TRACO 2014 - Ovarian cancer; Immune checkpoints

“en”:”>> OKAY, WE’RE GOING TO GET STARTED. SO THIS WEEK I’VE GOTTEN ALL OF YOU TRACES FOR THE TRACO COURSE. ONE OF WHICH IS GOING TO BE A FACILITY WHERE THEY HAVE THE ROBOTICS. THE TITLE THE USE OF ADVANCED TECHNOLOGIES AND SMALL MOLECULES FOR TRANSLATIONAL RESEARCH. SO A COUPLE OF INTRODUCTORY REMARKS. YOU WILL HEAR FROM ME VARIOUS VERSIONS OF THIS EXAMPLE, TRANSLATIONAL RESEARCH TEAM SPORT REALLY TAKES MULTIPLE DISCIPLINES TO ADVANCE BASIC RESEARCH DISCOVERIES TO. IN THE TALK TODAY YOU’LL SEE EXAMPLES, BIOLOGISTS WORKING, CHEMISTS. SO KEEP THIS IN THE BACK OF YOUR MIND. IT’S VERY DIFFERENT TYPE OF GAME WHERE YOU REALLY NEED MULTIPLE AREAS OF EXPERTISE COMING TOGETHER TO ADVANCE THESE BASIC DISCOVERIES.

I’LL BE TALKING ABOUT TYPES OF SCREENING AND IT HELPS YOU DEFINE SOME OF TERMS OR PROVIDE DEFINITIONS. I LOST MY SCREEN HERE SO I’M GOING TO TURN THIS WAY. SMALL MOLECULES, THESE ARE GENERALLY SYNTHETIC OR NATURALLY OCCURRING SUBSTANCES THAT ARE USUALLY BELOW THOUSAND MOLECULAR WEIGHT AND OF COURSE A LOT OF TRADITIONAL DRUGS YOU SEE IN USE ARE SMALL MOLECULES. OF COURSE NOW WE HAVE MORE AND MORE THE OTHER FLAVORS, PROTEINS, GENE THERAPIES, CELL THERAPIES AND SO ON. SO WHAT IS AN ASSAY. NOT EVERY TEST THAT ONE RUNS IN THE LAB CAN BE CALLED AN ASSAY.

ASSAY USUALLY CAN BE REPEATED MULTIPLE TIMES WITH VERY LOW POSSESSES ADEQUATE SIGNAL TO BACKGROUND AND CAN BE SCALED DOWN OR MINIATURIZED REPEAT TESTING WHICH BRINGS ME TO THE NEXT TERM HIGH-THROUGHPUT WHICH IS THE EXECUTION OF ASSAY MANY TIMES OVER TO TEST COMPOUND ACTIVITY AGAINST PARTICULAR TARGET. TARGET BEING ISOLATED PROTEIN, PATHWAY, YOU NAME IT. AND A LIBRARY IS A COLLECTION OF SMALL MOLECULES TYPICALLY OVER HUNDRED SMALL MOLECULES ARRIVED IN DIFFERENT FORMATS. USUALLY MICRO — PLATES FREQUENTLY DISSOLVED IN A STANDARD SOLVENT.

AND THIS ALLOWS YOU TO RUN THESE SCREENS IN PARALLEL FASHION GENERATING MANY DATA POINTS, EITHER SIMULTANEOUSLY OR OVER THE COURSE OF A COUPLE HOURS OR DAYS. I’M GOING TO ALSO TALK ABOUT NEW DEVELOPMENTS IN THE FIELD TELLING YOU ABOUT HOW WE DEVELOPED A HIGH-THROUGHPUT PLATFORM TO TEST DRUG CANDIDATES FOR REFURBISHING APPLICATIONS, FOR NEW INDICATIONS, AND ALSO TESTING FOR ADVANTAGEOUS DRUG-DRUG COMBINATIONS. BECAUSE AS YOU KNOW, DRUG COCKTAILS ARE NOW FREQUENTLY USED IN MANAGING, SAY, HIV. BUT HOW DO YOU GO ABOUT DISCOVERING NOVEL DRUG-DRUG COMBINATIONS RAPIDLY. LAST BUT NOT LEAST, THERE’S THE WHOLE ASPECT OF TOXICOLOGY, A LOT OF DRUGS FAILED BECAUSE OF TOXICITY. WE’RE WORKING IN THIS AREA TO DEVELOP BETTER PREDICTIVE METHODS TO PRIORITIZE COMPOUNDS, AND BY COMPOUNDS IN THIS CASE I MEAN DRUGS, DRUG CANDIDATES AND TYPICAL CHEMICALS THAT EXIST IN COMMERCE. BECAUSE FOR VERY FEW MOLECULES OUT THERE, THERE’S ANIMAL DATA ON HOW TOXIC OR SAFE THEY ARE.

SO THE WAY WE OPERATE THIS PROJECT IS THEY’RE ALL COLLABORATIONS. AGAIN TRANSLATION IS A TEAM SPORT. WE PROVIDE THE TECHNICAL EXPERTISE, KNOWLEDGE ABOUT THE PROCESS, KNOWLEDGE ABOUT ASSAYS, HIGH-THROUGHPUT SCREENING. BUT WE DO NOT PRETEND TO KNOW THE BIOLOGY OR THE PARTICULAR DISEASE THAT WE’RE STUDYING. THAT EXPERTISE COMES FROM THE OUTSIDE FROM THE COLLABORATORS WHO BRING TO US THEIR KNOWLEDGE, THEIR TEST SYSTEM AND THEIR DOWN STREAM EXPERTISE AS WELL AS SAY SEE NO GRAPH MODELS BEYOND HIGH-THROUGHPUT SCREENING. WE HAVE A FASHION THAT EXPANDS THE ENTIRE RANGE OF REDUCTIONISM IF YOU WILL, STARTING FROM THE SIMPLEST SYSTEM ISOLATED PROTEINS WHICH IS ENZYMATIC ASSAYS, PROTEIN PROTEIN INTERACTIONS. PROTEIN DNA INTERACTIONS AND SO ON. GOING TOWARDS PATHWAYS, LOOKING AT PARTICULAR PATHWAY TO IMBEDDING THE REPORTERS DOWN STREAM OR BY VARIOUS MEANS STAINING ZONE AND LAST BUT NOT LEAST THE HIGH THROUGHPUT LEVEL ON THE ASSAYS OR HIGH CONTENT SCREENS WHERE YOU DON’T NECESSARILY LOOK AT A PARTICULAR PATHWAY, YOU MANY TIMES DON’T KNOW THE TARGET BUT YOU’RE LOOKING FOR A PARTICULAR CHANGE IN THE CELL, WHETHER IT’S A TRANSLOCATION OR OTHER FEATURES.

SO SCREENING IS CAN BE DONE IN VARIOUS PLATE FORMATS. THESE REALLY REPRESENT THE PICTURES ON THE MOST COMMON PLATE TYPES. WE HAVE SET UP IN OUR PLACES REALLY THE MOST ADVANCED TECHNOLOGICAL VERSION OF HIGH THROUGHPUT SCREENING WHICH IS THE USE OF 1535 WELL PLATES. I’M NOT GOING TO GO THROUGH THE MATH HERE BUT YOU CAN EASILY SEE WHY IT IS IMPORTANT TO ADOPT THIS TECHNOLOGY FOR A LOT OF TYPES OF QUESTIONS THAT WE WANT TO ASK.

WE CAN ONLY ANSWER THEM OR APPROACH THEM BY USING THE 1536 WELL PLATE TECHNOLOGY. USING THE OTHER PLATES WILL LEAD TO VERY LONG SCREENS LASTING MANY MONTHS, OR THROUGH PRECIOUS REAGENTS THEY WILL MAKE THE SCREEN LITERALLY IMPOSSIBLE, EITHER TOO EXPENSIVE OR IMPOSSIBLE TO SET UP BECAUSE OF LACK OF SAY ENOUGH PRIMARY CELLS AND SO ON. YOU’RE REALLY SHRINKING THESE ASSAYS DOWN TO THE VOLUME OF TWO TO FIVE MICRO LITERS AS OPPOSED TO LARGE VOLUMES HERE. AND YOU’RE HANDLING A LOT LESS PLATES PER EXPERIMENT.

THIS IS IN THE SYSTEM SHOWN THROUGHOUT HERE. THEY MOVE THE PLATES AROUND, THEY REMOVE THE LIDS FROM THE PLATES AND TRANSPORT THEM FROM WORK STATION TO WORK STATION IN A COMPLETELY AUTOMATED MANNER. THERE ARE CELLS FOR THE COMPOUND LIBRARIES FOR RESOURCES. WE CAN INCUBATE SAY FOR MAMMALIAN CELL. YOU ARE LIMITED BY WHAT REAGENTS YOU HAVE AND HOW COMPLEX THE PROTOCOL IS AND HOW TO CUE THE PLATES AND MAKE SURE EVERYTHING RECEIVES THE SAME TYPE OF TREATMENT. EVERY PLATE IS TREATED EXACT SAME WAY WITHOUT WAITING OR SKIPPING STEPS AND SO ON. SO THIS IS ALL NICE AND OTHER FACILITIES HAVE THIS TECHNOLOGY. SO WHEN WE WERE SETTING UP THIS SYSTEM BACK IN 2005, WE WANTED TO REALLY ADVANCE THE FIELD REGARDLESS OF DISEASE AREA OR ASSAY METHODOLOGY.

SO WE ACQUIRED THIS SYSTEM BUT WE WERE STILL NOT COMPLETELY SATISFIED WITH THE WAY THE PROCESS WORKS. SO REALLY TRY TO IMPROVE THE PROCESS BY INTRODUCING SOMETHING THAT IS REALLY INTUITIVELY VERY OBVIOUS, WHICH IS WHY NOT TEST EVERY MOLECULE IN A DOSE RESPONSE FASHION. TRADITIONALLY HIGH THROUGHPUT SCREENING IS PERFORMED AT A SINGLE CONCENTRATION OF YOUR LIBRARY, WHETHER IT’S TEN MICRO MOLAR OR 20 MICRO MOLAR. STATISTICAL CUT OFFS TO THE DATA THAT IS OBTAINED FROM THE SCREEN AND ONE PICKS THESE HITS, SO-CALLED HITS BASED ON SAY EITHER GREATER THAN 50% ACTIVITY IN WHATEVER ASSAY YOU HAVE OR OTHER CRITERIA.

SPURIOUS NOISE FROM A SINGLE EXPERIMENTAL RESULT IS STILL CLOCKED AS A HIT. ONLY LATER WHEN YOU REOBTAIN THAT SAMPLE AND RETEST IT CAREFULLY, YOU REALIZE THAT IT WAS SINGLE POINT NOISE. MAYBE PIECE OF DUST HIT THE PLATE OR LIQUID DISPENSER ADDED DIFFERENT AMOUNT OF REAGENT. A LOT OF RESOURCES ARE WASTED AT THAT POINT. YOU ALSO MISS SHALLOW CURVES AND OTHER PECULIAR RESPONSES. SO WE ACTUALLY INVESTED A LOT OF EFFORT INTO DEVELOPING THIS DOSE RESPONSE BASE SCREENING, WHICH WE CALLED QUANTITATIVE HIGH THROUGHPUT SCREENING BECAUSE YOU REALLY ARE ADDING THE QUANTITATION DIMENSION TO IT. DILUTING THE COMPOUND LIBRARY WHICH IS FOUR ORDERS OF MAGNITUDE ACROSS. FOR THE ACTIVE COMPOUND YOU GET ACTUAL DOSE RESPONSE CURVES WHICH YOU CAN FIT THROUGH THE EQUATION AND ARRIVE OF COEFFICIENT, IC50’S AND SO ON. YOU CAN DELINEATE BETWEEN NICE PERFECT CURVES WHICH — WHICH ARE SHALLOW. YOU CAN HAVE COMPLETELY NEGATIVE OR NULL RESPONSE WITH ONE OUTLIER.

YOU CAN PRETTY QUICKLY L AUTOMATICALLY FIGURE OUT WHETHER SOMETHING IS SPURIOUS NOISE OR SOMETHING WORTH PURSUING. YOU CAN ALSO RANK THE COMPOUNDS BASED ON POTENCY AND BEGIN TO DERIVE STRUCTURE ACTIVITY RELATIONSHIPS BASED ON THE WHOLE RELATIONSHIP BETWEEN ACTIVITY DERIVED FROM THESE CURVES AND THE CHEMICAL STRUCTURES OF THOSE HITS. SO WE ALSO HAVE DEVELOPED A SYSTEM TO RUN SCREENS IN SAFETY LEVEL THREE ENVIRONMENT. THE ROBOTIC SYSTEM I SHOWED IN PREVIOUS SLIDE IS ONLY QUALIFIED TO RUN ASSAYS UP TO DSL2 LEVEL.

IN VARIOUS LAB BUILDING 33 WE SET ASSIST TELL TO ALLOW US TO RUN BSL-3 TYPE INFECTIOUS ORGANISMS. THESE PROTOCOLS ARE MORE COMPLICATED. A LOT OF TIMES THEY REQUIRE LONGER INCUBATION TIMES. SO THE TYPE IN THIS SYSTEM IS ONLY 354 WELL PLATE. WE DON’T SCREEN IN 1536 WELL PLATES IN THIS FACILITY. BECAUSE AGAIN, FOR EXAMPLE, FOR SLOW GROWING PARASITES YOU LITERALLY NEED PROTOCOLS THAT ARE FIVE TO SEVEN TO 20 DAYS. STILL THE SYSTEM IS CAPABLE OF PERFORMING ALL KINDS OF COMPLEX STEPS LIKE CEILING PLATES, INCUBATING READING MULTIPLE TYPES AND SO ON. WE USED IT TO RUN PILOT SCREENS ON MICRO BACTERIUM OR GEARING UP TO RUN A MERS TYPE SCREEN OF THAT FACILITY. EVEN THOUGH IT’S NOT PART OF THE INFRASTRUCTURE WE HELPED DEVELOP IT AND USE IT. ANOTHER ASPECT OF THIS WHOLE THING IS TEACHING THESE COLLABORATORS, PEOPLE LIKE YOU, WHAT AN ASSAY IS AND HOW TO THINK ABOUT CONFIGURING GOOD TYPE OF THE SCREENING ASSAY. A BAND ON THE WESTERN BLOCK IS NOT A SCREENING ASSAY FOR EXAMPLE. THINGS THAT REQUIRE CELL SORTING, THEY’RE NOT GOOD SCREENING ASSAYS AND SO ON. SO A LOT OF THIS HAS A SORT OF TEACHING ASPECT TO IT.

THIS WHOLE STARTED ABOUT TEN YEARS AGO WITH US ACTUALLY CONVINCING ELI LILLY LAWYERS TO RELEASE THEIR INTERNAL ASSAY DEVELOPMENT MANUAL. INITIALLY THIS WAS JUST A WEBSITE WITH A COUPLE OF VOLUNTEERS ADDING CHAPTERS TO THAT MANUAL AND BASICALLY A WEB LINK POINTING TO THE WHOLE MANUAL WITHOUT HAVING THE ABILITY CONVERTED TO PDF WERE PRINTED FOR COPYRIGHT REASONS. NOW IT’S GROWN INTO AN ACTUAL E-BOOK WITH A LARGE BODY OF EDITORS. AND WE’RE HOPING TO ADD NEW CHAPTERS TO IT THAT PERTAIN TO PRECLINICAL DEVELOPMENT AND ALSO IN THE FUTURE STEM CELL-BASED ASSAYS. SO, IN ADDITION TO SCREENING, THIS WHOLE PROCESS OF EARLY DISCOVERY COMING UP WITH THE TOOL MOLECULE OR IN SOME CASES YOU CAN CALL IT A LEAD OR DRUG CANDIDATE, REQUIRES THAT ONE PERFORM MEDICINAL CHEMISTRY OPTIMIZATION ON ONE OR MORE HITS.

SO WE HAVE AN INTEGRATED PIPELINE THAT DOES THAT WITH ADDITIONAL CHEMISTRY IN THE BUILDING AS PART OF THE TEAMS THAT WE HAVE AGAIN GOING BACK TO THE NOTION THAT TRANSLATION IS A TEAM SPORT. THE PURPOSE OF MEDICINAL CHEMISTRY IS FOLD, OF COURSE. ONE WANTS TO IMPROVE THE POTENCY OF THE MOLECULE AGAINST THE INITIAL TARGET. THIS IS AN EXAMPLE OF DEVELOPED AGAINST SPECIFIC PROTEASE ONE. USB1 DEALING WITH THE FIELD OF PROTEIN — CONTROL AS RELATES TO DNA DAMAGE RESPONSE — MEDICINAL CHEMISTRY IS TO IMPROVE THE COMPOUNDS. THINGS LIKE — MICROSOMAL PERMEABILITY, CELL PERMEABILITY, SELLS THAT DON’T RELATE TO THE AFFINITY OF THE MOLECULE TARGET BUT OBVIOUSLY ARE VERY MUCH NEEDED IN ORDER FOR ONE TO TRANSITION FROM A TEST EXPERIMENT TO SAY ANIMAL EXPERIMENT FOR TESTING IN HUMANS.

THE MOLECULE CANNOT BE VERY UNSTABLE SUCH THAT YOU WOULD HAVE VERY SHORT PATH LIFE. SOME MODIFICATIONS CAN BE MADE TO MAKE IT MORE STABLE, REMOVE SOME METABOLIC LIABILITIES AND SO ON. I WANT TO GIVE YOU ONE QUICK EXAMPLE OF AREA BIOLOGY WE WORKED ON FOR A WHILE, KIND OF TAKE YOU THROUGH THIS JOURNEY OF STARTING FROM UNDER STUDY TARGET CATEGORIES, FINDING A SMALL MOLECULE MODULE LATER TO BASICALLY CATEGORY TO ALLOW FOR CERTAIN EXPERIMENTS TO BE RUN FOR HYPOTHESES TO BE TESTED AND REALLY WHERE THIS JOURNEY TOOK US IN THE END.

SO BACK AROUND FOUR OR FIVE YEARS AGO, THERE WAS VERY LITTLE KNOWN ABOUT THE HISTONE MODIFYING OR INTERACTING PROTEINS. AND THESE ARE SEVERAL CATEGORIES LOOSELY DEFINED AS WRITERS, READERS AND ERASERS. WRITERS BEING EPIGENETIC MARKETS LIKE HISTONE METHYL TRANSPHRASES. READERS INTERACT WITH CERTAIN STATE ON HISTONE PROTEINS FOR EXAMPLE RECOGNIZING DIMETHYL VERSUS TRY METHYL AND OTHER PROTEINS FOR TRANSCRIPTION AND OTHER PROCESSES. SO THERE ARE MANY EXAMPLES HERE OF SUBFAMILIES — BEING THE MOST PROMINENT. AND THE ERASERS ARE THE ENZYMES THAT REMOVE MARKS SUCH AS HISTONE DEACETYLASE THAT REMOVES METALS FROM HISTONE TAILS ON ARGININES AND LYSINE. THERE WAS NO SCREENING ASSAY FOR ANY OF THESE. THERE WAS NO CONSTELLATION PHARMACEUTICALS ANY OF THE COMPANIES THAT WORKED IN THE FIELD OR NEAR THE FIELD. WE ACTUALLY SET UP SOME OF THE FIRST SCREENING ASSAYS FOR ALL OF THESE CATEGORIES AND RUN THE FIRST WELL BASED SCREENS. I’LL GIVE YOU AN EXAMPLE OF THE LAST CATEGORY, THE ERASERS.

SPECIFICALLY WE WORKED ON JUMONJI-DOMAIN HISTONE DEMETHYLASE. THIS SPARKED THE LARGE FAMILY OF DEPENDENT OXYGENASE . BY THE WAY THE HYDROYLASE IS PART OF THAT LARGE FAMILY. THE IMPORTANT THING ABOUT THIS ENZYME CLASS IS THEY TAKE TRY METHYLATED LYSINE TO SUBSTRATES. NO OTHER HISTONE DEMETALASE DOES THAT. ONE IS A PROMINENT MEMBER THAT DOES NOT USE TRI METHYLATED HIS TRONE SUBSTRATES START FROM THE LOWER DEGREE OF METHYLATION. SO AGAIN PRETTY COMPLEX REACTION. HOW DO YOU SET UP A SCREEN TO FIND THE ENZYMES. WE USED A SYSTEM UTILIZING — IS CONVERTED TO FORMALDEHYDE. SO WE COUPLED THE REACTION WITH FORMALDEHYDE DE– FLUORESCENT IN THE UV RANGE. WE WOULD USE EXCESS FORMALDEHYDE HIDRO — ON THE FLY IN LINE IN REAL TIME AND FOLLOWED THE FLORESCENCE AS A TIME COURSE TIME MEASUREMENT.

THIS IS WHAT THE PROTOCOL LOOKS LIKE. WE WERE ABLE TO MINIATURIZE THE REACTION TO FOUR MICRO LITERS TOTAL. AND REALLY THIS VERY SIMPLE SORT OF DIAGRAM ACTUALLY REPRESENTS ABOUT TWO MONTHS WORTH OF TESTING. WHAT CAN BE COMBINED TOGETHER AND WHAT NEEDS TO BE KEPT SEPARATE, SUCH THAT YOU CAN RUN THIS 24/7 WITHOUT THINGS PARTICIPATING AND WITHOUT ENZYMES DEGRADING AND SO ON. WE HAD TO RUN COMBINATION FOR TESTS LITERALLY GOING OVER THE COURSE OF SEVERAL WEEKS TO UNDERSTAND IT. NOW YOU CANNOT MEET SAY THE FIRST TWO COMPONENTS WITH THE ENZYME, YOU HAVE TO KEEP CERTAIN THINGS SEPARATE AND SO.

FINALLY THE PROTOCOL IS ABSOLUTELY SIMPLE. WE HAVE ENZYME, WE HAVE COMPOUNDS INHIBITORS AND THEN WE START THE REACTION BY ADDING EVERYTHING ELSE. WHICH WE COULD COMBINE. REALLY NICE STREAMLINE PROTOCOL WHICH ALLOWS YOU TO RUN THINGS IN THE PARALLEL FASHION UNINTERRUPTED FOR A LONG TIME. AND THEN AGAIN WE COLLECT FLUORESCENT DATA ON THE FLY. SO BACK IN THE DAY WHEN WE RAN THE SCREEN, WE HAD ABOUT QUARTER MILLION COMPOUNDS, AGAIN ALL TESTED IN DOSE RESPONSE. WE FOLLOWED THE SCREEN, GET A LOT OF HITS ACTUALLY, FOLLOWED THE SCREEN WITH LOT OF INFOMATIC ANALYSIS LOOKING AT AUTO FLORESCENCE, PROMISCUOUS-TYPE STRUCTURES AMONG THE HITS AND ALSO HAD SEVERAL EXPERIMENTAL STEPS INCLUDING LOOKING AT THE INHIBITORY PROPERTY OF THESE HITS USING MASS SPEC ASSAY WHICH DOES UTILIZE ENZYMATIC COUPLING OR ANY OTHER BAGGING OR LABELING OF THE SYSTEM.

AMONG THE VALIDATED INHIBITORS WAS THIS HYDROXY — IT WAS MICROMOLAR INHIBITOR AGAINST THE VARIOUS ISOFORMS WITHIN THE JUNONJI — SO IT WAS ACTIVE WITHIN THE FAMILY BUT SELECTIVE FOR DEMETHYLATION. WE COLLABORATED WITH THE STRUCTURAL GENOMICS CONSORTIUM BACK THEN TO DEPRIVE CRYSTAL STRUCTURE, TO REALLY ENABLE US TO UNDERSTAND THE MODE OF BINDING FOR THIS MOLECULE AND THEREFORE MAKE BETTER ANALOGS.

AND WE ALSO HAD INITIAL BETA THROUGH TRANSIENT INSPECTION SITE IMMUNOFLUORESCENT EXPERIMENT WITH THE — OF THE TARGET AND NOT SAY TRANSCRIPTION OR ANYTHING ELSE. SO IT WAS ACTING ON TARGET AND NOT THROUGH SOME SPURIOUS SIDE EFFECT. SO FURTHER EFFORT MAKING WELL OVER A HUNDRED ANALOGS OF THIS ADDITIONAL MOLECULE RESULTED IN THIS IMPROVED ANALOGUE WITH BETTER IN VITRO PROPERTIES. AND OF COURSE WE PUBLISHED MOST OF THIS EARLY WORK. AND AGAIN THE EARLY STORIES SURROUNDING EPI GENETICS WERE RELATED TO CANCER. THIS MOLECULE WAS SENT TO A DOZEN LABS WORLDWIDE TO TEST FOR VARIOUS CANCER MODELS.

BUT WHAT IS INTERESTING IS THE UNINTENDED USE OF THIS MOLECULE. SO IT’S A GREAT TOOL MOLECULE TO STUDY THE EFFECTS OF HISTONE DEMETHYLATION INHIBITION IN CELLS. SO IT WAS PURELY BY CHANCE THAT WE COLLABORATED WITH AN INTRAMURAL INVESTIGATOR HERE IN CAMPUS — FROM THE INFECTIOUS DISEASE TO TEST A NEW BIOLOGICAL HYPOTHESES. WHICH MAY BE THROUGH AFFECTING THE HOST HISTONE DEMETHYLATION MACHINERY ONCE AFFECT VIRAL INFECTION. SO THOMAS CHRISTY’S LAB HAS STUDIED HERPES VIRUS AND RELATED VIRUSES AND THEY DETERMINED THAT THIS IS A DNA VIRUS WHICH UPON INFECTION FORMS CHROMATIN-LIKE STRUCTURES OF DNA COMPACTED AND OF COURSE CARRIES THE REPRESSIVE HISTONE MARKS LIKE — TRI METHYL AND THEY USE THE MACHINERY TO REMOVE THOSE DEPRESSIVE MARKS FOR DEMETHYLATION. AND THEREFORE YOU REACTIVATE ITSELF FROM LATENCY OR TO PROCEED THROUGH WITH PRODUCTIVE INFECTION AND TRANSCRIPTION OF ITS IMMEDIATE EARLY GENES AND SO ON.

SO WE WANTED TO TEST WHETHER THE HISTONE DEMETHYLATION INHIBITORS WE HAD WOULD BE EFFECTIVE IN HERPES VIRUS INFECTION. BECAUSE AGAIN UNLIKE REPLICATION INHIBITORS, THESE WOULD BE ACTING UP STREAM AT AN EARLIER STAGE OF INFECTION AND HOPEFULLY WOULD BE MORE POTENT OR MORE EFFECTIVE IN STOPPING THE INFECTION OR SLOWING IT DOWN. THIS IS INDEED WHAT HAPPENED. ADDING THE JOUMONJI DEMETHYLATION INHIBITOR TO CELLS INFECTED WITH HERPES SIMPLEX VIRUS ONE LEADS TO REDUCTION IN TRANSCRIPTION OF EXACTLY THE IMMEDIATE EARLY GENES AS EXPECTED FROM THIS HYPOTHESES OF UTILIZATION OR THE CROSS MACHINERY. TREATMENT WITH INHIBITOR LEADS TO DRAMATIC REDUCTION IN VIRAL YIELD BASED ON LOOKING AT VIRAL COUNTS. ALSO LOOKING AT PLAQUE ASSAY LEADING TO ALMOST COMPLETE ERADICATION OF THE VIRAL PLAQUES. BY COMPARISON WE HAVE ACYLOVIR WHICH IS THE START OF TREATMENT FOR THE INHIBITOR. YOU CAN SEE THE JUMONJI INHIBITOR PERFORM IF NOT A LITTLE BETTER. THESE ARE IMAGES THAT ARE KIND OF HARD TO FOLLOW. BUT ON THE LEFT SIDE WE HAVE STUDIED IT SHOWS THE INHIBITORY EFFECT OF THE COMPOUND ON INFECTION THAT IS PROGRESSING AND SUPPRESSING THE CONTINUATION OF AN INFECTION. SO YOU WANT TO FOLLOW GREEN HERE WHERE TREATMENT OF THE COMPOUND LEADS TO PRACTICALLY NO GREEN.

AND ACYLOVIR STILL LEAVES SOME GREEN SPOTS WHICH ARE STAINING FOR THE REPLICATION PROTEIN FROM THE VIRUS AND LEAVES A LOT OF GREEN BASICALLY THE INFECTION IS PROGRESSING PRETTY RAPIDLY. ON THE RIGHT SIDE WE HAVE AN X ZERO EXPERIMENT WHERE MICE INFECTED WITH A VIRUS TO FINISH WITH THE INITIAL STAGE OF THEIR INFECTION. THE INFECTION ENTERS LATENCY. WHEN GANGLIA ARE TAKEN OUT OF THESE ANIMALS AND TRANSPLANTED INTO CULTURE MEDIUMS, THE LATENCY IS RELEASED. THERE’S BASICALLY A REACTIVATION PROCESS STARTING AND ONE IN THIS CASE CAN STAIN IN RED FOR THE VIRAL PROTEINS AGAIN. IN THIS CASE WE’RE LOOKING AT THE EFFECT OF ACYLOVIR OR THE JOUMONJI FOR STAINING. THESE INDIVIDUALS ONES ARE CLUSTERS BUT THE COUNT IS SHOWN HERE AND YOU CAN SEE THE TREATMENT WITH THE DEMETHYLATION INHIBITOR OR CLUSTER NEURONS LEAD TO ALMOST COMPLETE REDUCTION OR SUPPRESSION OF REACTIVATION FROM LATENCY WHEREAS TREATMENT WITH ACYLOVIR IS NOT EFFECTIVE EVEN THOUGH ACYLOVIR IS USED WITH A TWICE THE CONCENTRATION OF THE JUMONJI INHIBITOR.

AGAIN THIS IS IN CONCERT WITH THE WHOLE NOTION THAT WE’RE ACTING UP STREAM, PREVENTING THE REACTIVATION WHERE ACYLOVIR ACTS ON THE REPLICATION PORTION OF THE VIRAL LIFE CYCLE. SO WE DON’T HAVE THE ILLUSION THIS WILL BECOME A DRUG. IT HAS CERTAIN CHEMICAL LIABILITIES AND OTHER FEATURES THAT MAY NOT MAKE IT VERY DESIRABLE FOR FURTHER CLINICAL DEVELOPMENT THAT IT WAS REALLY A NICE TOOL TO SHOW THIS IS A VIABLE HYPOTHESES WHICH IS AFFECTING THE HOST SETTLER MACHINERY CAN BE USED TO ATTENUATE VIRAL INFECTION. SOMETHING PEOPLE COULDN’T DO BEYOND THE GENETIC MEANS AND SIR MEANS AND SUCH MOLECULE TOOL WAS AVAILABLE WE STARTED WITH THE FIRST INHIBITORS LOOKING AT MOSTLY CANCER APPLICATIONS. AND WE HAVE A VERY INTERESTING STORY WITH POTENTIAL ANTI-VIRAL APPLICATION. I’M GOING TO SWITCH GEARS AND TALK A LITTLE BIT ABOUT ABOUT ANOTHER ROUTE. WE HAVE ABOUT HALF A MILLION SUCH COMPOUNDS IN THE LIBRARY THAT ARE DIVERSE AND ALL THAT AND EMBARK ON ADDITIONAL CHEMISTRY AFTER SCREENING. AND CALLED ONE DAY FOR AN FDA APPROVAL OF THE DRUG CANDIDATE THAT ONE CAN ALSO START WITH APPROVED DRUGS AND LOOK FOR WHETHER THEY CAN BE USED TO TREAT THE DIFFERENT INDICATION, THE SO-CALLED NOTION OF DRUG REPURPOSING.

THERE ARE MANY SUCH SUCCESS STORIES OF DRUGS THAT ARE FINDING NEW USES. BUT HOW DO YOU REALLY GO ABOUT SYSTEMATICALLY DISCOVERING NEW USES FOR EXISTING DRUGS. WELL THERE ARE TWO COMPONENTS TO IT. ONE IS KNOWING WHAT THE DRUGS ARE, HOW MANY ARE OUT THERE AND THEN ASSEMBLING PHYSICAL COLLECTION OF THOSE IN TESTING IT. I’M NOT GOING TO DWELL ON THIS BUT IT TOOK ABOUT FIVE YEARS FOR OUR INFOMATICS TEAM TO SEARCH DATA BASES WORLDWIDE, LOOK AT DIFFERENT LANGUAGES, TRANSLATIONS, DEAL WITH AMBIGUITIES IN SPELLING. DEAL WITH AMBIGUOUS AMBIGUITIES WITH CHEMICAL STRUCTURES AND FIND OUT HOW MANY SMALL MOLECULES HAVE BEEN APPROVED FOR USES MAN. THESE INCLUDE CURRENTLY MARKETED DRUGS BUT ALSO INVESTIGATIONAL AGENTS FOR DEVELOPMENT FOR SAY BUSINESS REASONS. THIS WORK WAS PUBLISHED SOMETIME AGO AND YOU CAN GET DETAILS FROM THE PAPER BUT THERE’S ALSO A BROWSER ASSOCIATED WITH THE PAPER WHERE ONE CAN ACTUALLY LOOK UP THE DRUGS.

AND AGAIN WE’RE NOT TALKING JUST FDA DRUGS FOR EUROPEAN UNION APPROVED DRUGS. WE ALSO COVER OTHER MAJOR COUNTRIES. NOW WHAT IS THE NUMBER? THERE’S ALMOST 7,000 SUCH AGENTS. AGAIN NOT ALL OF THEM ARE CURRENTLY MARKETED DRUGS. AND AT N CAPS WE HAVE OBTAINED CLOSE TO 3600 OF THESE. THE FIRST THOUSAND OR SO ARE VERY EASY TO OBTAIN. YOU CAN BUY THEM AS A COLLECTION. THERE’S DNA CLINICAL COLLECTION — THESE ARE NO MORE THAN 1 100 MOLECULES. THE REMAINING 2000 OR SO, SOME OF THEM HAD TO BE SYNTHESIZED IN HOUSE OR OBTAINED ONE BY ONE BASIS.

IT’S REALLY A NICE PRECIOUS COLLECTION, AND WE SCREENED IT AGAINST MORE THAN 50 MODELS OF VARIOUS DISEASES INCLUDING RARE DISEASES AND NEGLECTED TROPICAL INFECTIONS. NOW THIS IS ONE PART OF REPURPOSING, BUILDING THE COLLECTION OF THE COMPREHENSIVE COLLECTION OF DRUGS. THE OTHER ASPECT IS USING COMBINATIONS OF DRUGS. FINDING COMBINATIONS THERE’S NOTHING NEW IT’S WITH EXPERT EXPERIMENTATION STARTING WITH THE PATHWAY AND LOOKING UP MOLECULES THAT MAY AFFECT IT AND TRYING TO COMBINE THEM WITH DRUGS. HOW DO YOU GO ABOUT DOING ETHICISTMATICALLY ON A LARGE SCALE. WE NEEDED A PLATFORM FOR THIS. AND AGAIN, THERE’S DUAL USE IN THIS. ONE IS TO FIND BETTER COMBINATIONS THAT HAVE HIGHER EFFICACY, CARRY LESS TOXICITY DUE TO LOWERING OF INDIVIDUAL DRUG DOSES. BUT THEN THE RESULTS FROM COMBINATION STUDIES CAN LEAD TO NEW HYPOTHESES AS TO USE CERTAIN PATHWAYS INSPIRED TO GIVE YOU SYNERGIES.

YOU MAY NOT ARRIVE AT A PARTICULAR NOVEL DRUG-DRUG COMBINATIONS BUT YOU MAY ARRIVE AT A NOVEL BIOLOGY BIOLOGY COMBINATION OR PATHWAY COMBINATION. SO RUNNING THESE SCREENS ON A MASSIVE SCALE CAN BE DOUBLY BENEFICIAL IF YOU WILL. SO THERE IS AGAIN SEVERAL COMPONENTS TO THIS. WE NEED HIGH VALUE LIBRARY, SMALL MOLECULES, EITHER DRUGS OR SOMETHING WE CALL MECHANISTIC INVESTIGATION PLATES. SO THESE ARE LIBRARIES OF SMALL MOLECULES THAT HAVE NOT BEEN IN MAN, THEY HAVEN’T BEEN THROUGH PRECLINICAL STUDIES THAT ARE VERY SELECTIVE BASED ON EXTENSIVE TESTING. SO WE FIND THOSE MOLECULES LITERALLY THROUGH LOOKING AT PAPERS ALMOST MANUALLY IN COPYING — EXAMINING QUALITY OF THE DATA OR PROCUREMENT. THE OTHER ASPECT TO IT IS TO FIND A WAY TO PLATE THESE DRUG-DRUG COMBINATIONS, TO DELIVER THESE COMBINATIONS IN HIGH THROUGHPUT MANNER. AND OF COURSE LAST BUT NOT LEAST, HOW DO YOU ANALYZE THE DATA. YOU NEED A PLATFORM TO MAKE SENSE OF ALL THESE DATA. SO WE START THESE BY SCREENING COMBINATIONS THROUGH AGENTS. BOTH DOSE IN CERTAIN RANGE, APPLYING ACOUSTIC DISPENSE TECHNOLOGY WHICH I WILL SHOW YOU IN A MOMENT. WE FOLLOW THESE SCREENS BY RUNNING THE KIDS THAT ARE MOST SYNERGY IC 11 BY 11 OR 12 BY 12 WITH HIGH RESOLUTION INFORMATION FOR SYNERGIES.

THIS IS THE TECHNOLOGY WE’RE USING. WE’RE USING ACOUSTIC DISPENSED TECHNOLOGY WHERE THE ACOUSTIC ENERGIES APPLY TO A WILDLY CONTAINED SOLUTION OF THE SMALL MOLECULE. FIRES A DROP AND FLIES UPWARDS AND LANDS INTO A RECIPIENT PLATE. THE DROPLETS HAVE LITERALLY EQUALLY PRECISE. AND YOU CAN LITERALLY PRINT ON THE BOTTOM OF THE PLATE DIFFERENT DOSES OF AGENT A AND B. THIS LOWER RIGHT SHOWING THE ZERO POINT SO THERE’S NO DRUG THERE, THERE’S ONLY DMSO VEHICLE. AND ALONG THESE AXIS ON THE ENDS, THE DRUGS ARE DOSED JUST BY THEMSELVES AND THEN IN THE MIDDLE YOU HAVE THE COMBINATION OF BOTH AGENTS. AND REALLY, THIS ALLOWS US TO HIGH THROUGHPUT. LET’S SEE IF I CAN MUTE THIS. THE SYSTEM IS COMPLETELY COMPACTLESS SO THE RECIPIENT IS ON TOP HERE AND THIS IS NOT A PLATE, IT’S JUST A LID FOR DEMONSTRATION PURPOSES. IT’S ON THE BOTTOM AND OF COURSE THE RECIPIENT PLATE IS TURNED RIGHT SIDE UP AND CAN BE PROCESSED FURTHER BY ADDING CELLS TO IT.

I HOPE YOU CAN SEE THIS HERE WHICH IN THE REAL CASE OF NITRATES OR COMBINATION SCREENING WOULD REPRESENT THE TWO AGENTS AT PARTICULAR MOLARITIES. AND WE CAN CUSTOMIZE THIS RANGE VERSUS THAT RANGE TO BE DIFFERENT, DEPENDING ON THE SINGLE AGENT POTENCY. AND WE CAN DO THIS ON THE TIME SPAN OF SAY TWO THREE BASE. WE CAN TEST ONE ONCOLOGY DRUG AGAINST ALL APPROVED DRUGS, SEVERAL THOUSAND COMBINATIONS IN RAPID SECESSION. THE INFOMATICS PLATFORM ALLOWS US TO LOOK AT VARIOUS INDUSTRIES OF SYNERGISM. YOU MAY KNOW THERE’S NOT A PERFECT WAY TO CALCULATE SYNERGY. THERE’S MANY MODELS OUT THERE. WE CAN RANK. IT’S BASED ON VARIOUS INDICES AND WE CAN LOOK AT THE ACTUAL MAP DATA ON THE FLY, SO THIS WAS ALL DEVELOPED IN HOUSE RECENTLY. IF YOU WANT TO KNOW MORE ABOUT THE PLATFORM.

THIS IS A PAPER WE PUBLISHED BACK IN THE WINTER WITH COLLEAGUES ON SOME OF THE FIRST SCREENS RUN USING THIS PLATFORM. WE’RE EXPANDING A PLATFORM NOW TO GO BEYOND ONCOLOGY TYPE APPLICATIONS, BUILDING SPECIFIC LIBRARIES FOR INFECTIOUS DISEASES AND LATER ON IMMUNOLOGY AND OTHER AREAS. BECAUSE AGAIN DEPENDING ON THE TEST SYSTEM, YOU MAY NOT BE ABLE TO SCREEN EVERYTHING AGAINST EVERYTHING. SO YOU KIND OF HAVE TO KEEP IT COMBINED AND YOU HAVE TO BUILD CUSTOM LIBRARIES OR SUBSETS TO RUN THOSE EXPERIMENTS. LAST TOPIC I WANT TO VISIT IS SOMETHING A LITTLE BIT MORE TO THE DRUG DISCOVERY PROCESS OR THE PROCESS OF RUNNING A SCREEN, PERFORMING MEDICINAL CHEMISTRIES, OPTIMIZING A MOLECULE AND PROCEEDING TO SAY ANIMAL AND CLINICAL TESTING.

AND IT IS ACTUALLY A PRETTY BIG PROBLEM WHICH IS TOXICITY OF MOLECULES. WHILE YOU’RE PREPARING A MOLECULE FOR ULTIMATE CLINICAL APPLICATION, YOU’RE MOSTLY OPTIMIZING BASIC PROPERTIES OF THE MOLECULE AGAINST THE TARGET OF INTEREST OR ROUTE OF DELIVERY AND SO ON. HOW DO YOU OPTIMIZE FOR LOWER TOXICITY. IT’S SOMETHING THAT IS REALLY HARD TO PREDICT AND TEST FOR ESPECIALLY IN HIGH THROUGHPUT MANNER AND CLEARLY REMAINS THE PROBLEM. THE RELATED PROBLEM AS PERTAINS TO TOXICITY IS THE FACT THAT FROM ENVIRONMENTAL STANDPOINT, FROM EXPOSURE STAND POINTED, THERE ARE TENS OF THOUSANDS OF CHEMICALS GENERATED BY CHEMICAL INDUSTRY THAT EXIST IN COMMERCE THAT ARE PART OF EVERY DAY MATERIALS OR EVERY DAY LIVES THAT DON’T HAVE ANY TOXICOLOGY DATA ASSOCIATED WITH THEM BECAUSE NOBODY REQUIRED THE MANUFACTURER TO GENERATE SUCH DATA. RUNNING ALL THESE TENS OF THOUSANDS OF CHEMICALS AGAINST TRADITIONAL METHODS FOR TOXICITY ASSESSMENT LIKE LONG TERM RATS OR OTHER STUDIES WOULD BE PROHIBITIVELY EXPENSIVE. SO ONE WAY TO LOOK AT THIS PROBLEM IS TO REALLY THINK HOW TO BREAK DOWN THE PROBLEM OF TOXICITY INTO COMPONENT QUESTIONS OR PATHWAYS AND APPLY HIGH THROUGHPUT METHODS TO BE ABLE TO ACCESS THE THOUSANDS OR TENS OF THOUSANDS CHEMICALS.

OR ANOTHER WAY TO LOOK AT IT IS THE ORGANISM, HUMAN OR ANIMAL IS EXPOSED TO THIS CHEMICAL, CHEMICAL GOES INTO VARIOUS TISSUES, ORGANS. ACTS ON VARIOUS MOLECULAR PATHWAYS, TARGETS WITHIN THE CELL. AND BEYOND HOMEOSTASIS, EVENTUALLY SOMETHING BREAKS AND YOU END UP WITH A TOXICITY OUTCOME ALSO REFERRED TO ADVERSE OUTCOME. WOULDN’T IT BE NICE TO DECONSTRUCT THE RAT INTO VARIOUS CELL-BASED ASSAYS, BIOCHEMICAL ASSAYS, HIGH CONTENT ASSAYS, GENERATE ALL THAT DATA USING PREFERABLY CHEAPER ASSAY METHODS, HIGH THROUGHPUT METHODS AND THEN COMPUTATIONALLY BUILD THE RAT BACK TOGETHER OR HUMAN. SO THIS IS THE GOAL OF A PROGRAM THAT WE STARTED A COUPLE YEARS AGO CALLED THE TOX 21. IT’S A CONSORTIUM N CAPS FOR ENVIRONMENTAL PROTECTION AGENCY. NIH INSTITUTE DOWN IN NORTH CAROLINA, IT’S A NATIONAL PROGRAM. AND FDA WHERE WE STARTED A COUPLE YEARS AGO LITERALLY GENERATING DATA ON VARIOUS ADVERSE OUTCOME PATHWAYS. COMBINING THE EXPERTISE OF THE DIFFERENT AGENCIES WHERE OF COURSE N CATS BRING TO THE TABLE THE EXPERTISE IN SCREENING, ACID MINIATURIZATION AND OTHER PARTNERS CONTRIBUTE OTHER TYPES OF EXPERTISE.

ON THE EPA ESPECIALLY THE LARGE COMPUTATIONAL TECHNOLOGY TEAMS LOOKING AT THE RESULTS GENERATED BY ROBOTICS SCREENING TO BEGIN TO BUILD THESE PREDICTIVE MODELS. THIS WAS DONE IN A COUPLE OF PHASES, INITIALLY WE JUST WANTED TO GET THE PROCESSES WORKED OUT. SO SMALL LIBRARY OF ABOUT 1400 CHEMICALS WAS TESTED SORT OF SEMI MANUALLY AGAINST VARIOUS ASSAYS. WE NOW GRADUATED TO A LIBRARY OF ABOUT 10,000 CHEMICALS. THESE ARE ALL TESTED AS 15 POINT DOSE RESPONSES IN TYPICAL GENERATING REALLY HIGH DENSITY DATA, VERY ACCURATE DATA, REDUNDANT DATA DELIBERATELY BECAUSE UNLIKE REGULAR HIGH THROUGHPUT SCREENING WHERE YOU JUST WANT A COUPLE HITS AND YOU HELPED TO PROGRESS THEM FOR DRUG DEVELOPMENT HERE, NEGATIVE DATA JUST AS IMPORTANT. MOST OF THE EARLY ASSAYS RUN AGAINST THIS COLLECTION OR SORT OF THE LOW HANGING FRUIT, THE SO-CALLED MOST IMPORTANT ASSAYS FOR TOXICOLOGY. ESTROGEN RECEPTOR AND RELATED. WE’RE ALWAYS ON THE LOOKOUT FOR NEW ASSAYS THAT LOOK FOR TOXICOLOGIES. WE ACCEPT NEW ASSAY PROPOSALS ALL THE TIME. AND AGAIN WE HAVE VARIOUS RESPONSE RELATED ASSAYS AND WE’LL BE MOVING FOR HIGH CONTENT AND OTHER SYSTEMS.

NEEDLESS TO SAY THIS IS DONE ROBOTALLY USING DEDICATED ROBOTIC SYSTEM. INCLUDING THE DISPENSE TECHNOLOGY AND OTHER INNOVATIONS. SO TO DATE, THE PROGRAM HAS BEEN VERY SUCCESSFUL. WE RECENTLY FINISHED POSTING THE REALLY LARGEST DATA SET OF WELL OVER 50 MILLION DATA POINTS. AGAIN ALL DOSE RESPONSES ON THE 10,000 MEMBER LIBRARY AGAINST THE FIRST SERIES OF ASSAYS. AND AGAIN, WE DON’T PRETEND TO KNOW EVERYTHING. WE’RE ACTUALLY USING THE ENTIRE COMMUNITY WORLDWIDE TO CROWD SOURCE THE DATA ANALYSIS AND MODEL BUILDING. WHAT DO WE MEAN BY THAT? WE PROVIDE DATA ON THE 10,000 COMPOUNDS INCLUDING THEIR STRUCTURES ACTIVITY AGAINST THE ASSAYS WE’VE RELEASED IN PUB CHEM AND WE’RE ASKING THE COMMUNITIES TO BUILD PROTECTIVE MODELS AND WE TEST THOSE MODELS ON A COUPLE HUNDRED COMPOUNDS WHICH WE HAVEN’T RELEASED THE DATA FOR. THAT’S WHAT WE’RE USING TO SCORE THESE MODELS. AND THE CHALLENGE ACTUALLY IS SCHEDULED TO END IN A COUPLE WEEKS, AND WE HAVE WELL OVER 2000 MODELS SUBMITTED FOR THE VARIOUS DATA SETS ACTIVITY MODELS. AND WE’RE ALSO WORKING CLOSELY WITH OTHER NON-U.S.

GOVERNMENT AGENCIES AND NON-PROFITS TO EXPAND THOSE EFFORTS. AND THIS IS JUST AN EXAMPLE OF ONE OF THE FIRST PAPERS DEALING WITH THESE LARGE SCREENS ON THE ESTROGEN RECEPTOR WHICH IS VERY IMPORTANT SYSTEM IS THE PATHOLOGY SO WE SCREENED IT FOUR DIFFERENT WAYS USING BETA REPORTER — FULL LENGTH RECEPTOR, LIGAND BINDING DOMAIN. SO REALLY FOUR DIFFERENT VERSIONS OF ALL THE INITIAL DATA COMPARED TO THIS PAPER. I’LL END AND ASK IF YOU HAVE ANY QUESTIONS? [APPLAUSE] >> [INDISCERNIBLE] >> SO, N CATS HAS LIKE MOST TWO SIDES OF IT. I’M ON THE INTRAMURAL SIDE. WE’RE THE PEOPLE WHO RUN EXPERIMENTS, RUN SCREENS AND WORK WITH OUTSIDE COLLABORATORS TO TAKE THEIR MODELS, BRING THEM IN HOUSE AND ESSENTIALLY SET THEM UP FOR SCREENING. N CATS ALSO HAS THE EXTRAMURAL COMPONENTS WITH THEIR GRANTS GOING OUT TO VARIOUS APPLICANTS TO DO WORK IN THE RARE DISEASE AREA. SO ON THE INTRAMURAL SIDE WE HAVE THE ENTIRE SPECTRUM.

WE WORK WITH DISEASE FOUNDATIONS TO SET UP NEW ASSAYS. WE DESIGN NEW ASSAYS FOR RARE GENETIC DISEASES, FOR EXAMPLE, OR TROPICAL INFECTIONS. SOMETIMES THIS TAKES TWO OR THREE YEARS TO GENERATE NEW CELL LINES, TO IMBED NEW REPORTERS, TO REALLY CAREFULLY LOOK AT THE BIOLOGY OF THE RARE DISEASE. WHETHER IT’S SILENT GENE OR SOME DISORDERS THAT IS CAUSED BY A POINT MUTANT.

THERE ARE REALLY VERY DIFFERENT FLAVORS OF RARE DISEASES, AND RARE CANCERS OF COURSE. WE RUN SCREENS ON THOSE ASSAYS. WE ALSO HAVE A LOT OF DRUG REPURPOSING EFFORTS, AND SOME OF THESE ARE ALSO TAKING PLACE ON THE EXTRAMURAL SIDE WHERE N CATS IS FUNDING CLINICAL TRIALS USING MOLECULES FROM PHARMACEUTICAL COMPANIES THAT HAVE BEEN BASICALLY PAUSED. THE COMPANY HAS STARTED DEVELOPING THE MOLECULE AND THEN AT ONE POINT HAS DECIDED TO STOP THE DEVELOPMENT SAY FOR BUSINESS REASONS, THEY RAN OUT OF MONEY OR WHATEVER BUT OTHERWISE THERE’S NOTHING WRONG WITH THE MOLECULE.

SEVERAL COMPANIES HAVE AGREED TO PROVIDE THESE MOLECULES FOR REPURPOSING TRIALS. SO ESSENTIALLY THE PHARMA COMPANY PROVIDES THE ACTIVE INGREDIENT INCLUDING PLACEBO IF NEEDED. THE OUTSIDE APPLICANT PROVIDES THE MODEL SYSTEM AND THE RATIONALE FOR WHY THIS DIABETES DRUG CANDIDATE SHOULD BE REPURPOSED FOR SOMETHING ELSE AND N CATS FUNDS THE CLINICAL TRIAL. IT’S A THREE PART COLLABORATION. IT TOOK SEVERAL YEARS TO DESIGN THE MASTER AGREEMENT. BECAUSE THERE ARE A LOT OF SUCH RELATIONSHIPS OUT THERE WHERE TESTING PHARMA COMPANIES, MOLECULES OR SOMETHING BUT USUALLY TAKES FOUR OR FIVE YEARS TO REACH AN AGREEMENT BETWEEN UNIVERSITY X AND PHARMA Y. SO IN THIS CASE, IT’S BRINGING A BIG ACHIEVEMENT IN OUR VIEW HAS BEEN TO HARMONIZE THE AGREEMENT AND REALLY BRING THE PARTIES TOGETHER AND LITERALLY TELL THEM YOU HAVE ONLY A COUPLE WEEKS TO FINALIZE AND SIGN OR ELSE YOU’RE NOT GETTING THIS GRANT. SO THAT’S REALLY THAT THERE. SO WE HAVE EFFORTS ON BOTH SIDES OF THE TABLE IF YOU WILL.

WE HAVE OUR OWN INTERNAL SCREENING EFFORTS AND WE WORK WITH A LOT OF DISEASE FOUNDATIONS IN INDIVIDUAL ACADEMIC INVESTIGATORS. BUT ALSO ON THE EXTRAMURAL SIDE THERE IS THIS SORT OF TARGETED REPURPOSE EFFORT AT PHARMACEUTICAL COMPANIES. >> [INDISCERNIBLE]. >> SO YOU KNOW WHEN YOU HAVE HERPES VIRUS IT STAYS SILENT AND LATENT AND EVERY ONCE IN A WHILE YOU GET THE COLD SORE AND YOU PUT AN OINT. AND THAT’S USUALLY ACYLOVIR. SO YOU HAVE THAT INFECTION REACTIVATION THROUGH APPLICATION OR REPLICATION INHIBITOR. WE THINK THAT BY APPLYING A JUMONJI INHIBITOR INSTEAD, WE WOULD BE MORE EFFECTIVE IN REDUCING THAT REACTIVATION, STOPPING IT EARLIER. SO THE HOPE IS TO UTILIZE THIS MECHANISM AS A SUPERIOR TO REPLICATION INHIBITION, IN THIS CASE. BUT OF COURSE, DEMETHYLATION OF HISTONES IS REALLY A WIDE RANGING PROCESS. YOU’RE NOT GOING TO BE ABLE TO APPLY JUMONJI INHIBITOR ON THE CHRONIC BASIS. SO IT WOULD HAVE TO BE EITHER FOR CANCER APPLICATION WHERE YOU KNOW THE PARTICULAR CANCER IS DEPENDENT ON HIGH ACTIVITY OF JUMONJI DEMETALASE ACE WHAT WOULD LITERALLY REQUIRE YOU TO USE THIS DRUG PROBABLY CASE WITHIN ONE DAY AND YOU’RE DONE IDEALLY.

BECAUSE THINGS HAPPEN SO RAPIDLY AND THEN THE INFECTION IS OVER BECAUSE IT ENTERS LATENCY AGAIN. SO YOU’RE NOT GOING TO ERADICATE HERPES VIRUS FROM THE HUMAN COAST. THAT’S NOT GOING TO PPEN. IT’S ALREADY THERE, BUT THE GOAL WOULD BE TO CONTROL THE REACTIVATION A LOT MORE EFFICIENTLY. THANK YOU. >> OUR NEXT SPEAKER IS SONIA JAKOWLEW. SHE CAME TO NIH AND STARTED WORKING WITH MICHAEL SPORN WHO IS FAMOUS FOR HIS INVESTIGATIONS ON TRANSFORMING GROWTH FACTOR BETA. HER TITLE IS TRANSFORMING GROWTH ACTIVATION AND GROWTH GENESIS. SHE’S CURRENTLY IN THE CENTER FOR CANCER TRAINING. AND SHE HAS AN OFFICE JUST ACROSS THE HALL FROM ME.

>> THANK YOU. I’M HAPPY TO BE HERE TO YOU. AND TODAY I’LL BE TALKING TO YOU ABOUT TRANSFORMING GROWTH FACTOR BETA AND NON-TUMOR GENESIS. LUNG CANCER HAS BECOME A VERY IMPORTANT DISEASE IN THE UNITED STATES. THIS YEAR LUNG CANCER HAS BECOME THE MOST COMMON CAUSE OF CANCER DEATH IN BOTH MEN AND WOMEN IN THIS COUNTRY. THERE HAVE BEEN OVER 220,000 NEW CASES THAT HAVE BEEN DIAGNOSED THIS YEAR AMONG BOTH MEN AND WOMEN. AND OVER 159 ,000 NEW DEATHS DUE TO THIS DISEASE. THERE’S A CLIMBING RISE IN THE NUMBER OF CASES THAT ARE AMONG PEOPLE WHO NEVER SMOKED BEFORE. IN THE FIVE YEAR SURVIVAL RATE IS STILL A DISMAL LESS THAN 15%. NOW MY LABORATORY BECAME INTERESTED IN TRANSFORMING GROWTH FACTOR BETA OR TGF-BETA FOR SHORT VERY EARLY ON. TGF-BETA IS A MULTIFUNCTIONAL REGULATOR OF CELL GROWTH. IT’S A POTENT INHIBITOR OF THE PROLIFERATION OF MOST NORMAL EPITHELIAL CELLS PARTICULARLY THE ENCROACHER. ISSUES WIDE SPREAD TISSUE EXPRESSION, AND HAS BEEN SHOWN TO PLAY A PIVOTAL ROLE IN MAINTAINING EPITHELIAL HOMEOSTASIS.

TGF-BETA HAS ALSO BEEN ASSOCIATED WITH VARIOUS TYPES OF CANCERS, INCLUDING HUMAN LUNG CANCER. AND IT SHOWS CONTEXT-DEPENDENT INHIBITION OR STIMULATION OF CELL PROLIFERATION AND NEOPLASTIC TRANSFORMATION AMONG NUMEROUS CELL TYPES. SO TGF-BETA IS A VERY ATTRACTIVE CANDIDATE FOR NEW THERAPEUTIC INTERVENTION APPROACHES, IF WE CAN FIND THEM. SO TGF-BETA ACTUALLY HAS ITS ROOTS IN ANOTHER PROTEIN GROWTH FACTOR CALLED SARCOMA GROWTH FACTOR. SARCOMA GROWTH FACTOR IS A POLYPEPTIDE THAT WAS FOUND TO BE CREATED BY — VIRUS TRANSFORMED MASS FIBROBLASTS THAT CAN STIMULATE NORMAL RATE FIBROBLASTS FROM COLONIES — THIS IS CALLED A TRANSFORMATION ASSAY — DEVISED IN THE EARLY 70’S AND USED TO DISCOVER THIS GROWTH FACTOR. TWO CLASSES OF TGF HAS BEEN ISOLATED FROM — SARCOMA VIRUS IN CELLS. IT WAS SHOWN TO BE ABLE TO COMPETE WITH — GROWTH FACTOR — AND THIS CLASS WAS CALLED TGF ALPHA. ANOTHER CLASS WAS FOUND TO NOT COMPETE FOR TGF BINDING BUT TO BE ABLE TO FORM COLONY THAT COULD BE ENHANCED BY THE APPLICATION OF EGF. THIS WAS CALLED TGF-BETA. SO ACTUALLY SARCOMA GROWTH FACTOR ACTUALLY COMPOSE OF TWO RAPID GROWTH FACTORS THAT ARE GENETICALLY LISTED, TGF ALPHA AND BETA.

THIS WAS PUBLISHED BY ROBERTS AND PORN IN THE EARLY 80’S. FOLLOWING THE — BECAME PUBLISHED FROM HUMAN PLATELETS AND HUMAN PLACENTA AS WELL AS BOVINE KIDNEY. AGAIN ALL THIS WAS DONE AT THE NCI. TO GIVE YOU SOME IDEA OF THE PURIFICATION, THIS IS FROM BOVINE KIDNEY. BOVINE KIDNEY WAS TYPICALLY OBTAINED FROM THE SLAUGHTERHOUSE, AND EXTRACTED WITH EIGHT LITERS OF TYPICAL — THEN AFTER EXTRACTION, THE — AND THE EXTRACT WAS THEN LEFT TO PRECIPITATE WITH ETHER ETHANOL OVER NIGHT. THE NEXT DAY — AND THEN APPLIED TO 80 LITER BIO GEL COLUMN FOR PURIFICATION. ONE LITER FRACTION WERE COLLECTED AND THESE WERE THEN LOCALIZED AND DISSOLVED FOR FURTHER CHROMATOGRAPHY PURIFICATION. SO THE YIELD WAS SIX MICRO GRAMS OF TGF-BETA ONE PROTEIN. THIS WAS RESULTED IN A 230,000 — PURIFICATION. TO GIVE YOU SOME IDEA OF THE E NORMALITY OF THE COLUMNS THAT WERE USED AT THAT POINT, THIS SLIDE SHOWS THE P60 COLUMNS THAT WERE USED AND THERE’S A 55 GAL GALLON.

TYPICALLY A MIXTURE OF MEDIUM SERUM AND RK CELLS IN THE SAMPLE WAS APPLIED TO — INDO BATES FOR ONE WEEK AT 37 DEGREES WITH CO2. AFTER ONE WEEK, THEY WERE STAINED AND THE COLONIES WERE COUNTED IN A — SYSTEM AND I HAVE A SHOT OF ONE OF THE IMAGE ANALYZERS THAT WAS USED. IF NO TGF-BETA IS PRESENT ON THE LEFT SIDE OF THE SLIDE YOU WOULDN’T SEE ANY COLONIES FORMED TO BE COUNTED. IF TGF-BETA IS PRESENT IN THE SAMPLE THEN COLONIES WERE NORMED AND — WOULD BE COUNTED. SO THIS SLIDE SHOWS A TYPICAL FINAL HPLC PURIFICATION PROTEIN. IT SHOWS ONE GRAM MIGRATING AT 25,000 MOLECULAR WEIGHT SINGLE BAND FROM A — DOCTORS MICHAEL SPORN AND ANITA ROBERTS IS CREDITED FOR GIVING BIRTH SO TO SPEAK TO THE TGF-BETA MOLECULE PROTEIN. AND FOLLOWING THE PURIFICATION OF TGF-BETA PROTEIN, THE AMINO ACIDS SEQUENCE OF THE PROTEIN WAS DETERMINED AND IT WAS DETERMINED THAT TGF-BETA EXISTED INITIALLY AS A PREPRO TGF-BETA FORM OF 391 AMINO ACIDS — WAS A LATENCY ASSOCIATED PEPTIDE SHOWN IN BLUE IN THE MIDDLE PORTION OF THE MOLECULE.

AND THE MATURE TGF-BETA POLY TEST SIDE CONSISTING OF 112 AMINO ACIDS — WAS ESTABLISHED IN THE EARLY 90’S AND IT WAS SHOWN TO BE CONSISTS OF TWO IDENTICAL MONOMERIC CHAINS THAT WERE HELD TOGETHER BY AN INTERCHANGE WITH HYDROPHOBIC POCKET IN THE CENTER PORTION. TGF-BETA IS ACTUALLY A MEMBER OF WHAT WE CALL THE TGF-BETA SUPER FAMILY. THIS SUPER FAMILY CONSISTS OF THE FIVE ISOFORMS OF TGF-BETA, ONE, TWO, THREE, FOUR AND FIVE, AS WELL AS THE — GENETIC PROTAIN AND GROWTH DIFFERENTIATION FACTORS OR — AS WELL AS THE ACTIVE INHIBITOR, VERY LARGE FAMILY OF — PROTEINS. TRANSFORMING GROWTH FACTOR BETA PER SE IS A 25,000 — HOMO DIMER. THREE HOMOLOGOUS ISOFORMS HAVE BEEN SHOWN TO THIS IN MAMMALS. THESE ARE TGF-BETA ONE, TWO AND THREE. IN ADDITION, TGF-BETA 4 HAS BEEN IDENTIFIED IN BIRDS AND IT HAS HIGH HOMOLOGY TO MAMMALIAN TGF-BETA ONE. AND TGF-BETA FIVE HAS BEEN IDENTIFIED IN AMPHIBIAN AND THAT HAS HIGH — ISOFORM HAS BEEN IDENTIFIED IN FISH AND IT SHOWS MOST HOMOLOGY TO TGF-BETA ONE IN MAMMALS.

THE PRINCIPAL SOURCES OF TGF-BETA ARE PLATELETS, BONE AND MEAN. AND MOST CELLS ARE KNOWN TO EXPRESS TGF-BETA IN ITS RECEPTORS. TGF-BETA IS USUALLY SECRETED IN WHAT IS CALLED A LATENT INACTIVE FORM. THEY MUST BE ACTIVATED IN ORDER FOR TGF-BETA TO PERFORM WITH VARIOUS FUNCTIONS. AND IN SUPER FAMILIES IT CONTAINS NOT ONLY TGF BUT OTHER COUSIN PROTEINS AS WELL. MEMBERS OF THE TGF-BETA FAMILY ACT AS CENTRAL CONTROL MODULES FOR MANY IMPORTANT BIOLOGICAL PROCESSES.

THESE INCLUDE DEVELOPMENT, IMMUNE SYSTEM FUNCTIONS, REPRODUCTION, ANGIOGENESIS, AGING AND — PROLIFERATIVE HOMEOSTASIS. SO TGF-BETA HAS SOME ROLE TO PLAY IN THAT PROCESS. MAJOR BIOLOGICAL RESPONSES BY TGF-BETA INCLUDE THE INHIBITION OF CELL PROLIFERATION, ARE REGULATION OF APOPTOSIS — STIMULATES THE ACCUMULATION OF EXTRACELLULAR MATRIX AND — THE CURRENT MODEL FOR TGF-BETA SIGNALING PATHWAY INVOLVES A RECEPTOR PROTEINS AND THE SMAD PROTEIN WHICH IS REFERRED TO THE SMAD DEPENDENT PATHWAY BECAUSE IT DEPENDS ON THE EXISTENCE OF SMAD PROTEINS.

TYPICALLY WHAT HAPPENS IS THE TYPE TWO TGF-BETA RECEPTOR IS PHOSPHORYLATED. IT BINDS THE TGF-BETA LIGAND AND THE LIGAND CAN BE EITHER THE TGF-BETA ISOFORM OR BAND P OR TGF WHATEVER. THIS THEN RECRUITS THE TYPE ONE TGF-BETA RECEPTOR TO MAKE ANOTHER MORE ENHANCE COMPLEX AND THE TYPE TWO RECEPTOR TRANSPHOSPHORYLATES THE TYPE ONE RECEPTOR. ONCE THIS HAPPENS, THEN, THIS COMPLEX IS ABLE TO THEN INTERACT WITH THE SMAD PROTEINS AND DEPENDS ON WHETHER YOU’RE TALKING ABOUT TGF-BETA ACTIVE OR — WHETHER THIS WILL BIND TO SMAD PROTEIN, SMAD 2 AND 3 OR SMAD 1, 5 AND 8.

THESE ARE THE SMAD RECEPTOR PROTEINS. AND ONCE THE SIGNAL IS TRANSDUCED HERE, IT’S THEN, THE RECEPTOR SMADS ARE ABLE TO THEN INTERACT WITH SMAD 4 AND SMAD 4 HAS THE ABILITY TO TRANSLOCATE THE SIGNALS TO THE NUCLEUS WHERE VERY TRANSCRIPTIONAL EVENTS THAT TGF-BETA PLAYS A ROLE. IT CAN BE INHIBITORY BY SMAD 7 OR SMAD 6. NOW CLINICALLY TGF-BETA HAS TUMOR SUPPRESSOR ACTIVITIES. FOR EXAMPLE THE PATHWAY COMPONENTS HAVE BEEN SHOWN TO CAUSE FAMILIAR PRE DISPOSITION TO CANCER.

THIS IS SHOWN TO OCCUR WITH SMAD 4 IN JUVENILE — SYNDROME. THE PATHWAY COMPONENTS HAVE BEEN SHOWN TO BE SOMATICALLY MUTATED OR DELETED IN SOME HUMAN CANCERS. THE TGF-BETA TYPE 2 RECEPTOR IN HUMAN — CANCER AS WELL AS SMAD 4 BEING PREDOMINANTLY MUTATED, SORRY, DELETED IN PANCREATIC CANCER. AND THIRDLY, THE EXPRESSION OF TGF-BETA COMPONENTS OR OVER EXPRESSION OF ENDOGENOUS PATHWAY INHIBITORS HAVE BEEN SHOWN TO BE ASSOCIATED WITH THESE DISEASE PROGRESSION. AND THIS INVOLVES THE TYPE ONE AND TYPE TWO TGF-BETA RECEPTORS AS WELL AS SMAD 7 AND TGF-BETA ASSOCIATED PROSTEINS CALLED — WHICH I WON’T HAVE TIME TO TALK ABOUT THIS AFTERNOON.

CLINICALLY TGF-BETA HAS ALSO BEEN SHOWN TO HAVE TUMOR PROMOTER ACTIVITIES. FOR EXAMPLE TGF-BETA 1 HAS BEEN SHOWN TO BE ELEVATED IN MANY ADVANCED HUMAN TUMORS AND HAS BEEN CORRELATED WITH METASTASIS OR POOR PROGNOSIS OF CANCER PROGRESSION. AND THESE INCLUDE SUCH TUMORS AS BREAST, COLON, STOMACH LIVER AS WELL AS THE LUNGS. SHOWN IN THE LOWER RIGHT SIDE OF THE SLIDE IS THE PANCREATIC, I’M SORRY THE PROSTHETIC ADENOCARCINOMA — TGF-BETA 1. HERE WE SEE THE BROWN STAINING FOR TGF-BETA INDICATING THAT IT IS AT THE INTERFACE BETWEEN THE TUMOR AND THE MICRO ENVIRONMENT. SO, TGF, WHAT IS THE ROLE OF TGF-BETA IN CARCINOGENESIS. IS IT A HERO OR VILLAIN. IT’S SEEN AS A MALIGNANT PHENOTYPE. IT’S ALSO A GROWTH INHIBITOR IN TUMOR STRESSOR UNDER SOME CASES. AND IT’S ALSO A PROMET AT — PROMETASTATIC IN OTHER CASES. IT USES THE ABILITY OF TGF-BETA TO SWITCH FROM BEING A TUMOR SUPPRESSOR TO A TUMOR PROMOTER PROONCOGENIC FACTOR DURING CANCER PROGRESSION. HOW DOES IT DO THIS? WELL, IN NORMAL — IN THE LUNGS, THE TUMOR SUPPRESSOR ACTIVITY OF TGF-BETA PREDOMINATES.

AND TGF-BETA ACTS AS A TUMOR SUPPRESSOR. CHANGES IN THE GENETIC AND EPI GENETIC TAKES PLACE, TGF-BETA LOSES RESPONSES. THE RESPONSIVENESS TO TGF-BETA IS LOSS AND THE EXPRESSION AND ANATION OF TGF-BETA GOES UP AS METASTASIS INCREASES. SO IN METASTATIC CANCER, THE PROONCOGENIC ACTIVITIES TGF-BETA BEGIN TO PREDOMINATED OR THE TUMOR — INCREASES. NOW I TOLD YOU ABOUT THE SMAD DEPENDENT PATHWAYS FOR TGF-BETA. THERE ARE ALSO SMAD INDEPENDENT PATHWAYS THAT EXIST FOR TGF-BETA AND THESE INCLUDE SUCH PATHWAYS AS THE — KINASE PATHWAY.

THE REST ARE — KINASE PATHWAY. THE — PATHWAY AND THE — PATHWAY. SO THESE ARE INDEPENDENT OF THE FUNCTION OF THE SMAD PROTEINS, OKAY. SO WHY IS THIS IMPORTANT? WELL, WE BECAME PARTICULARLY IMPORTANT IN THE K-RAS PATHWAY BECAUSE IN LUNG CANCER, THE K-RAS GENE SHOWS AN ACTIVATIONAL MUTATION IN AS MANY AS 50% OF HUMAN LUNG ADENOCARCINOMA. ONE ALLELE OF K-RAS HAS BEEN SHOWN TO INCREASE THE APPEARANCE OF LUNG LESIONS IN HUMAN CANCERS. THERE’S VALID — BETWEEN SMAD DEPENDENT PATTED WAY AND THE RAS SMAD SIGNALING PATHWAY AS WELL. ACTIVATION OF THE RAS PATHWAY CAN MODULATE SMAD OR WITHOUT SMAD. IN VITRO STUDIES HAVE SHOWN — EFFECTS OF RAS BUT THAT ACTIVATED RAS CAN OVERRIDE THE PROLIFERATIVE EFFECTS OF TGF-BETA ONE. THESE ARE THE VARIOUS PATHWAYS THAT CAN GO WRONG WITH TGF-BETA BEING A TUMOR PROMOTER. SO IF THE RAS KINASE PATHWAY IS ACTIVATED, THEN THE TUMOR SUPPRESSOR ACTIVITIES OF TGF-BETA CAN THEORETICALLY BE CIRCUMVENTED TO ALLOW TUMOR PROMOTIONS TO PREDOMINANT.

SO THE BROAD GOAL OF MY LABORATORY, ONE OF THE BROAD GOALS OF MY LABORATORY WAS TO DETERMINE THE ROLE OF TGV BETA IN THE DEVELOPMENT OF THE MALIGNANT TRANSFORMATION OF LUNG EVEN TELECELLS. THIS WAS PERFORMED IN THE EPITHELIAL CARCINOGENESIS SECTION CELL AND CANCER BIOLOGY BRANCH — OUR OBJECTIVES WERE THREE FOLD. FIRST TO EXAMINE TGF-BETA ONE DELETIONS AND K-RAS MUTATION ALONE AND IN COMBINATION ON LUNG TUMOR INCIDENCE IN PATHOLOGY. SECOND TO DETERMINE EARLY EVENTS IN THE DEVELOPMENT OF LUNG LESIONS AND THEIR PROGRESSION. AND THIRD, TO IDENTIFY POTENTIAL SIGNALING TRANSDUCTION PATHWAY CHANGES. THAT INCREASE WAS INCREASING TUMOR GENESIS. WE EMPLOYED FOUR MOUSE MODELS. THE AJ MOUSE THE C56 TGF-BETA 1 AND I’LL REFER TO THIS AS — FOR SIMPLICITY — AND THE TGF-BETA ONE HAS K-RAS ACTIVATABLE MOUSE. WE ASKED TWO QUESTIONS. DOES LUNG TUMOR GENESIS AFFECT THE TGF-BETA SIGNALING PATHWAY AND DOES THE TGF-BETA SIGNALING PATHWAY HAVE LUNG TUMOR GENESIS. WE EMPLOYED THE AJ MOUSE MODEL FIRST BECAUSE THIS MASS MODEL IS KNOWN TO BE SUSCEPTIBLE TO CHEMICALLY-INDUCED LUNG TUMORS — TUMORS DEVELOP IN A TIME DEPENDENT FASHION AND THEY GO THROUGH STAGES OF HYPERPLASIA, ADENOMA AND CARCINOMA — OCCUR IN BOTH HUMAN AND — SUCH AS OVER EXPRESSION OF K-RAS AND LOSS OF P53 EXPRESSION.

NOW, THE CARCINOGEN ETHYL CARBON MATE GOES TO AT LEAST TWO DIFFERENT TYPES OF METABOLIC EVENTS. THE FIRST IS A DETOXIFICATION PATHWAY WHICH EMPLOYS SIMPLE HYDROLYSIS USING — TO PRODUCE ETHANOL CO2 AND AMMONIA. THERE’S ALSO BIO ACTIVATION PATHWAY THAT UTILIZES — TWO FORMS OF VINYL CARBON MATEMALE — HIGH POX — EPOXIDE AND IT’S FOR THIS EPOXIDE TO BIND TO MOLECULES AND PROCEED WITH CARCINOGENESIS. SO, OUR STANDARD PROTOCOL WAS TO INJECT TWO MONTH OLD MICE WITH ETHYL CARBON MATE AND HAVE GROUPS OF MICE WITH MONTHLY INTERVALS UP TO 12 MONTHS. NOW SHOWN IN THIS SLIDE IS A CHEMICAL STAINING FOR TGF-BETA ONE IN THE TYPE ONE AND TYPE TWO RECEPTOR PROTEIN. AND LUNG TUMORS THAT FORMS AFTER TWO MONTHS, FOUR MONTHS AND EIGHT MONTHS OF ETHYL CARBON MATE. ONE CAN SEE BROWN STAINING FOR THE TGF-BETA ONE PROTEIN AS WELL AS THE TYPE ONE RECEPTOR PROTEIN AT ALL STAGES OF TUMOR GENESIS. IN CONTRAST EXPRESSION OF THE TYPE TWO RECEPTOR FOR TGF-BETA IS DEPRESSED AND REDUCED IN COMPARISON TO THE OTHER TWO PROTEINS.

SO THIS SLIDE SHOWS THIS A LITTLE MORE, A LITTLE BETTER IN COMPARING STAINING, IMMUNOSTAINING FOR THE TYPE ONE RECEPTOR WITH A TYPE TWO RECEPTOR. AND ONCE THESE VERY REDUCED EXPRESSION OF THE TYPE TWO RECEPTOR IN THE LUNG TUMOR COMPARED TO THE TYPE ONE RECEPTOR. AND IN COMPARISON, STANDING FOR THE FORMAL BRONCHIAL BY BOTH PROTEINS IS ALMOST IDENTICAL. WE ALSO LOOKED AT THE EXPRESSION OF THE MRNA FOR THE TYPE ONE AND TWO RECEPTOR. INTERESTINGLY WE SHOW LOSS OF THE TYPE TWO RECEPTOR MRNA IN THE TCC4 LINE WHICH IS THE — THAT WAS DERIVED FROM AFTER TREATMENT WITH ETHYL CARBON MATE. SO WE SHOWED DECREASED PRODUCTION OF THE PROTEIN IN THE TYPE TWO TGF-BETA RECEPTOR IN THE AGING MOUSE MODEL SYSTEM. WE ALSO TREATED THESE MICE WITH BENZO PYRINE AND ANOTHER LUNG CARCINOGEN AND SHOWED THAT ESSENTIALLY WE SEE BOTH — CHEMICAL STAINING FOR THE TYPE ONE RECEPTOR IN TGF-BETA LIGAND AND HYBRIDIZATION TO THE MRNA FOR THESE PROTEINS IN THE TUMORS. IN COMPARISON THE EXPRESSION OF THE PROTEIN AND MESSAGE FOR THE TYPE TWO RECEPTORS IS REDUCED IN THE TUMORS. WHILE THERE’S NORMAL EXPRESSION, EQUAL EXPRESSION OF THESE PROTEINS IN THE SURROUNDING BRONCHIAL.

SO, OUR MODEL SHOWS THAT TGF-BETA CAN BE COMPROMISED BY THE REDUCTION OF THE TYPE TWO RECEPTOR PROTEIN, WHICH AGAIN LEADS TO DECREASED ABILITY OF THE TGF-BETA MOLECULES TO ACT AS A TUMOR SUPPRESSOR AND MORE TO ACT AS A TUMOR PROMOTER IN THIS LUNG SYSTEM. OUR NEXT QUESTION WAS THE DOES DELETION OF TGF-BETA 1 AFFECT LUNG TUMOR GENESIS. WE USED THE C57 — NOW THE TGF-BETA ONE KNOCKOUT MOUSE WAS GENERATED IN THE — NON-INDUCED. AND THE MOUSE WAS BORN AND THRIVED LIKE ITS LITTER MATES. IT’S A GENERAL WASTING PHENOMENA AND SHORTLY THEREAFTER IT DIES. SO THE KNOCKOUT MOUSE IS NOT A GOOD MOUSE TO EMPLOY TO USE THE STUDY LUNG TUMOR GENESIS.

BUT THE — HETEROZYGOUS MOUSE SEEMS TO THRIVE AND REPRODUCE AND HAS NO PROBLEMS. SO WE TREATED THE TGF-BETA ONE HETEROZYGOUS MOUSE WITH THE CARCINOGEN TO LOOK AT LIVER TUMOR GENESIS INITIALLY. AND WE FOUND THAT THERE’S INCREASE INCIDENCE OF LIVER TUMOR GENESIS IN THE HETEROZYGOUS MOST COMPARED TO THE WILD TYPE. INTERESTINGLY WE ALSO OBSERVED THAT THERE WAS INCREASE LUNG TUMORS IN THE — MICE COMPARED TO THE WILD TYPE MICE. THAT WAS VERY UNEXPECTED. SO IN ORDER TO BETTER LOOK AT THIS IN LUNG TUMORS OR LUNG TUMOR GENESIS, WE THEN CROSSED THE AJ MOUSE WHICH WAS WILD TYPE FOR TGF-BETA WITH THE TGF-BETA 1HT MOUSE TO GENERATE THE AJ — F1 GENERATION WHICH INCLUDED PHENOTYPE THAT WERE HETEROZYGOUS AND WILD TYPE FOR TGF-BETA ONE. NOW, OUR PLAN WAS TO TREAT THESE MICE WITH CARCINOGEN AND LOOK AT THE LUNG TUMORS THAT WERE GENERATED.

AND SHOWN HERE IS A IMMUNO– HIGH PRIDIZATION FOR THE TGF-BETA 1 PROTEIN. AND HERE WE SHOW EVIDENCE THAT OUR SYSTEM, WE WANTED TO BE SURE THAT OUR SYSTEM WAS WORKING, SO THE HETEROZYGOUS SHOWS DECREASED STAINING FOR THE TGF-BETA ONE PROTEIN COMPARED TO THE WILD TYPE. AND DECREASE EXPRESSION OF THE TGF-BETA 1 MESSAGE IN THE HETEROZYGOUS MOUSE COMPARED TO THE WILD TYPE MOUSE. OKAY. WE ALSO SHOWED THAT A WITH NORTHERN — TO DECREASE THE EXPRESSION OF THE TGF-BETA 1 PROTEIN IN THE HETEROZYGOUS MOUSE FORM COMPARED TO THE WILD TYPE MOUSE, AND WE ALSO SHOWED THE SAME THING WITH USING KRG COMPETITIVE — IN THE HETEROZYGOUS MOUSE COMPARED TO THE WILD TYPE. SO WE PROCEEDED AS BEFORE. WE INJECTED A TWO MONTH OLD MICE WITH ETHYL CARBON MATE AND SACRIFICED MICE OF THE TWO GENOTYPES AT MONTHLY INTERVALS. ESSENTIALLY WE LOOKED AT HYPERPLASIA ADENOMA AND CARCINOMA THAT FORM AND WE SHOWED THAT IN ALL THREE TYPES OF TUMORS STAGES, WE SHOWED INCREASED TUMOR INCIDENCE IN MULTIPLICITY IN THE TGF-BETA 1 HETEROZYGOUS MOUSE SHOWN IN RED COMPARED TO THE WILD TYPE SHOWN IN GREEN.

AND THERE WAS ALSO DECREASE TUMOR LATENCY ESPECIALLY IN THE CARCINOMA THAT FORM, WHILE THE CARCINOMA STARTED FORMING AT FOUR MONTHS IN THE HETEROZYGOUS MOUSE, IT TOOK ALMOST 12 MONTHS BEFORE WE SEE CARCINOMA FORMING IN THE WILD TYPE MOUSE. SO WE LOOKED AT THE EXPRESSION OF THE TYPE TWO RECEPTOR PROTEIN IN THESE LUNG LESIONS AND SOON AT THE BOTTOM RIGHT WITH THE PINK ARROWS YOU SEE THE EXPRESSION IN TUMORS OF THE MICE COMPARED TO THE WILD TYPE CARCINOMA SHOWN ON THE LEFT. WE ALSO LOOKED AT THE RELATIVE LEVELS OF — IN THE LESIONS OF THE TGF-BETA 1 — HAS BEEN TREATED WITH THE CARCINOGEN ETHYL CARBON MATE INCREASING TUMOR GENESIS AND WERE ABLE TO SEE DECREASING AMOUNTS AND LESS OF THE TYPE TWO RECEPTOR MRNA WITH INCREASING LUNG TUMOR GENESIS AS TUMOR GENESIS RECEIVED IT FROM HYPERPLASIA TO ADENOMA AND THEN TO CARCINOMA. THE NEXT QUESTION IS DOES DELETION OF TGF-BETA 1 AND MUTATION OF K-RAS AFFECT LUNG TUMOR GENESIS IN COMBINATION. FOR THIS WE USED THE — THE K-RAS ACTIVATABLE MOUSE WE OBTAINED FROM — THE INTERPLAY OF THESE TWO GENES WAS GENERATED THE — BY CROSSING THE TGF-BETA ONE HETEROZYGOUS MOUSE WITH THE K-RAS MOUSE IN THE — BACKGROUND.

WE THEN GENERATED FOUR TYPES OF MICE. THE TGF-BETA ONE HETEROZYGOUS K-RAS WITH DOUBLE MUTANTS, THE TGF-BETA ONE WILD TYPE NEXT TO THE SINGLE MUTANTS. THE — HETEROZYGOUS AND THE WILD TYPE MOUSE LITTER MATE AS WELL. SO TO SHOW WHAT WE SAW AT THE LEVELS OVER HISTOLOGICAL PATHOLOGY, THESE ARE LUNGS THAT ARE TAKEN FROM SIX MONTH OLD MICE AFTER TREATMENT. AND WE SEE RIGHT MODULES THAT ARE LUNG TUMORS AND MICE WITH THE LATENT — K-RAS GENOTYPE. WE SAW ONLY VERY FEW SPONTANEOUS TUMORS APPEARING IN THE NON-LATENT — GENOTYPES. WE LOOKED AT THE EFFECT OF TGF-BETA GENE SOLUTION IN K-RAS MUTATION AND MOUSE SURVIVAL WITH THESE FOUR MICE GENERATED. AND WE SHOW THAT THERE WAS DEFINITE SIGNIFICANT DECREASE IN THE LIFE SPAN IN THE HETEROZYGOUS ACTIVATABLE MOUSE. AND THE WILD TYPE LATENT — MICE COMPARED TO THOSE MICE THAT DIDN’T CONTAIN THE K-RAS MUTATION. WE LOOKED AT THE PATHOLOGY OF THE LUNG LESIONS THAT FORMS IN THESE MICE, AND WE SAW INCREASE HYPERPLASIA AND ADENOMA IN THE WILD TYPE LATENT ACTIVATABLE MOUSE SHOWN IN GREEN COMPARED TO THE LATENT ACTIVATABLE MOUSE AS WELL AS INCREASED CARCINOMAS IN THE HETEROZYGOUS MOUSE.

OKAY, SHOWN IN RED. COMPARED TO THE WILD TYPE LATENT ACTIVATABLE MOUSE. WE STAIN FOR THE TGF-BETA 1 LIGAND AND THE TYPE 2 RECEPTOR PROTEIN. AND SHOWN THE — EXPRESSION OF THE TGF-BETA 1 PROTEIN AS WELL AS THE TYPE 2 RECEPTOR PROTEIN AND THE HETEROZYGOUS LATENT ACTIVATABLE CARCINOMAS. NOW, WE ALSO LOOKED AT THE EXPRESSION OF THE VARIOUS SMAD PROTEIN USING WESTERN BROAD ANALYSIS AND HERE SHOWN TYPICALLY IS COMPARISON OF WILD TYPE LATENT ACTIVATABLE WITH — WE SEE EXPEDITED REDUCTION OF THE TYPE 2 RECEPTOR AND INCREASED PRODUCTION OF THE SMAD PROTEIN IN THE HT — MICE BY ABOUT ONE MONTH COMPARED TO THE WILD TYPE LATENT ACTIVATABLE MOUSE LITERALLY. THE OTHER GENOTYPE SHOWED VIRTUALLY NO CHANGE IN THE PROTEINS. NOW, RATHER THAN SHOW YOU SEVERAL WESTERN BLOCKS PUTTING THIS ALTOGETHER FOR YOU, ESSENTIALLY USING WESTERN BROAD ANALYSIS WE SEE IN THE TGF-BETA 1 HETEROZYGOUS K MASS ACTIVATABLE MOUSE PRODUCTION OF THE TYPE TWO RECEPTOR PROTEIN, AND EXPEDITED PRODUCTION OF THE SMAD 3 PROTEIN.

BUT WE SEE REDUCED PRODUCTION OF BOTH THE SMAD 4 AND SMAD 7 PROTEIN. WE ALSO LOOK BECAUSE WE WERE INTERESTED IN THE RAS PATHWAY WE ALSO LOOKED AT RAS AND RAS 1 EXPRESSION AND BOTH THESE PROTEINS SHOWED EXPEDITED INCREASE IN THEIR PRODUCTION BY ABOUT A MONTH EACH TIME. WE ALSO CONDUCTED REAL TIME MARKET PTR AND SHOWED THERE WERE REDUCED EXPRESSION OF SMAD TWO, THREE, FOUR AND SEVEN IN THE ADENOMAS BUT WE DO EXPRESSION IN THE TYPE TWO RECEPTOR IN SMADS AND CARCINOMAS. SO NOT THE SAME THING AS GOING ON AT ALL STAGES OF CARCINOGENESIS. IT SEEMS TO BE A VERY COMPLEX SYSTEM THAT WE’RE TRYING TO SORT OUT. NOW, WE ALSO LOOKED AT THE APOPTOTIC INDEX IN THESE MICE, AND WE SHOW THAT THERE’S RESIST APOPTOSIS REDUCED LEVELS OF APOPTOSIS IN THE LATENT ACTIVATABLE ADENOMA COMPARED TO THE WILD TYPE LATENT ACTIVATABLE ADENOMA.

SO IN OUR SYSTEM USING THESE VARIOUS MOUSE MODELS SHOW THAT THERE ARE SEVERAL WAYS IN WHICH THE TGF-BETA SIGNALING PATHWAYS CAN BE IMPACTED IN ORDER TO TURN A KNOWN TUMOR SUPPRESSOR MORE INTO A LUNG TUMOR PROMOTER. WE SHOWED THAT THE TYPE TWO RECEPTOR CAN BE DECREASED LEADING TO LUNG TUMOR PROMOTION. ACTIVATED RAS KINASE CAN LEAD TO PROMOTION OF LUNG TUMORS AS WELL. EXPRESSION OF SMAD FOUR ALSO LEADS TO LUNG TUMOR PROMOTION AS WELL AS COMPROMISE APOPTOSIS LEADING TO LUNG TUMOR PROMOTION. SO RESPONSIBLE FOR CARCINOGENS TO COUNTERACT THE ACTIVITY OF WHAT’S KNOWN TO BE A GOOD TUMOR SUPPRESSOR TO MAKE IT REALLY A TUMOR PROMOTER, POWERFUL TUMOR PROMOTER. SO I’D LIKE TO ACKNOWLEDGE THE PEOPLE THAT WERE INVOLVED IN THIS STUDY IN MY LAB, JERRY — HE WAS A VERY GOOD POST BACK, AND POST DOCS — THEY’VE GONE ON TO BETTER AND HIGHER POSITIONS AS WELL.

AND I WOULD LIKE TO AGAIN ACKNOWLEDGE TYLER JACKS K RATION — I WILL BE HAPPY TO ANSWER ANY QUESTIONS IF YOU HAVE ANY. [APPLAUSE] YOU CAN ALL BE OVERWHELMED. OKAY. ONE QUESTION THAT I GET FROM SOME PEOPLE IS WHERE IS THIS WORK GOING NOW. WELL UNFORTUNA I’VE NOW MOVED INTO THE EXTRAMURAL FIELD AND I MONITOR AND MANAGE A NUMBER OF DIFFERENT GRANTS FOR NCI. AND THERE ARE VARIOUS PEOPLE INVESTIGATORS IN THE EXTRAMURAL SCIENTIFIC COMMUNITY TAKEN UP THIS WORK AND ARE PURSUING IT. SO IT’S A PROJECT THAT I THINK HAVE A LOT OF LEGS AND CAN BE MORE — PROJECTS ON ITS OWN. ANY QUESTIONS? YES. >> [INDISCERNIBLE]. >> I’M SORRY, CAN’T HEAR YOU.

>> [INDISCERNIBLE] >> YES, THERE ARE SOME THERAPEUTICS BEING WORKED ON, UNFORTUNATELY THEY’RE VERY TOXIC. SO THAT’S THE PROBLEM HERE, OKAY. THERE ARE A NUMBER OF SMALL MOLECULES THAT ARE BEING TESTED, BUT SO FAR THEY’VE NOT BEEN OF MUCH USE THROUGH TOXICITY AND DUE TO THE, THEY’RE NOT VERY EASY TO MANUFACTURE AS WELL. UNFORTUNATELY. OKAY, THANK YOU. YOU GET OUT EARLY.. “

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