK ABOUT TEN YEARS TO GET THE [INDISCERNIBLE] STRUCTURE OF TOPO [INDISCERNIBLE] WITH TOPO ONE AND YOU COULD SEE HERE THAT IT'S EXACTLY THE WAY THESE DRUGS INTERFERE WITH THE TOPO ONE CLEAVAGE COMPLEX.SO THIS IS WITH A SURFACE REPRESENTATION WHERE YOU COULD SEE THE SURFACE OF TOPO ONE.THE DNA NIECE GREEN AND THE DRUG IS IN RED.THE DRUG IS FOUND A SINGLE SPOT TO WHICH IT BINDS IN THE CLEAVAGE COMPLEX AND ONCE YOU TAKE AWAY THE SURFACE OF TOPO ONE, THE DRUG, YOU CAN SEE T EXACTLY BOUND, LIKE HERE N THIS -- THIS IS THE BREAK AND THAT'S WHERE THE DRUG IS.THIS MODEL IS ACTUALLY ACCURATE.THE DRUGS BIND IN THIS CLEAVAGE SITE.IN THE EARLY 90s OR LATE 90s WHEN IT WAS DISCOVERED THAT TOPO ONE WAS THE TARGET OF [INDISCERNIBLE], WE SET OUT TO DISCOVER NON-[INDISCERNIBLE] TOPO ONE INHIBITORS.AND THE REASONS WERE VERY SIMPLICITY, BUT I THINK THIS DID HOLD TRUE IS THAT -- VERY COMMONLY VERY DIFFERENT BIOLOGY MEDICINAL ACTIVITY.FOR INSTANCE N THE [INDISCERNIBLE] WORLD, YOU ALL KNOW [INDISCERNIBLE] GOUT AND [INDISCERNIBLE] TO TREAT LEUKEMIA.THEY BOTH TARGET THE ALPHA BETA [INDISCERNIBLE] DIMER, YET IN THE CLINIC ONE IS USED FOR GOUT T OTHER FOR LEUKEMIA, NO COMMON -- BUT AT THE MOLECULAR LEVEL, THEY'RE THE SAME MECHANISM.WE THOUGHT WE SHOULD FIND NEW TOPO ONE INHIBITORS THAT WOULD WORK DIFFERENTLY.ALSO , I MENTION TO YOU THAT THE CAMPTOTHECIM HAVE LIMITATIONS, WE LEARN THEY'RE HIGHLY BONE TOXIC, SUB STRAIGHT FOR THE DRUG [INDISCERNIBLE] AND CHEMICALLY UNSTABLE.THIS CHEMICAL INSTABILITY TELLING YOU THAT ONLY THE ACTIVE FORM IS REAK TONE AND THAT THIS ONE BINDS [INDISCERNIBLE] SO IT'S NOT VERY DESIRABLE.THEREFORE, WE FELT WE SHOULD DISCOVER THE NON-CAMPTOTHECIM INHIBITORS, WHICH HAS HAPPENED.THESE ARE THESE DRUGS THAT WE DISCOVERED WITH MARK AT [INDISCERNIBLE].OUR FIRST REPORT WAS IN 1998.IT TOOK WAS BETWEEN 1998 WHEN WE FIRST DISCOVERED THE FIRST INHIBITOR UNTIL ABOUT ONE OR TWO YEARS AGO TO GET TO THE CLINIC AND NOW THESE DRUGS ARE IN CLINICAL TRIAL. AT THE SAME TIME, THIS VERY SIMILARLY LOOKING [INDISCERNIBLE] IS IN TRIAL AS I MENTIONED BEFORE AND IS THE [INDISCERNIBLE] DRUG NOW WHICH IS [INDISCERNIBLE]. THE CRYSTAL STRUCTURE OF THESE TWO INHIBITORS HAVE BEEN ACHIEVED AND YOU COULD SEE HERE THE [INDISCERNIBLE] BOUND INTO TOPO ONE CLOOERJ COMPLEX.DRUGS IN GREEN, DNA'S IN BLUE, BREAK IS VERY VISIBLE HERE IN THE DNA BACKBONE.THE [INDISCERNIBLE] TO WHICH THE TOPO ONE IS LINKED TO THE THREE PRIME MAN IS SHOWN IN RED AND THE TOPO ONE IS SHOWN AS RIBBON DIAGRAMMED IN YELLOW.THE DRUG IS EXACTLY IN THE CLEAVAGE SIDE.NOT ONLY WE GOT CRYSTAL STRUCTURE FOR THE [INDISCERNIBLE] BUT AS OF TODAY, WE HAVE CRYSTAL STRUCTURE FOR ALL THE TOPO ONE-TARGETED DRUGS.THEY WORK IN THE SAME WAY SO SIDE VIEW THESE.THEY'RE LIKE WOODS IN A BOOK SHELF THAT STACK BETWEEN THE BASE PARES.IF YOU ARTHRITIC 90 DECISION YOU COULD SEE THE PERFECT STACKING WHERE THE BASE PAIRS ARE ON TOP AND UNDER THE DRUGS.IT'S A PERFECT PIE/PIE INTERACTION.PERFECT STACKING.COCRYSTAL STRUCTURE FOR [INDISCERNIBLE].BUT IT'S A LOT MORE INTERESTING THAN THIS.THESE DRUGS NOT ONLY STACK WITH THE DNA, THEY FORM A NETWORK OF HYDROGEN BOND WITH THE TOPO ONE, AND THAT'S WHAT MAKE THEM SO SPECIFIC.IN FACT, IF YOU LOOK @ @ THE CAMPTOTHECIM FROM THE PROTEIN SIDE, THERE ARE THREE HYDROGEN BOND IN THE CLEAVAGE COMPLEX TO THREE AMY KNOW ASTOED TOPOISOMERASE ONE AND THEY'RE INDICATED IN YELLOW BECAUSE -- SO YOU NEED TO HAVE PIE/PIE INTERACTION AND YOU NEED THE THREE HYDROGEN BONE INTERACTION.IF YOU LOSE ANY OF THESE THEN THEY BECOME RESISTANT.IT'S TOTALLY [INDISCERNIBLE] THING THAT NATURE HAS DESIGNED TO FIT PERFECTLY THE TOPO ONE DLEFRJ COMPLEX.AND THAT LED TO THIS NOTION THAT TOPO ONES WERE REVEALING ONE WAY BY WHICH NATURE HAD DESIGNED OR SELECTED COMPOUNDS TO TARGET VERY SPECIFIC MICROMOLECULAR MACHINES.IN THIS CASE IT WAS THE TOPO ONE DNA COMPLEX.WE REFER TO THIS AS THE [INDISCERNIBLE] INHIBITION CONCEPT.THE TOPO ONE WERE THE FIRST THAT LED TO THE UNDERSTANDING OF THIS PARTICULAR CONCEPT.AND IN FACT, THIS MOLECULAR CONCEPT WAS VERY RECENTLY STRENGTHENED BY THE DISCOVERY THAT THE PLANTS THAT PRODUCE CAMPTOTHECIM -- SO ALL THESE PLANTS FROM WHICH WE EXTRACT CAMPTOTHECIM HAVE TOPO ONE AND WE KNOW THE TOPO ONE FROM THIS IS PLANT SENSITIVE.FOR YEARS IT WAS A DILEMMA OF HOW COULD A PLANT PRODUCE A TOXIN THAT WOULD NOT KILL THE PARENT?WE ASSUMED LONG AGO THIS WAS BECAUSE THE DRUG WAS PRODUCED IN THE BARK.IN FACT, THIS WAS REVISITED BY A GROUP IN JAPAN PUBLISHED IN [INDISCERNIBLE] ONLY THREE YEARS AGO WHERE THEY LED TO SEQUENCE THE GENOMES OF ALL THE PLANTS THAT PRODUCE CAMPTOTHECIM.WHAT THEY FOUND IS ALL THESE PLANTS HAD A POINT MUTATION IN THE TOPO ONE AT THIS REDUCED SEVEN TO TWO AND THE BEAUTY IS YEARS BEFORE WE HAD IDENTIFIED AND SELECTED A HUMAN CANCER CELL LINE THAT WAS HIGHLY RESISTANT TO CAMPTOTHECIM AFTER EXPOSURE TO THE DRUG FOR A LONG TIME THAT HAD EXACTLY THE SAME MU MUTATION.IT MEANS THE DRUG IS SO TARGETED SA THAT ONLY WAY THE PLANT CAN MAKE IT A DRUG IS TO HAVE A TOPO ONE MUTATION IN ITS GENOME. NOW, THIS CONCEPT JUST THIS YEAR WAS FOUND TO BE TRUE FOR THE TOPO TWO INHIBITORS.IT'S BEEN LONG IN COMING, IS TO GET COCRYSTAL STRUCTURE OF ATOP POE SIDE CANCER DRUG WITH TOPO TWO.IT JUST CAME A FEW MONTHS AGO WHERE THIS STRUCTURE CAME.YOU HAVE HERE THE HOMO DIMER OF TOPO TWO, THE DNA'S INSIDE, VERY VISIBLE.THIS IS A LARGE MACHINE, UH?YOU ROTATE THIS 90 DEGREES SO YOU NOW LOOK AT THE TOP OF THE MACHINE AND YOU YOU COULD SEE THE DNA GOING THROUGH THIS, THIS IS THE G SEGMENT AND THESE ARE THE TWO ATAP POE SIDE MOLECULE, THERE ARE TWO IN THE DNA TOPO TWO COMPLEX AND WHEN YOU LOOK MORE CAREFULLY AS WHERE THE DRUG IS POSITIONED, IT'S EXACTLY POSITIONED WITHIN THE CLEAVAGE SITES.SO FOR ATOP POE SIDE WITH ONE, WHAT NATURE SELECTED IS A COMPOUND THAT SITS EXACTLY INTO THIS CLEAVAGE INTERMEDIATES AND PROVIDES THE QUICK RELY GAGS WHICH THEN DRAGS THE TOPO CLEAVAGE COMPLEXES ON DNA AND THEREFORE IN THE CASE OF THE NATURAL COMPOUND MAKES A TOXIN, IN THE CASE OF CANCER, MAKES AN ANTICANCER DRUG, AND IN BACTERIA, AN ANTIBACTERIAL DRUG.THIS HAS BEEN ALSO CONFIRMED FROM THE BACTERIA ANTEBIONIC TICS [INDISCERNIBLE] FOR BACTERIA TOPO TWO, THE TOPO FOUR WHERE WE HAVE EXACTLY SIMILAR CRYSTALS OR WE AS A COMMUNITY HAVE BEEN RELEASE WRD WE KNOW FOR A COUPLE OF YEARS AGO THAT'S EXACTLY THE SAME PRINCIPLE.WE GET THE SAME [INDISCERNIBLE] AND THIS IS NOT LIMBED TO [INDISCERNIBLE] DRUG.IT'S ALSO TRUE FOR MANY, MANY NATURAL PRODUCTS.COULD BE [INDISCERNIBLE] INHIBITOR, COULD BE [INDISCERNIBLE], SAME PRINCIPLE.THESE THINGS ARE NOT COMPETITIVE INHIBITOR.THEY JAM JERN JERN MACHINES BY [INDISCERNIBLE].SO WHAT I MEAN TO SAY IS THAT THE [INDISCERNIBLE] INHIBITORS ARE LIMIT LIMITED TO CAMPTOTHECIM OR ANTICANCER AGENTS BUT TRUE FOR MANY, MANY NATURAL PRODUCTS BY WHICH THE AGENTS BIND TO A [INDISCERNIBLE]SPECIFIC SITE, THE INTERFACE OF MICROMOLECULAR COMPLEXES AS THEY UNDERGO A MACRO MOLECULAR CHANGE.[INDISCERNIBLE] WHEN THE DNA'S BROKEN IT OPENS A LITTLE CAVITY BY WHICH THE [INDISCERNIBLE] PULLED IN.THE MACHINE IS JUST TRAPPED AND THAT'S IT'S LIKE YOUR [INDISCERNIBLE].THAT'S ENOUGH TO HAVE TROUBLE IN SOME WAYS IF YOU'RE ON A HIGHWAY AND THERE'S A FAST-MOVING CAR BEHIND YOU, IF YOUR ENGINE SUS DOESN'T GO RIGHT, IT'S GOING TO THE LEAD TO A COLLISION. AND THIS IS TRUE FOR MANY NATURAL PRODUCTS AS I'VE SAID.TOPO ONE, TOPO TWO INHIBITOR.[INDISCERNIBLE].THIS PRINCIPLE IS VERY GENERAL. SO I THINK AT THIS POINT WHAT I'LL DO IS LET YOU GO PROBABLY THROUGH THE SLIDES AND I'D RATHER ENTERTAIN SOME QUESTIONS FOR THE REST OF THE LECTURE.THE REST OF THE LECTURE WAS MORE ON THE TOPO ONE INHIBITORS IN CLINICAL TRIAL.YOU COULD JUST LOOK AT IT YOURSELF.I'D WILL B HAPPY TO ANSWER ANY E-MAIL YOU HAVE.THESE DRUGS ARE IN CLIN TALL TRIAL AT THE NCI HERE, AND WE [INDISCERNIBLE] BIOMARKER FOR THEM AND THIS IS IN THE HANDOUT AND I'LL BE HAPPY TO DISCUSS WITH YOU WHAT IS GOING ON WITH THIS.SO I'LL STOP HERE FORKER NOW.[APPLAUSE] ER NOW.[APPLAUSE] R NOW.[APPLAUSE] NOW.[APPLAUSE] JE, PLEASE.>> I HAVE A QUICK QUESTION.>> YOU CAN REPEAT.>> I NOTICE THAT IN A DIFFERENT [INDISCERNIBLE] [LOW AUDIO]. >> VERY, VERY GOOD QUESTION, AND THE SORT OF QUESTION IS WHY, WHAT IS DIFFERENT TOPOISOMERASE [INDISCERNIBLE].WHY DO THEY HAVE SUCH A DIFFERENT SPECTRUM OF CLINICAL ACTIVITY?[INDISCERNIBLE].THE ANSWER IS, I DON'T KNOW THE ANSWER.AND THE ANSWER IS THAT PROBABLY WE SHOULD LOOK MORE CAREFULLY AT THE REASON WHY THIS IS BECAUSE I DO NOT -- BASED ON THE MOLECULAR INTERACTION OF THESE DRUGS, THERE'S NO ABUSE THING.SO JUST ANY DRUG IS SUBJECT TO A LOT MORER THAN REACHING THE TARGET.EITHER INACTIVATION, DRUG UPTAKE, DEPENDING ON WHAT THE DRUG IS MADE OF.WE SEE THEM SIMILAR, [INDISCERNIBLE] THESE DRUGS HAVE APPENDIXES THAT THE CELL WILL USE THEM DIFFERENTLY TO TAKE THEM IN OR BREAK THEM APART.SO THIS IS THE DETAIL OF THE PHARMACOLOGY OF ANY OF THESE AGENTS AND, BUT YET CLINICALLY WOULD BE VERY IMPORTANT.SEEM TO BE TRIGGERED QUESTION ON THE BASIC STANDPOINT BUT PRACTICALLY IT'S A VERY IMPORTANT QUESTION.FOR EXAMPLE, THE [INDISCERNIBLE]THE NEW TOPO ONE INHIBITOR IN TRIAL, I WISH I COULD TELL YOU WHICH TUMOR WE SHOULD GO AFTER.I REALLY DO NOT KNOW YET HOW WE COULD DIFFERENTIATE THESE AGENTS FROM THE CAMPTOTHECIM.THE ONLY ANSWER WOULD BE IN THE CLINIC.UNFORTUNATELY WHATEVER WE TAKE IS TRY DIFFERENT TUMORS, DIFFERENT PATIENTS TO TELL YOU.OKAY.SORRY, SIR, LONG ANSWER, BUT I THOUGHT I COULD GET THE ANSWER FROM -- LET'S SEE F YOU TAKE THE NCI IS 60 CELL LINES FOR INSTANCE AND THOSE CELL LINES ARE, I THINK TEN TISSUES OF ORIGIN.WE HAVE BREAST, MELANOMA, RENAL, LUNG, YET, IF I PUT [INDISCERNIBLE] AND [INDISCERNIBLE] THEY STILL LOOK ABOUT THE SAME.SO IN PATIENTS EVEN MORE COMPLICATED THAN IN CELL LINES.IT'S SO EASIER TO ANSWER YOUR QUESTION YET.SO I THINK THE ONLY WAY WOULD BE TO DO THE CLINICAL TRIAL, UNFORTUNATELY, TO FIND HOW YOU SHOULD GO ABOUT IT.REALLY DON'T KNOW.>> [LOW AUDIO]. >> GOOD QUESTION. [LAUGHTER]>> [LOW AUDIO]. >> SO I THINK YOUR QUESTION IS OBVIOUSLY WHY ARE TOPO ONE INHIBITORS, WHAT IS THEIR SIDE EFFECT WHICH IS OF USE BECAUSE I SAID THESE ENZYMES ARE ESSENTIAL.SO THE MAIN SIDE EFFECT OF THESE AGENTS IS [INDISCERNIBLE] CELLS.SO WHAT YOU GET IS BONE MARROW TOXICITY TO SOME EXTENT INTESTINAL TOXICITY.THESE ARE THE SHORT-TERM EFFECTS.NEXT ASSOCIATED WITH THIS QUESTION, A LONG-TERM E FESHFECT, MU TOE MUTAGENIC.THERE ARE TEN PERCENT OF PATIENTS WHO ARE CURED WILL GET SECOND TEAR LEUKEMIA.THEY ARE MUTAGENIC BECAUSE IF YOU JAM THE [INDISCERNIBLE] OBVIOUSLY YOU'RE GOING DAMAGE THE CELL BECAUSE THE CELL THAT SURVIVED WILL CORRECT WRONGLY.WHAT'S AMAZING IS YOU COULD ASK, WHY WOULD THESE DRUG WORK DIFFERENTLY IN A CANCER CELL VERSUS NORMAL CELL.TO THAT WE HAVE A BETTER ANSWER THAN FOR THE PREVIOUS QUESTION.THERE ARE SOME VERY -- DOWN SFRAEM THE INITIAL LESION.THE INITIAL LESION THAT THESE DRUGS WILL INFLICT TO THE GENOME IS MAKE THE DNA STRAND BREAK AND MAKE A PROTEIN DNA [INDISCERNIBLE].SO IF YOU HAVE A CELL LINE WHICH IS -- AND WE KNOW THIS BRACKET EFFICIENT, BRINS -- IT WILL BE A LOT MORE SENSITIVE TO THE TOPO INHIBITORS THAN ANOTHER ONE.IF YOU HAVE A CELL WHICH HAS A [INDISCERNIBLE] DEFICIENCY, IT'S VERY SENSITIVE TO THESE AGENTS.THE REASON THESE DRUGS HAVE ANY USE, I THINK, IN THE CLINIC IS NOT SO MUCH THAT THE TARGET LEVEL IS DIFFERENT TO [INDISCERNIBLE] MAYBE LITTLE HIGHER IN TU HOR CELLS BUT THE MAIN REASON IS THE DUN STREAM PATHWAYS ARE INACTIVATED IN CANCER CELLS SO THESE THE DNA PATHWAYS OR CHECK POINT PATHWAYS AND THIS HAS PRACTICAL APPLICATION BECAUSE ONE WAY WE SHOULD CONSIDER USING THESE AGENTS IS LOOK AT THE GENETIC MAKEUP OF THE TUMOR.IF THE TUMOR IS BRACKET EFFICIENT, THEN YOU SHOULD GO WITH THESE AGENTS.IF THERE IS NO OF USE DEFICIENCY IN DNA PATHWAY OR CHECK POINT PATHWAY, IT'S UNLIKELY TO BE ACTIVE TO THE TUMOR THAN THE NORMAL TISSUES.YOUR QUESTION IS VERY, VERY PRACTICAL IMPLICATIONS, AND EVEN IN THE NCI 60 CELL LINE OR OTHER CELL LINE, WE COULD SEE THE DIFFERENCE IF THE CELL IS DEFICIENT YOU COULD -- SO THIS IS MOSTLY DOWNSTREAM AND THAT'S WHERE IT'S VERY IMPORTANT TO ASK THOSE QUESTIONS.YEAH. >> [LOW AUDIO]. >> THAT'S VERY GOOD QUESTION.SO MECHANISM RESISTANT WITH THIS AGENT IN THE LABORATORY, IF YOU PUSH THE CELL LINES TO BECOME RESISTANT, YOU WILL GHETTO POE MUTATIONS, NO DOUBT.IN THE CLINIC, NO. WHAT THE CELL WILL DO FIRST IS PROBABLY ACTIVATE THE DRUG [INDISCERNIBLE], AND THE OTHER THING IT WILL DO, THEY'LL JUST ACTIVATE SOME ALTERNATIVE PATHWAYS SUCH AS DNA REPAIR PATHWAYS OR SOME THINGS OF THAT KIND TO OVERCOME THE DAMAGE, BUT THEY WILL ONLY MUTATE THE GENE IF YOU REALLY PUSH AND WE NEVER REACH HIGH ENOUGH CONCENTRATION.THE CELLS HAVE MANY, MANY DIFFERENT WAYS TO OVERCOME THE CLINICAL RESISTANCE WITHOUT HAVING TO MUTATION TOPOISOMERASES.IN BACTERIA, YOU DO GET THAT.THEY WILL MUTATE THE REFLUX MACHINERY, BUT TYPICALLY THEY WILL MUTATE [INDISCERNIBLE].UH BUT IN CANCER, VERY RARELY. >> [LOW AUDIO]. >> CORRECT.>> [LOW AUDIO]. >> NVT AT ALL.>> THERE IS NO [INDISCERNIBLE] ACTIVITY FOR AGENTS THAT ACROSS CLASSES.THE TOPO ONE INHIBITORS DO NOT TARGET TOPO TWO OR THREE.THE TOPO TWO INHIBITORS DO NOT TARGET TOPO ONE OR THREE.AND THE ANTEBACTERIAL TOPO THREE DO NOT TARGET TOPO TWO.[INDISCERNIBLE].THEY DO NOT TARGET THE BACTERIA TOPO TWO.EACH OF THESE ENZYME FAMILY HAS A VERY SPECIFIC CLASS OF INHIBITORS. >> [LOW AUDIO]. >> GOOD YES. GOOD QUESTION.SO WE ARE ABLE TODAY TO ASK THOSE QUESTIONS BECAUSE WE HAVE ACCESS TO THE GENETIC MAKEUP OF TUMOR WHICH IS WE DIDN'T HAVE BEFORE, AND I WOULD NOT BE SURPRISED THAT IN SOME TUMORS THE LEVEL OF TOPO THREE MIGHT BE EXCESSIVELY HIGH OR NOT OR THAT ENZYME MAY BE MUTATE ORD A TARGET.BUT AT THIS STAGE, THIS IS A GOOD QUESTION TO WHICH WE COULD HAVE AN ANSWER. BECAUSE YOU JUST HAVE TO START LOOKING, BUT NOBODY FOR GOD'S SAKE IS LOOKING FOR TOPO THREE MUTATION IN CANCER, NOBODY. >> [LOW AUDIO]. >> WELL, I WOULD ARGUE THAT IF A CANCER CELL LINE OR CANCER IS HIGHLY [INDISCERNIBLE] IT WILL HAVE TO COPE IN HAVING A LOT OF TOPO THREE COMING ALONG AND IT MAY BECOME A LOT MORE DEPENDENT ON TOPO THREE THAN ANYTHING ELSE THAT WE KNOW OF, AND I'M JUST ANSWERING FROM WHAT I KNOW BUT THERE'S PROBABLE LAY LOT MORE IN THE BIOLOGY OF TOPO THREE THAT MAY BE MORE RELEVANT ESPECIALLY IF YOU THINK THAT TOPO THREE DOESN'T WORK ALONE THAT IT WORK WITH BLOOM AND IT WORKS WITH BURNER AND THOSE ARE CLEARLY [INDISCERNIBLE] IN CANCER. SO, I MEAN THE ANSWER IS NOT IN TO ME..THIS IS MORE OF A GOOD QUESTION THAN ANYTHING.SO FOR THE SAKE OF TIME WE'LL STOP.THANKS FOR THE INTERACTIONS. [APPLAUSE] >> OUR NEXT SPEAKER IS WYNDHAM WILSON.HE GOT HIS MEDICAL DEGREE AT STANFORD UNIVERSITY IN 1984, AND THEN HE DID A RESIDENCY IN MEDICAL ONCOLOGY AND IN 1995, HE JOINED THE MEDICINE BRANCH AS A SENIOR ONCOLOGIST, HE'S NOW A PRINCIPLE INVESTIGATOR AND GOING TO BE DISCUSSING LYMPHOMAS.WYNDHAM.>> SO I THINK IT WAS MAYBE FORTUITOUS I WALKED IN AND HEARD ABOUT TOPO TWO BECAUSE THAT'S PROBABLY ONE OF THE MOST IMPORTANT TARGETS THAT WE ACTUALLY NOW USE AGAINST LARGE CELL LYMPHOMA WHICH IS THE MOST COMMON TYPE.HOWEVER THE KIND OF WORK WE'VE BEEN DOING IS REALLY TRIED TO MOVE BEYOND STANDARD TARGETS LIKE THAT AND SO AFTER I GO OVER A LITTLE BIT OF A REVIEW OF WHAT LYMPHOMAS ARE AND SOME OF THEIR BIOLOGY, WE WILL START DELVING IN TO SOME OF THE DRIVER PATHWAYS THAT HAVE BEEN IDENTIFIED AND WAYS THAT WE'RE TRYING TO TARGET THOSE. SO, UM, FOR THOSE OF YOU WHO DON'T STUDY THIS FIELD, UM, I HAVE LISTED HERE THE MOST COMMON TYPES OF TUMORS, AND YOU CAN SEE IN MEN AND WOMEN IT'S OBVIOUSLY THE TWO HORMONE-DRIVEN TUMORS; PROSTATE IN MEN AND BREAST IN WOMEN.AND YOU CAN SEE THAT LYMPHOMA IS AROUND THE FOURTH TO FIFTH MOST COMMON TYPE.LOTS OF PEOPLE THINK THAT LYMPHOMA IS A SINGLE TUMOR TYPE.OBVIOUSLY, WE NOW KNOW EVEN WHEN YOU'RE LOOKING AT SOLID TUMORS LIKE PROSTATE CANCER THAT THERE ARE MANY DIFFERENT TYPES, BUT THERE'S EVEN MORE SO WHEN REGARDS TO LYMPHOMAS BECAUSE THEY ARE DERIVED FROM MANY DIFFERENT CELLS.AND SO THERE ARE SOME ARE IN UPWARDS OF 60-70 TYPES THAT WE NOW HAVE, AND MANY OF THEM DERIVE FROM LYMPHOCYTES THAT ARE AT DIFFERENT STAGES OF THEIR DIFFERENTIATION CYCLE. SO HOW OFTEN DO WE SEE THIS?IT'S NOW AROUND 50-60 THOUSAND CASES PER YEAR IN THE U.S., AND THAT WORKS OUT TO AROUND 14 PER HUNDRED THOUSAND.THIS IS A DISEASE THAT OCCURS AT ALL DIFFERENT AGE GROUPS, AND THERE ARE PEAKS IN CHILDREN AND THERE ARE PEAKS IN OLDER FOLKS TOO, BUT IF YOU JUST LOOK AT THE SHEER NUMBERS, YOU CAN SEE THAT IT IS A DISEASE THAT REALLY INCREASES IN FREQUENCY IN OLDER FOLKS GOING UP TO AS HIGH AS 68.HOWEVER, IT IS ONE OF THE MOST COMMON TYPES OF CANCERS THAT OCCUR IN CHILDREN AS WELL. AND THE OTHER THING THAT'S BEEN NOT WELL UNDERSTOOD IS THAT THE INCIDENCE OF THIS HAS BEEN GOING UP.NOW, MORE RECENTLY, IT'S BEEN FLATTENING OFF, HOWEVER, YOU CAN SEE HERE IN THIS DATABASE STARTING IN 1973 AND GOING OUT TO, WHAT IS THAT?'95, HOWEVER YOU'LL SEE THIS TREND ALSO GOING UP INTO THIS CENTURY AS WELL THAT THE INCIDENCE HAS BEEN GOING UP.MORTALITY HAS BEEN KEEPING PACE AS WELL.UP UNTIL AROUND 2000 BECAUSE WE REALLY DIDN'T HAVE THERAPIES THAT WERE THAT MUCH MORE EFFECTIVE IN THE 1990s THAN WE HAD IN THE 1970s. HOWEVER, OVER THE LAST TEN YEARS, WE HAVE HAD AN EXPLOSION OF THERAPIES.THE LEAD ONE BEING A MONO KONL ANTIBODY DIRECTED AGAINST CD 20.HOWEVER, WE'RE SEEING A WHOLE HOST OF OTHER AGENTS AS WELL AND SO WE'RE BEGINNING TO SEE THAT AMONG THE VARIOUS TYPES, PEOPLE ARE LIVING LONGER AND WE'RE INCREASING CURE RATES AS WELL. NOW, THIS IS A DISEASE THAT DOESN'T HAVE A SINGLE CAUSE, AND NO ONE'S REALLY UNDERSTOOD WHY THE INCIDENCE HAS BEEN GOING UP WITH AGE.I THINK A LOT OF US HAVE FELT THAT IT PROBABLY WAS GOING UP AS A RESULT OF ENVIRONMENTAL FACTORS.SOME OF THESE HAVE BEEN DRIVEN BY AIDS.HIV IS A RISK FACTOR FORKER THIS, HOWEVER, WHEN YOU CONSIDER THE NUMBER OF LYMPHOMAS AND CONSIDER THE NUMBER OF FOLKS WITH HIV, IT REALLY DOES NOT COUNT FOR THAT.IT'S PROBABLY IN LARGE MEASURE, MAYBE, RELATED TO CHEMICAL EXPOSURES.WE KNOW THAT PESTICIDES IS PROBABLY THE MOST FAMOUS ONE, AGENT, AGENT ORIGIN, HOWEVER, WE KNOW THAT ORGANIC COMPOUNDS ALSO INCREASE RISK OF THIS.HOWEVER, THERE ARE OTHER SETTINGS TOO, AND THESE SETTINGS WOULD BE UNDER THE RUBRIC OF ACQUIRED OR EVEN INHERITED IMMUNITY ISSUES.SOME OF THESE MAY BE DUE TO INCREASED IMMUNE STIMULATION SUCH AS YOU WILL GET IN A AUTO IMMUNE SETTING SUCH AS RHEUMATOID ART ROOITSZ, LUPUS, LUPUS [INDISCERNIBLE] WHERE YOU ACTUALLY HAVE A AUTO IMMUNE PROCESS THAT'S DRIVING P CELLS.WHEN YOU DRIVE A CELL TO REPLICATE, IT'S GOING TO INCREASE ITS OPPORTUNITY TO UNDERGO DNA CRITICAL DNA BREAKS, WHICH MAY LEAD TO EMERGENCE OF A LYMPHOMA.YOU CAN ALSO HAVE SETTINGS WHERE YOU CAN HAVE IMMUNODEFICIENCY, AND WE KNOW THAT IN IMMUNE SURVEILLANCE PLAYS A ROLE IN CLEARING OUT DAMAGED OR MUTATED PREMALIGNANT CELLS, AND SO PATIENTS THAT HAVE THE INABILITY TO HAVE NORMAL IMMUNE FUNCTION, IRRESPECTIVE OF WHETHER OR NOT THAT'S ASSOCIATED WITH DRIVING B CELLS ALSO HAS A INCREASED RISK. AND THEN THERE'S A WHOLE HOST OF VIRUSES, AND I THINK THE ONE THAT PROBABLY YOU ALL KNOW ABOUT MOST IS HIV, AND THIS IS REALLY KIND OF LIKE A DOUBLE HIT.HIV IS ACTUALLY ENDS UP, TURNING ON P CELLS IN AND OF ITSELF BECAUSE OF THE IMMUNE STIMULATION GOING ON, AND SO IT'S BOTH DRIVING P CELLS BUT IT'S ALSO DEPLETING THE PEACE ALARM, SO IT'S ALSO AFFECTING IMMUNE SURVEILLANCE AS WELL.AND THEN ON TOP OF THAT, AS THE T CELLS FALL, YOU BEGIN TO GET REACTIVATION OF VIRUSES THAT ARE ASSOCIATED WITH TRANSFORMATION SUCH AS EBV.IN FACT, WE'RE ALL VIRTUALLY CARRYING EBV BY THE TIME WE'RE IN OUR 20s AND THIS IS A VIRUS THAT RESIDES IN A DORMANT WAY WITHIN OUR B CELLS IN A VERY SMALL NUMBER OF THEM, AND OUR IMMUNE SYSTEM IS ABLE TO REGULATE THIS.HOWEVER, THERE ARE A HOST OF SETTINGS IN WHICH EBV IS NOT PROPERLY REGULAR LATED.ONE OF THEM, OF COURSE, WE'VE ALSO DISCUSSED IN IS HIV.ANOTHER ONE IS IN THE TRANSPLANTATION SETTING WHERE YOU'RE DOING SEVERE IMMUNE SUPPRESSION BE THAT BONE MARROW TRANSPLANT OR SOLID ORGAN, IT'S NOT UNCOMMON TO DEVELOP WHAT'S CALL A EBV DISORDER ALSO KNOWN AS POST TRANSPLANT [INDISCERNIBLE].THESE LARGE-CELL DRIVEN LYMPHOMAS DRIVE B CELLS TO BECOME LARGE.THEY REPLICATE AT A RELATIVELY HIGH RATE AND THEY CAN LOOET UNTIL A VERY RAPID WAY.THE FIRST VIRUS EVER TO BE IDENTIFY TO BE ASSOCIATED WITH CAUSING LYMPHOMA IS ACTUALLY HTLV-1, WHICH MANY OF YOU MAY KNOW GAL LOW ISOLATED HER IS ACTUALLY A COUSIN OF HIV, AND GAL LOW'S GROUP WAS THE ONE THAT SHOWED THAT IT CAUSED A RARE TYPE OF T CELL LYMPHOMA CALLED ADULT T CELL LEUKEMIA LYMPHOMA.ATL OR ATLL IS CAUSED BY THIS VIRUS.WE NOW KNOW OTHER RARE LYMPHOMAS SOMETIMES IN THE HIV SETTING, OTHER TIMES NOT SUCH AS PRIMARY FUSION LYMPHOMA IS VIRTUALLY ALWAYS ASSOCIATED WITH THE AIDS VIRUS.THERE'S BEEN SOME EVIDENCE, ALTHOUGH THIS HASN'T BORN OUT IN ANY KIND OF LARGE [INDISCERNIBLE] THAT HHVA MAY IN SOME CASES BE ASSOCIATED WITH MULTIPLE MYELOMA.HEPATITIS C HAS A DIFFERENT MECHANISM.RATHER THAN DIRECTLY INFECTING THE CELL AS DOES HHV-8, HTLV-1 OR EBV; HEPATITIS C ACTUALLY LEADS TO A IMMUNE SUPPRESSION AND A AUTO IMMUNE STATE WHERE IT CAN INDUCE THE EMERGENCE OF A LYMPHO PLASMA IS I DICK IMFOE MA.IF YOU REGULATE THE HEPATITIS C, YOU CAN ACTUALLY FIND THESE TUMORS REGRESSING.A SIMILAR ALBEIT PATHWAY HAS ALSO BEEN IDENTIFIED FOR A BACTERIA CALLED HELICAL FACTOR WHICH WHEN IT GETS ESTABLISHED IN THE TO STO MAC WHERE IT LIVES, IT CAN STIMULATE T CELLS THAT ARE DIRECTED AGAINST IT CAN ACTUALLY DRIVE LYMPHOMA, DRIVE T CELLS TO FORM WHAT LOOKS LIKE A MALT OR A [INDISCERNIBLE] ASSOCIATED LYMPHOMA.THERE ARE OTHER DISEASES SUCH AS MEDITERRANEAN LYMPHOMA THAT ARE ASSOCIATED WITH AN UNKNOWN PATHOGEN. SO I THINK ONE OF THE KEYS IN TRYING TO UNDERSTAND AND TRY TO PUT SOME CONSTRUCT AROUND THESE MULTIPLE DISEASES WITH MULTIPLE IDEA YOKES HAS BEEN THE CLASSIFICATION SYSTEMS.THESE CLASSIFICATION SYSTEMS HAVE NEEDED TO EVOLVE ALONG WITH OUR UNDERSTANDING ABOUT THE PATHOBIOLOGY OF THESE DISEASES, AND THIS JUST GOES THROUGH A NUMBER OF THOSE.YOU CAN SEE THAT IT REALLY BEGAN STARTING IN EARNEST BACK IN 1960 AND WE'RE AT 2008 WHERE WE'RE DEALING WITH THE SECOND EDITION OF THE WORLD HEALTH ORGANIZATION SYSTEM. BACK HERE IN 1960, THIS WAS ALL BASED ON WHAT THE CELLS LOOKED LIKE UNDER THE SCOPE.WHERE THEY LARGE, SMALL, GROWING IN LYMPH NOIND FOLLICLES?.DIDN'T REALLY KNOW, DIDN'T KNOW ANYTHING ABOUT B CELLS, T CELLS, MUCH LESS THE TYPES WITHIN THOSE, AND THIS ONE HERE IS STILL DRIVEN MORE OR LESS BY WHAT IT LOOKS LIKE UNDER THE SCOPE; HOWEVER, IT IS ALL SUBDIVIDED BY WHETHER OR NOT SIT A B CELL, WHETHER IT IS T CELL, AND NOW WE KNOW SPECIFIC [INDISCERNIBLE] EVENTS THAT ARE ASSOCIATED WITH CERTAIN TYPES, AND SO NOW WITHIN THE P CELL TYPE, THEY'RE NOW DIVIDED BY WEREN'T THEY HAVE CERTAIN OK GENIC EVENTS OR NOT. SO, UM, JUST KIND OF A VERY SIMPLICITY PRIMER ON PATHOLOGY.LYMPHOMA AS THE NAME IMPLIES IS A CANCER OF LYMPH FOE SITES.THEY ARE CLASSIFIED ACCORDING TO WHETHER OR NOT THEY ARE DERIVED FROM A T CELL OR A P CELL.MOST LYMPHOMAS, 85% ARE DRIVED BY FROM A P CELL, AND THE REMAINING ARE DRIVED FROM A T CELL AND THEN A SMALL NUMBER THAT ARE DERIVED FROM MACROPHAGES.THEY'RE STILL NOT LYMPHOMAS PER SE BUT ENCOMPASSES BEEN TWN LYMPHOMA CLASSIFICATION SYSTEMS OR NATURAL KILLER CELLS.WE DO TO SOME EXTENT SOME OF THE OLDER NAMES CAME BY WHERE IN THE LYMPH NODE ARCHITECTURE THEY WERE LOCATED.IF THE LYMPHO WA MAS WERE WITHIN THE MANTLE ZONE, THEY BECAME KNOWN AS MANTLE CELL LYMPHOMAS.WE PAY ATTENTION TO THE CRIOLOGY.ARE THEY BIG CELLS, SMALL CELLS?DO THEY HAVE A PROMINENT NUCLEI OR NOT?ARE THEY BLUE, NOT, ETC. ?MANY OF YOU HAVE HEARD THE TERM HODGKIN'S VERSUS NON-HODGKIN'S LYMPHOMAS.THE MAJORITY ARE NON-HODGKIN'S.WHY DID WE HAVE THIS NAME?WELL, IT DPRAM THE 19th CENTURY FROM THOMAS HODGKIN'S WHO ACTUALLY WASN'T THE FIRST PERSON TO IDENTIFY THE DISEASE KNOWN AS HODGKIN'S WITH ITS CHARACTERISTIC CELL, WHICH WAS LATER CALLED A REED STERMBRICK CELL, HOWEVER, ONE OF THE CHARACTERISTIC OF THESE CELLS WERE THAT THEY WERE BIG, THEY WERE HUGLY, AND AS WE BEGAN TO BE ABLE TO GET SOME NOTION ABOUT WHETHER OR NOT A TUMOR WAS A B CELL OR A T CELL FOR YEARS -- AND I'M TALKING UNTIL THE EARLY 1990s -- NO ONE EVEN NEW WHAT THIS HODGKIN'S CELL WAS.THEY DIDN'T KNOW IF IT WAS A T OR B CELL.I EVEN WENT TO A CONFERENCE AT STANFORD IN CELEBRATION OF THE RETIREMENT OF A GUY NAMED SAUL ROSENBURG WHO'S KIND OF ONE OF THE GIANTS IN THE FIELD OF TREATING THESE FROM THE 60s AND THE 70s AND 80s, AND HE THOUGHT THIS WAS SOME KIND OF A FUSION CELL.LONG STORY SHORT, WHAT WE NOW KNOW IS A HODGKIN'S IS NOTHING MORE THAN A B LYMPHOCYTE THAT IS DOWN REGULATED IT'S B CELL PROGRAM, AND THE B CELL PROGRAM HAS ALL THOSE CELL SERVICE ANTIGENS THAT WE USE TO IDENTIFY IT HAS A P CELL.SO IT DOESN'T EXPRESS THEM.WE COULDN'T FIND THEM FOR YEARS, AND SO NOBODY KNEW THEY WERE HODGKIN'S.SO, WE'RE NOW BEGINNING TO FINALLY -- THIS USED TO BE CALLED HODGKIN'S DISEASE.WE FINALLY GOT THEM TO DROP THE NAME DISEASE AND ADD LYMPHOMA ON IT.SO IT'S NOW KNOWN AS HODGKIN'S LYMPHOMA, AND I'M HOPING IN THE NEXT ITERATION OF DWHO, WE DROP THIS NON-HODGKIN'S NONSENSE BECAUSE IT IS SIMPLY A SUBTYPE OF A B CELL LYMPHOMA.AS I MENTIONED, WE'RE NOW UNDERSTANDING A LOT MORE ABOUT THE MOLECULAR BIOLOGY OF THESE, AND REALLY FOCUSING DOWN ON NUMBERS OF DRIVER MUTATIONS, AND THEN THESE CLASS FIE KAGSS ALSO TO SOME EXTENT TAKE IN CLINICAL FEATURES, BUT AS WE GET MORE, AS WE UNDERSTAND MORE ABOUT SUM OF ORIGIN AND BIOLOGY T CLINICAL FEATURES ARE NECESSARILY FALLING BY THE WAYSIDE. SO THIS JUST GIVES YOU A SCHEMATIC OF HERE WE HAVE THE HODGKIN'S MAKING UP ABOUT 17%, AND THE NON-HODGKIN'S MAKING UP 83%.I ALREADY MENTIONED 85% OF ALL LYMPHOMAS ARE DERIVED FROM B CELLS.15 A% FROM T CELLS. AND SO THIS JUST GIVES YOU A PIE CHART OF SOME OF THE MORE COMMON TYPES OF NON-HODGKIN'S LYMPHOMA.THE MOST COMMON TYPE MAKING A THIRD IS ONE I STUDY ALONG WITH LOU STOUT AND THIS IS SO CALLED DEFUSE LARGE B CELL LYMPHOMA.SECOND MOST COMMON IS FOLLICULA LYMPHOMA ALSO KNOWN AS LOW-GRADE LYMPHOMA BY LAY FOLKS AND THAT'S A QUARTER.THEEN THERE ARE MORE RARE TYPES, MARGINAL ZONE HERE, PERIPHERAL T CELL HERE, MANTLE CELL HERE.IT'S A VERY INTERESTING DISEASE, THIS MANTLE CELL AND THEN OTHERS HERE.THIS LIST IS VERY, VERY LONG AND THIS IS JUST KIND OF SOME OF THE MORE COMMON TYPES. SO THIS IS A SCHEMATIC OF THE OLD LYMPH NODE AND JUST KIND OF GIVING YOU SOME OF THE AREAS; THE PRIMARY FOLLICLE IS WHERE THE GERMAL CENTER REACTION GOES ON AND FOR THOSE WHO HAVE STUDY IMMUNOLOGY, YOU'LL KNOW THIS IS WHERE THE ANTIGEN SELECTION GOES ON WRAND THESE B CELLS HAVE TURNED UP BCL 6 AND THAT IN TURN HAS DIALLED DOWN P 53, DIALLED UP PROLIFERATION.SO THESE P CELLS ARE ALL EXCITED AND THEY'RE ALL GOING THROUGH RANDOM SCHEMATIC HYPER MUTATION, AND THEY HAVE A LOW AND THEIR THRESHOLD FOR GOING THROUGH SPONTANEOUS A POP TOE SIS EVEN THOUGH THEY ARE INDUCING A GEE KNOW TOXIC STRESS TO THEMSELVESES IS DIALLED DOWN BECAUSE IF THE P 53 WAS ALLOWED TO BE ON, THESE CELLS WOULD JUST BE RANDOMLY GOING INTO APOPTOSIS.SO THERE'S THIS REAL CAREFUL BALANCE BETWEEN ALLOWING THESE RANDOM SCHEMATIC MUTATIONS TO GO ON AND ALLOWING THAT CELL TO PROLIFERATE.NOW, AFTER A WHILE, IF THAT CELL DOESN'T GET A LOCK AND KEY SIGNAL, IT WILL, IN FACT, UNDERGO APOPTOSIS.AND SO WHAT HAPPENS IS, THERE'S THE ANTIGEN THAT ARE KIND OF FLOWING THROUGH THIS THING YOU KNOW BECAUSE THIS THING IS DRAINING OUR BLOOD AND INTO OUR BLOOD ALL THESE NASTY THINGS COME.BELIEVE IT OR NOT, YOUR GUT IS NOTHING MORE THAN A SIEVE CONSTANTLY THINGS FLOWING THROUGH IT INTO YOUR BLOODSTREAM.OUR BODY HAS A GREAT ABILITY TO FILTER THESE BACTERIA OUT AND GET THEM AT A VERY EARLY STAGE, BUT IT'S BECAUSE ALL THE IMLYMPH FLUID.-- IN THE ANTIGEN FITS ON TO THE RECEPTOR, THEN THAT SENDS A SIGNAL TO BEGIN THE IMMORTALLIZATION PROCESS OF THAT B CELL.THAT B BSHG CELL THEN WILL BEGIN ITS PROCESS TRANSITIONING OUT OF THE GERMAL CENTER AND IT WILL GO INTO WHAT'S CALLED A POST GERMAL CENTER STATE WHERE IT WILL BECOME MORE OF A PLASMA IS I TICK-LIKE CELL.A SO CALLED PLASMA BLAST AND THEN EVENTUALLY, IT WILL BECOME A PLASMA CELL THAT WILL THEN BE FEELING OUT ANTIBODIES THAT FIT THIS ANTIGEN THAT HAVE THROWN THROUGH THIS LYMPH NODE AND HAVE SAVED IT FROM ITS CERTAIN FATE OF DYING BECAUSE THE RIGHT KEY CAME ALONG TO STIMULATE ITS B CELL RECEPTOR. AND THIS PROCESS AND THE POST GERMAL CENTER PROCESS IS REALLY A KEY PROCESS TO REALLY -- GERMINAL -- TO THE BEGINNING STAGES OF LARGE CELL LIMB MOW FOE MA, LYMPHOMA.A LOT OF STUFF GOING ON HERE THAT IS PLAYING A CRITICAL ROLE IN THE POTENTIAL PATHOBIOLOGY OF THESE TUMORS. HERE, WE HAVE, IN FACT, YOU KNOW, THESE B CELLS THAT ARE FORMING AND IT'S, AGAIN, I'VE BEEN TALKING ABOUT NAIVE B CELLS AND THAIN THEY GO INTO THIS GERMAL CENTER REACTION WHERE THEY'RE UNDERGOING THIS RANDOM SCHEMATIC HYPER MUTATION AND ANTIGEN SELECTION AND ISOTYPE SWITCH IF THEY'RE, IN FACT, IMMORTALIZED, THEY WILL THEN LEAVE.SO JUST SO YOU KNOW THAT WE NOW KNOW FOR THE FIRST TIME -- AND THIS REALLY HAS ONLY COME ABOUT IN THE LAST TEN, 15 YEARS -- WE KNOW THE CELL OF ORIGIN.WHERE IN THIS PLASMA DIFFERENTIATION SCHEME MA DO THESE VARIOUS LYMPHOMAs COME FROM.FLICK LAR LYMPHOMAS ARE ALL DERIVED FROM A CELL THAT'S FROZEN OR GOT ITS HIT WITHIN THE GERMAL CENTER.WE KNOW BERKET LYMPHOMA COMES FROM A CELL WITHIN THE GERMAL CENTER.WE KNOW LARGE CELL, THOELG, WHICH WE USED TO THINK ABOUT AS A SINGLE DISEASE IS NOW MANY DIFFERENT TYPES, AND WE KNOW THAT ONE TYPE COMES FROM THE GERMAL CENTER, ANOTHER TYPE COMES FROM THE POST GERMAL CENTER, ANOTHER YET COMES FROM A B CELL THAT SPECIALIZED B CELL THAT LIVES IN THE THYMUS AND HAS AND DOESN'T AND HAS FEATURES THAT ARE UNIQUE TO IT AND ARE NOT OF GERMAL CENTER OR SO-CALLED POST GERMAL CENTER ORIGIN.SO, UNDERSTANDING WHERE THESE CELLS COME FROM AND UNDERSTANDING THE NORMAL BIOLOGY OF THEIR COUNTER PARTS GIVES US TREMENDOUS INSIGHTS IN TO UNDERSTANDING WHAT'S CAUSING THESE IDENTIFYING MOST CRITICALLY WHAT THE DRIVER PATHWAYS ARE AND THEN TARGETING THEM. BY THE WAY, HODGKIN'S IS UNCLEAR WHERE IT COMES FROM.IT HAS FEATURES OF THE GERMAL CENTER, BUT THERE ARE OTHER CELLS, IN FACT, THERE ARE OTHER TYPES OF HODGE KINS THAT COME PROBABLY FROM THE SAME BSHG CELL IN THE THYMUS AS DOES ONE OF THE SUBTYPES OF LARGE CELL. AND SO, UM, AT DIFFERENT STAGES IN THE B CELL DEVELOPMENT, THEY EXPRESS DIFFERENT ANTIGENS, AND THIS IS KEY BECAUSE IT IS THE EXPRESSION OF THESE ANTIGENS THAT WE USE IN PATHOLOGY TO TRY TO HELP US UNDERSTAND WHEN WE'RE LOOKING AT A TISSUE FOR THE FIRST TIME WHETHER OR NOT IT IS A B-CELL DERIVED TUMOR, BUT IT ALSO PROVIDES US AN OPPORTUNITY TO TARGET THINGS AS WELL.AND IN FACT, FIRST MONO CHROME L ANTIBODY EVER APPROVE BID THE FDA ANYTHING WAS MITUX EN.NOBODY KNOW WHAT IS THIS CD 20 ANTIGEN DOES.WE KNOW THE ANTIGEN CAN SIGNAL.WE KNOW THAT INHIBITING IT OR CROSS LIKING IT WILL INDUCE APOPTOSIS, BUT EVEN TO THIS DAY, TEN YEARS AFTER THE APPROVAL OF RITUX, MAP, [INDISCERNIBLE] ANTIBODY AGAINST CD 20SHGS WE STILL DON'T HAVE A GOOD UNDERSTANDING OF WHAT THIS ANTIGEN DOES.WHAT WE DO HAVE A GOOD UNDERSTANDING OF IS THAT RITUXMAP HAS MADE A TREMENDOUS IMPACT ON THE OVER ALL THERAPY OF LYMPHOMAS AND INCREASED THE CURE RATES OF LARGE CELL BY AROUND 10-15%, AND IT'S CERTAINLY INCREASED THE SURVIVAL OF COMMON LYMPHOMAS LIKE [INDISCERNIBLE] LYMPHOMA BY PROBABLY YEARS.THERE'S A LOT OF FOCUS NOW ON TRYING TO TARGET CD 19.YOU MAY HAVE SEEN THERE WAS THIS BIG SPLASH SHI SPREAD IN THE NEWSPAPERS TWO OR THREE WEEKS AGO FROM A [INDISCERNIBLE] ANTIRECEPTOR MADE BY CARL JUNE THAT INVOLVES CD 19, AND THEY ACTUALLY REPORTED THAT IN THAT BY USING THIS CAR THAT THEY WERE ABLE TO INDUCE COMPLETE REMISSIONS IN ONE PATIENT I THINK THEY HAD CLL AND IT WAS IN THE NEW YORK TIMES, NEW ENGLAND JOURNAL, ETC.SOME FOLKS ARE USING THIS TO MAKE A [INDISCERNIBLE] ANTIBODY AND A T CELL WITH A T CELL RECEPTOR ON IT.OTHERS HAVE ATTACHED RADIO ACTIVITY TO IT.OTHERS HAVE ATTACHED CYTOTOXIC CONJUGATES TO IT. I'M NOT GOING TO SPEND A LOT OF T CELLS, BUT THIS IS A SIMILAR LIST OF ANTIGENS AT DIFFERENT POINTS IN THE LIFE CYCLE OF A T CELL.OF COURSE, ALL OF YOU KNOW ABOUT CD 4 AND CD 8 CELLS, AND THERE IS A POINT AT WHICH ALL THESE T CELLS ARE SO-CALLED DOUBLE POSITIVE AND THEN THEY END UP LOSING ONE AND THEY BECOME EITHER A CD 4 OR CD 837 MOST PURPLE T CELL LYMPHOMA THAT HAS COMPRISE THE VAST MAJORITY OF LYMPHOMAS BELONG TO EITHER THE DRSHGS CD 4 OR CD 8 CLASS WITH MOST COMBLONG BLONGING TO THE CD 4 CLASS.THIS JUST GIVES YOU LONG LAUNDRY LIST OF A LOT OF DIFFERENT ANTIGENS THAT ARE EXPRESS BID DIFFERENT TYPES OF LYMPHOMAs AND THESE CAN BE USED BY PATHOLOGIST BY PATHOLOGIST WHO IS HELP THEM IDENTIFY WHAT TYPE OF LYMPHOMA THEY ARE DEALING WITH.HOWEVER, THEY, LIKE MOST TOOLS, HAVE TO BE DONE IN CONJUNCTION WITH OTHER THINGS AS WELL LOOKING AT SOME OF THE KNOWN AND IMPORTANT CRY TO CYTODEGENETICS THAT'S ASSOCIATED WITH DIFFERENT LYMPHOMA LYMPHOMAS.MANY OF HEARD ABOUT THE 1418 TRANSLOCATION.THIS INVOLVED IN BCL TWO [INDISCERNIBLE] GENE AND THIS IS FOUND IN ABOUT 90% OF PATIENTS THAT HAVE SO CALLED FOLECULAR LYMPHOMA.[INDISCERNIBLE] LEUKEMIA OR LYMPHOMA COUSIN CALLED SMALL LYMPHO IS I TICK LYMPHOMA WILL HAVE A DELETION OF 12.THEY CAN ALSO HAVE ABNORMALITIES IN 11 AND 13 AS WELL.LARGE CELL, THERE HAVEN'T BEEN ANY SPECIFIC KNOWN MUTATIONS THAT SEEM TO DRIVE LARGE CELL OVERALL UNTIL RECENTLY MY COLLEAGUE DR. LIEU STOUT IDENTIFIED A NUMBER OF THEM IN THE ABC TYPE THAT YOU DO NOT SEE LISTED HERE, MANTLE CELL IS ASSOCIATED WITH THE CYCLE IN D ONE.NOW, WHAT DO WE DO CLINICALLY WHENEVER WE SEE SOMEBODY FOR THE FIRST TIME?MOST OF YOU, I BELIEVE, ARE Ph.D.s, ETC, BUT WE DO A PHYSICAL EXAM.WE DO BLOOD WORK, A CT SCAN TO SEE WHERE THE DISEASE IS.A LOT OF THESE DISEASES BEING DISEASES OF LYMPHOCYTES LIKE TO HANG OUT IN THE MARROWS, SO WE EXEC THE BONE MARROW AND WE ALSO DO A NUMBER OF NUCLEAR MEDICINE STUDIES AS WELL.NOW, IN ORDER TO DETERMINE SOME ASPECTS OF TREATMENT, WE DO SOMETHING CALLED STAGING.THIS TELLS US WHETHER OR NOT THE DISEASE IS LOCALIZED, WHETHER IT IS REGIONALLY SPREAD, OR WHETHER IT'S WIDE-SPREAD.THIS WAS CRITICAL BACK IN THE 19 # 0s AND 70S -- 1960s AND 70s, WHEN RADIATION WAS REALLY A KEY PART OF THE THERAPY AND IN FACT WAS CURATIVE AND RADIATION ONLY WORKS WHEN YOU RADIATE THE DISEASED AREAS.SO ALL OF THESE SO-CALLED STAGINGS WERE REALLY BASED ON THE IDEA OF KNOWING WHERE THE DISEASE IS BECAUSE YOU WERE USING LOCAL OR LOCAL REGIONAL THERAPY.NOW THAT WE'RE USING CHEMOTHERAPY, REALLY, IT'S MUCH LESS CRITICAL EXPECT TO SAY THAT WHEN YOU HAVE A LITTLE BIT OF --IF YOU HAVE A LYMPHOMA AND A LITTLE BIT OF DISEASE VERSUS A LOT AND YOU HAVE A TYPE OF LYMPHOMA THAT IS POSSIBLY CURATIVE, IF THAT LYMPHOMA BELONGS TO A MOLECULARLY SENSITIVE GROUP, IT PROBABLY DOESN'T MAT FER YOU HAVE A LITTLE BIT OR A LOT BECAUSE IT'S SO SENSITIVE THAT YOUR THERAPY CURES IT ALL.HOWEVER, IF YOU HAVE A LYMPHOMA THAT IS POTENTIALLY CURATIVE BUT HAS A HIGHER LEVEL OF DRUG RESISTANCE SUCH THAT YOU CAN CURE SOME OF THEM BUT NOT ALL OF THEM, WE KNOW FROM STUDIES THAT HAVING A LITTLE BIT IS ASSOCIATED WITH HIGHER CHANCE OF BEING CUREDED THAN IF YOU HAVE A LOT, AND THE REASON FOR THIS COMES FROM THIS NOTION THAT'S BEEN KNOWN FOR VERY LONG TIME AND MAY BE RELEVANT TO WHAT YOU HEARD EARLIER TONIGHT, WHICH IS THIS IDEA THAT CHEMOTHERAPY KILLS A SET FRACTION OF CELLS, AND NOBODY KNOWS WHY THAT IS THE CASE, BUT IT'S EITHER BECAUSE THERE IS NESTED WITHIN THOSE CELLS THOSE THAT HAVE UNDERGONE ADDITIONAL MUTATIONS THAT HAVE MADE THEM RESISTANT TO CHEMOTHERAPY.SO IF YOU HAVE A LITTLE BIT OF TUMOR, THE CHANCES THAT YOU'RE GOING HAVE RESISTANT TUMOR THERE IS GOING TO BE SMALLER THAN IF YOU HAVE A LOT.NOW OTHER PEOPLE HAVE HYPOTHESIZED THAT THERE MAY BE PRESSURE DUE TO THE CHEMOTHERAPY THAT MAY IN AND OF ITSELF INDUCE MUTATIONS AS YOU GO ALONG.I THINK THAT'S LESS LIKELY.I THINK THAT WHEN YOU'RE DOING UP FRONT THERAPY AND PEOPLE THAT ARE POSSIBLY CURATIVE, IT IS VERY UNLIKELY THAT YOU'RE INDUCING MUTATIONS THAT ARE RELEVANT TO CURE AS YOU'RE TREATING THEM WITHIN THIS RELATIVELY SHORT PERIOD.DO YOU INDUCE MUTATIONS AGAINST TOPO TWO WHICH IS A KEY TARGET FOR TWO OF THE DRUGS WE USE IN LYMPHOMA.THE ANSWER IS, YOU ALMOST CERTAINLY DON'T INDUCE MEANINGFUL AMOUNTS OF TOPO TWO RESISTANCE, AND THAT'S NOT THE REASON WHY SOME PATIENTS MAY NOT BE CURED WITH THESE TYPES OF TUMORS.BUT, WE STILL HAVE TO CONSIDER THE AMOUNT OF TUMOR THERE BECAUSE IF YOU HAVE A LITTLE BIT OF TUMOR AND A SENSITIVE ONE, YOU CAN GET BY WITH GIVING A LOT LESS THERAPY THAN IF YOU HAVE A LOT TUMOR AND SOMETHING THAT'S SENSITIVE.AND IF YOU HAVE A LOT OF TUMOR AND SOMETHING THAT'S NOT SENSITIVE, USUALLY EVEN A LOT OF THERAPY DOESN'T WORK AND MAYBE YOU NEED TO GO TO MORE DRACONIAN THERAPIES SUCH AS ALOE GENETIC TRANSPLANT WHICH IS BRINGING TO BEAR A VERY DIFFERENT KIND OF THERAPY WITH WHICH IS IMMUNOTHERAPY OR SO CALLED CHI TOE TOXIC THERAPY BRING BG BROUGHT IN BY THE DONOR OF [INDISCERNIBLE].THIS GIVES YOU A LITTLE SNAPSHOT OF HOW LONG DO PEOPLE LIVE WITH THIS.THIS ISN'T FOCUSING ON WHETHER OR NOT YOU CAN CURE THEM, BUT YOU CAN SEE HERE IN THESE SO-CALLED INDOLENT DISEASES, THE VAST MAJORITY WHICH CANNOT BE CURED WITH CURRENT THERAPIES, YOU CAN SEE PEOPLE LIVE AT FIVE YEARS, A PRETTY LONG TIME.IN FACT, THIS IS REALLY MOSTLY IN THE PRERYE TUXIN' ERA.IF WE LOOK AT THE MEDIAN SURVIVAL TOR POINT AT WHICH 50% OF PEOPLE DIE WITH THESE TUMORS, THESE GROUPS, YOU KNOW, YOU'RE FINDING THAT, YOU KNOW, AT AROUND PROBABLY 12 YEARS, 12, 13 YEARS YOU'VE ONLY LOST HALF OF THE FOLKS WITH THIS, AND SO YOU'LL STILL HAVE PEOPLE LIVING.YOU'LL HAVE HALF YOUR FOLKS WITH THESE DISEASES EVEN THOUGH THEY'RE NOT CURATIVE LIVING WITH THEM, I'M NOT SAYING WITHOUT THERAPY BUT USING THERAPY WHEN NEEDED, LIVING WITH THEM BEYOND 12 YEARS.AGGRESSIVE, THOUGH, IS A VERY DIFFERENT STORY. WITH AGGRESSIVE LYMPHOMAS, IN GENERAL IF YOU DON'T CURE THEM, YOU DIE PRETTY QUICKLY, AND SO DEFUSE LARGE CELL, WHICH IS AGAIN THE MOST COMMON TYPE, WE USED TO ONLY BE ABLE TO CURE ABOUT A THIRD OF THESE, AND NOW WITH SOME OF THE NEWER THERAPIES USINGS AND EVEN LOOKING FURTHER INTO THE FUTURE USING SOME OF THE TARGETED WORK I'LL BE DISCUSSING HERE, WE'RE PUSHING THE CURE RATES OF THESE UPWARD TOSS 80-90%.MANTLE CELL LYMPHOMA, WE ARE CURING ALMOST NOBODY HERE.THIS IS KIND OF LIKE THE WORST OF THE WORST.THIS IS RELATIVELY RAPIDLY GROWING TUMOR.IT'S GOT A MEDIAN SURVIVAL WITH CONTEMPORARY THERAPY OF AROUND SIX YEARS, BUT WE DON'T CURE PEOPLE WITH THIS, GENERALLY.BUT WE'VE HAD A VERY INTERESTING RESULT AND I'M NOT GOING DISCUSS IT TODAY BUT I MIGHT MENTION ABOUT TEN YEARS AGO A COLLEAGUE OF MINE, GUY NAMED LARRY CLARK AND I DID A ID YOE TYPE VACCINE SCENE STUDY IN MANTLE CELL AND IT APPEARED TO BE NEGTIVE, BUT WE GENERATE A LOT OF INTERESTING DATA AND WE'VE NOW JUST UPDATED THAT DATA.NOW WE'RE OUT AT 11 YEARS AND LOW AND BEHOLD, WE HAVE FOUND THAT THE PATIENTS THAT DEVELOPED A ROBUST IMMUNE RESPONSE, A CD 4 CELL THAT PRODUCES A GNCSF RESPONSE THAT THOSE PATIENTS WERE LIVING SIGNIFICANTLY LONGER IE, OF THOSE THAT HAD THAT AT 11 YEARS 90% OF THOSE ARE LIVING WHERE AS THOSE WHO DIDN'T, ONLY ABOUT 30% ARE.SO IT MAY BE THAT VACCINE MAY BE USEFUL FOR THIS BUT SOMETIMES IT TAKES YEARS AND YEARS TO ACTUALLY FIND THAT OUT. SO THIS JUST GOES THROUGH A LIST OF THE ONES THAT ARE CURATIVE.I DON'T THINK YOU CARE ABOUT THAT.INCURRABLE.OKAY.THERAPY, HISTORICALLY, HAS BEEN FOCUSING ON GEE KNOW TOXIC STRESS, AND GEE KNOW TOXIC STRESS.FOR YEARS WE THOUGHT THE REASON WHY PEOPLE FAILED THERAPY WAS BECAUSE THEY DEVELOPED RESISTANCE AGAINST THE CYTOTOXIC AGENT.THE TALK EARLIER TODAY, HIS FOCUS WAS ON TOPO TWO AND EVERYONE IS LOOKING CAN TOPO TWO BECOME MUTATED?AND THAT'S WHY PEOPLE ARE RESISTANT.BACK IN 1990, WHEN I STARTED WORKING AFTER MY FELLOWSHIP, THE HOTTEST THING WAS MDR.YVES MENTIONED THAT.THE MULTI-DRUG RESISTANT PUMP THAT THE DRUG GETS INTO YOUR CELL AND THAT LITTLE PUMP JUST GETTING ALL REVVED UP AND KICKS IT OUT.IF YOU DON'T HAVE THE DRUG IN YOUR CELL, YOU'RE NOT GOING TO DO THE DAMAGE.WE SHOWED YEARS AGO, CLINICALLY THAT, IN FACT, LYMPHOMA CELLS ACTUALLY UP REGULATE, THEY ACTUALLY TURN THIS THING ON, AND IT GETS REALLY GOOD ABOUT KICKING THE CHEMOTHERAPY OUT.WE WERE VERY EXCITED ABOUT THIS.THIS IS HISTORY.THE BAD NEWS WAS THAT WHEN WE SHUT THE PUMP DOWN, NOTHING HAPPENED, CLINICALLY.WE SAW SOME INCREASE RESPONSES, BUT DID WE MAKE PEOPLE LIVE LONGER?DID WE CONVERT SOMEBODY FROM INCURRABLE TO A CURABLE PERSON?ABSOLUTELY NOT.WE DID NOT.AND, YOU KNOW, I THINK AT THAT TIME, IT REALLY WAS BECOMING VERY OBVIOUS.IT WAS PROBABLY THE DOWN-STREAM STUFF THAT REALLY MATTERED.AS THE GEE KNOW TOXIC STRESS IS THE FIRST STEP TO THEN INDUCING THAT CELL TO GET UPSET AND THEN TO BEGIN TO ACTIVATE ITS DEFENSE MECHANISMS WHICH IS TO ACTIVATE P 53 AND THE APOPTOSIS PATHWAYS.THE REASON WHY WE CAN CURE LYMPHOMAS OR TREAT THEM IS BECAUSE THEY ARE VERY, THEY ARE POISED TO ACTIVATE APOPTOSIS, WHEREAS A LOT OF SOLID TUMORS ARE NOT.YOU CAN SEE WHEN WE GO BACK TO THIS ORIGINAL THINGS WE WERE TALKING ABOUT, THE ENTIRE REGULATION OF APOPTOSIS THE DOWN REGULATION OF P 53 T DIALING DOWN, DIALING UP, THE USE OF THAT CELL, THE USE OF APOPTOSIS BY THAT CELL IS CRITICAL TO ITS ANTIGEN SELECTION PROCESS.SO THAT CELL IS VERY FINELY TUNED TO THIS AND ITS PART AND PARCEL OF THE BASIC BIOLOGY OF THESE CELLS, AND THERE ARE JUST A LOT MORE SENSITIVE TO GEE KNOW TOXIC STRESS AND THEY COMMIT SUICIDE WITH VERY LITTLE RELATIVE JE KNOW TOXIC STRESS.IT'S PROBABLY MODULATION OF THOSE PATHWAYS WHICH IS THE REAL REASON WHY WE CAN CURE SOME PEOPLE AND SOME NOT.I'LL SHOW YOU LATER ON HOW THAT WORKS. THIS IS JUST SOME OF THE ROADBLOCKS TO APOPTOSIS. SO JUST A LITTLE PRIMER ON WHAT DO THESE THINGS LOOK LIKE.THIS IS THE MOST COMMON TYPE OF LYMPHOMA.WHY DOES IT CALLED FOLECULA LYMPHOMA? BECAUSE IT FORMED IN FOLLICLES. THESE ARE THE GERMAL CENTERS IN WHICH THE ABNORMAL TUMOR CELLS HAVE JUST EXPANDED AND TAKEN OVER THE ENTIRE GERMAL CENTER.THE REASON FOR THAT IS THAT THESE CELLS CAME FROM THE GERMAL CENTER SO THEY STILL LIKE BEING IN THE GERMAL CENTER, AND SO WHAT THEY DO IS THEY SIMPLY INFILL STRAIT ALL THE GERMAL CENTERS AND TAKE OVER THE NORMAL CELLS AN YOU CAN SEE THEY KIND OF LOOK LIKE THESE SMALL ANGLATED CELLS. THIS IS JUST SHOWING YOU A SURVIVAL CURVE WITH IND LIN LIMB KNOW FOE MAS AND UP UNTIL 1996, THERE WAS NO PROCESS.SINCE RETUX INS COME ALONG WE'RE SEEING THESE CURVES GO LIKE THAT.RETUXIN' WE'VE ALREADY DISCUSSED SO WE'LL MOVE FORWARD.CD 20 IS GREAT BECAUSE IT'S ON A MATURE B CELL.THIS IS LOOKING AT THE RELATIVE CD 20 EXPRESSION OF DIFFERENT KINDS OF TUMORS AND IT TURNS OUT SUCH A POTENT TARGET THAT EVEN A CLL CELL THAT HAS RELATIVELY LITTLE COMPARED TO [INDISCERNIBLE] LYMPHOMA OR HARRY'S CELL STILL IS VERY SENSITIVE TO IT.IT DOESN'T TAKE LOT OF THE TARGET ON THE CELL IN ORDER FOR RITUXIN TO BE USEFUL.SO WE USE IT FOR VIRTUALLY ALMOST EVERY SINGLE CD 20 EXPRESSING LYMPHOMA OUT THERE. AND IT ACTUALLY WORKS WELL WHEN YOU GIVE IT BY ITSELF.RESPONSE RATES ARE ANYWHERE FROM 40-60%. LARGE CELL LYMPHOMA IS CALLED LARGE CELL BECAUSE THESE CELLS ARE LARGE. AND CHEMOTHERAPY CALLED R-CHOP IS THE STANDARD NOW SINCE R CAME ALONG THE MEDIAN SURVIVAL IS ABOUT 60% AFTER FIVE YEARS.SO THE REAL QUESTION IS -- AND THIS IS HISTORICAL DATA LOOKING AT IT BEFORE R-CHOP.YOU COULD SEE FIVE YEAR SURVIVAL WAS AROUND 35%.WHY DO PEOPLE DO SO VARIABLY WITH THIS?THIS IS WHAT WE'VE BEEN FOCUSING ON, AND SO WE REALLY BEEN FOCUSING ON TRYING TO UNDERSTAND THE MOLECULAR UNDERPINNINGS OF LARGE CELL LYMPHOMA. AND SO, ONE OF THE THINGS THAT LOU UH FOUND AND WE WERE WORKING WITH HIM IN THE LMP AT PP WHICH IS THE LYMPHOMA LEUKEMIA PROFILING PROJECT, AND ACTUALLY ONE OF THE VERY FIRST GENE EXPRESSION AND PROFILES EVER DONE IN TUMORS WAS, IN FACT, DONE IN LYMPHOMAS AND THIS WAS USING A HOME-GROWN CHIP THAT WAS BUILT BY LOU STOUT HERE WHERE HE TOOK A WHOLE HOST OF CDNAs THAT CODED FOR GENES THAT WERE ASSOCIATED WITH NORMAL LYMPHOCYTES, ACTIVATED LYMPHOCYTES, TUMOR LYMPHOCYTES, ETC, AN HE BUILT THIS ARRAY AND HE THEN THROUGH LARGE CELL TUMORS ON IT AND HE THREW NORMAL CELLS ON IT TOO.LOOKED AT RESTING B CELLS, LOOKED AT ACTIVATED BSHG CELLS, LOOKED AT NORMAL SDWRERMAL CENTER B CELLS AND WHAT HE FOUND WHICH WAS REALLY VERY STARTLING WAS THAT YOU COULD DIVIDE LARGE CELL LYMPHOMAS INTO TWO BASIC GROUPS.I THINK ALL OF YOU NOW KNOW ON THESE EXPRESSION ARRAYS THAT RED MEANS THE RNAs ARE EXPRESSED AT HIGHER FREQUENCY RELATIVE CONTROL AND GREEN AT LOWER, AND WHAT YOU CAN SEE IS, YOU CAN SEE THIS GROUP HAS A VERY SIMILAR EXPRESSION ARRAY TO THE NORMAL GERMAL CENTERS, AND ACTUALLY, TO A LOT MORE WORK IT TURNS OUT THAT THESE ARE IN FACT LARGE CELLS THAT ARE DERIVED FROM GERMAL CENTER-DERIVED B CELLS, AND THIS GROUP LOOKS VERY MUCH LIKE THIS ACTIVATED B CELL GROUP AND THEY WERE ACTIVATED BECAUSE THEY WERE TAKEN FROM THE BLOOD AND SO THEY WERE POST GERMAL CENTER OR THEY HAD ALREADY LEFT THE GERMAL CENTER.THEY WERE IMMORTALIZED AND THESE ACTUALLY COME FROM A B CELL THAT HAS ACTUALLY LEFT THE GERMAL CENTER BECOME IMMORTALIZED, BUT FROZEN AT A STATE OF DIFFERENT REN YAIGS SO THEY CAN'T GO ON TO BECOME PLASMA CELLS.HENCE THE NAME GCB AND ABC HAS BECOME PART OF THE NOMENCLATURE OF HOW WE BEGIN TO THINK ABOUT SOME OF THE SUBSETS OF LARGE CELL LYMPHOMA.IF WE LOOK AT SOME OF THE AND LOOK AT THIS MORE CAREFULLY, THE GERMAL CENTER, THEY HAVE THE BCL SIX TRANSCRIPTION FACTOR TURNED ON.THIS IS A NORMAL TRANSCRIPTIONAL FACTOR FOR GERMAL CELLS AND THIS IS KEY BECAUSE IT DIALS DOWN THE APOP TO TICK PATHWAYS, P 53, AND DIALLED UP PROLIFERATION, AND THAT'S NORMAL, AND SO THESE ARE -- THAT'S THE GERMAL CENTER WHEREAS THE ABC GROUP SHUTS DOWN AS THEY BECOME, AS THEY LEAVE THE GERMAL CENTER, THEY BEGIN TO, AND BECOME MORE PLASMA PLASTIC TYPE CELLS.THEY BEGIN TO SHUT DOWN BCL SIX AND TURN ON BLIMP ONE AND MOST IMPORTANTLY IRF 4.IT'S THE IRF 4 GENE WHICH REALLY LED DOWN THE PATH OF UNDERSTANDING THAT IN THE ACTIVATED BSHG CELL TYPE IT'S THE END OF KAPPA B PATHWAYS THAT SEEM TO BE DRIVING PATHWAYS IN THE ABC GROUP. AND THEN IT TURNS OUT THAT THERE WAS YET A THIRD TYPE OF LARGE CELL CALLED THE PRIMARY MEDIA STIERL LARGE CELL LYMPHOMA.ANOTHER DISEASE WE STUDY A LOT, AND THIS ACTUALLY IS DERIVED FROM A B CELL FROM THE THYMUS.THIS IS A DISEASE THAT SHOWS UP IN THE MEDIA STY NUMB BEING THE CHEST AREA HERE, IN YOUNG PEOPLE, LARGE MASSES AND THIS IS A VERY CLOSE COUSIN OF THE MOST COMMON TYPE OF HODGKIN'S LYMPHOMA CALLED [INDISCERNIBLE] HODGKIN'S LYMPHOMA.THREE QUARTERS OF HODGKIN'S LYMPHOMAS ARE KNOWLEDGE DISGROEZ GROESING HODGKIN'S LYMPHOMA DRIVED FROM A B CELL AND THIS AND THAT DISEASE ARE CLOSE COUSINS AND THERE IS A MISING LINK BETWEEN THEM WHICH WE CALL GRAY ZONE LYMPHOMAS WHICH HAS FEATURES OF BOTH HODGKIN'S AND MOOI PRIMARY [INDISCERNIBLE].WE NOW HAVE TWO DISEASES LARGE CELL AND HODGKIN'S LYMPHOMA AND WE NOW KNOW THEY REALLY ARE DISEASES THAT ARE VERY CLOSELY RELATED AND PROBABLY BELONG ALONG A CONTINUUM AND MAYBE THEY SHOULD BE RENAMED THIGH MIK AGGRESSIVE B CELL LYMPHOMAS AND WE SHOULD EVEN MOVE AWAY FROM SOME OF THESE OTHER TERM AND BEGIN TO RECOGNIZE PROBABLY WHAT DRIVES A LOT OF THE BIOLOGY HERE IS THE CELL OF ORIGIN AND NOT THESE OLD FASHIONED NAMES. AND SO A NUMBER OF ARK GENIC EVENTS HAVE NOW BEEN IDENTIFIED WITH THESE NEW SUBTYPES THAT HAVE BEEN MOLECULARLY IDENTIFIED.THE GERMAL CENTER, TYPE LARGE B CELL LYMPHOMA AS IN AROUND A THIRD OF THEM TRANSLOCATIONS OF BCL 2.IT IS ONLY FOUND IN THIS TYPE, NEVER FOUND IN ANY OTHER TYPE OF LARGE CELL. THEY ALSO HAVE AMPLIFICATION OF CREL, NEVER FOUND IN ANY OF THE OTHER SUBTYPES.AS A GROUP, THEY HAVE A HIGHER CURE RATE THAN THE ACTIVATED B TYPE WHICH IS THE POST GERMAL CENTER B CELL.THIS GROUP NOW HAS ACTIVATED OF -- NF KAPPA B.WE NOW UNDERSTAND WHY THIS HAPPENS.THEY HAVE A POOR SURVIVAL.PRIMARY [INDISCERNIBLE] AND THEY NOW HAVE AMPLIFICATION OF 9 P 24 INVOLVING SOME OF THE JACK TARGETS. NOW, THE OTHER THING THAT WE HAVEN'T TALKED ABOUT IS -- AND THIS IS SOMETHING ELSE YOU HAVE TO UNDERSTAND JUST FROM A GENERAL CANCER POINT OF VIEW IS THAT NONE OF THESE CELLS KIND OF LIVE THERE BY THEM THEMSELVES.THEY ALSO LIVE IN CONJUNCTION WITH MANY OTHER CELLS.STROE MA HAS BEEN SOMETHING THAT'S HARD TO GET HANDLE ON, BUT STROE MA IS ALSO A VERY IMPORTANT DRIVING FACTOR.IT'S PROBABLY THE TUMOR CELL IN MANY CASES THAT IS BRINGING ABOUT THE STROMAL REACTION.WE OFTEN THINK THAT WELL MAYBE THAT STROMAL REACTION IS THE BODY'S TRYING TO ATTACK THE TUMOR, BUT THE ALTERNATIVE EXPLANATION, IN FACT THEY MAY BOTH BE CORRECT BUT THE ALTERNATIVE EXPLANATION WOULD BE THAT MAYBE THE TUMOR IS KIND OF BRINGING THE KIND OF ENVIRONMENT IT LIKES AND IT ACTUALLY MAY BE A POSITIVE THING.WE DON'T REALLY KNOW.WE DO KNOW THAT CERTAIN STROMAL REACTIONS HAVE AN ADVERSE PROGNOSIS, BUT AGAIN, THAT DOESN'T ANSWER THE CART OR HORSE TYPE ISSUE. AND SO WORK HAS BEEN DONE IN TRYING TO UNDERSTAND WHETHER OR NOT THE STROEL STROMAL OR THE NON-TUMOR CELLS AS REFLECTED IN A MICROARRAY GENE EXPRESSION ARRAY MEAN SOMETHING AND THIS IS A STUDY DONE BY THE LMPP WHERE THEY LOOKED AT THE ABC AND THE GERMAL CENTER TYPES AND THEY ACTUALLY FOUND THAT THERE WERE TWO DISTINCT -- THEY WEREN'T DISTINCT -- YEAH, THERE WERE TWO STROMAL SIGNATURES THAT COULD BE IDENTIFIED.THEY WERE PRESENT AT VARIABLE AMOUNTS WITHIN THE VARIOUS TYPES AND EVEN VARIABLE AMOUNTS WITHIN EACH TYPE AS WELL.SO, AGAIN, IT'S NOT A BLACK AND WHITE THING.THAT'S THE DIFFICULTY WHENEVER YOU'RE DEALING WITH A MICROENVIRONMENT.IT'S A LITTLE BIT OF A MOVING TARGET. WHAT THEY FOUND WAS THEY ACTUALLY FOUND THAT THE STROMAL ONE SIGNATURE WAS ASSOCIATED WITH GENES THAT WERE ASSOCIATED WITH CELLS OF THE MACROPHAGE OR MYELOID LINEAGE AND ALSO EXTRA CELLAR MAY TREKS AND FOUND STROMAL TWO SIGNATURE HAD TO DO WITH INDOE THEEL Y'ALL CELL BIOLOGY, AND ALSO THE ASSOCIATED WITH ANGIOGENESIS AS WELL. AND THEY FOUND THAT THE WAY TO GET A SCORE -- BECAUSE A STROMAL ONE SIGNATURE WAS FAVORABLE AND THE STROMAL TWO SIGNATURE WAS NOT FAVORABLE -- WAS TO BASICALLY ADD THE TWO UP IN A COMPLEX ALG RHYTHM AND THAT WAY YOU COULD IDENTIFY WHEN YOU DIVIDED THE PATIENTS INTO CORE TILES DEPENDING UPON IF THEY HAD CORE TILE ONE THEY DID QUITE WELL.IF THEY HAD CORE TILE FOUR THEY DIDN'T DO AS WELL. SO ANYWAY, THIS WAS AN ATTEMPT TO TRY TO GET AT THE MICROENVIRONMENT.YOU CAN SEE IT'S A LITTLE BIT MESSY, BUT I WANTED TO SHOW IT TO YOU JUST TO ILLUSTRATE THAT WE AND OTHERS IN THE CANCER FIELD ARE LOOKING AT THIS, BUT IT IS A LITTLE BIT OF A QUAGMIRE.AND SO THIS IS LOOKING JUST LOOKING AT SOME OF THE KEY GENES THAT ARE UP IN THE STROMAL ONE SIGNATURE VERSUS THE STROEL STROMAL TWO SIGNATURE.AND INTERESTINGLY T GERMAL CENTER CELLS THAT GENERALLY DO BETTER BETTER HAVE MORE OF THIS AND LESS OF THIS. AND SO ONE STRATEGY WE SAID VERY SIMPLISTICICALLY IS, OKAY, IF THE STROMAL SIGNATURES, IF THE TWO IS NOT GOOD, MAYBE WE CAN TARGET THE STROMAL SIGNATURE AND SO WE SAY, WELL, WHAT COULD WE USE THE SPECIFICALLY TARGET THESE STROMA BECAUSE KEEP IN MIND EVERY TIME WE GIVE CHEMOTHERAPY WE'RE ALWAYS THINKING, WELL, WE'RE DIRECTING AGAINST THE TUMOR CELL, BUT THE REALITY S WE'RE GIVING THIS NON-SPECIFIC GEE KNOW TOXIC STRESS THERAPY -- MAKES YOU HAIR FALL OUT, MOUTH FALL OUT, GIVES YOU DIARRHEA, YOU KNOW, SO BELIEVE ME, THIS STUFF KILLS NORMAL CELLS TOO.SO HOW MUCH OF THE CHEMOTHERAPY ARE WE GIVING IS WORKING BECAUSE IT'S KILLING THE TUMOR CELLS AND HOW MUCH OF IT IS BECAUSE IT'S HITTING THE MICROENVIRONMENT?NOBODY KNOWS BUT WE SAID IF WE DON'T LIKE THE MICROENVIRONMENT LET'S BE SPECIFIC.WEB SAID WHAT COULD WE USE TO TRY TO KILL OFF THE MICROENVIRONMENT AND WHAT WE LOOK AT WAS USING A MONO [INDISCERNIBLE] ANTIBODY AGAINST A BROADLY EXPRESSED ANTIGEN CALLED CD 52 LOOKING AT RELATIVE EXPRESSION ON B CELLS, T CELLS AND [INDISCERNIBLE] AND YOU CAN SEE THAT IT'S PRETTY HIGH ON ALL THESE CELLS.THESE ARE KIND OF LAUNDRY LIST OF THE KINDS OF CELLS THAT YOU SEE WITHIN THESE TUMOR BIOPSIES.IF YOU LOOK AT IT KIND OF IN A DIFFERENT WAY, YOU KIND OF LOOK AT A KIND OF A AGGREGATE OF WHAT THE RELATIVE CD 52 EXPRESSION IS AND YOU CAN SEE FOR THE ABC LINE WHEN YOU DO THIS ALONG A MEDIUM, YOU CAN SEE THERE IS A DISTRIBUTION OF CD 52 EXPRESSING CD 52 EXPRESSION WITHIN THESE TUMOR BIOPSIES.SO WE JUST STARTED A STUDY OF TARGETING CD 52, ADDING IT TO CHEMOTHERAPY USING A DRUG CALLED [INDISCERNIBLE].AND WHAT WE HAVE INTERESTINGLY FOUND IS THAT TWO TUMOR TYPES THAT HAVE LOTS AND LOTS OF T CELLS AND MACROPHAGES, ONE BEING HODGKIN'S LYMPHOMA AND THE OTHER ONE BEING A T CELL-RICH LARGE CELL LYMPHOMA AND LYMPHOCYTE PREDOMINANT HODGKIN'S DISEASE, ALL OF THEM VERY RICH IN STROMAL ELEMENTS THAT CONTAIN THIS TARGET, WE HAVE A HANDFUL OF PATIENTS THAT CAME TO US FAILING THERAPY AND THEY'RE NOW IN COMPLETE REMISSION AND HAVE NOT FAILED AND HAVE NOT RELAPSED.SO, YOU KNOW, WE HAVE TO DO THIS TRIAL LONGER.WE HAVE TO WAIT LONGER, BUT IT'S A VERY INTERESTING EXAMPLE OF HOW YOU CAN TARGET, YOU KNOW, THE MICRO ENVIRONMENT AND POSSIBLY GET BENEFIT IN CERTAIN SUBTYPES OF LYMPHOMA WHERE THERE'S A LOT OF THE TARGETS THAT YOU WANT TO GO AFTER. AND PRESUMABLY, THAT PLAY AN IMPORTANT PART IN MAINTAINING THE INTEGRITY OF THE TUMOR. OKAY.SO, UM, LET'S JUST VERY BRIEFLY NOW WE'RE GOING THE END UP, I WANT TO TALK TO YOU ABOUT SOME OF THE REALLY INTERESTING WORK WE'RE NOW DOING AGAINST THE ABC LARGE CELL. THIS, ONCE AGAIN, SHOWS YOU A BLOW-UP PICTURE OF THE GENE EXPRESSION PROFILING OF ABC AND GERMAL CENTER.I'VE ALREADY MENTIONED TO YOU THAT THE NF KAPPA B TARGET GENES ARE HIGHLY EXPRESSED WITHIN THE ABC GROUP.NF KAPPA B IS THE TRANSCRIPTION FACTOR THAT COMES UP IN POST GERMAL CENTER LARGE CELLS AND IT'S GOT A LOT OF ANTIA POP TO TICK TARGETS IN ITS SIGHTS.AND SO IT REALLY DIALS DOWN THE SENSITIVITY OF THE TUMOR CELLS TO APOPTOSIS. AND SO WE ACTUALLY DID AN EXPERIMENT, AND WE DID THE FOLLOWING EXPERIMENT AND THIS WAS BASED THON LABORATORY WORK.LOU ASKED THE QUESTION; IF HE IN FACT WAS ABLE TO INHIBIT THE I KAPPA B KINASE WHICH IS RESPONSIBLE FOR PHOSPHORYLATING I PA KA PA B, WHICH THEN ALLOWS IT TO BE DEGRATED AND DISENGAGES FROM NF KAPPA BE AND THEN ALLOWS NF KAPPA B TO GO TO THE NUCLEUS.TO NF KAPPA BE FORMS A DIMER IN THE CYTOPLASM AND KEEPS IT THERE.THE WAY THE CELL REGULATED NF KAPPA BE TRANSCRIPTIONAL ACTIVITY IS IT PHOSPHORYLATES THE NF KAPPA BE WHICH IS A AN EKE LIB RI YUM WITHIN THE CYTOPLASM THEN IT GETS TARGETED THIS THE PROTEASOME DEGRADATION LEAVING THE NF KAPPA BE TO DO ITS THING ALLOWING IT TO BE TURNED ON.WHEN WE USED THIS INHIBITOR, HE COULD KILL ABC LINES THAT HAD LOT OF THIS NF KAPPA BE TURNED ON.IT SHUT DOWN IT AND CELL LINES DID NOT LIKE IT.[INDISCERNIBLE].WE DID AN EXPERIMENT IN HUMANS WHERE WE COULDN'T USE AN I KAPPA B KINASE BECAUSE IT'S ACTUALLY LETHAL, SO WE ACTUALLY INHIBITED THIS POINT HERE USING A DRUG CALLED [INDISCERNIBLE], AN INHIBITOR OF THE POETRY OWE SOEM.SO RATHER THAN INHIBITING PHOSPHORYLATION, WE INHIBITED ITS DEGRADATION SO IT COULD STAY HERE AND KEEP THE NF KAPPA B IN THE CYTOPLASM.THE EXPERIMENT WE DID WAS WE TOOK RELAPSE REFACTORY LARGE CELL LYMPHOMAS AND TRADED THEM WITH E POCK.WHEN YOU TAKE ABC AND GERMAL CENTER LARGE CELLS IN THE RELAPSE STATE AND GIVE THEM CHEMOTHERAPY, THEY BOTH DO HORRIBLY. AFTER WE DID ALL THIS, WE TOOK BIOPSY SPECIMENS AND ANALYZED THEM AFTER WE HAD ALL OF THE CLINICAL DATA AND ASKED OURSELVES WAS THERE A DIFFERENCE IN THE OUTCOME BETWEEN THE ABC AND GERMAL CENTER.IF [INDISCERNIBLE] WAS LECHING THE ABC THEN ALL THE SUDDEN WE SHOULD SEE A MUCH IMPROVED SURVIVAL WITHIN THE ABC GROUP.IT WAS ALL DONE BLINDLY.SO THIS IS JUST LOOKING AT THE SURVIVAL OF ALL OF THESE GUYS JUST TO GIVE YOU SOME IDEA THAT THE 50% OF THESE PEOPLE WERE DEAD WITHIN LIKE THREE MONTHS.THESE ARE RELAPSE.SO WHEN YOU'VE GOT INCURRABLE LARGE CELL, LOOK OUT BECAUSE YOU'LL BE DEAD VERY QUICKLY.BUT WHEN WE LOOKED AT THE OUTCOME ACCORDING TO WHETHER OR NOT YOU HAD ABC OR GECB ALL OF A SUDDEN WE'RE SEEING THE ABC GROUP IS LIVING A MEDIAN OF 11 MONTHS WHEREAS THE GERMAL CENTER GROUP THREE MONTHS.IF YOU LOOK AT THE ABILITY OF THE THERAPY TO CAUSE THE TUMORS TO SHRINK, 82% WITH ABC SHANK WHEREAS ONLY 12% WITH THE GERMAL CENTER SHANK.THE EXPECTED RESULT SHOULD HAVE BEEN THIS.NO DIFFERENCE.SLAING.SHRANK.THIS WAS THE FIRST EVIDENCE OF INHIBITING NFK KA PA B -- WE'RE OUT OF TIME, BUT WHAT I WOULD HAVE SHOWN YOU IS THAT WE NOW UNDERSTAND THE NF KAPPA BE BEING DOWN HERE THAT WE NOW KNOW MULTIPLE SPOTS HERE WHERE MUTATIONS ARE OCCURRING THAT'S ACTUALLY ACTIVATING THE NF KAPPA B AND WE'RE NOW TARGETING SOME OF THESE PROTEINS ABOVE AND BELOW THOSE POINTS AND WE'RE USING SPECIFICALLY ONE CALLED THE [INDISCERNIBLE] KINASE AND WE'RE SEEING EXCELLENT ACTIVITY THERE AS WELL.I'M GOING TO LEE IT AT THAT.THANK YOU. [APPLAUSE] IT AT THAT.THANK AT.THANK YOU.[APPLAUSE] E IT AT THAT.THANK YOU.[APPLAUSE] >> QUESTIONS? IN THE INTEREST OF TIME -- >>