I WOULD LIKE TO WELCOME YOU ALL
TO THE WEDNESDAY AFTERNOON
LECTURE SERIES.
MY NAME IS LAURA KALEY, I AM A
SENIOR INVESTIGATOR AND BRANCH
CHIEF OF THE SOCIAL AND
BEHAVIORIAL RESEARCH BRANCH IN
THE NATIONAL HUMAN GENOME
RESEARCH INSTITUTE.
TODAY'S LECTURE AND SPECIAL FOR
AT LEAST 2 REASONS, 1 REASON IS
THAT THIS IS AN NIH MARGARET
PITTMAN LECTURE WHICH I WILL
EXPAND UPON BRIEFLY IN A MOMENT
THE OTHER REASON IS THAT OUR
SPEAKER DR. GG LOZANO CAN
FINALLY JOIN US IN PERSON.
WE NATIONAL NOMINATED DR. LOZA2
YEARS AGO, SHE WAS SCHEDULED TO
SPEAK DURING LAST WALs VISIT
BUT POSTPONED SO THAT COULD SHE
COULD DELIVER HER TALK IN THE
FLESH.
SO HERE SHE IS, IT'S BEEN REALLY
EXCITING PLANNING FOR HER VISIT
AND BUILDING UP FOR THIS LECTURE
AND WE'RE REAL LE EXCITED TO
HAVE HER HERE.
DR. ROTINI WILL INTRODUCE OUR
SPEAKER TODAY BUT I WANTED TO
SAY A FEW WORDS ABOUT THIS
REMARKABLE PERSON, MARGARET
PITTMAN FOR APPROXIMATE WHOM
THIS LECTURE IS IN HONOR OF.
MARGARET PITTMAN CAME TO
MEDICINE EARLY IN OUR LIFE,
HELPING HER FATHER WHO WAS A
COUNTRY DOCTOR IN RURAL ARKANSAS
AND SHE BECAME SMITTEN WITH
BIOLOGY AFTER EARNING A
BACHELOR'S DEGREE AND BIOLOGY
AND MATHEMATICS SHE WENT INTO
TEACHING AND SHE USED THE MONEY
THAT SHE SAVED FROM TEACHING TO
GO TO THE UNIVERSITY OF CHICAGO
AND EARNED IN 1928 HER DOCTORATE
IN BACTERIAERIOLOGY.
DR. PITTMAN CAME TO THE NIH IN
1936 BY WAY OF THE ROCKEFELLER
INSTITUTE AND THE NEW YORK STATE
DEPARTMENT OF HEALTH.
AT THE NATIONAL INSTITUTES OF
HEALTH, HER CAREER PATH LED HER
TO BECOME THE VERY FIRST WOMAN
TO HOLD THE POSITION OF
LABORATORY CHIEF, HEADING THE
LABORATORY OF BACTERIAL PRODUCTS
DIVISION OF BIOLOGICS AND
STANDARDS FROM 1957 TO 1971.
DR. PITTMAN ISOLATED THE
INFLUENZA STRAIN, RESPONSIBLE
FOR MOST CHILDHOOD MENINGITIS,
AND HELPED IDENTIFY THE CAUSE OF
EPIDEMIC CONJUNCTIVITIS AND MADE
KEY OBSERVATIONS THAT LED TO THE
DEVELOPMENT OF THE SALMONELLA
VACCINE.
IN 1970, MARGARET PITTMAN WAS
RECOGNIZED WITH THE FEDERAL
WOMEN'S AWARD AND SHE SERVED AS
THE PRESIDENT OF THE SOCIETY OF
AMERICAN BACTERIAERIOLOGYISTS
AND OF THE WASHINGTON ACADEMY OF
SCIENCES.
ALTHOUGH DR. PITTMAN RETIRED IN
1971, SHE KEPT WORKING AT THE
NIH AS A GUEST UNTIL 1993.
THUS, SHE CONTRIBUTED TO THE
SCIENTIFIC COMMUNITY FOR ALMOST
50 YEARS.
THE MARGARET PITTMAN LECTURESHIP
CREATED IN 19 HONORS DR. PITTMAN
HONORS HER ACHIEVEMENTS AT THE
NATIONAL INSTITUTES OF HEALTH.
AS WELL THE MARGARET PITTMAN
LECTURE HONORS A RESEARCHER WHO
EXEMPLIFIES THE INTELLIGENCE
SCIENTIFIC EXCELLENCE AND DRIVE
OF DR. PITTMAN AND WHOSE
DEDICATED TO ADVANCING AND
IMPROVING THE CAREERS OF WOMEN
SCIENTISTS.
DR. LOZANO IS MOST CERTAINLY
DESERVING OF THIS HONOR.
SO ON BEHALF OF THE WOMEN
SCIENTIST ADVISORS, I WOULD LIKE
TO WELCOME DR. GG LOZANO TO THE
NIH AND WE ARE REALLY LOOKING
FORWARD TO HEARING MORE ABOUT
YOUR WORK.
WITH THAT I WOULD LIKE TO
INTRODUCE DR. CHARLD ROTINI WHO
WILL INTRODUCE DR. GIGI LOZANO.
>>THANK YOU SO MUCH FOR THAT
WONDERFUL COMEBTARY ABOUT
MARGARET PITTMAN.
IT IS INDEED MY HONOR TODAY TO
INTRODUCE OUR SPEAKER BUT BEFORE
I DO THAT, I--MY NAME IS
DR. ROTINI, AND I AM A MEMBER OF
THE NATIONAL WHOLE GENOME
INSTITUTE.
I HAVE QUICK ANNOUNCEMENTS, 1
HAS TO DO WITH CONTINUING
MEDICAL EDUCATION, THE CODE IS
44434.
AND I REPEAT, 44434.
OKAY, SO, PLEASE SIGN UP AND GET
YOUR CREDIT FOR ATTENDING THIS
WONDERFUL TALK.
ALSO, THERE'S A FEW WATCHING
REMOTELY, VIA THE NIH VIDEOCAST.
YOU CAN SEND LIVE FEEDBACK TO
THE SPEAKER, SIMPLY CLICK ON THE
BUTTON THAT SAYS SEND LIVE FEED
WHICH IS BELOW THE VIDEOCAST
WINDOW.
TYPE IN YOUR MESSAGE AND WE WILL
RELATE IT TO OUR SPEAKER AT THE
END OF HER LECTURE TODAY.
NOW, MY TREMENDOUS HONOR TO
INTRODUCE OUR GUEST DR. LOZANO
IS THE HERBERT L. OLIVE STRINGER
DISTINGUISHED CHAIR IN ONCOLOGY
IN HONOR OF SUE [INDISCERNIBLE]
STRINGER AT MD ANDERSON.
'S RENOWNED GENETICIST, SHE'S
RECOGNIZED FOR HER STUDIES OF
THE P53 TUMOR IN THE PATHWAYS
WHICH IS ON THE MIND IN THE
LARGE PERCENT OF HUMAN CANCER
VIA MUTATIONS AND DELETIONS OF
P53.
USING MASS MODEL DR. LOZANO AND
HER TEAM IDENTIFIED P53 PROTEINS
AS A TRANSCRIPTIONAL ACTIVATOR.
THEY SHOWED THAT MDM2 AND MD
M PROTEINS ARE INHIBITOR OF 13,
AND OVER TIME OUR LAB STUDIED
MULTIPLE MUTATIONS AND
EXTENSIVELY CHARACTERIZING THE
EFFECT ON THE TUMOR SUPPRESSION
AND TUMOR REGENESIS.
HER GROUND BREAKING WORK HAS
PROVIDED INVALUABLE TESTING
NOVEL THERAPEUTIC ISHT VENTIONS,
DR. LOZANO RECEIVED HER
UNDERGRADUATE DEGREE IN BIOLOGY
FROM THE UNIVERSITY OF TEXAS RIO
GRAND VALLEY, FORMERLY THE PAN
AMERICAN UNIVERSITY.
SHE RECEIVED HER Ph.D. IN
BIOCHEMISTRY FROM ROUGH ATOM
CHESTER UNIVERSITY CONDUCTED
POST DOCTORAL TRAINING AT
PRINCETON UNIVERSITY.
I SHOULD NOTE THAT GIGI, THAT'S
WHAT WE CALL HER, LET'S JUST SAY
THAT SHE SPENT SOMETIME HERE AT
THE NIH AND THAT IS WHERE WE
THINK SHE RECEIVED THE BUG OF
WHAT IS CALLED THE SCIENTIFIC
BUG AND THE RESEARCH BUG AND SHE
WAS AT NCI [INDISCERNIBLE] AND I
WAS JUST TALKING TO MICHAEL
GOTTESMAN, HE REMEMBERS ALL OF
THE STORY ABOUT GIGI AND
INTERRUPTION WITH NCI SO
HOPEFULLY AT SOME POINT WE CAN
TALK ABOUT THAT.
SO, JUST TO COMPLETE THE
INTRODUCTION, DR. LOZANO HAS
PICKED UP MANY ACCOLADES AND
HONORS ALONG THE WAY, SHE IS A
MEMBER OF THE AMERICAN RESEARCH
ACADEMY AND MEMBER OF THE
ACADEMY ARTS AND SCIENCES,
NATIONAL ACADEMY OF SCIENCES AND
NATIONAL ACADEMY OF MEDICINE AND
RECIPIENT OF THE LEADERSHIP ARK
WARD FOR ADVANCING WOMEN AND
MINORITY FACULTY AT MD CANCER
CENTER.
IN 2020 DR. LOZANO 1 OF THE MOST
INSPIRING, HISPANICS, LATINO
SCIENTISTS IN AMERICA.
THIS YEAR DR. LOZANO,
[INDISCERNIBLE] A WORD FOR
FACULTY LEADERSHIP AT MD
ANDERSON AND I WOULD BE VERY
REMISS NOT TO SAY THAT AT A
PERSONAL LEVEL AS A SCIENTIFIC
DIRECTOR OF NHGRI, DR. LOZANO
STAYS ON OUR BOARD OF SCIENTIFIC
COUNSELORS AND WE HAVE JUST
TAPPED HER SERVICES AGAIN TO BE
ON OUR BLUE RIBBON PANEL.
SO THE TITLE OF HER TALK TODAY
IS MUTANT P53 ACTIVITIES IN
MOUSE TUMOR MODELS.
GIGI, WELCOME BACK TO THE NIH.
>>THANK YOU CHARLES FOR THAT
WONDERFUL INTRODUCTION.
IT'S GREAT TO BE BACK.
I REALLY--NIH WAS MY VERY FIRST
LAB EXPERIENCE.
I LEARNED MOLECULAR BIOLOGY HERE
AND I THINK I'M JUST VERY GLAD
TO BE BACK APPROXIMATE SEE SOME
OLD FRIENDS AND TO TO FRIENDS U
FOR YOUR CONTRIBUTIONS TO MY
CAREER.
SO TODAY I WILL TALK ABOUT OUR
MODELS AND I CHANGED THE TITLE
JUST A LITTLE BIT TO INCLUDE
SOME WILD-TYPE P53 ACTIVITIES
BECAUSE, WE'RE TRYING TO REALLY
UNDERSTAND NOT JUST WHAT
WILD-TYPE PPEAF DOES BUT WHAT
HAPPENS WHAT WHEN IT'S NOT THERE
AND WHAT OTHER ACTIVITIES MUTANT
P53 MIGHT HAVE.
SO I THINK THIS WILL WORK.
OKAY, THESE ARE MY DISCLOSURES.
SO I'M GOING TO FIRST INTRODUCE
THE P53 PATHWAY.
HOPEFULLY--I WONDER IF I HAVE AN
ARROW?
PROBABLY NOT.
OKAY.
SO THE P53 PROTEIN IS PRESENT AT
VERY LOW LEVELS IN CELLS, IT'S
HARDLY DETECTABLE, BUT STRESS,
DNA DAMAGE, AND INAPPROPRIATE
ACTIVATION OF AN ONCA GENE WILL
UNLEASH A SERIES OF KINASES THAT
PHOSPHORYLATE P53 AND MAKE IT A
VERY STABLE PROTEIN.
AND THEN IN TURN P53 FUNCTIONs
AS A TRANSCRIPTION FACTOR,
TETRAMERIC TRANSCRIPTION FACTOR
THAT ACTIVATES HUNDREDS OF GENES
AND I ONLY SMOA SOME OF THEM ON
THIS SLIDE AND IN THESE GENES
HAVE IMPORTANT CELLULAR
FUNCTIONS.
PROBABLY THE FIRST 1 THAT WAS
CHARACTERIZED WAS THE ABILITY TO
INITIATE APOPTOSIS, SO P53 CAN
KILL THE CELL, IT CAN ALSO KILL
THE CELL BY ANOTHER PROCESS
CALLED FERROPITOSEIS, BUT IT CAN
ARREST THE CELL, SO IT CAN CAUSE
THE CELL TO ARREST OR UNDERGO
SENESCENSEL.
THIS CELL PSYCHE ALTHOUGH REST
IS NOT A DEAD END, SENESCENSEL
IS A DEAD END.
IN ADDITION, P53 HAS ABILITY TO
REGULATE ITS OWN LEVEL SO
THERE'S WAYS TO ACTIVATION TO
LEAD TO SURVIVAL OF THE CELL AND
IT'S THROUGH THE PROTEIN MDM2,
IT'S A P53 TARGET.
AND IN TURN MDM2 ENCODES LIGASE
THAT TARGETS P53 FOR
DEGRADATION.
SO I THINK WITH THIS MY OPENNIC
VIEW OF THE P53 PATHWAY, CAN YOU
BEGIN TO SEE THAT P53 HAS SHOW
MANY CHOICES IN THE CELL, IT CAN
KILL THE CELL, STOP THE CELL AND
SAY TIME TO GO ON.
AND EVEN THOUGH WE KNOW SOME OF
THE CIRCUMSTANCES WHERE P53
MAKES THESE DECISIONS, MOST OF
THE TIME, WE CAN'T GUESS WHAT
P53 IS GOING TO DO IN THE CELL.
SO THE FIRST FEW SLIDES BECAUSE
WE WERE INTERESTED IN TRYING TO
UNDERSTAND WHAT THE TARGETS ARE
AND COULD THEY HAVE POTENTIAL
ROLES AS TUMOR SUPPRESSORS IN
AND OF THEMSELVES.
IT'S BEEN VERY HARD TO REGRIEWS
P53S INTO THE TUMORS AND THE
IDEA WAS MAKE WE CAN REACTIVATE
AN IMPORTANT DOWN STREAM TUMOR
SUPPRESSIVE PATHWAY.
SO I HAVE TO ALSO INTRODUCE THE
MDM2 MOUSE BECAUSE THIS IS A
TOOL THAT WE USE IN THE LAB, SO
DILUTION OF MDM2 IS AN
EMBRYO--WHOOPS, WRONG SLIDE.
DELETION OF MDM2 IS EMBRYO
LETHAL.
OOPS, I'M PRESSING THE WRONG
BUTTON.
I THOUGHT I WAS PRESSING THE
RIGHT BUTTON.
OKAY, MDM2 LOSS LEADS TO EMBRYO
LEATHAL PHENOTYPE AT THE BLAST
SIS STAGE, THIS CELL IS
UNDERGOING APOPTOSIS, EVERY 1 OF
THOSE CELLS IS DEAD AND WE ASKED
THE QUESTION WHAT IS THE ROLE OF
PPEAR IN THIS PROCESS AND IF WE
DELETE P53, WE GET A PERFECTLY
VIABLE MOUSE, SO THIS REALLY
CEMENTED THE RELATIONSHIP
BETWEEN MDM2 AND PPARADOXICAL
AND IN FACT WE NOW HAVE
CONDITIONAL ALLELES AND WE'VE
TAKEN MDM2 OUT IN ANY TISSUE, IT
IS ALWAYS A CELL LETHAL AND IT'S
ALWAYS IN A P53 DEPENDENT
FASHION.
SO WHAT WE'VE DONE THEN IS WE'VE
USED A MODEL IN WHICH WE USE
TISSUE AND M
ACROPHAGES--TAMOXIFEN AND
WITHIN A FEW DAYS OF INJECTION,
ALL THE ANIMALS ARE DEAD SHOWN
HERE BY THE RED LINE AND AND
THIS IS A P53 DEPENDENT PROCESS
BECAUSE IF YOU REMOVE P53, THE
ANIMALS ARE PERFECTLY VIABLE.
SO THIS ALLOWS US THEN TO ASK,
WHAT HAPPENS--WHAT IS THE P53
PHYSIOLOGICAL RESPONSE UPON MDM2
DELETION.
AND WE DID THIS IN A SIMILAR
MANNER, BUT IN AN ACUTE WAY SO
THAT 24 HOURS AFTER THE LAST
INJECTION OF TAMOXIFEN IN WHICH
WE HAVE 50% OF THE CELLS THAT
HAVE LOST MDM2 WE CAN EVALUATE
THAT TRANSCRIPTIONAL PROGRAM.
AND THE FIRST THING WE DID WAS
JUST LOOK AT THE PATHOLOGIES OF
SEVERAL OF THE TISSUES IN THE
MOUSE, AND JUST 24 HOURS AFTER
RECOMBINING AND LOSING MDM2, WE
HAVE ALL THESE FAZINATING
PATHOLOGIES, THE HEART AND THE
OVARY, I'M PUSHING THE RIGHT
BUTTON--THE HEART AND THE OVARY
ARE PERFECTLY NORMAL, THEY DON'T
SHOW ANY TIME POINTSA THE THIS
POINT BUT CAN YOU SEE THE
INTESTINE, KIDNEY, PAN KRIST AS
HAVE ALL TYPES OF PHENOTYPES.
IN THE INTESTINE WE SEE LOSS OF
THE CRIPS, IN THE KIDNEY WE SEE
DILATED TUBULES AND SUGGESTING
DYSFUNCTION THERE AND IN THE
PANCREAS WE SEE THIS SAR
METADUCTILE TRANSPLACIA.
SO THEN WE LOOK AT ADDITIONAL
TARGETS OF P53 AND WE IDENTIFIED
HUNDREDS OF TARGETS SO FOR
EXAMPLE IN THE PANCREAS, WE
IDENTIFIED 206 P53 TARGETS.
IN THE OVARY WHICH HAD NO
PHENOTYPE, WE IESHES DENTIFIED
21 TARGETS AND THESE ARE P53
DIRECT TARGETS BECAUSE WE
BASICALLY TOOK THE RNA SEQ DATA
AND COMBINED IT WITH CHPSEQ DATA
THAT WAS AVAILABLE IN THE
LITERATURE AND THIS IS THE P53
TRANSCRIPTIONAL PHYSIOLOGICAL
PROGRAM.
SO I THINK THERE ARE 2 IMPORTANT
POINTS I WANT TO MAKE, THERE ARE
ONLY 7 GENES THAT ARE COMMON
BETWEEN THESE 5 TISSUES AND MDMD
IS 1 OF THEM.
SO IT'S INTERESTING THAT MDM2 IS
REGULATED BY P53 IN THE
PHYSIOLOGICAL SYSTEM IN EVERY
TISSUE THAT WE ANALYZED.
BUT IT'S PROBABLY DOING IS
BALANCING THAT P53 LEVELS, MAYBE
AT DIFFERENT TIMES DURING THE
CELL CYCLE.
POLK IS A POLYMERASE, EDA2 R IS
A CELL SURFACE RECEPTOR INVOLVED
IN CELL DEATH.
BUT TO ME THE OTHER FASCINATING
ASPECT OF THIS SLIDE IS HOW MANY
TARGETS ARE TISSUE SPECIFIC.
SO FOR EXAMPLE, AGAIN IN THE
PANCREAS, 135 TRANSCRIPTIONAL
TARGETING SYSTEM EXPOSURE TO
RADIATIONS ARE ONLY EXPRESS
INDEED THE PANCREAS, SO THAT
TELLS YOU THERE'S A HUGE
DIVERSITY OF TRANSCRIPTIONAL
TARGETS THAT P53 CAN REGULATE.
WE'VE DONE 1 ADDITIONAL
VALIDATION EXPERIMENT TO ASK IF
THIS IS A TRUE P53 RESPONSE BY
ERADIATING THE WHOLE MOUSE AND
ISOLATING THESE SAME TISSUES,
AND YOU CAN SEE HERE IN THE
MIDDLE IS A WILD-TYPE MOUSE THAT
WAS RADIATED, SO THOSE ARE THE
SAMPLES THAT THE TRANSCRIPTIONAL
TARGETS--OKAY, I'M NOT USING THE
POINTER ANYMORE.
SO THAT MIDDLE PORTION IS
WILD-TYPE P53 AFTER RADIATION
AND THOSE ARE THE LIKE LYNN G
AND ED82 R TARGETS THAT ARE
UPREGULATED IN A WILD-TYPE
ERADIATED MOUSE BUT NOT IN A P53
NULL MOUSE.
SO THIS IS NOT JUST--THIS IS THE
TRANSCRIPTIONAL PROGRAM, WITH
LOSS OF MDM2 AND IN THE PRESENCE
OF RADIATION.
SO I THE OTHER MAJOR FINDING WAS
REALLY WHEN WE TRIED TO ANALYZE
THIS ASENAR PHENOTYPE IN THE
FACIA.
BECAUSE AFTER 24 HOURS WE SEE A
COMPLETE SORT OF REWIRING OF THE
PANCREAS FROM NORMAL AT THE TOP
LEFT TO THIS DUCTILE STRACTURE
AND WE STAINED WITH THE DUCTILE
STAINING YOU CAN SEE THE BROWN
STAINING SO YOU CAN SEE A
PLASTICITY OF TRANSITION THAT
OCCURS AND THE DATA ARE
QUANTIFIED ON THE RIGHT.
SO THE QUESTION WE WANTED TO
ASK, WE WANTED TO DEVELOP A
MODEL THAT WE WERE JUST LOOKING
AT THIS TRANSITION IN THE
PANCREAS AND WE USED AN SNR
SPECIFIC KREE RECOMBIN ACE, THIS
IS A--THIS IS MST 1 CREE ER
TRANSGENE SO WE ADD TAMOXIFEN
AND WE ASKED OURSELVES DO YOU
SEE DELETION OF MDM2.
WHEN WE COMBEAR THE
RECOMBINATION FREQUENCIES FROM
FIRST MOUSE AND THIS MOUSE, THEY
ARE SIMILAR SO WE ARE DELETING
MDM 2 AND ACTIVATING P53 IN THE
PANCREAS.
BUT WE COMPLETELY LOST THIS SNR
TO DUCTILE METAPLACIA, SO NOW
THE TUMOR LOOKS--NOT TUMOR, THE
NORMAL MOUSE PANCREAS LOOKS
ABSOLUTELY NORMAL AND THE IMMUNE
COMPONENT IS ALSO VERY NORMAL.
SO, TO ME THIS IS AN IMPORTANT
FINDING BECAUSE IT'S THE FIRST
TIME THAT WE REALIZE THAT THERE
ARE THAT THE TRANSCRIPTIONAL
COMMUNICATION ISN'T TRUE FOR ANY
CELL IN THE PANCREAS AND THAT
THERE ARE IMPORTANT NONCELL
AUTONOMOUS EFFECTS THAT GIVE
RISE TO THIS PLASTICITY SO IT'S
1 OF THE FIRST TIMES THAT WE'VE
BEEN ABLE TO ACTUALLY SEE
COMMUNICATION BETWEEN P53
TRANSCRIPTIONAL PROGRAM IN
DIFFERENT CELLS.
SO TO SUMMARIZE THESE DATA, I'M
FASCINATED THAT P53, I MEAN IT
IS A TRANSCRIPTION FACTOR.
YOU EXPECT IT TO HAVE HUNDREDS
OF GENES THAT WERE REGULATED BUT
I DIDN'T EXPECT TO SEE SO MANY
REG IEWLGTED IN A TISSUE
SPECIFIC MANNER.
SO IT'S ALSO SHOWING US THAT P53
TRANSCRIPTION EFFECTS CELLS IN
THE NONCELL AUTONOMOUS FASHION.
AND SO 1 OF THE THINGS WE JUST
INITTIAITED IS A SINGLE CELL
ANALYSIS OF THE PANCREAS, TO
DETERMINE WHAT CELLS ARE
EXPRESSING WHICH TRANSCRIPTION
FACTOR AND HOW ARE THEY
COMMUNICATING WITH EACH OTHER.
BUT THE MAJOR PART OF MY TALK
TODAY IS ON MUTANT P53
ACTIVITIES AND WHAT IS MUTANT
P53, HOW IT MUTANT P53 CAUSING
CANCER SO THE FIRST FEW SLIDES
JUST BASICALLY TELL YOU HOW
IMPORTANT P53 IS AS TUMOR
SUPPRESSOR.
THAL GRAPH, WE CALL THE
MANHATTAN PLOT, WE LOOK LIKE
THERE'S A BUNCH OF SKYSCRAPERS,
IT WAS DEVELOPED BY A COLLEAGUE,
ACROSS THE X-AXIS, THERE'S 125
GENES THAT ARE MUTATE INDEED
CANCER, AND ACROSS THE Y-AXIS
THERE'S 36 DIFFERENT KINDS OF
CANCERS AND I THINK THAT YOU CAN
CLEARLY SEE IS THAT THAT ROW OF
SKYSCRAPERS ON THE RIGHT HAND
SIDE OF THE SLIDE IS WHERE P53
IS LOCATED SO IT'S MUTATE INDEED
MOST CANCERS.
AND WHEN P53 ISN'T MUTATED YOU
OFTEN HAVE PATHWAY OF THE OTHER
MECHANISMS.
THE OTHER FASCINATING ASPECT OF
THE P53 TUMOR SUPPRESSOR IS THAT
IT'S NOT USUALLY LOST, MISSENSED
MUTATIONS ARE THE MOST COMMON
TYPE OF P53 ALTERATION.
SO THIS A LINE DRAWING OF P53
AND THESE ARE THE KINDS OF
MUTATIONS THAT OCCUR IN BREAST
CANCER.
IT'S SIMILAR FOR OTHER CANCERS
BUT I'LL BE TALKING ABOUT BREAST
MODELS AND SO THAT'S WHY I'M
SHOWING THIS SLIDE.
THE LITTLE BLACK CIRCLES ARE
TRUNCATING MUTATIONS.
AND THEN THE GREEN CIRCLES ARE
ALL THE MISSENSE MUTATIONS AND
WHEN I DID THE CALCULATIONS THIS
IS ABOUT 60% MISSENSE MUTATIONS.
THERE ARE SEVERAL HOT SPOTS, THE
175, THE 248, AND THE 273 AMINO
ACIDS ARE VERY, VERY OFTEN
MUTATE INDEED CANCERS.
THE RGENINE ARE IN THERE, THEY
MOST OCCUR IN THE RED DOMAIN
WHICH IS THE DNA BINDING DOMAIN
AND THE 248 AND THE 273 ARE
RGENINEs THAT DIRECTLY BIND
DNA SO THOSE DISRUPT THE ABILITY
OF THE PROTEIN TO BIND DNA.
THE TRANSCRIPTIONAL ACTIVATION
IS AT THE TERMINUS OF THE
PROTEIN AND IT'S INTACT IN ALL
OF THESE IN A SENSE MUTATIONS.
AND THEN THE CAR BOXY TERMINUS
CONTAINS THE TETRAMERDOMAIN
BECAUSE P53 FUNCTIONS AS A
TETRAMERAND IN THAT DOMAIN YOU
ALSO SEE MISSENSED MUTATIONS
THAT DISRUPT TETRAMERRIZATION.
SO THERE'S SEVERAL FEATURES OF
THESE P53 MISSENSE MUTATIONS
THAT I WANT TO YOU KEEP IN MIND
AS I DISCUSS OUR DATA TODAY.
LOSS OF FUNCTION.
THESE ARE TRUE LOSS OF FUNCTION
ALLELES, THEY DO NOT EXPRESS A
PROTEIN THAT CAN BIND DNA AND
ACTIVATE TRANSCRIPTION.
THEY ALSO HAVE DOMINANT NEGATIVE
AND INHIBITORS EFFECTS ON
TISSUES.
SO BECAUSE IN MOST OF THESE D NA
BINDING MUTANTS, THE
TETRAMERRIZATION DOMAIN IS
INTACT, WHAT YOU HAD IS A MIXED
TETRAMERS THAT HAVE TILED TYPE
AND MUTANT PROTEINS SO THAT
MUTANT PROTEIN BASICALLY
INHIBITS THE ACTIVITY OF THAT
WILD-TYPE PROTEIN AND I WILL
SHOW YOU A VERY CLEAR EXAMPLE OF
THAT.
BUT THEN THE OTHER FEATURE
THAT'S OFTEN DISCUSSED IN THE
LITERATURE AND I THINK IT'S
REAL, I WAS ABLE TO CONVINCE
GLEN TODAY IS WAS LEGAL IS WHAT
WE CALL A GAIN OF FUNCTION.
BASICALLY WHAT THIS MEAN SYSTEM
THAT THE MUTANT P53S HAVE
ADDITIONAL ACTIVITIES THAT
CONTRIBUTE TO THEIR TUMOR GROWTH
AND OFTEN TIMES THESE ACTIVITIES
ARE DUE TO INTERACTIONS WITH
OTHER DNA BINDING PROTEINS AND
BASICALLY YOU UNLEASH AA
TRANSCRIPTIONAL PROGRAM THAT'S
NOT A P53 DEPENDENT
TRANSCRIPTIONAL PROGRAM.
SO THE FIRST FEW SLIDES ARE GERM
LINE MODELS WITH THESE P53
MISSENSED MUTATIONS BECAUSE WE
REALLY--THE FIRST, THE R1728S
ARE EQUIVALENT TO THE 175 AND
THAT'S THAT CONTACT MUTANT AND
THEN THERE'S A STRUCTURAL MUTANT
AND THEN WE HAVE THE 245 AND THE
270 MUTANTS, AND WE REALLY
WANTED TO ASK A VERY SIMPLE
QUESTION, ARE ALL MUTANT PP53S
THE SAME?
SO THIS IS A MODEL IN WHICH WE
CAM PAIR THE 3 DIFFERENT ALLELES
IN THE SAME BACKGROUND, C57
BLACK 6 AND WE ASKED DO THESE
THEY ALL GET TUMORS BUT DO THEY
GET THE SAME KIND?
SO THE FIRST EXPERIMENT WE DID
IS ACTUALLY LOOK TO SEE WHETHER
THE WILD-TYPE PROTEIN ACTIVITY
VARIED DEPENDING ON THE PRESENCE
OF THE MUTANT P53.
SO THIS IS AN EXPERIMENT RAW
DATA IN VIVO WE'VE TAKEN A WHOLE
NEW MOUSE, WITH THE DIFFERENT
GENOTYPES, RADIATED THE MOUSE
AND LOOKED AT THE ABILITY OF
THAT P53 TO ACTIVATE
TRANSCRIPTION.
SO THE FIRST GRAY BAR IS THE
ERADIATED TISSUE AND THIS IS
JUST PROBABLY THE THYMUS AND
THAT SHOWS THAT FOR EXAMPLE HERE
IN THE BAX, P53 TARGETING SYSTEM
SET ACTIVATED 8 FOLD ABOVE
BACKGROUND AND THEN YOU SEE THE
CONTROLS WHICH IS THE WHITE BAR
IS THE HETEROZYGOUS MOUSE AND
THEN THE 3 DIFFERENT MUTANTS FOR
ANALYZED SUBSEQUENTLY TO THAT.
AND I THINK AS YOU LOOK ACROSS
THE ENTIRE SLIDE, YOU CAN SEE
THAT THOSE BARS WITH THE MUTANT
P53, AN LOG TYPE ALLELE ARE
ALWAYS LOWER THAN THE
HETEROZYGOUS WHITE BAR AND WHAT
THAT SAYS IS THAT IN RESPONSE TO
DNA DAMAGE, THAT MUTANT PPEA IS
ACTUALLY DAMPENING THE EFFECTS
OF THAT WILD-TYPE P53.
SO THIS CAN TRIBUTE TO AN
INCREASED TUMOR PHENOTYPE
BECAUSE YOU'VE GOT LESS
WILD-TYPE P53.
AND IN FACT, WHEN WE LOOK AT THE
SURVIVAL OF THESE ANIMALS, THESE
ANIMALS GET TUMORS, I WILL TELL
YOU WHICH KIND IN A MINUTE, BUT,
YOU CAN CLEARLY SEE ON THE LEFT
WHEN WE LOOK AT A LITTLE OVER A
YEAR, THE SAMPLES THAT HAVE THE
BLUE AND THE BLACK, THE 245 AND
THE 270, THESE ARE THE CONTACT
MUTANTS, THEY GET CANCERS
EARLIER THAN THE--THAN THE
HETEROZYGOUS MOUSE AND THE 172
MUTATIONS, SO THERE IS A
DIFFERENT IN TERMS OF TUMOR
ONSETS IN VIVO.
AND IN ADDITION THE KINDS OF
TUMORS THEY GET ARE ALL VERY
SIMILAR AND THEY GET LYMPHOMAS,
SARCOMA AND THEY GET CARCINOMAS,
REMEMBER WE'RE IN THE SAME
GENETIC BACKGROUND BUT THE OTHER
MAJOR PHENOTYPE THAT WE SEE WITH
THE MUTANTS, ALL 3 MUTANTS IS A
METASTATIC PHENOTYPE AND TYLER
JAX'S LAB AND MY DESCRIBED THIS
AS THE MUTANT P53 GAIN OF
FUNCTION PHENOTYPE YEARS AGO.
SO, THE DIFFERENT MUTANTS, I
THINK I SUMMARIZED, OKAY THIS IS
1 MORE SLIDE FROM THESE DATA IS
1 OF THE OTHER CONCEPTS, I WANT
YOU TO TAKE HOME FROM THIS, TALK
IS THAT MUSEUM TABT PROTEIN HAS
TO BE PRESENT IN LIE LEVELS IN
ORDER TO DAMPEN THE WILD-TYPE
P53 AND IN ORDER FOR IT TO
CONTRIBUTE TO THE GAIN OF
FUNCTION OF THE METASTATIC
PHENOTYPE.
SO WHAT WE'VE DONE ON THIS
EXPERIMENTOT RIGHT IS WE
COMPARED ANIMALS THAT ARE
HOMOZYGOUS FOR THESE DIFFERENT
ALLELES AND WE BEND THEM INTO
WHETHER THEY EXPRESS LESS THAN
40% OF THE STABLE MUTANT P53 ON
THE LEFT OR VERY HIGH LEVELS OF
P53 ON THE RIGHT.
YOU CAN SEE A TISSUE STAINED
WITH P53 BY IMMUNOHISTOCHEMISTRY
AND ON 91% OF THOSE CELLS HAVE
VERY STABLE MUTANT P53 AND WHEN
WE BEND THESE IN 2 CATEGORIES
LESS THAN 54% STABLE IN P53
CELLS OR GREATER THAN 60% IN
SALE CELLS THERE'S A BIG
DIFFERENCE IN SURVIVAL AS WELL
AND THE SAMPLES THAT HAVE STABLE
P53 IN RED, THOSE MICE ARE
GETTING CANCER SOONER THAN THE
OTHER ANIMALS THAT HAVE LESS
STABLE P53.
SO THIS IS 1 OF NUMEROUS PIECES
OF DATA THAT SUGGEST THAT
STABILITY IS CONTRIBUTING TO THE
LETHAL EFFECTS IN THESE CELLS.
SO JUST TO SUMMARIZE THIS BIT OF
THE TALK.
WE'VE MADE GERM LINE MUTATIONS
THAT MIMIC FROM THE SYNDROME AND
ALL 3 OF THOSE WHEN STRESSED
DAMPEN THE ACTIVITY OF WILD-TYPE
P53.
THE WILD-TYPE ARE MORE
ADDRESSIVE, THEY HAVE WORSE
OUTCOMES THAN THE 172 OF THE P53
KNOWN ALLELE AND THAT'S THE
STABILITY OF THE MUTANT P53
LEADS TO THE DECREASE SURVIVAL
OF THOSE ANIMALS.
SO WE STUDIED THE GERM LINE AD
NAUSEUM LOOKING AT P53 YOU
HADITATIONS AND ALLELES BUT WE
REALLY WANT TO STUDY A SOMATIC
MODEL BECAUSE SOMATIC HAPPENS
MORE OFTEN THAN GERM LINE AND
WHAT WE REALLY WANTED TO DO IS
DEVELOP AN ANIMAL MODEL ON THE
RIGHT WHERE YOU CAN MAKE A
MUTANT P53 ONLY IN A FEW CELLS
SURROUNDED BY A WILD-TYPE
COMPARTMENT AND FOR US IT
WAS--WE REALLY WANTED THIS MOUSE
TO BE ABLE TO UNDERSTAND THE
TUMOR MICROENVIRONMENT
INTERACTIONS AND SO IN THE
ANIMAL THEY WILL SHOW YOU IN A
FEW MINUTES, THE NUTRIFILLS, THE
CAPS, THE IMMUNE SYSTEMS, THE
T-CELLS ARE ALL WILD-TYPE FOR
P53.
SO THIS IS HOW WE GENERATED
THOSE ANIMALS.
WE CALL IT THE P53 WM-ALLELE
BECAUSE IT CONVERTS WILD-TYPE
P53 TO MUTANT P53 AND THE WAI WE
GENERATE THESE ALLELES IS
BASICALLY UPSTREAM OF THE POINT
MUTATION WHICH IS AN ASTERISK
AND WE INSERTED BY WILD-TYPE
CDNA IN THE POPULATION SEAT
EVERSITE.
SO NORMALLY THIS WOULD EXPRESS A
WILD-TYPE P53 PROTEIN AND THEN
IN A FASHION, YOU WOULD REMOVE
THOSE WILD-TYPE SEQUENCES AND
EXPRESS THE MUTANT PROTEIN AND
WE DID THIS BOTH WITH THE
STRUCTURAL MUTANT, THE 172 IN
THE CONTACT MUTANT AND THE 245
WHICH IS THE MOST AGGRESSIVE
ALLELE WE HAVE IN THE LAB.
SO THIS IS JUST 1 OF MANY SLIDES
THAT I WON'T SHOW YOU, THAT
BASICALLY SHOWS THAT THE ANIMALS
THAT INHERENT 1 OF THESE
ALLELES, EITHER THE 172 IN BLUE,
OR THE 245 IN RED HAVE NO
DIFFERENCES TO A NORMAL MOUSE.
SO THESE ARE NOT TUMOR PRO
MODELS THAT SUGGEST THAT YOU'RE
REALLY MAKING A WILD-TYPE P53 IN
THESE MICE.
OKAY, SO THE FIRST EXPERIMENT I
WILL DESCRIBE IS 1 IN WHICH
WE'VE DONE--A BREAST MODEL AND
WHAT WE'VE DONE IS WE'VE
INJECTED AN ADENO VIRUS IN THE
CREE INTO THE DUCT OF THE MEMORY
GLAND.
AND IF WE USE LOW TITER, WE CAN
GET TD ON THE RED CELLS AND ON
THE RIGHT IF WE USE A HIGHER
TITER, WE GENERATE A WHOLE BUNCH
OF CELLS THAT ARE POSITIVE FOR
CREE AND TDTMTO, AND THE OTHER
POINT I WANT TO MAKE WITH THE
SLIDE IS WE INJECT ALWAYS
FEMALES WHEN THEY'RE 10-12 WEEKS
OLD AND WE USE A BACKGROUND THAT
IS 50% C57 AND 50% BALBC, AND
REASON WE USE THE BALB C MICE IS
THAT THEY'RE SUSCEPTIBLE TO
BREAST CANCER WHEREAS THE C57
STRAIN WE'VE BEEN WORKING WITH
ALL ALONG, RARELY GETS BREAST
CANCER SO SOMETHING ABOUT THIS 1
THAT YIELDS TO BREAST CANCER.
SO THESE ARE THE DAILY BASIS
THEA.
AGAIN, THESE STUDIES SHOW THERE
ARE DIFFERENCES BETWEEN THE
ALLELES, SO THE 172 MICE ON THE
LEFT FOR EXAMPLE, HETEROZYGOUS
MICE BECAUSE WE WANTED TO MIMIC
THE SOMATIC MUTANT P53 WITHOUT
LOH, WE NEVER SAW ANY TUMORS.
IF WE ERADIATE THAD MICE AND
GENERATED RANDOM MUTATIONS WE
BEGIN TO SEE TUMORS, BUT LOOK AT
THE RIGHT HAND SIDE OF THAT
GRAPH, THE 245 HETEROZYGOUS ALL
BY ITSELF AT LOW DOSE AND AT
HIGH DOSE, YIELDED BREAST
CANCERS.
ALL OF THE MICE THAT HAD MUTANT
P53 HA METASTASIS BUT AGAIN THE
245 WAS THE MOST METASTATIC
LESION AND THE OTHER FASCINATIG
ASPECT OF THE STORY IS ON THE
RIGHT THERE, SO YOU DON'T ALWAYS
APPROXIMATE SEE LOH WITH THESE,
WITH THE 245 BUT YOU ALWAYS SAW
LOH WITH THE 172.
SO TO ME THIS SUGGESTS THAT
THERE ARE DIFFERENCES IN THE
ABILITYINGS OF THESE MUTANT
PROTEINS TO DAMPEN WILE TYPE P53
AND SOMETIMES YOU NEED TO LOSE
THAT,A LEGAL AND SOMETIMES YOU
DON'T.
SO YOU NEED TO DAMPEN THE P53
PATHWAY AND YOU PROBABLY DO IT
WITH MULTIPLE MECHANISMS.
THE OTHER I THINK FASCINATING
PHENOTYPE THAT WE'RE STRUGGLING
WITH AND TRYING TO UNDERSTAND IS
THAT RIGHT COLUMN HERE IS SO
THAT THE 245 MUTANT GAVE US 3
DIFFERENT KINDS OF BREAST
CANCER, BLACK, BAR IS TRIPLE
NEGATIVE BREAST, AND THEN IN
DARK BLUE IS HER TO ENRICHED AND
LIGHT BLUE IS LIKELY LUMINAL B,
SO TO ME THIS IS FASCINATING
BECAUSE WE START OUT WITH THE
SAME P53 MUTATION IN ALL OF
THESE CELLS, AND YET TUMORS
EVOLVE IN DIFFERENT DIRECTIONS
AND I DON'T NEED TO TELL THIS
AUDIENCE THAT YOU KNOW TRIPLE
NEGATIVE BREAST CANCER IS HARD
TO TREAT BUT HERE IN LUMINAL B
THERE ARE HORMONE THERAPIES TO
TREAT THOSE TUMORS SO WE
EMBARKED ON IA BIG GENOME STUDY
TO TRY TO UNDERSTAND THE RNA AND
THE DNA TRANSCRIPTOMES TO SEE
WHAT ARE THE CHANGES THAT ARE
DRIVING THESE 3 DIFFERENT KINDS
OF BREAST CANCERS.
SO TO CONCLUDE THIS PART OF THE
STUDY, MUTANT P53 MODELS DEVELOP
METASTATIC BREAST CANCER, I MEAN
THIS IS JUST A BEAUTIFUL MODEL
OF WHAT HAPPENS IN HUMANS.
THE 245 AS I JUST INDICATED,
LEADS TO DEVELOPMENT OF 3
DIFFERENT KINDS OF BREAST
CANCERS AND THE DATA I DIDN'T
HAVE TIME TO SHOW YOU BECAUSE I
DO WANT TO GET TO THE OTHER PART
OF MY TALK IS THAT, THE
METASTASIS IN THIS MODEL, ARE
SEATED VERY EARLY.
THE INITIAL STUDY I THINK
INCLUDED 20 MICE EXPW WE'VE NOW
EXPANDED THAT STUDY TO ABOUT 100
ANIMALS WITH THE 245 MUTATION
AND WHICH ACI HAVE FIESED MOST
OF THESE MICE AND SOME OF THEM
WE SACRIFICED BECAUSE THEY HAD
DIFFICULTY BREATHING AND THE
VETERINARIAN SAID WE HAVE TO
SACRIFICE THE MICE, SO WHEN WE
SACRIFICE THE MICE WE HAVE A LOT
OF LESIONS IN THE LUNG YOU BUT
WE CAN'T FIND A PRIMARY BEST
TUMOR SO WE THINK THIS IS JUST
ANOTHER EXAMPLE OF EARLY
DISSEMINATION OF THAT PRIMARY
CANCER.
SO THE LAST MODEL I WILL DISCUSS
WITH YOU TODAY IS 1 IN WHICH WE
USED THIS SYSTEM TO BASICALLY
TURN ON THE MUTANT P53 AND THEN
TAKE IT AWAY AND WE WANT TO ASK
THE QUESTIONS, ARE THOSE TUMORS
DEPENDENT ON HAVING THAT MUTANT
P53.
SO, WE START OUT WITH THE
WILD-TYPE P53, THESE ARE THESE
WM ALLELES, WE THEN IN THIS
MODEL WE USE K14 CREE, SO WE'RE
EXPRESSING CREE RECOMBIN ACE IN
THE ENTIRE EPITHELIUM OF THE
BREAST AND THE--THE SALIVARY
GLANDS AND THE SKIN AND THEN A
YEAR LATER WE GET BREAST CANCER
AND WE ALSO GET HEAD AND NECK
CANCERS BUT WE HAVEN'T STUDIED
THOSE, SO FOR THE BREAST MODELS
BECAUSE WE'VE ALSO TURNED ON
CAS9 WHEN WE TURNED ON K14 CREE,
SO NOW WE'RE ABLE TO COME IN,
WITH A GUIDE RNA, THE ADENO
VIRUS EXPRESSING A GUIDE RNA TO
TAKE OUT THE MUTANT P53 AND WE
CAN ASK WHAT HAPPENS TO THIS
CELL WHEN YOU REMOVE THAT MUTANT
PPEA.
SO IMPORTANTLY, THESE MICE ONLY
EXPRESS 1 MUTANT P53 AND 1 KNOWN
ALLELE, SO THERE'S NO WILD-TYPE
P53, THERE'S NO LOH, WE DID IT
FROM THE BEGINNING, MUTANT P53S
IS ONLY EXPRESSED IN THE SYSTEM.
AND SOME OF THESE HAVE TMTD, SO
WE CAN MONITOR THE PRIMARY TUMOR
DEVELOPMENT AND THE METASTASIS.
SO THE K14 CREE MODEL GIVES US
TRIPLE NEGATIVE BREAST.
ALL THE MICE ARE DEAD WITHIN
LIKE A YEAR AND A HALF.
THEY GET 2 KINDS OF BREAST
CARCINOMAS 1 IS THE ADEN O
CARCINOMA AND THE OTHER IS A
SARCOMAATOID LIKE TUMOR AND THEY
HAVE VERY DIFFERENT ETIOLOGIES.
SO WHAT WE'VE DONE THEN IS
INJECTED THESE TUMORS AFTER THEY
DEVELOPED WITH THE ADEN O
ASSOCIATED VIRUS EXPRESSING A
GUIDE RNA TO THE MUTANT P53, SO
DEPLETING THE TUMORS OF MUTANT
P53 AND ON THE LEFT I HAVE A.
Q.OF THAT SHOWS THE
CONTROLS IN A, SO THIS
IS HA HAPPEN WHEN IS
MUTANT P53 IS STILL
BEING EXPRESSED AND
YOU CAN SEE THAT THE
TUMOR VOLUME CONTINUES
TO INCREASE IN PURPLE,
OR THE TUMORS THAT NOW
HAVE DEPLETED MUTANT
P53 AND I THINK YOU
CAN SEE A BIG
DIFFERENCE BETWEEN
THOSE BARS, SO SOME OF
THE TUMORS HAVE A
DECREASED VOLUME AND
SOME HAVE A LARGE
DECREASE IN OW QUICKLY
THAT TUMOR IS GROWING.
ON THE RIGHT YOU SEE THE TUMOR
VOLUME WITH TIME, AGAIN IN
PURPLE ARE THE MICE THAT HAVE
DEPLETED P53 AND IN GREEN ARE
THE MICE THAT HAVE HIGH LEVELS
OF THAT MUTANT PROTEIN.
IN ORANGE ARE 2 INTERESTING
SAMPLES THAT LOOK LIKE MUTANT
P53, THEY'VE NOT--THEY'RE
CONTINUING TO GROW.
THIS WAS A--THESE WERE 2 SAMPLES
FOR WHICH THE MUTANT P53 WAS NOT
STABLE.
SO YOU NEED TO HAVE A STABLE
MUTANT P53 FOR THE TUMORS TO BE
DEPENDENT ON THAT MUTANT P53.
NEXT IS JUST A SURVIVAL KUEVER
SO IN PURPLE ARE THE ANIMALS FOR
WHICH WE REMOVE THAT P53 PROTEIN
AND YOU CAN SEE THAT THEY'RE
LIVING A LOT LONGER.
THIS IS PROBABLY AN
UNDERESTIMATE OF HOW LONG THEY
LIVE BECAUSE SOME OF THESE
ANIMALS GET SECONDARY TUMORS AND
YOU HAVE TO SACRIFICE THEM
BECAUSE OF THE SECONDARY TUMOR
AND NOT THE PRIMARY TUMOR THAT
WE TREATED.
THE OTHER MUTATION, THE 245,
IT'S THE SAME STORY, WE DELETE
THE MUTANT P53 AND YOU CAN SEE
IN PURPLE THAT THESE TUMORS
EITHER REGRESS OR DRASTICALLY
SLOW DOWN AND CAN YOU SEE ON THE
RIGHT, AND SURVIVAL KUEVER, YOU
ALSO EXTEND SURVIVAL OF THESE
MICE WHEN YOU REMOVE THAT MUTANT
P53 PROTEIN.
AND THIS IS THE EXPERIMENT WHERE
I WANT TO SHOW YOU THE
RESPONDERS VERSUS THE
NONRESPONDERS SO HAVE YOU
THIS--THIS IS A SECTION THROUGH
THE TUMORS.
THE AAV CONTROL IS ON THE LEFT
AND EVERYTHING IN RED IS P53, SO
THE 2 RESPONDERS IN THE MIDDLE,
CAN YOU SEE THE HUGE HOLE THAT
DOESN'T HAVE ANY STAINING AND
THAT'S WHERE WE DEPLETED THE
MUTANT P53 IN BOTH OF THOSE
TUMORS REGRESSED, THE VOLUMES
REGRESSED AND ON THE RIGHT YOU
HAVE A NONRESPONDER SO THAT YOU
CAN CLEARLY SEE THERE'S NO
STABLE MUTANT P53 IN THIS
RESPONDER SO THIS RESPONDER
LOOKS LIKE IT HAD NO P53 TO
START WITH SO IT WASN'T
DEPENDENT ON THE MUTANT P53.
SO I THINK THAT THIS IS AN
IMPORTANT EXPERIMENT, BECAUSE I
THINK IN THE CLINIC WE NEED TO
THINK ABOUT NOT JUST THE POINT
MUTATION BUT WHETHER IT'S BECOME
A STABLE PROTEIN OR NOT.
SO WE MIGHT HAVE TO REFER BACK
TO IMMUNOHISTOCHEMISTRY TO
DETECT MUTANT P53 STABILITY.
SO THE MUTANT PPARADOXICAL
PROTEINS BOTH THE 172 AND THE
245, ARE REQUIRED FOR
MAINTENANCE OF THESE TUMOR
PHENOTYPES BECAUSE WHEN WE TAKE
THESE 2 PROTEINS OUT, TUMORS
REGRESS, AND MOST OF THESE
TUMORS HAVE STABILIZED THE
MUTANT P53, A FEW DO NOT AND I
THINK THAT'S ANOTHER IMPORTANT
QUESTION IN THE FIELD THAT WE'VE
NOT BEEN ABLE TO FULLY
UNDERSTAND WHAT IS IT IN THESE
TUMORS THAT IS CAUSING THE
STABILITY OF THAT MUTANT P53.
YOU KNOW, IS THERE SOME STRESS,
DAMAGES, INFLAMMATION, ARE THOSE
CONTRIBUTING TO THE INSTABILITY
OF P53 PROTEIN AND THEN IF THE
TUMORS HAVE AN UNSTABLE P53
THEY'RE NOT DEPENDENT ON THAT
MUTANT P53 FOR CONTINUED GROWTH.
SO NOW THAT WE KNOW THAT TAKING
OUTLET MUSEUM TABT P53 CAUSES
THESE TUMORS TO REGRESS, WE'RE
REALLY INTERESTED IN TRYING TO
UNDERSTAND HOW THAT HAPPENS, SO
IN THIS EXPERIMENT, WHAT WE'VE
DONE IS WE'VE IN PURPLE AGAIN WE
TREATED THESE TUMORS WITH THE
ASSOCIATED GUIDE RNA THAT
DEPLETES MUTANT P53 AND YOU CAN
SEE THAT THE TUMORS DON'T GROW
AS QUICKLY.
THIS IS JUST 6 DAYS, SO IT'S
VERY EARLY ON, BECAUSE WE WANTED
TO CATCH THE EARLY EVENTS TO TRY
TO UNDERSTAND WHAT THE TRIGGER
WAS.
SO IN THE MIDDLE PANELS WHAT WE
JUST DID SOME H& E AND LOOKED
AT THESE AS THESE TUMORS AND
WHEN WE DEPLETE MUTANT P53, WE
CAN SEE THESE VERY LARGE LIPID
LIKE STRUCTURES AND THEY'RE
QUANTIFIED ON THE RIGHT, SO WHEN
WE DEPLETE P53 WE SEE THESE
LARGE STRUCTURES VERY
REMINISCENT OF A PROCESS CALLED
CELL APOPTOSIS, SO WE STAINED
THESE WITH 4 HNE WHICH IS
BASICALLY A MARKER FOR LIPID
PEROXIDATION AND I THINK CAN YOU
CLEARLY SEE ALL THAT BROWN
STAINING IN THE MIDDLE WHEN WE
DEPLETE MUTANT P53 WE HAVE A
HIGH LEVEL OF LIPID
PEROXIDATION, SO THESE THAT
SUGGEST TO US THAT MAYBE WE'RE
DEALING WITH A CELL DETH
MECHANISM SO NEXT EXPERIMENT WAS
SIMPLY TO ISOLATE THOSE TUMOR
CELLS AWAY FROM ALL THE REST OF
THE MICROENVIRONMENT TO SEE WHAT
WAS ACTUALLY HAPPENING IN THOSE
CELLS SO WE DID SOME SINGLE CELL
ANALYSIS, WE'VE IDENTIFIED 3
CLUSTERS, 1, 11 AND 14 THAT WERE
THE TUMOR CELLS AND YOU CAN SEE
IN THE CLOAL THESE CLUSTERS ARE
PRESENT AT HIGH LEVELS AND WHEN
WE DEPLETE THESE, THESE CLUSTERS
ARE LOST, SO THESE ARE THE
CLUSTERS THAT ARE THE TUMOR
CELLS THAT EXPRESS THE MUTANT
P53 PROTEIN SO OF COURSE YOU DO
THESE ANALYSIS AND YOU LOOK AT
THE TRANSCRIPTOME AND IN THESE
SAMPLES WITH THE DEPLETION, WE
SAW UPREGULATION OF REACTIVE
OXYGEN SPECIES SO AGAIN, THIS IS
A MECHANISM THAT INCREASED R. O.
S. LEADS TO THERAPY AND
OPERATING GLOBALLY TO THETIC
RESPONSE, SO WE'RE BEGINNING TO
THING THAT THERRAPITOSEIS.
SO WHAT IS IT?
WE'RE LEARNING A LOT ABOUT
THERRAPOTIIS.
IT'S 2 OF THE SPECIES CAUSES
LIPID PEROXIDATION, IT'S AN IRON
DEPENDENT PROCESS AND BASICALLY
IT KILLS THE CELL.
SO, ON THE LEFT CAN YOU SEE A
LIPID MOLECULE WITH A LOT OF
HYDROXYL RADICALS AND THAT WILL
LEAD TO FERROPITOSEIS, AND
THERE'S A GLUTEO THIGH OWN
TRANSFER ACE WHICH REMOVES THESE
RADICALS AND ALLOWS THE CELL TO
SURVIVE SO IT'S A VERY CRITICAL
PROTEIN IN THE CELL THAT
MAINTAINS THE PEROXIDATION AT A
LEVEL THAT'S TOLERATED.
SO THE NEXT EXPERIMENT I WILL
SHOW YOU IS 1 IN WHICH WE'VE
INHIBITED GBX 4, SO BASICALLY WE
INDUCED FERROPITOSEIS IN THESE
CELLS.
SO IT'S AN INTERESTING
EXPERIMENT.
WE HAVE IN GREEN CELLS EITHER
EXPRESS THE MUTANT 172, OR THE
MUTANT 245 AND THEN YOU USE
CRSPR TO REMOVE IT FROM THESE
CELLS IN MY POST DOCKER CAME TO
ME AND SAID, I HAVE CELLS WITH
DELETION P53 AND SAID IN VIVO
THOSE CELLS ARE DYING HOW DID
YOU GET THOSE CELLS TO LIVE AND
IT TURNS OUT THEY'RE VERY
FRAGILE, WE HAVE TO CHANGE THE
MEDIA EVERY DAY BECAUSE IT TURNS
VERY YELLOW.
AND SO, THE TOLERATED LOSS OF
P53 IN CULTURE BUT THEY'RE VERY
FRAGILE AND WHEN WE ADD RSL 3,
WE CAN SEE THE CELLS HAVE NO
P53, DELETION ARE VERY SENSITIVE
TO FERROPITOSEIS AND I THINK THE
DRAMATIC SAMPLE ON THE FAR RIGHT
IS 1 IN WHICH, THE CELL THAT HAS
THE MUTANT P53 IS RESISTANT AND
THE CELL THAT HAS LOST P53 IS
JUST--IT DIES VERY QUICKLY.
SO WE'VE ALSO INHIBITED
FERROPITOSEIS IN VIVO.
SOPHISTICATEDY THIS IS AN
EXPERIMENT THAT I'VE PREVIOUSLY
SHOWN WHERE YOU CAN SEE IF YOU
DEPLETE MUTANT P53 IN PURPLE
THESE ANIMALS LIVE LONGER SO
WHAT WE'VE DONE IS WE'VE
DEPLETED MUTANT P53 AND THEN WE
ADDED LACK ROUGH ATOM STATIN
WHICH IS AN INHIBITOR OF
FERROPITOSEIS, THE DATA ARE
QUANTIFIED IN THE MIDDLE, SO
WHEN WE PREVENT FERROPITOSEIS
FROM HAPPENING, THOSE ANIMALS
ARE DYING SOONER AND THAT DATA
THEN IS SHOWN ON THE RIGHT.
THE MUTANT P53 DEPLETED TUMORS
CONTINUE TO--THEY LIVE LONGER,
THE MICE WITH MUTANT P53
DEPLETED LIVE LONGER BUT IF YOU
INDUCE FERROPITOSEIS, THEY'RE
DYING.
AND I THINK THIS IS VERY CLEAR.
EACH 1 OF THESE IS A--IT'S NOT A
SIN GENETIC MOUSE, IT'S A REAL
MOUSE THAT GEPPED A TUMOR AND
THEN ONCE YOU SAW THE TUMOR WE
TREATED IT WITH THE LIPO STATIN.
SO THE HYPOTHESIS THAT WE'RE
WORKING WITH IS THAT P53OXIDATES
THIS STRESS AND PROTECTS ITS
FROM FERROPITOSEIS, WE REMOVE
THE P53 MUTANT IN THE CELL
TISSUE, THE CELLS ARE DYING BY
FERROPITOSEIS.
SO HOW ON TO TEST THIS.
SO HOW THE FIRST THING WE DID WE
WENT BACK TO OUR SINGLE CELL RNA
SEQ DAT IS AND WE LOOKED AT THE
REACTIVEOX GENERATED SPECIES,
THE GENES THAT ARE RESPONSIBLE
FOR ROS, AND THERE ARE 2 GENES
THAT STUCK OUT, PIDRX 6 AND MGST
3, AND PIDR6 A GLUTATHIONE
DEPENDENT SO IT'S GOING TO WORK
SIMILARLY TO GPX 4 AND REMOVE
THOSE EYE DROKS ILLEGALS
RADICALS AND MDGIT IS A MIKE
STUDIES OF MULTIPLE ENDOCRINIA
GRIEWT O THE IPAD OWN AS A
TRANSFER ACE GREENE GENE SO BOTH
OF THESE ARE SIMILAR TO GPX 4 IN
THAT THEY'RE REMOVING THE OXYGEN
RADICALS FROM THE LOW SYSTEM.
ON THE LOWER LEFT ON THE SLIDE,
WHAT WE'VE DONE IS WE'VE
COMPARED THE MUTANT P53
EXPRESSING CELLS AND THOSE WITH
MUTANT P53 DEPLETION AND
BASICALLY VALIDATED THE DATA
FROM RNA SEQ, SO DEPLETION OF
YOU HADITANT P53 CAUSES DOWN
REGULATION OF THESE 2 IMPORTANT
GLUTEO THIGH OWN ENZYMES.
SO IN THESE CELLS, NOW, IN SIN
GENERATED EIC MICE, WE'VE
KNOCKED DOWN PRDX 6 AND THE
KNOCK DOWN DATAA ARE ON THE LEFT
AND WE GOT PRETTY GOOD KNOCK
DOWN ASK ON THE RIGHT WE MONITOR
TUMOR GROWTH.
SO CAN YOU CLEARLY SEE IN THE
RED AND GREEN BARS THAT IF WE
KNOCK DOWN THESE 2 ENZYMES WE'VE
SENSITIZED THE CELLS TO
FERROPITOSEIS.
SO WHAT WE THINK P53 IS DOING IS
BASICALLY BY ALLOWING EXPRESSION
OF THESE 2 GENES IT'S
MAINTAINING THE STAINING OF THE
PEROX DAITIONZ OF THE LIPID
RADICALS IS TOLERATED BY THE
CELL.
SO IF YOU REMEMBER THE GAIN OF
FUNCTION SLIDE, 1 OF THE WAYS
THAT MUTANT P53 CAN CONTRIBUTE
TO GAIN OF FUNCTION IS BASICALLY
BY INTERACTING WITH OTHER
TRANSCRIPTION FACTORS.
SO WE TOOK OUR RNA SEQ DATA AND
WE ASKED--THERE'S DIFFERENT
DATABASES THAT YOU CAN USE TO
BASICALLY ASK, IS THERE ANY
SIMILARITY BETWEEN ALL OF THESE
TRANSCRIPTOMES AND SO WHAT WE
IDENTIFIED IN THIS CASE IS 4
DIFFERENT--4 DIFFERENT PROMOTER
SEQUENCES THAT WERE PRESENT IN
THESE GENES THAT ARE UPREGULATED
UPON DEPLETION OF MUTANT P53,
AND THE 1 THAT CAUGHT OUR
ATTENTION IN THIS CASE, IS THIS
NRF 2 PROTEIN, AND NRF 2 IS A
DNA BINDING PROTEIN AND ABOUT A
THIRD OF THE GENES THAT WERE
DIFIENTIALLY EXPRESSED UPON
DEPLETION OF MUTANT P53 HAD AN
NRF 2 MOTIF.
AND NRF 2 IS IN THE
FERROPITOSEIS PATHWAY AND I WILL
SHOW YOU WHERE IT FITS IN A
MINUTE.
SO THE NEXT QUESTION WE ASK IS
BECAUSE THIS IS THE HYPOTHESIS
WE'RE WORKING WITH IS THAT
MUSEUM AT THAT POINT P53
INTERACTING WITH OTHER MUTANT
TRANSCRIPTION FACTORS TO DRIVE
THE PROGRAM SO THESE ARE 2
DIFFERENT ASSAYS IN WHICH WE
LOOK TO SEE IF MUTANT COULD
INTERACT WITH NRF 2, SO ON THE
LEFT IS IMMUNO PRECIPITATION
EXPERIMENTS IN CELLS, THAT
EXPRESS THE 172 MUTANT PROTEIN
AND YOU CAN SEE THAT IF WE
IMMUNO PRECIPITATE WITH P53 AND
THE RIGHT MOST LANE, WE CAN
DETECT NRF 2.
AND THEN ON THE RIGHT WAS SOME
INVITRO DATA WHERE BASICALLY WE
EXPRESS THESE PROTEINS, INVITRO
PURIFIED THEM AND AGAIN IN THE
RIGHT MOST PANEL YOU CAN SEE
THAT A EFTHIMIOS TAG NRF 2
PROTEIN IS INTERACTING WITH THE
245 W TAG.
SO, IN--AND WE'VE DONE THIS FOR
BOTH THE 245 AND THE 172 MUTANT
SO IT DOESN'T MATTER WHICH
MUTANT YOU'RE EXPRESSING IN THIS
SYSTEM, THAT MUTANT PROTEIN IS
INTERACTING WITH NRF 2.
SO, THIS IS A--THIS IS NOT A CHP
ASSAY, IT'S A NEW FANGLED CUT
AND RUN ASSAY WHERE AGAIN YOU'RE
ASKING THE SAME QUESTION, IS THE
MUTANT PROTEIN, MUTANT P53 AND
NR2 PRESCRIBINGENT ON THE SAME
DNA SEQUENCES SO WE TOOK 2 OF
THESE A. R. E. ELEMENTS FROM THE
PRDX 6 GENES, SITE 1 EXPW 2 AND
CAN YOU SEE THAT NRF 2 ON THE
LEFT IS BOUND TO THE SEQUENCES
AND ON THE RIGHT, YOU CAN SEE
THAT WHEN MUTANT P53 IS PRESENT,
IT'S ALSO BOUND TO THE SEQUENCE.
AND SO THIS WAS, I THINK VERY
CONVINCING DATA ALTHOUGH WE'RE
HAVING A HARD TIME CONVINCING
THE REVIEWERS THAT MUTANT P53 IS
INTERACTING WITH NRF 2.
SO HERE'S WHAT WE THINK IS
HAPPENING IN THE CELL, SO IN THE
LOWER LEFT HAND CORNER, YOU HAD
MUTANT P53 INTERACKING WITH NRF
2 AND IT'S DRIVING EXPRESSION OF
THESE 2 PROTEINS IN GST 3 AND
PRDX 6.
IN THESE PROTEINS, IN THE MIDDLE
GREEN, FUNCTION SIMILAR TO GPX
4, THEY'RE RESPONSIBLE FOR
REMOVING SOME OF THESE HYDROXY
RADICALS ON LIPIDS.
AND ALLOWING THE CELLS TO
SURVIVE.
SO WHAT HAPPENS IF YOU
TRANSITION TO 1 SIDE OF THE
SLIDE IS YOU REMOVE THE MUTANT
P53 AND YOU NO LONGER HAVE
EXPRESSION OF THESE 2 PROTEINS,
CAN YOU SEE FROM THE SINGLE CELL
DATA IN THE TRANSCRIPTOME DATA
THAT THESE 2 PROTEINS WERE DOWN
MODULATED UPON DOWN MODULATION
OF P53.
SO WE THINK YOU OFFSET THE
BALANCE, NOW YOU ONLY HAVE GPX 4
TRYING TO DEAL WITH THIS
INCREASED REACTIVE OXYGEN
SPECIES AND RADICALS AND IT'S
NOT SUFFICIENT AND SO REMOVING
THAT MUTANT P53 IS TAKING CELLS
DOWN THAT PATHWAY.
NOW 1 ARK DITIONAL SIDE STORY
THAT I WON'T GIVE YOU A WHOLE
LOT OF DETAILS BUT AGAIN IT'S
FASCINATING WITH RESPECT TO THE
COMPLEXITY OF THE SYSTEM, SO I
TOLD YOU AT THE BEGINNING THAT
THESE MICE GET THESE ADEN O
CARCINOMAS AND THEY ALSO GET
THESE SARCOMA LIKE TUMORS SO IN
BOTH INSTANCES WHEN WE REMOVE
THE MUTANT P53, WE GET DEPLETION
OF THE TUMOR VOLUME AND THE
ANIMALS LIVE LONGER BUT FOR THE
ADEN O CARCINOMAS, I'VE SHOWN
YOU ALL THE DATA FOR THE ADEN O
CARCINOMA, THIS SEEMS TO BE
FERROPITOSEIS, BUT IF WE LOOK AT
THE SARCOMA DATA IT'S NOT
FERROPITOSEIS, EVEN THOUGH THOSE
TUMORS THAT ARE DRIVEN BY THE
SAME P53 MUTATION, THEY DEVELOP
DIFFERENTLY.
THEY GIVE RISE TO A DIFFERENT
PATHOLOGY, AND WHILE THEY'RE
DEPEND OANT P53, THEY'RE NOT
DEPENDENT BECAUSE OF A
FERROPITOSEIS.
SO THAT JUST GIVES YOU 1 MORE
EXAMPLE OF THE COMPLEXITY OF
WORKING WITH P53.
I THINK THERE'S GOING TO BE A
LOT OF TISSUE SPECIFICITY.
SO HOPEFULLY I'VE GIVEN YOU A
SMATTERING OF SOME OF THE
STUDIES THAT WE'VE DONE.
I'M NASAA.COMINATED BY WILD-TYPE
P53 ACTIVITIES.
WHAT IS THE TRANSCRIPTIONAL
PROGRAM THAT P53 CAN INITIATE IT
IN DIFFERENT CELL TYPES.
WE ARE HOPING THAT MIGHT LEAD TO
THE ABILITY TO ACTIVATE THAT
PATHWAY DOWN STREAM AND
POTENTIALLY CONTRIBUTE TO TUMOR
SUPPRESSION IN SOME WAY?
AND THEN I TALKED ABOUT A LOT OF
THE DIFFERENT P53 MISSENSED
MUTATIONS, SOME OF THEM IN THE
GERM LINE, AND THEY'RE
DIFFERENT, THEY'RE NOT THE SAME
AND I THINK THE OTHER
FASCINATING ASPECT OF ALL OF
THESE MODELS THAT WE'VE
GENERATED IS WE NEVER KNOW WHAT
TUMOR TYPE WE'RE GOING TO GET.
SO I THINK TRYING TO UNDERSTAND
THE EVOLUTION OF THOSE TUMORS,
TRYING TO UNDERSTAND SOME OF THE
CONTRIBUTING LESIONS, AND SO
SOME OF THE STUDIES ONGOING IN
THE LAB ARE TRYING TO UNDERSTAND
THOSE EARLY LESIONS AND WHAT ARE
THE CHANGES THAT HAPPEN EARLY ON
THAT CONTRIBUTE TO DISEASE.
LIKE I SAID, WE'RE SEQUENCING A
BUNCH OF THESE TUMORS TO TRY TO
UNDERSTAND THE GENOME AS IT
EVOLVES, AND THEN LASTLY THERE
WAS 1 MORE, WE REALLY WANT TO
UNDERSTAND THE METASTATIC
PROCESS BECAUSE TAKEN--THEY
ULTIMATELY WHAT KILLS THE
PATIENT BESIDES DRUG RESISTANCE,
MICHAEL, AND SO, WE'RE
SEQUENCING THOSE IN THE LUNG AND
LIVER TO SEE WHAT CHANGES.
IT'S NOT AN EASY PROBLEM.
I THINK METASTASIS IS A VERY
DIFFICULT PROBLEM TO STUDY,
WE'VE STUDIED IT IN TISSUE
CULTURE WAY TOO LONG AND I THINK
WE NEED TO STUDY IT IN IN VIVO
SYSTEMS WHILE WATCHING THE
PROCESS BUT AGAIN WE NEED TO
MAKE THESE WITH TDTOMATO AND
SACRIFICE A LOT OF MICE EARLY IN
THE PROCESS TO TRY TO UNDERSTAND
THAT EVOLUTIONARY PROCESS, SO
WITH THAT, I WILL JUST KIND OF
THANK THE PEOPLE IN MY LAB, THIS
IS A NEW PICTURE EVER SINCE JUST
A FEW WEEKS AGO.
WE DROPPED THE MASK MANDATE AT
MD ANDERSON AND WE QUICK TOOK A
PICTURE.
[INDISCERNIBLE] DID ALL THE
STUDIES WITH THE FERROPITOSEIS
AND IT WAS REALLY HER WORK
LOOKING AT THE SEQUENCES,
LOOKING AT THE SAMPLES THAT
SUGGESTED THAT IT WAS A
MECHANISM BY WHICH MUTANT PPEA
WAS MAINTAINING THESE TUMORS,
[INDISCERNIBLE] IS A LONG-TERM
TECHNICIAN IN MY LAB, SYDNEY WHO
FINISHED PERPh.D. AND MOVED ON
SO WASN'T HERE FOR THE PICTURE
DID THE TRANSCRIPT OHM STUDIES
AND I WORK WITH 2 INCREDIBLE
PATHOLOGISTS, ADEL AND JAMES WHO
NEVER HESITATE TO LOOK AT TUMORS
THAT WE BRING THEM.
SO WITH THAT, I WILL STOP HERE
AND TAKE QUESTIONS.
[ APPLAUSE ]
>>THANK YOU, ANYONE'S FREE TO
USE THE MIC.
LET ME START WITH A QUESTION
FROM ONLINE.
IT'S FROM [INDISCERNIBLE].
AND CAN YOU COMMENT ABOUT
ISOCHROMOSOMES 17 Q IN MYELOIDNY
O PLASMS.
THESE TUMORS ALWAYS LOSE 1 COPY
OF TP 53, DELETED, BUT WE NEVER
FOUND MUTATIONS IN THE SECOND
COPY.
THE BEHAVIORS OF THESE LEUKEMIA
ARE AS BAD AS THE TP53BIA
LEGALLIC LOST 1S?
>>YEAH, YEAH, SO I KNOW THAT'S
TRUE, NONE OF OUR MODELS GET
LEUKEMIA, SO I THINK THAT
ESPECIALLY THE GERM LINE MODELS,
I THINK THAT THE OTHER CELL
TYPES ARE MORE SUSCEPTIBLE TO
P53 MUTATIONS IN THE BACKGROUNDS
THAT WE'RE WORKING WITH, IT'S A
FASCINATING PROBLEM AND I JUST
DON'T KNOW THE ANSWER.
IT'S DOING SOMETHING.
AND THOSE ARE THE WORST
LEUKEMIAS, WHEN THEY--WHEN THEY
HAVE A MUTANT P53, SO, AGAIN, I
THINK IT'S CONTEXT IS VERY
IMPORTANT AND I THINK WE NEED TO
LOOK AT THE TRANSCRIPT OHM IN
THE CONTEXT OF THAT MUTANT P53
AND HOW IT'S CHANGING.
BUT I'VE NOT KNOCKED ON AML BUT
I DO WANT TO.
WE'LL GET THERE.
>>YEAH, BEAUTIFUL.
AND LIKE AN IMMUNOLOGIST SO I
WOULD HAVE AN IMMUNOLOGY RELATED
QUESTION.
HAVE YOU LOOKED IN THIS TUMOR
INTO A RAG DEFICIENCY OR HOW
MUCH IS THE IMMUNE SYSTEM
IMPLICATED?
SO [INDISCERNIBLE] IT'S A VERY
LONG TIMELINE SO HAVE YOU ALSO
LOOKEDDA THE HOW MANY MUTATION
YOU ACCUMULATING IN THESE MODELS
AND HOW MANY CAN INDUCE A
SPECIFIC IMMUNE RESPONSE?
>>OKAY, SO WHAT WE'VE DONE IS
WE'RE JUST IN THE PROCESS OF
SEQUENCING SO WE'RE GETTING ALL
THAT DATA BUT THE SINGLE CELL
DATA SUGGESTED THAT THERE WAS AN
IMMUNE RESPONSE WHEN WE REMOVE
THE MUTANT P53 SO WE'RE TRYING
TO FIGURE THAT OUT BUT WE
HAVEN'T DONE ANY EXPERIMENTS TO
ACTUALLY SAY HOW THAT IMMUNE
SYSTEM IS CONTRIBUTING TO THE
DECREASE IN THE TUMOR VOLUME AND
INCREASED SURVIVAL.
SO IN RELATION TO THAT, DO YOU
SEE LIKE IN HOW MANY CELLS, IS
THERE LIKE A TUMOR MOUSE WHICH
BECOMES CRITICAL IN TERMS OF
LIKE WHETHER THERE IS REJECTION
OR THERE IS LIKE REDUCTION OF
THE TUMOR GROWTH WHERE YOU
INDUCE, SO LIKE IF THE IMMUNE
RESPONSE IS INVOLVED, DO YOU
THINK THAT HOW MANY CELLS WOULD
THEN BE MUTATE SIDE RELEVANT?
>>YEAH, YEAH.
THAT'S A GREAT QUESTION AND 1 OF
THE REASONS THAT WE DEVELOPED
THE SOMATIC MODEL WITH THE ADEN
O DUCTAL INJECTION OF CREE IS
JUST TO MAKE A FEW CELLS BECOME
A TUMOR CELL.
SO I DON'T KNOW THE ANSWER TO
THE QUESTION, BUT I THINK WE CAN
USE THE MODEL TO TRY TO ADDRESS
THOSE QUESTIONS OVERTIME WHICH
IS I THINK WHAT YOU'RE ASKING,
YEAH.
>>GLEN?
>>GREAT TALK, SO I HAVE A
RELATED QUESTION OR MAYBE A
DEEPER QUESTION, SO YOU KNOW
YOU'VE DONE A FANTASTIC JOB OF
LOOKINGA THE INTRINSIC OR CELL
AUTONOMOUS MODELS BUT YOU COULD
ALMOST ELECTRIC AT EVERY PART
OFIOURE TALK AND ASK A QUESTION
ABOUT THE TUMOR
MICROENVIRONMENT.
SO FOR EXAMPLE, DO YOU KNOW IF
THE DIFFERENT MUTANTS HAVE
DIFFERENT EFFECTS ON EITHER THE
IMMUNE SYSTEM OR OTHER
MICROENVIRONMENTAL COMPONENTS?
DOES THAT PLAY A ROLE?
WHAT ABOUT AT THE METASTATIC
SITES?
AND OF COURSE WHEN YOU TURN IT
ON AND OFF, BUT THESE--THERE'S
PROBABLY DIFFERENCES.
BECAUSE THEY'LL CHANGE
SECRETIONS AND ALL KINDS OF
STUFF.
>>RIGHT.
WE'RE SEQUENCING ALL OF THESE
SAMPLES.
INVITE ME BACK NEXT YEAR.
NO, GIVE ME A COUPLE YEARS.
[LAUGHTER]
>>GIGI THANKS SO MUCH FOR THE
LOVELY TALK.
SPEAKING ABOUT DRUG RESISTANCE,
1 PREDICTION IS THAT FOR YOUR
MUTANT P53, THAT HAS SOME WAY OF
BLOCKING FOR APOPTOSIS, YOU
WOULD EXPECT THAT THE TUMORS
WOULD BE MORE RESISTANT TO THE
NORMAL STIMULANTS, ARAFTIN FOR
EXAMPLE, FOR AFERROPITOSEIS, ARE
THEY RESISTANT TO THE
FERROPITOSEIS STIMULATORS?
>>SO WE HAVEN'T DONE THE RAS
EXPERIMENT IN VIVO.
WE'VE ONLY DONE IT IN THE TISSUE
CULTURE CELLS, AT LEAST I DON'T
THINK SO.
>>IN TISSUE CULTURE ARE THEY
RICYST ANT?
>>BUT THAT'S AN EXPERIMENT WE
NEED DO.
>>AND WE HAD A CONVERSATION
BEFORE ABOUT THE FACT IT THERE
ARE SOME MUTANT P53S THAT
IMSTIEWLT PGFG, MY FAVORITE
GENE, DO YOU KNOW IF THE 1
YOU'RE LOOKING AT IS A
STIMULATOR AND IF SO, THEN THE
CELLS SHOULD BE MORE GENERALLY
RESISTANT TO ANTICANCER?
>>RIGHT, RIGHT.
WE HAVE ALL THE SEQUENCING DATA
WE NEED TO LOOK AT FOR EXAMPLE
THOSE KINDS OF QUESTIONS.
YEAH.
>>HI, I CONQUER WITH EVERYBODY
ELSE, WONDERFUL TALK, MY
QUESTION IS WHAT HAPPENS IN THE
CONTEXT OF A TUMOR VIRUS WHERE
YOU HAVE WILD-TYPE P53, AND THEN
A MUTANT THAT'S UNABLE TO ENGAGE
THE VIRAL PROTEIN LIKE T-ANTIGEN
OR WHAT HAVE YOU.
>>INTERESTING QUESTION.
SO YES, YES, SO TANTIGEN TUMOR
MODELS GENERATED YEARS AGO AND
IT WAS THE FIRST IN FACT THE
FIRST CANCER MODEL THAT WAS
GENERATED AND WE KNOW THAT
T-ANTIGEN SEQUESTERS WILD-TYPE
P53 PREVENTS IT FROM
FUNCTIONING, OKAY.
AND I DON'T REMEMBER EXACTLY
WHERE IN P53 T-ANTIGEN BINDS BUT
I'M GOING TO ASSUME IT'S THE
AMINO OR CAR BOXY TERMINUS
BECAUSE THAT'S WHERE MOST
INTERACTIONS OCCUR AND HAVE YOU
THE BYPASSING BOUND TO DNA AND
SO I THINK WHAT WOULD HAPPEN AND
I DON'T KNOW IF ANYBODY'S DONE
THE EXPERIMENT IS THAT THE
INTERACTIONS OF THAT T-ANTIGEN
WITH MUTANT P53 IS WOULD
STABILIZE THAT MUTANT PROTEIN
AND THEN YOU WOULD GET, IT
WOULDN'T JUST BE LOSS OF P53
WILD-TYPE P53 ACTIVITIES YOU
WOULD HAVE A MORE STABLE PROTEIN
THAT WOULD CONTRIBUTE TO A MORE
AGGRESSIVE PHENOTYPE.
IT WAS THE PREDICTION.
>>RIGHT.
AND THAT WOULD BE THE CASE FOR
EVERY ADEN O HPV WHARKS HAVE YOU
BECAUSE I NOTICED IN YOUR 125
GENES THAT WERE MODULATED,
CERVICAL, VIRAL AND BURKEETS.
>>RIGHT.
>>IN FACT WE PUBLISHED A NICE
REVIEW A FEW YEARS BACK ABOUT
ALL THE MECHANISMS THAT CAN
ENACT VAIF P53 AND THAT'S
SOMETHING THAT WE NEED TO THINK
ABOUT BECAUSE YOU KNOW HPV, E6,
I THINK, DEGRADES WHILE TYPE P53
SO TO TREAT THOSE CANCERS YOU
NEED TO REMOVE HPV SO THAT THE
P53 CAN COME IN AND DO ITS WORK.
OTHER MECHANISMS INCLUDE
EXPRESSION OF THE MDM2 OR 4, AND
YOU SEE THOSE IN A LOT OF
SARCOMAS.
SO AGAIN, THERE ARE MDM2
INHIBITORS AND MDM4 BEING
DEVELOPED IN THE CLINIC TO
DISRUPT THAT MDM2 INTERACTION,
THEY'VE BEEN A LITTLE BIT TOXIC
IN THE TOXICITIES WERE PREDICTED
FROM OUR MODELS, BONE MARROW,
YOU KNOW IT'S A HIGHLY
PROLIFERATIVE TISSUE, YOU
DISRUPT THAT MDM2 P53
INTERACTION AND YOU DPET
DEPLETION OF BONE MARROW.
AND THEN, SO IT'S THE VIRAL
OVEREXPRESSED WITH P53 MUTATIONS
AND SEVERAL MECHANISMS AND I
THINK WE NEED TO BE COGNIZANT OF
HOW P53 WAS DISRUPTED IN ORDER
TO BE THINKING ABOUT HOW WE'RE
GOING TO TREAT THE PATIENTS BUT
WE'VE DONE A FEW STUDIES IN
ANIMAL MODELS WHERE WE
REINTRODUCED WILD-TYPE P53, IF
THERE'S NO P53 TO START WITH,
IT'S VERY GOOD AT SUPPRESSING
THAT GROWTH BUT IF YOU HAVE
MUTANT P53, IT CAN'T SUPPRESS.
, THATTA WHAT YOU HAVE WITH THE
VIRUS.
>>YES, SORE IF YOU HAVE HIGH
LEVELS OF MDM2 YOU CAN'T GET
ENOUGH WILD-TYPE IN THERE TO
SUPPRESS GROWTH.
SO THOSE ARE ALL THINGS WE NEED
TO CONSIDER WHEN WE MOVE FORWARD
CLINICAL TREATMENTS.
>>YEAH.
>>THANKS.
>>YES?
>>I NOTICED THAT I MEAN YOU
REALLY PRESENT THE STORY IN SOME
WAYS OF P53 AND PARTICULARLY THE
MUTANT AS A REDUCKS MODULATOR IN
THE CELL BUT I WONDER IF THE
OTHER SIDE OF THE EQUATION, THE
IRON AND THE HANDLING OF IRON
AND THE CELL, THE TRANSPORT OF
IRON INTO THE CELL OR OUT OF
TRANSFERRING DOES THAT CHANGE IN
ANY WAY WITH THESE MUTANTS.
>>THAT'S A GREAT QUESTION.
WE HAVEN'T MEASURED IRON IN THE
SYSTEM.
I DON'T KNOW HOW TO DO IT BUT I
THINK WE COULD FIND OUT.
YEAH, THAT'S A GOOD QUESTION
BECAUSE, THAT IS REQUIRED, SO
MAYBE APPROACHING THE PROBLEM.
>>YEAH.
>>EQUATION THAT--
>>YEAH, I HADN'T THOUGHT OF
THAT, THANK YOU.
>>THANK YOU.
>>WE WILL GO BACK AND THINK
ABOUT EXPERIMENTS TO DO.
>>ONE MORE ONLINE, THIS IS FROM
A COLLEAGUE ROWLAND OWEN WHO IS
I THINK TRIES TO MAKE ME TONGUE
TIED SO HERE'S MY BEST HERE
FLUTE O THE IPAD OWN CAN
RECHARGE NUCLEOTIDES O TIDE
REDUCT ACE, HAVE YOU LOOKEDDA
THE DNTPSPIEZ POOLS IN ANY OF
THESE P53 MUTANTS?
>>THAT'S A GREAT QUESTION.
BECAUSE THERE'S OLD DAT THAT
WILD-TYPE P53 ACTIVATES REDUCT
ACE AND CAUSES CHANGES IN THE
DUCT ACE BUT WE DON'T HAVE
WILD-TYPE IN OUR SYSTEM, WE
HAVEN'T LOOKED SO WE NEED TO
LOOK.
YES?
>>I'M NOT SAYING THAT WHAT
YOU'RE PRESENTING WASN'T
INTERESTING, I LOVED IT BUT I'M
STARTING TO GET CURIOUS ABOUT
THOSE NONP53 TUMORS AND IF
THERE'S A--IF YOU'VE SEEN OR A
POSSIBILITY THAT YOU INITTIAIT
THE TUMOR WITH THESE MUTANT
FORMS BUT THEN CAN YOU GET LOSS
OF THAT AND COMP PENSATORY LOSS
AND SAY FERRO--FER, OPITOSEIS.
DO YOU SEE IN THE TUMORS THERE'S
MUTATIONS IN IN FERROPITOSEIS
PATHWAY SNR.
>>THAT'S--WE DIDN'T SEEQUENCE
THOSE TUMORS BUT I THINK THAT'S
A GOOD POINT BECAUSE YOU SHOULD
ALWAYS SEEQUENCE THE OPPOSITE,
THE 1S THAT AREN'T--NO, WE
HAVEN'T DONE THAT.
>>YOU JUST MIGHT SEE--
>>MAKES PERFECT SENSE, YOU
DON'T JUST SEQUENCE THE 1S THAT
GIVE YOU THE RESULTS YOU WANT,
YOU SEQUENCE THEM TO GLEAN MORE
FROM THE 1S YOU DON'T.
WE WILL GO BACK.
, IT MAY COST MORE MONEY.
>>THANK YOU.
>>GIGI, THANK YOU REFLECTIVE OF
MY IGNORANCE, IN THE FIELD OF
P53,.
>>ALL QUESTIONS ARE GOOD.
>>IS THERE ANY ROLE IN STEM
CELL HOMEOSTASIS AND EACH STEM
CELL, P53 TREAT ANY ROLE?
AND I'M THINKING OF THOSE TUMORS
THAT ARE HYPOTHESIZED TO BE
DERIVED FROM STEM CELL AG
GRAVATION?
>>SO THE P53 NULL MOUSE FOR THE
MOST PART IS VIABLE, SO AND IT
LOOKS NORMAL IN THE RIGHT SIZE
AND IT'S TUMOR PRONE, BUT IT
LOOKS DEVELOPMENT NORMAL SO IN
THAT SCENARIO, I WOULD SAY NO
BUT THERE'S AN INTERESTING
PHENOTYPE IN THE LIVER, AND THAT
IS IN YOU DELETE MDM2 IN THE
LIVER AND I TOLD YOU IT WAS CELL
LETHAL SO IT WAS--IT WAS TD THE
ALBUT MIN PROMOTER SO YOU DELETE
IT IN THOSE CELLS AND WHAT
HAPPENS IN THAT SCENARIO IS THAT
THE REMAINING CELLS REGENERATE A
LIVER.
SO THEY GET RID OF THE CELLS
THAT HAVE 2 HIGH LEVELS OF P53,
BUT THAT SENDS A SIGNAL TO THE
REST OF THE LIVER TO REGENERATE.
AND SO I DO THINK THERE'S A
WILD-TYPE ROLE IN REGENERATION.
I THINK THAT IT'S--IT'S NOT AT
THE CELL LEVEL LIKE YOU WERE
IMPLYING, IT'S AT THE ORGANISMAL
LEVEL.
WE HAD SIMILAR DATA AGING AGO IN
THE INTESTINE BECAUSE WE WOULD
TAKE OUT MDM2 IN THE INTESTINE
AND IN THE YOUNG MOUSE WE PUNCH
ALL THESE HOLES IN THE
INTESTINE, THEY WERE SICKLY, A
WEEK LATER THEY'RE FINE AND WHEN
WE LOOK AT THE MOUSE, EVERY CELL
THAT HAD DEPLETED MDM2 WAS GONE
AND WE HAD THIS CRYPT FUNCTION
THAT INCREASED AND SO,--SO THERE
IS A COMP PENSATORY MECHANISM TO
REGENERATE A TISSUE WHEN P53
DEPLETES THE TISSUE.
>>THANK YOU.
>>IT'S A FASCINATING PHENOTYPE.
>>SO PLEASE JOIN ME IN
CONGRATULATING OUR SPEAKERS AND
THANKING THEM FOR A WONDERFUL
TALK.
[APPLAUSE ]
>>THANK YOU ALL.
ENJOYED BEING HERE IN PERSON.