>> THANK YOU, WE'LL MOVE ON TO

OUR SECOND SPEAKER, DR.

DR. YARDENA SAMUELS WHO IS HEAD

OF THE TRACK INVESTIGATOR AND

HEAD OF THE MOLECULAR CANCER

CENTER AND HEAD OF NHGRI'S

CANCER GENETICS BRANCH.

HER TOPIC IS GENETIC OF

MELANOMA, SEARCHING FOR NEW

THERAPEUTIC TARGETS, SHE GOT HER

BACCALAUREATE AT AUTOPSY

SERIESED KINGDOM AND EARNED HER

MASTERS FROM HEBREW UNIVERSITY

IN ISRAEL.

HER Ph.D. IN CANCER RESEARCH

IS FROM LONDON'S IMPERIAL

COLLEGE AND SHE COMPLETED A POST

DOCTORAL FELLOWSHIP IN CANCER

GENETICS AT JOHNS HOPKINS.

HER MEMBERSHIPS INCLUDE THE

AMERICAN ASSOCIATION OF CANCER

RESEARCH, AND THE MELANOMA

RESEARCH ARIANCE.

THE LABORATORY RESEARCH FOCUS IS

IDENTIFICATION OF GENERATED

THETIC CHANGES THAT UNDERLIE

MELANOMA IN COLLABORATION WITH

STEVE ROSENBERG AND NCI, THE

RESEARCH TEAM HAS ESTABLISHED A

UNIQUE TUMOR BANK CONSISTING OF

100 TUMOR SAMPLES AND HIGHLIGHTS

OF HER RESEARCH INCLUDE

IDENTIFICATION THAT RB4 IS THE

MOST HIGHLY MUTATED TYROSEEN

KINASE AND MELANOMA AND

DEMONSTRATES THAT GLUTEA MINE

SIGNALING PATHWAY IS A HIGHLY

SIGNIFICANT PATHWAY THAT PLAYS A

ROLE IN MELANOMA PROGRESSION.

IT'S NOW MY PLEASURE TO WELCOME

HER TO THE PODIUM.

>> WELL, THANK YOU VERY MUCH FOR

THAT KIND INTRODUCTION, IT'S A

PLEASURE TO BE HERE AND I'LL BE

TALKING INDEED ABOUT SEARCHING

NEW THERAPEUTIC TARGETS FOR

MELANOMA.

SO I HAVE NO FINANCIAL

RELATIONSHIPS TO DISCLOSE AND

THESE ARE THE GOALS OF THIS

TALK.

>> AND SO IT'S WELL ACCEPTED

THAT CANC SER A GENETIC DISEASE

AND 1 OF THE BEST EXAMPLES FOR

THIS IS COLORECTAL CANCERS SINCE

IT DEVELOPED MORPH LOGICALLY

CLEAR DISTINCT PHASES STARTING

FROM NORMAL EPITHELIAL CELL, ON

TO EARLIER ADENOMAS, AND

ADENOMAS, LATE MEL AN OHM AS,

ULTIMATELY LEADING TO METASTASIS

AND THESE HAVE BEEN LINKED TO

MOLECULAR CHANGES SOME OF WHICH

ARE SEEN IN THIS SLIDE AND THIS

HAS BECOME REALLY A PARADIGM FOR

GENETIC DEVELOPMENT IN SOLID

CANCERS.

AND WE'VE BEEN INVESTIGATORRING

THIS INDEPTH AND HOWEVER, WE

STILL BELIEVE THAT THE DATA IS

THE TIP OF THE IBERG AND THERE'S

MORE TO--ICEBERG AND THERE'S

MORE TO BE FOUND OUT.

WE AIM TO DO THIS FOR SPORADIC

METASTATIC MELANOMA.

OVER 8000 PEOPLE DIED OF THIS

DISEASE IN 2011 IN THE U.S.

THE ALARMING PART IS THAT THE

MEDIAN PATIENT SURVIVAL IS ONLY

6 MONTHS, THEREFORE IN DIAGNOSIS

OF LATE STAGE DISEASE IN MOST

CASES SO WE BELIEVE IN

IDENTIFYING A NOVEL

GENETICOTERATIONS MAY PROVIDE

OPPORTUNITIES FOR DRUG

DEVELOPMENT.

AND SO, SIMILAR TO WHAT I SHOWED

YOU FOR COLORECTAL CANCER THIS

IS THE HISTOLOGICAL PROGRESSION

FOR MELLAGEOMA--MELANOMA

DEVELOPMENT WHERE IT RESIDES

WITHIN THE EPIDERMIS AND IT

DEVELOPS INTO THE NEV ISOTOPE

AND RADIAL GROWTH PHASE AND IN

VERTICAL GROWTH PHASE AND

METASTATIC MELANOMA, AND SOME OF

THE MUTATIONS ASSOCIATED WITH

THESE STAGES, I'VE SEEN THIS

SLIDE, THE BEST 1 OF COURSE AND

KNOWN BRASE MODEL MUTATIONS AND

ALTHOUGH MORE OF THESE ARE

KNOWN, WHEN I STARTED MY PROGRAM

HERE AT NHGRI, AND IT WASN'T A

COMPREHENSIVE ANALYSIS OF THE

GENETICS OF MELANOMA, AND SO

THOSE WERE WERE TO PERFORM

MUTATION ARKINAL SIS OF THE

GENOME, WITH AIM TO FUNCTIONALLY

ANALYZE THE MOST HIGHLY AND MOST

INTERESTING MUTATIVE GENES, AND

WE TRY TO TRANSLATE IN

INFORMATION BACK INTO THE

CLINIC.

WE ESTABLISHED A TUMOR BANK

TOGETHER WITH DR. STEVE

ROSENBERG AT THE NCI AS WELL AS

DR. GERB AS WELL AS M. D.

ANDERSON AND THE COLO CANCER

CENTER AND THE MAJORITY OF THE

STUDIES ARE HERE FROM FROM THE

NCI.

YOU SEE THE NUMBERS HERE SO WOO

HAVE METASTATIC DNA AS WELL AS

MATCHED NORMAL DNAs AND MOST

OF THE TIME THIS IS BLOOD, THE

OCT BLOCKS, ORIGINAL TUMOR WAS

RESECTED AND FROZEN DOWN FOR ALL

OF THE ROSENBERG SAMPLES THAT

MATCHED LOW PASSAGE CELL LINES

WHICH WERE EXTREMELY USEFUL

ESPECIALLY IF YOU WANT THE TO

START EVALUATING FUNCTIONALLY,

THE EFFECTS OF THE MUTATIONS,

YOU CAN MAKE MATCHED RNAs FROM

THESE CELL LINES AS WELL AS

PROTEIN LIAISON SATES.

AND FOR ALL OF THESE SAMPLES WE

HAVE CLINICAL INFORMATION.

THIS IS A SOMATIC MUTATION

ANALYSIS, WHERE HAVE YOU THE

TUMOR, SOMETIMES THE CELL LINE,

YOU EXTRACT THE DNA, SEQUENCE

THE GENES OF INTEREST, AND YOU

DO THE SAME FOR THE NORMAL

TISSUE, AS I SAID MOSTLY BLOOD

AND YOU COMPARE THOSE SEQUENCES

AND WE ONLY FOLLOW UP ON SOMATIC

MUTATIONS.

THE MUTATIONS ARE IN THE TUMOR

SO I'LL GIVE YOU AN EXAMPLE OF

THIS KIND OF ANALYSIS, AS YOU

PERFORMED FOR I TYROSEEN KINASE

FAMILY.

SO THIS IS THE HUMAN KINOME AND

ON THE UPPER LEFT HAND CORNER

YOU SEE THE KINASEIS, AND WE

DECIDE TO SEQUENCE THESE BECAUSE

IT'S KNOWN THAT THEY'RE HIGHLY

MUTATE INDEED CANCER

AND--MUTATED IN CANCERS AND IT'S

GONE TO BE INHIBITION, NOW THESE

HAVE BEEN SEQUENCED IN 2007 BUT

THEY ONLY SEQUENCED 6 MELANOMAS

AND SO THE MUTATION RATE THAT

THEY OBSERVED WAS TOO LOW TO

DEFINITELY IMPLICATE THESE GENES

TO TUMOR GENESIS.

SO NOW THAT WE HAD THIS UNIQUE

TUMOR WE DECIDE TO REVISIT THIS

FAMILY IN THAT TUMOR BANK.

SO THIS IS THE KINOME SCREEN

EVERY 1 OF OUR GENETIC SCREEN

SYSTEM MADE UP OF 2 PHASES,

DISCOVERY PHASE AND VAILEDDATION

PHASE AND COLLABORATION HERE,

THE SEQUENCING CENTER, WE SENSE

REQUESTED 29 SAMPLES, LOW NUMBER

OF SAMPLES FOR ALL THE TYROSEEN

KINASES.

IT'S A LOT OF SEQUENCING AND IF

MUTATIONS TAKE PLACE THEY WOULD

BE FOUND IN THE DOMAIN, AT THIS

TIME WE SEARCH FOR 1 SOMATIC

MUTATION OR MORE AND THERE WERE

19 GENES, MOVED ON TO

VAILEDDATION PHASE WHERE WE

SEQUENCED IN MY LAB, 80 CELLS

AND IN THIS CASE WE SEQUENCED

ALL OF THE CODING AXONS AND THEN

WE ANALYZE THIS WHICH WAS

SOMATIC AND INTERESTING GENES

MOVED ON TO FUNCTIONAL ANALYSIS.

AND THE 1 THAT WAS INTERESTING

WHICH WAS BEFORE WHICH WAS

MUTATED IN 90% OF THE CASES.

THIS IS EGFR, RB2 BOTH OF WHICH

ARE KNOWN GOOD TARGETS AND THESE

EITHER HOMODIME RISE ON THE

MEMBRANE AND UPON LIAISON GAPPED

BIPEDDING THEY TRANSPHOS

FORALATE EACH OTHER FORMING GOT

IT FOR VARIOUS SIGNALING

POLARIZED MOLECULES.

SO WE DECIDE TO CLONE 7 OF THE

MUTATIONS SINCE WE IDENTIFIED

AND WE CHOSE THESE 7 BASED ON

THE KRYSTAL STRUCTURE THAT WAS

MADE BY DR. LEAKY HERE AT JOHNS

HOPKINS UNIVERSITY, AND ALSO

BASED ON CONSERVATION THROUGH

THE ERP MEMBERS WHAT WE DID

ISLET BIOCHEMICAL ASSAY, WE LOOK

AT KINASEœ

MUTANTS VERSES WHILED TYPE OR

BEFORE, AND WHAT YOU SEE IS THAT

ALL THE MUTANTS HAVE INCREASED

KINASE ACTIVITY COMPARED TO THE

WILD-TYPE DESPITE THE FACT THAT

THEY EXPRESS SIMILARLY.

SO THESE MUSEUM AT THAT POINTS

HAVE INCREASED BASAL ACTIVATION.

AND THIS IS A CLASSIC NIH CELLS

AND WHAT WE SAW WAS THAT THESE

SAME EXACT 7 MUTANTINGS FORMED

AN INCREASED NUMBER OF FOCI

BEFORE AND STRIKINGLY SIMILAR TO

THE ONCOGENIC RASE MODEL, AND

THE NEXT QUESTION WAS, IS THIS

ARE THESE IMPORTANT IN THE CELL

WHICH IS IN WHICH THEY'RE

HARBORED AND THIS PASSAGE CELL

LINE COMES IN SO WE GO BACK TO

THE PASSAGE CELL LINES AND USE

THE SHRNA TECHNOLOGY TO KNOCK

DOWN THE ENDOGENOUS ERB4 WITHIN

THE CELLS.

WE COULD CHECK VARIOUS

PHENOTYPES AND FIRST WE CHECKED

OF COURSE WAS WHETHER THIS

AFFECTS GROWTHo[[ AND WHAT YOU SEE

HERE IS THAT WHEN YOU KNOCK OUT

BEFORE, IN CELLS THAT THE

WILD-TYPE YOU SEE NO EFFECT IN

GROWTH, THEY GROW SIMILARLY TO

THE CONTROL IF YOU DO THIS

BEFORE, IT REDUCES COMPARE TO

CONTROL, AND THESE RESULTS WERE

SEEN IN SEVERAL OF THESE

MELANOMA CELL LINES.

SO MUTANT BEFORE IS PROVIDING A

CELL SIGNAL TO THESE CELLS

OTHERWISE KNOWN AS ONK O GENE

ADDICTION.

SO THE NEXT STEP WAS IS THIS A

GOOD TARGET.

AND IT'S A SMALL MOLECULE

INHIBITOR, FDA APPROVED FOR

BREAST CANCER PATIENTS AND WHEN

WE EXPOSED A MANY MELANOMA CELL

FOR THE WILD-TYPE BEFORE, AND

THE MUTANT FOR ABOVE AND THE

PATENT TO LE PAT NIB AND THE

PLOT OF THE EC 50 GRAPH, CAN YOU

SEE THE FELLS THAT WERE MUTANT

BEFORE, FOR WERE MORE SENSITIVE

TO THE PRESENCE OF LE PAT NIB

COMPARED TO THE WILD TYPE

BEFORE.

SO THE MUTANT IS SENSITIZING

THESE TO LE PAT NIB AND WE

EXTENDED THIS TO ADDITION CELL

LINE SPECIALIZATION OF SPECIFIC

ENDOTHELIAL WE DO SEE A RANGE OF

SENSITIVITY MORE SENSITIVE IN

THE WILD-TYPE, THERE'S

HETEROGENERATED AIT AND THERE IT

SEEMS TO BE A GOOD POTENTIAL

PARINGET AND THE HYPOTHESIS

BEING THAT ABOUT 20% OF

CAR-MELANOMA PATIENTS, AND

EXPOSURE OF THE CELLS WOULD MAKE

THAT SENSITIVE TO LE PAT NIB AND

BASED, DR. URDA RUDE NOVARTIS,

BECAME THE PI HERE AT THE

SURGERY BRANCH FOR A CLINICAL

TRIAL, WHERE WITH THE HELP OF

DR. [INDISCERNIBLE] AT THE NCI,

THERE'S CLEAR CERTIFIED IRB 4

SEQUENCING, CTEMIS PROVIDING

HEPATITIS EAT NIB AND THIS IS A

CLINICAL TRIAL INVOLVING NCI AS

WELL AS MEMORIAL SLOAN-KETTERING

AND THIS IS RECRUITING PATIENTS

AND AT THIS POINT WE DON'T HAVE

ANY REPORTS, BUT HOPEFULLY WE'LL

HAVE POSITIVE THINGS TO SAY

ABOUT THIS.

SO IF THESE ARE THE MUTATIONS

THAT WE IDENTIFY INDEED THE

FIRST STUDY, THESE ARE THE

MUTATIONS THAT HAVE BEEN ARKS

DENTIFIED SO FAR THROUGH THE

CLINICAL TRIAL AND YOU SEE THE

FREQUENCY THE SAME OR EVEN

HIGHER WE'RE STILL FINDING THESE

MUTATIONS.

THESE MUTATION VS BEEN

IDENTIFIED SINCE OUR

PUBLICATION, THROUGH WHOLE EXOHM

AND GENOME STUDIES, THE 1S IN

PURPLE HAVE BEEN IDENTIFIED BY

NICK HAYWOOD IN AUSTRALIA AND

THESE ORANGE 1 HAS BEEN IDENTIFY

BOOED LEVI GARRA WAY.

THE POINT HERE IS SAYING THAT

THE FREQUENCY IS STILL HIGH, AND

WE'RE ALSO STARTING TO SEE

MINIHOT SPOTS SO RECURRING

MUTATIONS IN PARTICULAR

LOCATIONS, WHICH IS A

CHARACTERISTIC OF ONK O GENES

THIS, IS SOME PRELIMINARY DATA

THROUGH COLLABORATION WITH

DR. LANNUOUS WHERE HE LOOKED AT

ERB BEFORE IN THE SERUM IN SOME

OF THESE PATIENTS HE FOUND

BEFORE AND HE FOUND AN INCREASE

IN THE BEFORE ECTODOMAIN IN

THESE SIERRA COMPARED TO HEALTHY

INDIVIDUALS IN THE T-TESTING TO

GIVE A SIGNIFICANT P-VALUE.

SO THIS IS VERY INTERESTING

BECAUSE MAYBE WE COULD HAVE A

BIOMARKER TO DETECT THESE

PATIENTS RATHER THAN SEQUENCING

THE IRB 4.

OKAY, SO I'LL SHIFT GEARS AND

I'LL START TALKING ABOUT THE

MELANOMA LANDSCAPE BECAUSE NOW

WE CAN START USING DIFFERENT

TECHNOLOGIES, WE CAN USE WHOLE

GENOME AND WHOLE EXOHM

SEQUENCING.

AND SO THIS IN COLLABORATION

WITH THE FOUNDATION OF NIH,

ELLIOTT AND STEPHEN PARKER.

OUR PILOT, WAS TO FIRST COMPARE

LOW PASSAGE CELL LINE TO THE

ORIGINAL TUMOR FROM WHICH IT WAS

FORMED.

SO MELANOMA CELL LINES AND

RELATIVELY EASY TO FORM COMPARED

TO OTHER SOLID CANCERS, BUT THE

MAIN QUESTION IS THE SOMATIC

MUTATIONS SIMILAR IN THESE CELL

LINES TO THIS ORIGINAL FRESH

TUMOR SO WE DID WHOLE GENOME

SEQUENCING AS WELL AS WHOLE

GENOME SEQUENCING OF THE NORMAL,

OF THE BLOOD.

THE MAIN POINT TO BE MADE IS

THIS WHEN YOU INTERSECT THE

SOMATIC MUTATIONS, 90% ARE

CONCORDANT WHEN YOU LOOK AT THE

COPEINA OF THE MCDS, AT THE

CODING REGIONS.

SO THIS SUGGESTS THAT THAT IT IS

FINE TOO USE THESE LOW PASSAGE

CELL LINES, ARRIVED GENOMIC DNA

IN ORDER TO LOOK FOR SOMATIC

MUTATION SAYS BECAUSE THESE ARE

MAINTAINED.

WHEN YOU LOOK AT COPY NUMBER

VARIATIONS THESE ARE LESS

CONKORT ANT, IT'S LESS THAN 80%.

BUT WE'RE FOCUSING NOW ON

SOMATIC MUTATIONS.

SO, TO CONTINUE LOOKING AT A

LANDSCAPE WE DECIDE TO DO WHOLE

EXOHMS SINCE IT'S MORE PRACTICAL

THAN WHOLE GENOMES IT'S MUCH

LESS EXPENSIVE.

SO WE USE LO PASSAGE DNA TO

PERFORM WHOLE EXOHM SEQUENCING

OF TUMORS AND THE MATCH NORMALS,

WITH ADJUVANT CHOICE SELECT AND

LUMENNA SEQUENCING AND THE THIS

IS DONE BY SEQUENCING.

WE'VE GOT 12 GIGA BASE PER

SAMPLE AND THE MEAN DEPTH 180 X

OR GREATER, WE GOT OVER 90%

COVERAGE, WE ONLY HAD 2.4%

FALSE-NEGATIVE RATE AND 81%

SENSITIVITY.

THIS IS JUST THE STAGES FOR THE

DISCOVERY SCREEN.

I TOLD YOU WE CAPTURED 14

MELANOMA SAMPLES, WE GOT OVER

5000 SOMATIC MUTATIONS HOWEVER

ONCE YOU ASSEMBLE THE DATA AND

FILTER IT THROUGH VARIOUS

DIFFERENT FELTERS YOU FIND ABOUT

4000 SOMATIC MUTATIONS THAT ARE

DIVIDED INTO MISSENSE, NONSENSE,

INSERTIONS, DELETIONS AND SINON

MOUSE MUTATIONS, SO MUTATIONS

THAT DO NOT AFFECT AMINO ACID

SEQUENCE AND IT'S IMPORTANT TO

CAPTURE THIS BECAUSE THEN CAN

YOU LOOK AT THE RATIO OF THE

NONSINON MOUSE TO SINON MOUSE

MUTATIONS AND IN THIS CASE, IT

WAS 221 R 1.

WHICH IS WHAT YOU WOULD EXPECT

TO CLEAR BY CHANCE, THIS IS WHAT

TO EXPECT BY CHANCE, SUGGESTING

MOST OF THESE MUTATIONS ARE

PATHOGEN MUTATIONS MEANING THEY

HAVE A NEUTRAL EFFECT, THEY

SHOULD NOT EFFECT THE TUMOR

GENESIS PROCESS.

OKAY, SO THE CHALLENGE HERE IS

PASSENGERS AND WHICH ARE DRIVERS

AND WHICH HAVE A ROLL TO PLAY IN

TUMOR GENESIS.

AND THIS IS AN OPEN QUESTION,

MANY PEOPLE ARE WORKING ON THIS,

AND NO CLEAR ANSWERS BUT WHAT WE

USE ARE STATISTICS

BIOINFORMATICS AND FUNCTIONAL IS

STUDIES.

SO WHEY MEAN BY STATISTICS IS TO

LOOK FOR RECURRENT MUTATED

GENES, MEANING HOT SPOTS, EXACT

SAME MUTATION OCCURRING SEVERAL

TIMES IN DIFFERENT SAMPLES, SUCH

AS BRAUGHT, HILY MUTATED GENES,

NONSINON MOUSE TO SINON MOUSE

RATIOS AND THEY SHOULD OCCUR

ABOVE THE BACKGROUND MUTATION

RATE.

SO IF YOU START LOOKING FOR

RECURRENT HOT SPOT MUTATIONS

MEANING THE SAME EXACT MUTATION

WOULD OCCUR IN THESE 2 SAMPLES

OR MORE, SO OF COURSE WE FOUND

BRASE MODEL, THAT IS A POSITIVE

CONTROL, IT'S KNOWN TO BE HOT

SPOT MUTATION, BUT I WAS

SURPRISED WE FOUND 9 NOVEL GENES

WITH REOCCURRING MUTATIONS SO WE

VALIDATED THESE IN A LARGER

NUMBER OF SAMPLES AND WE FOUND

TRAPS WHO HAVE 6 MUTATIONS

EXACTLY IN THE SAME POSITION.

THE LIKELIHOOD OF THIS HAPPENING

IS EXTREMELY LOW, YOU SEE THE

P-VALUE THIS, IS BEING SELECTED

FOR AND IT PROBABLY AS A

FUNCTIONAL EFFECT.

NOW THIS PARTICULAR MUTATION WAS

ALSO IDEBTIFIED--IDENTIFIED BY

RUTH HAL BAN IN YALE AND AS WELL

AS A COLLEAGUE HERE AT NCI.

AND THIS IS A YOU THIS CLEAR

PROTEIN, FUNCTIONS AS PART OF A

TRANSFER ACE AND IT'S DISRUPTION

CAUSES DEFECTS IN CELL PSYCHE

ALTHOUGH PROGRESSION AND OUR

FUNCTIONAL STUDIES IN MELANOMA

CELLS SUGGEST THE SAME.

SO IF WE LOOK AT THE OTHER SIDE,

SEARCHING FOR HIGHLY MUTATED

GENES WE FOUND 16 SUCH GREENES

TAKING INTO ACCOUNT THE SIZE OF

THE GENE AND THE BACKGROUND

MUTATION RATE AND SO, AGAIN WE

LOOK AT THESE 16 GENES AND

VALIDATED HEM IN A LARGER NUMBER

OF SAMPLES, AND THESE ARE THE 16

IN THIS TABLE.

SO FORTUNATELY WE SEE B-RASE

MODEL UP HERE ON THE TOP, A GOOD

POSITIVE CONTROL BUT ALL OTHER

GENES WERE NEVER LINKED TO

MELANOMA BEFORE AND THESE ARE

THE P-VALUESVALUES AND THIS IS THE

PERCENT OF THE TUMORS THAT WERE

AFFECT INDEED IN DISCOVERY

SCREEN.

WHEN WE VALIDATED THIS IN THE

LARGER SAMPLE SAID OF 52, YOU

SEE THE PERCENTAGES ARE

CONCORDANT TO WHAT WE FOUND IN

OUR DISCOVERY SCREEN, SUGGESTING

THAT OUR METHODOLOGIES IS

WORKING SO WE DECIDE TO FOCUS ON

GREEN 2 A, THE REASON IS IT'S

THE SECOND MOST HILY MUTATED

GENE AND IT'S NOVEL.

SO WE FOCUSED ON IT AND SCALED

IT EVEN MORE SAMPLES, AND THIS

IS THE SCHEMEATIC OF ALL THE

MUTATIONS THAT WE IDENTIFIED,

YOU SEE THERE'S A VERY LARGE

NUMBER AND YOU SEE THEY'RE

SCATTERED THROUGHOUT THE

PROTEIN, WE HAVE NYE 5 NONSENSE

MUTATIONS MEANING THESE WOULD BE

TRUNCATING THE PROTEIN AND WE

ALSO HAVE A FEW PLACES WHERE YOU

HAVE RECURRENT MUTATION, THE

SAME EXACT MUTATION AND ACTUALLY

THIS PARTICULAR MUTATION WAS

PREVIOUSLY SEEN IN COSMIC WHICH

IS A WELL KNOWN RELIABLE CANCER

DATABASE AND THESE 1S IN RED

WERE RECENTLY PUBLISHED BY GAR O

WAY IN NATURE LAST WEEK, AGAIN

SHOWING RECURRENT MUTATIONS

ALONG GREEN 2 A.

NOW GREEN 2 A IS ANATROPIC

GLUTAMATE RECEPTOR UNIT AND I

WILL TOUCH BEG YOUR PARDON

GLUTAMATE PATHWAY AGAIN LATER IN

MY TALK.

SO THIS IS A SCHEMEATIC OF GREEN

2 A AND OTHER SUBUNIT GREEN 1

AND THE ADDITIONAL TUE IT IN

THIS SCHEMEATIC AND THIS IS

REALLY CALCIUM CHANNEL.

UPON GLUTAMATE AND GLUE MARIOUS

SEEN BINDING, THE CHANNEL OPENS

AND CALCIUM ENTERS THE CELL,

ACTIVATING AND ENACTIVATING

PATHWAYS AND ULTIMATELY WELL

WELL--LEADING TO APOPTOSIS.

NOW WE DECIDE TO CLONE A FEW
– OF

THE MUTATIONS IN GREEN TABOO A

IN HERE WITH RED ARROWS AND

FIRST CHECK THE BINDING OF

WHILED TYPE AND MUTANT GREEN 2

A, WITH WILD-TYPE GREEN 1.

BY OVEREXPRESSION AND WHAT WE

SAW HERE, IS THAT WHEN HAVE YOU

THE WILD-TYPE THAT BINDS GREEN

1, THE MUTANT VS REDUCED A NO

BINDING TO GREEN 1 EVEN THOUGH

THEY ARE EXPRESS INDEED THE

LIAISON SATE.

SO, SINCE THIS IS INVOLVED IN

CALCIUM SIGNAL NOTHING

COLLABORATION WITH SILV RA, WE

DECIDE TO LOOK AT CALCIUM.

SO IF YOU OVEREXPRESS THESE IN

CELLS AND ACTIVE RATES WITH

NMDA, THIS IS THE PROFILE YOU

GET FOR CALCIUM ENTRY.

IF YOU OVEREXPRESS THE GREEN 2 A

MUTANTS YOU SEE THE DISRUPTION

OF THE CALCIUM ENTRY INTO THE

CELLS.

SO OUR MODEL NOW IS IF THESE

DIFFERENT STARS WE NOW HAVE

REDUCED CALCIUM ENTRY INTO THE

CELLS, LEADING TO SURVIVAL.

WHICH IS WHAT YOU WOULD EXPECT

IN A CANCER SCENARIO, SO THIS IS

JUST PRELIMINARY DATA SHOWING

WHEN YOU KNOCK DOWN GREEN 2 A IN

CELLS THAT ARE MUTANT FOR GREEN

2A.

YOU DO NOT SEE AN EFFECT IN

GROWTH.

HOWEVER IF DO YOU THIS AND FILL

A WILD-TYPE, YOU SEE AN

INCREASED GROWTH.

THE AND THIS IS ACTUALLY CONSIST

WENT GREEN 2 A BEING A TUMOR

SUPPRESSOR.

SO THIS IS PRELIMINARY BUT

CERTAINLY AN INTEREST OF OUR LAB

TO FOLLOW UP ON.

SO I'D LIKE TO REMIND YOU THAT

IN ADDITION TO LOOKING AT

SPECIFIC GENES WE ACTUALLY LOOK

AT MOST OF THE HUMAN GENES, MOST

OF OF THE AXONS SO WE CAN LOOK

AT PATHWAYS, AND THE PATHWAY

THAT SEEMED TO BE MOST

SIGNIFICANT WAS THE GLUTAMATE

SIGNALING PATHWAY.

SO YOU SEE AT A P-VALUE RIGHT

HERE AND GREEN 2 A WHICH I TOLD

YOU ABOUT HIGHLY MUTATED AS WELL

AS SOME OF ITS SUBUNITS ARE

BEFORE WHICH I TOLD YOU ABOUT

BEING MUTATEDDED AND THERE HAS

BEEN A NATURE MEDICINE PAPER

SHOWING THAT THEY INTERACT AND

B4 PHOS PORALATES GREEN 2 A

AFFECTING ITSELF NEGATIVITY.

NOW WE ALSO SEE THESE TO BE

MUTATED AND THIS IS A GLUTAMATE

RECEPTOR AND PRELIMINARY DATA

SUGGESTS THAT IT ALSO BINDS

IRB4.

SO WE'RE FOCUS NOTHING THIS

PATHWAY SINCE WE THINK IT'S RELL

VABT FOR MELANOMA SO IN PERRA

LEAL TO OUR AXON SCREENS WE HAVE

BEEN LOOKING AT THE G-PROTEIN

COUPLED RECEPTORS THIS IS THE

LARGEST HUMAN GENE FAMILY SO WE

HAD TO USE CAPTURE METHODS AND

AND SECOND GENERATION SEQUENCING

AND 2 PHASES AND THE GENE THAT

SEEMED TO BE INTERESTING AND I

ALREADY MENTION TODAY IS GRIM 3.

SEEM TO BE MUTATED IN 16% AND

15% OF CASES IN 2 DIFFERENT

COHORTS.

SO AGAIN WE HAVE A GLUTAMATE

RECEPTOR HERE, AND IT HAS THE

PARTICULAR MINIHOT SPOT SO YOU

SEE THIS EXACT SAME MUTATION

THAT OCCURS IN HERE IN THE 2

DIFFERENT COHORTS AND AGAIN THE

P-VALUE OF THE LIKELIHOOD OF

THIS HAPPEN SUGGEST LOW.

--HAPPENING IS LOW, BUT IT

PROBABLY HAS A FUNCTIONAL ROLE.

TO SHOW YOU A QUICK FUNCTIONAL

ANALYSIS, YOU SEER WHEN THESE

ARE CHECK INDEED MELANOMA CELLS

AND ACTIVATED BY AN AGONIST, YOU

SEE THAT MIC PHOSPHORYLATION IS

INCREASED, SPECS ESPECIALLY BY

THE GRIM 3 HOT SPOT RIGHT HERE.

--SO 1 STUDY WE DID WAS LOOKAL

PULMONARY MICROMETASTASIS

FORMATION AND YOU SEE THAT THIS

IS INCREASED AFTER TAIL VAIN

INJECTION OF CELLS THAT HAVE

GRIM 3 MUTATIONS.

SO IF HAD IS A PAPER ON GRIM 2

A, YOU SEE ANOTHER PAPER,

FACULTYING ABOUT THE GLUTAMATE

PATHWAY, SPECIFICALLY IN THIS

CASE, GRIM 1, SHOWING THAT IT

INDUCES A MELANOMA IN MICE THIS,

IS ANOTHER PAPER SHOWING AS WELL

BY PEOPLE IN THE NIH SHOWING

THAT GRIM 5, AND ANOTHER GRIEWT

MATE RECEPTOR WITH THE GENIC

MICE AND THIS OUR GRIM 3 PAPER

AND THE COMMON DENOMINATOR

BEING, AGAIN, THE FLUTE MATE

PATHWAY EMPHASIZING ITS

IMPORTANCE.

SO WHAT ARE WE PLANNING TO DO,

AND SO WE PLANNING TO FURTHER

DWELVE INTO THE MELANOMA G

GENOME.

MAINLY BECAUSE WHEN YOU LOOK AT

THE FREQUENCY VERSES OTHER SOLID

CANCERS YOU DO YOU SEE IT'S

EXTREMELY HIGH.

IT'S SIMILAR TO LUNG CANCER

PROBABLY DUE TO THE UV ETIOLOGY

THAT MELANOMA HAS SO WE REALLY

NEED MORE SAMPLES TO BE

SEQUENCED IN ORDER TO FIND OUT

WHICH OF THE--ARE THE PASSENGERS

AND WHICH ARE THE DRIVERS.

THAT'S WHAT WE'RE DOING.

WE HAVE 18 WHOLE EXOHMS, WE HAVE

10 WHOLE DENOMES AND WE CAN

MERGE THE DATA AND WE WHO-FIC WE

DO THAT WE CAN SEARCH FOR

RECURRENT MUTATIONS AND WE'RE

FINDING EVEN MORE NTHIS CASE,

THE 38, AND AGAIN SEARCH FOR

HIGHLY MUTATED GENES AND WE

FOUND 15, THE GOOD NEWS IS THAT

7 OF THESE 15 HAVE PREVIOUSLY

BEEN LINKED TO MEL AN OHM AND

THESE ARE THE 7.

BRASE MODEL AGAIN ON THE TOP, SO

THIS SUGGESTS THAT OUR

METHODOLOGIES IS WORKING.

IT ALSO SUGGESTS THAT WE'RE

REACHING A PLATEAU, AT LEAST

WITH THIS PARTICULAR MELANOMA

COHORT.

SO WE WILL FURTHER ANALYZE BRASE

MODEL HAS RECENTLY HAD AN FDA

APPROVED DRUG.

AND WE'RE LOOKING FOR TARGETABLE

DRIVERS AND THOSE THAT DO NOT

HAVE THE BRASE MODEL MUTATIONS

SINCE WE'RE LOOKING AT GENOMES

WE CAN LOOK AT STRUCTURAL

IMPLICATIONS, REARRANGEMENTS, WE

CAN LOOK AT NONGENIC TARGETS

SUCH AS REGULATORY REGIONS,

ENTRONS AND THE GOLD STANDARD WE

BELIEVE IS DOING FUNCTIONAL

ANALYSIS, SINCE WE'RE DOING THIS

IN LOW THROUGH PUT AT THIS

POINT, WE'RE FOCUSING ON GREEN 2

A BEFORE AND THE GLUTAMATE

PATHWAY AND WE BELIEVE IN

GENETIC INTEGRATION ACROSS

MELANOMA DATA SETS; THE MANY

GROUPS DOING THIS AT THIS POINT,

SO WE'RE INTEGRATING DATA, TCGA

HAS TAKEN OUT MELANOMA AS WELL.

SO THEY'RE RELEASING THEIR DAT

AND WE REALLY HOPE THAT

INTEGRATION SUCH AS THE 1 SEEN

HERE WHERE WE'RE ALREADY

INTEGRATE WIDE NICK HEYWARD AND

CAN YOU SEE FROM THE OVER

THOUSAND MUTATIONS, MUTATED

GENES THAT THEY FOUND, 446 ARE

FOUND OUR SAMPLES THAT WE HOPE

AND THIS WILL HELP US LOOK FOR

COMMON DENOMINATORS AND

THEREFORE DRIVERS.

SO, IF NOW, THE DISEASE IS BEING

CATEGORRORRIZED BY CLINICAL AND

PATHOLOGICAL ASPECTS.

WE HOPE THAT IN THE FUTURE

SOMATIC MUTATION SIGNATURES,

TELL BE ADDED IT TO THIS

PARTICULAR CATEGORIZATION IN

ORDER TO HELP DEVELOP MORE

TARGETED THERAPIES.

AT THIS POINT I WOULD LIKE TO

ACKNOWLEDGE MANY PEOPLE,

SPECIALLY IN MY LAB, AND THE IN

THE 3 STORY AND THIS WAS THE

FIRST EXOHM PAPER AND THIS IS

ALL THIS INTEGRATION WHICH IS

ALSO DONE BY STEPHEN PARKER AND

OF COURSE A VERY, VERY, BIG

THANK TO YOU DR. ROSENBERG AND

HIS GROUP, WITHOUT THESE SAMPLES

WE WOULD NOT BE ABLE TO DO THIS

WORK AS WELL AS SAMPLE

PROVIDERS, AND MANY OTHER PEOPLE

AT THE NIH.

ELLIOTT MARGUILES AND STEPHEN

HELPING US WITH THE WHOLE

GENOMES.

THANK YOU FOR LISTENING AND I'LL

BE HAPPY TO TAKE QUESTIONS.

DISCLOSURE FRAMEWORK

[ APPLAUSE ]