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TODAY WE HAVE NATASHA
CAPLEN TALKING FIRST.
SHE GOT HER Ph.D. FROM THE
UNIVERSITY OF LONDON, AND
SUBSEQUENTLY IN 1996 SHE JOINED
NHGRI AND
STARTED WORKING ON RNA AND WE
THOUGHT THAT WAS A VERY
IMPORTANT AREA SO THEN WE HIRED
HER AND SHE JOINED THE NTSB
CENTER FOR CANCER RESEARCH IN
2004, A SENIOR SCIENTIST.
SHE NOW HEADS THE GENE
SILENCING SECTION AND GENETIC
BRANCH, HER TITLE FUNCTIONAL AN
ANNOTATION BY RNA INTERFERENCE.
>> THANK YOU, SIR.
I KNOW EVERYONE CAN HEAR ME IN
HERE.
ARE WE OKAY?
THUMBS U UP.
WE'RE GOOD.
WHAT I'M GOING TO TALK ABOUT
TODAY IS WHAT I CALL A STORY OF
TRANSLATIONAL RESEARCH AN GIVE
YOU HISTORICAL BACKGROUND AND
WORK THROUGH SOME OF THE WORK
THAT'S CURRENTLY GOING ON IN MY
LABORATORY AND THEN SOME
PUBLISHED WORK FROM OTHER
GROUPS, TO HIGHLIGHT HOW PEOPLE
ARE USING RNA AND WHAT ITS
APPLICATION HAS BEEN.
I CALL A TRANSLATIONAL RESEARCH
STORY, THAT'S BECAUSE IT
STARTED AS A -- IS THIS THE
POINTER?
YEAH.
THIS IS A PHENOMENA THAT WE NOW
KNOW WAS ACTUALLY BEING
OBSERVED IN PLANTS AND FUNGI IN
THE 1990'S BUT NOBODY KNEW WHAT
IT WENT, IN CERTAIN SITUATIONS
WHEN PARTICULARLY GENES THAT
WERE INTRODUCED IN MULTIPLE
COPIES INTO IN THIS CASE PEA
PETUNIAS, THEY SAW ACTUALLY A
SINUSSING, THE GENE EXPRESSION
WAS SUPPRESSED.
IT WAS UNCLEAR THE MECHANISM
BEHIND THIS OBSERVATION, UNTIL
THE LATE 1990'S WHEN CRAIG
MELLOW AND ANDY FIRE PUBLISHED
THE SEMINAL OBSERVATION, IF YOU
STRANDED RNAUBLE RANDED RNA
AGAINST GFP YOU SILENCE THE
GENE SUPPRESSION IN THE
NEMATOME WORM, YOU WERE
PRODUCING DOUBLE STRANDED DAN
BEING PROCESSED THROUGH WHAT WE
NOW IS THE RNA INTERFERENCE
PATHWAY.
THEN YOU HAVE THE STUDIES WHICH
I WAS PART OF, SHOWING THIS WAS
A PHENOMENA, A MECHANISM THAT
OCCURRED NATURALLY IN M MAMMALIAN
CELLS, WE HAVE VERY
SOPHISTICATED RESPONSES TO
DOUBLE STRANDED RNA THROUGH OUR
IMMUNE RESPONSE, MYSELF AND
TOM, WHEN HE WAS IN GERMANY,
SHOWED YOU CAN INDUCE THIS
EFFECT IN MAMMALIAN CELLS, AND
THIS OBSERVATION WAS TAKEN TO
CLINICAL TRIALS AND STILL
CONTINUING, WE SAW BEING ABLE
TO SCALE UP THIS EXPLOITIVE
PATHWAY, WHOLE GENOME SCREENING
ANALYSIS IN THE LATE 2000'S AND
CURRENTLY.
I WANT TO MAKE SURE EVERYONE IN
THE ROOM HAS A BASIC OF WHAT
I'M TALKING ABOUT IN TERMS OF
THE MECHANISM.
THIS IS NOT GOING TO BE A
MECHANICKIST PARTICULAKISTIC TALK, THIS I S TO
GIVE EVERYONE BACKGROUND.
WHAT WE NOW KNOW IS THAT THIS
RNA EXPERIENCE IS A NATURAL
OCCURRING GENE SILENCING
MECHANISM THAT'S BEEN STUDIED
THROUGH ANALYSIS OF MICRO RNA,
NATURAL, BUT THESE ARE THE MOST
WELL STUDIED OF ALL OF THE
MICRO RNA.
MICRO RNA GENES ARE EXPRESSED
IN THE GENOME AS TRADITIONAL
POLE 2 GENES, MANY POLY
SISTRONIC, A FEW ARE STILL
INDIVIDUAL GENES, SORT OF A
PLETHORA OF FORMS YOU COULD
FIND IN CODING RNA, BUT THESE
ARE REGULARLY -- I PRESSED THE
WRONG BUTTON, PROCESSED THROUGH
THE PROCESS THAT CONTAINS OTHER
PROTEINS THAT HAVE NOT BEEN
FULLY IDENTIFIED.
THIS ALLOWS FOR THE PROCESSING
OF THE UNIQUE HAIR PIN
STRUCTURE YOU SEE MOST MICRO
RNA'S FORMED, TRANSPORTED INTO
THE SIGH TO CYTO PLASM.
ONCE IT'S BEEN FURTHER
PROCESSED BY ANOTHER ENZYME,
AGAIN WITHIN THE COMPLEX.
THE RNA INDUCED SILENCING
COMPLEX, PREDOMINANT FAMILY OF
PROTEINS THAT FORM THE KEY
CATALYTIC DOMAIN OF THIS
COMPLEX, PART OF THE ARGONNAL
FAMILY OF PROTEINS.
WHEN IN THE CONTEXT OF MICRO
RNA REGULATION OF GENE
EXPRESSION DIFFERENT FAMILY
MEMBERS CAN SUBSTITUTE.
I'M TALKING ABOUT THE EFFECT OF
ONE PROTEIN, WHICH INVOLVES THE
CLEAVAGE OF THE TRANSCRIPT.
I FIRST WANT TO FINISH
DESCRIBING HERE, SO MOST IN A
NATURALLY OCCURRING SENSE
SETTING, MY RO THROUGH FORMATION OF
THE MICRO RNA CONTAINING RNA
INDUCING SILENCING COMPLEX,
THIS WILL INVOLVE MRNA
DEGRADATION OF THE PROTEIN
ENCODING GENE THAT IT INTERACTS
AT THE THREE PRIME UTR, VERY
SPECIFIC.
THERE ARE SOME REPORTS OF
INTERACTIONS WITH OTHER PARTS
OF THE TRANSCRIPT,
PREDOMINANTLY THREE.
THIS IS INTERACTION REQUIRES A
MINIMAL SEQUENCE OVERLAP WHICH
IS PREDOMINANTLY DRIVEN BY THE
FIVE PRIME END OF THE MICRO RNA
SEQUENCE.
AND SO YOU END UP WITH THIS
SORT OF -- SEVERAL MISMATCHES.
THIS IS A MISMATCH INTERACTION
WITH PROTEIN ENCODING
TRANSCRIPT.
THIS IS KEY FOR REGULATION,
OVERA THIRD OF PROTEIN ENCODING
GENES, A MAJOR REGULATORY
MECHANISM.
I'M ONLY GOING TO TOUCH ON THIS
A LITTLE MORE TODAY BECAUSE OF
THE TRANSLATIONAL PRESENTATION
SO I'M GOING TO MAINLY FOCUSED
ON THIS SIDE WHICH IS WHERE
WE'RE USING ARTIFICIAL
MEDIATORS, SMALL INTERFERING
RNA'S, INTERACTS NOW WITH ONE
MEMBER OF THE FAMILY, TO INDUCE
THIS A TRAS TARGET TRANSCRIPT
CLEAVAGE.
THE MEDIATOR, ARTIFICIAL, HAS
TO HAVE AN EXACT MATCH IN TERMS
OF SEQUENCE, BETWEEN THE SIRNA
AND TARGET TRANSCRIPT, THIS CAN
INTERACT WITH ANY POINT IN THE
TARGET TRANSCRIPT, ANYWHERE,
ANY PART OF THE TARGET
TRANSCRIPT, SO WHAT CAN HAPPEN
IS SO THE DIFFERENCE IS THAT WE
HAVE IN THIS CASE WE'RE DEALING
WITH GENE REGULATION, IN A
NATURAL SATING SAT SETTING AND USE THIS
FOR LOSS OF FUNCTION ANALYSIS.
WHAT DO I MEAN BY THAT?
SO WHY DO WE CARE ABOUT LOSS OF
FUNCTION ANALYSIS?
THIS IS A REALLY IMPORTANT
REASON FOR THIS.
SO OUR ABILITY TO PROFILE THE
CANCER GENOME ON A LARGE SCALE
HAS BEEN REVOLUTIO REVOLUTIONIZED IN
RECENT YEARS.
YOU'LL HEAR A LOT ABOUT HOW WE
CAN EXPRESS PROFILE AT A
GENOMIC LEVEL, RNA LEVEL, ON A
LARGE SCALE.
WE KNOW WHAT CAN LOOK AT COFFEE
NUMBER, ANALYZE NONCODING RNA'S
BUT THERE'S SOMETHING MISSING,
AND THIS HAS ALLOWED A LOOK AT
THE CHANGES WHICH UNDERLINE
CANCERS WE NEED TO
FUNCTIONALLIZE THEM.
MUCH OF THIS IS TRACTABLE FOR
DIAGNOSIS, BUT
FUNCTIONALLIZING, UNDERSTANDING
THE MECHANIZE THE BASIS,
REMAINS A CHALLENGE.
WE NEED TO HAVE A WAY OF DOING
THIS ON A LARGER SCALE.
THIS IS 'HERE LOSS OF FUNCTION
ANALYSIS COMES IN.
CURRENTLY, THOUGH THERE ARE
METHODS AND ALTERNATIVES THAT
ARE STARTING TO BE DEVELOPED,
UP UNTIL CURRENTLY, RNA
EXPERIENCE IS THE MOST WIDELY
USED APPROACH FOR CONDUCTING
GENE SPECIFIC LOSS OF FUNCTION
STUDIES IN MAMMALIAN.
I'LL SUMMARIZE FROM THE
PREVIOUS SLIDE.
WE HAVE THE FIRNA AND THIS
COULD BE AN FRNA OR FHRNA.
I'LL EXPLAIN WHAT THOSE ARE IN
MORE DETAILS, USUALLY NOW BEING
ABLE TO BE PURCHASED
COMMERCIALLY, THEY HAVE BEEN
PRE-DESIGNED TO MATCH A
PARTICULAR TARGET TRANSCRIPT,
YOU'RE FORMING THE COMPLEX AND
YOU'RE GOING TO INHIBIT NEW
PROTEIN PRODUCTION.
SO YOU ARE NOT GOING TO INDUCE
AN IMMEDIATE EFFECT.
IT WILL TAKE TIME.
IT'S GOING TO BE DEPENDENT ON
THE HALF-LIFE OF PROTEIN YOU'RE
LOOKING AT, BECAUSE YOU'RE
INHIBITING NEW PRODUCTION.
SO IF YOU HAVE A VERY LONG
STABLE PROTEIN YOU'RE TRYING TO
SILENCE YOU'RE NOT GOING TO
NECESSARILY SEE ANY EFFECT OR
YOU'RE GOING TO HAVE TO HAVE A
FORMAL WAY, USE, FOR EXAMPLE, A
ALLOWSAIR PIN RNA THAT
A LONGER TERM SILENCING OF YOUR
GENE.
THE BOTTOM LINE IS YOU'RE
HOPING THAT BY INHIBITING
PROTEIN PRODUCTION, YOU'RE
GOING TO INDUCE SOME SORT OF
LOSS OF CELLULAR FUNCTION,
WHETHER IT'S A BROAD PHENOTYPE,
PRECISETO A MUCH MORE PRECISE
ASPECT.
WHAT ARE PEOPLE USING IT FOR
INCLUDING WORK IN PL MY
LABORATORY?
FOR GENE ANALYSIS, YOU CAN DO
IT FOR LOOKING AT THE ENTIRE
PATHWAY OR NETWORK, SEEING IF
YOU HAVE DIFFERENT PROTEINS
INTERACT WITHIN THAT.
AN IMPORTANT USE OF RNA LOSS OF
FUNCTION IS IMPROVING MOLECULAR
TARGET IDENTIFICATION AND
CORRECT -- FURTHER VALIDATION
OF MOLECULAR TARGETS.
THERE'S LOOKING AT HOW
DIFFERENT SMALL COMPOUNDS,
SMALL INHIBITORS WORK FOR
IMPROVING THERAPEUTIC
EVALUATION.
I'M GOING TO TOUCH ON SOME OF
THE VARIOUS FEATURES OF HOW IT
WORKS IN MY LAB, WE'VE TOUCHED
ON APPLICATIONS SUCH AS THESE.
BEFORE I GET INTO SOME
EXAMPLES, I WANT TO MAKE SURE
THAT EVERYBODY'S ON THE SAME
PAGE AS TO WHAT AN EXPERIMENT
WOULD LOOK LIKE IN THE LAB, SO
I WANT TO TAKE A QUICK SHOW OF
HANDS, I DO THIS EVERY YEAR,
HOW MANY PEOPLE HAVE DONE AN
RNAI EXPERIMENT IN THE LAB?
ONLY TWO.
I'M NOT GOING TO BITE.
IF YOU'VE DONE IT, IT DIDN'T
WORK, OR THERE WAS A PROBLEM,
MAYBE IT'S JUST FALLING OUT OF
FAVOR, I DON'T KNOW.
SO RNAI INDUCES A KNOCKDOWN IN
THE JARGON A LOT OF PEOPLE USE.
IT'S NOT ONE I CARE TO USE BUT
IT'S A WAY OF DIFFERENTIATING
IT FROM A KNOCKOUT, WHICH IS
WHAT WE'RE USED TO WHEN THIS
FIELD DEVELOPED WHERE WE WERE
USED TO SEEING KNOCKOUT
ANIMATION, COMPLETE ABLATION,
LOSS OF FUNCTION.
NOW THAT MEANS TO DO THAT YOU
HAVE TO AUTOMIZI WHAT YOU'RE
DOING IN THE LABORATORY, MAKE
SURE THE MODEL SYSTEM IS RIGHT
FOR ASKING THE QUESTION YOU
WANT TO ANSWER, THAT MIGHT
SOUND LIKE THE OBVIOUS
STATEMENT BUT ONE ISSUE WE'VE
COME ACROSS A LOT, I'M GOING TO
DESCRIBE ONE OF THESE STUDIES,
THAT YOU HAVE A LIST OF GENES
THAT WE KNOW ARE OVEREXPRESSED
IN A TUMOR, FOR EXAMPLE, AND
YOU WANT TO SILENCE THOSE
BECAUSE THE THOUGHT IS ONE OF
THESE IS THE DRIVER OF THAT
PROCESS.
IF YOUR MODEL SYSTEM, YOUR CELL
LINE, DOESN'T LOOK LIKE THE
EXPRESS PROFILES YOU HAVE AS
THE TUMOR, YOU'RE NOT GOING TO
GET THE RESULT YOU WANT.
THIS IS ONLY AS GOOD AS THE
MODELS THAT WE HAVE FOR IT.
ONE OF THE LIMITATIONS IS
CELLULAR MODEL.
EFFICACY AND SPECIFICS
PHYSICALITY IS IMPORTANT.
EFFICACY IS DEPENDENT ON WHAT
SORT OF PROTEIN YOU'RE
SILENCING BY THIS MECHANISM.
SO SOME PROTEINS, IF YOU JUST
TWEAK THE AMOUNT OF PROTEIN IN
THE CELL, YOU'RE GOING TO SEE
SOME EFFECT.
YOU DON'T HAVE TO GET TO A
HETEROZYGOTE STATE.
YOU COULD TAKE OUT 99.9% OF THE
PROTEIN AND THAT .1% IS STILL
GOING TO BE SUFFICIENT TO
PERFORM THE JOB REQUIRED
BECAUSE IT'S JUST EITHER SO
MUCH OF IT OR IT'S FUNCTION --
THERE'S FUNCTIONAL REDUNDANCY,
THAT MEANS IT'S NOT ESSENTIAL,
IT MIGHT BE IMPORTANT BUT
DOESN'T MEAN IT'S ESSENTIAL FOR
THAT FUNCTION.
LOTS OF THINGS.
YOU HAVE TO BE VERY CAREFUL
ABOUT WHAT EFFICACY AND DEGREE
OF SILENCING MEANS.
PEOPLE THROW UP NUMBERS LIKE I
SAW 70% SILENCING OF MY GENE,
MEASURED BY PROTEIN DECREASE OR
WHATEVER IT IS.
THAT'S MEANINGLESS UNLESS YOU
KNOW HOW MUCH PROTEIN YOU'VE
GOT.
WE CAN SILENCE, FOR EXAMPLE.
ONE OF THE PROTEINS, WE CAN
SILENCE AT 99%, OR THE TH 99% OF THE
RNA IS GONE, WE SILENCE IT.
1% LEFT, OKAY?
THE THING IS THAT PROTEIN IS --
THAT TRANSFER IS AT EXPRESSED
AT THOUSANDS OF COPIES PER
CELL.
EVEN THOUGH WE GOT 99% OF THEM
GONE, THERE'S STILL MORE
TRANSCRIPT LEFT FOR THAT, THAN
MOST OTHER PROTEINS LEFT, MOST
PROTEIN CODING TRANSCRIPTS
LEFT.
YOU HAVE TO THINK ABOUT WHAT IT
IS YOU'RE SILENCING.
POSITIONALLY NOW WHAT'S
DEVELOPED OVER THE YEARS, WE'RE
DELIVERING SYNTHETIC SRNA'S,
WHICH ARE ARTIFICIALLY
GENERATED, SO THESE ARE
EFFECTSIVELY SOPHISTICATED
NUCLEOTIDES, WHAT MY LABORATORY
USES.
IT'S NOT THAT I'M -- THERE'S NO
FOR OR AGAINST IT, WE TEND TO
DO SYNTHETIC SRNA'S TO ANSWER
THE QUESTIONS THAT WE'RE
ASKING.
WHEREARE MANY QUESTIONS WE'RE
IT'S APPLICABLE, IT JUST
DEPENDS ON THE CIRCUMSTANCES.
WHAT IS KEY TO A GOOD RNAI
BASIC EXPERIMENT IS HAVING ALL
THE ASSAYS IN PLACE TO BE SURE
YOU HAVE QUANTIFYING KNOCKDOWNS
AND LOOKING AT THE RIGHT
FUNCTION.
THAT MIGHT SOUND STUPID BUT A
LOT OF PEOPLE COME TO ME
WANTING TO KNOW WHAT SRNA'S OR
SHRNA'S, WHAT THAT
INCORPORATION THEY SHOULD ORDER
BEFORE THEY HAVE AN ASSAY FOR
THE GENE.
I'M LIKE GOING AWAY, MAKE SURE
YOU CAN ACTUALLY MEASURE YOUR
GENE OF INTEREST BY QRTPCR AND
THEN COMBINE YOUR RESOURCES.
ASSAYS COME FIRST.
I ACTUALLY SAY DO THIS, AND
THIS FIRST, AND THEN WORRY
ABOUT WHAT YOU'RE GOING TO BUY.
IT TENDS TO GO THE OTHER WAY
AROUND IN MY EXPERIENCE.
WHAT DOES THE EXPERIMENT IN MY
LAB LOOK LIKE?
THIS IS SLIGHTLY OUT OF DATE
APPLICABLE.Y AFTER
WE'RE TAKING HERE THE KNOCKDOWN
OF A GENE THAT MANY OF YOU ARE
GOING TO BE FAMILIAR WITH,
INVOLVED WITH SIGNALLING, AND
TWO FRNA'S, SHOWING THE
THIS IS, MRNA LEVELS, THIS IS
COLO-*** CARCINOMA.
WE'VE REDUCED BY A HIGH DEGREE
48 HOURS TYPICALLY AFTER
TRANSFECTION ON.
AT A PROTEIN LEVEL, THIS IS
WHAT WE'RE SEEING HERE OVER
TIME.
LOOKING HERE AT 48, 72, 96
HOURS, LOOKING TO SEE WHAT IS
THE DECREASE OVER TIME IN THE
PROTEIN AND WE CAN SEE HERE WE
QUANTIFIED HERE THE SILENCING
AT EACH TIME POINT.
VIEWING THE TIME POINTS IS
USEFUL IF YOU DO NOT KNOW THE
STABILITY OF YOUR PROTEIN AND
WHEN YOU ANTICIPATE SEEING
FUNCTIONAL EFFECTS, SO WE
USUALLY DO RNA AT 48 HOURS,
PROTEIN AT 72 HOURS, THAT'S OUR
STARTING POINT AND WE WORK FROM
THERE.
NOW, BEFORE I GO INTO ACTUAL
DATA AND EXPERIMENTS, I WANTED
TO PUT OUT THE DIRTY LAUNDRY OF
RNAI BECAUSE YOU HAVE TO BE
HONEST ABOUT THIS.
I REALIZE IT'S HARD TO SEE SOME
OF THIS.
THIS IS THE CONCEPT OF RNAI AND
TARGET EFFECT.
ONE OF MY COLLABORATORS, WHO IS
PART OF THE RNAI SCREENING
FACILITY THAT I'LL TALK ABOUT,
HAD A PROVOCATIVE TITLE AT THE
NIH FESTIVAL LAST WEEK, YOUR
SRNA'S ARE RUBBISH BUT I CAN
MAKE THEM BETTER.
HERE BEEN WORKING WITH THE
GENOME TAKE AND TALKING ABOUT
HOW YOU CAN USE APPROACHES TO
ADDRESS IT.
SIDEOU'RE DOING BENCH SIZE
EXPERIMENTS YOU HAVE TO TAKE A
DIFFERENT APPROACH.
HE'S GOT A LOT OF PEOPLE WITH
HIS POSTER.
A PROVOCATIVE TITLE IS USEFUL.
THIS OVER HERE IS ILLUSTRATING
WHAT I SHOWED EARLIER, WHAT WE
WANT IS OUR SRNA TO HAVE A
PERFECT MATCH TO ITS TARGET
TRANSCRIPT, SEQUENCE DRIVEN BUT
THIS IS -- WHAT I DIDN'T SAY
EARLIER, THIS IS ONLY 21 NUCLEO
TIDES, WE DON'T HAVE MUCH REAL
STATE, 21 NUCLEOTIDES.
PIKE ROMICRO RNA'S USE THIS PART.
SEVEN AT THE BEGINNING, TO FIND
THIS SEQUENCE THAT IT'S GOING
TO MATCH TO.
WHEN YOU PUT IN AN SIRNA INTO A
CELL OR AN SHRNA INTO A CELL
THAT CELL DOESN'T KNOW WHETHER
IT'S AN S.I. OR MICRO RNA AND
DOES NOT CARE WHETHER YOUR
EXPERIMENT WORKS OR NOT.
IT WILL TREAT IT AS A SEQUENCE
AND TRY TO MATCH IT TO ANY
TRANSCRIPT IT CAN MATCH, OKAY?
AND IF YOU UNFORTUNATELY HAVE
AN EIGHT -- SEVEN OR EIGHT
NUCLEOTIDE THAT HAPPENS TO
MATCH THAT RISK COMBINED, IT
WILL ACT LIKE A MICRO RNA.
YOU'RE TRYING TO FAVOR BY
DESIGN AND BY CAREFUL
EXPERIMENTATION GOING IN THIS
DIRECTION.
BUT YOU WILL HAVE THE POTENTIAL
SEEING OF THINGS GOING IN THIS
DIRECTION, SO YOU HAVE MICRO
RNA-LIKE EFFECT.
WE SUMMARIZED A POST-DOC THAT
NOW RUNS THE SKEI FACILITY, WE PUT
THIS TOGETHER AS PART OF THE
REVIEW A FEW YEARS AGO.
IT HASN'T CHANGED IN TERMS OF
HOW ONE ATTACKS THIS PROBLEM AT
THIS POINT.
FIRST THING TO SAY, PEOPLE WILL
TRY AND SELL YOU THINGS THAT
SAY, THIS SOLVES YOUR PROBLEM.
OKAY.
DON'T -- NONE OF THEM DO.
NONE OF THEM SOLVE THE PROBLEM.
THEY MIGHT MITIGATE IT A LITTLE
BUT THEY CERTAINLY DON'T SOLVE
IT.
SOME OF THE MODIFICATIONS HELP,
SO YOU CAN -- YOU HAVE TWO
TYPES OF TARGET EFFECTS, SOME
ARE SEQUENCE INDEPENDENT.
I'M NOT TALKING ABOUT THESE.
YOU PUT IN SO MUCH RNAI FACTOR,
THE WHOLE SYSTEM GETS GUMMED
UP.
THAT'S REALLY ONLY SEEN -- IT'S
NOT AN ISSUE IN STREE VITRO.
PEOPLE THAT DO RNA THERAPEUTIC
WORRY ABOUT THAT.
SOME CELL LOAN LINES MAKE A RESPONSE
TO INTRODUCTION OF NUCLEOTIDES,
YOU WANT TO USE THE LOWEST DOSE
POSSIBLE, WE NEVER USE ANYTHING
OVER 20.
SO IT'S PRACTICAL.
AS A REVIEWER IF I SEE 100 OR
ABOVE I DON'T EVEN REVIEW THE
PAPER.
IT GOES STRAIGHT BACK.
IT'S WAY TOO MUCH.
YOU CAN'T CONTROL FOR THAT.
SO THE SEQUENCE, YOU CAN HAVE
SOME CASES OF IMMUNE RESPONSE
THAT AGAIN IS REALLY ONLY
CONFINED TO SPECIFIC CELL TYPES
AND PHENOTYPES, AND THE RNA AND
THERAPEUTICS PEOPLE WORRY ABOUT
THAT.
THIS IS WHAT I'M TALKING ABOUT,
WE HAVE UNATTENDED TARGETS
THROUGH PARTIAL COMMENT
ELEMENT, USING RNA AT THE
LOWEST CONCENTRATION AND USING
VERY CAREFUL CONTROLS AND
MULTIPLE, MULTIPLE S.I.'S OR
SHRNA'S TO CONFIRM PHENOTYPES.
I WILL DESCRIBE THAT AS I GET
INTO INDIVIDUAL CASES.
SO HOW DO WE USE SRNA?
WE -- THE SUMMARY OF HOW WE USE
SRNA'S IS SORT OF HERE.
WE USE TWO DIFFERENT
COMPLEMENTARY APPROACHES, WE
EITHER DO IT ON A SCREENING
BASIS WHERE WE CAN SCREEN
THOUSANDS, THE FACILITY CAN DO
THE WHOLE GENOME OF SIRNA.
WE ALWAYS DO EVERYTHING WITH
ONE SIRNA.
WE CAN ASSAY PARTICULAR
PHENOTYPES, DOWNSTREAM OF THESE
EFFECTS.
ANY ASSAYS THAT CAN BE USED,
CELL BASED THAT CAN BE USED FOR
DRUG SCREENING, CAN BE ADAPTED
FOR RNAI STUDIES.
THE ALTERNATIVE PATHWAY WE
TAKE, APPROACH THAT WE TAKE, IS
TO ACTUALLY STUDY INDIVIDUAL
GENES BUT IN A COMPREHENSIVE
MANNER.
WE MIGHT SILENCE AN INDIVIDUAL
GENE AND THEN DO MICRO OR SOME
OTHER EXPRESSION PROFILING TO
GET A SENSE OF WHAT IT IS THAT,
THE LOSS OF FUNCTION OF THAT
GENE ASSERTING ON A PARTICULAR
CELL PHONE.
I'LL SHOW EXAMPLES OF THOSE
NOW.
ALL RIGHT.
SO WE CAN ALSO ADAPT THE WHOLE
PROCESS FOR DOING THERAPEUTIC
INTERVENTION SO INSTEAD OF
DOING -- WE CAN ADD A DRUG,
I'LL SHOW YOU BRIEFLY A STUDY
WHETHER WE USED A DRUG IN
ADDITION TO THIS.
AND THEN WE CAN ALSO EXPAND OR
END POINTS AS WELL.
WE CAN DO THE SAME THING WITH
MICRO RNA'S BUT I'M NOT GOING
TO DO THAT.
I'M GOING TO TALK ABOUT SOME
ACTUAL REAL WORLD STUDIES.
AND THIS PRESENTATION IS PART
OF ONE I USED OFTEN FOR
PRESENTATIONS IN OTHER
SCENARIOS, WE'RE TRYING TO
DEFINE THE FUNCTIONAL GENOME OR
CERTAINLY THE CANCER ONE, AND
WE CAN DO THIS ONE GENE AT A
TIME, IT COMES DOWN TO ONE
FELLOW AT A TIME.
I'LL TALK ABOUT WORK IN BREAST
CANISTER IN STAN LIPOWITZ'S
LAB, WORK STARTED HERE WITH
JOHN WEINSTEIN, NOW AT M.D.
ANDERSON AND WE'RE WRAPPING UP
NOW, WORK WITH MY FORMER
POST-DOC SCOTT MARTIN AND NEW
WORK WITH PEDIATRIC ONCOLOGY.
I'M GOING TO USE THESE TO SHOW
EXAMPLES OF THE TIMES OF THINGS
I TALKED ABOUT, HOW WE USED
RNAI TO INVESTIGATE CANCER.
A STUDY ON BREAST CANCER, JUST
TO GIVE YOU BACKGROUND, WE KNOW
THIS IS A HETEROGENEOUS
DISEASE, 65% OF PATIENTS HAVE
TUMORS E.R. AND/OR P.R.
POSITIVE.
20% HAVE AN AMPLIFIED.
THESE ARE SOME FIRST LINE
TARGETED THERAPIES.
15% OF CASES, WHAT IS DESCRIBED
AS TRIPLE NEGATIVE BREAST
CANCER, THEY DO NOT HAVE THESE
MARKERS BUT HERE IS MARKERS FOR
EPITHELIAL CELLS, THE ONLY
SURGERY CAN ASK FOR IS
AND DRUG TREATMENT SO WE NEED
TARGETED THERAPY.
SO MY GROUP HELPED WORK WITH
STAN LIPOWITZ'S GROUP, LIND YEA
AND SERETIA.
IT WAS A SMALL SCREEN, BUT AT
THE TIME IT WAS WHAT WE CAN DO,
NOW PUBLISHED IN 2009, 2010.
WHAT WE'RE SEEING HERE IS
ACTUALLY THE GROWTH OF
PERCENTAGE -- PERCENTAGE GROWTH
REDUCTION IN A TRIPLE NEGATIVE
BREAST CANCER CELL, MDA AND
B-231.
WE RANKED TO A STRONG POSITIVE
CONTROL BUT WE ALREADY DEFINED
IN THIS CELL TYPE, SILENCING OF
RM-2, ONE OF THE SUBUNITS OF
THE RIBONUKNUCLEOTIDE.
WHEN WE SILENCE THESE, WE SAW
THIS LEVEL OF REDUCTION IN CELL
VIABILITY, IT WAS QUITE
EXTREME.
THE TOP HIT WAS WE WON.
STAN'S GROUP CHARACTER
ADVERTISE THIS IN MORE DETAIL.
YOU'RE SEEING WHAT I SHOWED
EARLIER, SILENCING OF PROTEIN
LEVEL, AND WHAT WE HAVE HERE IS
WE'VE SHOWN THE BREAST CANCER
CELL LINES, WE HAVE SIGNIFICANT
REDUCTION IN CELL GROWTH, WHICH
IS FROM SI NEGATIVE, SHOWN
HERE, ONLY 20% OF THE CELLS
LEFT HERE.
WHICH IN COMPARISON WITH THIS
CELL LINE, A NONTUMOR MAMMARI
EPITHELIAL.
THAT SUGGESTS THERE'S SOMETHING
THAT'S ALTERED IN THE GENETIC
BACKGROUNDS OF THESE CELL LINES
THAT MEANS THESE ARE MORE
DEPENDENT ON ACTIVITY.
YOU HAVE A SENSE OF SOME CELL
ACTIVITY AND THIS IS COPIED
WITH AN INHIBITOR, ACTIVELY BY
MANY GROUPS THAT HAVE BUILT ON
THIS AND SIMILAR OBSERVATIONS.
THIS IS ACTUALLY THE SAME WITH
THE DRUG.
WE SEE THE SAME EFFECTS.
THIS IS PUBLISHED SO EVERYONE
CAN GO BACK TO THIS STUDY.
I'M GOING TO MOVE THROUGH SOME
OF THESE.
OKAY.
I'M GOING TO MOVE TO THE NEXT
ONE, COLO-*** CANCER, A
DIFFERENT WAY OF LOOKING AT
THIS.
SO COLO-*** CANCER A
RELATIVELY WELL CHARACTERIZED
CHANGED CANCER, WE HAVE A FAIR
SENSE OF SOME GENETICS CHANGES
THAT OCCUR OVER TIME BUT
THERE'S A NEED TO UNDERSTAND
THE CONSEQUENCES OF THE
MOLECULAR CHANGES BEING
CHARACTERIZED THERE.
ONE OF THE CHANGES IS
ILLUSTRATED HERE, THIS IS A
STUDY FROM JODY AND THOMAS
REED, A COUPLE OF STUDIES, WHAT
YOU'RE SEEING HERE IS THE DNA
COPY NUMBER, IN COLO AND
PRIMARY TUMORS, 31 OF THEM,
GREEN REPRESENTS A LOSS ACROSS
THE WHOLE GENOME, FROM JUST A
CHROMOSOME ONE TO CHROMOSOME Y,
A LOSS IN GREEN.
YOU SEE BLOCKS WHERE YOU HAVE
AGAIN A NUMBER.
I WANT TO DRAW YOUR ATTENTION
HERE TO THIS PARTICULAR ONE ON
CHROMOSOME 13.
THERE'S RECURRING GAIN IN A
LARGE POSITION OF CHROMOSOME 13
WITH CURRENTLY IN BOTH COLON
AND IN *** TUMORS, BUT AS
YET IT'S BEEN UNDEFINED WHAT
13 THATE ON CHROMOSOME SP THAT
MIGHT BE DRIVING OR AS A
CONSEQUENCE OF THIS GAIN,
CONTRIBUTING TO COLO-***
CARCINOGENESIS.
I'LL WALK YOU THROUGH THIS
PARTICULAR SLIDE.
WHAT STARTED OUT WAS THE READ
GROUP CAME TO ME WITH A LIST OF
GENES THAT HAD AN INCREASE IN
COPY NUMBER AND INCREASE IN
GENE EXPRESSION.
AND THAT'S IS U SUMMARIZED HERE,
TAKING OUT THAT PORTION OF
CHROMOSOME 13 WITH A
CORRELATION BETWEEN
AMPLIFICATION AND GENE
EXPRESSION.
THIS IS THE GENES THAT HAVE
BEEN AMPLIFIED, MOST OF THEM
ARE OVE OVEREXPRESSED.
WHAT WE NEEDED TO DO WAS GET
DOWN TO A LIST OF CANDIDATE
GENES, 116 AT THAT TIME, AND WE
DECIDED THE THING TO DO WAS PCR
VALIDATE THESE IN A SERIES OF
CELL LINES.
WE TOOK COLO-*** CANCER CELL
LINES AND LOOKED TO SEE WHICH
OF THESE SHOWED SIMILAR
PATTERNS OF UNDER AND OVER
EXPRESSION OF THE GENES FOUND
WITHIN THAT REGION.
SO THAT WE COULD CHOOSE JUST
ONE OR TWO OF THESE FOR OUR
FUNCTIONAL STUDIES.
AND SO WE GOT DOWN TO 67 GENES
THAT LOOKED POSSIBLE, AND BY
THEN GOING TO JUST CHOOSE THOSE
ONES THAT WE COULD TRANSPECT,
HIGH EFFICIENCIES, WE GOT DOWN
TO
44 IN TWO CELL LINES, THIS
AXIS AND THIS AXIS.
WE'RE LOOKING AT CELL
VIABILITY, A BROAD PHENOTYPIC
EFFECT, ONE THAT CANCER
BIOLOGISTS ARE MOST INTERESTED
IN.
SOMETIMES AS YOU'LL SEE WE WANT
TO DO SOMETHING MORE
SOPHISTICATED.
WHAT WE'RE DOING HERE IS
LOOKING TO SEE IF WE HAD A
REDUCTION IN CELL GROWTH THIS
WAY, IN BOTH CELL LINES.
WE FOUND A BUNCH OF SRNA'S THAT
REDUCED THIS EFFECT.
WE DID SECONDARY SCREENS THAT
GOT US DOWN TO 17 GENES.
AND THEN WE CHOSE FROM THAT,
SIX THAT LOOKED LIKE THEY WERE
HIGHLY REPRODUCIBLE, VERY SMALL
BUT GIVING SOME EFFECT WHEN WE
SILENCED THEM IN THE CELL
LINES.
BUT AS I SAY, THERE IS A
PROBLEM.
THIS IS A VERY BROAD PHENOTYPE.
IT WAS HUGE VARIATION BETWEEN
THE CELL LINES THAT WE
ANALYZED, THERE'S NO CONTROL
CELL LINE, THERE'S NO
NONTUMORGENIC REPRESENTATION OF
COLON OR *** TISSUES.
THAT'S A PROBLEM.
WHEN YOU SAW THE PREVIOUS STUDY
OF BREAST CANCER WE HAVE A CELL
LINE THAT GROWS ON PLASTIC WITH
A I WILLED NUMBE LIMITED NUMBER OF CHANGE S,
AND DOES NOT FORM TUMORS BUT
IT'S MAMMARY.
THIS IS AN ISSUE FOR A LOT OF
THE WORK WE'RE DON DONE ON THIS.
THE TROUBLE WHEN YOU LOOK AT
THAT, AMPLIFIED I AND ALL THESE
GENES ARE SKPWROEF SKPWROEFR OVEREXPRESSI ONED,
YOU'RE TRYING TO SAY ONE GENE
THREE,144, NOT TWO OR 3, IT'S
THERE WAS SOME PROBLEMS WITH
THIS APPROACH BUT WE GOT
SOMETHING OUT OF IT.
ONLY ONE OF THESE GENES HAS
EVER BEEN ASSOCIATED WITH
COLO-*** CANCER BEFORE.
WHAT WE DECIDED TO DO WAS TAKE
AGNOSTIC APPROACH, SILENCE
THE CELLS AND JUST SEE AT A
TRANSCRIPTIONAL LEVEL WHAT
HAPPENS TO GENES WHERE THESE
HAVE ALL BEEN SILENCED.
WE GENERATED THIS DATA SET, AND
THIS HAS JUST BEEN PUBLISHED IN
CANCER RESEARCH.
WHAT WE'RE TRYING TO LOOK AT
HERE, THIS IS LNX-2, YOU SEE
WHAT WE'VE SILENCED, LNX-2 WITH
TWO SRNA'S AND DONE EXPRESS
PROFILING, 72 HOURS LATER, AND
WE SEE THESE ARE THE GENES THAT
ARE DEREGULATED BY SIRNA, SIGH
SEE HIGH CORRELATIONS, WE CAN
PICK OUT THE GENES BEING
2EREGULATED FOLLOWING LNX-IT
SILENCING AND LIMIT TARGET
EFFECTINGS.
IT'S A PRACTICAL WAY OF LOOKING
IN AN UNBIASED MANNER WHAT'S
HAPPENING TO THESE CELLS.
AND YOU WHA WHAT YOU CAN SEE IN RED
HERE ARE THE TWO CORRESPONDING
LNX-2'S.
IT'S INTERESTING TO SEE WHAT'S
HERE AND WHAT'S HERE.
WE DID THIS FOR ALL OF THESE
GENES BUT I'M GOING TO FOCUS ON
THE LNX-2 STORY HERE.
WHAT WE FOUND, THIS IS A MORE
TRADITIONAL REPRESENTATION OF
THE DATA THAT YOU'RE USED TO,
SEEING A MAP SHOWING 337 GENES
THAT ARE DOWN REGULATED, 343
THAT ARE UP-REGULATED FOLLOWING
SILENCING OF LNX-2 AND THE
QUESTION IS WHAT IS THIS
TELLING US?
IT HELPS US TO KNOW WHAT WE DO
KNOW ABOUTLNX-2.
ALSO THOUGHT TO SERVE AS A
MEMBRANEFOR THE MEMORIAL RAIN
PROTEIN, PART OF A NOT
SIGNALLING PATHWAY, KNOWN AS A
NOT SIGNALLING PATHWAY, ALSO IN
COLO-*** CANCER, THIS SEEMED
FOR A GOOD CANDIDATE TO EXPAND
A STUDY IN MORE DETAIL BUT NOT
PREVIOUSLY LINKED TO COAL OH
COLO-*** CAN SEAR.
WE KNOW LNX-2 IS SUPPOSED TO
INTERACT, AND WE WOULD
ANTICIPATE SEEING CHANGES IN
DOWNSTREAM MARKERS OF NOTCHED
SIGNALLING.
IF WE DO LOOK AT OUR DATA FROM
EXPRESS PROFILING WE FIND ALL
OF THESE MAJOR AND DECREASE IN
NOTCH ONE, WE SEE NO CHANGES IN
ONE BUT WE SEE CHANGES IN NOTCH
ONE AND DOWNSTREAM TARGETS.
SO THIS INDICATES THAT, YES,
WHEN WE SILENCIN SILENCING LNX-22 WE SEE
DOWNSTREAM EFFECTINGS,
AT PROTEIN LEVEL.S, CONFIRMED
WHAT ELSE CAN WE GET OUT OF
THIS?
A POST-DOC IN MY LAB NOW AT THE
UNIVERSITY OF COLORAD CHICAGO CREATED
LISTS OF TRANSCRIPTION FACTORS
DEREGULATED IN COLO-***
CANCER AND CURATED A LIST OF
TARGETS ESTABLISHED FOR THEM
AND LOOKED TO SEE IF THERE WAS
ENRICHMENT FOR ANY PARTICULAR
DOWNSTREAM TARGETS OF ANY OF
THESE COLO-*** CANCER
ASSOCIATED TRANSCRIPTION
FACTORS.
I DRAW YOUR ATTENTION TO THIS
ONE HERE.
THIS IS THE DATA ARE TO LNX-2,
ENRICHMENT PACED ON P VALUE.
ARE THE TARGETS OF THESE
TRANSCRIPTION FACTORS ALTERED
WHEN WE SILENCE LNX-2?
IF YOU SEE GREEN, THIS IS HUGE
ENRICHMENT FOR THE
DOWN-REGULATED PARTS OF THIS
GENE, OR THIS PROTEIN I SHOULD
SAY, TRANSCRIPTION FACTOR, TCF
7-L2.
TCF-P-L2 IS A MAJOR EFFECTOR
OF -- THIS IS SIGNALLING, I'M
SORRY.
SO IT'S WELL ESTABLISHED THIS
IS A NAG NEGATIV SIGNIFICANT PATHWAY THAT 'S
DEREGULATED IN COAL O COLO-***
CANCER.
WE DO SEE WHEN WE SILENCE
LNX-2, WE SEE COMPLETE ABLATION
OF PROTEIN LEVELS.
WE SEE THAT WE CAN ALSO
SIGNIFICANTLY REPORT AN ASSAY
WHEN SIGNALLING, THE TOP FLASH
ASSAY, ALL I WANT TO DRAW YOUR
ATTENTION TO IS WE SEE THIS
HUGE DECREASE IN THAT ASSAY
SYSTEM.
MORE IMPORTANTLY, WE COULD GO
BACK TO A TUMOR, WE HAVE TO DO
THIS WITH PUBLIC DATA SETS, IF
WE SAKE THE GENES THAT WE FOUND
2,AT ARE REGULATED BY LNX-IT,
USING OUR GENE EXPRESSION DATA,
THE KNOWN TARGETS OF TC 7L 2,
THAT INDEED IS THE CASE.
THIS EXPRESS MODULE CLEARLY
SHOWS THAT THERE'S A DIFFERENCE
FROM THIS MODULE GENERATED FROM
OUR DATA BETWEEN TUMOR AND
MUCOSA.
THIS SAID TO US THERE IS A LINK
THROUGH LNX-2 TO MAJOR PATHWAYS
DEREGULATED IN COLO-***
CANCER, THE UPREGULATION OF
THIS GENE IN TUMORS MAY BE
AFFECTING THE ACTUAL OUTCOME OF
MAYBE INFLUENCING THE BIOLOGY
OF THIS CANCER.
SO THAT WAS COAL OH ELECTRIC
OVER AMPLIFICATION OF GENES.
THERE HAVE BEEN STUDIES OF
PROFILES, OVEREXPRESSION OF
GENES IN DIFFERENT GENOMES, IF
WE TARGET THIS WE'LL BE ABLE TO
TREAT THESE TUMORS.
THIS IS A CASE WHERE YOU SEE,
COMING BACK TO A GENE I
MENTIONED EARLIER, RM-2, RM-2
AS I SAY IS PART OF THE
RIBONUCLEAR, ESSENTIAL FOR
MPT'S, REQUIRED FOR REPLICATION
DNA REPAIR.
WHAT WE SAW HERE IS IT
CONSISTENTLY UPREGULATED IN
TUMORS, THIS IS UPREGULATION IN
*** CANCER, IN COLON CANCER,
IN CELL LINES, RED MEANS
OVEREXPRESSION, IN 25 LINES.
IT'S STRAIGHT FORWARD.
THE CELLS REQUIRE MORE DMPT AND
UPREGULATE RM-2 TO DO IT.
CELLS DO SEEM TO BE -- CANCER
CELLS SEEMS TO ARE
PREFERENTIALLY DEPENDENT ON ITS
ACTIVITY.
YOU MAY FIND A SWEET SPOT IF
YOU COULD TARGET IT MORE
EFFECTIVELY.
THERE'S NO SPECIFIC DRUGS FOR
IT.
THERE'S A SPECTRUM BUT IT'S AN
INDIRECT EFFECT.
WE'RE SHOWING HERE IF YOU
SILENCE RM-2, YOU SEE THIS IS
THE REDUCTION IN RNA LEVELS, A
PERCENTAGE OF CONTROL, AND WE
SEE THIS CHANGE IN CELL
VIABILITY OVER TIME.
RM-2 IS PART OF THE
RIBONUCLEOTIDE COMPLEX.
IT'S NOT JUST AN ANTICANCER
TARGET BUT AN ANTIVIRAL TARGET.
WE KNOW THERE ARE EFFECTS ON
RM-1, AND THEN RM-2, THERE ARE
IS SOME O EFFECT AND IT'S EFFECT.
I'M GOING TO COME BACK TO THAT
TARGETING IN A MOMENT.
SO WE CAME UP WITH AN
INHIBITION, WE GOT A PROFILE OF
WHAT OCCURS IN THESE CELLS,
FOLLOWING KNOCKDOWN OF RM-2 TO
GET A SENSE OF WHAT WAS
HAPPENING.
SO THE THERE'S AN ATTEMPT TO LIMIT
WITH DRUGS.
IT'S UNCLEAN.
THEY DO LOTS OF THINGS.
THIS IS A WAY OF US
CONTRIBUTING TO THE COMMUNITY
EXACTLY WHAT SHOULD HAPPEN WHEN
YOU SILENCE RM-2.
FROM THAT WE CAN GET A SENSE OF
WHAT IT OCCURRING WHEN WE
SILENCE RM-2, AND AGAIN THIS IS
PUBLISHED WORK.
WE CHANGING PARTICULARLY HERE A
LOT OF GENES ASSOCIATED WITH
PROTEINS AND CONNECTING GRAND
CYCLONES,ROUND THE SIGH LOANS
INCLUDING MYTOTIC ASSOCIATED
GENES.
THE KEY THING WAS THIS ACTUALLY
CONTRIBUTES TO A FIELD THAT'S
ONGOING.
I WANT TO HIGHLIGHT THIS
TRANSLATIONAL IMPLICATION TO
THE WORK WE DO AND OTHERS IN
THIS FIELD, CAN RM-2 BE
SILENCED?
IT'S A TARGET FOR DECADES
PEOPLE HAVE BEEN TRYING TO
ATTACK.
AND I JUST WANT TO HIGHLIGHT
THIS PAPER THAT CAME OUT IN
2010, AND WORK IS CONTINUING,
WHERE FROM MARK DAVIS' LAB AT
CAL TECH, THEY TOOK NANO
PARTICLES, AND SO IT WAS PUT
INTO A SOPHISTICATEDDE SOPHISTICATED NANO
PARTICLE AND DELIVERED TO
PATIENTS WITH MELANOMA.
WHAT YOU HAVE HERE IS THE DATA
FOR THREE DIFFERENT PATIENTS.
ACTUALLY, NO, THIS IS TWO
PATIENTS.
THEY DID THREE, WHAT THEY WERE
LOOKING FOR, EXCUSE ME, HERE
WAS FOR DECREASE IN RM-2 AND
PRE-AND POST ADMINISTRATION OF
THE NANO PARTICLE CONTAINING
RM-2 AND EVEN BETTER IN THE
SECOND PATIENT.
THIS IS THE THIRD PATIENT WHERE
THEY DID -- THEY JUST HAVE
MRNA, HERE THEY HAVE MRNA AND
PROTEIN.
AND SO THIS SHOWS ACTUALLY IN
VIVO THEY COULD GET GENERATION
OF A KNOCKDOWN.
THIS IS PATIENTS THEMSELVES.
SORRY ABOUT THIS.
THEY WERE ABLE TO GET WHAT
YOU'RE LOOKING AT HERE, RM-2
STAINING, SO PROTEIN IN PATIENT
C, THIS IS THE ANTIBODY TO RM-2
AND SIGNIFICANT REDUCTION IN
ANTIBODY.
WHAT THEY USED FOR THIS
DELIVERY FOR THIS NANO PARTICLE
WAS A TRANSFERENCE REFLECTOR,
SO YOU ACTUALLY WERE SHOWING
THERE WAS UPTAKE HERE AS A
CONSEQUENCE OF TARGETING WITH
THAT.
NOW, THERE ARE LOTS OF GROUPS
WORKING ON RNAI THERAPEUTIC
APPROACHES.
I WON'T GO INTO DETAIL, THIS IS
ANOTHER STUDY MORE RECENTLY.
IT WAS PUBLISHED IN "CELL" IN
2012 WHERE THEY USED DIFFERENT
WAYS OF CHANGING THE SRNA TO
MAKE IT MORE DELIVERABLE, THIS
WAS TARE TARGETING T-10 IN THE LIVER
AND LOOKING FOR REDUCTION OVER
TIME.
IT COMES BACK WHERE YOU'VE GOT
SILENCING OF P-10 FOR QUITE
SOME TIME BY SUB-CU AND IV,
SYSTEMIC DELIVERY.
LOTS OF PEOPLE ARE WORKING ON
DELIVERS SR IN,NA OR MICRO RNA, I
WANTED TO TOUCH ON THAT BEFORE
RETURNING TO SOME OTHER WORK.
SO I WANT TO RUN THROUGH A
COUPLE OF THINGS TOWARDS THE
END, WHICH IS TRYING TO TAKE
THIS WHERE WE -- MOST OF WHAT
WE'VE DONE IN THE THERAPEUTIC
FIELD IS TO USE SRNA'S, RNAI TO
MAKE THE DRUGS WE HAVE NOW
BETTER OR BETTER UNDERSTAND
THEM.
SO THIS IS WHERE WE'RE
COMBINING EITHER -- USUALLY AN
RNAI SCREENING DOWN TO
ANALYSIS, WE TAKE SILENCING
GENES OVER A ERRED POOH OF TIME
ADDING THERAPEUTICND
INTERVENTION.
WE'LL TALK ABOUT TWO STORIES.
ONE IS LOOKING AT BACTERIALLY
DERIVED OR ENZYMEATIC DRIVE,
AND THE OTHER IS A DNA DAMAGING
AGENT.
SO THE REASON FOR DOING THIS IS
SORT OF OUTLINED HERE.
WE WANT TO USE THIS SO WE CAN
PERHAPS FIND BETTER WAYS OF
LOOKING AT COMPLEMENTARY
BALANCES, BY DOING SINNE SYNERGISTIC
TARGETING.
I'VE BEEN SHOCKED.
I DON'T HAVE EXACT NUMBERS BUT
I'M TOLD BY VERY, VERY RELIABLE
SOURCES THAT THE AMOUNT OF COST
FOR TRIALS TRYING TO FIGURE OUT
COMBINATIONINGS THAT WE SHOULD
COMBINATIONS THAT WE SHOULD PUT
TOGETHER IN INDIVIDUALS IS
CHUTELY PHENOMENAL.
NO KANGE CANCER PATIENT WILL GET ONE
DRUG.
THEY ARE GOING TO GET
MULTIPLES.
WE HAVE TO KNOW WHAT
COMBINATION AND AS WE GO INTO
THIS AREA OF PERSONALIZED
MEDICINE OR PRECISION MEDICINE,
WHATEVER YOU WANT TO CALL IT,
THAT'S GOING TO BECOME MORE
IMPORTANT BECAUSE WE SIS TANS
A MAJOR ISSUE.
WE'RE GOING TO KNOCKDOWN PUP
MULTIPLE PATHWAYS AND DO THEM
TOGETHER.
TESTING THAT IS DIFFICULT.
THERTHERE ARE WAYS PEOPLE ARE
TESTING MATRISES OF COMPOUNDS.
WE'RE TRYING TO TAKE OUT USER
DRUG, KNOCK OUT A GENE AND MAKE
THOSE BETTER AND GIVE US A
SENSE OF WHERE WE SHOULD START.
YOU CAN USE LOWER CON TRYINGS
CONCENTRATIONS OF TWO DRUGS
TOGETHER.
WE CAN OVERCOME DRUG RESISTANCE
OR OVERCOME SIDE EFFECTS AS A
BURDEN OF USING VERY HIGH
LEVELS OF DRUGS, PARTICULARLY
RELEVANT FOR EXAMPLE FOR WHAT
WE WERE DOING.
THE CLINICAL APPLICATIONS, THE
FIRST VIGNETTE IS A CASE OF
THAT.
SO THIS STUDY WAS PUBLISHED A
WHILE AGO.
I THOUGHT I COULD TELL THE
STORY BECAUSE I THINK IT'S
REALLY COOL, FRANKLY.
IT WAS ONE OF THE FIRST ONES I
DID WHEN I MOVED TO NCI.
IT'S GOING TO BE CLOSE TO MY
HEART FOR MANY YEARS I THINK.
THE WORK CAME OUT OF AN
OBSERVATION JOHN WEINSTEIN
MADE, I SAW A STRONG
CORRELATION IN ONE SUBSET OF
THE NCI-60 CELL LINES USED FOR
SCREENING AT THE NCI, A STRONG
CORRELATION BETWEEN THE
SENSITIVITY OF CELLS TO THIS
DRUG, AL, ANA, AND THE EXPRESSION
OF THIS GENE, SHOWN WITH
DIFFERENT PARAMETERS.
YOU CAN TELL HOW OLD THEY ARE.
THEY ARE EARLY CGH.
HE SAW AHE LOOKS AT, YOU SAW A
CORRELATION BETWEEN THIS DRUG
AND THIS GENE.
ONE IS AN F.D.A. APPROVED DRUG
FOR 30 YEARS FOR LEUKEMIA, AND
IT DEPLEATS THE BLOOD LEVELS OF
THIS NONESSENTIAL AMINO ACID BY
HYDROLIZING SERUM, EY AMMONIA AND
ASPARTIC ACID.
IT'S A CIVILE SIMPLE WAY OF DOING IT.
IT'S NO RECOMEBINANT.
IT'S A NONESSENTIAL AMINO ACID,
BECAUSE WE HAVE THE SYNTHETASE
BEGIN.
WHAT WE WANT TO DO WAS LOOK AT
IS THERE AN EFFECT OF HOW MUCH
YOU HAVE AND HOW YOU RESPOND TO
WE HINTED AND OVARIAN CANCER,
THAT'S WHERE WE'VE SEEN THE
ORIGINAL RELATIONSHIP AND COULD
DO THE EXPERIMENTS THERE MUCH
EASIER THAN IN A-L-L CELL
LINES.
WHAT WE'RE SEEING HERE IS
SILENCING OF THE MRNA AFTER 48
HOURS IN THREE CELL LINES THAT
HAVE DIFFERENT LEVELS.
THIS IS LEVELS ON THE SCALE,
THIS IS SENSITIVITY DOWN HERE.
WE SEE KNOCKDOWNS IN ALL 43
CELL LINES, A PERFECT BASIS FOR
YOUR EXPERIMENT.
WE CONFIRM THIS AT A PROTEIN
LEVEL.
THIS IS PLOTTING HERE THE
AMOUNT OF PROTEIN OVER TIME,
THIS IS A VERY NICE EXAMPLE OF
WHAT HAPPENS OVER TIME IN AN
SRNA BASED EXPERIMENT IN THE
LAB.
PROTEIN WILL DEPLETE OVER FIVE
DAYS AND THEN WILL RETURN AS
CELLS DIVIDE.
WE HAVE SUFFICIENT WINDOW HERE
TO DO OUR ANALYSIS.
SO AFTER 48 HOURS, YEAH, AFTER
48 HOURS WE ADDED OUR DRUG AND
LOOKED TO SEE WHAT HAPPENED 48
OR 72 HOURS LATER.
USING AN ASSAY.
WHAT WE SEE HERE IS IN THE
CELLS, A FIVE-FOLD, AND IN THE
CELL LINE THAT EXPRESSES
ALREADY VERY LOW LEVELS, IF WE
TAKE AWAY WHATEVER ELSE THERE
IS WE NOW GET A 500-FOLD
POTENTIATION OF THIS DRUG.
THIS IS QUITE, YOU KNOW, A
PHENOMENAL EFFECT.
SO WHAT WE NOW KNOW IS THAT
WHAT WE'RE ABLE TO DO IS
ACTUALLY WHEN IT IS ACTUALLY
ADDED TO CELLS, IT ACTUALLY
UP-REGULATES THE GENE, THOSE
CELLS THAT DON'T HAVE IT VERY
MUCH, BY SILENCING IT WE
ATTENUATE IT FURTHER.
WE GET A DOUBLE WHAMMY.
WE CANNOT JUST KNOCK IT DOWN
BUT SUPPRESS UP-REGULATION,
WORKING FOR THE LAST FEW YEARS
TO FIGURE OUT THAT MECHANISM.
THAT WILL HOPEFULLY BE WRAPPED
UP SOON.
THERE'S AN NCI PATENT ON THIS
THAT LINKS THE TWO.
THE LEVELS COULD BE USE AS A
BIOMARKER.
I'LL FINISH OFF TALKING ABOUT A
DNA DAMAGING AGENT THAT WORKS
BY INHIBITING THE ABILITY OF
CELLS TO RELEGATE AFTER
REPLICATION AND THROUGH THE
FORMATION OF DOUBLE-STRANDED
DNA BREAKS.
WHAT WE DID WAS A STUDY, AND
I'M GOING TO SKIP THROUGH THIS
ONE, BUT WE DID AN RNA --
SORRY, WE DID AN RNAI SCREEN TO
LOOK FOR GENES THAT
POTENTIALATED THE EFFECTS OF
THIS DRUG, THINGS THAT WE KNOW,
THE CPT AND ITS CLINICAL
ANALOGS USEDDED A HIG AT HIGH
DOSES.
WE FOUND ONE PROTEIN THAT WHEN
WE SILENCE, WE SIGNIFICANTLY
POTENTIATE ITSELF WE HAVE A
REPRESENTATION.
SCREENING DATA WITH NO CPT
ADDED, SO THERE'S NO EFFECT BUT
WHEN WE ADD CPT WE SEE A
SIGNIFICANT CHANGE IN THE
TRANSFORMED Z-SCORE.
WHAT IS THIS PROTEIN?
IT ENCODES TACK ONE, DOWNSTREAM
OF THE RECEPTOR, AND INTERACTS
THROUGH OTHER SIGNALS, THIS
SEEMS TO BE THE PREDOMINANT ONE
WE WERE INTERESTED IN AT THIS
POINT, AND WHAT IS KNOWN IS
IT'S PART OF THE SIGNAL.
CPT WHEN IT INDUCE INDUCES DAMAGE, IT
TRIGGERS AND SUPPRESSES THAT --
IT WILL ATTEMPT TO SUPPRESS
APOPTOSIS.
SO ACTUALLY CELLS THAT EXPRESS
CPT ARE DYING KNEW NONAPOPTOTIC
PATHWAYS.
WHAT WE'RE SPECULATING IS THAT
BY SILENCING TACK ONE WE'RE
ABLE TO STOP THIS RESPONSE AND
YOU GET YOUR -- YOU'RE DRIVING
MORE APOPTOSIT, NOT BLOCKING
IT.
WE WERE ABLE TO ACTUALLY SHOW
THAT WE CAN TARGET AN
PROTEIN OF TACK ONE AND GET THE
SAME EFFECT.
WE SEE THESE SENSITIVE CELLS AS
WELL.
THIS IS A FAIRLY BROAD SYSTEM
WITH DNA DAMAGE.
SO IN THE LAST COUPLE MINUTES,
I WANT TO MOVE TO HIGHLIGHT
VARIOUS FEATURES OF HOW WE USE
RNAI TO ADDRESS ISSUES PEOPLE
ARE USING, WE WANT TO ADDRESS
IN TRANSLATIONAL CANCER
BIOLOGY, WE WANT TO GET BIG.
WHAT I THOUGHT ABOUT IS DONE ON
A SMALL SCALE BUT WE CAN NOW GO
TO WHOLE GENOME TRIALS.
IIN LATE 2010 WE DEVELOPED A
COLLABORATIVE SCREENING
FACILITY NOW PART OF THE
NATIONAL CENTER FOR ADVANCING
TRANSLATIONAL SCIENCES, BASED
IN SHADY GROVE.
AND WHAT THEY ARE ABLE TO DO
BECAUSE THEY HAVE A ROBOTIC SET
UP, IS THEY CAN DO VERY LARGE
SCREENING PROJECTS.
THE JOKE THOUGH IS -- WELL,
IT'S NOT A JOKE.
IT TAKES SIX MONTHS TO BE ABLE
TO PROBABLY GET ONTO THIS
MACHINE, IT TAKES THE MACHINE A
WEEK TO DO THE WHOLE GENOME,
AND YOU'LL SPEND ABOUT TWO
YEARS TRYING TO FIGURE OUT WHAT
IT ALL MEANS.
SO THIS IS WORKING REALLY WELL
GETTING INTO IT AND GETTING OUT
OF IT IS THE HARD BIT.
THE TEAM HAS DEVELOPED
SOPHISTICATED SOFTWARE TO
ANALYZE THIS DATA.
THIS IS AN EXTENSION OF THE
SCREEN I JUST DESCRIBED, WHICH
IS WHY I WANTED TO DESCRIBE
THIS ONE AND NOT THE OTHER
PROJECT.
WHERE WE TOOK ONE OF OUR PILOT
STUDIES, WE DID THE SAME SCREEN
THAT WE HAD DONE ON A SMALL
SCALE, NOW DONE ON A MACHINE
LOOKING AT 100,000 DATA POINTS,
NO CPT AND EC-10 OF CPT AND
EC-30 OF CPT AND LOOK TO SEE
WHAT WE COULD GET OUT OF THIS.
WE GET THIS.
WE WERE REALLY PLEASED THAT
MAP-3K 7 WAS ONE OF OUR BIG
HITS.
WE GOT A BUNCH OF OTHER THINGS
FROM THE PATHWAY AS WELL, AS
WELL AS KNOWN DNA DAMAGE AND
OTHER GENES YOU WOULD EXPECT TO
BE THERE.
WE'RE FOLLOWING UP ON THAT.
LAST, I'M GOING TO GO TO A
REALLY BIG SCALE, THIS IS
TALKING ABOUT A PROJECT I'M
CURRENTLY INVOLVED IN, MASSIVE
COLLABORATION ARE PETEA PEDIATRIC
ONCOLOGY AND PATRICK AT
VANDERBILT WHO STARTED THIS,
LOOKING AT TRYING TO DO A WHOLE
GENOME SCREEN TO LOOK AT
ACTIVITY TO BETTER UNDERSTAND
MECHANISM BEHIND EWING SARCOMA,
HIGHLY AGGRESSIVE IN CHILDHOOD.
IT HAS A CLASSICAL GENETIC HAUL
HALLMARK, 85% OF CASES.
THIS IS THE INITIATING GENETIC
EVENT IN THESE TUMORS.
IT'S NECESSARY FOR MALIGNANT
TRANSFORMATION BUT MANY
DOWNSTREAM THINGS HAVE TO
OCCUR.
YOU END UP WITH A TRANSCRIPTION
FACTOR WHICH HAS A
TRANSITIVATING DOMAIN AND WE
HAVE A DISPLAY WITH A DNA
BINDING DOMAIN, ABERRANT,
ACTIVATING AND REPRESSING WHOLE
SETS OF GENES.
IF YOU INHIBIT THIS FUSION BY
SRNA OR ANTI-SENT, YOU CAN
REDUCE SUBPROLIFERATION IN
VITRO AND MICE, BUT IT'S A
TRANSCRIPTION FACTOR.
WE NEED A BETTER WAY TO
UNDERSTAND THE BIOLOGY TO
HOPEFULLY FIND BETTER THINGS TO
TARGET AND THAT COULD BE TURNED
INTO A MORE SUITABLE DRUG
TARGET.
WE'VE CONDUCTED A WHOLE GENOME
SCREEN USING A REPORTER ASSAY,
THAT WAS ORIGINALLY DEVELOPED
FOR DRUG SCREEN, AND THIS
CORRESPONDS WITH DAD DATA FROM
143,000 DATA POINTS AND WE CAN
FIGURE OUT FROM THIS WHAT IT
LOOKS LIKE, SPECIFICALLY
MODULATING THE EXPRESSION OF
THE EWS-1.
I'M GOING TO SHOW YOU THIS IS
LOOKING AT THE ENTIRE SCREENING
WHERE WE LOOK AT THE DIFFERENCE
BETWEEN THE TWO DIFFERENT
REPORTER ASSAYS AND GET A SENSE
OF WHAT IS SELECTIVELY ALTERING
THIS RESPONSE, AND WE
FORTUNATELY GOT THE SRNA'S TO
EWSR-1 AND FLY-1, THEY SELL OUT
NICELY ON THE SCREEN.
WE'VE STILL GOT 200 OTHER GENES
IN FOLLOW-UP.
THIS IS JUST SHOWING THE 43,
AND THIS IS PROVING A VERY HARD
PHASE.
TO WORK OUR WAY THROUGH
BUT MOIS MOST WILL PROVE TO BE ACT
SIT.
ACCURATE.
I'VE TAKEN YOU THROUGH A RUSHED
JOURNEY, ALL THE THINK THINGS PEOPLE
ARE USING RNAI FOR IN A
TRANSLATIONAL SETTING, BASIC
RESEARCH MECHANISMS THAT PEOPLE
ARE STUDYING, AND OTHERS WILL
BE TALKING ABOUT IN A MOMENT.
AS YOU CAN IMAGINE, THIS IS --
I'VE HAD HUGE NUMBER OF
COLLABORATORS AT CCR AND I
HIGHLIGHTED, I HOPE, MOST OF
THE PEOPLE WE'VE WORKED WITH
BUT I'M GOING TO HIGHLIGHT FROM
MY LAB SCOTT MARTIN AND JASON
PICK, I DISCUSSED A LOT OF WORK
THEY DID.
THANK YOU.
[APPLAUSE]
>> QUESTIONS?
[INAUDIBLE]
>> SO WHAT THAT HAD TO BE BUILD
INTO THE ANALYSIS, GENE BUEHLER
WAS RECRUITED FROM MERCK, HE'S
ALGORITHMICCOUPLE AL GO RATE
APPROACHES.
WE PUT THAT IN THE ANALYSIS UP
FRONT.
I WOULD SAY ON THE WHOLE,
MOST -- THE PROBLEM IS THE BAND
WIDTH OF THE NUMBERS OF HITS
THAT WE GET IS SUCH THAT I
REALLY CAN'T ANSWER YOUR
QUESTION BECAUSE WHAT WE -- WE
CAN'T -- WHEN WE GO BACK TO CCR
INVESTIGATORS, ALL ACROSS THE
WHOLE NIH, WE GO BACK WITH THE
LIST OF GENES TO DO, PEOPLE
PICK TWO OR THREE.
SO I ACTUALLY CAN'T ANSWER YOUR
QUESTION.
I WOULD SAY THAT IT'S PRETTY
HIGH, A SUCCESS RATE.
WE DO SECONDARY SCREENS WITH
SRNA'S, ALL THOSE, BUT THE
BOTTOM LINE, IT'S NOT JUST THE
BAND WIDTH TO DO EVERYTHING.
WE DON'T REALLY KNOW.
>> OUR NEXT SPEAKER IS ENRIQUE
ZADARE, Ph.D.
CURRENTLY HE'S WITH THE CANCER
GENETICS PROGRAM AT NIH
FREDERICK, HE'S IN THE TUMOR
ANGIO GENESIS SECTION, ANG,O
>> THANK YOU, TERRY.
I'M OF THE TUMOR ANGIOGENESIS
SECTION.
I'M GOING TO BE TALKING
POSTALLY ABOUT TUMOR
ANGIOGENESIS FOR THE NEXT
HOPEFULLY 45 MINUTES.
THANK YOU FOR BEING HERE.
ENJOYERY COLD OUTSIDE SO ENJOY
THE WARMTH OF THE ROOM.
I WOULD LIKE TO BEGIN MY
PRESENTATION BY DEFINING WHAT
ANGIOGENESIS IS.
I USE A SLIDE THAT COMPARES TWO
PROCESSES THAT ARE RELATED BUT
THAT ARE DIFFERENT.
ONE IS ANGIOGENESIS, THE OTHER
IS VASCULARGENESIS,
ANGIOGENESIS IS FORMATION OF
VASCULATURE FROM PREEXISTING
VESSELS.
I HAVE A LITTLE SOMETHING HERE
WHICH DEMONSTRATES THAT.
THIS IS FROM A PUBLICATION OF
STUDIES DONE.
YOU CAN SEE A VESSEL AND HOW
ADDITIONAL VESSELS ACTUALLY
GROW DURING EMBRYO DEVELOPMENT
OF THE FISH.
THIS IS WHAT WE CALL
ANGIOGENESIS.
A DIFFERENT PROCESS IS VASCULAR
GENESIS, THE FORMATION OF
VASCULATURE, DIFFERENTIATED
ANGIOBLASTS, BOTH PROCESSES ARE
IMPORTANT IN EMBRYO
DEVELOPMENT.
ANGIOGENESIS OCCURS IN ADULTING
ORGANISMS.
REPORTS OF VASCULAR GENESIS
OCCUR IN ADULTS, NOT AS PROM
SAYS AS ANGIOGENESIS.
FORMATION OF VASCULATURE FROM
PREEXISTING VESSELS.
ANGIOGENESIS, I LIKE TO TALK
ABOUT PHYSIOLOGIC AND PATH O.J.
PATHOLOGIC ANGIOGENESIS.
ANGIOGENESIS IS A VERY
IMPORTANT PROCESS,
PHYSIOLOGICALLY SPEAKING, NOT
ONLY DURING EMBRYO DEVELOPMENT
BUT THROUGH A NUMBER OF
DIFFERENT PROCESSES, SOME OF
WHICH ARE DETAILED HERE, YOU
KNOW, FEMALE REPRODUCTION
SYSTEM, HEALING IS IMPORTANT
FOR WOUND HEALING, WOUND
HEALING IS
ANGIOGENESIS-DEPENDENT PROCESS.
WE HAVE PATHOLOGIC ANGIO
CAROLINANGIOGENESIS.
IT'S A TIGHTLY REGULATED
PROCESS.
WHEN THIS PROCESS IS
DEREGULATED, IT'S NORMAL
ASSOCIATED WITH A DISEASED
STATE.
AND SO WE CAN HAVE BOTH THAT
BECOME PATHOLOGICAL SITUATIONS.
SO IN SOME CASES, WE WANT TO
STIMULATE ANGIOGENESIS.
WE HAVE A HEART ATTACK, AND IN
THE RECOVERY OF THE HEART
ATTACK, WOUND HEALING FOR
INSTANCE, WE WANT TO INDUCE
ANGIOGENESIS, ANGELS THE THEY ARE
DEPENDENT.
IN SOME CASES THE THERAPEUTIC
GOAL IS TO BLOCK ANGIOGENESIS.
THERE IS A NUMBER OF THESE
DISEASES, MOST NOTABLY TUMOR
GROWTH M METASTASIS WHICH WE SEE
LATER IN 1971 ACTUALLY A
HYPOTHESIS WAS PUT FORWARD THAT
WE WOULD BE ABLE TO RETARD AND
DELAY TUMOR GROWTH.
THIS IS A VERY SIMPLIFIED STORY
OF TUMOR PROGRESSION, OKAY?
HERE WE HAVE OUR HAPPY NORMAL
CELLS THAT FOR REASONS THAT
NOWE NOT GOING TO DISCUSS
BECOMES A TUMOR CELL.
THIS TUMOR CELL IS TUMORGENIC
AND IN THE RIGHT CONTEXT COULD
BECOME A CLINICALLY RELEVANT
TUMOR BUT NEEDS TO GO THROUGH A
SERIES OF STEPS.
THE CELL DIVIDES AND REACHES A
CERTAIN SIZE.
THAT SIZE OF THE TUMOR IS
DORMANT TUMORS.
I LIKE TO CITE THIS SEMINAL
PUBLICATION FROM BLACK AND
WELCH IN 1993, THE FOCUS OF THE
PUBLICATION WAS NOW, WHAT I'M
GOING TO DESCRIBE NOW, IT'S
MENTION THE IN THE PUBLICATION,
THE TWO DOCTORS LOOKING AT
TRAUMA PATIENTS FOR SMALL
LESIONS, YOU KNOW, MALIGNANT
LESIONS, AND WHAT THEY FOUND
WAS I THINK SURPRISING, FOR
INSTANCE, AGE 40 TO 50, 39% OF
WOMEN PRESENT THESE LESIONS IN
THE BREAST.
AGE 60 TO 70, 46% OF MALES
PRESENT THESE LESIONS IN THE
PROSTATE.
HOWEVER, ONLY ABOUT 1% OF THESE
LESIONS ARE EVER DIAGNOSED.
MEANING 1% OF THESE PEOPLE GO
TO THE DOCTOR SAYING I HAVE A
CANCER, OKAY?
ALL THIS 38% REMAINING, THOSE
CANCERS, THEY NEVER DEVELOP.
THE CANCERS ARE THERE BUT THEY
NEVER EVOLVE INTO A CLINICALLY
RELEVANT ENTITY.
WHICH IS A GOOD THING.
SO THESE NUMBERS, THERE'S NOW A
NUMBER OF PUBLICATIONS THAT
SUPPORTED THE SAME OBSERVATION,
AND THESE NUMBERS ARE ACTUALLY
GOING DOWN AND THESE NUMBERS
ARE GOING UP, MEANING THIS IS A
NORMAL THING.
WE ALL HAVE THESE THINGS WHEN
WE REACH A CERTAIN AGE.
SO THERE IS THEN DATA TO
SUPPORT THIS DORMANT TO CANCER,
FULLY TUMORGENIC CELLS THAT
COULD DEVELOP INTO FUMER TUMORS BUT
DON'T.
THERE'S INTEREST IN STUDIES
THAT THEY TAKE LESIONS AND PUT
THEM IN A DIFFERENT ENVIRONMENT
AND THE LESIONS ARE ABLE TO
CLINICALLY RELEVANT
TUMORS.
SO WHAT THESE NEED IN ORDER TO
GROW INTO A TUMOR, THERE'S A
NUMBER OF CHARACTERISTICS WE
CAN DETERMINE THE ENVIRONMENTS
IN THE LESIONS THAT ALLOW THE
CELLS TO BE ABLE TO RECRUIT
VESSELS.
TO INDUCE ANGIOGENESIS, THERE
ARE A NUMBER OF DIFFERENT
STIMULUS THAT CAN OCCUR IN THE
TUMOR-LIKE ENVIRONMENT.
ONE IS HIG HYPOXIA.
WHEN CELLS REACH A CERTAIN SIZE
BECAUSE THEY ARE NOT
VASCULARIZED BECOME HIG HYPOCIX, A
STIMULUS FOR ANGIOGENESIS.
THIS IS HOW ANGIOGENESIS OCCURS
AND IT'S AT THAT POINT ONCE THE
TUMOR GETS VAS VASCULARIZED IT CAN
GROW AND REACH A CLINICALLY
RELEVANT SIZE AND METASTASIZE.
I WANT TO EMPHASIZE, I
MENTIONED BEFORE, I JUST
MENTIONED TUMOR MICRO
ENVIRONMENT.
THIS IS NOT ONLY A GAME BETWEEN
ENDOTHELIALAND THE EN ENDOUGH
CELLS, THERE'S PARTICIPATION IN
GENESIS PROCESS THATUATION
I'LL MENTION MORE IN A MINUTE.
AS I SAID BEFORE, ANGIOGENESIS
IS TIGHTLY REGULATED.
UNDER NORMAL CONDITIONS THERE
ARE STIMULATORS OF ANGIO
GENESIS AND INHIBITORS THAT ARE
BALANCED.
AND ANGIO GENESIS IS
CONTROLLED.
FOR INSTANCE, MY 4-YEAR-OLD
DAUGHTER HAS A CONSTANT WOUND
IN HER KNEE.
THANK YOU FOR ANGIOGENESIS,
THIS REGULATED PROCESS AL THOSE
THESE KNEES TO HEAL CONSTANTLY
DURING THE SUMMER.
PERFECT BALANCE BETWEEN
STIMULATORS AND INHIBITORS OF
ANGIO GENESIS.
THERE'S A BALANCE AND THE TUMOR
STAYS.
IF THERE IS AN IMBALANCE IN
THESE FACTORS, EITHER TOO MUCH
STIMULATORS OR TW TOO FEW
INHIBITORS, ANGIO GENESIS
OCCURS AND THE CELLS CAN GROW
TO CLINICALLY RELEVANT TUMORS.
I LIKE THIS SLIDE.
IT ALSO MENTIONS OLDER PLAYERS
WITHIN THE TUMOR MICRO
ENVIRONMENT, INFLAMMATORY CELLS
RECRUITED TO THE TUMOR CITE AND
PARTICIPATE IN THE TUMOR ANGIO
GENESIS PROCESS, ENDOTELI,
AEROBICSL, FIBROBLASTS, T-CELLS
WHICH ARE IMPORTANT, NOT JUST
TUMOR CELLS OR THE ENDOTHELIAL
CELLS.
THIS IS A REPRESENTATION OF THE
IDEA THAT WE STUDIED A NUMBER
OF YEARS AGO WHERE WE LOOKED AT
THE INFLUENCE OF CERTAIN
INFLAMMATORY CELLS IN THE TUMOR
MICRO ENVIRONMENT, THIS IS
SUMMARIZED IN THIS SLIDE WHERE
WE HAVE TUMOR CELLS WHICH
BECAUSE OF THE DISTANCE FROM
THESE VESSELS OVER HERE, THEY
ARE IN A HIG HYPOXIC ENVIRONMENT
WHICH ALLOWS FACTORS WHICH ARE
ANGIO GENESIS STIMULATORS,
CREATING A GRADIENT TOWARDS
THIS PREEXISTING VESSEL, AND IN
THESE MIGRATION OF INFLAMMATORY
CELLS TO THE TUMOR MICRO
ENVIRONMENT WHERE WEE THEY CAN
RELEASE ADDITIONAL FACTORS, AND
THAT IS WHEN THESE ANGIO
GENESIS PROCESS IS ENHANCED BY
TUMOR ELSE WHY AND AL CELLS AND ALL THE C ELLS
IN THE TUMOR MIK MICRO ENVIRONMENT.
THE VESSELS THAT REACH THE
TUMOR, THE TUMOR VESSELS
BASICALLY, ARE DIFFERENT THAN
THE NORMAL VESSELS.
I WANTED TO SHOW THIS.
IT ILLUSTRATES VERY WELL HOW
THE VASCULATURE INVADES THE
TUMOR.
THIS IS A CASE OF A
14-YEAR-OLD, WITH A KIDNEY, THE
RIBS, THE CYTOMA IS HERE.
YOU CAN LOOK AT THE VESSELS.
YOU CAN SEE HOW ALL THESE
VESSELS ARE INVADING.
ALL THIS IS VASCULATURE THAT
WAS NOT THERE BEFORE.
IT'S GROWN TOWARDS THE TUMOR,
INVADING THE TUMOR WHICH IN
THIS CASE IS VERY INVOLVED.
AND ONE OF THE THINGS THAT YOU
ALREADY SEE HERE IS NOT ONLY
THAT THE TUMOR HAS A LOT OF
VESSELS, BUT THESE VESSELS LOOK
DIFFERENT THAN THE NORMAL
VESSELS LIKE THESE VESSELS OVER
HERE.
ONE OF THE THINGS THAT YOU CAN
OBSERVE ALREADY IS THAT THEY
ARE TORTUOUS, THEY ARE VERY
TORTUOUS VESSELS.
THIS IS SOMETHING THAT IS BEING
STUDIED, AND INDEED NORMAL
VASCULATURE AND VESSELS HAVE A
NICE COVERAGE, NOT SURE YOU'RE
FAMILIAR WITH THIS COVER, THE
VESSELS COVER THE ENDO THELIAL
CELLS AND STABILIZE VESSELS.
THEY HAVE REDUCED EXPRESSION,
THEY ARE TIGHT.
NORMAL VESSELS ARE NOT LEAKING,
NOT LOSING BLOOD.
THEY ARE TIGHT VESSELS.
AND THIS IS INCONTRAST TO TUMOR
VESSELS WHICH ARE VERY
TORTUOUS, YOU KNOW, THE
MORPHOLOGY IS DIFFERENT.
THEY HAVE LOST COVERAGE, AND
THEREFORE THEY ARE VERY LEAKY.
TUMOR CELLS ARE VERY LEAKY.
INTERESTINGLY, THIS IS
SOMETHING THAT I'LL TALK ABOUT
LATER, THEY ALSO EXPRESS
MARKERS THAT ARE NOT EXPRESSED
IN THE NORMAL VASCULATURE.
AND THIS IS WHAT ARE CALLED
TUMOR ENDOTHELIAL MARKERS, THE
MAIN SUBJECT OF STUDY IN OUR
GROUP.
YOU CAN ALREADY UNDERSTAND THAT
IF THESE VESSELS, THE TUMOR
ENDOTHELIAL MARKERS EXPRESS, WE
HAVE A MEANS AND A WAY TO
DEVELOP THERAPEUTICS THAT ARE
ABLE TO FAR TARGET THOSE VESSELS.
AN CA EXAMPLE, NORMAL VESSELS AND
TUMOR VESSELS, VERY EASY TO
DIFFERENTIATE JUST BY THE
MORPHOLOGY OF THE VESSELS.
ANOTHER EXAMPLE USING THE
ELECTRO MICROSCOPE, STRIKING
DIFFERENCES BETWEEN THE NORMAL
VESSELS OVER HERE AND THE TUMOR
VESSELS, AND JUST
REPRESENTATION OF HOW LEAKY THE
VESSELS ARE, SOME OF THESE
TUMORS ARE FULL WITH BLOOD,
THEY FORM THESE BLOOD LAKES,
IT'S JUST BECAUSE THE VESSELS
WITH EXTREMELY LEAKY AND
CONTINUOUSLY LEAK BLOOD.
SO BASED ON THIS, AS I
MENTIONED BEFORE, JUDAH FAULK
MAN IN 1971 IN THIS PAPER, IN
ENGLAND JOURNAL OF MEDICINE,
PROPOSED THAT TUMOR
GROWTH IS DEPENTEN DEPENDENT ON ANGIO
GENESIS AND IF WE CAN' WE CAN TARGET
THESE WE'LL BE ABLE TO DELAY
TUMOR GROWTH.
IT'S BEEN LIKE 42 YEARS UNTIL
TODAY.
4'RE GOING TO KEEP THESE HAD 2
YEARS AND TELL YOU THERE'S BEEN
A LOT OF EFFORT TO DEVELOP
ANGIO THERAPEUTICS, AND ONE OF
THE MOST PROMISING FIELDS IN
ONCOLOGY, SO CAN WE CURE CANCER
WITH ANGIO GENETIC THERAPY?
I GUESS THE ANSWER IS YES, THIS
WAS DEMONSTRATED IN AS EARLY AS
1997 IN JUDAH FAULKMAN'S LAB.
THEY SHOW WITH THIS PARTICULAR
ANGIO GENIC COMPOUND, THEY GROW
TUMORS AND WHEN THEY STOP
ADMINISTRATION THE ANTIANGIO
GENIC COMPOUND THE TUMOR
GROWS
, THEYTHEY WERE
SHALL ABLE TO SHOW THESE IN
TUMOR TYPES.
IF YOU BLOCK ANGIO GENESIS,
YOU'RE ABLE TO BLOCK TUMOR
GROWTH AND REGRESS THE TUMOR.
NOW, THESE ARE MODELS, THESE
ARE MICEMEN.
WHEN PEOPLE ASKED JUDAH CAN YOU
CURE CANCER, HE SAID SURE, IN
MICE.
A COMPLETELY DIFFERENT STORY IN
HUMANSMENT WHE.
THIS IS ONE OF THE THINGS I
WANT TO TALK ABOUT, I HAVE A
NUMBER OF SLIDES THAT WILL
ILLUSTRATE THE PROBLEMS OR
CHALLENGES OF TRANSLATING THIS
PARTICULAR THERAPEUTIC INTO THE
CLINIC.
THIS IS ONE OF THE THERAPEUTICS
THAT FAILED IN THE CLINIC, EVEN
THOUGH RESULTS IN MODELS WERE
SO STRIKING.
YES?
[INAUDIBLE QUESTION]
>> WELL, YOU HAD TO READ THE
PAPER.
THIS IS HOW THEY HAVE DONE THE
STUDIES, THIS ADMINISTRATION OF
THE DRUG.
OKAY.
AS I MENTIONED BEFORE, THERE'S
BEEN A TREMENDOUS EFFORT TO
DEVELOP THERAPEUTICS, TARGETING
ANGIO GENIC COMPOUNDS, AND THIS
IS A SUMMARY, TEMPORAL LINE OF
ALL THOSE ANTIANGIO GENIC
THERAPEUTICS THAT HAVE BEEN
TARGETED.
IT'S NOT COMPREHENSIVE.
THERE ARE MORE THAN THE ONES
SHOWN THEIRMEN.
THESE COVERED A SPECTRUM OF
DIFFERENT MOLECULES IN THE
CELL, AN DODGE NEWS
INHIBITORS, THAT CAN ARE TAB
TARGETED.
PHARMA IS INTERESTED AND
INVESTS HEAVILY IN ANGIO GENIC
RESEARCH, GOOD FOR THE PATIENT
EVENTUALLY.
SO THERE'S A NUMBER OF PATHWAYS
IN THE PREVIOUS SLIDE THAT CAN
BE TARGETED IN ORDER TO TRY TO
GET ANGIOGENESIS.
I HAVEN'T MENTIONED IT'S QUITE
COMPLEX, IT CAN BE SEEN AS AN
ORDERLY SERIES OF STEPS THAT
ARE REQUIRED BASICALLY FROM THE
MOMENT WHERE ENDOTHELILA CELLS
GET ACTIVATED AND NEED TO MOVE
AROUND AND ALIGN THEMSELVES,
INTERACT WITH EACH OTHER, SO
IT'S QUITE A COMPLEX PROCESS.
FROM THE PERSPECTIVE OF PEOPLE
LIKE ME THAT TRY TO BLOCK THIS
PROCESS, ALL OF THOSE STEPS
REPRESENT AN OPPORTUNITY TO
BLOCK ANGIOGENESIS, REQUIRED
AND THEREFORE ALL OF THEM, YOU
KNOW, ARE AN OPPORTUNITY TO
BLOCK ANGIOGENESIS.
THERE'S A LOT OF TARGETS THAT
CAN BE USED.
THIS IS A SUMMARY OF F.D.A.
APPROVED ANTIANGIOGENIC
THERAPEUTICS, IT'S NOT UP TO
DATE BUT IT'S A GOOD SUMMARY.
ONE THING I WANT TO HIGHLIGHT,
THERE'S A LOT OF PATHWAYS THAT
CAN BE TARGETED, MOST OF THE
EFFORT, YOU KNOW, TO TODAY,
HAVE BEEN HE CAN TO USE FOCUSED ON VASCUL AR
ENDOTHELIAL GROWTH FACTORS,
PRODUCED BY TUMOR CELLS AND
OTHER CELLS AS WELL.
AND THERE ARE A LOT OF
DIFFERENT OF THESE COMPOUNDS
THAT TARGET VASCULAR ENDOTHELIL
GROWTH FACTOR IN DIRECT OR
INDIRECT MANNER.
HERIS THIS THERAPEUTIC, YOU
PROBABLY HEARD OF AN ANTIBODY
THAT TARGETS VASCULAR
ENDOTHELIAL GROWTH FACTORS, A
MULTI-BILLION DOLLAR DRUG, AND
WAS BASICALLY THERAPEUTIC TO
PROVE ANTIANGIOGENIC THERAPY
COULD BE USED IN THE CLINIC.
A LOT OF EFFORTS CURRENTLY --
WHICH ARE CURRENTLY UNDERGOING
ARE FOCUSED ON VASCULAR
ENDOTHELIL GROWTH FACTORS.
I DID A SEARCH ON PubMed.
ANGIOGENESIS PAPERS KEEP
INCREASING.
THIS IS THE LAST ONE FOR LAST
YEAR, 2012, WE'RE REACHING 1400
A YEAR.
FROM ALL THOSE, 50% OF THOSE
PAPERS ARE DEDICATED TO THE
VGF.
IF YOU PUT THE OTHER ANGIO
GENESIS COMPOUNDS COMBINED,ER
THEY ARE IN 10% OF THE PAPERS.
THERE'S A LACK OF DIVERSITY, A
LOT OF PEOPLE FOCUSED ON GROWTH
FACTORS AND A LOT OF
OPPORTUNITIES, THERAPEUTIC
OPPORTUNITIES THAT NEED TO BE
EXPLORED.
WHICH WOULD BE DOWN HERE ON
THIS GRAPH.
THAT'S THE THERAPY THAT WORKS,
THE ANSWER IS YES, AT LEAST IN
MODELS AND ALSO IN THE CLINIC,
AS I SHOWED BEFORE AND AFTER.
SO THIS IS JUST AN EXAMPLE,
IT'S IN COLON CANCERS.
PRIOR TREATMENT WITH
THERAPEUTICS, AND 4 48 HOURS.
YOU CAN SEE THIS ANTIBODY
REQUIRES 48 HOURS, 98% OF THE
VASCULATURE IS GONE.
YES, IT DOES WORK IN THE
MODELS, WE USE IT ALL THE TIME,
IT CONSISTENTLY WORKS WELL TO
BLOCK TUMOR GROWTH.
SOME EXAMPLES OF THOSE
THERAPEUTICS THAT I SHOWED
BEFORE THERE ARE OTHER
THERAPEUTICS OR DRUGS THAT
TARGET GROWTH FACTOR.
WHAT HAPPENS IN THE CLINIC, I
ALREADY WAS TELLING YOU BEFORE
THERE ARE NUMEROUS CHALLENGES
TO TRANSLATE WHAT WE SEE IN THE
MODELS INTO THE CLINIC.
THESE ARE SOME OF THE
SUCCESSFUL PHASE 3 CLINICAL
TRIALS WITH SOME DRUGS THAT I
SHOWED YOU IN THE PREVIOUS
SLIDE.
THERE ARE A LOT DONE, EITHER
ALONE OR IN COMBINATION WITH
ALL KINDS MUCH THERAPEUTICS,
AND AS YOU CAN SEE THERE IS A
NUMBER OF CLINICAL TRIALS WHERE
THERE IS A BENEFIT IN SURVIVAL
BUT NOT OVERALL SURVIVAL.
THE MESSAGE FOR THIS SLIDE,
YES, IT HELPS SOMETIMES IN BUT
IN A LOT OF CASES WE DON'T SEE
OVERALL SURVIVAL.
THE OTHER ISSUE THAT IS WORTH
MENTIONING HERE IS THAT THE
BENEFITS ACHIEVED SO FAR IS
OFTEN MEASURED IN WEEKS, OR IN
MONTHS, TWO MONTHS, THREE
MONTHS, FOR THE PATIENT.
SO IT IS A SUCCESS.
AVASTIN WAS THE FIRST F.D.A.
APPROVED THERAPEUTIC THAT DID
CLINIC IT'SIN THE CLINTON IT'S
EFFECTIVE AND CAN PURE
PATIENTS.
THE AVERAGE, JEER AL OVERALL IT'S
MEASURED IN WEEKS OR MONTHS.
IN MY OPINION, I THINK IN THE
OPINION OF A LOT OF PEOPLE IN
THE ANGIO GENESIS FIELD, IT
DIDN'T LIVE UP TO THE
EXPECTATION BASED ON CLINICAL
STUDIES.
THERE IS ALSO A LOT OF
SUCCESSFUL TRIALS.
AGAIN, THIS AND OTHER
ANGIOGENIC COMPOUNDS, ALONE OR
WITH OTHER THERAPEUTICS,
THERE'S A LOT OF CLINICAL
TRIALS IN WHICH WE DON'T SEE
PROGRESSIVE FREE SURVIVAL OR
OVERALL
SURVIVAL.
SO ON TOP OF THIS THERE ARE
PRE-CLINICAL INDICATIONS, TO
PAPERS PUBLISHED IN THE SAME
NUMBER OF CELLS, IN 2009,
SUGGESTING TREATMENT OR CHRONIC
TREATMENT WITH THERAPEUTICS
THAT WILL TARGET VGF PATHWAYS
COULD HAVE DELETERIOUS EFFECT
IN TERMS OF POETENTIATING,
WHICH IS COUNTER INTUITIVE, MA
METASTASIS, ANGIOGENESIS, AND I
ALREADY SAID METASTASIS.
SO IT'S OBVIOUSLY CONCERNING,
AND THERE ARE OTHER MUST
PUBLICATIONS THAT CORROBORATED
THIS DATA, WHETHER OR NOT THIS
IS SEEN IN THE CLINIC I THINK
IS AS ARGUABLE AND A MATTER OF
DISCUSSION AT THE MOMENT.
THERE'S A BUNCH OF PUBLICATIONS
THAT FOCUS ON THE FACT THAT
WHEN YOU STOP ANTIANGIO GENIC
THERAPY, TUMORS GROW LIKE
CRAZY.
CRAZY.
YOU CAN STOP DURING
ADMINISTRATION, ONCE YOU STOP
THAT THERAPY, THE TUMORS COME
BACK EVEN MUCH MORE
AGGRESSIVELY THAN THEY WERE
INITIALLY.
THERE ARE OTHER PUBLICATIONS
LIKE THIS ONE.
THIS ONE TALKS ABOUT RELATED
MORTALITY WITH AVASTIN.
SO AVASTIN SEEMS TO TO HAVE
UNINTENDED CONSEQUENCES IN THE
CLINIC.
GENICERAL, ALL ANTIANGIO
THERAPEUTICS ARE HYPER TENSIVE,
A PROBLEM WITH ALMOST ALL
ANTIANGIO GENIC THERAPEUTICS,
YOU FIND THIS OVER AND OVER
AGAIN IN LIT LITERATURE.
MOST OF THIS IS I MANAGEABLE,
SOME ARE NOT MANAGEABLE.
YOU MAY HAVE LIKE ABOUT FOUR
TIMES THE CHANCES OF DEVELOPING
A PULMONARY HEMORRHAGE, ABOUT
FOUR TIMES THE CHANCES OF
DEVELOPING GASTROINTESTINAL
HEMORRHAGE, AND OTHER
COMPLICATIONS WHICH ARE
OBVIOUSLY NOT MANAGEABLE.
SO THERE ARE PROBLEMS.
THERE'S A LOT OF WORK BEING
DONE TO UNDERSTAND, IS THIS A
PROBLEM, EXCLUSIVELY WITH THIS,
OR OTHER TYPES OF ANTIANGIO
GENIC THERAPEUTICS AS WELL,
APPLIED TO THE VAST MAJORITY OF
PATIENTS?
I'D LIKE TO SUMMARIZE BASICALLY
THE CHALLENGES WITH ANTIANGIO
GENIC THERAPY, AN OBVIOUS GAP
BETWEEN THE CLINIC AND MODEST
CLINICAL BENEFITS.
ANGIO GENESIS IS A FUNDAMENTAL
PROCESS BOTH FOR HOMEOSTASIS
AND EMBRYO DEVELOPMENT, AND
THEREFORE IS AN EXTREMELY
REDUNDANT PROCESS.
IF YOU BLOCK ONE PATHWAY,
THERE'S ANOTHER PATHWAY THAT'S
GOING TO TAKE OTHER.
IF YOU BLOCK THOSE TWO, ANOTHER
PATHWAY WILL TAKE OVER.
THIS REPRESENTS A TREMENDOUS
CHALLENGE IN TERMS OF
DEVELOPMENT.
WE'VE ALREADY TALKED ABOUT
ADVERSE SIDE EFFECTS.
THE MECHANISM OF ACTION IS
INCOMPLETELY UNDERSTOOD.
IN MY OPINION IT'S ONE OF THE
MOST IMPORTANT THINGS.
WE UNDERSTAND A LOT OF WHAT
HAPPENS IN PRE-CLINICAL MODELS,
WE GO TOSYSTEMS, WHEN WHICH GO
PATIENTS WE DON'T UNDERSTAND
WHY THIS IS ABLE TO CURE
PATIENTS SOMETIMES.
WE DON'T UNDERSTAND IT VERY
WELL.
SO THIS IS ONE OF THE, IN MY
OPINION, ONE OF THE MAJOR
PROBLEMS RIGHT NOW.
WHICH IS RELATED TO THE LAST
ONE THAT I -- RIGHT HERE, THE
LACK OF BIOMARKERS.
THERE ARE NOT GOOD ANGIO BEN
JUSGENESIS BIOMARKERS.
WE DON'T HAVE BIO BIOMARKERS FOR
STRATIFIED PATIENTS.
WE DON'T KNOW WHICH PATIENTS
WILL GET CURED.
WE NEED A BETTER UNDERSTAND HOW
THE ANTIANGIO GENIC TREATMENT
WORKS IN PATIENTS.
WE NEED BIOMARKERS TO HELP US
STRATI IF,FY PATIENTS.
AND ONE PROBLEM IS THAT
VASCULAR ENDOTHELILA GROWTH IS
OUR BODIES.EOSTASIS INATS IS IN
IT'S NOT UNTHINKABLE THAT
TREATMENT WITH THE DRUG IS
GOING TO AFFECT THE PATIENT,
SOMETIMES IN DRAMATIC WAYS.
SO WHAT WE NEED IS WE NEED
TARGETS, NEW TARGETS, THAT ARE
SPECIFIC FOR THE TUMOR
VASCULATURE AS OPPOSED TO
PHYSIOLOGIC PROCESSES.
SO DURING THE YEARS I'VE BEEN
INVOLVED IN A NUMBER OF EFFORTS
TO TRY TO TACKLE THESE NEEDS,
I'LL SHOW YOU SOME OF THOSE.
FIRST, OLDER TARGETS, ANGIO
GENESIS TARGETS, APART FROM
SOME OF THE CLASSIC ANGIO
COMPOUNDS.
THIS IS A STUDY THAT IS A BIT
OLD NOW, BUT I LIKE IT BECAUSE
WHAT IT SHOWS IS THAT -- THIS
PART IS SHOWING THE ESTABLISHED
MICRO VASCULATURE FUNCTION,
ESTABLISHED MICROSHED PIKE ROW
VASCULATURE FUNCTION, AND
OTHERS WE NEVER HEAR OF, NOT
THE CLASSIC GENES FOR MICRO
VASCULAR FUNCTION.
THERE ARE MORE OF THESE GENES
THAN THESE OVER HERE.
SO THE ANSWER IS YES, THERE'S
PLENTY OF TARGETS.
THERE'S PLENTY OF GENES RELATED
TO -- THER THEY ARE IMPORTANT THAT
WE'RE NOT LOOKING AT.
THE QUESTION IS HOW DO YOU FIND
WHICH ARE THESE GENES AND WHICH
ARE IMPORTANT, WHICH OF THESE
GENES ARE MORE INTERESTING FROM
A THERAPEUTIC PERSPECTIVE?
SO ONE OF THE, IN MY OPINION,
CRUCIAL MOMENTS FOR ANGIO
THERAPEUTIC A PER THROUGH
PERSPECTIVE OCCURRED IN 2000
WHEN THE P.I. IN OUR GROUP
PUBLISHED A PAPER, A HALLMARK
IN THERAPEUTIC ANGIO GENERAL
HE LOOKED AT TUMOR VASCULATURE
COMPARED TO NORMAL VASCULATURE
TO UNDERSTAND WHAT GENES ARE,
YOU KNOW, DIFFERENTIALLY
REGULATED IN TUMOR MICRO
VASCULATURE.
HE TOOK AN INTERESTING
APPROACH.
HE ACTUALLY DID SOME DIFFERENT
APPROACHES.
THIS IS ONE OF THEM.
HE USED A NORMAL VASCULATURE IN
NORMAL RESTING VAST L VASCULATURE,
REGENERATED LEVER AS AN EXAMPLE
OF PHYSIOLOGIC AND ANGIO
GENESIS, YOU CUT A LIVER, IT
GROSS BACK, THAT IS DEPENDENT
ON ANGIO
GENESIS.
AND I HAD TUMOR BEARING LIVES.
LIVERS.
HE DID USE ANALYSIS TO COMPARE
BETWEEN THE VASCULATURE AND HE
CAME UP WITH A NUMBER OF WHAT
HE CALLED TUMOR ENDOTHELIAL
MARKERS, MARKERS OVEREXPRESSED
IN TUMOR VASCULATURE, WHEN
COMPARED TO NORMAL, AND HE DID
A LOT OF WORK NOT ONLY TO
VALIDATE THAT BUT ALSO TO
UNDERSTAND WHICH ONES WERE MORE
INTERESTING FROM A THERAPEUTIC
PERSPECTIVE.
HE CAME UP WITH A NUMBER OF
GENES MEMORY RECEPTORS, HE
MOREHT THOSE WOULD BE MORTAR
TARGETABLE THAN OTHER GENES.
HERE ARE SOME PUBLICATIONS THAT
TALK ABOUT ALL THESE, BUT I'M
SUMMARIZING IN A MINUTE.
ONE OF THOSE GENES, MARKER 8,
HE WAS ABLE TO DEVELOP A
THERAPEUTIC FOR THIS PARTICULAR
GENE, THIS GENE IS EXPRESSED,
HE DEMONSTRATED IT'S EXPRESSED
IN THE TUMOR VASCULATURE, HE
DID ANALYSIS OF THE EXPRESSION
INS HAVE CH VASCULATURE IN DIFFERENT
NORMAL TISSUES, WE COULD NEVER
FIND IT THERE, EXCLUSIVELY IN
THE TUMOR VASCULATURE.
WHAT WAS INTERESTING HE ALSO
DEVELOPED A KNOCKOUT FOR THIS
PARTICULAR GENE, THE KNOCKOUT
SHOWS THE VIEWLY N ABSOLUTELY NO PHENOTYP E
EXCEPT TEETH THAT ARE
MISALIGNED.
THE KNOCKOUTS ARE VIABLE AND
NORMAL.
WHEN YOU GROW A TUMOR IN THESE
MICE, THE TUMOR GROWTH IS
GREATLY DELAYED.
SO THIS WOULD BE THE GROWTH OF
THE TUMOR, AND THIS IS THE
GROWTH OF THE TUMOR IN THE
KNOCKOUT.
SO THIS RESULT IS NOT
SURPRISING, IT'S NOT SURPRISING
BECAUSE WHAT HE WAS LOOKING AT,
THIS TUMOR ENDOTHELIAL MARKER
IS OVEREXPRESSING THE TUMORS,
NOT SURPRISING THAT IF IT
DOESN'T PLAY A ROLE IN NORMAL
PHYSIOLOGY, THE ANIMALS ARE
PERFECTLY NORMAL.
YOU CAN ONLY SEE THE EFFECT
WHEN IN THE CONTEXT OF TUMOR
GROWTH, HE WENT AND DEVELOPED
ANTIBODIES TO TARGET THIS
PARTICULAR TUMOR MARKER, THE
ANTIBODY IS L-2.
HE ASSERTS A SIMILAR RESPONSE
WITH THE KNOCKOUT ANIMAL.
EVEN A SIMILAR DEGREE OF
INHIBITION OF TUMOR GROWTH
INHIBITION, SUGGESTING HE'S
ABLE TO BLOCK PRETTY MUCH ALL
THE TEM-8 PRESENCE WITHIN THE
TUMORMEN.
HE WENT AHEAD AND DID DIFFERENT
COMBINATIONS WITH OTHERS AND
SHOWED THAT TEM-8 ACTS WITH
OTHER THERAPEUTICS OR OTHER
ANGIO GENIC COMPOUNDS TO
SUPPRESS TUMOR GROWTH.
HE WAS ABLE TO ACHIEVE
TUMOR-FREE ANIMALS IN SOME OF
THE STUDIES HE DID.
ONE EXPERIMENT I WISH I WOULD
HAVE ADDED IN HERE IS
BEAUTIFUL, IN THE SAME ANIMAL,
HE PUT IN ONE SIDE ONE TUMOR,
IN THE OTHER SIDE OF THE ANIMAL
HE MADE A WOUND.
OKAY?
WOUND HEALING IS VERY MUCH
DEPENDENT ON ANGIOGENESIS.
HE TREATED THE ANIMAL WITH THE
ANTIBODY, AGAIN FOR TEM-8.
HE WAS ABLE TO REGRESS THE
TUMOR IN THE ANIMAL BUT THE
WOUND HEALED AT THE SAME SPEED
IN ANIMALS THAT WERE UNTREATED.
AGAIN, ONE FUNCTIONAL BASICALLY
EXAMPLE THAT THE ANTIBODIES
BASICALLY HAS AN EFFECT ON
TUMOR VASCULATURE BUT NOT
NORMAL VASCULATURE.
WHAT OTHER APPROACHES CAN WE
TAKE TO DEVICE TARGETS TO
TARGET SPECIFICALLY THE TUMOR
MICRO ENVIRONMENT?
THERE'S ALWAYS PRETTY MUCH A
PROCESS OF SOME SORT, IN THE
PREVIOUS PRESENTATION SHE WAS
TALKING ABOUT THE MASSIVE
STRAINS WITH RNAI.
TRYING TO DEVICE A WAY TO COME
UP WITH AN INVITVR OH
MEANINGFUL FROM TUMOR ANGIO
GENESIS USED TO SCREEN LARGE
NUMBERS OF DRUGS, AND THIS IS A
TWO-FORMATION AS SAY, NOT SURE
YOU'RE FAMILIAR WITH THIS
ASSAYS, IT CONSISTS OF
GENERATING A THREE DIMENSIONAL
MATRIX OF A GEL THAT IS
COMPOSED OF AN EXTRA-CELLULAR
MATRIX FROM A MOUSE TUMOR.
WE HE CA EXTRACT AND GENERATE, WE ADD
ENDOTHELIAL CELLS, ON THEIR
MATRIX.
THIS IS WHAT HAPPENS WHEN YOU
DO THAT.
THERE YOU GO.
THE CELLS WHICH OFFICIALLY ARE
HOMOGENOUSLY DISTRIBUTED WILL
REARRANGE TO FORM THE CORDS YOU
SEE THAT MIMIC THE VASCULATURE
IN THE TUMOR MICRO ENVIRONMENT.
THIS IS A TWO-FORMATION ASSAY.
WE DIDN'T INVENT IT.
IT'S BEEN AROUND FOR A LONG
TIME.
THE THIS WE COULD USE THE
ASSAY TO DO SCREENINGS
BASICALLY OF DRUGS.
TO MAKE A LONG STORY SHORT THIS
IS JUST AN EXAMPLE OF SOME OF
THE SCREENINGS THAT WE DID.
HERE ON THE LEG YOU HAVE AN
ASSAY WHERE WE DIDN'T TREAT
THESE COMPOUNDS, THESE CELLS,
AND THAT'S NOT GOING TO WORK
BUT BASICALLY IT WAS DOING
EXACTLY THE SAME THING AS IN
THE PREVIOUS IMAGE, AND THIS IS
ONE OF THE COMPOUNDS THAT WE
WERE ABLE TO FIND.
THIS IS ONE OF THOSE ASSAYS
TREATED WITH ONE OF THOSE
COMPOUNDS, BASICALLY THE CELLS
ARE ABLE TO MOVE AROUND FREELY
IN THE MATRIX.
IT'S NOT BLOCKING MIK MIGRATION OR
VIABILITY, BUT IT IS BLOCKING
THE ABILITY OF THE CELLS TO
FORM TUMORS.
WE WENT AHEAD AND TESTED
COMPOUNDS FOR CLINICAL MODELS,
AND THIS IS ONE OF COMPOUNDS I
SHOWED YOU BEFORE, ACTUALLY IN
THIS PARTICULAR EXPERIMENT WE
WERE TESTING TWO OF THESE
COMPOUNDS, THIS IS THE GROWTH
OF THE NORMAL TUMOR, AND THIS
IS THE GROWTH DOWN HERE OF TWO
COMPOUNDS COMPARED HAVE
AVASTIN.
SO AS YOU CAN SEE, WE WERE ABLE
TO FIND -- AS A MATTER OF FACT,
THIS IS WHERE THE TWO COMPOUNDS
THAT WE FOUND, IN THE
SCREENINGS, WE PICKED TWO.
WE DID THE WHOLE SCREEN, PICKED
TWO, THESE ARE THE FIRST TWO
COMPOUNDS WE TESTED IN VIVO.
TO INDICATE THE ASSAY IS
ROBUST, IN TERMS OF FINDING
SMALL MOLECULES, IN THIS CASE
SMALL MOLECULES THAT WERE
AFFECTED IN PRE-CLINICAL MOD
ALSO.
MODELS.
THESE ARE TWO EXAMPLES OF
FINDING DRUGS THAT ARE
SPECIFIC, MORE SPECIFIC FOR THE
TUMOR ENVIRONMENT, CAN BE
POTENTIALLY TRANSLATED INTO THE
CLINIC.
I DID PROMISE THAT I WOULD
FINISH.
IT'S TEN OF SIX.
I WILL TAKE ANY QUESTIONS THAT
YOU MIGHT HAVE AT THIS POINT.
[APPLAUSE]
YES?
[INAUDIBLE QUESTION]
>> IN TERMS OF WHAT?
[INAUDIBLE]
WE
>> WE HAVEN'T HAD COMPARISONS
OF BOTH OF THEM.
TEM-8 THERAPEUTICS HAVE NO
TOXICITY WHATSOEVER.
ONE OF THE CHALLENGES WITH THIS
TARGET IS TO REALLY FIND
WHETHER OR NOT THEY ARE
EXPRESSED ANYWHERE ELSE IN THE
BODY.
THERE MIGHT BE AN B OBSCURE
PLACE, YOU KNOW, A CELL TYPE
THAT WE DIDN'T LOOK AT, YOU
KNOW, THAT'S EXPRESSING TEM-8
WHERE IT'S FUNCTIONAL AND
NECESSITY FOR HOME HOMEOSTASIS, WE
DO THE WOUND AND WE DO THE
TUMOR IN THE SAME ANIMAL AND
TREAT THE ANIMALS, REALL REALLY
DEMONSTRATED CENTE THERAPEUTICS FOR
TEM-8 ARE NOT TOXIC.
A PAPER LAST YEAR FOUND THIS
GROUP OF PATIENTS THAT HAVE GAB
GABBA SYNDROME THAT PRODUCED
RETARDATION, ALOPECIA,OH
PEOPLE ARE NORMALLY NORMAL
OTHERWISE.
THEY HAVE MUTATIONS ON THE
TEM-8 GENE, THEY DON'T HAVE
TEM-8 AND THEY ARE NORMAL.
THAT'S A GOOD INDICATION THAT
TEM-8 IS NOT REQUIRED FOR
NORMAL DEVELOPMENT AND FOR, YOU
KNOW, HOMEOSTASIS IN ADULTS.
IT'S A GOOD INDICATION
THERAPEUTICS FOR TEM-8 WOULDN'T
SHOW TOXICITY IN PATIENTS.
YES?
[INAUDIBLE QUESTION]
>> THAT'S A VERY GOOD QUESTION
AND WE'RE WORKING VERY HARD TO
UNDERSTAND THAT QUESTION.
NAME8, I ALSO PUT ANOTHER MAIM
IN, AN ANTHRAX RECEPTOR ONE.
IT'S A RECEPTOR FOR ANTHRAX.
I DID NOT EVOLVE TO BE AN AN
ANTHRAX RECEPTOR, IT'S PROBABLY
GENES,NT TO OTHER CHAINS,
KNOCKING DOWN OF TEM-8 IS NOT
REQUIRED.
[INAUDIBLE QUESTION]
ONE OF THE INTERESTING
EXPERIMENTS, BRAD LOOKEDDED A
WHERE THIS HAPPENS, HE FOUND NO
EXPRESSION OF TEM-8.
THAT'S WHY IT'S SO ATTRACTIVE
TO US, BECAUSE IT'S REALLY
EXPRESSING TUMOR VASCULATURE,
WE CAN'T FIND IT ANYWHERE ELSE.
[INAUDIBLE QUESTION]
>> WE HAVEN'T DONE THAT.
YEAH, WE HAVEN'T DONE THAT.
WE CAN OVER EXPRESS IN VITRO,
THE CELLS DON'T SEEM TO SHOW
PHENOTYPE IN TERMS OF GROWTH,
MORPHOLOGY, ANY OF THOSE OVERT
THINGS THAT YOU WOULD BE ABLE
TO SEE.
[INAUDIBLE QUESTION]
>> WELL, THIS IS KIND OF LIKE,
IN MY OPINION, LIKE A
HISTORICAL REASON.
THIS IS AN ENTIRE FIELD IN THE
SEARCH FOR ONE RGET THAT WE
CAN TRANSLATE TO THE CLINIC.
SO AT GENENTECH, DESIGNING AN
ANTIBODY, IT WAS THE FIRST
F.D.A.-APPROVED DRUG, AND THEY
SHOWED IT CAN'T WORK IN THE
CLINIC, THAT ATTRACTED MASSIVE
INTEREST, SO THAT'S DONE A LOT
OF GOOD FOR THE FIELD IN TERMS
OF WE'VE SHOWN THAT WE CAN, YOU
KNOW, TRANSLATE THIS INTO THE
CLINIC AND IT'S AFFECTED, BUT
AT THE SAME TIME IT'S PROBABLY
NOT THE BEST TARGET, BECAUSE OF
ALL THESE THINGS WE HAVE
DISCUSSED TODAY.
BUT IT'S PROBABLY A HISTORICAL
REASON FROM MY PERSPECTIVE.
[INAUDIBLE]
[INAUDIBLE QUESTION]
>> THE END STUDY RESULTS ARE
HARD TO REPRODUCE, BUT YES,
MOVE THEYNG TO MOVE THEY ARE
THERAPEUTICS TO THE CLINIC.
MORE QUESTIONS?
THANK YOU VERY MUCH.
[APPLAUSE]