Repetitive DNA sequences in health and disease: gift wrappings for precision medicine

WELCOME TO THE NIH DIRECTORS WEDNESDAY AFTERNOON LECTURE SERIES I AM FROM THE NATIONAL CANCER INSTITUTE AND I HAVE THE HONOR OF HOSTING DR. KEN RAMOS TODAY AS WELL AS INTRODUCING HIM FOR HIS LECTURE DR. RAMOS WAS BORN IN NEVADA AND RECEIVED BACHELOR OF SCIENCE DEGREE AND CHEMISTRY FROM THE UNIVERSITY OF PUERTO RICO MEDICAL SCIENCE CAMPUS HE RECEIVED Ph.D. IN BIOCLINICAL PHARMACOLOGY FROM THE UNIVERSITY OF TEXAS AT AUSTIN AND HIS M.D. DEGREE AT THE UNIVERSITY OF KENTUCKY DR. RAMOS CURRENTLY HOLDS SIMILAR POSITIONS, HE’S ASSOCIATE VICE PRESIDENT FOR PRECISION HEALTH SCIENCES INTERIM DEAN OF UNIVERSITY OF ARIZONA COALITION OF MEDICINE, PHOENIX, EXECUTIVE DIRECTOR CENTER FOR APPLIED GENETICS AND GENOMIC MEDICINE, PROFESSOR OF MEDICINE UNIVERSITY OF ARIZONA HEALTH SCIENCES AND HE DOES NOT SLEEP, RIGHT? DR. RAMOS RECEIVED INTERNATIONAL RECOGNITION IN THE FIELDS OF APPLIED GENETIC AND GENOMIC MEDICINE PRECISION MEDICINE AND VITAMIN HEALTH SCIENCES AND TOXICOLOGY HE IS AN ELECTED MEMBER OF THE NATIONAL ACADEMY OF MEDICINE AS WELL AS ASSOCIATE OF THE NATIONAL ACADEMY OF SCIENCES TODAY HE WILL TALK TO US ABOUT REPETITIVE DNA SEQUENCES IN HEALTH AND DISEASE FOR PRECISION MEDICINE I HOPE YOU ALL CAN JOIN US FOR A RECEPTION IN THE NIH LIBRARY IMMEDIATELY AFTER HIS TALK PLEASE JOIN ME IN WELCOMING DR. RAMOS TO THE STAGE [APPLAUSE] >> THANK YOU OPHILIA FOR YOUR INTRODUCTION AND CERTAINLY IT IS A PLEASURE FOB ME TO BE HERE TO SHARE WITH YOU SOME OF THE THINGS THAT OCCUPY SOME OF MY TIME I DID GET TIRED LISTENING TO ALL THE THINGS THAT I DO, HOPEFULLY I DO WELL BUT AS ALL OF YOU KNOW, WE ALWAYS FIND TIME TO DO THE THINGS THAT CARRY US WITH PASSION AND CERTAINLY SCIENCE AND MEDICINE DOES THAT FOR ME THE STORY THAT I’M ABOUT TO TELL YOU TODAY IS ONE THAT BEGAN IN THE EARLY ’90s FOR ME WHEN WE MADE THE OBSERVATION REPETITIVE SEQUENCES WHICH WERE SEQUENCES WE BELIEVED TO BE DORMANT IN THE GENOME COULD BE REAWAKENED IN THE CONTEXT OF STRESS THOSE FINDINGS OF COURSE WERE VERY MUCH IN KEEPING WITH THE FINDINGS OF (INDISCERNIBLE) WHEN SHE REPORT AD DECADE, TWO DECADES BEFORE THAT ON THE STRESSFUL RESPONSE OF MACE AND OF COURSE PROBABLY MANY OF YOU KNOW, THE REDEFINITION OF WHAT WE THINK IN TERMS OF IMAGES AND STABILITY OF THE GENOME OVER THE COURSE I THINK OF OUR STUDIES, WHAT BECAME APPARENT AND HOPEFULLY SOMETHING YOU WILL APPRECIATE FROM SOME OF THE HIGHLIGHTS AND EXAMPLES THAT I WILL GIVE YOU, IS HOW IMPORTANT THESE SEQUENCES WILL TURN OUT TO BE IN THE CONTEXT OF PRECISION MEDICINE WHICH IS THE BUZZ WORD WE NOW USE THESE DAYS TO TALK ABOUT THE INTERSECTION OF MOLECULAR GENETICS, APPLIED GENETICS, AND DELIVERY OF PATIENT CARE SO I WILL TRY TO PLACE THOSE THINGS INTO CONTEXT, AND GIVE YOU SOME OF THE EXAMPLES OF HOW WE TACKLED THAT PARTICULAR COMPONENT IN CHOOSING THE TITLE, I LIKE TO RECALL REPETITIVE SEQUENCES IN PARTICULAR LONG INTERSPERSED NUCLEAR ELEMENT SEQUENCES OF THAT PARTICULAR FAMILY, GIFT WRAPPINGS FOR PRECISION MEDICINE THE REASON I CALL THAT, USE THAT DESCRIPTION IS BECAUSE SURPRISINGLY PERHAPS DISAPPOINTINGLY, WHEN WE THINK OF PRECISION MEDICINE TODAY WE REALLY THINK OF BIOLOGY THAT WILL COVERS ROUGHLY 2% OF OUR GENOME AND DESPITE ALL OF THE ADVANCES THAT WE HAVE REALIZED AS A FUNCTION OF THAT, THAT FINDING REMINDS US THAT THERE’S 98% GENOME THAT WE NEED TO BEGIN TO TRY TO UNCOVER TO BEGIN TO SCRATCH THE SURFACE HOW WE’RE GOING TO APPLY WHAT WE DO IN THE LABORATORY IN THE CONTEXT OF PATIENT CARE SO THE INTENT IS TO GIVE YOU SOME EXAMPLES THAT BASICALLY SPEAK TO THAT COUPLE OF DISCLOSURES BEFORE WE MOVE FORWARD

WE ACTUALLY FILED PATENT FOR TEST AND TREAT PARADIGM BUILDING ON FINDINGS WHICH WILL BE CAPTURED HERE AND WE HAVE BEEN WORKING TO ESTABLISH PRIVATE PARTNERSHIPS TO ADVANCE NOVEL THERAPEUTICS IN THIS SPACE OF THIS PARTICULAR SET OF PROBLEMS THIS SLIDE BASICALLY CAPTURES SOMETHING CLOSE NEAR AND DEAR TO YOUR HEART, IT’S ESSENTIALLY THE HUGE ACCOMPLISHMENT THAT WE MADE AS A SCIENTIFIC BODY IN COMPLETING THE HUMAN GENOME PROJECT AND ALL THE WORK THAT’S ENSUED FOLLOWING COMPLETION OF THAT EXERCISE THIS TRULY HAS BEEN ONE OF THE GREATEST BEASTS IN EXPLORATORY BIOLOGY AND CERTAINLY I THINK HAS GIVEN US AN OPPORTUNITY TO DEVELOP THE BLUEPRINT, THE BOOK ESSENTIALLY THAT HAS GUIDED MUCH OF WHAT WE DO IN THE CONTEXT OF APPLIED GENETICS AND ITS INTERSECTION WITH THE FIELD OF MEDICINE FROM THAT INTERSECTION WE HAVE BEEN ABLE TO REALIZE ESSENTIALLY THREE MAJOR BUCKETS OF ACTIVITY THAT I THINK GUIDE WHAT WE DO WE APPLIED TO INCREASE DIAGNOSTIC ADMISSION SOME OF THE EXAMPLE WE CAN SITE IN THAT PARTICULAR CONTEXT RELATE TO PRECISION ONCOLOGY WHERE STANDARDS OF CARE ARE NOW BEING DEFINED ON THE BASIS OF GENOMIC INSIGHT YOU CAN THINK OF BREAST CANCER PERHAPS AS A POSTER CHILD OF PRECISION ONCOLOGY THE PERSPECTIVE WHERE IN THE COUNTRY YOU ARE SOME FORM OF MOLECULAR DIAGNOSTICS WILL BE UTILIZED ROUTINELY TO CARRY OUT DECISIONS TRIAGE AND MANAGEMENT OF PATIENTS AFFLICTED WITH THAT CONDITION SECOND IMPORTANT APPLICATION OF INTERSECTION OF GENOMICS SCIENCE AND MEDICINE IS IN THE CONTEXT OF PHARMACO GENOMICS CERTAINLY A SCIENCE THAT’S NOT VERY NEW, I RECALL 40 YEARS AGO TAKING A COURSE IN PHARMACOGENETICS BUT THE LEVEL OF INSIGHT WHICH WE COVER PHARMACOGENETICS HAS TOTALLY COMPLETELY REDEFINED ITSELF OVER THE COURSE OF THE LAST 40 YEARS SO NOW WE’RE BEGINNING TO REALIZE TARGETED THERAPIES THAT NOT ONLY CAN BE USED FOR IMPROVED EFFICACY, IN MANAGING SPECIFIC CONDITIONS, BUT PERHAPS MORE IMPORTANTLY IN DECREASING TOXICITY AND MINIMIZATION OF ADVERSE OUTCOMES FOR THE PATIENTS THAT GET TREATED WITH TARGETED THERAPIES I THINK AN AREA THAT’S GOING TO PROVIDE OPPORTUNITY FOR CONTINUED EXPANSION AS BOTH PUBLIC AND PRIVATE SECTORS CONTINUE TO WORK TOGETHER, TO BE ABLE TO ADVANCE THAT PARTICULAR SPACE THEN LAST BUT NOT LEAST, THE IDEA OPTIMIZED CLINICAL MANAGEMENT OF PATIENTS REQUIRE STRATIFICATION AND MOLECULAR DIAGNOSTICS BECAUSE ONE OF THE BIGGEST PROBLEMS THAT WE HAVE FACED IN THE FIELD OF MEDICINE AND WE CERTAINLY ENCOUNTER AS WE ENGAGE IN CLINICAL TRIALS IS THE FACT THAT ONE SIZE DOESN’T FIT ALL, THE MANTRA POSITION MEDICINE IDEA THAT EACH OF US IS UNIQUE IN OUR OWN WAY AND THAT WE NEED TO BE ABLE TO CAPTURE THAT LEVEL OF RESOLUTION IN ORDER TO PROVIDE OPTIMIZED WAYS IN MANAGING SPECIFIC CONDITIONS A SHIFT AWAY FROM REMEDIATIONS OF PROBLEMS IN THE CONTEXT OF HEALTH TO A PARADIGM THAT FOCUS ES ON PREVENTION SO ONE OF THE MORE SOMBERING ASPECTS OF THIS WHOLE STORY IS THE FACT AS INDICATED IN MY INTRODOWNINGTORY REMARKS TO YOU THAT A LOT OF WHAT WE DO, A LOT OF WHAT WE THINK WE UNDERSTAND REALLY CONSTITUTES 2% OF WHAT I’M SHOWING HERE IN THIS SLIDE AS 55% OF A GENOME AND THAT THE REST OF THAT GENOME COMES FROM UNDERSTANDING OF REGULATORY BIOLOGIES VERY, VERY LIMITED SO THE FOCUS OF MY TALK TODAY IS GOING TO BE STRICTLY ON 17% OF THAT 45% THAT WE UNDERSTAND VERY POORLY AND WE CALL REPETITIVE DNA WITHIN OUR GENOME AND FOR THAT MATTER THE GENOMES HAVE PRETTY MUCH ALL SPECIES TURNS OUT THAT 17% OR 19 DEPENDING ON REFERENCE YOU USE OR 20 EVEN NOW WITH SOME OTHER CLINICIANS, THAT HAVE COME

ALONG, CONCEPT TO A SET OF SEQUENCES WE CALL LONG INTERSPERSED NUCLEAR ELEMENTS ONE THE MAJORITY OF THOSE SEQUENCES ARE SEQUENCES THAT HAVE BEEN SILENCED DURING THE COURSE OF EVOLUTION, PRIMARILY THROUGH TRUNCATION OF SEQUENCES THAT REGULATE THE ACTIVITY OF THESE SEQUENCES AND AS OF TODAY UP TO MAYBE 100 OF THESE SEQUENCES REMAIN ACTIVE IN THE HUMAN GENOME TURNS OUT THAT OF THOSE SEQUENCES THAT DO REMAIN ACTIVE BECAUSE THEY HAVE RETAINED STRUCTURAL INTEGRITY TO CARRY OUT TRANSCRIPTIONAL ACTIVATION AS WELL AS PASTING BACK REINSERTION BACK INTO THE GENOME, ARE THE SEQUENCES THAT I BELIEVE WE’RE ABLE TO CAPITALIZE ON IN TRYING TO ADVANCE THE GOALS OF PRECISION MEDICINE THESE ARE THE GIFT WRAPPINGS I WAS ALLUDING TO IN MY TITLE NOTICE THE REMAINDER OF THOSE SEQUENCE ARE SEQUENCES WE CALLED MOBILE REPETITIVE DNA SEQUENCES THAT UNDER THE RIGHT CONTEXT UNDER THE RIGHT CONDITIONS, THE SEQUENCES CAN MOBILIZE BUT WHAT MAKES IT 17% ALIGN SEQUENCE AS GROUP WITHIN THE LARGER 45% SO UNIQUE IS THE LINES ARE THE ONLY ONES THAT CAN DO SO AUTONOMOUSLY SO IS THAT AUTONOMY OF MOBILIZATION THAT MAKES THEM THE I GUESS THE PREMIER REPETITIVE SEQUENCE OF GENOME IN THE CONTEXT OF MOBILE REPETITIVE SEQUENCES SO THE STORY I WILL SHARE WITH YOU IS ONE THAT’S TAKEN ROUGHLY 20 YEARS IN EVOLUTION AND I WILL BEGIN GIVING YOU SNIPPETS OF TRANSLATIONAL STUDIES THAT SET THE STAGE FOR CLINICAL VALIDATION BOTH IN RETROSPECTIVE AS WELL AS PROSPECTIVE STUDIES WITH THE IDEA THAT HOPEFULLY WE CAN TAKE ADVANTAGE OF OUR UNDERSTANDING OF THAT BIOLOGY TO TREAT AND MANAGE DISEASE CONDITIONS IN WHICH SEQUENCES APPEAR TO BE IMPORTANT SO BACK IN THE ’90s WE REPORTED, MADE THE OBSERVATION THAT WHEN YOU STUDY THE WHOLE GENOME EXPRESSION OF TRANSCRIPTOME IF YOU WOULD OF CELLS UNDERGOING TRANSDIFFERENTIATION, A PROCESS THAT ESSENTIALLY GUIDES MANY PA PATHOLOGISTS WE THINK OF TODAY INCLUDING CANCER, FIBROSIS, CHRONIC OBSTRUCTIVE DISEASE, ATHEROSCLEROSIS, DEMENTIA, THERE’S GENETIC REPROGRAMMING IN THOSE PATHOLOGICAL CONDITIONS AND WHEN YOU TRY TO GAIN INSIGHT INTO THE GENETIC REPROGRAMMING EVENTS THAT DRIVE TRANS-DIFFERENTIATION OF CELLS, LINE ONE SEQUENCES APPEAR TO PLAY A VERY HUGE ROLE BACK IN THE DAY WHEN WE COMPLETED THESE EXPERIMENTS AND WE CLONE AND SUBCLONE THESE SEQUENCES, WHAT WE DISCOVERED WAS THAT 50% SEQUENCES THAT APPEAR UPREGULATED IN CONTEXT OF THAT TRANS-CHILDRENIATION RESPONSE WHETHER EPITHELIUM TO MAZE KIEM OR MAZE KIEM TO EPITHELIUM APPEAR — MESENCHYME TO LINE ONE EPITHELIUM, THE LINE 1 IS MADE THESE SEQUENCES MUST BE EITHER SECONDARY PLAYERSES IN THE TRANSDIFFERENTIATION RESPONSE OR POTENTIALLY DRIVERS OF THAT RESPONSE FOR THOSE OF YOU WHO MAY NOT BE IN THIS FIELD, HERE IS A QUICK SCHEMATIC, SCHEMATIC GIVING YOU THE MAJOR MAMMALIAN TRANSPOSABLE ELEMENTS IN HUMANS SUFFICE IT TO SAY THE SEQUENCES THAT WE ARE PRIMARILY INTERESTED IN ARE THE ONES, THE NON-LONG TERMINAL REPEAT FAMILY WHICH L-1 IS THE MAJOR FAMILY MEMBER REMEMBER THAT THIS IS TO BE DIFFERENTIATED FROM LONG TERMINAL REPEAT SEQUENCES, THE VAST MAJORITY OF WHICH UP TO 98% WHICH ARE INACTIVE IN THE HUMAN GENOME AND ONLY INACTIVE IN RODENTS AND OTHER SPECIES ALSO IMPORTANT TO THE BIOLOGY OF LINES THE ACTIVITY OF ALLO ELEMENTS OF INTEREST TO SOME IN THE AUDIENCE IS HEAVILY REGULATED THROUGH THE MACHINERY ENCODED FOR LINE ONE ELEMENTS WHEN WE MADE THE DIFFERENCEIATION THAT TRANSDIFFERENTIATION WAS COUPLED TO THE UPREGULATION OF THESE SEQUENCES WE KNEW LITTLE ABOUT THE SEQUENCES IN THOSE DAYS WHEN YOU DID A A

LITERATURE SEARCH ON IT, UNFORTUNATELY NOT PUBMED BACK IN THOSE DAYS THESE ARE THE DAYS OF INDEX MED ICUS, PLOWING THROUGH PAGES AND PAGES, WHAT YOU FIND IS THERE’S HARDLY ANYTHING IN THE LITERATURE, IF YOU DID THAT SAME SEARCH TODAY TAKING ADVANTAGE OF ELECTRONIC SEARCH ENGINE YOU FIND THOUSANDS OF PUBLICATIONS FOR YEAR IN THIS PARTICULAR TOPIC WHEN YOU PUT ALL THE SMALL PIECES OF INFORMATION AVAILABLE TOGETHER, WHAT YOU COULD COME UP WITH IS THE IDEA REPETITIVE SEQUENCES WORK A HYBRID OF REPETITIVE INERT SEQUENCES WITHIN THE GENOME WITH CODIFICATION MACHINERY THAT WE HAVE GROWN TO BE SO FAMILIAR WITH IN TERMS OF CODIFICATION FOR PROTEINS LINE ELEMENTS CARRIED WITHIN THEM A FIVE PRIME TRANSLATED REGION THAT FUNCTIONS AS REGULATORY REGION AND HAD TWO OPEN READING FRAMES ONE OTHER READING FRAME ENCODING FROM NOW WHAT WE UNDERSTAND IS A PROTEIN CRITICAL TO ASSEMBLY FOR NUCLEAR PROTEIN PARTICLES THAT GET SHUTTLED BACK TO NUCLEUS FOR REVERSE TRANSCRIPTION AND OR TOO, WHICH IS AN OPEN READING FRAME, ADJACENT TO OPEN READING FRAME ONE, WHICH ENCODES FOR INCITEATIC ACTIVITY OF REVERSE TRANSCRIPTTASE FOR MAKING OF DNA AND FOR PRIMING OF REVERSE TRANSCRIPTION THREE PRIME UTR WITH POLYTAIL ARCHITECTURE REMIND YOU OF CONVENTIONAL GENE CODIFICATION STRUCTURE AND WHAT’S INTERESTING IS THAT IT’S ONLY THROUGH THE COURSE OF EVOLUTION WHERE REVERSE TRUNCATION OF THESE SEQUENCES ONLY ALLOWS FOR COPYING IN REVERSE MODE OF THE SEQUENCES THAT ARE INERT WHEN PASTED IN ELECTRONIC KATEED FASHION IN GENOME BECOME WHAT WE USED TO CALL THE JUNK DNA THAT DOES NOT ENCODE FOR ANYTHING FOR THE MOST PART WE DID NOT UNDERSTAND ITS BIOLOGY AS ALLUDED TO ALREADY, A HUNDRED OF THESE SEQUENCES REMAINED FULL LENGTH AND PRESUMABLY ACTIVE IN THE HUMAN GENOME AND IMPORTANT AND RELEVANT FOR REMAINDER OF THE TALK IS THE FACT THAT MANY OF THESE SEQUENCES ARE HIGHLY POLYMORPHIC IN HUMANS SO THE ASSUMPTION THAT WE MANY EXPERIMENTS WE DESIGN, IF EXPERIMENTS ARE POLYMORPHIC IN HUMANS AND UPREGULATED OR DEREGULATED DURING THE TRANSREGULATION SERVE TO EXPLAIN SOME OF THE DIFFERENTIAL RESPONSE THAT WE SEE I DID NOT WANT TO BE IN INCOMPLETE, I WANTED TO INTRODUCE YOU TO PERHAPS NEWEST SET OF DISCOVERIES MADE BY THE LAB WITH A NEW OPEN READING FRAME THAT WAS DISCOVERED IN PRY MED, IT’S IMPORTANT BECAUSE IT APPEARS TO INFLUENCE MOBILITY OF LINE 1, ENCODES IN THE ANTI-SENSE ORIENTATION AND THAT WILL BE CRITICAL BECAUSE THE ANTI-SENSE CODIFICATION OF SEQUENCES HAS BEEN REGARDED AS ONE OF SILENCING MECHANISMS UTILIZED BY THE L 1 MACHINERY TO REGULATE RATES OF TRANSCRIPTION IN CELLS I THINK ONE OF THE COMMENTS THAT WAS MADE BY COLLEAGUE IN THE AUDIENCE I WAS CHATTING WITH IS THE IDEA THAT THESE SEQUENCES ARE NOT SO ACTIVE AND WE ARE ALIVE AND WELL OF COURSE THE ANSWER TO THAT IS YES, NATURE IS WISE AND NATURE HAS ENGINEERED MANY MULTIPLE WAYS WHICH THE SEQUENCES ARE CHECK IN CHECK AND NOT ALLOWED TO DO THE HAVOC THEY COULD DO IN THE GENOME WHEN SEQUENCES ARE REAWAKENED IF YOU WOULD, THEY’RE REAWAKENED THROUGH DICORRUPTION OF EPIGENETIC SILENCING THAT INVOLVES BOTH HISTONE CODE AS WELL AS DNA MACHINERY, DNA METHYLATION MACHINERY TO MAINTAIN THE DNA OF THE L 1 SEQUENCE SILENCED INJURY, STRESS, TRANSDIFFERENTIATION FORCES SEQUENCES TO BE REAWAKENED, WHEN THEY REAWAKE A NUMBER OF THINGS HAPPEN THE CONVENTIONAL UNDERSTANDING OF THE BIOLOGY IS THAT THESE SEQUENCING IF ALLOWED, BY THE CONTEXTUAL FRAMEWORK OF THE CELL INVOLVED IN THE INSULT WOULD REINSERT BACK TO THE GENOME, WHAT’S BELIEVED TO BE RANDOM INSERTION THROUGHOUT THE GENOME AND THAT INSERTION GOES INTO INERT REGION OF THE GENOME IT WOULD PROBABLY BE A SILENT MUTATION IF IT GOES INTO A REGION OF RELEVANCE AND FUNCTIONALITY OF A GENOME THEN DISEASE COULD ENSUE SINCE THE EARLY ’80s YOU FIND EXAMPLES IN THE LITERATURE OF DISEASES THAT HAVE BEEN IMPLICATED WITH ABERRANT, IF YOU WOULD, LINE ONE MOBILIZATION THROUGHOUT THE GENOME, HEMOPHILIA BEING THE FIRST THAT WAS DOCUMENTED BY THE LAB WE

HAVE ACTUALLY PUBLISHED WORK THAT IMPLICATES A NON-INSERTIONAL BIOLOGY AND SOME WHAT I HAVE TO SAY FOR THE REMAINDER OF THE TALK WILL BE IMPORTANT FOR THAT CONTEXT SO I WILL RESERVE COMMENTS FOR THAT WE SPENT A NUMBER OF YEARS CHARACTERIZING THE SILENTING MACHINERY THAT GOVERNS THE REGULATORY EXPRESSION OF LINE 1 ELEMENTS SINCE THAT BECAME CRITICAL TO OUR UNDERSTANDING OF TRANSDIFFERENTIATION MACHINERIES IN CELLS SO SUFFICE IT TO SAY WE COULD SPEND TIME TALKING ABOUT THE CONTENT OF THE SLIDE, THIS IS ONE OF THOSE SLIDES THAT IN ONE FRAME YOU CAPTURE 15 YEARS WORTH OF EFFORT BUT SUFFICE IT TO SAY THE MAJOR DISCOVERY MADE BY THE GROUP IS RETINAL BLASTOMA PROTEIN AND MACHINERY OF ORCHESTRATED BY RETINAL BLASTOMA APPEARS TO BE A MAJOR MASTER REGULATOR OF A SILENCING ACTIVITY OF LINE 1 RETRO ELEMENTS AND MAMMALIAN CELLS AND IN THE CONTEXT OF THAT TRANSDIFFERENTIATION RESPONSE THAT I ALLUDED TO EARLY ON, WHAT HAPPENS IS THAT THE ABILITY OF RETINAL BLASTOMA TO REMAIN IN COMPLEX INTERACTION WITH THE MACRO MOLECULAR SILENCING MACHINERY BECOMES DISRUPTED THROUGH CHANGES IN DNA TRANSFERASE ACTIVITY AS WELL AS CHANGES IN HISTONE METHYLATIONS AND ACETYLATIONS IN WAYS THAT ALLOW FOR THE CHROMATIN TO OPEN UP AND THAT ALLOW FOR TRANSCRIPTIONAL ACTIVATION OF THE RETRO ELEMENT WHICH IN TURN ENCODES FOR PROTEIN IS SHOWED IN THE PREVIOUS SLIDE, DOES FORMATION OF THOSE NUCLEAR PROTEIN PARTICLES INSIST PREFERENTIAL L-1 ENCODED PROTEINS FORMING THE PARTICLE THAT DIDN’T GET SHUTTLED TO THE NUCLEUS FOR REVERSE TRANSCRIPTION AND REINSERTION AND THAT IF YOU WANT TO RESILENCE THE ELEMENT YOU HAVE TO REGAIN REGULATORY CONTROL IN THE CELL AS YOU WOULD IMAGINE BASED ON THIS VERY QUICK SYNOPSIS OF THE COMPLEXITY OF LINE ONE SILENCING, WHAT YOU CAN SURPRISE, ANYTHING THAT DISRUPTS CELL CYCLE REGULATORY CONTROL, ANYTHING THAT MODULATES THE ACTIVITY OF THE TRANSCRIPTION FACTORS INTERACTING WITH RETINAL BLASTOMA, ANYTHING THAT MODULATES THE EPIGENETIC MACHINERY IN WAYS THAT INTERACT DURING THELY WITH THAT SILENCING MACRO MOLECULAR COMPLEX IS A POTENTIAL IMPACT OF LINE ONE SEQUENCES HERE IS AN EXAMPLE OF THE IMPORTANCE OF RETINAL BLASTOMA IN THAT CONTEXT SO THIS IS AN EXPERIMENT IN WHICH WE TOOK CLONES OF CELLS IN WHICH ALL MEMBERS OF RETINAL BLASTOMA FAMILY HAVE BEEN SILENCED WE BASICALLY CHALLENGED THESE CELLS IN CULTURE WITH A CHEMICAL CARCINOGEN KNOWN TO REACTIVATE LINE ONE EXPRESSION AS YOU CAN SEE THERE’S MARKED UPREGULATION OF LINE ONE EXPRESSION IN THE CONTEXT OF RB LOSS, YOU CAN REPRODUCE THE EXPERIMENT THROUGH NATIVE RB YOU CAN DO PHOSPHORYLATION ACTIVITY OF RB REPRODUCE EMPERIMENT MODULATION OF P-53 ACTIVITY, ANYTHING THAT YOU WILL ULTIMATELY REALIZE RB INTERACTION WITH THAT DNA SEQUENCE WILL CERTAINLY BE CONTRIBUTORY TO UPREGULATION OF LINE ONE SEQUENCE THEN YOU WONDER ABOUT WHAT THE BIOLOGICAL IMPACT OF THAT, BUT AT THE TIME WE COMPLETED MUCH WORK TRYING TO FIGURE OUT THIS LEVEL OF COMPLEXITY AT THE LEVEL OF REGULATORY CONTROL FOR TRANSCRIPTION OF THIS ELEMENT, ONE THING OCCUPIED ON OUR MIND IS WE UNDERSTOOD LITTLE ABOUT THE REPROGRAMMING BIOLOGY INVOLVED IN TRANSDIFFERENTIATION SO ONE THING WE DID IS TAKE ADVANTAGE OF COMPUTATIONAL BIOLOGY APPROACHES UTILIZING VERY SIMPLE BINARY MODELS FOR PREDICTIVE BIOLOGY, CERTIFIED STRATEGIES AND WE DEFINED WHAT WE BELIEVE TO BE A RELEVANT ARCHITECTURE OF LINE ONE REGULAR RATORY NETWORK THE INTENT OF THESE EXPERIMENTS WAS ESSENTIALLY THE IDEA THAT IF WE UNDERSTOOD PROTEINS AND GENES THAT INTERACTED WITH LINE IN CONTEXT OF TRANSDIFFERENTIATION WE MIGHT BE ABLE TO BEGIN TO REVERSE ENGINEER IF YOU WOULD, THE REGULATION OF CONFERENCEIATION PROGRAMMING IN WAYS TO TAKE ADVANTAGE OF DISEASE, SUFFICE IT TO SAY IN THE INTEREST OF TIME WE VALIDATED THE COMPUTATIONALLY DERIVED NETWORK I HAVE SHOWN HERE IN THE SLIDE THESE ARE SOME OF THE CRITICAL PLAYERS, I’M NOT GOING TO HAVE TIME TO GO INTO THE UNIQUE

INTERACTIONS THERE, CERTAINLY HAPPY TO TALK TO YOU SPECIFIC EXAMPLES BUT FOR NOW I THINK TAKE HOME MESSAGE, THIS REGULATORY NETWORK APPEARED TO BE HONED IN TO THREE SPECIFIC DOMAINS OF BIOLOGICAL ACTIVITY THOSE INCLUDE INFLAMMATION, INFLAMMATORY BIOLOGY, INTERACTIONS WITH CYTOKINES AND INTERACTION WITH THINGS THAT REGULATE CYTOKINES EXTRA CELLULAR MATRIX MACHINERY OF CELLS WHICH IS VERY IMPORTANT REGULATORY SIGNALING PATHWAY FOR CONTROL OF DIFFERENTIATION BEHAVIOR IN CELLS AND METABOLISM SO WHEN YOU PUT TOGETHER THOSE THREE DOMAINS, YOU START THINKING OF DISEASES WHICH THIS MIGHT BE IMPORTANT CANCER IMMEDIATELY OF COURSE, SURFACES TO THE TOP IN MY CASE GIVEN CLINICAL INTEREST AND BACKGROUND IN PULMONOLOGY, I STARTED THINKING OF REGULATORY CONDITIONS OF THE LUNG WHICH PERHAPS LINE ONE MIGHT BE PLAYING AN IMPORTANT REGULATORY ROLE THE REMAINDER OF OUR TALK WILL HIGHLIGHT TRANSLATIONAL VALIDATION PROSPECTIVE RETROSPECTIVE VALIDATION OF LINE ONE ACTIVITY IN THE CONTEXT OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE AND POTENTIAL TO CONTRIBUTE TO CANCERS OF THE LUNG SO WHY LUNG AS POTENTIAL TARGET? TURNS OUT THE LUNG IS A PORTAL OF ENTRY FOR MANY AGENTS IN THE ENVIRONMENT INJURY ASSOCIATED WITH ENVIRONMENTAL IMPACTS IN LUNG EPITHELIAL CELL AS WELL AS OTHER CELL TYPES PRESENT IN THE LUNGS INVOLVE INFLAMMATORY SIGNALING PATHWAYS BECAUSE A LOT OF THE CONDITIONS IN THE LUNG ARE DICTATED AND DEFINED BY UNCONTROLLED INFLAMMATION IN RESPONSE TO THAT RESPONSE, SOMETHING THAT OCCURRED HEAVILY, THAT IS EPITHELIUM MESENCHYMAL TRANSLATION, A TERM VERY FAMILIAR TO PEOPLE IN THE FIELD OF CANCER AND DEVELOPING ARTHRITIS, EPITHELIAL MESENCHYMAL TRANSITION RECAPITULATES THE TRANSDIFFERENTIAL THAT I ALLUDED TO WHEN I PRESENTED THE SLIDE WE STUDIED 20 YEARS BEFORE SO IT TURNS OUT THAT IN RESPONSE TO UNCONTROLLED INFLAMMATION IN THE LUNG, THERE IS EPITHELIAL MESENCHYMAL TRANSITION AND THAT TRANSITION HAS BEEN DIRECTLY IMPLICATED TO CHRONIC OBSTRUCTIVE PULMONARY DISEASE OR NON-SMALL CELL LUNG CARCINOMAS OF THE LUNG WHICH CONSTITUTE THE MAJORITY OF THE CANCERS IN THE LUNG, # 5% OF THEM IMPORTANTLY — 585% OF THEM — 85% OF THEM IMPORTANTLY TO THE WORK I SHOW YOU THIS RESPONSE IS DIRECTLY COUPLED TO DISREGULATION OF TRANSFORMING GROWTH FACTOR BETA SIGNALING THROUGH CANONICAL PATHWAYS AS WELL AS NON-CANONICAL ONE TERE’S EXTENSIVE UPREGULATION OF LINE ONE SEQUENCES IN MET TA STATIC LUNG CANCER AND MANY DISEASES OF THE LUNG EXHIBIT HETEROGENEITY BACK TO THE POINT BEFORE ABOUT THE HIGHLY POLYMORPHIC NATURE OF SEQUENCES AN WHETHER THAT BEHAVIOR MAY CONTRIBUTE TO DISEASE HERE IS THE RESULT OF AN EXPERIMENT WHICH WHAT WE DID WAS WE FORCED EXPRESSED LINE 1 IN TO BRONCHIAL EPITHELIAL CELLS, CELLS THAT COME FROM THE LARGER BRONCHI, PROBABLY TO THIRD DEGREE LEVEL WITHIN THE BRONCHIAL TRAIT AND CELLS THAT CONTRIBUTE PRIMARILY TO OCCURRENCE OF EITHER CONTRACTILE ACTIVITY IN THE CONTEXT OF COPD INFLAMMATION IN THE CONTEXT OF COPD OR CENTRAL TUMORS IN THE CONTEXT OF NON-SMALL CELL LUNG CARCINOMAS, SO THESE FORCE EXPRESSION WAS ASSOCIATED WITH INDUCTIONS EPITHELIAL MESENCHYMAL TRANSITION KEEPING WITH THE IDEA THAT THE REPROGRAMMING ASSOCIATED WITH TRANSDIFFERENTIATION MIGHT BE DIRECTLY COUPLED TO THE OVEREXPRESSION OF THESE SEQUENCES THERE IS CONTRIBUTION OF BOTH INSERTIONAL BIOLOGY KNEW THAT GENESIS — MUTAGENESIS BIOLOGY FOR TARGETS AS WELL AS NON-BIOLOGY, SUFFICE IT TO SAY TO THIS TUCK THE MAJORITY OF THE ACTIONS OF LINES TO MODULATE EPITHELIAL MESENCHYMAL PROGRAMMING IN EPITHELIAL CELLS IS UNRELATED TO INSERTIONAL MECHANISM AND ALL THE ACTIVITIES ACTUALLY SEEN IN THE ABSENCE OF INTEGRATION BACK INTO THE GENOME SO WE STARTED ASKING OURSELVES QUESTIONS ABOUT THE RELEVANCE OF THIS BIOLOGY TO THE PATHOGENESIS OF COPD, CLINICALLY SPEAKING,

THIS IS A VERY IMPORTANT QUESTION TO ASK BECAUSE CHRONIC OBSTRUCTIVE PULMONARY DISEASE HAS BECOME THE THIRD KILLER IN THE U.S. AS WELL AS REMAINDER OF THE WORLD IN MANY WAYS CONNECTED TO LIFESTYLE FACTORS ENVIRONMENTAL EXPOSURE, SMOKING, BUT INTERESTINGLY EVEN TODAY WITH THE ADVANCE MEDICINE THAT WE HAVE ALL THERAPIES FOR CHRONIC OBSTRUCTIVE PULMONARY DISEASE ARE PALLIATIVE THERAPIES, WE ARE TRADING SYMPTOM, WE ARE NOT DIRECTING THESE THERAPIES TO UNDERSTANDING MECHANISTIC BIOLOGY MORE TO PATHOLOGY OF DISEASE SO WE ARE TRYING TO UTILIZE OUR UNDERSTANDING OF THE CRITICAL ROLE OF LINE REGULATING THE REPROGRAMMING BEHAVIOR OF CELLS IN THE CONTEXT OF HEALTH, TO DISEASE TRANSITION TO BRONCHIAL EPITHELIAL CELLS AS TARGETS FOR THERAPEUTIC INTERVENTION IN CONTEXT OF COPD I THINK IT’S IMPORTANT BECAUSE A LOT OF THE BIOLOGY THAT YOU HAVE SEEN PATIENTS WITH COPD IS HIGHLY CONSISTENT WITH THE BIOLOGY YOU SEE DURING ALTERED LINE ONE SIGNALING IN CELLS SO I WILL GIVE YOU A COUPLE OF EXAMPLES OF AN ASSAY WE HAVE DEVELOPED AND SOME OF THE RESULTS OF RETROSPECTIVE EXPERIMENTAL STUDY SO ONE OF THE CHALLENGES IN STUDYING LINE ONE SEQUENCES OF COURSE THE SIGNAL TO NOISE RATIO YOU HAVE MULTIPLE SEQUENCES IN THE GENOME YOU SPECIFY ZIP CODES IF YOU WOULD, ADDRESS LOCATIONS TO KNOW WHICH LINES WORKING WITH IMPORTANTLY CAUSE AND EFFECT WITH MANY OTHER GENETIC RELATIONSHIPS BECOMES CRITICAL SO WE WANT TO DEVELOP A SIMPLE BLOOD TEST THAT WOULD BE SUGGESTIVE OR REPRESENTATIVE OF WHAT THE ACTIVITY OF LINES IN THE GENOME WERE SO WE DEVELOPED AN ELISA ASSAY THAT BASICALLY ESTABLISH ON LINE 1 ACTIVITY WE HAVE CONTACTORRIZED THIS RF 1P ELISA ASSAY IN THE CONTEXT OF BIOMARKER DEVELOPMENT PATHWAY, WALKING ALL THE WAY FROM ITS DISCOVERY AS A CANDIDATE BIOMARKER ALL THE WAY TO HUMAN POPULATION TESTING AND I WILL SHOW YOU SOME OF THE TIDBITS OF RESULTS HAPPY TO ANSWER SOME QUESTIONS RELATED TO THAT SO WHAT WE DID, IN THIS PARTICULAR SET OF STUDIES WE TOOK A VERY LARGE COHORT OF CHRONIC COPD PATIENTS, COPD DEFINE ON THE BASIS OF EXPIRATORY VOLUMES IN THE LUNG, A CLINICAL DEFINITION USED TO UTILIZE THESE TYPES OF PATIENTS AND WE STRATIFY THESE PATIENTS ON BASIS OF SEVERITY OF COPD AND THAT’S WHERE THE L-1 DATA YOU SEE ON THIS SLIDE ALLUDES TO AS YOU CAN SEE, AS YOU CORRELATE BLOOD LEVELS OF TESTIFY PROTEIN ENCODED BY LINE 1 WITH SEVERITY OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE, THESE LEVELS ACTUALLY GO UP IF YOU WERE TO — THIS IS ABOUT DATA FROM ABOUT 600 SUBJECTS WE’LL CONTINUE TO EXPAND UPON THAT BECAUSE WE HAVE BECOME INTERESTED IN DETERMINING NOT ONLY CORRELATION BETWEEN LEVELS OF PROTEIN AND SEVERITY OF DISEASE, BUT LONGITUDINAL TRAJECTORY OF LEVELS RELATIVE TO PROGRESSION IN TO MALIGNANCY FOR THE LUNG I WILL SPEAK TO THAT IN A FEW MINUTES THAT PARTICULAR SET OF QUESTIONS BECOME CRITICAL GIVEN HIGH GENETIC MIXTURE THAT WE HAVE IN U.S. POPULATIONS IT BECOMES CRITICAL BECAUSE GENETIC MIXTURE THAT CHARACTERIZES THE AMERICAN SOCIETY IS SOMETHING THAT FOR THE MOST PART HAS BEEN LARGELY IGNORED IN MOST GENETIC STUDIES WE HAVE BROAD CATEGORIZATIONS OF ETHNICITY MANY OF THOSE CATEGORIZATIONS ARE PROBABLY INCORRECT BECAUSE THEY FAIL TO TAKE INTO ACCOUNT GENETIC HETEROGENEITY AND THIS IS CRITICAL TO LINE ONE BIOLOGY BECAUSE OF THE POLYMORPHIC NATURE AND I WILL SHOW YOU RESULTS THAT WILL SPEAK DIRECTLY TO THAT SO THE IDEA IS THAT WE CHARACTERIZE THE HETEROGENEITY OF THE U.S. POPULATION IN THIS CASE, CHRONIC COPD PATIENTS, TOE UNDERSTAND HOW THERAPEUTIC INTERVENTIONS TO CONTROL OF LINE ONE BUT REGULATE TRAJECTORY OF DISEASE IN THESE PATIENTS IF IN FACT L 1 IS DRIVING THE TRAJECTORY OF DISEASE, WHICH WE BELIEVE IT IS LINE ONE CAN BE USED AS A MEANS OF STRATIFYING THE POPULATION IF YOU STRATIFY THE POPULATION ON THE BASIS OF LINE ONE, ON LINE 1 PLAYING A CONTRIBUTOR ROLE TO TRAJECTORY OF DISEASE AND YOU INTERVENE WITH THAT STUDIES REGULATION YOU SHOULD BE

ABLE TO MAKE THAT, THAT’S THE CONCEPTUAL FRAMEWORK GUIDING THE WORK WE DO NOW I ACTUALLY BELIEVE THAT IN THE SPACE OF PRECISION MEDICINE STRATIFICATION OF POPULATIONS IS ONE OF THE GREATEST CONTRIBUTIONS THAT CAN BE MADE IN CONTEXT OF LINES THIS IS GOING TO BE REALLY IMPORTANT BECAUSE EVEN THOUGH WE HAVE GOT A HUNDRED NULL SEQUENCES THOUGH WE HAVE A HUGE SEA OF SIGNAL TO NOISE RATIO IN THE GENOME, ONLY NINE OF THOSE LINES APPEAR TO BE ACTIVE TO THE LEVEL THAT DISREGULATES BIOLOGY AND CONTRIBUTE TO DISEASE SO WHAT APPEARS LIKE INSURMOUNTABLE TASK OF NAVIGATING THROUGH 600,000 SEQUENCES, BECOMES MANAGEABLE WHEN YOU ONLY HAVE TO WORRY ABOUT TEN TURNS OUT HIGHLY POLYMORPHIC FOR SEQUENCES IS LOCATION IS HERE AND THESE ARE THE SEQUENCES THAT WE ARE ACTUALLY NOW EXPLORING IN CONTEXT OF CHRONIC OBSTRUCTIVE DISEASE, WE HAVE PARTICULAR INTEREST IN GENES AND LOCI RESIDING IN CHROMOSOME 6 WHICH IS AN IMPORTANT CHROMOSOME IN THE CONTEXT OF LUNG PATHOLOGY SO THE RESULTS OF A EXPERIMENT IN WHICH WE COMPARED FOUR CHRONIC OBSTRUCTIVE PULMONARY DISEASE PATIENTS TO CONTROL ARE SHOWN HERE IN THIS SLIDE FOR EACH ONE OF THOSE LOCI I SHOWED IN THE PREVIOUS SLIDE SUFFICE IT TO SAY TURNS OUT THAT COHORT WAS POLYMORPHIC FOR THESE SEQUENCES AND THOSE PRESENT IN CHROMOSOME 6 APPEAR TO PLAY AN IMPORTANT ROLE IN REGULATION OF SEVERITY OF DISEASE SO THIS IS AN AREA WE’RE CONTINUING TO MAKE HUGE STRIDES IF YOU REMEMBER I ALLUDED TO THE FACT THAT PATIENTS WITH COPD THREEFOLD HIGHER RISK TO PROGRESS TO MALIGNANCY OF THE LUNG WE KNOW A BUNCH OF SMOKERS THAT NEVER GET LUNG CANCER SO THAT DIRECTLY SPEAKS TO THE HETEROGENEITY OF THE POPULATION AND STUDIES NOW ARE FOCUSED ONLY TRYING TO — ON TRYING TO UNRAVEL THE POTENTIAL CONTRIBUTION OF LINE GIVEN ABILITY TO REGULATE OF TRANSDIFFERENTIATION IN THE LUNG, INDUCING GENOMIC INSTABILITY, GIVEN ABILITY TO CAUSE HAVOC IN THE GENOME TO DRIVE TRANSITION OF PATIENTS FROM INTO CHRONIC OBSTRUCTIVE PULMONARY DISEASE AND COMPLETE THAT JOURNEY INTO MALIGNANCY OF THE LUNG THE IDEA IS TO IF YOU UNDERSTAND THAT BIOLOGY YOU CAN CAPITALIZE ON THAT FOR PHARMACEUTICAL AGENTS THAT INTERFERE WITH THAT PROGRESSION THE IDEA WOULD BE YOU TREAT COPDER WITH MEDICATIONS THAT WOULD PREVENT UNCONTROLLED TRANSDIFFERENTIATION TO PROGRESS AND IF YOU IN COMBINATION WITH SILENCING OR RESILENCING OF LINE ONE SEQUENCES YOU MAY CHANGE TRAJECTORY OF DISEASE SO AN EXAMPLE THAT I ELECTED TO PRESENT TO YOU IS AN INTERVENTION WE TESTED THE ABILITY OF TGF BETA ONE INHIBITORS DRUGS ARE CLINICALLY USED IN THE MARKET INHIBITION OF LINE ONE ACTIVITY CAUSED BY AGENTS AND ABILITY TO PREVENT TRANSDIFFERENTIATION AND IMPROVE CLINICAL OUTCOMES OF PATIENTS WE HAVE A CLINICAL TRIAL GOING ON NOW BEGINNING TO EXPLORE THE USEFULNESS OF THIS APPROACH WE’RE UTILIZING MULTIPLE WAYS OF TACKLING LINE ONE UPSTREAM THROUGH REGULATION OF TGF BETA BECAUSE WE DEMONSTRATED IN OUR STUDIES THAT TGF BETA LIES UPSTREAM OF LINE ONE IN REGULATION OF EMT BIOLOGY AND WE’RE ALSO TACKLING THE LINE ONE DIRECTLY BY WAY OF THERAPEUTIC MODIFICATION OF LINE ONE SEQUENCES TO PREVENT ABILITY TO REVERSE TRANSCRIBE SO THE SUCCESS OF THOSE EXPERIMENTS IS STILL PRELIMINARY BUT I THINK WE’RE ENCOURAGED ENOUGH BY RESULTS OF THOSE STUDIES THAT WE THINK THAT THIS IS GOING TO BE AN AREA WHERE NO SMALL MOLECULES ARE DEVELOPED IN WAYS TO TACKLE LINE ONE AND TO BE MORE PRECISE IN THE WAY WE’RE MAKING INTERVENTIONS THAT GUIDE CLINICAL DECISION MAKING FOR MANAGEMENT OF THOSE CONDITIONS SO SOME OF YOU MIGHT ASK YOURSELVES, WHY IS THIS IMPORTANT?

THE ANSWER TO THAT LIES IN THE NEWEST REPERTOIRE GIVEN TO PULMONOLOGISTS AND TO ONCOLOGISTS IN SCREENING FOR LUNG CANCER NOT MANY LUNG CANCERS ARE SCREENED FOR EXCEPT PERHAPS NOTORIOUS DIFFERENCE FOR BREAST CANCER BUT MOST OTHER CANCERS, SCREENING IS ACTUALLY DISCOURAGED AND LUNG CANCER IS ANOTHER EXCEPTION BUT LUNG CANCER SCREENING HAS ONE HUGE ACHILLES HEEL TO IT, AND THAT IS THAT THE SCREENING IS CONFINED TO THE RIGHT AGE BRACKET, PATIENTS AGE 55 TO 80, WHO HAVE A 30 PACK SMOKING HISTORY OR WHO HAVE ONLY QUIT THE PAST 15 YEARS, IE AT HIGHER RISK OF MALIGNANCY AND LOW DOSE CT SCREEN THAT IDENTIFIES NODULES IN THE LUNG IT TURNS OUT THAT INCIDENCE OF SOLITARY NODULE IDENTIFICATION AND AS PART OF THE SCREENING PRESENTS A HUGE CLINICAL PROBLEM FOR THE PULMONOLOGIST AND PROBABLY ONCOLOGIST BECAUSE 95% OF THOSE NODULES IDENTIFIED ARE FALSE POSITIVES SO THIS INVOKES THE COMPLEXITY THAT WE HAVE TO DEAL WITH IN TERMS OF ARE WE DOING OURSELVES A SERVICE BY SCREENING PATIENTS IN WAYS TO MOSTLY PROVIDE FALSE POSITIVE RESULT, INCREASE COSTS T THE HEALTHCARE SYSTEM, INCREASE PATIENT DISSATISFACTION, INCREASE PSYCHOLOGICAL DISCOMFORT AND END UP IN A CLINICAL TRAJECTORY THAT PROBABLY IS NOT CONDUCIVE TO INCREASED PROGRESS SO ONE OF THE THINGS THAT WE ARE DOING IS, WE’RE COMBINING LINE ONE MEASUREMENTS MEASUREMENTS WITH LOW DOS E CT PREDICTIVE POWER OF THIS SCREENING EXERCISE ACTUALLY IMPROVES AS A FUNCTION OF THAT SO THAT TAKES FULL CIRCLE INTO THE SPACE OF PRECISION PULMONOLOGY WITH THE THREE AREAS THAT I ALLUDED TO IN ONE OF MY INTRODUCTORY SLIDES THAT IS THE IMPORTANCE OF TAKING ADVANTAGE OF GENOME WIDE EVALUATIONS FOR DIAGNOSTIC PRECISION IN THIS CASE, SCREENING OF PATIENTS FOR POLYMORPHIC PATIENTS THAT MIGHT BE MORE PREDICTIVE OF LINE ONE HEIGHTENED LINE ONE ACTIVITY IN THE GENOME, YOU CAN DO THAT CERTAINLY TAKING ADVANTAGE OF SEQUENCING BUT YOU CAN DO IT TAKING A PROXY DEVELOPMENT OF TARGETED THERAPIES THAT INCREASE EFFICACY BECAUSE IT’S TARGETED AT THAT APOLOGY AND DECREASE TOXICITY THIS IS CERTAINLY TRUE IN THE MANAGEMENT OF LUNG CANCER MALIGNANCIES WHICH ARE METASTATIC WHICH LINE ONE APPEARS TO BE PLAYING A VERY HUGE ROLE, TURNS OUT MAJORITY IS DIRECTED TOWARDS REGULATION OF MIGRATORY AND MALIGNANCY PHENOTYPES STRATIFICATION OF POPULATIONS IN THE CONTEXT OF REPETITIVE SEQUENCES WHICH IS AN AREA WE HAVE LARGELY IGNORED MAINLY BECAUSE IT’S ONLY REFLECTING THE LACK OF UNDERSTANDING WE HAVE HAD UP UNTIL TECHNOLOGY COOPERATED TO GUIDE US THROUGH NEW JOURNEYS OF DISCOVERY WITH THAT I WOULD LIKE TO — I HAVE BEEN TOLD 45 MINUTES IS THE RIGHT MARK AND ACKNOWLEDGE OF COURSE THE FACT THAT ONCE ACHIEVEMENTS ARE NEVER — OUR OWN ACHIEVEMENTS, THEY ARE AN EXTENSION OF THOSE, I WOULD LIKE TO ACKNOWLEDGE MY FAMILY AT HOME, MY LABORATORY, COLLABORATORS FUNDING AGENCIES AND ALL OF YOU FOR YOUR ATTENTION I LOOK FORWARD TO ENGABLINGENING DIALOGUE AND TALKING ABOUT WHAT I HAVE DISCUSSIONth CUSS WITH YOU TODAY — DISCUSSED WITH YOU TODAY THANK YOU VERY MUCH [APPLAUSE] >> WE HAVE TIME FOR SOME QUESTIONS, PLEASE USE THE MICROPHONES >> CONGRATULATIONS FOR — (INDISCERNIBLE) LUNG DISEASE SO YOU MENTION THERE ARE SEQUENCE VARIANTS AND THERE ARE HUNDRED YOU HAVE ALREADY IDENTIFIED USING CALLED ELISA TECHNIQUE FIND THOSE VARIANTS OR DO YOU HAVE TO DO THE SEQUENCING TO LOOK FOR THOSE VARIANTS THAT AFFECT LUNG CANCER IN SOME OF THESE PATIENTS? >> GREAT QUESTION THE LAST ASSAY IS A CRUDE READ

OUT OF INCREASE ACTIVITY OR LOW ACTIVITY FOR LINE ONE, IF YOU WANT TO KNOW VARIANTS ITSELF YOU HAVE TO DO IT BY SEQUENCING >> YOU MENTIONED SOME GENDER VARIATION IS THERE SOME PROTECTIVE EFFECT FOR WOMAN? >> ACTUALLY, THE COUNTER TO THAT THE SEX DIFFERENCE FOR LINE ONE IS REALLY INTERESTING IT PROBABLY HAS DEGREE OF CONNECTION WITH X CHROMOSOME SILENCING AND THE BIOLOGY OF CYST AND CONNECTION TO LINES LONG STORY SHORT, TURNS OUT TO BE THAT MALES OVEREXPRESS LINE ONE MORE READILY THAN FEMALES SO FEMALES ARE BETTER ADEPT AT MAINTAINING LINE ONE SEQUENCES SILENCED BUT WOMEN THAT ARE POLYMORPHISM FOR HIGH EXPRESSION VARIANTS OR THAT GET EXPOSED TO THINGS THAT DEREGULATE STABILITY OF LINE ONE SEQUENCES, ONCE DESTABILIZED THE RESPONSE OF WOMEN IS HEIGHTENED CMPARED TO MALES THIS IS REALLY INTERESTING IN THE CONTEXT OF COPD BECAUSE THE SAME BIOLOGY IS C OBJECTIONPD WHERE YOU MOSTLY SEE MALE COPD PATIENTS BECAUSE THEY ARE THE ONES THAT BEGAN SMOKING EARLIER BUT WHEN YOU SEE FEMALES PRESENTING WITH COPD, THE SERVERITY OF DISEASE IS ENHANCED SO THE OTHER ASPECT TO GENETICS OF LINE ONE VARIATION THAT IS CRITICAL, NATIVE AMERICAN ANCESTRY APPEARS PROTECTIVE OF LINE ONE REACTIVATION SO WE’RE BEGINNING TO EXPLORE THE CONNECTION BETWEEN GENETIC ANCESTRY AND REGULATION OF SEQUENCES WE DON’T KNOW IF THE DIFFERENCE BETWEEN NATIVE AMERICANS AND OTHER ETHNIC GROUPS IS GENETIC OR EPIGENETIC SO WE’RE BEGINNING TO PLOW IN THAT DIRECTION BUT YOUR OBSERVATION IS QUITE ON TARGET >> COUPLE OF QUESTIONS ABOUT MECHANISM SO IF YOU’RE NOW TRANSCRIPTIONALLY ACTIVATING ELEMENTS ARE YOU ALSO POTENTIALLY ACTIVATING RECOMBINATION MECHANISMS? >> BY ALL MEANS WHEN ONE OF THE CONSEQUENCES OF LINE ONE ACTIVATION IS THE FACT THAT YOU ARE INCREASING RATES OF RECOMBINATION FOR LINES THAT BECOMES SORT OF THREE EDGE SWORD NOT ONLY LIENS, IT’S THE ALLOS THE SDAs THAT YOU NEED TO WORRY ABOUT LUCKY FOR US I THINK WHAT HAPPENS IS THAT THERE’S SO MANY — THERE’S 12 MECHANISMS THAT WOULD HALT RECOMBINATION IN THE CONTEXT OF LINES THAT YOU ACTUALLY HAVE TO HAVE EVERY STAR ALIGNED IN THE SKY FOR FULL RECOMBINATION CYCLE TO BE COMPLETED ‘S ONE OF THE MAJOR DEVASTATING CONSEQUENCES OF IT >> THERE’S INCREASING EVIDENCE THAT REPETITIVE ELEMENTS FORM TRANSCRIPTIONAL FUSION WITH ADJACENT PROTEIN CODING AXONS IS THAT LIKELY A MECHANISM THAT’S AT PLAY HERE AS WELL? IF YOU TRANSCRIPTIONALLY ACTIVATE OR REPRESS THE ELEMENT IT NOW ACTIVATES DOWNSTREAM AXONS OF AN ONCOGENE OR SOMETHING ELSE? >> INDEED A POSSIBILITY WE’RE BEGINNING TO LOOK IT TO THAT INTERACTOME COMPRESSION YOU HAVE ALLUDED TO — COMPLEX YOU ALLUDED TO THAT’S A GREAT QUESTION >> THANK YOU ALL FOR BEING HERE PLEASE JOIN US FOR A RECEPTION BY THE LIBRARY THANK YOU [APPLAUSE]