TRACO 2015: Small Molecules – TGF Beta

TRACO 2015: Small Molecules – TGF Beta


>>GOOD NEWS, THE GOVERNMENT HAS A CONTINUING RESOLUTION TILL DECEMBER 11, SO, TRACO, WE NOW HAVE THE GREEN LIGHT. LATER THIS WEEK I’LL BE SENDING E-MAILS TO ALL THE PEOPLE TWO SIGNED UP TO VISIT THE CORES AND TUMOR BOARDS AND YOU’LL GET YOUR ASSIGNMENTS, AND TODAY OUR FIRST SPEAKER IS ANTON SIMEONOV, HE GOT HIS Ph.D. FROM THE UNIVERSITY OF SOUTHERN CALIFORNIA, SUBSEQUENTLY DID A POSTDOCTORAL FELLOWSHIP AT THE SCRIPPS INSTITUTE, AND THEN CAME TO THE NIH AND CURRENTLY HE’S THE SCIENTIFIC DIRECTOR OF NCATS, HIS TITLE “CHALLENGES AND OPPORTUNITY IN SMALL MOLECULE TRANSLATIONAL RESEARCH.”>>THANK YOU FOR COMING IN TODAY. WE’RE CONTINUING THE OPERATIONS HAVING MADE THE PLANS FOR WHO IS ACCEPTED AND WHO IS NOT. SO LET’S SEE IF WE’RE GOING TO BE ABLE TO PROJECT AGAIN RIGHT, SO WHAT I WOULD LIKE TO TALK TO YOU ABOUT TODAY IS HOW NCATS IS ADDRESSING THESE PROBLEMS AND WHAT IT MEANS FOR US AND HOW WE SPECIFICALLY WORK ON SMALL MOLECULES TO ADVANCE TRANSLATION. NCATS IS ONE OF THE — I DON’T MEAN TO INTERRUPT. WE’RE NOT VIEWING THEM IN FREDERICK JUST YET. >>CAN YOU SEE THEM NOW? >>HANG ON ONE SECOND>>THERE WE GO, THANK YOU. >>NCATS MISSION. >>YES.>>OUR MISSION IS CAPTURED IN THIS ONE SENTENCE, TO CATALYZE THE GENERATION OF INNOVATIVE METHODS, IT’S A MOUTHFUL. WHAT IT MEANS TO INDICATE, THERE ARE MANY BARRIERS THAT PREVENT YOU FROM GOING FROM A SCIENCE PAPER DESCRIBING NEW PATHWAY OR MOLECULAR PATHWAY IN DISEASE AND THERAPEUTIC INTERVENTION, WHETHER IT’S TO A SMALL MOLECULE DRUG, THERE ARE MANY HURDLES THAT NEED TO BE OVERCOME BETWEEN THE POINT OF DECLARING VICTORY AS IN FIRST AUTHOR IN SCIENCE AND HELPING PATIENTS THROUGH THE DISCOVERY. IT’S A VERY LENGTHY FAILURE-PRONE PROCESS, EXTREMELY EXPENSIVE. NCATS IS TRYING TO IMPROVE THAT PROCESS, INCREASE EFFICIENCY, LOWER COSTS AND BY DOING SO HELP BOTH RISE. WHAT ARE SOME OF THE TRANSLATIONAL PROBLEMS? THERE ARE TWO KINDS OF PROBLEMS IN TRANSLATIONAL SPACE, SCIENTIFIC IN NATURE, ORGANIZATIONAL IN NATURE, AND THE LIST IS ACTUALLY PRETTY LONG. HERE ARE REALLY THE TOP PROBLEMS THAT WE’RE ATTEMPTING TO SOLVE. THERE IS REALLY NO GOOD WAY TO PREDICT TOXICOLOGY OR TOXICITY OF A CHEMICAL OR DRUG CANDIDATE. A LOT OF DRUGS FAIL IN THAT STAGE AFTER HAVING ACCRUED HUGE EXPENSES FOR DEVELOPMENT. THERE’S STILL NO GOOD WAY TO PREDICT WHETHER SOMETHING WILL BE TOXIC ONCE IT ENTERS HUMANS. SO THIS IS A HUGE PROBLEM, LEADS TO A LOT OF ATTRITION IN DEVELOPMENT OF BIOLOGIC AND SMALL MOLECULE DRUGS. SAME APPLIES TO PREDICTIVE EFFICACY. YOU MAY HAVE EFFICACY OF YOUR MOLECULE AGAINST MOLECULAR TARGET IN THE TEST TUBE, BIOCHEMICAL ASSAY. CAN YOU PREDICT WHETHER THAT MOLECULE WILL BE EFFICACIOUS IN HUMANS, TO ACTUALLY RELIEVE SYMPTOMS? WHAT ARE THE MODELS THAT ARE MISSING TO MAKE THAT PREDICTION A LITTLE BETTER? A LOT OF DRUGS FAIL AT LATE STAGE CLINICAL TRIALS, BECAUSE THEY DON’T MEET THE EFFICACY MILESTONES FOR THE DISEASE. THEY WORK ON MECHANISM, THEY DO EVERYTHING, THEY ARE NOT TOXIC, THEY JUST DON’T MOVE THE NEEDLE. AND THERE ARE MANY OTHER PROBLEMS IN THIS SPACE, INCLUDING PROBLEMS RELATED TO CLINICAL TRIALS. HOW TO IMPROVE CLINICAL TRIALS, HOW TO HARMONIZE INSTITUTIONAL REVIEW BOARDS, LITTLE PROBLEMS THAT ARE PROBLEMS OF EVERY INSTITUTE HERE AT NIH, EVERY DRUG DEVELOPMENT ORGANIZATION AND BECAUSE THEY ARE SO COMMON TO EVERYBODY, PRACTICALLY NOBODY’S WORKING ON THEM. THAT’S WHAT NCATS TRIED TO ADDRESS. THERE ARE ORGANIZATIONAL PROBLEMS IN TRANSLATIONAL SPACE. DATA RELEASE, DATA TRANSPARENCY, PEOPLE TEND TO GUARD DATA, THAT’S GOING TO ENSURE THEIR TENURE APPROVAL OR LATEST GREATEST PAPER. IP MANAGEMENT, INCENTIVES FOR TEAM SCIENCE, GOING AGAINST THE SCIENCE PAPER TO THE THERAPEUTIC REQUIRES PRETTY STRONG TEAM EFFORT. YOU HAVE TO DROP YOUR EGO, BE COMFORTABLE BEING MIDDLE AUTHOR ON A PAPER. SO HOW DO YOU ACTUALLY GET INCENTIVES FOR BEING THE TEAM PLAYER? ALSO, COLLABORATIVE STRUCTURES, PUBLIC/PRIVATE PARTNERSHIP MODELS, WE’RE ATTEMPTING TO CHANGE THOSE BECAUSE TRADITIONAL WAYS TO INCENTIVIZE COLLABORATIONS BETWEEN ACADEMIA AND INDUSTRY ARE NOT WORKING VERY WELL. SO ALL OF THESE ARE PROBLEMS OF ORGANIZATIONAL NATURE, SO IT’S A PRETTY LARGE SPACE. AGAIN, WE’RE APPROACHING IT FROM DISEASE AGNOSTIC STANDPOINT. SO TO FOCUS ON SMALL MOLECULES, THIS IS KIND OF A NAIVE VIEW OF HOW YOU GO ABOUT DISCOVERING A DRUG, RIGHT? YOU HAVE PRESUMABLY MOLECULAR TARGET, YOU ACTUALLY HAVE TO CREATE A TEST SYSTEM TO EVALUATE YOUR DRUG CANDIDATES AGAINST THAT TARGET OR PATHWAY, SO YOU CALL THAT AN ASSAY, REDUCIBLE, RELATIVELY SIMPLE AND SUBJECT TO AUTOMATION SO YOU WANT AS MANY TIMES OVER AND NOT LOSE TOO MUCH TIME ON IT. THERE’S STAGES OF TESTING OF VARIOUS CHEMICALS, EITHER LIBRARIES TO DISCOVER INITIAL CHEMICAL MATTER SO THIS IS THE PROCESS OF HIGH THROUGHPUT SCREENING, OR IF YOU HAVE A STARTING POINT YOU CAN IMMEDIATELY START MAKING CHEMICAL MODIFICATIONS TO MEDICINAL CHEMISTRY TO MOVE VARIOUS PROPERTIES, SUCH AS AFFINITY TOWARDS THE TARGET, TO IMPROVE SOLUBILITY, PERMEABILITY, HALF-LIFE IN BLOOD, PROPERTIES THAT MAKE THAT MOLECULE A GOOD CANDIDATE FOR TESTING IN ANIMAL MODELS AND THEN ULTIMATELY IN HUMANS. SO YOU CAN SEE THE COSTS GOING UP BY INCREMENTS OF TEN-FOLD TYPICALLY, AND OF COURSE WHEN YOU TRAVERSE THE WHOLE PATH YOU’RE LOOKING AT PROBABLY MORE THAN A BILLION DOLLARS INVESTED. SO THE SCIENCE PAPER AGAIN IS RIGHT AROUND HERE, AND SOMEBODY HAS TO DO ALL THIS AND DO IT MORE AND MORE EFFICIENTLY FROM NOW ON IF WE WANT TO ACTUALLY REVERSE THIS TREND OF THERE BEING RELATIVELY FEW NEW DRUGS INTRODUCED TO PATIENTS OVER THE PAST COUPLE YEARS OR EVEN MORE THAN A DECADE. EFFICIENCY OF DRUG DISCOVERY HAS BEEN GOING DOWN PER UNIT OF INVESTMENT. SO WHAT ARE WE TRYING TO DO AT NCATS TO IMPROVE THAT PROCESS AND I’M GOING TO GIVE YOU VARIOUS EXAMPLES FROM DIFFERENT STEPS OF THIS PROCESS, HOW WE ADDRESS THEM THROUGH VARIOUS PROGRAMS. SO WE OPERATE USING THE COLLABORATIVE MODEL. WE HAVE PROBABLY OVER 300 COLLABORATIONS RIGHT NOW, SO EVERY PROBLEM WE TRY TO SOLVE IS NOT A SELF INVENTED PROBLEM THAT WE SOLVE INTERNALLY AND MOVE ON. IT IS A PROBLEM THAT SOMEBODY ELSE EXPERIENCES, WHETHER IT’S IN INFECTIOUS DISEASES, ONCOLOGY, BASIC BIOLOGY, SO TO DEMONSTRATE THE SOLUTION WE ACTUALLY HAVE TO HAVE A COLLABORATOR WHO IS EXPERT IN THAT PARTICULAR DISEASE OR BIOLOGY AREA. SO IN TURN WE PROVIDE TO THEM THE TECHNOLOGY ACCESS AND PROCESS IMPROVEMENT. SO TO DISCOVER SMALL MOLECULES YOU HAVE TO SET UP VARIOUS ASSAYS, AND RUN VARIOUS TESTS ON SMALL MOLECULES AS I MENTIONED EARLIER, SO THESE ARE THE VARIOUS ASSAY TYPES THAT WE CAN RETURN AT NCATS. WE CAN APPROACH THE SYSTEM FROM A HIGH LEVEL PHENOTYPIC ANGLE, LOOKING AT CELL MORPHOLOGY, VARIOUS ORGANELLES CAN BE STAINED AND MON TOWARD FOR CHANGES. WE CAN FOCUS ON SPECIFIC PATHWAY BY EMBEDDING REPORTERS SUCH AS LUCIFERASE BETA-BACTAMASE OR LOOK AT A SPECIFIC PROTEIN AND INTERACTION WITH THE SMALL MOLECULE DRUG CANDIDATES, SUCH AS USING ENZYMATIC REACTION READOUTS, PROTEIN PROTEIN AND SO ON. WE RUN THESE TESTS IN 1536-WELL PLATES, THIS IS REALLY THE CUTTING EDGE TECHNOLOGY THAT WE HAVE ADOPTED, ABOUT TEN YEARS AGO ACTUALLY, AND WE’VE BEEN PERFECTING IT WITH TIME. I’LL MENTION WHAT IMPROVEMENTS WE’VE MADE. WHY DO YOU NEED TO GO TO SUCH LOW VOLUMES AND SUCH HIGH DENSITY PLATES? IT IS TO SAVE TIME AND REAGENTS. LIBRARY THAT IS SAY A MILLION COMPOUNDS OR EVEN MORE, IF YOU RUN IT IN 96-WELL PLATES, YOU’LL CONSUME A LOT OF REAGENTS, IT WILL TAKE QUITE A BIT OF TIME TO EXECUTE THE SCREEN. IN 1536 YOU CAN RUN THINGS A LOT MORE RAPIDLY, AND BECAUSE OF THAT YOU CAN ACTUALLY ASK DIFFERENT QUESTIONS, AS YOU RUN THROUGH THESE EXPERIMENTS, AND I’LL GIVE YOU AN EXAMPLE OF HOW WE CHANGE THE PARADIGM. SO ALL THIS TESTING IS EFFECTED IN THIS AUTOMATED SYSTEM. YOU CAN THINK OF THE THREE ORANGE ARMS AS THREE TECHNICIANS, THE MICRO TIGHTER PLATES, CANDIDATES, BARCODES, PICK IT UP AND MOVE IT FROM WORK STATION TO WORK STATION, AND THEN WE HAVE VARIOUS UNITS THAT ALLOW US TO ADD REAGENTS TO THESE PLATES, REPRODUCIBILITY ALIQUOTS, 37 DEGREES, BASICALLY CONTROLLED ENVIRONMENT, AND WE HAVE THE LIBRARIES OF SMALL MOLECULES AVAILABLE ON RANDOM ACCESS BASIS, AND ALL SORTS OF DETECTORS TO MEASURE THE OUTCOME OF THESE ASSAYS. SO THE SYSTEM RUNS PRETTY MUCH 24/7. WE’VE MODIFIED IT SEVERAL TIMES TO ESSENTIALLY INTEGRATE LATEST AND GREATEST PLATE READERS, BECAUSE THAT’S REALLY THE TECHNOLOGY THAT CHANGES THE FASTEST. WE’VE RUN WELL OVER 154 COLLECTION SCREENS ON OVER HALF A MILLION COMPOUNDS THROUGHOUT THE YEARS. WHAT DID WE CHANGE? WHAT DID WE IMPROVE IN THIS PROCESS? SO KEEP REFERRING TO THIS NOTION OF IMPROVEMENT, RIGHT? HOW DO WE MAKE SURE THAT ALL BOATS RISE? HOW DO WE IMPROVE THE PROCESS? HOW DO WE INCREASE QUALITY, LOWER COST AND SO ON, ONE THING WE THOUGHT ABOUT BEGAN A DECADE WHEN WE WERE INSTALLING THIS EQUIPMENT, CAN WE RUN SCREENING IN DIFFERENT WAYS TO MAKE IT MORE EFFICIENT. SCREENING IS RUN IN SINGLE CONCENTRATION. YOU TEST YOUR DRUG CANDIDATE AT ONE MOLARITY AND SPLICE CUTOFFS TO DATA AND PICK HITS, SO CALLED, AND THEN YOU RETEST THEM AND A LOT OF THEM DROP OUT BECAUSE THEY TURN OUT TO BE SINGLE POINT ARTIFACTS. WOULDN’T IT BE NICE TO ACTUALLY TEST COMPOUNDS IN MULTIPLE DOSES THE WAY ONE WOULD APPROACH A PHARMACOLOGICAL QUESTION, WHAT IS THE POTENCY OF THE MOLECULE, OBVIOUSLY DOSE IS IMPORTANT IN NOT ONLY BASIC BIOLOGY BUT IN DRUG APPLICATIONS. YOU DON’T JUST TAKE A DRUG AT ANY DOSE. THERE IS RECOMMENDED DOSE. SO LONG STORY SHORT, WE ACTUALLY WORKED FOR ALMOST A YEAR TO DESIGN A NEW PROCESS TO INCORPORATE SERIAL DILUTIONS. IN THIS LIBRARY PREPARATION, THE DIFFERENT COLORS, DIFFERENT COLORS ARE DIFFERENT COMPOUNDS, DIFFERENT INTENSITIES ARE DIFFERENT DILUTION POINTS. YOU RUN THIS LIBRARY AT ANYWHERE BETWEEN 7 AND 15 CONCENTRATIONS, SPANNING FOUR ORDERS OF MAGNITUDE, MOLARITY FROM KNOWN NANOMOLAR TO DOUBLE OR TRIPLE DIGIT NANOMOLAR AND CONSTRUCT BASED ON SCREENING DATA. WE HAD TO DESIGN SPECIFIC PLATING PROCESSES TO MAKE THIS POSSIBLE AND EFFICIENT AND HAD TO WRITE PROBABLY NEW CODE TO DEVELOP BROWSERS, INFORMATICS PIPELINE TO CALCULATE, SAY, HALF A MILLION CONCENTRATION RESPONSE CURVES, HALF A MILLION EQUATIONS TO BE PERFORMED AUTOMATICALLY WITHIN SAY 20-SOMETHING MINUTE TIME SPAN WITH MINIMAL INTERVENTION. YOU CANNOT DO THIS IN EXCEL. YOU CANNOT DO IT IN GRAPH PAD PRISM, WE HAD TO DESIGN A NEW INFORMATICS PROCESS TO DO THAT. SO YOU CAN GENERATE BY DOING CELL PHARMACOLOGICAL OR TOXICOLOGICAL ACTIVES WITH THEIR POTENCY, WITH THEIR SPECIFIC CURVE SHAPE, WHETHER IT’S THE TRADITIONAL SHAPE OR BIPHASIC, WHATEVER WE CAN DISTINGUISH FOR THE ENTIRE LIBRARY, MADE POSSIBLE BY USING THIS AUTOMATED SYSTEM AND VERY LOW ASSAY VOLUMES. THAT’S THE ONLY WAY YOU CAN ACTUALLY GENERATE SO MANY BETA POINTS WITHOUT BREAKING THE BANK. FALSE POSITIVE AND FALSE NEGATIVE DECREASE DRAMATICALLY. WE OBVIOUSLY DEVELOPED THIS PROCESS, PROVED IT WORKED, AND WE PUBLISHED IT NOT JUST IN SUMMARY FORM BUT ALSO PROVIDING SOURCE CODE, EQUIPMENT PROGRAMS, SO THAT EVERYBODY CAN REPLICATE IT OUT THERE. SO AGAIN ANOTHER COMMON MOTIF IN THIS IMPROVEMENT IN SPACE, WE DON’T HOLD ANYTHING BACK. WE TRY TO GIVE IT TO THE COMMUNITY, THAT’S THE ONLY WAY TO IMPROVE THE PROCESS FOR EVERYBODY, NOT JUST US INTERNALLY. OUTREACH IS A HUGE DEAL FOR US. WHAT IS THE SCREENING ASSAY? WHAT IS AN AUTOMATABLE ASSAY, WESTERN BLOCK IS NOT THE SCREENING ASSAY FOR EXAMPLE EVEN THOUGH IT’S USED MANY TIMES A DAY ON THIS CAMPUS AND EVERY LAB AROUND THE WORLD. IT IS NOT A SCREENING ASSAY. SO TO TEACH PEOPLE HOW TO APPROACH THIS, WE ACTUALLY WORKED WITH ELI LILLY, WE’RE ABLE TO CONVINCE THEIR LAWYERS TO RELEASE INTERNAL ASSAY GUIDANCE DOCUMENTS WHICH WE THEN EDITED OVER THE YEARS AND NOW WE HAVE A PRETTY LARGE EDITORIAL GROUP AND IT’S NOW AN E-BOOK ON THE NCBI BOOKSHELF, WITH MORE AND MORE CHAPTERS BEING ADDED AND ABOUT 20,000 OR SO HITS EVERY MONTH, AND GROWING. SO IF YOU WANT TO LEARN ABOUT ASSAY DEVELOPMENT, ASSAY ARTIFACT, FALSE POSITIVES, YOU NAME IT, IT’S ALL THERE. AND WE’RE ADDING CHAPTERS ON TESTING OF BIOLOGICS AND SO ON. IT’S ACTUALLY PRETTY GOOD RESOURCE. SO WHAT IS THE SPACE WE OPERATE IN? WE CLEARLY DON’T WANT TO COMPETE WITH PHARMACEUTICAL COMPANIES ON DEVELOPMENT COMMONLY NEEDED DRUGS FOR, SAY, DIABETES OR OTHER SUCH INDICATIONS. AN AREA OF UNMET NEED WHERE ONE REALLY NEEDS IMPROVEMENT IN PROCESSES IS THIS NEGLECTED DISEASES AREA, AND NIH IS A PUBLIC HEALTH ORGANIZATION, OBVIOUSLY VERY INTERESTED IN CLOSING THIS GAP. SO THERE ARE ABOUT 7000 DISEASES AFFECTING HUMANS AND PROBABLY NO MORE THAN FIVE OR SIX HUNDRED OF THOSE, 10%, HAVE A THERAPEUTIC INTERVENTION ASSOCIATED WITH THEM. THE REST ARE JUST SITTING THERE AND DOCTORS ARE TRYING TO BASICALLY TREAT SYMPTOMS AND NOT THE DISEASE. AND THERE ARE ACTUALLY TWO TYPES OF NEGLECTED DISEASES. THE ONES THAT ARE LOW PREVALENCE, ALSO CALLED RARE OR ORPHAN DISEASES, SO THESE ARE USUALLY GENETIC DISEASES. AFFECTING VERY, VERY FEW PATIENTS WORLDWIDE. SOME OF THEM ARE ULTRA RARE, AFFECTING PROBABLY 500 PATIENTS, WORLDWIDE. OR EVEN LESS IN SOME CASES. AND THEN THERE’S THE OTHER KIND, WHERE A LOT OF PEOPLE SUFFER FROM THESE, AND THEY ARE USUALLY INFECTIOUS DISEASES IN KIND, THE POPULATION RESIDES IN THIRD WORLD DEVELOPING COUNTRIES AND CANNOT PAY FOR THE MEDICINE, SO YOU’RE LOOKING AT SOME VERY TRADITIONAL DISEASES LIKE MALARIA, BUT ALSO SCISTOSOMIASIS AND OTHERS THAT DON’T MAKE HEADLINES EVERY DAY. TWO AREAS OF NEGLECT, OR UNMET NEED, I’LL GIVE YOU YOU AN EXAMPLE OF HOW WE ADDRESS GAPS IN TRANSLATIONAL DEVELOPMENT. ON THE SIDE OF EARLY DISCOVERY, WE ACTUALLY WORKED A COUPLE YEARS AGO ON SCHISTOSOMIASIS CAUSED BY THIS WORM SHOWN HERE, A NASTY INFECTION, SHOWN IN THE BLOWNUP IMAGE. THE WORM PENETRATES THE SKIN OF PEOPLE WHO BATHE IN RIVERS OR LAKES AND ULTIMATELY MAKES ITS WAY INTO THE LIVER, AND RESIDES THERE FOR A PRETTY LONG TIME. IT CAN ACTUALLY SURVIVE IN THE HUMAN HOST FOR PROBABLY 20, 30 YEARS, AND GO UNDETECTED AND GRADUALLY GROW INSIDE THE LIVER AND THESE KIDS LOOK LIKE THEY ARE PREGNANT. THAT’S BECAUSE THEIR LIVES ARE 7 TO 8 TIMES THEIR NORMAL SIZE. A HUGE NUMBER OF INFECTED INDIVIDUALS, MOSTLY SUB-SAHARAN AFRICA, PARTS OF CHINA, UP UNTIL RECENTLY JAPAN AS WELL, BUT VERY KNEW DEATHS RELATIVELY SPEAKING. IT’S NOT A FAMOUS A DISEASE AS MALARIA OR RELATED DISEASES LIKE TB THAT MAKE THE HEADLINES, MORBIDITY IS PRETTY BIG. PEOPLE CAN’T REALLY WALK TO GO TO WORK, THEY JUST SIT THERE AND SLOWLY EXPIRE. SO INTERESTINGLY, BECAUSE THE PARASITES CAN REMAIN IN HUMAN HOST FOR SO LONG, THEY CLEARLY MUST HAVE DEVELOPED SOME DETOXIFICATION MECHANISMS AGAINST INNATE IMMUNE SYSTEM OXYGEN SPECIES. THIS IS THE DEVELOPMENT THAT CAUSED US TO RUN SCREEN FOR IT, THIS COLLABORATOR DAVID WILLIAMS IS FACULTY AT RUSH UNIVERSITY IN CHICAGO, DISCOVERED THE REDOX PATHWAY AND PUBLISHED THE PATHWAY AND TARGET. LONG STORY SHORT, TURNS OUT UNLIKE HUMANS, WHERE DETOXIFICATION OF REACTIVE OXYGEN SPECIES CAN BE PERFORMED BY TWO ENZYMES, IN SCHISTU YOU HAVE ONE ENZYME, SO THIS ENZYME NATURALLY PROVIDES A BOTTLENECK IN THE PARASITES DETOXIFICATION MECHANISM AND WE SET UP A SCREEN USING ENZYME SYSTEM FOLLOWING NADPH FLUORESCENCE IN REALTIME, DETERMINED WHAT THE HITS WERE DOING AND OUT OF THE SCREEN OF A RELATIVELY SMALL COLLECTION BACK THEN WE GOT VARIOUS HITS, DIFFERENT CHEMO TYPES, AND INTERESTINGLY COMPOUND WAS NOT THE MOST POTENT AGAINST THE ENZYME. YOU SEE HERE 1.5, 12 TO 2, AS OPPOSED TO OTHERS THAT ARE NANOMOLAR, THIS TURNED OUT TO BE THE COMPOUND WITH THE STRONGEST EFFECT ON CULTURED WORMS, SO WE WERE LUCKY TO HAVE THROUGH THIS COLLABORATION THE MODEL, THAT ALLOWED US TO TREAT WORMS IN MEDIA WITH THE COMPOUNDS, AND LITERALLY LOOK FOR MORPHOLOGICAL CHANGES AND DEATH. SO THIS WAS THEN FOLLOWED BY TESTING THIS COMPOUND IN VIVO, THIS COMPOUND IS A KNOWN NL DONOR, NOT APPROVED BUT BEING TESTED FOR VARIOUS OTHER INDICATIONS. SO MICE CAN BE INFECTED WITH SCHISTO WORMS, THAT CAN SERVE AS A NATURAL SIMPLE RELATIVELY MODEL FOR THE INFECTION, AND WITH THIS SET OF IMAGES SHOWS UNTREATED LIVER FULL OF LARGE SCHISTO EGGS, LARGE SIZE, UPON TREATMENT WITH THE FUROXAN MOLECULE AT DIFFERENT STAGES OF WORM DEVELOPMENT YOU CAN SEE INVARIABLY THE WORM BURDEN IS BROUGHT DOWN TO ALMOST ZERO LEVEL, AND ALSO THE LIVERS ARE BROUGHT BACK TO A MORE NORMAL SIZE AND PRACTICALLY FREE OF EGGS. THE COMPOUND WAS ALSO ACTIVE AGAINST DIFFERENT SPECIES OR ISOFORMS, BROADLY ACTING, THERAPY ACTIVE ONLY AGAINST ADULT WORMS. SO THIS WAS AN INTERESTING STORY. OF COURSE YOU SEE WHERE IT WAS PUBLISHED, GENERATED ALL KINDS OF INITIAL PUBLICITY BACK THEN WHEN WE RAN THIS STUDY. MOST IMPORTANTLY, IT WAS SEEN AS A GOOD EXAMPLE OF HOW TRANSLATION CAN ADDRESS THESE AREAS OF UNMET NEED THAT IS NEGLECTED TROPICAL INFECTIONS IN A CASE, AND WE WERE ABLE TO TAKE THIS TO THE BANK IN ALMOST THE LITERAL SENSE. CONGRESS ACTUALLY AUTHORIZED THE CREATION OF THIS THERAPEUTICS FOR RARE AND NEGLECTED DISEASE PROGRAM, NOW PART OF NCATS, TO DEAL SPECIFICALLY WITH THE RISKY DRUG CANDIDATES, WHETHER BIOLOGICS OR THERAPEUTIC OR SMALL MOLECULES FOR THE PURPOSE OF TREATING RARE AND NEGLECTED DISEASES. SO THE TREND PROGRAM IS STILL PART OF NCATS, AND IT HANDLES DRUG CANDIDATES THAT WERE INITIALLY DISCOVERED BY EITHER ACADEMIA ORE SMALL BIOTECHS STALLED FOR BUSINESS REASONS OR COMPANY RUNNING OUT OF MONEY OR COMPANY NOT HAVING THE EXPERTISE AT A PARTICULAR STAGE OF DEVELOPMENT. THAT’S WHERE OUR PROGRAM COMMENTS INTO PLAY WITH HELP WITH A COUPLE SPECS OF DEVELOPMENT SO THE PROJECT CAN CROSS THE VALLEY OF DEATH, SO CALLED. TO GIVE YOU AN EXAMPLE WHAT WE’VE DONE FOR ANOTHER RARE DISEASE WITHIN THE TREND PROGRAM, THIS IS AS YOU CAN SEE PRETTY WELL KNOWN SICKLE CELL DISEASE, A RARE DISEASE, THE FIRST ONE FOR WHICH THE GENETIC CODE WAS DISCOVERED BACK IN THE DAY, STILL NO GOOD TREATMENT FOR IT. THE SMALL COMPANY, AESRX DISCOVERED THIS SMALL AND UGLY LOOKING CHEMICAL, HYDROXY METHYL FURFURAL BINDING TO SICKLE HEMOGLOBIN, REDUCING CYCLINE, A PROOF-OF-CONCEPT IN VITRO, NO INVESTOR WAS WILLING TO PUT MONEY INTO THE COMPANY BECAUSE THE MOLECULE LOOKED SO UGLY. THERE WERE CONCERNS ABOUT TOXICITY, FORMULATION, AND THE COMPANY WAS LITERALLY STUCK. THEY HAD AGAIN GOOD EVIDENCE FOR EFFICACY BUT THEY COULD NOT MOVE ANY FURTHER. THIS IS AGAIN, THE VALLEY OF DEATH, THE AREA OF QUOTE/UNQUOTE BORING WORK WHERE SOMEBODY HAD TO HELP THE COMPANY WITH TOXICOLOGY AND FORMULATION. SOMETHING THAT DOES NOT LEAD TO SCIENCE PAPER, SOMETHING THAT IS NOT REALLY THE FINAL CLINICAL TRIAL, BUT IT’S A REQUIRED UNIT OF WORK TO HELP THAT PROGRAM BECOME ATTRACTIVE FROM A COMMERCIAL STANDPOINT, AND THAT’S WHAT OUR TEAM DID. WE HELPED THEM RUN ANIMAL TOX WORK ON CMC WHICH STANDS FOR CHEMISTRY MANUFACTURING QUALITY COALS AND HELP FOR REGULATORY ASPECTS, INTERACTION WITH FDA. LONG STORY SHORT, THE COMPANY WAS ACQUIRED BY BAXTER, THEIR BLOOD DIVISION, NOW BAXTER IS ACTUALLY BANK ROLLING THE DEVELOPMENT OF THIS MOLECULE AND ACTUALLY SENDING IT TO CLINIC AND SO ON. THIS IS AN EXAMPLE WHERE WE’RE ACTING PRETTY MUCH AS A CATALYST. WE TOOK THE PROJECT FOR VERY LITTLE TIME, JUST HELPING THE COMPANY WITH TWO OR THREE SPECS ALONG THIS DRUG DEVELOPMENT PATH, AND NOW THEY ARE OFF ON THEIR OWN, AND WE’RE DONE, AND WE CAN MOVE ON TO HELPING ANOTHER COMPANY OR ACADEMICIAN. THIS IS AN EXAMPLE OF HOW WE WORK IN THE LATER STAGE OF DRUG DEVELOPMENT. I WANT TO SHIFT GEARS AND TALK ABOUT DIFFERENT APROFESSOR TO DISCOVER DRUGS FOR NEW INDICATIONS, WHICH IS TO REPURPOSE EXISTING DRUGS. SO TRADITIONAL PROCESS REQUIRES ONE TO RUN SCREENS OF LARGE LIBRARIES, AND THEN SPEND A LOT OF TIME OPTIMIZING THE MOLECULES, DOING ALL THIS PRE-CLINICAL DEVELOPMENT AND HOPEFULLY GAINING FDA APPROVAL. WHAT IF ONE CAN START WITH EXISTING DRUGS, WHICH HAVE BEEN SHOWN TO BE NON-TOXIC IN HUMANS, SAFE FOR USE IN HUMANS, AND SIMPLY TEST THEM FOR NEW THERAPEUTIC HYPOTHESIS, MAYBE CHANGE THEIR FORMULATION, OR DOSAGE A LITTLE BIT, AND GAIN APPROVAL FOR THIS INDICATION, WITHIN ABOUT ONE TO TWO YEARS, KEEPING A LOT OF STEPS AND A LOT OF COSTS, SO THIS IS THE CONCEPT OF DRUG REPURPOSING, AND THERE ARE SEVERAL THOUSANDS SUCH DRUGS. TO ACTUALLY ENABLE THIS PROCESS, ONE NEEDS OBVIOUSLY SCREENING SYSTEM, SOME THERAPEUTIC HYPOTHESIS, BUT ONE ALSO NEEDS DRUGS COLLECTION, AND SO FOR US THE FIRST QUESTION WAS HOW MANY DRUGS ARE OUT THERE? YOU MIGHT SAY, WELL, CAN’T YOU LOOK THEM UP? ISN’T THERE A WEBSITE OF APPROVED DRUGS? THE ANSWER IS NO, ACTUALLY. EVEN FDA DOESN’T HAVE A FULL-BLOWN WEBSITE THAT TELLS YOU WHAT IS APPROVED, WHAT IS INVESTIGATIONAL AND ALL THAT. LET ALONE THE REST OF THE WORLD. SO OUR INFORMATICS TEAM SPENDS ABOUT FOUR YEARS GOING TO WEBSITES, DATABASES, DISAMBIGUATING SPELLINGS OF DIFFERENT DRUGS, GETTING TO CASS NUMBERS AND BUILDING THE DATABASE AND BROWSER PUBLISHED A COUPLE YEARS AGO. AND THEN WE ACTUALLY EMBARKED ON BUILDING THE PHYSICAL COLLECTION OF ABOUT 4000 DRUGS WHICH WE’RE STILL BUILDING, AND WE’RE USING FOR REPURPOSING SCREENS. WE’RE ALSO WORKING IN THIS OTHER AREA WHICH IS SORT OF COMPANION AREA OF DRUG REPURPOSING, TO LOOK FOR NOVEL DRUG COMBINATIONS. SO THIS IS NOT A NEW CONCEPT. IT IS WELL KNOWN FROM AREAS OF AIDS TREATMENT, THE HIV COCKTAIL, TWO OR THREE DRUGS, THEY HAVE BEEN PROVED AND HAVE SHOWN TO BE PRETTY EFFICACIOUS. WHY WOULD YOU WANT TO HAVE DRUG COMBINATIONS? YOU MAY GAIN ADVANTAGES IN TERMS OF EFFICACY, THAT IS IMPROVED EFFICACY, OR YOU CAN ALSO LOWER THE DOSE OF EACH INDIVIDUAL DRUG TO LOWER THE TARGET RELATED TOXICITIES. ALL KINDS OF OPPORTUNITIES THERE. YOU CAN ALSO USE COMBINATION STUDIES TO — AS A HYPOTHESIS-BUILDING EXERCISE, TO UNDERSTAND WHY THERAPEUTIC AGENTS OR SMALL MOLECULE AGENTS ACT SYNERGISTICALLY. BASICALLY IT GIVES YOU AN ENTRY OF BIOLOGY INVESTIGATION. HOW DO YOU SET THIS UP AS A HIGH EFFICIENCY HIGH-SPEED EXERCISE FINDING THE DRUG COMBINATIONS? SO WE WORKED ON THIS, AGAIN THIS IS A PLATFORM TECHNOLOGY THAT WE DEVELOPED THE PAST COUPLE OF YEARS, SO TO DO THIS YOU NEED A LIBRARY, AS I MENTIONED EARLIER WE’RE BUILDING THE LARGEST SUCH COLLECTION OF APPROVED DRUGS AND INVESTIGATIONAL AGENTS. WE NEED HIGHLY EFFECTIVE WAY TO MIX THESE DRUGS AND DO IT RAPIDLY, AND COMPREHENSIVELY AND REPRODUCIBLY. THE WAY WE’RE APPROACHING THIS, WE FIRST GENERATE SINGLE AGENT RESULTS TO GUIDE US AS TO THE APPROXIMATE POTENCY OF THESE DRUGS IN THAT PARTICULAR ASSAY. THEN WE STAMP, USING ACOUSTIC DISPENSE, LIKE A PRINTING TECHNIQUE, 6 X 6 MATRICES, SIX OF DRUG A AND SIX OF DRUG B. WE DON’T COMBINE. WE GET 36 DATA POINTS SPANNING CERTAIN MOLARITY RANGE DICTATED BY THE HYPOTHESES OF INDIVIDUAL AGENT, WE DON’T WANT TO UNDERSHOOT OR OVERSHOOT, IT’S IMPORTANT TO HAVE A SYSTEM TO PLATE THESE IN CUSTOM CONCENTRATION MATTER. IT’S A HUGE DEAL. IT CANNOT BE DONE USING TRADITIONAL PIPETTING EQUIPMENT. YOU WILL RUN OUT OF DELIVERY RANGE, YOU’LL SPEND A LOT OF TIME ACTUALLY MAKING SERIAL INTERMEDIATE DILUTIONS AND ERRORS WILL PROPAGATE, THIS ALLOWS US TO DO IT EXTREMELY RAPIDLY AND EFFICIENTLY. SO COMBINING, FOR EXAMPLE, LET’S JUST SAY CISPLATIN, SOMEBODY’S FAVORITE ONCOLOGY AGENT, WITH THE ENTIRE PARMACOPIA, 4000 COMBINATIONS, EACH HAVING 36 DATA POINTS, IT TAKES US ABOUT 3 1/2 HOURS TO STAMP THOSE PLATES SO WE CAN COMBINE THAT PROCESS WITH PREPARING CELLS, EITHER USING PRIMARY PATIENT CELLS, FOR EXAMPLE, IN LEUKEMIA CASES ONE CAN DO THAT, OR TUMOR LINE, WHATEVER YOU’RE STUDYING, AND RUN THIS ASSAY ON FRESH AND PREPARED COMPOUNDS AND GET THAT DATA WITHIN LITERALLY AN OVERNIGHT PERIOD. WE HAVE AUTOMATED DATA ANALYSIS METHOD TO BUILD THESE HEAT MAPS AND CALCULATE VARIOUS INDICES OF SYNERGISM, AND WE FOLLOW THIS PRIMARY SCREEN BY ARRAYING THE HITS WITH 10 X 10 CONCENTRATION COMBINATIONS, THAT WAY WE GET MORE DENSE COVERAGE OF ACTUAL CURVES AND CAN VERIFY THE SYNERGISM. SO NOT TO BORE YOU WITH DETAILS, YOU CAN LOOK UP THE PROCESS IN THIS PAPER, IT WAS ACTUALLY INITIALLY FUNDED THROUGH A COLLABORATION WITH NCI, WITH LOU STELT’S GROUP. WE PRESENTED THE RESULTS FROM THIS FIRST SCREEN BUT ALSO THE ENTIRE DATA BROWSER AND ACOUSTIC DISPENSER SOURCE CODE. SO AGAIN, WE DIDN’T KEEP ANYTHING PROPRIETARY. WE’RE GIVING EVERYTHING TO THE WORLD SO THAT OTHERS CAN REPLICATE THIS METHOD. THIS IS THE ONLY WAY TO ADVANCE TRANSLATION IN THE TRUEST SENSE IS TO GIVE DATA METHODS TO OTHERS. SO I MENTIONED PREDICTIVE TOXICOLOGY AS ONE OF THE MAIN TRANSLATIONAL PROBLEMS IN DEVELOPING SMALL MOLECULE AND LARGE MOLECULE DRUGS. SO HERE IS THE PLOT THAT KIND OF GIVES YOU FURTHER EVIDENCE FOR WHY THIS IS NOT A CELL PROBLEM YET. SO ABOUT A THIRD OF ALL DRUG FAILURES ARE DUE TO PRE-CLINICAL OR CLINICALLY MANIFESTED TOXICITY ISSUE. NOTICE FOR EXAMPLE PHARMACOKINETICS AND BIOAVAILABILITY HAS BEEN LARGELY SOLVED AS A DRUG DEVELOPMENT PROBLEM. TOXICITY HAS NOT. THIS IS A PRETTY SERIOUS IMPETUS FOR ONE TO TRY AND WORK ON BETTER METHODS TO IMPROVE TOXICITY OF A CHEMICAL DRUG CANDIDATE. THE OTHER REASON FOR WANTING TO WORK IN THIS AREA IS ACTUALLY ENVIRONMENTAL REASONS. SO THERE ARE OVER 80,000 CHEMICALS THAT EXIST IN COMMERCE, AND FOR MOST OF THEM PROBABLY 79,000 OF THEM, THERE’S PRACTICALLY NO TOXICOLOGICAL DATA AGAINST ANIMAL, HUMAN, ANYTHING. WHY? BECAUSE THERE’S NO MANDATE TO GENERATE SUCH DATA, AND BECAUSE IT’S EXTREMELY EXPENSIVE TO GENERATE SUCH DATA, BECAUSE YOU HAVE TO USE MANY RATS AND FOLLOW THEM FOR MANY YEARS AND SO ON. SO ALL THESE CHEMICALS ARE AROUND US. WE GET EXPOSED TO THEM BISPHENOL A, YOU NAME IT, THERE’S VERY LITTLE DATA TO INFORM EPA AND THE GENERAL PUBLIC TO DANGERS ASSOCIATED WITH THEM. ALSO THERE’S ENORMOUS PUSH TO REDUCE USE OF ANIMALS IN LAB RESEARCH, SO HOW DO WE APPROACH THIS PROBLEM? YOU CAN THINK OF THE ATTEMPT AS TRYING TO DECONSTRUCT THE HUMAN OR THE RAT INTO COMPONENT PATHWAYS, AND TEST THE CHEMICALS USING RELATIVELY CHEAP AND SCALABLE BIOCHEMICAL CELL-BASED ASSAYS TO DETERMINE THEIR EFFECTS ON THEIR PATHWAYS, AND THEN BY BUILDING THIS VERY LARGE DATABASE TO ATTEMPT TO RECONSTRUCT ON SYSTEMS BIOLOGY LEVEL THE EFFECT OF THESE CHEMICALS IN VIVO. SO THIS IS THE AMBITIOUS PROGRAM WHERE IT STARTED A COUPLE YEARS AGO CALLED TOX21, A COLLABORATION BETWEEN NCATS, NIH NATIONAL TOXICOLOGY PROGRAM, TO IDENTIFY PATTERNS OF COMPOUND INDUCED BIOLOGICAL RESPONSES IN ASSAY SYSTEMS THAT HAVE RELEVANCE TO TOXICITY OUTCOMES. ESTROGEN RECEPTOR DISRUPTION, MEMBRANE POTENTIAL MODULATORS, INDUCERS OF DNA DAMAGE RESPONSE AND SO ON. THE INTERMEDIATE GOAL BEING TO PRIORITIZE COMPOUNDS AND ULTIMATELY TO DEVELOP PREDICTIVE MODELS FOR BIOLOGICAL RESPONSE IN HUMANS. BY DOING SO, WE HOPE TO MINIMIZE ANIMAL TESTING AND TO IMPROVE PREDICTIVE POWER OF THESE EARLY PRE-CLINICAL DEVELOPMENT ACTIVITIES, SO COMPOUNDS BE FLAGGED AS BEING POTENTIALLY DAMAGING TO HUMIDITIES BEFORE THEY GET TO CLINICAL TRIALS OR RELEASED INTO THE ENVIRONMENT. TO DO THIS, WE ACTUALLY HAVE TO DEVELOP A WHOLE PIPELINE OF TESTING, AND IT STARTED WITH ASSEMBLING COLLECTION OF ABOUT 10,000 CHEMICALS, AND THE VARIOUS CHEMICALS WERE CONTRIBUTED BY THE DIFFERENT AGENCIES, BASED ON THEIR LONG-STANDING INTERESTS AND PREVIOUS PROGRAMS. THERE WAS DELIBERATELY OVERLAP BETWEEN THE PROPOSED MOLECULES, BECAUSE WE WANTED TO EVALUATE INTERVENDON OR DIFFERENCES BECAUSE DEPENDING ON WHERE YOU SOURCE THEM THEY WILL HAVE DIFFERENT IMPURITY SIGNATURE AND YOU WANT TO KNOW WHAT THE DIFFERENT VERSIONS DO IN THE ASSAY BECAUSE THAT’S REALLY THE MORE REALISTIC REPRESENTATION OF WHAT THE CHEMICAL WILL DO WHEN RELEASED IN THE ENVIRONMENT, FOR EXAMPLE. WE INCLUDED ALL SORTS OF POSITIVE CONTROLS, FAILED DRUGS, ALSO THE DRUG COLLECTION THAT WE’RE ASSEMBLING FOR DRUG REPURPOSING, AND THIS LIBRARY WAS ACTUALLY PLATED AS A TRIPLICATE SET, THREE SEPARATE INSTANCES OF THE LIBRARY WERE CREATED WITH COMPOUNDS MOVED AROUND THE PLATE. WE INCLUDED A LOT OF INTERNAL CONTROLS, 88 SINGLE SOURCE COMPOUNDS IN DUPLICATE ON EACH PLATE TO FOLLOW SYSTEMATIC TRENDS ACROSS SCREENING, AND WE TESTED EVERY MOLECULE AS 15-POINT DOSE RESPONSE TO GENERATE REALLY DENSE DATA COVERAGE. SO WE’VE BEEN DOING THESE SCREENINGS FOR THREE YEARS, THIS IS THE EXAMPLE OF THE OUTPUT OF ONE OF THE FIRST SCREENS GIVEN ITS IMPORTANCE WAS ESTROGEN RECEPTOR ALPHA USING VARIOUS OUTPUTS, LUCIFERASE, LIGAND OR FULL-LENGTH TO FIND OUT WHAT WE CAN GET AWAY WITH GOING FORWARD BECAUSE OBVIOUSLY THE MORE VERSIONS ONE PATHWAY YOU RUN, OR ONE RECEPTOR, THE MORE EXPENSIVE THE WHOLE OPERATION. SO THIS BEING ONE OF THE FIRST PROJECTS WE WANTED TO DO IT EXHAUSTIVELY AND THEN FIGURE OUT WHAT IS SORT OF POSSIBLE TO SKIP OR SIMPLIFY. SO THIS IS THE INITIAL REVIEW OF THE DATA, AND INITIAL ANALYSIS, AND OF COURSE ALL THE DATA ARE MADE PUBLIC. BUT WE DON’T EVEN BEGIN TO PRETEND THAT WE CAN MAKE SENSE OF THIS DATA. THIS IS REALLY BIG DATA. AND WE PUBLISHED ACTUALLY OVER 30 NUCLEAR RECEPTOR SIGNALING SCREENS AND STRESS PATHWAY SCREENS AND BASICALLY WE TURNED TO CROWD SOURCING TO SORT OF GET WISDOM FROM ALL THIS, ALL THIS DATA. SO WHAT WE DID WAS WE PROVIDED ABOUT 50 MILLION DATA POINTS, ALL INDIVIDUAL DATA POINTS, INPUT THEM TO THE ENTIRE COMMUNITY WITH NO RESTRICTIONS AND PROVIDED STRUCTURES OF THESE 10,000 CHEMICALS THAT WERE TESTED, SO FOR EVERY CHEMICAL YOU KNEW ITS NAME, CASS NUMBER, STRUCTURE, AND THEN ALL THE DATA DOWN TO SINGLE ACTIVITY DATA POINT. THE GOAL WAS FOR PEOPLE TO SUBMIT PREDICTIVE MODELS TO PREDICT THE RESPONSE OF A NEW CHEMICAL BASED ON ITS STRUCTURE IN THAT PARTICULAR ASSAY. SO IT’S NOT THE AMBITIOUS GOAL OF PREDICTING RESPONSES IN HUMANS, YET IT’S TO PREDICT RESPONSE IN THE SAME ASSAY, STILL PRETTY AMBITIOUS FOR THE STATE OF THE FIELD. SO WE HAD A TREMENDOUS RESPONSE ACTUALLY, FROM ALL OVER THE WORLD. WE USED 300 NEW COMPOUNDS THAT WE HAD TESTED IN THE ASSAY FOR WHICH WE HAD NOT PROVIDED THE DATA TO THE COMMUNITY TO SCORE THESE MODELS. AND IT’S INTERESTING TO LOOK AT THE LIST OF WINNERS, AND CONCENTRATE PROBABLY JUST ON THE CITY AND COUNTRY HERE. YOU WILL SEE NO SINGLE WINNER FROM THE UNITED STATES. SO LET THIS SINK IN. THIS IS AN EXCELLENT ARGUMENT FOR WHY IT’S GOOD TO SHARE YOUR DATA. YOU JUST DON’T KNOW WHO IS GOING TO MAKE THE BEST SENSE OUT OF IT. VERY SMALL COUNTRIES, VERY SMALL UNIVERSITIES, ALL THESE WINNING TEAMS FROM AUSTRIA, HUNGARY AND OTHER PLACES. SO IT’S A LESSON LEARNED. IT’S GOOD TO SHARE YOUR DATA. RUN SOME INITIAL ANALYSES, PUBLISH THE INITIAL FINDINGS, BUT DO SHARE YOUR DATA BECAUSE THAT’S REALLY THE BEST WAY TO ADVANCE THE FIELD. WE SHOULD ALL LEARN TO BE MORE SELFLESS IN THIS REGARD. SO WE INTEND TO USE THIS CROWD SOURCING MODEL FOR OTHER PROBLEMS GOING FORWARD, BOTH ON THE TOX SIDE AND DRUG EFFICACY SIDE. SO IN THIS SORT OF FIRST AND SECOND PHASE OF TOX21 WE BUILT A PLATFORM TO RUN VERY LARGE SCALE TESTING OF THOUSANDS OF CHEMICALS, HOPEFULLY GRADUATING TO TENS OF THOUSANDS OF CHEMICALS ACROSS MULTIPLE CELL-BASED ASSAYS, MADE PUBLIC, PROTOCOLS, DISCUSSING METHODOLOGY AND ENGAGING OVERSEAS PARTNERS AND AGENCIES TO RECRUIT BETTER ASSAYS. WE’RE STILL JUST TREATING CELLS IN TWO-DIMENSIONAL CULTURE, FOR ONLY A COUPLE DAYS AND TAKING VERY SIMPLE READOUTS. WE INTEND TO INCREASE THE ROLE OR USAGE OF IPSC IN FUTURE TESTING, I’LL TELL YOU ABOUT THAT IN A SECOND. WE’RE MAKING EFFORTS TO INCREASE THE SOPHISTICATION OF DATA GATHERING BY SETTING UP SCREENS, LOOKING AT MULTIPLE PATHWAYS SIMULTANEOUSLY, TAKING SNAPSHOTS OF THE WHOLE TRANSCRIPTOME OF A CELL AS A RESULT OF TREATMENT WITH A CHEMICAL. WE’RE WORKING ON NEXT GENERATION TRANSCRIPTIONAL PROFILING TO ALLOW US TO DO IT CHEAPLY AND EFFICIENTLY AND WE’RE ALSO WORKING ON WAYS TO EMBED METABOLIC PROCESSING INTO THESE ASSAYS, BECAUSE RIGHT NOW THEY ARE VERY SIMPLE. YOU ADD CHEMICALS TO A CELL, THERE IS NO LIVER CELL, NO METABOLISM, WE NEED TO IMPROVE ON THAT AS WELL. AS FAR AS SETTING UP THE INITIAL TECHNOLOGY, WE’RE PRETTY MUCH DONE WITH THE ROBOTICS AND INFORMATICS PART, AND COMPOUND SOURCING AND QC PART. WE’RE MAKING EFFORTS AS PART OF A COLLABORATION WITH THE NIH COMMON FUND, OFFICE OF THE NIH DIRECTOR, TO SET UP A STEM CELL TECHNOLOGY FACILITY AT NCATS TO WORK ON COMMON TECHNOLOGICAL HURDLES IN THIS FIELD, SPECIFICALLY PROBLEMS THAT PREVENT ONE FROM USING IPS-DERIVED DIFFERENTIATED CELTS FOR CELL THERAPY. AND REALLY TWO MAIN PROBLEMS IN THIS AREA IS LACK OF GOOD REFERENCE METHODS TO CHARACTERIZE CELLS, WHETHER YOU HAVE ACTUALLY MADE FULLY DIFFERENTIATED MATURE CELL OUT OF YOUR IPS CULTURE, SO WE’LL BE WORKING ON SETTING THESE STANDARDS, DEVELOPING DATABASES, OF CELLULAR SIGNATURES TO INFORM THE COMMUNITY AS TO THE STATUS OF THEIR CELLS AS THEY PROCESS THROUGH DIFFERENTIATION PROTOCOLS. THE OTHER PROBLEM IS IMPROVEMENT TO PROTOCOLS THEMSELVES. RIGHT NOW THERE ARE A LOT OF PROTOCOLS TO MAKE THEM, EXPAND THEM, DIFFERENTIATE THEM. HARDLY REPRODUCIBLE, FOR SURE EXTREMELY EXPENSIVE AND NON-SCALABLE. SO WHAT WE’RE GOING TO BE DOING IS RUNNING HIGH THROUGHPUT SCREENS TO FIND SMALL MOLECULE DRUGS, NOVEL MOLECULES TO REPLACE EXPENSIVE FACTORS AND MEDIA COMPONENTS TO IMPROVE THE EFFICIENCY REPROGRAMMING, POTENTIALLY SPEED OF REPROGRAMMING AND DIFF DEFINITELY LOWER COST AND HELP REPRODUCIBILITY AND USE IN TOXICITY EVALUATION, IN THIS CASE LOOKING AT CELLS, DIFFERENTIATED TO THE SAME FINAL IMAGE BUT COMING FROM DIFFERENT INDIVIDUALS AND REALLY THIS FIELD OF TOXIC GENOMICS, AND AGAIN IT’S BEFORE WE’RE GOING TO RUN THESE PROJECTS AS COLLABORATIONS AND MAKE ALL DATA METHODS AND COMPOUNDS PUBLICLY AVAILABLE TO EVERYBODY OUT THERE USING THEIR RESEARCH. SO JUST TO GIVE YOU AN EXAMPLE OF HOW BIG THIS PROBLEM IS, WE’RE COLLABORATING WITH INVESTIGATOR AT THE NATIONAL EYE INSTITUTE KAPILBHRAT WORKING ON BOAT CAL FOR PROTOCOL FOR CELL THERAPY, PATIENTS WITH AGE-RELATED MACULAR DEGENERATION, TO CONVERT TO IP S CELLS TO EXPAND THEM AND RETURN THEM TO THE PATIENT’S RETINAL AREA IN THE FORM OF A SPECIFIC INJECTION, THIS IS THE PROTOCOL TO MAKE OUR RPEs. IT’S A THREE-MONTH PROTOCOL WITH MANY INTERVENTION STEPS, AND IT’S FAIRLY EXPENSIVE AND HARD TO SCALE UP AND REPRODUCE. SO WHAT WE INTEND TO DO IS TAKE THIS PROTOCOL AND DISSECT IT INTO LITERALLY DOZENS OF SCREENS, SMALL MOLECULE SCREENS AT WEEK ONE, WEEK TWO, THIS STEP AND SO ON, ARRIVING AT THIS MATRIX OF DATA TO TRY TO FIND SMALL MOLECULES THAT WILL IMPROVE EFFICIENCY. IF ANY ORE ALL OF THOSE REPLACE AN EXPENSIVE COMPONENT, YOU NOTICE IT’S A PRETTY COMPLEX PROBLEM TO SOLVE, SO IT WILL BE DONE THROUGH MULTIPLICITY OF SCREENS, NOT JUST ONE SCREEN. LAST THING I’D LIKE TO MENTION IS SOMETHING WE’RE DOING ON THE EXTRAMURAL SITE OF NCATS THROUGH GRANT PROGRAM, WHICH RELATES TO THIS QUESTION OF IMPROVEMENT OF PREDICTIVE MODELS. SO THIS IS THE TISSUE CHIP PROGRAM WHICH IS DESIGNED TO GENERATE THIS IN VITRO PLATFORM THAT RECAPITULATES MAJOR COMPONENTS OF HUMAN PHYSIOLOGY IN MICROFLUIDIC CHIP OR CARTRIDGE FORMAT IN A IS TO HAVE LIVER ON THE FORMAT TO HAVE LUNG OR LIVER OR GUT ON THE CHIP AND SO ON, THE SYSTEMS ARE ALIVE AND PERFORMING WHAT THEY ARE SUPPOSED TO PERFORM AS FAR AS PHYSIOLOGY, AND THEN TO BEGIN TO CONNECT THEM TO EACH OTHER TO TAKE IT TO THE NEXT STAGE. SO THE GOAL IS TO HAVE TISSUE BE VIABLE IN THESE SYSTEMS FOR AT LEAST FOUR WEEKS TO HAVE THESE BE MODULAR AND RECONFIGURABLE SO THEY CAN BE CONNECTED, AND IN THE END TO HAVE SOME ASSURANCE FOR COMMUNITY-WIDE ACCESS. AGAIN I’M NOT GOING TO GO INTO DETAIL AS TO HOW THIS IS REALIZED. ONE HAS TO ACCOUNTED FOR ALL THESE ELEMENTS OF COMPLEXITY WHEN ONE BUILDS A HUMAN ON A CHIP. CELLS HAVE TO BE THE RIGHT KIND, OBVIOUSLY SOME OF THEM COME FROM PRIMARY CELLS, OTHERS COME FROM IPS CELLS AND SO ON, ONE NEEDS TO TAKE CARE OF THE SCAFFOLD STRUCTURE, PATTERNING, ENSURING PERFUSION, ENERVATION, AND THEN HAVING HAVING READOUT, HOW DO YOU MEASURE YOUR GUT ON A CHIP IS WORKING, WHAT DO YOU ACTUALLY MEASURE? ALL OF THESE HAS BEEN GOING ON FOR A WHILE. IT’S ACTUALLY A COLLABORATION WITH DARPA, THE DEFENSE ADVANCE RESEARCH PROJECT AGENCY THE PROGRAM IS ABOUT HALFWAY THROUGH, THE ORIGINAL ORGANS HAVE BEEN GENERATE AND VALIDATED, NOW THE PROGRAM IS ENTERING THE PHASE OF INTEGRATION, CONNECTING DIFFERENT ORGANS, SO FOR EXAMPLE HAVING GUT ON A CHIP WITH LIVER ON THE CHIP, WITH KIDNEY ON THE CHIP, FEEDING IT LIVER TOXIC DRUG AND ACTUALLY DEMONSTRATING DRUG IS METABOLIZED, AND THAT IT EXHIBITS FOR EXAMPLE LIVER TOCKS EFFECT. SO THIS WOULD BE THE USE OF THIS PLATFORM. LAST SLIDE, YOU CAN LEARN MORE FROM THE WEBSITE, THERE’S VIDEOS AND DETAILS FOR THE VARIOUS ORGANS. AND IT’S ACTUALLY PRETTY FASCINATING SYSTEM. ONE GRANTEE WAS EVEN ABLE TO RECAPITULATE FEMALE REPRODUCTIVE SYSTEM WITH THE 28 CYCLE ON THE CHIP, FOR ETHICS REASONS THEY CAN NOT USE HUMAN CELLS, SO IT WAS ACTUALLY A MOUSE SYSTEM DESIGNED TO MIMIC THE HUMAN BIOLOGY WITH A 28-DAY CYCLE. I’LL LEAVE IT AT THAT. THERE’S PLENTY OF INFORMATION THERE. THE HOPE IS THAT WITHIN ABOUT A YEAR OR TWO, WE’LL START SEEING CONVERGENCE OF ALL THESE TECHNOLOGIES, WHERE THE ROBOTICS SCREENS WHICH CAN ADDRESS TENS OF THOUSANDS OF COMPOUNDS CAN PRODUCE VARIOUS HITS WHICH CAN THEN BE FED INTO ORGANS ON A CHIP OR HUMAN ON A CHIP TO BOTH BETTER UNDERSTAND THE MECHANISM OF ACTION OF THESE HITS FROM THE CELL SCREENS AND TO VALIDATE THEIR IN VIVO EFFECT WHETHER IT’S TOXIC EFFECT, OR DRUG EFFICACY TYPE EFFECT. SO WE’RE EAGERLY AWAITING THE PRODUCTIZING OF THESE ORGANS AND PHYSIOLOGICAL SYSTEMS, THERE ARE HALF A DOZEN START-UP COMPANIES THAT HAVE BEEN SPUN OFF FROM THE VARIOUS ACADEMIC INSTITUTIONS THAT WORKED ON THE VARIOUS ORGANS. I’LL STOP HERE. THANK YOU FOR YOUR ATTENTION AND I’LL BE HAPPY TO ANSWER QUESTIONS. [APPLAUSE] >>WHAT TYPE OF (INAUDIBLE) PROJECTS ARE YOU DOING NOW.>>COMBINATION SCREENING PLATFORM WAS DEVELOPED IN COLLABORATION WITH NCI, THEY SENT THIS PROBABLY ABOUT 15 DIFFERENT COMBINATION SCREENS TO WORK ON, WITH VARIOUS INVESTIGATORS, LEE HELMOND, VARIOUS MODELS, REALLY. A LOT OF READOUTS REVOLVE AROUND CELL DEATH AND VIABILITY BECAUSE THEY ARE RELATIVELY SIMPLE. WE’RE WORKING ON OTHER READOUTS AS WELL. WE’RE ALSO TRYING TO COMBINE IMMUN0TOXIN WITH SMALL MOLECULE DRUGS. IT’S NOT AS SIMPLE AS COMBINING TWO SMALL MOLECULE DRUGS BECAUSE OF DIFFERENT FORMULATION REQUIREMENTS FOR, SAY, THERAPEUTIC ANTIBODY, BUT WE’RE WORKING ON THAT AS WELL. SO THIS IS REALLY WHERE DRUG-DRUG COMBINATION PLATFORM HAS TAKEN OFF, MOSTLY ONCOLOGY PROMISE, ALSO STARTING INFECTIOUS DISEASE PROJECTS WITH DRUG-DRUG COMBINATION PLATFORM. WE ALSO HAVE A LOT OF EFFORTS IN THE AREA OF RARE TUMORS, PEDIATRIC CANCERS, PROBABLY OVER 80 DIFFERENT PROJECTS IN CANCER FIELD. BOTH TARGET BASED AND PHENOTYPIC.>>THANK YOU.>>ALL RIGHT, THANK YOU. >>ONE ADDITIONAL ANNOUNCEMENTSOME NEXT MONDAY IS COLUMBUS DAY. AND THAT IS A GOVERNMENT HOLIDAY. SO NEXT WEEK WE’LL BE HAVING TRACO ON TUESDAY, FOUR TO SIX, INSTEAD OF MONDAY. OUR NEXT SPEAKER IS SONIA JAKOWLEW, Ph.D. AT RUTGERS UNIVERSITY, SUBSEQUENTLY DID A POSTDOCTORAL TRAINING IN FRANCE, AND CAME BACK TO THE NCI AND STARTED WORKING, NOW IN THE CANCER TRAINING BRANCH, HER TITLE TRANSFORMING GROWTH FACTOR BETA AND LUNG TUMORIGENESIS. SONIA?>>THANK YOU THIERRY FOR INVITING ME TO THIS LECTURE. TODAY I’M TALKING ABOUT A GROWTH FACTOR TRANSFORMING GROWTH FACTOR BETA, A VERY SERIOUS CANCER PROCESS HAVING LUNG TUMORIGENESIS, MY SEMINAR WILL INCLUDE A BRIEF BACKGROUND HISTORY ABOUT TGF-BETA IN LUNG TUMORI GENESIS AND PRESENTING EXPERIMENTAL DATA FROM MY LABORATORY. SO LUNG CANCER HAS BECOME THE MOST COMMON CAUSE OF CANCER DEATH, LAST YEAR 224,000 CASES OF LUNG CAN DIAGNOSED AMONG MEN AND WOMEN IN THIS COUNTRY. THERE WERE OVER 150,000 DEATHS DUE TO THIS HORRENDOUS DISEASES. MOST CASES OCCUR IN FORMER SMOKERS, LUNG CANCER TAKES A VERY LONG TIME TO DEVELOP AND USUALLY YOU DON’T SEE THIS UNTIL YOU’VE BEEN SMOKING FOR SOME YEARS, OR HAVE QUIT. AND DESPITE OUR AGGRESSIVE ANTI-SMOKING CAMPAIGN EFFORTS, AND THE VARIOUS NEW THERAPIES AND TREATMENTS FOR LUNG CANCER THE FIVE-YEAR SURVIVAL RATE IS STILL LESS THAN 15%. SO TRANSFORMING TGF-BETA IS A MULTI-FUNCTIONAL REGULATOR OF CELL GROWTH, A POTENT INHIBITOR OF MOST NORMAL EPITHELIAL CELL PROLIFERATION, WIDESPREAD TISSUE EXPRESSION PARTICULARLY IN MAMMALS. TGF-BETA HAS BEEN SHOWN TO PLAY A PIVOTAL ROLE IN EPITHELIAL HOMEOSTASIS. SO TGF-BETA MIGHT BE AN ATTRACTIVE CANDIDATE FOR NEW THERAPEUTIC INTERVENTION APPROACHES IN LUNG CANCER. SO TO UNDERSTAND TGF-BETA I HAVE TO TAKE YOU BACK TO THE BEGINNING AND TGF-BETA HAS ROOTS IN ANOTHER GROWTH FACTOR CALLED SARCOMA GROWTH FACTOR, WHICH WAS A POLYPEPTIDE THAT WAS DEMONSTRATED TO BE SECRETED BY MURINE SARCOMA VIRUS TRANSFORMED MOUSE FIBROBLASTS THAT COULD STIMULATE NORMAL RAT FIBROBLASTS TO FORM COLONIES IN A SOFT AGAR, THE TRANSFORMATION ASSAY THAT WAS USED TO IDENTIFY THIS GROWTH FACTOR BY DELARCO AND TODARO IN THE LATE 70s. FOLLOWING THE DISCOVER OF SARCOMA GROWTH FACTOR, TWO CLASSES OF TRANSFORMING GROWTH FACTOR WERE ISOLATED, ONE SHOWN TO BE (INAUDIBLE) THIS WAS CALLED TGF-ALPHA. THE SECOND CLASS DID NOT COMPETE FOR TGF BINDING BUT WAS ENHANCED AND CALLED TGF-BETA, SARCOMA GROWTH FACTOR IS MADE UP OF TWO DIFFERENT GROWTH FACTORS, TGF-ALPHA AND TGF-BETA, REPORTED IN A PAPER IN 1982. FOLLOWING THE DISCOVERY OF TGF-BETA, THE PURIFICATION WAS DEMONSTRATED FROM AT LEAST THREE DIFFERENT SOURCES, IN 1983. HUMAN PLATELETS, AND PLACENTA AS WELL AS BOVINE KIDNEY. THIS WAS DISCOVERED BY THREE INVESTIGATORS AT THE NCI. TO GIVE YOU SOME IDEA OF ABOUT THE PURIFICATION THAT WAS INVOLVED IN PURIFYING TGF BAITA TYPICALLY 100 GRAMS OF BOVINE KIDNEY OBTAINED FROM THE SLAUGHTERHOUSE WAS EXTRACTED WITH 8 LITERS OF ETHANOL, SEVENRY TRI– CENTRIFUGED, AND THE NEXT MORNING REDISSOLVED IN ACETIC ACID AND APPLIED TO 80 LEADER, 1 LITER FRACTION, REDISSOLVED FOR FURTHER CHROMATOGRAPHY, THE FINAL YIELD FROM WAS 6 GRAMS OF TGF-BETA. THE ENORMITY OF THIS PURIFICATION, THESE ARE TYPICAL COLUMNS, AND THE PINK ARROW SHOWS THE 55-GALLON DRUM. SO THIS WAS MASSIVE, AND THEY WERE TRYING TO PURIFY ENOUGH TO WORK WITH. THE ASSAY FOR TGF-BETA WAS TYPICALLY THE GROWTH OF NORMAL SIDNEY CELLS, TO DO THIS A MIXTURE OF MEDIA, SERUM AND NRK CELLS WERE APPLIED, INCUBATED AT 37 DEGREES FOR A WEEK, PLATES WERE STAINED AND COLONIES WERE COUNTED, WITH AN ONNICON IMAGE ANALYSIS SHOWN AT THE BOTTOM OF THE SLIDE. IF NO TGF-BETA WAS PRESENT IN THE SAMPLES, NO COLONIES WOULD GROW, AND NOTHING WOULD BE COUNTED. IF TGF-BETA IS PRESENT IN THE SAMPLE, THE COLONY WOULD FORM AND THEY WOULD BE COUNTED, IF THEY WERE OVER A CERTAIN SIZE THRESHOLD. SO SHOWN HERE IS A FINAL HPLC PURIFICATION IN TGF-BETA, AFTER THIS PURIFICATION MEASURE, AND TGF-BETA MIGRATES AS A — LET’S SEE IF WE CAN GET THIS TO WORK. OKAY. AS A 25,000 SIGNAL BAND SO EUREKA, TGF-BETA IS BORN AT THE NCI, AND DOCTORS SPORN AND ROBERTS ARE CREDITED WITH THE DISCOVERY. FOLLOWING THE PURIFICATION OF TGF-BETA THE AMINO ACID SEQUENCE WAS DEVELOPED, AND IT WOULD SHOW TGF-BETA ACTUALLY EXISTS AS A PRE-GROWTH MOLECULE, WITH THE MATURE TGF-BETA CONSISTING OF 112 AMINO ACIDS AT THE C TERMINUS. LAB TESTS WERE NECESSARY TO DEVELOP INTO THE PROPER CONFIGURATION. NOW, IN THE EARLY ’90s THE CRYSTAL STRUCTURE OF TGF-BETA, AND I SHOULD SAY THIS IS TGF-BETA ONE WERE DEVELOPED, SHOWN TO BE A DIMER COMPOSED OF TWO IDENTICAL MONOMERIC CHAINS, HELD TOGETHER BY INTERCHAIN DISULFIDE BOND BETWEEN THE TWO MONOMERS. TGF-BETA IS ACTUALLY A MEMBER OF WHAT IS NOW CALLED THE TGB BETA SUPER FAMILY, IN ADDITION TO TGF-BETA WHICH CONSISTS OF THREE ISOFORMS THERE ARE TWO ADDITIONAL TGF-BETAS THAT HAVE BEEN IDENTIFIED, TGF-BETA 4 IN A CHICKEN AND 5 IN XENOPHOBES, 4 AND 5 THOUGHT TO BE HOMOLOGUES FOR MAMMALIAN ISOFORMS. SHOWN IN THIS CARTOON IS THE BASIC STRUCTURE OF TGF-BETA. IT’S A 25,000 MW DISULFIDE-BONDED HOMODIMER, 3 HIGHLY HOMOLOGOUS ISOFORMS IN MAMMALS, PRINCIPAL SOURCES ARE PLATELETS, BONE AND SPLEEN BUT IT’S VIRTUALLY FOUND IN EVERY TISSUE IN THE BODY. MOST CELLS EXPRESS TG F BETA IN RECEPTORS, USUALLY SECRETED IN THE LATENT INACTIVE FORM, WHICH HAS TO BE ACTIVATED BEFORE TGF-BETA CAN EXHIBIT ITS PROPERTIES. AND SUPERFAMILY CAN CONTAIN NOT ONLY THE TFG BETA ISOFORMS, BUT ACTTIVIN. THEY PLAY A PLACE IN BIOLOGICAL PROCESSES VIEWING DEVELOPMENT, IMMUNE SYSTEM FUNCTION, REPRODUCTION, ANGIOGENESIS, AGING, TISSUE REPAIR AND RESPONSE TO INJURY, METABOLIC REGULATION AND PROLIFERATIVE HOMEOSTASIS. NOW, THERE ARE MAJOR BIOLOGICAL RESPONSES THAT ARE REGULATED BY TGF-BETA. IT CAN INHIBIT PROLIFERATION UNDER CERTAIN CIRCUMSTANCES AND CAN REGULATE APOPTOSIS, DIFFERENTIATION AND IMMUNE CELL FUNCTION. UNDER CERTAIN CIRCUMSTANCES THEY CAN BE STIMULATEIVV OF EXTRACELLULAR MATRIX AND PROMOTE CHEMO TAXIS. THE MODEL HAS BEEN DEVELOPED OVER SEVERAL YEARS, IT STARTS ON THE UPPER LEFT, THE TGF-BETA LIGAND, THE DMPs, ACTIVE, BINDS TO TYPE II RECEPTOR SHOWN IN PINK, WHICH IS PHOSPHORYLATED, THIS COMPLEX RECRUITS AND BINDS THE TYPE ONE TGF-BETA RECEPTOR IN PINK TO FORM A SUPER COMPLEX IN THE TYPE 1 RECEPTOR. NOW AS A COMPLEX IT IS NOW READY TO BIND AND INTERACT WITH ANOTHER SET OF PROTEINS CALLED — HMM. CALLED THE SMADS, AND DEPENDING ON WHETHER YOU HAVE ACTTIVN, IT WILL BIND TO A DIFFERENT SET OF SMADS. NOW, THE SMAD PROTEINS INVOLVED LIVE IN (INAUDIBLE) INVOLVED WITH BMPs ARE SMAD 1, DISTINCT PROTEINS. WHAT THEY DO IS WHEN THEY BIND TO THE COMPLEX, THEY ARE THEN ABLE TO — THESE SMADS ARE PHOSPHORYLATED, AFTER PHOSPHORYLATION THEY ARE ABLE TO BIND WITH A COMMON SMAD. SMAD 4 HAS UNIQUE PROPERTIES. OF IT CAN TRANSLATE INTO THE NUCLEUS, CARRY THE CELL, REGULATING TRANSCRIPTION FACTORS. NOW, THE WHOLE THING CAN BE SHORT CIRCUITED BY INHIBITORY SMADS. OOPS, SORRY. MENT . SIX AND SEVEN CAN NEGATE THE SYSTEM WHICH OFTEN HAPPENS IN CASES. NOW, CLINICALLY, TGF-BETA HAS BEEN SHOWN TO BE A TUMOR SUPPRESSOR. IT’S BEEN SHOWN THAT THERE ARE GERMLINE MUTATIONS IN SPECIFIC TGF PATHWAY COMPONENTS CAUSING FAMILIAL REDISPOSITION TO CANCER, SMAD 4 HAS BEEN SHOWN TO BE HEAVILY MUTATED IN A CONDITION CALLED JUVENILE POLYPOSIS SYNDROME. IN ADDITION, SEVERAL PATHWAY COMPONENTS ARE DELETED OR SO MATICALLY MUTATED IN SOME HUMANS. SMAD 4 HAS BEEN SHOWN TO BE HEAVILY DELETED IN HUMAN PANCREATIC CANCER. THIRDLY, REDUCED EXPRESSION OF SEVERAL TGF-BETA PATHWAY SIGNALING COMPONENTS HAVE BEEN SHOWN TO OCCUR AS WELL AS OVEREXPRESSION OF ENDOGENOUS PATHWAY INHIBITORS HAVE BEEN SHOWN TO BE ASSOCIATED WITH TUMORIGENESIS, INCLUDING THE TYPE 1 AND TYPE 2 TGF-BETA RECEPTORS AS WELL AS INHIBITOR SMAD PROTEIN AND SKI. CLINICALLY TGF-BETA HAS BEEN SHOWN TO BE A TUMOR PROMOTER, ELEVATED IN MANY TYPES OF ADVANCED HUMAN TUMORS AND CORRELATE WITH METASTASIS AND/OR POOR PROGNOSIS. THIS HAS BEEN SHOWN IN A WIDE VARIETY OF DIFFERENT ONES, INCLUDING LUNG. ON THE LOWER RIGHT SLIDE IS SHOWN PROSTATIC ADENOCARCINOMA, STAINED WITH ANTIBODIES SPECIFICALLY FOR TGF-BETA ONE. YOU CAN SEEN THE STAINING SHOWN LIE THE BLACK ARROW, THE INTERFACE BETWEEN THE TUMOR AND MICROENVIRONMENT. IF YOU LOOK AT THE LIGHT ARROW, AT THE TOP, YOU DON’T SEE ANY STAINING WITH TGF-BETA 1 BECAUSE THIS IS NORMAL TISSUE. SO WHAT’S GOING ON AND WHY IS THIS SO CONFUSING? IS TGF-BETA IN CARCINOGENESIS HERO OR IS IT A VILLAIN? WELL, IT TURNS OUT TO BE BOTH. TGF-BETA CAN BE A PROXIMAL INHIBITOR OF MALIGNANT PHENOTYPE AND A POTENT GROWTH INHIBITOR AND TUMOR SUPPRESSOR, OR PRO METASTATIC FACTOR, EVERYTHING DEPENDS ON CONTEXT AND ENVIRONMENT IT’S PUT IN. UNIFYING HYPOTHESIS HAS BEEN DEVELOPED TO EXPLAIN HOW IT CHANGES FROM BEING TUMOR SUPPRESSANT TO PRO ONCOGENIC FACTOR IN TUMORIGENESIS PROGRESSION. SO IN THE NORMAL EPITHELIUM, THE TUMOR SUPPRESSOR ACTIVITIES OF TGF-BETA PREDOMINATE, BUT AS CHANGES OCCUR IN THE GENETIC AND EPIGENETIC CONTEXT AS TUMORIGENESIS PROGRESSES, TGF-BETA LOSES RESPONSIVENESS AND TGF-BETA EXPRESSION AND ACTIVATION INCREASES, SO WHEN YOU GET MORE TOWARD THE INVASIVE METASTATIC CANCER THE PRO ONCOGENIC ACTIVITIES PREDOMINATE OVER THE TUMOR SUPPRESSOR ACTIVITIES. AND YOU HAVE PRO ONCOGENIC PHENOMENA. SO IN ADDITION TO THE TGF-BETA SMAD DEPENDENT PATHWAYS THAT I JUST SPOKE TO YOU ABOUT, THERE ARE OTHER SMAD-INDEPENDENT PATHWAYS THAT WORK AT THE SAME TIME IN BOTH NORMAL AND TUMOR CELLS. AND THIS INCLUDES SUCH PATHWAYS AS THE MAP KINASE PATHWAY, RAS, RHOA PHOSPHATASE. WE’RE INTERESTED IN THE RAS BECAUSE THE K-RAS PROTOONCOGENE STUDY HAS BEEN CANCER, SHOWING ACTIVATIONAL MUTATION IN 25% OF HUMAN LUNG ADENOCARCINOMAS, MUTATION OF THESE IN ONE ALLELE OF K-RAS INCREASES THE APPEARANCE OF LUNG LESION, AND IT’S BEEN SHOWN THERE IS TREMENDOUS CROSS-TALK BETWEEN SMAD-DEPENDENT PATHWAY AND RAS/MEK PATHWAY AND CAN MODERATE HOW TGF-BETA1 SIGNALING OCCURS THROUGH THE SMAD PATHWAY AND FINALLY IN VITRO STUDIES HAVE SHOWN TGF-BETA1 DOMINATES OVER THE MITOGENIC EFFECTS OF RAS BUT ACTIVATED RAS CAN OVERRIDE. THEY CAN HAVE AN INTERPLAY IN LUNG CANCER. SO WE DECIDED TO LOOK AT THE TUMOR SUPPRESSOR AND TUMOR PROMOTER ACTIVITY, AND ON THE TOP LEFT IS THE NORMAL SITUATION, WHERE IF EVERY IS RECEIVED NORMALLY IN NORMAL CELLS TGF-BETA ACTS AS TUMOR SUPPRESSOR. IN TUMOR CELLS IF YOU HAVE DECREASED EXPRESSION OF THE RECEPTOR, ONHYPERACTIVATED MAP KINASE OR DECREASED LEVEL ALSO OF SMAD PROTEIN OR COMPROMISED EFFECTIVE FUNCTION IN ANY SUPPRESSOR ARMS OF THE PATH, YOU MIGHT HAVE A SITUATION WHERE YOU HAVE TUMOR PROMOTION. SO THE GOAL OF MY LABORATORY WAS TO DETERMINE THE ROLE OF TRANSFORMING GROWTH FACTOR BETA IN THE DEVELOPMENT AND MALIGNANT TRANSFORMATION OF LUNG EPITHELIAL CELLS, A MODEST GOAL, OKAY? SO THIS WORK WAS DONE IN THE EPITHELIAL CARCINOGENESIS SECTION OF THE CELL AND CANCER BIOLOGY BRANCH OF CCR. THREE OBJECTIVES, FIRST TO EXAMINE EFFECTS OF DELETION AND K-RAS AND LUNG TUMOR INCIDENCE AND PATHOLOGY. THEN TO DETERMINE THE EARLY EVENTS IN THE DEVELOPMENT OF LUNG LESIONS AND HOW THEY PROGRESS ONTO TUMORS. CARCINOMAS. THIRDLY TO IDENTIFY POTENTIAL SIGNAL TRANSDUCTION PATHWAYWAY CHANGES THAT OCCUR WITH TUMORIGENESIS. WE EMPLOYED FOUR MOUSE MODELS, A/J, HETEROZYGOUS. WE ASKED TWO QUESTIONS, DOES LUNG TUMORIGENESIS AFFECT THE TGF-BETA SIGNALING PATHWAY AND DOES THE TGF-BETA SIGNALING PATHWAY AFFECT LUNG TUMORIGENESIS. WE FIRST UTILIZED THE A/J MOUSE MODEL, SUSCEPTIBLE TO CHEMICALLY INDUCE THE LUNG TUMORS WITH SUCH CHEMICALS AS ETHEL CARBONATE. THE LUNG TUMORSOR DEVELOP IN A TIME DEPENDENT MANNER AND PROGRESS THROUGH STAGES OF HYPERPLASIA, ADENOMA AND CARCINOMAS. THE CARCINOMAS THAT DEVELOP HAVE BEEN SHOWN TO BE HISTOLOGICALLY SIMILAR TO HUMAN LUNG ADENOCARCINOMA, AND VERY IMPORTANTLY THE SAME MOLECULAR MUTATIONS OCCUR IN HUMAN AND MOUSE LUNG TUMORS, SUCH AS OVEREXPRESSION OF RAS AND LOSS OF P53. SO TO GIVE YOU A LITTLE BIT OF BACKGROUND ABOUT ETHEL CARBAMATE, IT CAN BE METABOLIZED IN TWO PATHWAYS, FIRST THE DETOXIFICATION ON THE LEFT, ESTERASE CONVERTS THE MOLECULE INTO HARMLESS ETHANOL AND AMMONIA, THERE’S A BIOACTIVATION PATHWAY, AND REDUCES ETHEL CARBAMATE TO VINYL CARBAMATE AND THIS IS AIBLE TO BIND TO VARIOUS MACROMOLECULES AND CAUSE A LOT OF PROBLEMS. SO WE INITIALLY STARTED OUR STUDY BY INJECTING ETHEL CARBAMATE TWO 2-MONTH-OLD AGING MICE, LETTING THEM SIT AND SACRIFICING THEM AT MONTHLY INTERVALS UP TO 12 MONTHS. AND HERE I SHOW YOU A TYPICAL IMMUNOSTAINING PATTERNS IN THESE LUNG TUMORS, AND THEN THE LEFT PANEL, MIDDLE PANEL, YOU CAN SEE INTENSE BROWN STAINING FOR TGF-BETA1 LIGAND AND TYPE 1 RECEPTOR IN ALL THE STAGES OF TUMORIGENESIS. HOWEVER, IF YOU LOOK AT THE TYPE II RECEPTOR IN THE RIGHT-HAND PANELS, YOU CAN SEE THAT THERE’S DECREASED STAINING FOR THE TYPE II RECEPTOR IN THE TUMORS, INDICATING THERE’S DECREASED REDUCTION OF A TYPE II RECEPTOR IN THESE TUMORS. THIS IS BETTER SHOWN IN THIS SLIDE ON THE LEFT, WHERE WE COMPARE STAINING FOR THE TYPE 1 RECEPTOR, THE TYPE II RECEPTOR, AND WE CAN SEE BROWN STAINING IN THE ADENOCARCINOMA, AND BRONCHIAL, WE CAN SEE REDUCED STAINING FOR PRECEPTOR PROTEIN IN THE TUMORS WITH TYPE I RECEPTOR. WE ALSO LOOKED AT THE MESSENGER RNA FOR TYPE I AND TYPE II, AND I POINT YOUR ATTENTION TO THE PCC4 LINE ON THE FAR RIGHT. THIS CELL LINE WAS DERIVED FROM AN ETHEL CARBAMATE INDUCED MOUSE LUNG TUMOR AND WE SEE DECREASED MESSAGE FOR THE TYPE 1 RECEPTOR AND TYPE II RECEPTOR IN CELL LINES. WE ALSO INVESTIGATED THE EXPRESSION OF THE PROTEIN AND MESSENGER RNA FOR TGF-BETA 1, TIE 1 AND TYPE II RECEPTORS AND AGING MOUSE TUMORS AS WELL, AND THE TOP PANEL SHOW INTENSE STAINING FOR THE TGF-BETA 1 LIGAND IN TYPE 1 RECEPTOR BUT DECREASED STAINING FOR TYPE II RECEPTOR, MIDDLE PANEL SHOWS ALSO ABUNDANT EXPRESSION OF THE MESSENGER RNA, FOR THE PROTEINS, FOR TGF-BETA1 AND TYPE I RECEPTOR BUT REDUCED STAINING FOR THE TYPE II RECEPTOR. THE BOTTOM STEP PANELS SHOW IMMUNOSTAINING FOR ALL THREE PROTEINS, AND NORMAL SURROUNDING BRONCHIAL TISSUE SO WE’RE SEEING IN THESE MICE AS WELL DECREASED EXPRESSION OF THE TYPE I — I’M SORRY, TYPE II RECEPTOR MRNA PROTEIN AND THESE ADVANCED TUMORS. SO OUR MODELS SHOW THERE WAS REDUCED EXPRESSION OF THE TYPE II RECEPTOR WITH THE LUNG TUMOR PROMOTION. NOW, WE ASKED THE ADDITIONAL QUESTION, DOES DELETION OF TGF-BETA1 AFFECT LUNG TUMORIGENESIS? FOR THIS WE USED THE C57BL MOUSE, THE BLACK SIX KNOCKOUT MOUSE WAS GENERATED AS AT THE NCI IN THE ’80s, AND THE MOUSE SHOWN IN THE UPPER — THE KNOCKOUT MOUSE WHICH BOTH ALLELES HAVE BEEN DELETED IS BORN, AND IS SMALLER THAN ITS WILD-TYPE LITTERMATE. BY 21 DAYS AFTER BIRTH THEY SUCCUMB AND DIE QUICKLY, THIS MOUSE WOULD NOT BE A GOOD MODEL FOR USE IN THE TUMORIGENESIS STUDY. BUT UNLIKE THE KNOCKOUT MOUSE, THE HETEROZYGOUS LITTERMATES IN WHICH ONLY ONE ALLELE OF TGB BETA IS SURVIVED, THRIVES AND GOES TO ADULTHOOD. IN COLLABORATION WITH LAKEFIELD AT THE NCI WE COLLABORATED TO FIRST LOOK AT — WE TREATED WITH DIETHEL KNOCK (INAUDIBLE) AND WE DID SEE IN THE LIVER THERE WAS INCREASED TUMORIGENESIS IN THE LIVER, BUT UNEXPECTEDLY AND SURPRISINGLY WE ALSO SAW INCREASED TUMORIGENESIS IN THE LUNG, EVEN MORE ELEVATED THAN THE LIVER. WE’RE SCRATCHING OUR HEADS TRYING TO FIGURE OUT WHAT’S GOING ON. IN ORDER TO GET THE SYSTEM INTO A BETTER SYSTEM FOR LOOKING AT THE LUNG AND NOT THE LIVER, WE DECIDED TO CROSS THE C57BL/6 MOUSE WITH THE WILD TYPE AGING MOUSE, TO MAKE A GENERATION OF TGF 11 HETEROZYGOUS IN THE BACKGROUND, AND IN THIS SLIDE WE DID A PRELIMINARY IN SITU HYBRIDIZATION TO SO DECREASED STAINING AND EXPRESSION OF THE TGF-BETA MESSAGE IN THESE MICE, THESE ARE — I’M SHOWING THE NORMAL MOUSE BRONCHOILES. WE DID THE SAME THING WITH COMPETITIVE SHOWING THE HETEROZYGOUS MICE CONTAIN FEWER COPIES THAN THE WILD TYPE LITTERMATES, SO OUR MODEL WAS WHAT WE HOPED IT WOULD BE, SO AGAIN WE INJECTED THESE MICE WITH ETHEL CARBAMATE, TWO MONTHS OF AGE, SACRIFICED MICE AT MONTHLY INTERVALS USING THE TWO, AND LOOKED AT THE TUMORS. FORGIVE ME, IT’S A BUSY SLIDE. BUT THERE IS INCREASED HYPERPLASIA AND ADENOMA IN THE TGF-BETA 1 HETEROZYGOUS MICE SHOWN IN RED COMPARED TO THE WILD TYPE MICE IN GREEN. OKAY? AND I WANT YOU TO FOCUS MORE SPECIFICALLY ON THE CARCINOMA BECAUSE IT SEEMS LIKE IT TAKES UP TO — WELL, IN THE HETEROZYGOUS MICE, BEGINS CARCINOMA BY FOUR MONTHS OF AGE BUT TAKES AT LEAST 12 MONTHS BEFORE WE SAW ANY CARCINOMAS AND VERY FEW CARCINOMAS IN THE WILD TYPE MICE. SO THERE’S IS INCREASE THE TUMOR INCIDENCE AS WELL AS MULTIPLICITY AND DECREASED TUMOR LATENCY IN THE TGF-BETA1 HETEROZYGOUS COMPARED TO WILD TYPE LITTERMATE IN TERMS OF TUMORS. NOW, WE ALSO WERE INTERESTED IN THE TYPE II RECEPTOR PROTEIN IN THESE LUNG LESIONS, WE STAINED FOR THE TYPE 2 RECEPTOR AND AGAIN IN THE CARCINOMAS, SPECIFICALLY THE CARCINOMAS, WE SEE REDUCED STAINING IN THE HETEROZYGOUS COMPARED TO THE WILD TYPE LITTERMATES. TGF-BETA PATHWAY IS BEING AFFECTED IN LONG TERM REGENESIS, WE WERE INTERESTED IN LOOKING AT WHAT HAPPENED WITH TUMOR GENESIS AND LOOKED AT THE VARIOUS STAGES OF TUMORIGENESIS, HYPERPLASIA,ED A ADENOMA CARCINOMA, VARIOUS TYPE II RECEPTOR WITH INCREASING TUMORIGENESIS. OUR NEXT QUESTION WAS DOES DELETION OF TGF-BETA1 AND MUTATION OF K-RAS IN COMBINATION AFFECT LUNG TUMORIGENESIS? AND WE USED THE TGF-BETA 1 HT WAS MOUSE, REQUIRING AT LEAST ONE STEP TO ACTIVATE THE TUMORIGENESIS SYSTEM. SO WE CROSSED THE MOUSE WITH BLACK SIX WITH K-RAS LATENT ACTIVATABLE MOUSE, WHICH WAS IN THE BACKGROUND SV129 TO MAKE THE GENERATION OF A HETEROZYGOUS K-RAS LATENT ACTIVATABLE DOUBLE MUTANT, THE K-RAS LATENT ACTIVATABLE SINGLE MUTANT HETEROZYGOUS AND WILD TYPE, FOUR GENOTYPES TO BE CONCERNED WITH, AND WE LET THESE MICE GO FOR ABOUT FOUR MONTHS, AND THEN WE OPENED THEM UP AND LOOKED AT THEIR LUNGS. AND AT THE TOP TWO PHOTOGRAPH, A AND B, YOU CAN SEE NODULES ON THE SURFACE OF THE LUNGS, AND THE DOUBLE MUTANT AND THE K-RAS SINGLE MUTANT, BUT IN THE HETEROZYGOUS MOUSE AND WILD-TYPE MOUSE SHOWN IN THE BOTTOM TWO PANELS YOU CAN SEE NO NODULES. IN FACT, IT’S VERY RARE THAT YOU SAW ANY KIND OF NODULE THERE. SO WE LOOKED TO SEE THE EFFECT OF TGF-BETA1 GENE DELETION AND K-RAS FOR GENOTYPE AND WE OBSERVED THE DOUBLE MUTANT SHOWN IN A AND THE K-RAS SINGLE MUTANT SHOWN IN B SHOWED DECREASED LIFESPANS IN THE DOUBLE MUTANT AND SINGLE K-RAS MUTANT MICE COMPARED TO THE HETEROZYGOUS MOUSE AND THE WILD-TYPE MOUSE ON THEIR OWN. WE LOOKED AT THE PATHOLOGY OF THE LUNG LESIONS IN THESE MICE AND INCREASED HYPERPLASIA AND ADENOMA IN THE A AND B SINGLE K-RAS MUTANT MICE, BUT IN CONTRAST WE SAW INCREASED CARCINOMA IN A DOUBLE MUTANT LUNGS, SO IT TAKES A WHILE FOR THE CARCINOMAS TO BUILD UP BUT WHEN THEY DO, THE DOUBLE MUTANT REALLY IS EXPLOSIVE IN TERMS OF TUMORS. WE STAINED FOR THE TGF-BETA 1 AND TYPE 2 RECEPTORS IN THE LUNG LESIONS, I DRAW YOUR ATTENTION TO PANELS ON THE RIGHT WHERE YOU SEE THE PINK ARROWS, AND IT SHOWS REDUCED EXPRESSION OF LIGAND IN TYPE II RECEPTOR IN THE DOUBLE MUTANT TUMORS COMPARED TO THE SINGLE MUTANT K-RAS LATENT ACTIVATABLE MOUSE IN THE CARCINOMAS. NOW, WE’RE ALSO INTERESTED IN WHAT’S HAPPENING DOWNSTREAM, SO WE LOOKED AT IN ADDITION TO THE K-RAS PROTEINS INVOLVED IN SIGNALING, AND HERE WE SHOW A WESTERN BLOT WHERE THERE ARE EXPEDITED REDUCTION OF THE TYPE II RECEPTOR AND INCREASED PRODUCTION OF THE SMAD 3 PROTEIN IN THE DOUBLE MUTANT ON THE RIGHT COMPARED TO THE SINGLE K-RAS MUTANT ON THE LEFT. VERY LITTLE HAPPENS IN THESE. THE PROTEINS ARE EXPEDITED IN TERMS OF REDUCTION, SMAD PROTEIN IS EXPEDITE THE IN TERMS OF PRODUCTION SO THERE’S A DIFFERENCE HERE. WE SEE REDUCED PRODUCTION OF SMAD 7 AND 4 AND EXPEDITEDDED PRODUCTION OF K-RAS IN THE RAS PATHWAY. SO KNOWING THAT TGF-BETA ALSO CAN IMPACT APOPTOSIS WE LOOKED AT APOPTOSIS IN THE DOUBLE MUTANT AND SINGLE MUTANT K-RAS LUNG TUMORS, AND SHOWED ESSENTIALLY REDUCING APOPTOSIS IN THE DOUBLE MUTANT AS SHOWN IN RED, COMPARED TO THE K-RAS SINGLE MUTANT. SO OUR SYSTEM SHOWS USING THESE VARIOUS MOUSE MODELS THERE’S DECREASED EXPRESSION OF A TYPE II RECEPTOR WHICH CAN LEAD TO LUNG TUMOR PROMOTION, AS WELL AS ACTIVATED RAS MAP KINASE CAN LEAD TO LUNG TUMOR PRODUCTION, DECREASED PRODUCTION OF SMAD 4, DIDN’T SLOW YOU MUCH OF THIS BUT DECREASED SMAD 4 PRODUCTION AND COMPROMISED APOPTOSIS, OR CONTRIBUTING LUNG TUMOR PRODUCTION. SO OUR SYSTEM IS GEARED FOR LUNG TUMOR PROMOTION. SO THE CONTEXT IN WHICH TGF-BETA REALLY IS PUT IN TO MAKE A LARGE DIFFERENCE TO WHAT IT CAN AND CANNOT DO, SPECIFICALLY WHEN IT CAN ACTIVATE A TUMOR SUPPRESSOR, WHEN IT CAN ACT AS TUMOR PROMOTER. SO I’D LIKE TO ACKNOWLEDGE THE PEOPLE WHO WERE INVOLVED IN THIS WORK WHO HAVE SINCE MOVED ON. AND WE’VE GOT THE K-RAS NOISE FROM THE TYLER JACKS LAB AT M.I.T. I’LL ENTERTAIN ANY QUESTIONS YOU MIGHT HAVE AT THIS TIME. [APPLAUSE] OKAY, THANK YOU. YES?>>(OFF MIC.)>>NO, BECAUSE THE INACTIVE CANNOT REALLY PARTICIPATE IN TUMORIGENESIS. IT HAS TO BE ACTIVATED. (OFF MIC) NO, IT HAS TO BE ACTIVATED TO BE SECRETED.>>(OFF MIC.)>>THERE ARE A NUMBER OF COMPOUNDS THAT CAN ACTIVATE SUCH AS ACID, INCREASED HEAT, DECREASED Ph.D. AND IT’S NOT REALLY UNDERSTOOD WHAT REALLY ACTIVATES IT INTERNALLY IN THE BODY, YEP. YES?>>(OFF MIC.)>>WELT IN OUR SYSTEM WE CREATED WITH A LIVER CARCINOGEN AND A LUNG CARCINOGEN, SO THEREFORE W E GOT THE LIVER AND LUNG BASICALLY. I SUPPOSE IT CAN BE TURNED ON BY OTHER TYPES OF TISSUE PROMOTERS AS WELL.>>(OFF MIC)>>THAT’S A GOOD QUESTION. I DON’T THINK IT’S BEEN LOOKED AT. I WOULD SUSPECT THAT THERE PROBABLY IS, DEPENDING ON WHAT THE ENVIRONMENT IS, YOU KNOW. I THINK THERE WOULD BE. OKAY. THANK YOU.

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