WELL, WHY DON'T WE GO AHEAD AND
GET STARTED.
THANK YOU ALL FOR COMING DOWN.
THOSE OF YOU WHO DON'T KNOW ME
I'M DAN Mc VICAR FROM UP IN
FREDERICK AND IT'S A REAL
PLEASURE FOR ME THIS AFTERNOON
TO INTRODUCE OUR SPEAKER DEBBIE
MORRISON.
I THINK MANY OF YOU KNOW DEBBIE
AND KNOW AND KNOW HER HISTORY
VERY WELL BUT I'M GOING TO
REPEAT IT FOR YOU ANYWAY.
SO SHE'S INITIALLY DID HER
UNDERGRADUATE WORK IN NASHVILLE,
AND THEN COMPLETED HER Ph.D.
AT VANTER BUILT WITH RICHARD
MOYER AND PUBLISHED A CELL PAPER
THERE, NOT BAD FOR A GRADUATE
STUDENT LOOKING AT THE USE OF
CELLULAR RNA POLYMERASE BY P. PX
VIRUSES.
BUT THEN SHE MOVED TO THE
DANA-FARBER AS A POST DOC AND
JOINED TOM LAMBERT'S LAB THERE
AND MADE HER STUDY ON THE REST
OF HER CAREER ON WHICH IS
TYROSINE PHOSPHOR LEAN AND
SIGNALS.
SHE STARTED WORKING ON R,A ,--RF
AND YOU WILL HEAR A LOT ABOUT
THAT TODAY, PUBLISHED A SECOND
CELL PAPER, TALKING ABOUT
TYROSINE PHOSPHORYLATION BY PDGF
RECEPTOR AND ITS ACTIVATION OF
--RAF, TO GIVE YOU AN IDEA OF
THE FIELD IT WAS TYROSINE AND
PHOSPHORYLATION SIGNALING WAS
STARTING TO BE ACCEPTED AND IN
FACT, I THOUGHT IT WAS REALLY
FUN ACTUALLY THAT ON HER CV, SHE
HAS A TITLE TYROSINE
PHOSPHORYLATION IS SIGNAL
TRANSDUCTION, AT THE TIME YOU
HAD TO MAKE THAT ARGUMENT BUT
YOU DON'T HAVE TO DO THAT
ANYMORE.
BUT THAT WAS A LONG LINE OF
SEMINOLE WORK REALLY ON RAF 1
FROM DEBBIE'S LAB.
SHE JOINED THE LAB UP IN
FREDERICK WHICH GEORGE WAS THE
DIRECTOR, RIGHT?
GEORGE VANNEDDER WOOD WHO MANY
OF YOU PROBABLY KNOW, SHE WAS
THEN PROMOTED UP TO SENIOR
INVESTIGATOR AND TENURED WITHIN
THE NCI UP THERE.
DEBBIE'S WORK AND REALLY
REACHING AND FANTASTIC.
SHE'S BEEN FEATURED MULTIPLE
TIMES BY THE CCR LEADERSHIP AND
INNER MILESTONES AND THINGS LIKE
THAT AND SHE HAS MULTIPLE NIH
DIRECTOR AWARDS FOR HER
DISSECTION OF THE SIGNALING AND
RAS AND R,A F AND, WOOING OUT
DETAILS OF THAT PATHWAY, SHE'S
ALSO BEEN RECOGNIZED IMPORTANTLY
WELL OUTSIDE OF THE CCR, SHE WON
THE PFIZER RESEARCH AWARD FROM
THE UNIVERSITY OF MONITOR TRIAL,
A SIGNALING, LRI TRANSDUCTION
AWARD FROM THE ROYAL COLLEGE OF
SURGEONS IN LONDON AND THE KARL
BIER AWARD FROM THE UNIVERSITY
OF WISCONSIN MADISON AND OF
COURSE MANY OF YOU KNOW THAT JUF
IN 2002 SHE WAS ELECTED TO THE
NAGGAL ACADEMY OF SCIENCES.
SORRY?
WHAT DID I SAY?
TWO.
TWENTY-TWO.
YOU SHOULD HAVE BEEN ELECTED IN
2002--WE THAT WOULD SHOW YOUR
AGE, WE DON'T WANT TO DO THAT.
THANK YOU FOR THE CORRECTION.
>> DEBBIE'S LAB CONTINUES TO BE
A WORLD LEADER IN THE FUNCTION
OF RAF AND THEY HAVE FOCUSED ON
THE KEY SIGNAL TRANSDUCTION OF
LOCATION, LOCATION, LOCATION, SO
SHE'S BEEN CRITICAL IN
DISSECTING THE ROLE OF SCAFFOLD
PROTEINS AND HOW THEY COORDINATE
THESE ACTIVATION COMPLEXES AND
CONTINUES TO INCREASE OUR
FUNDAMENTAL UNDERSTANDING OF RAS
AND RAF SIGNALING AND IN MANY
CASES NOW THROUGH CLOSE RAS
INITIATIVE AND THE RAS OPERATIVE
HERES HERE AT NIH AND TODAY SHE
WILL TELL US ABOUT HER ONGOING
WORK AND THE TITLE OF HER TALK
IS UNDERSTANDING RAF KINASE
DISEASE REGULATION IN HUMAN
DISEASE STATES EMPLOY.
THE DETAILS DO MATTER!
SO YOU WILL HEAR DETAILS.
THANKS SO MUCH DEBBIE.
>> WELL, I WANT TO SAY FIRST OF
ALL, IT'S A REAL HONOR TO BE
HERE TO GIVE THE GRAND ROUND AND
IT'S GOD TO SEE THE FACES THAT I
DON'T SEE ALL THE TIME UP IN
FREDERICK.
SO I HOPE WHEN NIGH TALK'S OVER
AND YOU WALK ARK WAY, YOU WILL
REALIZE HOW IMPORTANT THE
DETAILS FOR FOR UNDERSTANDING
RAF REGULATION.
SO MY LAB HAS REALLY HAD A LONG
STANDING INTEREST IN STUDYING
THE SIGNAL TRANSDUCTION PATHWAY
THAT'S REGULATED BY THE RAF
GTPACES, OFTEN IT'S REFERRED TO
AS A MOLECULAR SWITCH TO TURN ON
SIGNALING IN THE CELL AND MANY
TIMES THAT ACTIVATING SWITCH IS
WHEN THE CELL RECEIVES SIGNALS
FROM RECEIPTOR TYROSEEN KINASES
ON THE CELL SURFACE AND RAS ONCE
ITS ACT VAILTED WITH INTERACT
WITH A NUMBER OF EFFECTIVE
CASCADINGS BUT 1 OF ESSENTIAL
CAS CADE IS THE ERK CASCADE
WHICH IS COMPRISED OF THE RAF,
MEK AND ERK PROTEIN KINASES.
NOT ONLY DOES THIS PLAY A
PATHWAY ROLE IN NORMAL GROWTH
AND DEVELOPMENTAL SIGNALS, A
NUMBER OF THE COMPONENTS OF THE
PATHWAY HAD FOUND TO BE MUTATE
INDEED HUMAN KANSASER AND IN
PARTICULAR KRAS IS MUTATED AT
90% OF THE PANCREATIC CANCER AND
BRAF IS MUTATED AT ABOUT 60% OF
THE MALIGNANT MELANOMAS.
BUT IN ADDITION TO BEING MUTATE
INDEED CANCER, THESE PROTEINS
CAN CONTAIN GERM LINE MUTATIONS
THAT WILL CAUSE A GROUP OF
RELATED DEVELOPMENTAL DISORDERS
THAT ARE KNOWN COLLECTIVELY NOW
AS THE RAS-OP A THIES.
AND BECAUSE OF THE FUNCTION OF
THIS PATHWAY IN YOU HAD NANOG
DISEASE STATES THERE'S BEEN A
LOT OF INTEREST TO FIGURE OUT
HOW CAN WE TARGET THIS PATHWAY?
AND SO, FOR THERAPEUTIC AND
INTERVENTION, BOTH IN CANCER AND
IN THESE DEVELOPMENTAL
DISORDERS.
BUT I THINK THE 1 THING THAT
WE'VE REALLY LEARNED IS
RESEARCHERS OVER THE YEARS, IS
THAT IN ORDER TO TARGET THE
PATHWAY, IT'S REALLY IMPORTANT
THAT YOU UNDERSTAND HOW IT
NORMALLY FUNCTIONS AND
UNFORTUNATELY, OR MAYBE
FORTUNATELY FOR ME BECAUSE IT'S
GIIVE ME JOB SECURITY IS THE
RAFS ARE REALLY VERY COMPLEX
WHEN IT COMES TO THEIR
REGULATION.
AND SO JUST TO SHOW YOU A
SCHEMATIC FOR THOSE OF YOU WHO
DON'T AND THINK ABOUT THE RAFS
24/7, THEY'RE A GROUP OF PROTEIN
KINASES AND THEY CONTAIN A
CATALYTIC DOMAIN AT THE
C-TERMINAL REGION AND A
REGULATORY DOMAIN AT THE END
TERMINUS AND THIS HAS A RAS
BINDING DOMAIN, ASSISTING RICH
DOMAIN AND A C3 ARHINE, AND IF
WE THINK ABOUT THEIR INTRINSIC
CATALYTIC ACTIVITY, BRAF HAS THE
HIGHEST INTRINSIC ACTIVITY
FOLLOWED BY CRAF AND THEN ARAF.
AND WHAT WE'VE LEARNED MAINLY
HISTORICALLY A LOT OF OUR
INFORMATION THROUGH THE YEARS
ABOUT THE RAFS CAME FROM STUDIES
OF BIOCHECKICAL STUDIES WITH
MUTE ANTS OR WITH THE STUDY OF
INDIVIDUAL PROTEIN DOMAINS.
AND SO WHAT WE LEARNED WAS THAT
AND I THINK THIS WAS PROBABLY
THE FIRST REAL BREAK THROUGH IN
UNDERSTANDING RAF WAS THAT IT
CAN DIRECTLY INTERACT WITH RAS
AND THAT'S MEDIATED THROUGH THE
RAS BINDING DOMAIN BUT ONCE RAS
INTERACTS, HAS A HIGH AFFINITY
BINDING TO THE RBD, IT CAN ALSO
MAKE CONTACTS WITH THE CYSTINE
RICH DOMAIN.
THE CYSTINE RICH DOMAIN WAS
PROBABLY FIRST CHARACTERIZED
BASED UPON ITS ABILITY TO
INTERACT WITH FOSTER NURSED
FOCUSED ON TILE SERIES POINTSINE
IN THE MEMBRANE, AND THEN THERE
ARE ALSO 1433 BINDING SITES AND
1433 OF-THEY'RE DIMERS,
CONSTITTIVE BINDERS THAT
INTERACT WITH THE PROTEINS AND
THE RAFS CAN ALSO DIMERIZE AND
THEY CAN INTERACT WITH THEIR
DOWN STREAM SUBSTRATE WHICH IS
MEK.
SO TO KIND OF PUT THIS
ALTOGETHER IN CONTEXT, WHAT WE
KNOW IS THAT IN A QUIE ESTIMATE
THADENT CELL, BOTH BRAF AND CRAF
ARE FOUND IN THE CYTOSOL AS
INACTIVE MONITOR MERS AND I WILL
JUST POINT OUT HERE THAT BRAF
AND CRAF ARE THE PRIMARY DISEASE
DRIVERS.
BRAF IS PROMINENT DRIVER IN
HUMAN CANCER, CRAF HAS NOT BEEN
CHARACTERIZED SO MUCH AS BEING A
CANCER DRIVER BUT BOTH OF THEM
ARE DRIVERS OF THE DEVELOPMENTAL
DISORDERS AND ARAF HAS NOT
REALLY BEEN PICKED UP AS A
DRIVER OF THE RAS-OP A THIES AND
ISN'T REALLY KNOWN TO BE A
PRIMARY DRIVER IN CANCER EITHER.
SO LESS IS KNOWN ABOUT ARAF.
BUT BRAF AND CRAF ARE ACTIVATED
IN RESPONSE TO RAF'S ACTIVATION
AND RTK SIGNALING AND THEY EXIST
AS MONOMERS IN A QUIESCENT CELL.
BUT THEN WHEN SIGNALS ARE
RECEIVED BY THE CELLS AND RAS
BECOMES ACTIVATED IN ITS ON
STATE, IT CAN DIRECTLY INTERACT
WITH THE RAF RBD AND THEN THAT
ALLOWS THE CRD TO INTERACT WITH
BOTH THE MEMBRANE AND TO CONTAKE
RAS AS WELL.
WE ALSO KNOW THAT 1433 COMES OFF
OF THIS END TERMINAL SITE WHICH
EXPOSES IT TO BE
DEPHOSPHORYLATED BY A COMPLEX OF
FOSTER
PHOSPHATASE COMPLEX THAT'S KNOWN
AS SMP AND THERE'S BEEN A LOT OF
STUDIES ON THE SMP COMPLEX AND
ITS ROLE IN KRAS DRIVEN CANCERS
AS WELL.
BUT WE ALSO KNOW THAT THE RAF
KINASE DOMAINS CAN DIMERIZE AND
THEIR DIMERIZATION RESULTS IN
ACTIVATION.
AND I JUST WANT TO SHOW YOU HERE
SOME OF THE PRIMARY DATA THAT
KIND OF DEPICTS THAT, AND HERE
WE'RE LOOKING AT SERUM STAR
CELLS THAT HAVE EITHER BEEN
SERUM STARVED OR TREATED WITH
EGF AND WE'RE LOOKING AT EITHER
THE WILD-TYPE ERAF PROTEIN OR
CRAF OR A MUTATION IN THE
DIMERIZATION MOTIF.
SO THERE'S A CRITICAL RTKR MOTIF
THAT IS ESSENTIAL FOR THE RAF TO
DIMERIZE AND IF YOU MUTATE THIS
RGENINE, TO HISTIDINE, WHAT YOU
FIND IS YOU DISRUPT ALL
DIMERIZATION AND IN RESPONSE TO
GROWTH FACTOR SIGNALING YOU SEE
NO IPT GREATER CREASE
IN--INCREASE IN ACTIVITY WHERE
NORMALLY YOU SEE AN INCREASE IN
ACTIVITY AND RESPONSE TO BRAF
AND CRAF AND YOU CAN SEE THAT IT
CORRELATES WITH INCREASED
DIMERIZATION.
BRAF HAS A LITTLE BIT OF ABILITY
WE TEND TO SEE SOME JEIMER
DIMERIZATION, IT DOES GO UP, THE
SAME IS TRUE BUT IT'S LESS FOR
CRAF.
BUT THE 1 THING I WILL SAY THAT
WE DON'T COMPLETELY UNDERSTAND
RIGHT NOW IS THAT THERE IS
REALLY A PREFERENCE FOR BC
DIMERS IN RESPONSE TO RAS
ACTIVATION AND THAT'S SOMETHING
THAT'S GOING TO CONTINUE TO KEEP
US BUSY AND TRYING TO UNDERSTAND
HOW THERE IS SELECTIVITY FOR THE
DIFFERENT RAF FAMILY MEMBERS AND
PROMOTING DIMERS.
BUT FOR THIS, IN GROWTH FACTORS
SIGNALING, IT'S THE BC DIMERS.
SO, WHAT WE KNOW IS THAT
DIMERIZATION IS ABSOLUTELY
CRITICAL FOR ACTIVATION AND DOWN
STREAM SIGNAL TRANSDITION TO MEK
AND ERK, THE THING GOES ON AND
TELLS THE CELL TRANSMITS SIGNALS
TO TELL THE CELL TO DO A VARIETY
OF DIFFERENT THINGS.
BUT NOT ONLY IN GROWTH FACTOR
SIGNALING IS DIMERIZATION
CRITICAL IT ALSO PLAYS A ROLE IN
HUMAN DISEASE STATES AS WELL.
SO YOU CAN IMAGINE THAT
DIMERIZATION IS CRITICAL IN
ORDER FOR RECEPTOR TYROSEEN
KINASES OR RAS PROTEINS THAT ARE
MOOITATED SO IF ANY
CONSTITTATIVELY RAS PROTEIN OR
RECEPTOR IS GOING TO RECRUIT THE
RAFs AND THEY ARE NEEDED FOR
THE DOWN STREAM SIGNALING TO MEK
AND ERK.
AND ALSO IT'S CRITICAL FOR CLASS
2 AND CLASS 3 DISEASE ASSOCIATED
RAF MIEWITANTS AND NEAL ROSEN
SEVERAL YEARS AGO, I THINK
REALLY HAD A TOUR DEFORCE EFFORT
TO TRY TO CHARACTERIZE ONCA
GENIC RAF MUTANTS.
AND THE BRAF THE 1 THAT PROBABLY
EVERYONE IS MOST FAMILIAR WITH
IS V600 E, IT'S THE PRIMARY
DRIVER IN MALIGNANT ELANNOMA AND
WHAT IS REALLY UNIQUE ABOUT THIS
MUTATION, THE VAILING TO GLUE
TISSUE AND MACROPHAGESSIC ACID
IS THAT SINGLE MUTATION HAS
ALLOWED A SALT BRIDGE TO FORM
THAT ALLOWS THE CATALYTIC DOMAIN
TO ASSUME THE ACTIVE
CONFIRMATION IN THE ABSENCE OF
DIMERIZATION.
AND I WILL SHOW THAT A LITTLE
BIT LATER BUT FOR THE MAIORITY
OF THE OTHER MUTANTS THAT ARE
CALLED CLASS 2, THEY HAVE THIS
ABILITY TO SELF-DIMERIZE AND IN
THAT CASE, DIMERIZATION IS
REQUIRED FOR THEM TO TRANSMIT A
SIGNAL.
AND I THINK 1 OF THE VERY FIRST
BIG SURPRISES IN THE RAF FIELD
CAME WITH THE DEVELOPMENT OF
SOME OF THE TARGETED INHIBITORS,
BRAF NIB, AND THEY WERE GREAT,
THEY WERE SUPPRESSING BSE
ACTIVITY AND BLOCKING SIGNAL
TRANSMISSION TO MEK.
BUT WHAT WAS COMPLETELY
UNEXPECTED AT THE TIME WAS THAT
HE'S INHIBITORS HAD THIS
PARADOXICAL ACTIVATING EFFECT
AND WHAT I MEAN BY THAT WAS THAT
WHEN THEY BOUND TO EITHER A
HETERODIMER OR THE SELF-DIMERS,
WHAT THEY ACTUALLY DID WAS THEY
STABILIZED THE DIMERS, AND THEY
HAD THIS PROPERTY OF ONCE THEY
WERE BOUND IT CAUSED NEGATIVE
COOPERATIVITY AND ALLOWING THE
INHIBITOR TO BIND TO THE OTHER
PROTOMER, SO THE INHIBITOR COULD
BIND HERE BUT THEN YOU HAD
REALLY REDUCED ABILITY FOR THE
INHIBITOR TO GET INTO THE PRO
TOMMER, AND SO SIGNALING STILL
WENT TO MEK AND ERK.
IN FACT, IT WAS AUGMENTED AND
THIS IS 1 OF THE PRIMARY
CONTRIBUTORS TO THE DRUG
RESISTANCE TO VINMIRATANIB IN
THE LAB AS WELL.
SO IT RAISES THE QUESTION, THAT
DIMERIZATION IS CRITICAL FOR
NORMAL SIGNALING THROUGH RAF BUT
IT CAN ALSO AFFECT THE EFFICACY
OF RAF INHIBITORS AND CO PROMOTE
DRUG RESISTANCE.
SO THERE ARE BETTER WAYS TO
TARGET THE RAFS?
AND WE THOUGHT IT WOULD BE
IMPORTANT AND I GUESS--OUR LAB
WASN'T THE ONLY 1, EVERYONE
WORKING ON RAF WANTED TO KNOW
WHAT IS THE STRUCTURAL CHANGES
THAT OCCUR WHEN BRAF TRANSITIONS
FROM AN INACTIVE MONITOR MERTO
AN ACTIVE DIMER.
AND SO WE HAD A PHENOMENAL
COLLABORATION WITH JULIANA
MARTINEZ FIESCO, AND PING CHANG
ON STRUCTURAL BIOLOGY UP IN
FREDERICK.
AND WHAT WOE DID IS WE TOOK OUR
BRAF CONSTRUCT AND WE GENERATED
HUMAN CELL LINES THAT WERE
STABLY EXPRESSING THIS TAGGED
PROTEIN.
AND IT TURNS OUT THAT ACTUALLY
THIS WAS VERY IMPORTANT TO HAVE
IT IN HUMAN CELLS WHERE IT BE
PROPERLY MODIFIED IN THE CELL
AND WE COULD SERUM STARVE IT AND
WE COULD ALSO GROWTH FACTOR
TREAT IT.
AND WE WERE ABLE TO SOLVE THE
STRUCTURE, [INDISCERNIBLE]
STRUCTURE OF 3 RAF COMPLEXES.
SO THIS IS SHOWING THE DIMER
COMPLEX AND THERE HAD BEEN A
NUMBER OF THE DIMER COMPLEXES
THAT HAVE BEEN REPORTED BUT THIS
IS ACTUALLY THE FULL LENGTH
PROTEIN BUT THE ONLY THING THAT
WAS RESOLVED WAS THE KINASE
DOMAIN AND 1433 DIMER POUND TO
EACH PROTOMER OF THE RAF DIMER.
THE ENTIRE REGULATORY DOMAIN WAS
NOT RESOLVED AND WE AREN'T THE
ONLY 1S WHO SOLVE THIS
STRUCTURE, JOHN KIRRIAN AND MIKE
[INDISCERNIBLE] ALSO, AND FOR
ALL OF THESE COMPOWBDS THIS WAS
NOT RESOLVED LIKELY DUE TO
FLEXIBILITY IN THE ABSENCE OF
ITS NORMAL MEMBRANE BINDING
PARTNERS.
BUT WHAT WAS REALLY EXCITING FOR
US IS THAT WE WERE ABLE TO
IDENTIFY 2 AUTOINHIBITTED
COMPLEXES.
ONE THAT CONTAINED--I HAVE TO
FIND IT, 1 THAT CONTAINED MEK,
AND 1 THAT DID NOT, BUT THE
OTHER REGIONS EXCEPT FOR THE MEK
BINDING WERE REALLY VIRTUALLY
IDENTICAL.
AND WHAT WAS UNIQUE, MIKE YEK
HAS ALSO PUBLISHED A
[INDISCERNIBLE] MONOMERBUT OURS
ENDED UP BEING HIGHER RESOLUTION
AND WE WERE ABLE TO UNIQUELY
RESOLVE THE RBD WHICH IS SO
CRITICAL FOR RAF ACTIVATION AND
WE WERE ABLE TO DETERMINE ITS
POSITION AND ORIENTATION.
SO NOW THAT WE'VE GOT THE
COMPLEX, WHAT CAN IT TELL US
ABOUT THE ACTIVATION MECHANISMS.
WE WERE ABLE TO DECIDE FOR THAT,
THAT THE KINASE DOMAIN IS IN THE
ACTIVE STATE, AND SO FOR KINASE
OFFICKIAN ADOS, THAT MEANS THIS
CRITICAL ALPHA C HELIX IS IN THE
OUTPOSITION AND ITION AND WHAT -
BUT IN,A DITION TO THAT IT'S
NOT JUST THE KINASE DOMAIN, YOU
ALSO HAVE THIS DIMER OF 1433
HERE AND ACTUALLY THE 1433 DIMER
OBSCURES THAT CRITICAL RTKR
MOTIF THAT'S REQUIRED FOR
DIMERIZATION AND ANOTHER THING
THAT I THINK I WANT TO POINT OUT
THAT I THINK PEOPLE DON'T FULLY
APPRECIATE SOMETIMES IS THIS
ALPHA C HELIX IS IMMEDIATELY
ADJACENT TO THIS RTKR MOTIF,
THEY ARE SIDE BY SIDE.
AND SO MOVEMENT OF THIS ALPHA C
HELIX OR INTERACTIONS HERE CAN
TRANSFER BACK AND FORTH.
AND SO, WE KNOW THE CYSTINE RICH
DOMAIN IS IN THE CENTER OF THE
COMPLEX AND YOU CAN SEE HERE
THAT IT CONTACTS ACTUALLY MAKES
CONTACTS WITH BOTH 1433 POLYMERS
AND THE KINASE DOMAIN AND THE
RBD MAKES CONTACTS WITH THIS
PROTOMEROF 1433 THAT'S BOUND TO
THE C-TERMINAL SITE.
SO, WHEN YOU HAVE ACTIVATION, WE
KNOW THAT THE RBDs ARE
RELEASED AND 1433 IS RELEASED
FROM THE END TERMINAL AND IS
THAT SITE'S DEPHOSPHORYLATED
THERE'S A REARRANGEMENT.
THERE'S THE DIMER INTERFACE IS
EXPOSED AND ONCE YOU HAVE THESE
DIMERIZATION, IT'S THOUGHT TO
THEN CAUSE THIS ALPHA C HELIX TO
DROP DOWN TO MELT THIS
INHIBITORY TURN.
AND YOU HAVE 1433 REARRANGING
AND BINDING TO BOTH PROTOMERS OF
THE RAF DIMER AND THE WORK FROM
GENENTECH GROUP HAS REALLY SHOWN
THIS IS ESSENTIAL FOR KEEPING
THIS KINASE DOMAINS DIMERIZED
DURING CATALIAISONS, AS IT BINDS
MEK, PHOSPHORYLATES IT AND
RELEASED, IN EK THAT YOU NEED
THE 1433 THERE TO KIND OF HOLD
THE 2 TOGETHER EMPLOY KIND OF
THINK OF IT LIKE A SEE SAW GOING
BACK AND FORTH FROM 1433 HOLDS
THEM TOGETHER, BUT YOU CAN ALSO
SEE, I MEAN, THERE'S A LOT THAT
WE DON'T UNDERSTAND.
AND SO WE WERE STARTING TO
WONDER, IS THERE WAYS THAT WE
CAN FILL IN SOME OF THESE
MISSING STRUCTURES, SO WHEN THE
RAFS BEGIN TO YOU KNOW INFOLD OR
THE AUTOINHIBITTED COMPLEX KIND
OF UNRAVELS, WHAT HAPPENS FIRST
YOU KNOW DOES 1433 COME OFF,
DOES KINASE COME OUT?
AND WE THINK IT MADE BE
IMPORTANT AGAIN BECAUSE IT MAY
IDENTIFY NOW TARGETS AND
APPROACHES FOR CANCER THERAPY.
AND IN PARTICULAR WE WANT TO
KNOW WHAT IS THE EXACT FUNCTION
OF THE RAS RAF INTERACTION
BECAUSE IT'S KIND OF
CONTROVERSIAL IN THE FIELD RIGHT
NOW IS ALL RAS DOES IT LOCALIZE
RAF TO THE MEMBRANE OR DOES IT
PLAY A MORE ACTIVE ROLE IN
DISASSEMBLING THE AUTOINHIBITTED
COMPLEX IS WHAT IS THE
CONTRIBUTION OF THE PLASMA
MEMBRANE, AND HOW IMPORTANT IS
THIS PHOSPHATASE COMPLEX.
WELL, WE'VE BEEN REALLY
FORTUNATE NOW THAT WE'RE PART OF
THIS ADMIRAL PROJECT AND IT'S AN
AI DRIVEN, IT STANDS FOR AI
DRIVEN MULTISCALE INVESTIGATION
OF RAS, RAF ACTIVATION LIFE
CYCLE AND IT IS COLLABORATION
BETWEEN THE NATIONAL CANCER
INSTITUTE, THE NCI, RAS
INITIATIVE AND ALSO TAKES
ADVANTAGE OF THE COMPUTER
CAPABILITIES PRESENT AT LAWRENCE
LIVER MOORE NATIONAL LABS WHICH
ARE AMONGST THE MOST POWERFUL
AND ACTUALLY GLOBALLY SO WE'RE
GOING FROM STRUCTURES IN KNOWN
BIOLOGY, WE'RE DOING LARGE SCALE
AND LONG COMPUTATIONAL MODELS
AND THEN COMING BACK AND TESTING
AND VALIDATING THE MODELS.
AND SO WHEN WE BEGAN THIS, WE
KIND OF START WIDE OUR STRUCTURE
AND SO, THIS IS KIND OF A SPACE
FILLING MODEL OF THAT AND WHAT
YOU CAN SEE IS THE RBD IS
SITTING ON TOP OF THIS 1433
PROTOMER, AND IF WE LOOK AT THE
RESIDUES INVOLVED IN THIS
INTERACTION AND IT WAS QUITE A
LARGE CONTEXT SURFACE AREA, YOU
CAN SEE IT ALSO HAS A DEGREE
CONSIDERABLE ELECTROSTATIC
CHARGE COMPLIMENTARY.
AND THE DYNAMIC SIMULATIONS THAT
WERE PERFORMED AT THE
LIVERMOORE LAB WERE ABLE TO
IDENTIFY A NUMBER OF POTENTIAL
HYDROGEN BONDS THAT CAN FORM
HERE BETWEEN THIS IS THE ALPHA
HELIXES OF THE RBD LOOP 2 AND
ALPHA HELIXES 8 AND 9 IN THE
1433 PROTOMER.
BUT, IN THIS PARTICULAR
ORIENTATION, WHAT YOU CAN ALSO
SEE IS THAT THE 4 CRITICAL BASIC
RESIDUES THAT MAKE IONIC
INTERACTIONS WITH RAS ARE
LARGELY EXPOSED.
AND WE CAN SEE IN OUR STRUCTURE
THAT THERE WAS A POCKET FOR RAS
TO FIT IN HERE SO WHEN WE WEPT
ON TO DO THE MODELING AS RAS
WOULD ENGAGE WITH THE BASIC
RESDUES, WHAT WE SAW IS THAT IT
WOULD GENERATE ASTERIC CLASS AT
THIS INTERFACE AND IN ADDITION
WHILE THESE ACIDIC RESIDUES ARE
VERY COMPLIMENTARY WITH THE
BASIC CHARGES AND THE RBD WHAT
YOU CAN SEE IS THE 2 RESIDUES
DOWN HERE TO THE GLUTEAMILLIOIC
ACID RESIDUES WOULD CAUSE A
REPULINGS HERE AGAIN AT THE
INTERFACE SO BOTH A CLASS AND A
ELECTROSTATIC REPULINGS.
SO OUR QUESTION WAS, DOES THE
RAS RBD CONTACT ACTUALLY
INITIATE THE DISASSEMBLY OF THE
AUTOINHIBITTED COMPLEX.
AND SO, WE WONDERED, YOU KNOW
CAN WE DEVELOP AN INVITRO RAF
ACTIVATION ASSAY TO CONFIRM THE
IMPORTANCE OF VARIOUS COMPONENTS
THAT FACILITATE THE MONITOR
MERTO DIMER TRANSITION AND CAN
THIS HELP ORDER THE STEPS THAT
ARE REQUIRED FOR THIS
TRANSITION.
SO BASICALLY WHAT WE DID WAS WE
TOOK OUR PURIFIED BRAF MONOMERS
FROM THE HUMAN CELLS THAT WE HAD
AND WE INCUBATED THEM IN TEST
TUBES, EITHER KRAS THAT WAS
FULLY PROCESSED AND GTP LOADED,
LIPO STUDIES OF MULTIPLE
ENDOCRINES THAT CRATE 30 PRGT
FOSTER NURSED FETILE SERIES
POINTSINE OR IS THE SMP COMPLEX
WE HOPED WOULD BE ABLE TO
PHOSPHORYLATE THAT PROTEIN 1433
BINDING SITE OR A COMBINATION.
AND THEN WE ADDED KINASE BUFFER
AND DID OLD SCHOOL P32 ATP.
AND THEN RAN A GEL AND ANALYZED
IT FOR THE PHOSPHORYLATION OF
MEK THAT WE THEN ADDED TO THE
SAMPLES.
AND ACTUALLY, I THINK THE DESIGN
OF THIS ASSAY WAS REALLY
CRITICAL IN THAT IT'S ONLY
MONITORING WHAT IS HAPPENING IN
THE TEST TUBE BECAUSE WE'RE ONLY
MONITORING WHETHER THE BRAF
ACTIVITY ALL BY ITSELF OR DOES
IT CHANGE IN THE PRESENCE OF ANY
OF THESE ADDITIONS.
AND SO THESE ARE THE RESULTS
THAT WE FOUND THAT BASICALLY
THERE'S A LOW BASAL ACTIVITY OF
THE AUTOINHIBITTOR MONOMERAND WE
SEE ABOUT A 2 OF THIS 3 FOLD
INCREASE WHEN WE ADD IN KRAS, WE
CONSISTENTLY SEE A FALLER EFFECT
WITH THE LIPO STUDIES OF
MULTIPLE ENDOCRINES AND VERY
LITTLE EECT WITH THE SMP COMPLEX
AND WHAT I THINK'S REALLY NICE
ABOUT THIS, YOU CAN ACTUALLY, WE
CUT OUT AND COUNT THE MEK THAT'S
PHOSPHORYLATED BUT BEFORE WE DO
THAT WE JUST ECPOSE IT AND SO
VISUALLY YOU CAN JUST SEE, YES,
THERE'S A MAJOR INCREASE IN RAF
ACTIVITY IN THE PRESENCE OF
ADDING FULLY PROCESSED KRAS.
AND WE COULD ALSO IN THE SAMPLES
THAT WE'RE ADDING, BECAUSE WE
RAN EVERYTHING ON A GEL, WE CAN
PROBE TO SEE, OKAY, DID SEE SMP
ACTUALLY DEPHOSPHORYLATE THE
SITE THAT WAS IT SUPPOSED TO AND
YOU CAN SEE THAT WE ARE SEEING A
REDUCTION IN THE PHOSPHORYLATION
THERE.
AND EVEN THIS EXPERIMENT GAVE US
2 IMPORTANT PIECES OF
INFORMATION THAT BRAF AT LEAST
IN A TEST TUBE, CAN BE ACTIVATED
WITHOUT DEPHOSPHORYLATION OF
THIS 1433 BINDING SITE AND THAT
DEPHOSPHORYLATION OF THIS
BINDING SITE ON ITS OWN ISN'T
ENOUGH TO ACTIVATE BRAF.
SO WE WANTED TO DO THE PROPER
CONTROLS, WE WANTED TO MAKE SURE
THE ACTIVATION WE'RE SEEING WITH
KRAS IS ACTUALLY DEPENDENT ON
THE ACTIVATION STATE OF KRAS.
SO WHEN IT'S INACTIVE WHEN IT'S
GDP-BOUND BUT HERE IS AN THE
HYDROLYZABLE ANALOGUE OF GTP AND
YOU CAN SEE AGAIN, WE'RE SEEING
ACTIVATION BUT WITH GDP, IT'S
NOT, YOU AND SEE IT OVER HERE AS
WELL.
ANOTHER THING THAT WAS I THINK
REALLY IMPORTANT TO US IS OKAY
SO RAS IS ACTIVATING BUT IS
DIMERIZATION REQUIRED.
IS RAS PROMOTING THE DIMERS TO
FORM, AND SO WE ADDED IN THE
CONTROL OF MUTATING THE CRITICAL
DIMER INTERFACE WITH THIS
RGENINE TO HISTIDINE MUTATION
EMPLOY AND WHAT WE FOUND WAS
THAT FOR BRAF TO BE ACTIVATED BY
RAF IT HAD TO BE DIMERIZATION
COMPETENT.
AND 1 OTHER THING WE DID WAS THE
TITRATION CURVE WE'RE ADDING
INCREASING AMOUNTS OF KRAS AND
AS YOU CAN SEE, THAT THERE IS A
DOSE RESPONSE EFFECT HERE AND
CAN YOU VISUALLY SEE IT HERE IN
THE GEL THAT WE'RE GETTING
INCREASING LEVELS OF ACTIVATION
WITH THE MORE RAS THAT WE ADD.
SO WE WANTED TO GO BACK AND SAY
TO LOOK AT WHAT ABOUT THE PFs
CONTAINING LIPO STUDIES OF
MULTIPLE ENDOCRINES, MAYBE WE
WERE JUST AT THE WRONG
CONCENTRATION, SO WE ALSO DID A
DOSE RESPONSE AND INCREASING
LEVELS OF THE LIPOSOMES, AND
WHAT WE FOUND WAS THAT WE KIND
OF PLATEAUED, BUT WE NEVER SAW
MORE THAN A 2 FOLD INCREASE IN
ACTIVATION.
BUT THEN, WHEN WE MIXED THE LIPO
STUDIES S
OMES WITH THE RAS WHAT WE
SAW WAS AN AUGMENTING EFFECT
HERE OF THE ACTIVATION LEVEL
THAT WE'RE ACHIEVING IF WE HAVE
FULLY PROCESSED KRAS PLUS THE
LIPOSOMES.
BUT REMEMBER, THESE ASSAYS ARE
DONE IN THE TEST TUBES SO
THEY'RE IN SOLUTION, SO WE'RE
KIND OF DEPENDING ON THEM MIXING
AROUND IN THE TEST TUBE, SO WE
THOUGHT IT WOULD BE IMPORTANT TO
TRY TO RECAPITULATE A MORE
AUTHENTIC ENVIRONMENT.
SO IN COLLABORATION WITH SUZANNE
SANDIN IN THE RAS INITIATIVE,
SHE WAS ABLE TO GENERATE FOR US
SOME LIPID NANO DISKS THAT
CONTAINED KRAS, WITH KRAS
TETHERED ON TO THE NANO DISK.
AND SO, THESE ARE THE RESULTS
FROM THAT EXPERIMENT THERE.
AS YOU CAN SEE, IF THE NEW NANO
DISK DID NOT HAVE KRAS PRESENT
WE SAW VERY LITTLE ACTIVATION
BUT THEN FOR THOSE THAT HAD 3
MOLECULES OF KRAS, IT WAS UP TO
REALLY ALMOST 7 FOLD INCREASE IN
ACTIVITY AND IF SHE ADDED MORE
KRAS MOLECULES, PROTEINS, SHE
DOUBLED IT TO 6 KRAS THERE AND
THEN WE SAW AN EVEN FURTHER
INCREASE AND AGAIN, THIS IS A
RAS DEPENDENT EFFECT WE'RE
SEEING IN THE CONTEXT OF THE
LIPID THAT IS PRESENT ON THE
NANO DISK.
SO PUTTING ALL THIS TOGETHER, I
THINK WE HAVE LEARNED A LOT
ABOUT IN OF THE MOLECULAR
MECHANISMS AND SO, IF YOU LOOK
WE ARE OUR AUTOINHIBITTOR
MONOMERAND THEN WHEN YOU HAVE
RAS, BECOMES GTP LOADED AND
ACTIVE IN THE RBD CAN INTERACT,
IT CAUSES THIS CLASH WITH THIS
PROTOMEROF 1433 THAT GENERATES A
CLASH AND CAUSES THE REPULINGS
OF 1433 THAT STARTS TO UNRAVEL
THIS AUTOINHIBITTED COMPLEX.
AND THAT THEN ALLOWS SMP TO COME
IN AND DEPHOSPHORYLATE THAT.
AND I THINK EVEN THOUGH SMP
WASN'T CRITICAL IN OUR TEST
TUBE, I DO FULLY BELIEVE THAT IT
IS IMPORTANT IN THE CELL BECAUSE
REMEMBER, IN THE CELL WHEN YOU
HAVE GROWTH FACTOR SIGNALS
COMING IN, THERE'S A LOT GOING
ON AT THE MEMBRANE, IT'S NOT
JUST RAS SIGNALING, YOU HAVE
ACTIVATION OF OTHER KINASES AND
PHOSPHATASES BUT WHAT THIS DOES,
BY DEPHOSPHORYLATING THIS SITE,
THIS ACTUALLY PROVIDES THE
DIRECTIONALITY OF SIGNALING.
IT DOESN'T ALLOW THIS 1433 TO
COME BACK AND REBIND AND IT
CAUSES THE DIRECTIONALITY TO BE
FORCED INTO THE DIMER FORMATION.
SO I'M GOING TO SWITCH GEARS
COMPLETELY NOW.
SO WE'VE TAKEN A LOT WE LEARNED
ABOUT THE MOLECULAR MECHANISM
BUT I WANT TO TALK ABOUT ITS
ROLE IN HUMAN CANCER AND THE
OTHER RAS-OP A THIES AS WELL.
AND BRAF IS A DRIVER IN BOTH.
SO FOR BRAF MOST OF THE
ONCOGENIC DRIVERS OCCUR WITHIN
THE CATALYTIC DOMAIN AND THESE
ARE MUTATIONS THAT ARE OFTEN
STRONG GAIN OF FUNCTION AND
THEY'VE BEEN SHOWN TO BE
EMBRYONIC LETHAL.
BUT FOR THE
RAS-OPOTHAY MUTATIONS THEY TEND
TO BE WEAKER AND THE REAL HOT
SPOT IS THIS PSYCHIATRISTIN RICH
DOMAIN.
BUT I WANT TO MAKE A POINT HERE
TOO, I THINK IT'S IMPORTANT TO
THIS AUDIENCE THAT SOME OF THE
MUTANTS THAT ARE OBSERVED IN THE
RAF'S OP A THIESS HAVE BEEN
FOUND TO OCCUR WITH OTHER
MUTATIONS WITH OTHER UPSTREAM
COMPONENTS OF THE RAS PATHWAYS.
SUCH AS RTKs, RAFs AND
NSWOBDERFUL--WONDERFUL KNOW 1
EMPLOY SEE WE CAN SEE SOME OF
THESE WEAKER GAIN OF FUNCTION
RAF MUTATIONS, CO DEARING WITH
SOME OF THE OTHER UPSTREAM
COMPONENTS AS WELL.
AND I THINK WHAT WE'RE--WHAT I
HOPE TO SHOW YOU ABOUT WHAT
WE'VE LEARNED ABOUT THE RAS OP A
THIES, WILL BE IMPORTANT
INFORMATION WHEN WE'RE IN THIS
ERA OF PERSONALIZED MEDICINE AND
YOU LOOK AT THE MUTATIONS THAT A
TUMOR HAS AS YOU START TO DECIDE
WHAT THERAPIES OR WHAT TARGETED
THERAPIES YOU WANT TO USE.
SO THE RAS-OP A THIES, THEY'RE
DEVELOPMENTAL, THEY'RE OCCURRING
IN 1-2000 LIVE BIGGERS.
THERE ARE A NUMBER OF DIFFERENT
SYNDROMES.
CRAF HAS BEEN FOUND MOST OFFIN
IN [INDISCERNIBLE] SYNDROME AND
BRAF IS A PRIMARY DRIVER IN
CARDIO FACIALIAL SYNDROME, AND
ALTHOUGH THEY'RE DISTINCT THEY
CAN BE BROADLY CHARACTERIZED BY
DISTINCT CARDIO FACIAL FEATURES,
OFTEN CONGENITAL HEART DEFECS,
SHORT STATURE, AUTISM TRAITS AND
FOR SOME OF THEM AN INCREASED
RISK OF CERTAIN CHILDHOOD
CANCERS.
SO WE WONDERED WHY IS BRAF CRD A
HOT SPOT FOR THE RASOP A
THY MUTATIONS.
SO NOW YOU NEED TO KNOW WHAT IS
THE CRD IN IT'S CALLED THE
CYSTINE RICH DOMAIN.
THERE ARE 2 TYPES.
THERE'S A TYPICAL AND A AN
ATYPICAL.
EVERYONE PROBABLY KNOWS THEM
MOST BECAUSE THEY'RE FOUND IN
PROTIEN KINASE C, AND IN PROTIEN
KINASE C, THEY'RE TYPICAL
DOMAINS AND THEY BIND DIAZAL
GLYCEROL BUT THE ATYPICAL 1S DO
NOT, THEY'RE FOUND IN NUMEROUS
SIGNALING MOLECULES, THEY CAN
IRPT ACT WITH LIPIDS AND
PROTEINS AND THEY'RE OFTEN
INVOLVED IN REGULATING CATALYTIC
ACTIVITY AND LOCALIZATION.
SO WHAT DO WE KNOW ABOUT THE RAF
CRD, WELL IT DOESN'T BIND DAZAL
GLYCEROL IT WAS FIRST
CHARACTERIZED AS BEING A FOSTER
NURSED FOCUSED ON TERILE CYSTINE
BINDING PARTNER AND IT WAS
THOUGHT TO HELP FACILITATE THE
INTERACTION WITH THE MEMBRANE
AND ALLOWING IT ALSO TO CONTACT
RAS.
BUT WHEN WE SAW THE
AUTOINHIBITTED CONFIRMATION AND
WE SAW THE CENTRAL POSITION OF
THE CRD, YOU KNOW WE WONDERED IF
IT ALSO PLAYS A ROLE IN RAF
AUTOINHIBITION.
AND I THINK WHAT WE'RE LEARNING
1 THING THAT'S--I THINK IS
BECOMING A TREND IS SOME OF
THESE REGULATORY DOMAINS IN RAF
ACTUALLY HAVE DUAL FUNCTIONS.
THEY FUNCTION TO HELP MAINTAIN
THE AUTOINHIBITTED STATE BUT
THEY ALSO HAVE AN OPPOSING
FUNCTION AND PROMOTING
ACTIVATION.
AND WE WANTED TO KNOW DOES THE
RAS OP A THY MUTATIONS MUTATIOR
THESE FUNCTIONS.
SO WHEN YOU LOOK AT THE CYSTINE
RICH DOMAIN, YOU LOOK AT 2
GROUPS.
ONE IS THIS LOOP 1 WHICH
INTERACTING WITH THE MEMBRANE
AND CONTACT FOSTER NURSED FET
ILLEGALS SIRRINE.
ELEVEN% ARE FOUND IN THIS REGION
BUT THE REMAINDER ARE FOUND
ELSEWHERE IN THE DOMAIN AND THEY
TEND TO OR SUBSTITUTION THAT
INTRODUCE A BASIC RESIDUE WITH
THIS Q257 R ACCOUNTING FOR 67%
OF THE CRD MUTATIONS AND SO, HOW
DO THEY FUNCTION?
SO WE HAD 3 ASSAYS THAT WE
WANTED TO MONITOR, THE FIRST
BEING THE ABILITY TO BIND FOSTER
NURSED FET ILLEGALS SERIES
POINTSINE SINCE THAT WAS KIND OF
THE MOST WELL KNOWN SO WE WORKED
WITH ANDY STEPHENS GROUP IN THE
RAS MINISHIAATIVE TO DO
PLACEMENT RESIDENCE, ANALYSIS OF
LIPID BINDING SO THIS WE'RE
USING JUST OUR ICE ISOLATED
CYSTINE RICH DOMAIN OF BRAF AND
LOOKING FOR ITS ABILITY TO BIND
NANO DISKS THAT CONTAINED 30%
FOSTER NURSED FOCUSED ON TILE
SERIES POINTSINE AND WE CAN
MONITORING THE BINDING THERE.
OF BUT IF WE FLOW THE SAME
PURIFIED CRD OVER A NANO DISK
THAT HAS 0 FOSTER NURSED FET
ILLEGALS SERINE, AND HAVE
CLONING WE SEE LITTLE BINDING SO
IT IS SPECIFIC FOR THE PHOSPHO
FETAL SERINE.
BUT WE DIDN'T SEE ANY MUTATIONS
IN THE LOOP 1 BUT WE FOUND THAT
THE 1S THAT INTRODUCE THIS
POSITIVE CHARGE WE DID SEE A
SIGNIFICANT INCREASE IN THE
INTERACTION WITH THE FOSTER
NURSED FOCUSED ON TITLE SERINE,
SO IF WE GO BACK AND LOOK AT THE
STRUCTURE AND THIS IS A SURFACE
CHARGE DISTRIBUTION OF THE
CYSTINE RICH DOMAIN AND WE'RE
LOOKINGA THE IT NOW AS THE
MEMBRANE BINDING VIEW, AND SO
YOU CAN SEE HERE'S THE
HYDROPHOBIC POCKET AND IT'S
ACTUALLY VERY HYDROPHOBIC--BUT
THE INTRODUCTION OF RASOP A
THY MUTATIONS HERE ACTUALLY MAKE
THE SURFACE OF THIS DOMAIN MORE
POSITIVE 3 CHARGED WHICH WE
THINK IS CAUSING A MORE
POSITIVELY CHARGED SURFACE WOULD
BE CONDUCIVE FOR THE
INTERACTIONS WITH THE NEGATIVELY
CHARGED PLASMA MEMBRANE.
SO THAT KIND OF, I THINK, I'VE
REALLY ENJOYED SO MUCH ABOUT
THIS WORK IS THAT YOU CAN PUT
STRUCTURE AND FUNCTION TOGETHER
AND YOU CAN KIND OF GET, OKAY,
THIS MAKES SENSE, THIS IS THE
REASON WHY THESE MUTATIONS ARE
HAVING THIS EFFECT AND
ESPECIALLY IF YOU THINK THIS
Q257 R WHICH IS THE PREDOMINANT
MUTATION AND HOW CLOSE IT IS TO
THE HYDROPHOBIC POCKET.
SO NEXT WE LOOK AT
AUTOINHIBITION, AND FOR THIS WE
KNEW, IT WOULD GOING TO BE
REALLY CRITICAL TO COME UP WITH
A LIFESTYLE ASSAY, SO WE
DEVELOPED LIFESTYLE PROXIMITY
BASED ASSAY TO MONITOR OUR
INHIBITION AND IT WAS BASED ON
THESE PREVIOUS FINDINGS THAT IF
YOU JUST EXPRESS THE ISOLATED
KINASE DOMAIN OF BRAF IN A CELL,
IT CAN DIMERIZE ON ITS OWN AND
TRANSMIT A SIGNAL TO PMEK, BUT
IF YOU EXPRESS ANISEOLATED REG
DOMAIN WITH THE KINASE DOMAIN,
THEY CAN ACTUALLY FORM AN
AUTOINHIBITTED MONOMERKIND OF
ACTING IN TRANS, AND BLOCK
SIGNALING TO PMEK.
SO WE WANTED TO KNOW IF WE
TAGGED 1 WITH AN ENERGY DONOR
AND AN ENERGY ACCEPTOR COULD WE
THEN DETECT AUTOINHIBITION?
AND SO THAT'S SHOWN HERE, SO WE
HAD CONSTANT LEVELS OF THE
KINASE DOMAIN, AND AS YOU CAN
SEE, INCREASING AMOUNTS OF THE
REG DOMAIN, AND AS WE ADDED THE
REG DOMAIN, WE ARE DETEBTING A
BRETT SIGNAL IN A DOSE DEPENDENT
MANNER AND WHAT I THINK IS
REALLY IMPORTANT IS THAT YOU CAN
SEE A DOSE DEPENDENT
TRANSMISSION AND THE SIGNAL TO
MEK.
SO YOU CAN SEE THE KINASE
SIGNALING TO MEK WITHOUT THE REG
DOMAIN BUT IT GOES AWAY WHEN THE
REG DOMAIN COMBINE.
SO THAT'S GREAT.
WE CAN DETECT AUTOINHIBITION BUT
WHAT'S CRITICAL CAN WE DETECT
DISRUPTION OF AUTOINHIBITION?
SO WE ADDED A FEW EXTRA
CONTROLS.
SO WE KNOW THAT THE REG DOMAIN
CAN INTERACT WITH RAS, IF THERE
THEY'RE SEPARATED THEN THAT
SHOULD FREE THE REG DOMAIN AND
WOULD SEPARATE THE 2 AND WE
WOULDN'T SEE THE SIGNAL AND
THAT'S ACTUALLY WHAT WE SAW WAS
A REDUCTION IN THE BRETT SIGNAL.
WE ALSO NEEDED V600 DISRUPTED
THE MUTATION STATE AND WE SAW
THAT AGAIN AND WE AWE A
DISRUPTION IN AUTOINHIBITION
EMPLOY SO WE INCORPORATED ALL OF
OUR CRD MUTATIONS AND WITH ALL
OF THE EXCEPTION OF THIS E275-K
MUTATION THEY ALL DISRUPTED
AUTOINHIKING HIB THORSITION
INDICATING THAT THE CRD DOES
INDEED PLAY A CRITICAL ROLE IN
AUTOINHIBITION AND AGAIN IF WE
LOOK AT THE STRUCTURE, WE CAN
SEE THAT THESE RESIDUES ARE IN
CONTACT WITH BOTH THE 1433
PROTOMERAND WITH BRAF BUT THE
275 ALONE IS KIND OF OUT THERE,
IT'S NOT MAKING INTERACTIONS SO
IT SHOULD BE AUTOINHIBITTED.
AND SO THIS KIND OF DIVIDED THE
CATEGORIES OF THE MUTANTS INTO
THOSE THAT RELIEVE
AUTOINHIBITION WHICH ARE ALL 4
OF THESE AND THIS 1 BEING THE
ONLY 1 THAT JUST HAS THE EFFECT
OF INCREASING FOSTER NURSED
FOCUSED ON AT THE TIME ILLEGALS
SIRRINE BINDING SO WE'RE
ANYTHING TO BIOLOGICAL FUNCTION
NOW, AND GO THROUGH THIS PRETTY
QUICKLY, WE CAN FOCUS ON ASSAYS
LOOKING AT PROLIFERATION AND ALL
OF 1S THAT RELIEVED
AUTOINHIBITION, ACTUALLY INDUCED
FOCUS FORMATION AND IT WAS
SEVERELY REDUCED FOR THIS MUTANT
WHICH ONLY HAS INCREASED BINDING
AND IF WE LOOK AT CELLS AT THE
PMEK LEVELS AGAIN IT CORRELATED
WELL.
BUT WE WERE REALLY FORTUNATE TO
BE ABLE TO COLLABORATE WITH THE
ZEBRAFISH FACILITY BECAUSE WE
WANTED A MORE BIOLOGICAL ASSAY.
SO WE'RE USING ZEB RAW DATA FISH
AND WE'RE INYECTING MUTANTS INTO
THE EMBRYOS AND WE HAVE 3 ASSAYS
THAT WE'RE LOOKING AT.
ONE IS THE ELONGATION OR THE
MEASUREMENT OF THE EMBRYOAXIS
AND IT IS, IF YOU HAVE ENHANCED
SIGNALING OF THE PATHWAY WHAT
YOU GET IS ELONGATED EMBRYOS AND
THIS IS A SIGN OF DEFECT ANDS
CONVERGENT EXTENSION MOVEMENTS.
WE ALSO LOOKED AT 3 DAYS AT HARD
EDEMA AND WITH THAT SHOWN HERE
AS--HEART EDEMA AND WITH THAT
SHOWN HERE MEASURING HEART
DEFEBTS AND FAILURE, AND WE ALSO
LOOKED AT THE [INDISCERNIBLE]
ANGLE AND THIS IS KNOWN TO BE A
MEASURE OF HEAD WIDTH, EYE
SPACES WHICH IS A CHARACTERISTIC
OF THE RASOP A THIES.
SO WE TOOK REPRESENTATIVES FROM
EACH OF OUR CLASSES, HERE YOU
CAN SEE THE EXPRESSION IN THE
EMBRYOS AND WAWE FOUND THAT WAS
THAT THE 1S THAT RELIEVED
AUTOINHIBITION ACTUALLY W
ERE--HAD SIGNIFICANT DEFECT
ANDS THERE WAS DEFECTS FOR THE
EE275 K AS WELL BUT IT WAS MUCH
LESS SIGNIFICANT.
IF WE LOOK AT HEART EDEMA,
AGAIN, THE 2 THAT MUTANTS THAT
DISRUPT AUTOINHIBITION, WE SAW
SIGNIFICANT DEFECTS AND AGAIN,
IT WAS REDUCED FOR THE MUTATION
THAT ONLY HAD INCREASED BINDING,
PS-BINDING AND I THINK FINALLY,
WE LOOKED AT [INDISCERNIBLE]
ANGLE AND AGAIN, HERE WE
ACTUALLY SAW NO INCREASE AT ALL
WHERE WITH BOTH OF THE MUTANTS
THAT HAVE AUTOINHIBITION
RELIEVED, YOU DO SEE THE
INCREASE.
AND WE'RE FORTUNATE THAT THESE
MUTATIONS HAVE BEEN REPORTED IN
CHARACTERIZED AND I KNOW THAT
THEY'RE NOT A LOT OF THE
NUMBERS, BUT WHAT YOU FIND IS
THAT THIS GROUP OF ASSAYS DO
CORRELATE WELL WITH WHAT HAS
BEEN OBSERVED WITH THE PATIENTS
AND IN PARTICULAR, THIS WIDE SET
EYES IS OBSERVED IN ALL THE
PATIENTS THAT HAVE THE MUTANTS
THAT DISRUPT AUTOINHIBITION IS
IT'S NOT OBSERVED IN THE
PATIENTS THAT HAVE THE E275 K
MUTATION AND OVERALL, THESE
PATIENTS, THE SEVERITY OF THE
DEFECTS OR THE PHENOTYPE IS
GREATLY REDUCED IN COMPARISON TO
THESE.
SO I THINK THIS IS REALLY
TELLING US THAT RELIEF OF
AUTOINHIBITION IS REALLY THE
MAJOR DETERMINANT FOR THESE
RASOP A THY CRD MUTATIONS AND
HOW SEVERE THE DEFECTS ARE GOING
TO BE.
SO I THINK WHAT I WOULD LIKE--I
HOPE THIS HAS CONVINCED YOU THAT
THE STUDY OF THIS DISEASE
ASSOCIATED MUTATIONS CAN REVEAL
IMPORTANT REGULATORY INFORMATION
AND IT DEMONSTRATES THE CRITICAL
ROLE OF THE CRD AND HOW TAKING A
MULTIFACETED APPROACH OF LOOKING
AT THE STRUCTURE, DOING
BIOCHEMISTRY, HAVING LIVE CELL
ASSAYS AND THEN GOING TO
AN ANIMAL MODEL SYSTEM THAT DOES
FULLY KIND OF REPRESENT OR IS A
GOOD MODEL FOR THE HUMAN DISEASE
CAN BE VERY BENEFICIAL SO WITH
OUR ASSAYS IN PLACE, WE HAVE
BEEN INTERACTING WITH THE NCI
ADVANCING RASOP A THIES, WHICH
IS A COLLABORATION BETWEEN THE
CLINICAL GENETICS BRANCH, THE
PEDIATRIC ONCOLOGY BRANCH AND
THE NCI RAS INITIATIVE AS WELL
AND IT'S TAKING A GENETICS FIRST
APPROACH TO IDENTIFY NEW MUTANTS
AND WE HAVE THESE ASSAYS, THE
ZEBBIA FISH ASSAYS IN PLACE AND
IN FACT, MARIO IS IN OUR
LABORATORY IN THE LCDS AND
UTILIZING THESE ASSAYS NOW IN
ANALYZING SOME NEW RAS MUTANTS,
HRAS MUTANTS.
AND WE ALSO WANT TO USE IT TO
DEVELOP NEW THERAPIES SO I WILL
GIVE A SHOUT OUT TO ALSO THE
MOLECULAR TARGETS, PROTEIN WHICH
IS LOCATED IN FREDERICK, AND IT
HAS THE LARGEST AND MOST DIVERSE
COLLECTION OF THE NATURAL
PRODUCTS AND THERE ARE OVER
200,000 PARTIALLY PURIFIED
FRACTIONS FROM OVER 80,000
ORGANISMS BOTH AQUATIC, MARINE,
AND OTHERS.
AND WE IDENTIFIED A NATURAL
PRODUCT THAT IS A COMPOUND
THAT'S ISOLATED FROM THE EDIBLE
PLANTS YOU SEE CANNED WHAT,
WE'RE CALLING IT A DIMER
DISRUPTER AND IT'S JUST SHOWING
IT CAN DISRUPT BRAF, CRAF DIMERS
AND SUPPRESSES SIGNALING TO
PMEK, AND WHEN WE INCUBATE OUR
EMBRYOS WITH THE DIMER
DISRUPTER, HERE YOU CAN SEE THE
ELONGATIONS THAT'S INDUCED BY
THIS RAS OPERATING GLOBALLYATHY
MUTANT AND THAT IT'S SUPPRESSED
HERE IF IT'S IN THE PRESENCE OF
THE DIMER DISRUPTER.
AND SOMETHING THAT WE'RE REALLY,
REALLY EXCITED ABOUT, WE
THOUGHT, WELL IF IT'S
DISRUPTING, DIMERIZATION HERE,
MAYBE LET'S ANY BACK AND RETHINK
ABOUT THE RESISTANCE TO
[INDISCERNIBLE] RAF AND
MELANOMA.
BECAUSE WHAT WE LEARNED IN MEL
AN OHM IS THAT THE REASON
RESISTANCE IS DEVELOPED IS OFTEN
DUE TO MUTATIONS AND UPTREME
COMPONENTS, LIKE THE ACQUISITION
OF RAS MUTATIONS OR THE
OUTGROWTH OF A MUTATION ACTUALLY
IN A V600 E WHICH IS A
TRUNCATION MUTANT THAT
CONSTITTATIVELY DIMERIZES AND SO
AGAIN, WHEN YOU HAVE RESISTANCE,
WHAT HAPPENS IS YOU GET RESTORE
STIGINALLING AND ALL OF THESE
MECHANISMS REQUIRE RAF
DIMERIZATION.
SO CAN WE CO TREAT WITH THE RAF
DISRUPTERROR INHIBIT THE
ACQUISITION OF RESISTANCE TO
[INDISCERNIBLE] AND THIS IS
SHOWING THE EXPERIMENTS ARE
DONE, YOU CULTURE, THIS IS SKML5
HERE AND WE'RE GROWING THEM
CONSTITTATIVELY IN TREATMENT, I
THINK HAD IS A 3-4 WEEK
TREATMENT AND YOU GET THE
EMERGES ENSEL OF RESISTANT CELLS
BUT AS YOU CAN HERE IN THE
COMBINATION OF THE DIMER
DISRUPTER, WE'RE ACTUALLY
PREVENTING THE RESISTANCE CELLS
FROM COMING OUT WHERE BY ITSELF,
IT REALLY HAS VERY LITTLE
EFFECT, AND WE FOUND THIS TO BE
THE CASE IN A NUMBER OF THE
MELANOMA LINES THAT WE LOOKED AT
AND WE'RE WORKING NOW TO TRY TO
OPTIMIZE, YOU KNOW THE STRUCTURE
FUNCTION ACTIVITY OF THESE
MOLECULES.
SO I'LL STOP THERE AND THANK THE
PEOPLE THAT DID THE WORK AND SAY
THAT DOING SCIENCE TAKES A
VILLAGE AND I'M REALLY FORTUNATE
AND THANKFUL THAT MY VILLAGE HAS
BEEN THE NCI BECAUSE AS YOU CAN
SEE HERE I'VE REALLY BENEFITED
FROM OUR COLLABORATORS AND THE
STRUCTURAL BIOLOGY LAB, THE
MOLECULAR TARGETS, THE R,A S
INITIATIVE AND OUR NOW
COLLABBATION WITH THE LAWRENCE
LIVERMOORE, AND I WANT TO SHOUT
OUT THAT DAVE DURAN IN MY LAB
THE 1 WHO REALLY WORKED WITH AND
SOLVED THE STRUCTURES.
DAN'S BEEN DOING KEEN ACE
ASSAYS, RUSSELL DID ALL OF THE
RASOP A THY ANALYSIS TOGETHER
WITH OUR ZEBRAFISH CORE
FACILITY, I WILL STOP THERE AND
ANSWER ANY QUESTIONS AND THANK
YOU.
[ APPLAUSE ]
>> REALLY NICE.
SO I WILL TAKE INTRODUCERS
PREROGATIVE.
SO IF I'M FOLLOWING THE MODEL
YOU SHOULD PERHAPS SEE, YOU GUYS
FIGURED THIS MODEL OUT SO THE
TUMOR SHOULD HAVE MAYBE AND
WOULD HAVE MUTATIONS ON THE
1433, THAT WOULD PREVENT IT FROM
THAT AUTOINHIBITTORY COMPLEX,
RIGHT?
SORE IS DUS RUPGZ OF THAT AND
INTERACTION NOT ENOUGH TO EXPOSE
THE LOOP FOR DEPHOSPHORYLATION?
>> SO THERE'S GOING TO BE THE
SELECTIVE PRESSURE, I GUESS FOR,
OKAY, SO YOU'RE TALKING ABOUT ON
A MUTATION IN 1433.
>> RIGHT.
>> WELL 1433 IS KNOW WOOF THOSE
IT HAS A DUAL FUNCTION.
IT'S REQUIRED TO STABILIZE THE
AUTOINHIBIT AND MONOMERBUT IT IS
REQUIRED FOR DIMER STABILITY
BECAUSE IF YOU MUTATE THOSE
C-TERMINAL SITES--
>> IT MIGHT KICK AWAY BUT IT
WON'T HOLD--
>> NO, IT WON'T HOLD.
IN FACT MUTATION OF THAT SITE
CAN BE A DOMINANT NEGATIVE IF
YOU OVEREXPRESS IT IN CELLS.
BUT, I WILL SAY, WHICH IS A
LITTLE--IT'S NOT YOUR QUESTION,
BUT FOR CRAF THAT 1433 BINDING
SITE THAT'S INVOLVED IN THE
AUTOINHIBITION, AND INTERNAL 1
THAT'S THE CRAF HOT SPOT IN THE
RASOP A THIES, AND THAT'S A
COMMON MUTATION PICKED UP IN
DRUG RESISTANCE AS WELL, IN SO
THEN WHY DO WE HAVE RAF A
ANYWAY?
IT DOESN'T SEEM TO DO ANYTHING?
>> WELL WE FOUND OUT--
>> WELL YOU SHOW TODAY WASN'T
INTERACTIVE, IS IT DOMINANT
NEGATIVE?
>> NO, IT'S NOT A DOMINANT
NEGATIVE, SO THERE'S A QUESTION
OF ARE WE NOT LOOKING IN THE
RIGHT PLACE OR IN THE RIGHT
CONDITION IN.
>> RIGHT, RIGHT.
ALL RIGHT, OTHER QUESTIONS?
>> DEBBIE BEAUTIFUL, BEAUTIFUL
TALK AND RESEARCH.
QUESTION WITH OUR DIMERIZATION
RISK ASSESSMENTS DID YOU WANTER
DID YOU
WANT--DISRUPTER, 1 OF THE
QUESTIONS IS [INDISCERNIBLE]
GROUP HAS SHOWN BEAUTIFULLY THAT
THE MEK INHIBITORS WORK WELL
WITH A CERTAIN GROUP OF NF1
MUTANT PATIENTS BUT YOU GD LOOK
AND SEE THE PHENOTYPE WHEN YOU
INTERFERE WITH THE DIMERIZATION
OF THE RAF TO WHAT DEGREE DOES
IT MIMIC OR IS IT DISTINCT FROM
PI3--I'M SORRY NOT PI3 KINASE
BUT MEK INHIBITION OR THOSE
KINDS OF EXPERIMENTS TO TRY TO
HAVE IMPACT WITH OTHER RASOP A
THIES.
>> YOU KNOW RIGHT NOW WE'RE
TRYING TO FIND THE EXACT--IT'S
DISRUPTING DIMERIZATION, BUT HOW
IS IT DISRUPTING DIMERIZATION IN
AND WE'RE WORKING AND DOING SOME
OF THESE CANNED WHAT EXPERIMENTS
RIGHT NOW LOOKING FOR IF IT
FINDS DOES IT TABLIZE THE--IS IT
THERMAL STABILITY AND DOING MASS
SPEC. ON IT?
IT DOESN'T SEEM TO BE A DIRECT
BINDER OF THE RAF OR OF THE MEK
PATHWAY, BUT IT DOES SEEM TO
HAVE EFFECTS MODULATING THE 1433
INTERACTION WHICH IS CRITICAL
FOR DIMERIZATION, TABLE
DIMERIZATION, BUT AGAIN THIS IS
IN THE EARLY STAGES OF SORTING
THAT OUT.
BUT MY QUESTION WAS THAT, TOO.
IS IT, YOU KNOW IS IT BINDING TO
RAF AND DISRUPTING OR IS IT
HAVING EFFECTS SIMILAR TO THE
MEK INHIBITOR BUT IT SEEMS TO BE
DIFFERENT.
>> BEAUTIFUL STORY, BEAUTIFUL
TALK, FANTASTIC OVER THE YEARS.
QUICK QUESTION, MAYBE YOU
MENTIONED THIS AND I MISSED IT,
DOES THE DIMER DISRUPTOR
INTERFERE WITH KRAS CELL
PROLIFERATION OR GROWING IN.
>> THAT'S A GOOD QUESTION.
SO YES AND NO.
SO IT CAN HAVE AN EFFECT IF IT'S
IN MELANOMA WHERE THERE'S NOT A
LOT OF UPSTREAM SIGNAL FREE
RADICALS GENERATED OTHER RTKs,
BUT WE'VE--AND THAT'S WHY WE'VE
KIND OF WANTED TO FURTHER
INCREASE ITS EFFICACY.
IF YOU HAVE LIKE IN A COLORECTAL
CANCER WHERE YOU'VE GOT AND YOU
HAVE A RAS MUTATION BUT THERE'S
A LOT OF SIGNALING COMING FROM
THE EGF RECEPTOR, IT'S NOT VERY
EFFECTIVE, SO, IT SEEMS, YOU
KNOW, IT'S IN THE MELANOMAS
WHERE REALLY, YOU DON'T HAVE A
LOT OF SIGNALING UPSTREAM OF RAF
SO IT'S NOT THAT POTENT.
>> SO BY EXTENSION WOULD YOU
ENVISION THE DIMER DISRUPTER TO
BE AN OBLIGATE COLD DRUG AS
[INDISCERNIBLE] INHIBITOR
TREATMENTS, HER 2 INHIBITORS, IS
THAT WHAT YOU'RE THINKING?
>> WELL, YOU KNOW THAT'S
SOMETHING WE HAVEN'T DONE.
THOSE ARE EXPERIMENTS ACTUALLY
THAT'S ON WELL, EXPERIMENTS WE
WANT TO DO.
YES.
WE'RE LOOKING AT IT.
WE STARTED IT WITH THE RAF
INHIBITOR, THE CO TREATMENT OF
THE RAF INHIBITOR, WE NEED TO
GO, WE NEED TO DO THE EGF
RECEPTOR, SOMETHING UPSTREAM.
>> OTHER QUESTIONS IN QUESTIONS?
ALL RIGHT.
LET'S THANK DEBBIE.
THANK YOU SO MUCH.