IT'S MY PLEASURE TO WELCOME
DR. STEPHANIE HELENA TO THE
NATIONAL CANCER INSTITUTE.
DR. HELENA IS THE PROFESSOR OF
HEMEATOLOGY AND CHIEF OF THE
SECTION OF HEMEATOLOGY AT THE
YALE SCHOOL OF MEDICINE AND I
IPT GREATER VIETED HER BECAUSE I
CONSIDER HER A STUNNING EXAMPLE
OF A PHYSICIAN SCIENTIST WHOSE
BOTH A CARING PHYSICIAN AND AN
OUTSTANDING SCIENTIST WHO
COMBINED BASIC TRANSLATIONAL AND
CLINICAL STUDIES TO ADVANCE OUR
UNDERSTANDING OF HEMEAT O LOGIC
MALIGNANCIES.
SHE RECEIVED HER DOCTORAL DEGREE
FROM THE EVERHART MEDICINE AND
AFTER THAT SHE JOINED CHILDREN'S
HOSPITAL LOS ANGELES WHERE SHE
STUDIED HEMATOPOIETIC STEM CELL
BIOLOGY.
IN 2000 SHE JOINED THE YALE
INTERNAL MEDICINE RESIDENCY
PROGRAM IN THE PHYSICIAN
SCIENTIST RESEARCH TRACK AND IN
2002 SHE BEGAN A HEMEATOLOGY
FELLOWSHIP WHERE SHE STUDIED
NUTRIFILL WITH MARY, AND THEN
ALSO STUDIED WITH DR. KRAUSE.
IN 2010 SHE TEABED HER OWN LAB
RAARE TOY IN THE SECURITIZATION.
OF HEMEATOLOGY AND IN 2019 SHE
BECAME THE INTERIM SECTION CHIEF
OF HEMEATOLOGY AND IN 2020, WAS
LABEL--WAS NAMED SECTION CHIEF,
WAS NAMED THE SECTION CHIEF OF
HEMEATOLOGY.
SHE ESTABLISHED THE HEMEATOLOGY
TISSUE BANK AND TOOK ON THE ROLE
OF ASSISTANT MEDICAL DIRECTOR OF
THE CLINICAL TRIALS RESEARCH
SUPPORT LABORATORY IN THE YALE
CANCER CENTER.
SHE'S ALSO CHIEF OF THE DIVISION
OF CHANSALATION HEMEATOLOGY AND
DIRECTS THE DE LUCA CENTER FOR
HEMEATOLOGY RESEARCH IN THE YALE
CANCER CENTER.
HER SCIENTIFIC INTERESTS FOCUS
ON HEMEAT O POETICESIS,
MYELODYSPLASTIC SYNDROME AND
AAMERICA L.
AND IN COLLABORATION WITH
COLLEAGUES AT YALE AND ELSEWHERE
SHE'S DEVELOPED A
XENOTRANSPLANTATION MODEL FOR
MDS, ADVANCED OUR UNDERSTANDING
OF R NA SPLICING FACTORS IN MDS
AND ALSO HEMEAT ON POETICESIS
AND LEUKEMIA.
SHE'S RECEIVED SIGNIFICANT
RECOGNITION FOR HER WORK.
IN 2015 SHE RECEIVED THE SIR
WILLIAM AWARD FROM THE
INTERURBAN CLINICAL CLUB AND IN
2022 SHE RECEIVED THE EMERGING
LEADER IN INTERNAL MEDICINE
AWARD FROM THE YALE SCHOOL OF
MEDICINE AND IN 2024 SHE WAS
ELECTED TO MEMBERSHIP IN A
CONNECTICUT ACADEMY OF SCIENCE
AND ENGINEERING SO PLEASE JOIN
ME IN WELCOMING DR. STEPHANIE
HELENA TO THE NCI.
[ APPLAUSE ]
>> THANK YOU SANDY FOR THIS
AMAZING INTRODUCTION, VERY KIND.
[INDISCERNIBLE] COVID OR FLU,
BUT OTHERWISE IT'S JUST
ALLERGIES AND THEY DON'T SEEM TO
GO AWAY, BUT I UPON TO SAY AS A
PHYSICIAN, I REALLY, REALLY YOU
KNOW WHEN I STARTED RESEARCH, I
REALLY HAD NO CLUE AND I THINK
TO BE A PHYSICIAN WHO ALWAYS
DOES RESEARCH IT TAKES THIS
VILLAGE AROUND YOU AND SANDY
[INDISCERNIBLE] WAS 1 OF THOSE
PEOPLE ENCOURAGING PHYSICIANS
LIKE ME, YEAH, JUST WORK ON RNA,
IT'S AN ARK MAZING FIELD AND
THAT IS I'M INCREDIBLY GRATEFUL
FOR THE SCIENTISTS, INVITING
SOMEBODY LIKE ME INTO DOING
SCIENCE AND DISEASE, SO I HAVE
KIND OF 2 STORIES FOR YOU AND I
KNOW THIS IS ALSO CLINICAL, I
HAVE A BIT OF CLINICAL
BACKGROUND.
I KNOW THERE ARE TRAINEES IN THE
ROOM, IF YOU WANT TO ASK
QUESTIONS, PLEASE INTERRUPT ME
AND WE CAN IMPROVISE AT THE END
AND CUT A FEW SLIDES.
OKAY, LET'S SEE HOW I MOVE
FORWARD.
>> OKAY, SO MY LAB IN PARTICULAR
IS INTERESTED IN MYELODYSPLASIA
BECAUSE IT'S ALSO A CLINICAL
PROBLEM I'M INTERESTED WHEN I
SEE THESE PATIENTS I ALSO WANT
TO KNOW WHAT WE CAN DO FOR THEM
AND TREATMENT ISN'T GREAT AND IF
YOU LOOK HERE AT MDS
MYELODYSPLASIA, THERE'S OVER
20,000 PATIENTS IN THE U.S. PER
YEAR, IT'S CAUSED BY GENETIC,
EPIGENETIC, TRANSKRINGZ AND
SIGNALING AND SPLICING
ABNORMALITIES AND MANY PATIENTS
HAVE CYTOPENIA, AND HEART
DISEASE OR WHATEVER DUE TO MDS
AND A SUBSET PROGRESS TO AML.
IF YOU SEE HERE, SURVIVAL ISN'T
GREAT AND IT AFFECTS OLDER
PEOPLE ON THE RIGHT THAT IS
ALWAYS RELATIVE.
SO THE HEMATOPOIETIC STEM CELL
DISORDER SO ALL CELLS IN THE
BLOOD CAN BE AFFECTED BUT
PREDOMINANTLY MYELOID
ILLUMINATEIAGE IS SHOWN HERE
ASSOCIATED WITH INCREASED AGE,
EXPOSURE TO PREVIOUS CHEMO
THERAPY AND RADIATION, SO IF YOU
CURE PATIENTS FROM THE CANCER,
THEY'RE NOW AT RISK FOR MDS, AND
THEN PROGRESSION TO SECONDARY
AML AND IN 2011, BEING AN
OPPORTUNE TIME FOR MY LAB, THESE
MUTATIONS WERE DISCOVERED TO
OCCUR IN OVER 50% OF THE
PATIENTS WITH MDS AND SEVERAL
OTHER PATHWAYS ARE ALSO AFFECTED
BY OUR FOLKS TODAY ARE ON
SPLICING.
SO MY TALK HAS KIND OF 2 SIDES,
1 IS REALLY TALKING ABOUT OUR
WORK ON LIMITATIONS AND I HOPE I
WILL GET TO TELL YOU ABOUT THE
XENOTRANSPLANTATION MODEL WE
DEVELOPED TOGETHER WITH RICHARD
[INDISCERNIBLE].
SO JUST A FACTOR ON THE
MUTATIONS, THEY WERE DISCOVERED
IN 2011, YOU WONDERED WHY NOT
BEFORE BUT THIS WAS A BIG EFFORT
TO SEQUENCE THE MDS GENOME, THEY
ARE REALLY KEY FACTORS OF THE
SPLICING MACHINERY AND MAYBE
THAT'S WHY THEY WERE ALSO KIND
OF--LIMITATIONS WERE IGNORED
BECAUSE PEOPLE THOUGHT IT WAS
IMPOSSIBLE.
AND THEY OCCURRED IN OVER 50% OF
THE PATIENTS WITH MDS OR CHRONIC
MONOSITTIC LEUKEMIA AND IN 20%
OF THE PATIENTS WITH SECONDARY
AML BUT ALSO WITH THE CHRONIC
LEUKEMIA AND EVEN THIS A HANDFUL
OF SOLID TUMORS AND WE DON'T
KNOW WHY THE SELECTION SO
THERE'S STILL A LOT OF WORK ON
TO DO.
WHAT'S INTERESTING IS THAT I
THEY ARE MUTUALLY EXCLUSIVE SO
PATIENTS GENERALLY OVER HAVE 1
OF THE PLIESING MUTATIONS, IF
THERE ARE 2, THEY ARE IN THE
SAME CELL, THE VERY FREQUENTY IS
HIGH, AND REALLY THEY ARE IN THE
DOMINANT CLONE, AND WE KNOW THAT
THEIR DRIVER MUTATIONS FOR MANY
MOUSE STUDIES FOR EXAMPLE, SO
JUST A BRIEF, BRIEF, VERY SIMPLE
PRIMER ON SPLICING RIGHT, SO IN
THE GENE, YOU HAVE REGULATORY
SEQUENCES, ENHANCES PROMOTERS
YOU HAVE THE TEAM AND THEN THE
YEEN IS EXIT AND ENTRON, THEY
SPLICE THESE ENTRONS OUT TO
CREATE THE MRNA, OR THE
RNATHAT'S UNCAPPED,
[INDISCERNIBLE] EXPORTED INTO
THE CYTOPLASM WHERE IT'S THEN
TRANSLATED INTO PROTEINS.
AND YOU KNOW AUTOSPLICING IS AN
AMAZING REGULATORY MECHANISM
WHERE YOU CAN NOW CREATE PROTEIN
DIVERSITY FROM A TRANSCRIPT
THAT'S DIFFERENTIALLY SPLICED
WHEN THE SPLICING IS
DISREGULATIONED, THEN YOU KNOW
YOU LOSE PROTEINS, YOU GAIN
PROTEINS, SO THAT IS PART OF THE
DISEASE PROCESS.
GENERALLY WE THINK OF THESE
MUTATIONS AS HOT SPOT MUTATIONS
SO THEY'RE VERY SPECIFIC
RESIDUES IN THE DIFFERENT
PROTEINS THAT MUTATED AND WHAT
WE KNOW SO FAR IS THAT THEY ARE
ALL KIND OF INTERACT WITH THE
RNA, ZRSR2 IS A BIT DIFFERENT,
THE CELLS HAVE TO HAVE A
WILD-TYPE ALLELE AND WE THINK OF
THEM AS GAIN OF MUTATION
FUNCTIONS.
MEANING THE PROTEIN FUNCTION IS
LOT BUT THERE'S SOMETHING NEW
ADDED TO WHAT THE PRACTICES TEEN
- IS NOW DOING.
AND WHEN WE SPLICE THESE
MUTATIONS TO FIND CLINICAL MINO
TYPES SO AS THE MUTATIONS OCCUR,
THE CANNED WHAT UV MELANOMA AND
MUTATIONS OCCUR IN MDS BUT IN
LUNG CANCER AS THEY OCCUR IN MDS
AND CHRONIC MILE O SIDIC
LEUKEMIA BUT I DON'T THINK
THIEVE BEEN SEEN INSIDE TUMORS.
THIS IS JUST AN OLDER
CLASSIFICATION, AND YOU CAN SEE
HERE, YOU KNOW HOW FREQUENT
THESE SPLICING FACTORS ARE, THE
MUTATIONS ARE THE MOST COMMON
AND THEY'LL CURE AGAIN, THERE'S
SUBTYPE SPECIFICITY WITH AN MDS,
I KIND OF TRIED TO PULL OUT LIKE
THE KEY DIFFERENTIALLY PLIESED,
YOU KNOW TRANSCRIPTS AND YOU
KNOW RIGHT, THEY VARY BETWEEN
THE DIFFERENT SPLICING FACTORS,
THERE ARE FEW THAT MAYBE
OVERLAPPING BUT OF COURSE WE ALL
WANT TO KNOW WHY LIMITATIONS
WHAT DO THEY HAVE IN COMMON AND
MAYBE THAT'S A COMMON MECHANISM
THAT WE CAN TARGET, AND WHAT YOU
SEE HERE IT'S AGAIN, THIS TILL A
SIMPLIFICATION OF PLIESING AND
YOU VALID TO HAVE
[INDISCERNIBLE] IN THE AUDIENCE
TO GO IN NOR DETAIL BUT YOU SEE
THESE SPLICING FACTORS, ALL HAVE
A FUNCTION EARLY IN THE WHOLE
SPLICING PROCESS AS I DEFINE THE
EXOHMS THAT ARE INCLUDED OR
EXCLUDED, THE SPLICE SIDES, AND
REALLY INITIAL ATTACK AT THESE
ENTRON EXON JUNCTIONS.
AND SO, EARLY ON, SO WE WERE
WORKING ON REALLY, REALLY
COMPLICATED FUSION PROTEIN IN MY
LAB AND I WAS INTERESTING IN THE
BINDING PROCESS AND THESE
MUTATIONS WERE PUBLISHED AND
THAT'S WHAT WE JUMPED ON BECAUSE
I REALLY WANTED TO BE AN ADULT
HEMEATOLOGYIST AND NOT A
PEDIATRIC HEMEATOLOGYIST, AND WE
CHOSE THE [INDISCERNIBLE]
BECAUSE IT BINDS RNA, IT BINDS
EXONS AND THOSE ARE MORE
CONSERVED BETWEEN MOUSE AND
MUSEUM MAN.
AND JUST A QUICK PRIMER ON WHAT
WE DID, RIGHT?
, SO, AS THE TUMORS ARE KNOWN TO
HAVE THE RNA BINDING DOMAIN AND
KIND OF LIKE THESE PIECES OF RNA
AND 1 SIDE AND ON THE OTHER SIDE
AND THIS REGION HERE IS CALLED
THE LINKER SO IT LOOKED LIKE
THESE MUTATIONS ARE JUST IN A
LINKER REGION, BUT THAT'S NOT
TRUE BECAUSE YOU CAN SIGH HERE,
THESE 2 RNA SEQUENCES, AND
ACTUALLY IT'S A CONIF I WERATION
CHANGES COME SF 2 WHEN RNA'S
THERE AND THESE 2 LINKERS AND
END TERMINAL REGION ARE REALLY
KIND OF LIKE ARMS THAT GO AROUND
THE RNA AND IT MAKES SENSE IF
YOU HAVE A MUTATION HERE, A
CHANGE MAY BE HOW RNA IS BOUND
AND THAT'S WHEN WE DID IN
COLLABORATION WITH YOUR
[INDISCERNIBLE], AND GOT ON TO
SHOW THAT THERE IS SIGNIFY
SEQUENCE SPECIFIC DIFFERENCE IN
BINDING AFFINITY.
WE PERFORMED YOU KNOW BACK
THEN HITS CLIP AND NOT SO
EFFICIENT TECHNOLOGY TO LOOK AT
HOW WILD-TYPE AND MUTANT, HAVE
BIND RNA AND THEN WE SEE A
SEQUENCE MOTIVE DIFFERENCE AND
SO IN MUTANT TO CC& G
CONTAINING EXONS ARE
PREFERENTIALLY INCLUED AND
[INDISCERNIBLE] ARE EXCLUDED.
SO YOU HAVE ABERRANT SPLICING.
AND THEN AT THE SAME TIME, WHILE
BRADLEY AND DAUBNER WERE WORKING
ON [INDISCERNIBLE], AND OMAR
GENERATED THE MOUSE MODEL SO WE
PUT ALL OUR DATA TOGETHER AND
THE SRSF2 MUTATIONS DID BIND AND
SPLICE, IT IS INCLUSION OF
SOPHISTICATED CALLED POISON EXON
THAT CHANGES THE FRAME AND
RESULTS IN DECREASED TRANSLATION
OF EZH2, ALSO CONSIDER IN
MDECKER S BUT THEY'RE MUTT YULE
LE EXCLUSIVE SO OTHER BIOLOGY
MADE SENSE, I ALWAYS FELT THIS
WAS LIKE THE FINAL PUBLISHING WE
HAVE TO DO, RIGHT?
YOU HAVE A REALLY COMPLEX
PROCESS, YOU AND YOU FIND 1
ALTERNATIVE SPLICED EVENT THAT
TELLS A BEAUTIFUL STORY, SO WE
STILL DON'T KNOW SO MUCH ABOUT
HOW THE SPLICING FACTOR
MUTATIONS FUNCTION BUT IT WAS A
GREAT START.
SO OF COURSE EARLY ON, YOU KNOW
PEOPLE WERE ASKING, OKAY, CAN WE
TREAT PATIENTS, CAN WE TREAT
PATIENTS AND FROM THE FACT THAT
YOU KNOW THAT THESE MUTATIONS
ALWAYS HETEROZYGOUS, HE KNOWS HE
RELIES ON THE WILD-TYPE ALLELE,
THEY'RE MUTUALLY EXCLUSIVE SO
ONLY 1 SPLICING MUTATION
TOLERATED, LATER ON PEOPLE PROVE
THAT IN-MOUSE MODELS AND SO CAN
YOU ACTUALLY KIND OF LIKE, YOU
KNOW PERTURB THE WILD-TYPE
NORMAL SPLICING PROCESS AND WHAT
MUTANT CELLS WOULD BE
PREFERENTIALLY SENSITIVE.
AND YOU KNOW THERE WAS AN
INHIBITOR OF SF 3 B 1 AND SO
PEOPLE THOUGHT LIKE OKAY, COME
AND USE THE SFB 3 B 1 INHIBITOR
AND THEN CAN WE SPECIFICALLY
TARGET MUTANT MDS AND I THINK IN
MOUSE MODELS AND CELL LINES IT
LOOKED ALL GREAT.
WHEN I WENT INTO CLINICAL TRIAL,
UNFORTUNATELY IT DID NOT WORK.
THERE'S ALSOA A LOT OF FEAR
OVER, YOU KNOW TOXICITY, THIS
WAS LIKE AN OLDER COMPOUND THAT
HAD LED TO BLINDNESS WHICH
DIDN'T HAPPEN IN THIS TRIAL BUT
REALLY WAS NO MUTANT SPLICING
SIGNATURE SO RIGHT, SO NOW THIS
DRUG IS NOT ON THE MARKET,
ALTHOUGH PEOPLE AGAIN CAREFULLY
LOOKING AT THE DATA AND
HOPEFULLY FINDING MAYBE A SUBSET
OR COMBINATIONS OF OTHER
MECHANISMS WHERE IT CAN BE USED
IN THE TREATMENT OF MDECKER S
BECAUSE WE DON'T HAVE ANYTHING
GOOD TO GIVE TO PATIENTS.
SO AT THAT SAME TIME, YOU KNOW
RIGHT, ONCE YOU DEVELOP A REALLY
GOOD APPROACH, ECNOLOGY, IT WAS
OBVIOUS FOR US, OKAY, LET'S
TACKLE U2 FAC1, BECAUSE THAT'S
WHAT WE CAN DO IN THE LAB, WE
DIDN'T WANT TO REINVENT THE
WHILE, WE DIDN'T WANT TO MOVE ON
TO MANAGE NEW.
SO WE LOOKED AT MUTANT 1 BIND
AND HOW DO THEY ALTER BINDING
AND THERE WAS THIS VERY SIMPLE,
YOU KNOW HYPOTHESIS THAT PEOPLE
HAD FROM IN INVITRO DATA AND
ALSO RNA SEQUENCING DATA WHERE
IT SAID, YOU KNOW, SO IT MUST
JUST NOT BIND AND THEN HAVE YOU
EXON EXCLUSION.
I SAID WELL, LET'S LOOK AT OUR
TECHNOLOGY AND WHEN I SAY LET'S
LOOK, I TOLD YULIA CAN YOU
PLEASE LOOK AND SO JULIA IS AN
AMAZING POST DOC, AND JUNIOR
SCIENTIST IN THE LAB ACTUALLY
LOOKING FOR FACULTY POSITION,
SHE'S UTTERLY AMAZING, SHE'S A
TRIPLE THREAT THAT EVERYONE
WANTS TO HAVE IN THE LAB,
BIO-INFORMATICS, AMAZING AT THE
BENCH, FANTAST COLLABORATION AND
TUMOR BIOLOGY WHO FORTUNATELY
WORKED IN MY LAB FOR 3 YEARS AND
I WAS A PROFESSOR IN ITALY SO WE
HAVE JOINT LABS ACROSS THE
ATLANTIC.
SO AGAIN, IF YOU LOOK AT U21 AND
USF2, SO HERE THESE FINGER
DOMAINS THAT HAVE THE POINT
MUTATIONS AND IN THE MIDDLE IS
YOUR END DOMAIN WHERE PEOPLE
HAVE SHOWN BEAUTIFULLY IT'S
THESE 2 ZINC FINGERS THAT
COMBINE AND ARE ACTUALLY THE RNA
BINDING SURFACE WHICH IS
GORGEOUS.
THE PROBLEM IS THAT IT ALWAYS
OCCURS IT'S LIKE AN OBLIGATORY
HETERODIMER.
AND PEOPLE ALWAYS THOUGHT, WELL
WE KNOW U2 AF2 BINDS BUT IT
HADN'T BEEN PROVEN IN VIVO.
SO WE DECIDED, YOU KNOW TO USE
ADVANCED TECHNOLOGY OVER
[INDISCERNIBLE] CLIP ENHANCED
CROSS LINKS AND IMMUNO
PRECIPITATION, AND PULL OUT
THOSE THAT HAVE A FLAG TAG.
AND WE LABEL THESE RNAs THAT
WE PULL OUT WITH P32 AND WHEN WE
SEQUENCE TWINED OF THE END OF
THE READ FROM THE REVERSE
TRANSCRIPTION IS EXACTLY THE
CROSS LINKING SIDE.
AND BECAUSE WE KNEW THAT YOU
KNOW WE HAVE THIS HETERODIMER,
YULIA DECIDED TO CUT THIS
MEMBRANE, THIS IS THE RIA THAT'S
BOUND, LABELED AS P32 TO CUT THE
MEMBRANE JUST UNDERNEATH THE
SIZE OF U2 AF2, WHICH IS 65
KILODALTONS.
AND THEN THE BOUND RNETWORKA
SHIFT IS BOUND THESE PROTEINS
UP.
SO SHE CUT THE MEMBRANE TO
RELOOK AT, OKAY, WHAT DOES U2
AF1 RECEIVE THE DIFFERENCES?
SO UP HERE WE DIDN'T KNOW WHAT
WE WERE GOING TO GET, WERE WE
GETTING U2 AF2 OR
HETEROBINDINGING.
SO HERE YOU SEE THE
[INDISCERNIBLE] TRACK AND YOU
CAN SEE THE VERY BRIGHT CLEAR
PEEK OF U2 F1 BINDING AT THE 3
PRIME SPLICE SIDE AND THEN RIGHT
IN THIS LARGER BAND THAT WE
PULLED OUT, YOU CAN SEE THAT
THERE'S U2 AF1 BINDING AND NOW
YOU SEE U2 AF2 BINDING OVER THE
TRACT.
IF YOU LOOK AT WHAT DO THE
MUTATIONS DO, SO ORANGE THE S34
F-MUTATION IN THE FIRST FINGER
AND THE R-MUTATION THE
SEQUENCING FINGER, YOU CAN SEE
THEY'RE DIFFERENT, THEY'RE NOT
THE SAME SO CAN YOU SEE THAT
WITH F34 F WE GAIN A MINUS 3 P,
OVER THIS C VERSUS U-3 POSITION
AND WITH Q87 WE GAIN A VERY
SUBTLE PLUS 1 PEAK.
AND THEN THE OTHER THING YOU CAN
SEE THAT THE MUTATIONS, THEY NOT
ONLY AFFECT THE U2 F 1 BINDING
BUT MABEL THEY ALSO AFFECT HOW
THE WAVE INTERACTING WITH THE R,
INA AND THEN IF WE ARE NOT
COMBINE THIS WITH THE SEQUENCING
DATA AND THAT HAD ALREADY BEEN
PUB LESHED BEFORE, IS THERE IN
BINDING WE ACTUALLY DO NOT HAVE
A SEQUENCE SPECIFICITY TO
WHETHER YOU--S34 F-MUTANT BINDS
OR DOESN'T BIND, IT BINDS
EQUALLY WELL, IT DOESN'T MATTER
WHAT THE SEQUENCE IS AT THE 3,
OR WHAT THE SEQUENCE IS AT THE +
1 BUT WHEN YOU LOOK AT ACTUAL
SPLICING, IF YOU HAVE U AT THAT
MINUS 3 POSITION, THE EXON IS
ACTUALLY PREVENTIVE
REPRESENTIALLY EXCLUDED IF YOU
HAVE A C IT'S PREFERENTIALLY
INCLUDED.
SO SPLICING DOESN'T NECESSARILY
MEAN SPLICING IN THE OUTCOME SO
A FEW MORE WORK ON DO.
SO THIS IS NICE AND IT'S NOT
LIKE ALL OR NOTHING OR BLACK AND
WHITE.
SO JULIA REALLY INTEGRATED
BINDING AND SPLICING AND IT
SEEMS THAT U2 AF1 MUTATIONS LOOK
LIKE MORE OF GAIN OF BYPASSING,
LOTS OF SPLICING MODELS, THAT'S
THIS QUADRANT HERE, MORE BINDING
BUT THERE'S LESS EXIT AND
INCLUSION AND THIS IS AN EXAMPLE
OF BOTH, YOU KNOW THE U2 F 1 AND
THEN THE COMBINED BINDING AND
YOU CAN SEE THAT THERE'S A
REDUCED BINDING THAT IS NEEDED
FOR THE INCLUSION OF THE EXON
AND THEN WHEN YOU LOOK AT U2 AF1
MUTATIONS, THAT LOOKS MORE LIKE
A LOSS OF BINDING, LOSS OF
SPLICING MODEL KIND OF SHOWN
HERE, RIGHT, SO THERE'S LESS OF
A PEAK AT THIS 1 POSITION AND
ALSO YOU AGAIN HAVE KIND OF LESS
BINDING OF U2 F2, BUT OF COURSE
YOU HAVE DOTS IN ALL THE
QUADRANTS.
SO IN THE NEXT STEP, YULIA
LOOKED AT OKAY, WE WANTED TO
UNDERSTAND WHAT ARE THE DIRECT
EFFECTS OF THESE MUTATIONS SO WE
START BY INTEGRATING, BINDING
AND SPLICING AND LOOKING AT
PATHWAYS THAT WE WOULDN'T LOOK
AT SECONDARY EFFECTS BECAUSE ANY
EFFECT BY THESE CELLS GROW FOR
SOMETIME, SO IF YOU JUST LOOK AT
PLIESING, THERE MAY BE MANY
SECONDARY EFFECTS AND IN
PARTICULAR, WE SEE DIFIENTIAL
SPLICING OF OTHER RNA BINDING
PROTEINS, WELL THERE'S KIND OF
THIS CASCADE, BUT BY LOOKING
DREBLGHTLY AT BINDING AND
SPLICING, YOU KNOW MAYBE ABLE TO
LOOK AT DIRECT EFFECTS, AND WHAT
YOU FOUND IN AN ENRICHMENT FOR
THIS, REGULATION OF ORGANELLE
ASSEMBLY, OR CYTOPLASMIC STRESS
GRANULES AND BEFORE THIS THREAD,
MANY OTHER PATHWAYS WERE
IDENTIFIED BUT IN OUR DATA,
BYPASSING, SPLICING, THIS IS A
REALLY PREDOMINANT NODE STRESS
GRANULES BUT ALSO CALLED
[INDISCERNIBLE] BODIES AND OTHER
NUCLEAR BODIES, ASSEMBLIES.
SO, WHAT OUR STRESS GRANULES, I
GREAN THINK IT'S TOTALLY
BEAUTIFUL SCIENCE AND I'M VERY
TBLAD THAT JULIA AS A PUNISHING
HD IS KIND OF GOING INTO THAT
FEE AND I'M LOOKING AT IT LIKE
WOW, THIS IS TOO COMPLICATED FOR
ME, BUT STRESS GRANULES AND MANY
OTHER GRANULES, THEY ARE
ORGANELLES AND THEY RIGHT, SO
THEY'RE LIKE PHYSICAL CHEMICAL
TIMING, WHATEVER, CONGRAMERATES
OF CERTAIN PROTEINS AND RNAs
AND I THINK THE BEAUTY AND
STRESS GRANULES IS THAT THEY CAN
BE--PROBABLY--IT'S PROBABLY
ENERGY EFFICIENT TO REGULATE
TRANSLATION AND CELLULAR
FUNCTION THAT WAY AND IT'S
PROBABLY ALSO, IT CAN HAPPEN
FAST BECAUSE YOU DON'T HAVE THIS
ACTIVE TRANSPORT ACROSS
MEMBRANES.
AND [INDISCERNIBLE] SMART, SO
PEOPLE HAVE SHOWN THAT STRESS
GRANULES SEEM TO PROVIDE AN
ADVANTAGE TO CANCERS AND SO
COULD STRESS GRANULES BE A
MECHANISM BY WHICH THESE MUTANT
CELLS GAIN THAT ADVANTAGE IN
ELDERLY PATIENTS BONE MARROW
OVERTIME SO THE FIRST THING SHE
HAD TO SHOW THAT YES THERE ARE
MORE STRESS GRANULES SO THIS IS
INDUCIBLE CELL LINES, AND TO USE
1 STRESSOXIDATIVE STRESS WITH
[INDISCERNIBLE] AND YOU CAN SEE
THERE ARE MORE STRESS GRANULES
IN THE MUTANT CELLS AND THE
WILD-TYPE CELLS, THESE CELL
LINES WEOXON NOWSLY EXPRESS, YOU
HAVE A MUTANT F1 AND YOU TOOK
PATIENT SAMPLES FROM PATIENTS
WITH SECONDARY AML FROM MDS WHO
ARE EITHER WILD-TYPE OR MUTANT
FOR UGF 1 BUT FOR OTHER SPLICING
FACTORS AND SEE CAN SEE STRESS
GRANULES.
SO, IN ANOTHER--BECAUSE AGAIN 1
CAN SEE LONG INVITRO OF THE CELL
LINES, ET CETERA BUT SHE ALSO
WANTED TO KNOW OKAY, CAN SHE
FIGURE OUT WHAT THESE MUTATIONS
DO, IN THE SINGLE CELL LEVEL IN
THE HEMATOPOIETIC HIERARCHY AND
SHE DID 10 X SEQUENCING ON 5 MDS
PEASHTS WITHOUT ANY SPECIFIC
MUTATIONS AND 3 MDS PASHTS WITH
[INDISCERNIBLE] F MUTATIONS AND
SHE ACTUALLY USED 10 X 5 PRIME
LIBRARY, WHICH IS NICE BECAUSE
THIS S34 F MUTATION, RIGHT, IS
VERY, VERY 5 PRIME AND WE'RE
HOPING THAT MAYBE EVEN IN THE
SINGLE CELL, SCHWARTZ THAT THEY
WOULD GET, THAT YOU WOULD BE
ABLE TO SEE WIDER VERSUS MUTANT
READS SO SHE CAN DO THAT RIGHT,
SO THE IN GRAY, ARE THE CELLS
FROM PATIENTS WHO DON'T HAVE
MUTATIONS, IN THIS DARK ORANGE
ARE CELLS FROM PATIENTS WITH S34
FMARKED FOR IDENTIFICATION
MUTATIONS WHERE FOR EACH CELL
SHE COULD SEE AT LEAST 1 MUTANT
READ AND THEN SLIDE ORANGE ARE
CELLS WHERE THERE WERE TO HAVE
READS BUT NONE OF THEM MUTANT
BUT IF YOU DON'T HAVE ENOUGH
READS, YOU CAN'T NECESSARILY
EXCLUDE THAT THEY'RE MUTANT AND
SORRY, I'LL KEEP CLICKING HERE
AND THEN, RIGHT FROM HER BINDING
SPLICING [INDISCERNIBLE], AND
THEN PUBLISH DATA SETS
PROTEOMICS AND DATA SETS ON ICE
GRANULES, SHE DESCRIBED A STRESS
GRANULE SCORE AND SHE CAN APPLY
THIS TO THE SINGLE CELLS.
I THINK I YUSMED A SLIGHT SO I
THINK I WILL GO BACK TO THAT SO
YOU CAN SEE THAT THE CELLS WITH
MUTANT READINGS REALLY HAVE A
HIGH STRESS GRANULE SCORE
COMPARED TO WILD-TYPE CELLS AND
THESE CELLS WHERE WE DON'T KNOW
OTHER WILD-TYPE MUTANT THEY FALL
IN BETWEEN AND SORRY I JUMPED
HERE, AND WHAT YOU SEE HERE NOW
IS RIGHT, SO THIS REGION HERE
HAS THE PROGENITOR CELLS, GRANUE
LOW CITES, MONOCYTES AND THE
MUTANT CELLS SEEM TO BE ARRESTED
IN THAT MORE PROGENITOR CELL,
AND IF ANYTHING THEY SEEM TO
HAVE MORE MONOSITTIC AND WE SEE
THIS IN PATIENTS WITH ANEMIA AS
IT FITS THEIR PHENOTYPE.
AS A SUMMARY OF THE FIRST PART,
RIGHT, SO IT SEEMS THAT SPLICE
MUTATIONS, USUALLY 1 MUTATIONS
RESULT IN THE STRESS GRANULE
RESPONSE AND IS THAT MAYBE A
MECHANISM THAT ALLOWS THESE
CELLS TO SURVIVE IN AN ADVERSE
ENVIRONMENT WITH AGING AND THEN
THE OTHER QUESTION IS, WAS IT
COMMON TO OTHER LIMITATIONS,
TOO, MAYBE ARE THEY TARGETABLE
IN IS THIS PROCESS TARGETABLE IN
SO THEN WE WENT BACK TO OUR SSF2
MUTANT CELL ANDS DATA AND I
DIDN'T SHOW THIS EARLIER BUT
WHEN WE INITIALLY DID OUR RNA
SEQUENCING, ANALYSIS, WE WERE A
LITTLE BIT AT A LOSS BECAUSE WE
SAW THE DIFFERENTIAL SPLICING OF
MANY OTHER R, INA BINDING
PROTEINS, MANY OF THESE ARE
INVOLVED IN YOU KNOW IN STRESS
GRANULES, FOR EXAMPLE, AND OTHER
MEMBRANES ORGANELLES, AND THEN,
JULIA ANALYZED ALL THE SRF 2
MUTANT PLIESING CELL LINE AT
PATIENT DATA SETS AND ALSO FOUND
AGAIN SOME ENRICHMENT IN THIS
STRESS GRANULE SIGNATURE, AND
THEN AGAIN SHE SEQUENCED--USED
THE SAME PATIENTS BUT ALSO
SEQUENCE SF 2 MUTANT PATIENTS
AGAIN WITH THE 3 BIEM
SEQUENCING, SO RIGHT S34 F IS
THE ESEN 34, P95 AND AN AMINO
ACID, 95, SO MUCH, MUCH, HARDER
TO ACTUALLY FIND READS THAT GO
THAT FAR INTO, YOU KNOW INTO THE
SEQUENCE.
BUT EVEN THERE, THERE'S A
SUGWREOF THE YEN THAT THERE MAY
BE AN INCREASED STRESS RESPONSE
SO THAT COULD BE A COMMON
MECHANISM.
SO THEN THE QUESTION IS HEY,
DOES IT HAVE ANY BIOLOGIC
FUNCTION AND ONCE SHE TAKES THE
WILD-TYPE OR THE CELLS AND
EXPOSES THEM TO
ARE--ADMINISTRATIVE SONNITE AND
LOOKS AT CELL VIABILITY, THERE
SEEMS TO BE SOME ADVANTAGE IN
THE MUTANT CELLS.
AND THERE IS AN INHIBITOR OF
THIS INTEGRATED TRESES RESPONSE
CALLED ISRIP AND IT FUNCTIONS
HERE, IT INHIBITS THE FUNCTION
OF PHOSPHORYLATED EIF2 ALPHA
WHICH IS IN THE STRESS RESPONSE
THAT WOULD LEAD TO STOP OF
TRANSLATION.
AND I LEARNED READING ABOUT
ISRIB, THAT I THINK IT'S BEEN
USED, YOU KNOW PEOPLE WHO WANT
TO NOT AGE OR ANYTHING LIKE THAT
BUT ALSO IN ALL SORTS OF THINGS.
SO WE DON'T KNOW IF THIS IS
VIABLE DRUG OR NOT THAT PEOPLE
COULD TAKE WITH THESE DISEASES,
BUT HE TESTED IT INVITRO IN OUR
CELL LINES AND THE EFFECT IS
VERY, VERY SUBTLE, HENS THIS
REPRESENTATION BUT THERE SEEMS
TO BE A DIFFERENTIAL EFFECT OF
THIS INHIBITOR ON MUTANT
COMPARED TO WILD-TYPE CELLS.
SO THIS IS WHAT SHE HAD SO FAR.
AND THEN OBVIOUSLY, THE OBVIOUS
QUESTION AND THOMAS ASKED ME,
WELL IS IT DIRECT, IS IT
INCORRECT AND WE DON'T KNOW.
BUT THE FIRST PLACE TO ELECTRIC
NOW IS OKAY, WHAT IS IN THESE
STRESS GRANULES?
AND I THINK IT'S DIFFICULT TO
ISOLATE LITTLE ORGANELLES IN TE
MEMBRANES, HOW DO YOU ISOLATE
THEM AND ARE THIS PHYSICAL
CONGLOMERATE BUT SHE GOT A
PROTOCOL FROM [INDISCERNIBLE]
LAB WHO'S THE WORLD EXPERT IN
THIS FIELD AND IT'S A VERY
COMPLICATED PROCESS WITH LOTS OF
SENTRIFICATIONS, LABELING OF
THESE STRESS GRANULES AND THEN
YOU CAN PULL THEM OUT WITH 1 OF
THE STRESS GRANULE, KIND OF LIKE
MARKET PROTEINS GPP31, SO SHE
DID THIS ON CELLS WITH OR
WITHOUT ARSONITE STRESS, AND
SUBMITTED INPUT AND THE IP
PRODUCT BOTH MASS SPEC AND
RNA-SEQ AND THIS IS RECENT DATA
AND WE STILL DOESN'T HAVE AN
ANSWER.
BUT SHE WAS ABLE TO YOU KNOW
PULL OUT 37 DIFFERENTIALLY 37
PROTEINS, THAT WERE PRESENT IN
S34 F MUTANT CELLS, TRESES
GRANULES, VERSUS WILD-TYPE.
AND THEY WERE ALSO ENRICHED OVER
JUST INPUT.
SO THAT'S THE FIRST START AND
THEN, SAME ANALYZING THESE
RNAs, SO THEY'RE--SHE HAS
DIFFERENTIAL GENE EXPRESSION
WITH RNAs ENRICHED IN THE
STRESS GRANULES THAT ARE
DIFFERENTIALLY EXPRESSED IN THE
WILD-TYPE AND THEN ENRICHED IN
THE GRANULES OVER TOTAL SIDE OF
RTHIS,A IN THE CELL, AND SHE
ALSO HAS SPLICING DIFFERENCES,
SO NOW I THINK FOR HER, THIS IS
A PROJECT SHE WILL TAKE ON IN
HER OWN LAB WILL COME TO NOW
FIGURE OUT WHAT THEY ARE AND I
THINK DOES EVERYBODY HAVE A
COLLABORATION HERE WITH THE NCI.
BECAUSE NOW I THINK THE NEXT
WORK WILL BE TO FIND THESE
PROTEINS RNAs AND THEN ALTER
THEIR EXON INCLUSION, EXCLUSION
TO SEE DOES IT ALTER STRESS
GRANULE BIOLOGY.
AND AGAIN, WHAT'S EXCITING I
THINK, MKS, AML, ARE ALL SOLID
TUMORS AND THEY MAY USE SIMILAR
MECHANISMS IN A SIMILAR WAY AND
P-GRANULES THEY'RE INVOLVED IN
MANY CANCER HALLMARKS, SO
HOPEFULFULLY WE WILL BE ABLE TO
FIND A MECHANISM THAT MAYBE
ULTIMATELY NOT OHM LEAD TO
TREATMENT FOR MDS, BUT ALSO
OTHER LEUKEMIAS, AND SO, I'M
SWITCHING NOW, BECAUSE THIS IS
VERY, VERY, RNA HEAVY AND SANDY
INVITED ME BUT I ALSO SAID I'M A
CLINICIAN, LET ME MAYBE SHOW
SOMETHING ELSE THAT WE'RE DOING
IN THE LAB.
SO, WHEN I WAS A POST DOC AND I
WANTED TO STUDY IN SANDY'S LAB,
I HAD 2 PROBLEMS, I HAD NO
SAMPLES.
THERE WERE NO SAMPLES FOR MILE
MYELODYSPLASIA.
AND AT THAT POINT THERE WERE NOT
CONVINCING MOUSE MODELS, BUT I'M
A CLINICIAN, AND SO I ATTENDED
IN-PEASHT SERVICE AND OF COURSE
THAT'S ALWAYS THE WORST, BECAUSE
THE PEOPLE WHO DO WELL ARE NOT
IN THE HOSPITAL AND EVERY TIME I
COME OFF SERVICE, I'M LIKE WHAT
CAN WE DO?
SO I REALLY WANTED TO DEVELOP A
MODEL TO STUDY MDS AND AT THE
TIME THAT I KIND OF WAS A POST
DOC TO THE LAB, WAS VERY
FORTUNATE THAT IN A RICHARD
FLAVELL, WAS DEVELOPING WITH
ANTHONY [INDISCERNIBLE] A POST
DOC IN HIS LAB, THESE MICE WHERE
THEY WANTED TO HAVE A HUMANIZED
IMMUNE SIZE TO STUDY HIV AND
THEN HUMAN RAT CELLS TO STUDY
MALARIA BECAUSE IT WAS IN PART
FUNDED BY THE GATES FOUNDATION
TO IMPROVE GLOBAL HEALTH AND FOR
THAT, THEY DECIDED TO HUMANIZE
THE BONE MARROW BECAUSE THEY
NEEDED TO BE ABLE TO INGRAFT
HEMATOPOIETIC STEM CELLS AND
ALREADY, WE KNEW AT THAT POINT
THAT MDS, YOU CAN TAKE AN A.
L. L. OR MANY A. M. L.S AND PUT
THEM IN THE WORSE DEFIC MOUSE
MODELS AND YOU CAN GET
INGRAFTMENT AND YOU CAN KEEP
THIS OVERTIME BUT MDS, IF PEOPLE
HAD 0.5% INGRAFTMENT, THEY WERE
HAPPY THEY HAD INGRAFTMENT AND
YOU COULD STUDY IT A LITTLE BIT.
SO WHAT RICHARD'S GROUP DID, WAS
TOO LOOK AT, YOU KNOW CYTOKINES
AND HEMEAT O POETICESIS AND WHAT
CYTOKINES ARE NEEDED AND TO LOOK
AT WELL, WHERE IS A CROSS
REACTIVITY BETWEEN MOUSE AND
HUMAN, RIGHT IN AND MOST
IMPORTANTLY THEY WERE LOOKING AT
OKAY, WHICH CYTOKINES FROM THE
MOUSE DO NOT WORK ON HUMAN
CELLS?
AND AMONG THOSE THROMPOETIN, AND
OTHERS, AND THEY PAIRED UP WITH
REGEN RON WHO DEVELOPED THESE
TECHNOLOGIES SO IT WAS LIKE JUST
BEFORE CRISPR SOLVED EVERYTHING
AND THEY INTRODUCE THE ENTIRE
HUMAN GENOMIC LOCUST INTO THE
MOUSE GENOMIC LOCUST.
SO NOW THE ENTIRE CODING AND
ALSO NONCODING BUT EVER SEQUENCE
OF THE HUMAN GENES WERE
PHYSIOLOGICALLY EXPRESSED UNDER
THE MOUSE, YOU KNOW REGULATORY
SEQUENCES.
AND THIS IS KIND OF JUST A
LITTLE BIT OF A STORY OF THESE,
YOU KNOW HUMANIZED--IMMUNO
DEFICIENT MODELS, RIGHT, SO I
REMEMBER WHEN I WAS IN--AT CHLA,
YOU KNOW PEOPLE WERE WORKING ON
NUDE AND SKID MICE, SO WE FIRST
PRIORITY YOU HAVE TO HAVE A
MOUSE THAT IS IMMUNO DEFICIENT
SO THE MOUSE IMMUNE CELLS DO NOT
ATTACK THE HUMAN CELLS AND YOU
KNOW STARTED WITH GETTING RID OF
T-CELLS, T AND B CELLS, MAYBE
INHIBITING A LITTLE BIT
MONOCYTES MACROPHAGES AND THEN
EVENTUALLY ALSO GETTING RID OF
NK CELLS, SO NOW THE HUMAN CELLS
CAN GET IN BUT AGAIN, RIGHT, SO
THEY WERE STILL LACKING
DIFFERENTIATION FACTORS THAT,
WOOED ON THEM, SO THEN A VERY
POPULAR MODEL IS NSGS MODEL AND
THAT EXPRESSES STEM CELL
FACTORED GMCSF AND INTERLEUKIN
3, BUT THOSE ARE TRANSGENIC MICE
SO THOSE FACTORS ARE JUST
EXPRESSED ANYWHERE, THEY'RE NOT
REGULATED, THEY'RE EXPRESSED ALL
THE TIME SO ACTUALLY IF YOU
INGRAFT HEMATOPOIETIC STEM CELLS
INTO THE MICE FOR SOMETIME, YOU
GET GET HEMEAT O POEESIS BUT
THEN YOU GET EXHAUSTION BECAUSE
OF THESE CELL LINES.
AND THEN THE LAB LABELED THESE
MICE MR. G, IT'S A GOOD ACRONYM
AND THESE THAT STAND FOR THE
CYTOKINES THAT ARE INTRODUCED,
THE RAD GAMMA KNOCK OUT
PHENOTYPE, IMMUNO PHENOTYPE AND
THERE'S ALSO REPLACEMENT OF
ALPHA WITH HUMAN ALPHA, AND
ALPHA BINDS CD47, IT'S KIND OF
THIS DON'T EAT ME SIGNAL AND
IT'S VERY IMPORTANT IN THE XENO
TOLERANCE.
SO MANY POST DOCS WORKING ON
THIS IN RICHARD'S LAB, REALLY
CHARACTERIZED EVERY SINGLE
REPLACEMENT, AGAIN, RESULTING IN
THE CYTOKINE KNOCK OUT MOUSE
MODEL THAT ALSO INCLUDES
INTERLEUKIN 6, THERE ARE OTHER
VERSIONS FOR EXAMPLE,
INTERLEUKIN 15 THAT THEY QUICKLY
REALIZED THAT IF YOU HAVE MCSF
AND YOU HAVE MONOCYTES AND
MACROPHAGES, THOSE ACTUALLY THEN
MAKE HUMAN IL15 THAT'S NEEDED
FOR NK CELL DEVELOPMENT, YOU
DON'T NEED TO PUT IN EXOGENOUS
INTERLEUKIN 15 AND SO THEN,
EARLY ON I WAS STILL A POST DOC
BUT IT WAS EVENTUALLY FINISHED
BY WOREALLY MAIRKSZING PEOPLE IN
MY LAB, BILLY AND
[INDISCERNIBLE] AND TOGETHER
WITH ANTHONY [INDISCERNIBLE], WE
TOOK MDS OF ALL LO GRADE AND
HIGH RISK AND THEN ALWAYS
COMPARED INGRAFTMENT AND MR. G
MICE COMPARED TO THE GOAL
STANDARD THEIR, MSG MICE AND HAD
MUCH, MUCH, IMPROVED INGRAFTMENT
AND REALLY OVER50% OF THE MICE
INGRAFTMENT OVER LIKE 10%, SO
YOU CAN IMAGINE, YOU CAN REALLY
ALSO TEST DRUGS BECAUSE CAN YOU
ACTUALLY MEASURE SOMETHING WITH
SOME RELIABILITY.
IMPORTANTLY, WE COULD RECEDE
DISPLASSIA, BECAUSE YOU KNOW YOU
GET ARITHROUGH ATOM POETICESIS,
THE HALLMARK, WITH THE
MUTATIONS, AND I REMEMBER, YOU
KNOW PERFORMING A BONE MARROW
ON THE PATIENT, SEEING THE BONE
MARROW IN PATHOLOGY, AND THEN
THE PEASHT GRACIOUSLY WRITE,
CONSENTED TO GIVING US A SAMPLE,
INGRAFTING IT INTO MICE AND
LOOKING AT THE MOUSE, AND I'M
LIKE WOW, THIS IS EXACTLY THE
SAME PHENOTYPE.
WE ALSO SEQUENCED, THE PATIENTS
AND THE MICE TO SHOW THAT WE ARE
INGRAFTING THE MUTANT STEM
CELLS, AND THEN ULTIMATELY WE
ALSO PERFORMED THERAPEUTIC
TESTING WITH MUTANT IDH
INHIBITORS COMPARING THAT FOR
EXAMPLE IN ELABORATE OR PERP
INHIBITOR WHICH A COLLEAGUE HAD
SHOWN WORKS IN IDH MUTANT CELLS
THAT END UP HAVING A DNA REPAIR
DEFECT.
SO WE'RE VERY EXCITED ABOUT THAT
AND THEN OF COURSE, ONCE YOU
HAVE SUCH A MODEL THAT'S QUITE
GOOD, YOU THINK IT'S NOT GOOD
ENOUGH.
AND 1 BIG DEFICIT IN ALL
HUMANIZED MOUSE MODELS IS THAT
WHILE YOU STILL HAVE THE MOUSE
INNATE IMMUNITY, SO THE MOUSE
GRANUE LOW SIGHTS BUT THE
MACROPHAGES ARE ACTUALLY A
PROBLEM, AND SO IN THESE MICE
YOU ACTUALLY DON'T HAVE
CIRCULATING RED CELLS, AND
INKRED EBLLY ARK MAZING POST DOC
IN RICHARD'S LAB WHOSE GOT
FACULTY
FACULTY TRY TO FIGURE OUT AGAIN
DELETING ALL THINGS FROM THE
MICE, CROSSING IT TO MCSF,
KNOCKOUT MICE, ET CETERA TO SEE
WHAT WE CAN GET, CAN WE GET
CIRCULATING HUMAN RED CELLS, AND
THEN 1 EXPERIMENT THAT KIND OF
TOLD THEM WHAT TO GO AFTER WAS
TO TAKE HUMAN RED CELLS, LABEL
THEM WITH FLUORESCENT DIE,
INYECT THEM INTO THE MICE AND
TAKING OTHER ORGANS AND THEN
LOOKING WHERE ARE THESE CELLS,
SO SOME GOT THE QUESTION THE
SPLEEN AND WE ALL THOUGHT THAT
WAS THE MAIN SITE WHERE MOST OF
THE CELLS WERE SEQUESTERED IN
THE LIVER LIVER EMPLOY SO THERS
THIS MOUSE.
I ALWAYS HAVE TO WRITE THIS DOWN
BECAUSE I CAN NEVER SAY IT THAT
IS KNOCKED OUT FOR FUMER OLDER
PEOPLE ASEITATE-ASIGNIFYTAL
HYDROLACE [INDISCERNIBLE].
SO THEY HAVE A METABOLIC DEFECT
IN TYROSEEN METABOLISM AND SO
YOU ACTUALLY HAVE TO KEEP THEM
ON SPECIAL WATER SO THEY DON'T
GENERATE TOXIC METABOLITES THAT
TISSUE CULTURE MEDIAS THEIR
LIVER CELLS.
WELL, IT TURNS OUT YOU CAN
ACTUALLY TAKE HEPATOCYTES,
EITHER MOUSE HEPATOCYTES OR
HUMAN HEPATOCYTES AND INJECT
THEM INTO THE SPLENIC VEIN, AND
THEN THESE CELLS WERE LARGE IN
THE LIVER, SO YOU CAN INJECT
HUMAN HEPATOCYTES INTO THESE
SURGERY INTO THESE MICE, AND YOU
WITHDRAW THIS WATER, SO WITH
BITS NOW, YOU'RE CREATING THE
MOUSE HUMAN HEPATOCYTES AND THEY
WILL ACTUALLY TAKE OVER AND THEN
YOU CAN MEASURE HUMAN ALBUT MIN
IN PLASMA AND KIND OF WHAT IT
SETS A CERTAIN LEVEL, THEY'RE
PROBABLY MOSTLY HUMAN
HEPATOCYTES AROUND AND SO THEN
WE CAN TAKE THESE HUMAN LIVER
HUMANIZED MR. G MICE AND INGRAFT
HUMAN CD34 CELLS JUST
RETROORBITALLY, AND NOW WE HAVE
HUMAN AND MICE THAT HAVE HUMAN
LIVER AND HEMATOPOIETIC STEM
CELLS INGRAFTED AND REALLY TO
OUR DELIGHT, THESE MICE NOW HAVE
CIRCULATING HUMAN RED CELLS.
AGAIN THIS IS A LOG SCALE, BUT
YOU KNOW REALLY THERE ARE UP TO
10% CIRCULATING HUMAN RED CELLS,
IN THESE MICE.
YOU CAN'T REPLACE ALL THE RED
CELLS, THE HUMAN RED CELLS ARE
MUCH BIGGER THAN THE RED CELLS
AND THE MICE NEED THEIR OWN RED
CELLS THAT CIRCULATE PROPERLY,
MOST LIKELY.
AND THAT WAS--WELL, SURPRISINGLY
ALSO THAT WE ACTUALLY HAD HIGHY
INGRAFTMENT IN THE BONE MARROW
AND ALSO GENERALLY MORE ERYTH
ROUGH ATOM PORESIS IN THE BONE
MARROW, SO SUGGESTING IN THE
LIVER, MANY OTHER PROTEINS THAT
MAY BE 1 DAY WE COULD SCREEN, TO
UNDERSTAND, WHAT OTHER PROTEINS
MADE IN THE LIVER THAT ARE
ACTUALLY IMPORTANT FOR HEMEAT O
POETICESIS.
SO, WE WANTED TO KNOW, YOU KNOW
WHAT THE MECHANISM IS FOR THIS
IMPROVED, YOU KNOW CIRCULATION
OF HUMAN RED CELL, AND 1 VERY
OBVIOUS THING WAS ACTUALLY TO
LOOK FOR COMPLEMENT AND SO
REALLY THE LIVER HUMANIZED MICE
HAVE LESS MURINE C3 AND THERE'S
ALSO LESS COMPLEMENT COATING ON
THE HUMAN RED CELLS SO YOU
REALLY JUST DON'T HAVE
DESTRUCTION OF THE LABELING OF
THE RED CELLS WITH COMPLEMENT
AND THEN THEIR PHAGOCYTOSIS BY
MOUSE MACROPHAGES AND SO I THINK
THIS RIGHT--I KNOW HAD IS CANCER
BUT ACTUALLY ATL, BUT CLASSIC
[INDISCERNIBLE] AND ALSO OUR
PATIENTS WITH SICKLE CELL
DISEASE ARE GETTING CARE IN THE
CENTER, AND WHICH IS PHENOMENAL
AND THE RIGHT THING TO DID.
SO WE TOOK THESE CELLS AND CD34
CELLS WITH PATIENTS FROM SICKLE
CELL DISEASE, A MONOGENIC DEC WE
IGNORED FOR FAR TOO LONG AND
THEN WHAT YOU SEE NOW HERE, ARE
ACTUALLY SECTIONS FROM LUNG,
LIVER, AND KIDNEY WITH THE HUMAN
SPECIFIC HEMOGLOBIN A ANTIBODY
AND IF YOU COMPARE LUNGS FROM
PATIENTS FROM MICE INGRAFTED
WITH THESE CELLS, OR CD34 CELLS
WITH PATIENT FROM SICKLE CELL
DISEASE, CAN YOU SEE VASE O,
CLIEWGZ, SO THESE ARE RED CELLS
STUCK IN THE LUNG VASCULATURE,
YOU CAN SEE RED CELLS KIND OF
STUCK IN THE CAPILLARIES IN THE
LIVER AND ALSO IN THE GLUE MARUE
LIE AND YOU KNOW IN THE KIDNEYS
SO WE CAN ACTUALLY NOW STUDY,
THESE OCCLUSION FROM HUMAN
CELLS.
SO AGAIN WE'RE NEVER SATISFIED.
SO THAT IS ALSO COMPLICATED
MODEL TO GENERATE BUT YOU KNOW 1
THEORY IS THAT IN MDS,
INFLAMMATION PLAYS A ROLE,
RIGHT?
AND WE KNOW INFLAMMATION PLAYS A
ROLE AND ET CETERA, AND SO TO
INGRAFT HUMAN WITH THESE MICE WE
HAVE TO RADIATE THE MICE.
THERE ARE BEAUTIFUL PUBLICATIONS
SHOWINGS THAT IF YOU MUTATE THE
STEM CELL FACTOR RECEPTOR IN
MICE THAT YOU MAY NOT NEED TO
RADIATE BUT THAT IS ENOUGH OF A
COMPETITIVE DISADVANTAGE TO THE
MURINE STEM CELLS AND PORESIS
THAT YOU CAN NOW INGRAFT HUMAN
CELLS WITHOUT IRRADIATION, SO
VIA CRISPR, AGAIN, WE GENERATED
THESE MICE WITH THESE W41
MUTATIONS SO THESE ARE ACTUALLY
MUTATIONS THAT WERE DRIEBED IN
B6 MICE CALLED THE WHITE SPOTTED
MICE SO IN EFFECT THE RECEPTOR
IS ALSO IMPORTANT TO THE NANO
SIGHTS SO THESE MICE ARE
ACTUALLY BLACK BUT THEY HAVE A
LITTLE WHITE SPOT ON THEIR
STOMACH, THAT WAS EASY FOR GENO
TYPING, THE MSG IS WHITE SO WE
SELECTED COMPLICATED GEANY
TYPING TO MAKE THESE MICE AND SO
WITH THIS MOUSE MODEL WE STARTED
A COLLABORATION WITH LEE GRIMES
AND [INDISCERNIBLE], AND HE'S A
PHENOMENAL INFORMATICS PERSON TO
MODEL MYELODYSPLASIA IN THESE
MICE AND IT REALLY WAS THE IDEA,
WITH HOW FAITHFUL REALLY ARE
THESE MODELS, WE HAD DONE LIKE A
CRUDE ASSESSMENT AND WITH LEE,
AMY AND GABRIEL AMAZING PEOPLE,
SEAN, JENNIFER AND WEI, SO LEE
IN THE PAPER, HIS PAPER ON THIS
PHENOMENON METHOD JUST PUBLISHED
IN IMMUNOLOGY, HE DEVELOPED A
VERY COMPREHENSIVE SIDE SEQ
PANEL FOR HEMEAT O POETICESIS SO
EVERY ANTIBODY IS TITRATED.
AND SO, AND YOU SEE, HIS GROUP
SEQUENCED HEPATITIS E C CONTROL
[INDISCERNIBLE], CD34 CELLS, MSC
CELLS AND, IT NC AND THIS IS THE
WHOLE WITH BEAUTIFUL
REPRESENTATION.
THIS IS NOW AN MDS SAMPLE FROM
JOHNS HOPKINS AND WE TOOK THE
SASM AND THE G-MICE, TO AFTER 16
WEEKS AFTER INGRAFTMENT,
HARVESTED THE BONE MARROW TO
SEQUENCE AND YOU CAN SEE THAT
WHY THIS IS NOT PERFECT, IT'S
QUITE A GOOD REPRESENTATION, AT
THE SINGLE CELL LEVEL OF THE MDS
POPULATIONS AND HERE'S PROBABLY
A LITTLE BIT CLEARER SO THIS IS
THEN SEQUENCING RIGHTFULLY SO
ALL YEENS, THESE ARE ALL
SUBPOPULATIONS AND YOU CAN SEE
THAT THE MDS XENOGRAFTS LOOK
LIKE THE PASHT IN TERLS OF
DIFFERENT YALE EXPRESSION
ANALYSIS.
SO NOW WE HAVE SO 50ICATEED
MODEL WHERE WE CAN INGRAFT, AND
1 OF THE GOALS NOW IS TO INGRAFT
MICE AND TREAT THEM WITHAISE O
CYTODEAN, THE STANDARD OF CLAIR
THAT I THINK ANY MDS CLINICIAN
DOESN'T LIKE AT ALL BECAUSE WE
DON'T KNOW WHAT IT DOES OR HOW
IT WORKS AND MANY PATIENTS
RELAPSE EVEN AN TRANSPLANT AND
ONCE PATIENTS ARE REFRACTORY TO
ISOTOPE AZACYTIDINE, AND THERE'S
NOTHING ELSE THAT WORKS.
SO MICHELLE RUDEK YOU KNOW
DEVELOPED THE REGIMEN IN THE
MR. G MICE, THEY ARE SENSITIVE,
AND WE WERE ABLE TO YOU KNOW
MEASURE BOTH INCORPORATION OF
THE NUTRITIGHT INTO DNA AND THEN
ALSO SHOW DECREASE IN
METHYLATION RIGHT HERE.
SO THAT'S AN ONGOING PROJECT.
I THINK I BRIEFLY SHOWED THIS,
WE HAD USED THESE MICE BEFORE
BECAUSE YOU KNOW WE HAD FOUND
THIS MECHANISM, THAT IDH MUTANT
MDS, AML IS ACTUALLY SENSITIVE
TO P A RP INHIBITION BECAUSE OF
A DNA REDETECTED AND THESE MICE
WERE INGRAFTED AND MDS AML AND
THEN THEY ARE--WE ACTUALLY
PERFORMED BONE MARROW
ASSPIRITUAL STRUGGLESSATIONS
BEFORE TREATMENT AND SACRIFICE
MICE AFTER TREATMENT AND THIS IS
SHOWING IN THESE 2 PATIENTS,
RIGHT, IN THE SPECIFIC MUTANT
IDH INHIBITOR, OR THE LAB WORKS
REALLY WELL, AND THEN THIS IS A
PATIENT NOW WHO HAD SECONDARY
AML, AND WHEN ON TREATMENT WITH
AZATINIB FOR A WREER AND THEP
PROGRESSED TO AAMERICA L, AND
THEN WAS NO LONGER SENSITIVE TO
AZACITINETWORK IB, BUT WAS
SENSITIVE TO [INDISCERNIBLE].
AND THEN WENT TO TO A SMALL
TRIAL, AND THIS IS A WAKE UP
CALL THAT WE CAN DO A LOT OF
THINGS IN THESE MODELS IN
INVITRO IN THE PATIENT, GIVING
PATIENTS PREP WHEN THEY HAVE
AMERICA DS, IT LOOKED VERY
DIFFERENT AND I WOULD SAY, IT
PROBABLY DOESN'T WORK AND WE
HAVE A LOT MORE WORK TO DO TO
FIGURE OUT CAN 1 FOR EXAMPLE
INTRODUCE THESE TREATMENTS FOR
THESE PATIENTS OR MAYBE NOT,
BECAUSE MDS, CYTOPENIC IS NOT A
GOOD RUG TO USE.
BUT WE'RE REALLY POISED TO USE
THESE MODELS, USE OUR ADVANCES
IN UNDERSTANDING MUTATIONS TO
HOPEFULLY GET SOME TREATMENT TO
PATIENTS AND EVEN IF WE ONLY,
YOU KNOW CONTRIBUTE SOME STONES
TO THAT BIG MOSAIC WITH THE
RESEARCH COMMUNITY, IT'S A VERY
EXCITING THING TO DO.
AND AGAIN, SO THIS WORK IS DONE
BY ABSOLUTELY AMAZING PEOPLE IN
MY LAB.
FORMER MEMBERS OF THE LAB AND
COLLABORATORS REALLY ALL OVER
THE WORLD AND OBVIOUSLY AMAZING
FUNDING, FUNDING SOURCES AND
THEN, IT'S ALWAYS IMPORTANT TO
HAVE FUN IN THE LAB, THIS IS
[INDISCERNIBLE] PROSENTING A
POSTER AT THE MOILOID MEETING
THIS IS HANNAH [INDISCERNIBLE]
AND JULIA, AND THE
[INDISCERNIBLE] MEETING THIS WAS
A DESSERT HE DESIGNED FOR THE
MEETING AND I CAN'T TELL YOU HOW
IT TASTED, I DIDN'T GET TO HAVE
IT, AND NEW HAVEN HAS GOOD PIZZA
AND SO THANK YOU FOR BEING HERE
TODAY.
[ APPLAUSE ]
>> OKAY, THAT WAS GREAT.
THE MOUSE MODEL WAS REALLY
EXCITING, I HAVE A QUESTION
ABOUT THE SPLICING FACTOR
MUTATIONS.
I THINK WHAT'S STRIKING TO ME IS
THE FACT THAT THERAPY AND
HETEROZYGOTE AND YET, THE MUTANT
PROTEIN HAS AN OUTSIZED ROLE, OR
OUTSIZED FUNCTION.
DO YOU THINK THAT THAT'S
BECAUSE--THESE PROTEINS TEND TO
BE HIGHLY EXPRESSED, DO YOU
THINK IT'S BECAUSE OF A FUNCTION
OF A MUTANT PROINE, OR LESS
WILD-TYPE PROTEIN HAS BEEN MADE.
HAVE YOU OR ANYBODILE CHECKED IF
YOU INHIBIT THE MUTANT PROTEIN
DO YOU GET A RESTORATION OF
FUNCTION?
>> OKAY, SO MICE THAT ARE
HETEROZYGOUS FOR SHE'S SPLICING
FACTORS ARE DOING PRETTY WELL,
SO THEY DON'T HAVE MUCH OF A
PHENOTYPE SO I THINK THAT'S 1
ASPECT.
YOU KNOW 1 OF THE REASONS WE
WERE EXCITED THAT MUTANT 1
ACTUALLY BINDS THE RNA IS MAYBE
NOW AND DIFFERENTLY MAYBE NOW
THERE'S SOMETHING YOU COULD
INHIBIT,.
>> WITH THE DRUG DEVELOPERS, I
WOULDN'T KNOW WHERE TO START TO
TRY TO DO THAT.
BUT I THINK, SO, IN THE CELL
LINES YOU KNOW WE EXOGLY EXPRESS
THESE PROTEINS, AND IT'S AULENTY
VIRAL VECTOR SO WE TRY TO
TRANSDUCE THESE CELLS AND IT WAS
1 SO WE GET 1 INTERNATIONAL
CLASSIFICATION GRAIN BUT THEN
IT'S STILL AULENTY VIRAL LTR AND
SO FOR THE SFR2, THERE'S RNA,
THERE'S LOTS OF MUSEUM TABT SSF2
BUT AT THE PROTEIN LEVEL THERE
ISN'T THAT MUCH MORE.
I THINK THE MOST WE GET
OVEREXPRESSED AT THE PROTEIN
LEVEL IS 1.5 X OR SO, SO I THINK
THERE'S A VERY, VERY TIGHT
REGULATION OF STILL SPLICING
FACTOR STOICHIOMETRY, AND
SO,--SO I THINK IT'S A GAME, I
THINK IT'S A GAIN OF FUNCTION
AND NOT NECESSARILY--BUT IT
COULD ALSO BE A COMBINATION.
>> SO HAS ANYONE TRIED TO
OVEREXPRESS THE WILD-TYPE
PROTEIN?
>> SO ALL OUR WORK IN CELL LINES
WE OVEREXPRESS WILD-TYPE MUTANT
SEPARATELY, WE HAVE NOT DONE
THAT TOGETHER.
>> OKAY, THANKS.
>> HAY, THANK YOU SO MUCH, THAT
WAS FASCINATING.
I HAVE A QUESTION ABOUT STRESS
GRANULES, SO WHEN YOU PULL DOWN
STRESS GRANULES AND WHICH RNAs
WERE UPOR COUNSEL REGULATED AND
I WONDERED IF YOU LOOK TO SEE A
CORRELATION FOR STRESS GRANULES
THAT WERE DOWN REGULATED IF THEY
WERE DISPLACED DRCH LITE AND
BECAME SHORTER SINCE THE LEPGHT
OF RNAs TEND TO CORRELATE HOW
ENRICHED OR NOT ENRICHED THEY
ARE IN THE STRESS GRANULES.
NYEAH, THAT'S WHATEE JULIA'S
LOOKING AT NOW SHE WAS JUST AT
THE RNA SOCIETY MEETING THAT'S
WHAT SHE CRANKED THIS DATA FOR,
I'VE SEEN IT LIKE 5 MINUTES SO
WE DON'T HAVE THAT YET, BUT
YEAH, YEAH, YOU'RE RIGHT, SO
THAT'S FIRST YOU WILL HAVE TO
LOOK AT AND YOU KNOW IT'S A
LITTLE BIT SCARY BECAUSE WE MAY
OR MAY NOT FIND SOMETHING.
>> THANK YOU.
>> REALLY GOOD QUESTION.
>> GREAT TALK.
SO IT'S EXCITING TO SEE THIS IS
[INDISCERNIBLE] STRESS GRANULES
AND SO, WHEN YOU LOOK AT THE
OVERLAP AFFECTED VENTS BETWEEN
[INDISCERNIBLE] MUTATIONS, THIS
IS GOING ON THIS WAY THINKING
INDEPENDENTLY, THIS MUTATIONS
MAY AFFECT DIFFERENT
TRANSCRIPTS,.
>> YEAH, YEAH, GOOD QUESTION.
SO WE HAVEN'T ANALYZED PATIENT
DATA SETS BASED ON THAT.
YOU KNOW IN OUR--IF I REMEMBER
CORRECTLY IN OUR--BECAUSE OUR
SRF2 KIND OF LIKE SPLICING
ANALYSIS DATA WAS A WHILE AGO,
THERE WEREN'T ACTUALLY A TON OF
EVENTS SO I THINK NOW, THAT'S
WHAT JULIA WILL HAVE TO DO IS
REALLY, SO WE ACTUALLY BACK THEN
WE PUBLISHED SRF 2 TO
[INDISCERNIBLE] CLIP AND THAT'S
NOT SUCH A GREAT TECHNIQUE SO WE
ACTUALLY HAVE SSF2 ECLIP DATA
FROM LIKE 4 YEARS AGO THAT WE
HAVEN'T ANALYZED WHICH IS
EMBARRASSING BUT NOW WE'RE DOING
THAT.
I CAN'T AT ALL, BUT I THINK IT
WILL BE A REALLY INTERESTING.
SO THIS--YOU KNOW THE STRESS
GRANULES SIGNATURE, RIGHT, SHE'S
TAKING, SHE TOOK ALL THESE DATA
SETS, AND I THINK SHE'S PUTTING
THAT TOGETHER AS A LITTLE THING
FOR THE RNA SOCIETY, RIGHT, SO
YOU PULLED OUT ALL THE OTHER
REFRESH YOUR RECOLLECTION NAs
AND PROTEINS THAT HAVE BEEN
ISOLATED AND STRESS GRANULES, BY
VARIOUS GROUPS AND A LOT OF THAT
FOR EXAMPLE, I THINK LIKE
[INDISCERNIBLE] AND 2 AND 3
CELLS SO THERE'S STILL MORE THAT
CAN BE DONE.
HER STRESS GRANULE SIGNATURE,
SHE HAS TO NOW LOOK AT THAT
SPECIFICALLY FOR SSRF 2, BECAUSE
A LOT IS TILL INFLUENCED BY HER
DATA JUST BECAUSE SHE DOESN'T
HAVE THE BINDING, SPLICING,
YEAH.
YEAH, I DON'T KNOW.
I THINK IT WOULD BE NICE IF
THERE WERE A COMMON MECHANISM,
BECAUSE IT'S INTERESTING, RIGHT
BECAUSE WE HAVE PAPERS
SUGGESTING THAT SSF2 UGF 1 FOR
EXAMPLE, ALSO RESIDE IN MORE
R-LOOPS, BUT EVEN THE MUTATIONS
FOR [INDISCERNIBLE], THEY'RE NOT
THE SAME, THEY CURVE, THEY HAVE
DIFFERENT PHENOTYPES, THEY DO
DIFFERENT THINGS, SO WHAT'S
COMMON BETWEEN THOSE 2?
I DON'T THINK WE KNOW.
>> THANKS.
>> ANY OTHER QUESTIONS?
ANYBODY ONLINE?
I HAVE NO IDEA HOW THIS WORKS.
OKAY, SO, IF THERE ARE NO
FURTHER QUESTIONS, LET'S THANK
STEPHANIE FOR A GREAT
PRESENTATION.
[ APPLAUSE ]