>> ALL RIGHT, GOOD MORNING
EVERYONE, ONLINE AND HERE, THANK
YOU FOR BEING HERE, THIS IS THE
FINAL SEMINAR ORGANIZED BY THE
PROTEOMICS INTEREST GROUP IN
THIS SEASON, AND FOR OUR SPEAKER
WE HAVE DR. DAVID SCHWEPPE, HE
BEGAN HIS RESEARCH CAREER
WORKING FOR A DOCTOR AT WILLIAMS
COLLEGE AND THEN PURSUED
DOCTORAL WORK AT MEDICAL SCHOOL.
THEN HE DID POST DOCS AT THE
UNIVERSITY OF WASHINGTON AND
HARVARD MEDICAL SCHOOL.
IN 2020, DEVIN ESTABLISHED HIS
RESEARCH GROUP IN THE DEPARTMENT
OF GENOME SCIENCES AND HIS FOCUS
IS ON THE INFORMATION
PROTEOMICS, AND THEY QUANTIFY
PROTEINS AS A READ OUT SPANNING
MICROBIAL STUDIES.
THE RESEARCH BUILDS ON THE
STUDYING DISCOVERY AND
PROTEOMICS PLATFORMS.
ALONG WITH TECHNOLOGY
DEVELOPMENT FOCUS THE WORK GROUP
TO BUILD APPLICATIONS AND
RESOURCES TO DISSEMINATE
PROTEOMICS DATA SETS TO THE
RESEARCH COMMUNITY AND THIS
EFFORTS INCLUDE WORK WITH THE
CROSS THINKING MAZ SPECTROMETRY
AND EXVIVO DATABASE AND BIOFLEX
HUMAN PROTEIN COMPLEX YOLKS
STUDY.
HIS TALK TODAY IS MOLECULAR
PHENOTYPING OF THERAPEUTIC
RESPONSES AND MAMMALIAN AGING
WITH QUAWPTITATIVE PROTEOMICS
AND I HAVE TO MENTION THAT HE'S
TRAVELED AND VISIT IS MADE
POSSIBLE GENERAL SPONSORSHIP.
>> THANK YOU FOR THAT GENEROUS
INTRODUCTION.
I DID A LOT OF WORK FOR
INTELLIGENT DATA ACQUISITION AND
I'M NOT GOING TO TALK ABOUT ANY
OF THAT BUT YOU CAN BELIEVE WE
USED IT TO DO MOST OF THE WORK
THAT I'M GOING TO SHOW YOU
TODAY.
SO WHAT I WILL HOPEFULLY
CONVINCE YOU OF TODAY IS THAT MY
LAB STUDIES A REALLY SIMPLE
QUESTION, RIGHT?
WHAT HAPPENS TO CELLS WHEN WE
PERTURB THEM AND THE MANNER WITH
WHICH I STUDY THIS, I WOULD NOT
BE HERE IF NOT, IS HOW DO CELLS
REMODEL THEMSELVES AND
PARTICULARLY THEIR BIOLOGICAL
BIOCHEMICAL MACHINERY UPON THAT
PROTURBATION.
AND WE CPS ABOUT THIS IN THE
SIMPLEST POSSIBLE CASE AND 1 WE
THINK OF PRETTY CONSISTENTLY IN
NIGH LAB, WHICH IS WHAT HAPPENS
TO PROTEOME WHEN WE ADD A DRUG
TO CELLS OR TO LIAISON
SUITABILITY ZONESS?
RELATIVELY SIMPLE QUESTION,
RIGHT?
THIS SMALL MOLECULE IS TARGETED
AND SHOULD HIT 1 PROTEIN, MAYBE
A SMALL FAMILY OF PROTEINS AT
BEST, AND WE CAN THINK IF THIS
IS REALRY TRUE IN THE MENTALITY
RIGHT HERE WHERE WE HAVE A BUNCH
OF STUFF BASICALLY HAPPENS, AND
REALLY WE'RE GOING TO FOCUS ON 2
THINGS FOR THE MOST PART, AN
INITIAL SET OF INTERACTIONS AND
SOME DOWN STREAM SET OF EFFECTS
AND THE BENEFIT AND UTILITY OF
USING PROTEOMICS IS ACTUALLY
THAT IF WE DO THIS RIGHT AND IF
WE DO THIS A LOT WITH MANY
SAMPLES AND MANY DRUGS, WE CAN
ACTUALLY BUILD UP LARGE SCALE
NETWORKS AND BEGIN TO UNDERSTAND
MORE METHODS OF ACTION AND
THOROUGHLY UNDERSTAND THE
PROCESS BETWEEN INITIAL
INTERACTION BETWEEN SMALL
MOLECULES AND PROTEIN AND
EVERYTHING THAT HAPPENS AFTER
THAT.
ALL RIGHT, SO THAT'S HARD,
RIGHT, OF COURSE, EVERYONE KNOWS
PROTURBATIONS CAN DRIVE
THOUSANDS OF PROTEIN CHANGES.
ONE OF MY FAVORITE SET SUGGESTS
ON ARE SMALL MOLECULE INHIBITORS
BECAUSE THEY REALLY JUST MUCK UP
CELLS.
YOU TREAT -- MOST CANCER CELLS
PICK JUST ABOUT ANY TISSUE, ANY
SUBTYPE AND YOU CAN CHANGE
ALMOST EVERYTHING GOING ON WITH
IT BY MESSING WITH THE
EPIGENOME.
SO IN THIS CASE, IF WE MESS WITH
HISTONE ACETYLATION, INCREASE
HISTONE ACETYLATION, WE GET
CLARNLG SCALE CHANGES ON THE ORD
EVALUATION PROCESS OF THOUSANDS
OF PROTEINS THAT CHANGE, RIGHT?
THAT'S NOT THE MOST HELPFUL
THING RIGHT BECAUSE WHAT DO YOU
TELL A COLLABORATOR, WE WERE
JUST TALKING THIS MORNING, WHAT
DO YOU TELL A COLLABORATOR WHEN
YOU GIVE THEM A LIST OF 2000
THINGS THAT HAVE CHANGED?
WHAT ARE WE GOING TO FOCUS ON?
WHAT ARE THEY GOING TO FOCUS ON?
WHAT WILL SOMEONE BUILD THEIR
THESIS ON?
AND THE QUESTION BECOMES TO NOW
WHAT, 2 GENERAL STORIES CUSTOMER
PROTECTION THE FIRST LOOKING AT
DRUG MECHANISMS OF ACTION AND SO
DIGGING DEEPER TO TRY TO
UNDERSTAND ON-TARGET
INTERACTIONS WE KNOW SHOULD BE
HAPPENING AND OFF-TARGET
INTERACTION WE BELIEVE ARE
HAPPENING AND THE CAUSE OF
ADVERSE EFFECTS AND
SECONDULARLY, I WILL TALK ABOUT
HOW PROTERBATIONS AFFECT CELLS
IN LETTING US STUDY THEM IN
DEVELOPMENT FEATURE, AND WE WILL
START WITH THE SIMPLEST CASE,
AND DRUG PROTERBATION AND
LOOKING AT A DRUG SPECIFIC
RESPONSE, WE TREAT CELLS,
PROTEOME RESPONSES IN A SPECIFIC
WAY TO THAT DRUG, CERTAIN SET OF
PROTEINS HERE CHANGE IN
MEANINGFUL WAYS, VIOLA, WE CAN
DRIVE UNDERSTANDING FROM THAT.
WE CAN ALSO GO THE OTHER WAY IF
WE WANT TO DO A LOT OF
EXPERIMENTS RIGHT, ESSENTIALLY A
PROTEIN SPECIFIC RESPONSE SO
INSTEAD OF LOOKINGAL EVERYTHING
THAT HAPPENS THAT YOU TREAT WITH
THE DRUG, WHAT HAPPENS TO 1
PROTEIN WHEN YOU TREAT WITH LOTS
OF DRUGS.
SO 1 BIG QUESTION THAT WAS BEING
ASKED
ASKED BY DYLAN MITCHELL WAS WHAT
IF YOU JUST COMBINE THESE?
SO WHAT YOU DO PROTEOMICS ON A
LARGE NUMBER OF SMALL MOLECULE
DRUG TREATMENTS AND LOOK FOR
PROTEIN SPECIFIC EFFECTS ACROSS
THESE?
SO THAT LED TO THE DEEP COVER
MOA PROJECT AND I'M NOT GOING TO
GET FULLY INTO THAT, I WILL
MENTION MORE ABOUT THE SPECIFICS
IN 1-SECOND, BUT 1 OF THE BIG
THINGS I WANT TO HIGHLIGHT ABOUT
THIS PROGEC, WAS THAT SOMETHING
THAT DYLAN PULLED OUT OF THIS IS
THIS IDEA THAT EVEN THOUGH IF
YOU LOOK AT THIS LIST OF
PROTEINS THAT ARE RESPONDING TO
POLO LIKE KINASE 1, IT'S NOT
CHANGING SIGNIFICANTLY, BUT WE
SEE A LOT OF OTHER PROTEINS
CHANGING AND CHANGING
CONSISTENTLY WITH 1 OR 2
STRUCTURALLY INHIBITED
INHIBITORS SO THE PROTEOME
CHANGES, WE NOT SEEING THE
DIRECT MECHANISM TO BE THE DREK
MECHANISM BUT WE CAN USE THAT TO
BUILD UP NETWORKS OF COORDINATED
PROTILES AND THAL CASE ARE NODES
IN THIS CASE OR DRUGS, AND EDGES
BETWEEN THOSE NODES THEN, ARE
THE LEVEL OF CORRELATION,
BETWEEN PROTEOME RESPONSES TO
THESE INDIVIDUAL DRUGS, AND THEN
WE CAN SEE, RIGHT, SO WE KNOW
VOLASERTIB, AND PLK 1 ARE
INHIBITORS THEY HAVE A BIG
PURPLE EDGE BETWEEN THEM.
WE CAN SEE THE BRD INHIBITORS
HAVE NICE HIGHLY COORDINATED
EDGES BETWEEN THEM AND WE KNOW
THAT BRD 1 AND PLK1 INHIBITORS
HAVE ESSENTIALLY OFF TARGET
EFFECTS WITH EACH OTHER AND SO
WE SEE CORRELATED PROFILES WITH
THESE 2, SO THE INTERESTING
THING ABOUT THE DATA SET IS THE
IDEA THAT IF YOU HAVE SOME NEW
COMPOUNDS, YOU HAVE NO IDEA WHAT
IT HITS OR WHAT IT DOES, IF YOU
TREAT CELLS, AND YOU SEE A
CORRELATIVE RESPONSE, IN THIS
CASE, SAY TO BOTH OF THESE PLK 1
ENHIB THORSITTORS, THAT'S A GO
CHANCE THAT A NEW INHIBITOR.
IF YOU'RE NEW COMPOUND IS IN
ACCORD ASBESTOS WITH THE PLK1,
IT'S A CHANCE IT'S A GOOD
COMBINATION OF THE PLK1, AND
BRD4 INHIBITOR.
SO WE COO MAKE A CORRELATION
LOOKING AT HOW THEY RESPOND.
WE CAN DO RELATIVELY SIMPLE
WORK, EVERYONE HERE HAS DONE
PROTEOMICS LIKELY RIGHT, BUT IF
WE DO ENOUGH OF IT, WE USE THESE
PROFILES AND THE INHERENT
INFORMATION, WE CAN GAIN USEFUL
MECHANISMS OF ACTION DATA FOR
NEW COMPOUNDS, SO THE REQUESTY
IS THEN, HOW DO WE BEGIN TO
UNDERSTAND THESE OFF TARGET
INTERACTIONS, AND THE EFFECTS
THAT THEY HAVE, SO 1 TIDBIT, SO
THIS IS A SUMMARY FROM DYLAN'S
PAPER, OF DEEP COVER MOA PAPER
THAT CAME OUT OF THE LAB, RIGHT,
AND THE EAGLE EYED WILL SEE
THAT'S THE INTELLIGENT DATA
ACQUISITION PLATFORM THAT I SAID
I WILL NOT TALK ABOUT BUT WILL
BE IN THERE, RIGHT?
875 COMPOUNDS TREAT OFFICE OF
DIVERSITY CELLS AT 10
MICROMOLAR, HUGE STUDY, A LOT OF
PROFILES.
A SIMILAR 1 CAME OUT OF A
COLLEAGUE'S LAB, THE EXPRESSION
BASED ANALYSIS FOR THE EQUIPPED
GENERAL PLATFORM, FOR THE 144
DRUGS FOR A VARIETY OF DIFFERENT
CONCENTRATIONS OF SMALL
MOLECULE, PROTEOME TREATMENT AND
READ OUT.
AND THESE WERE FANTASTIC STUDIES
BUT 1 CAVEAT TO BOTH OF THESE IS
THAT EACH 1 IS DONE IN A SINGLE
CELL LINE, RIGHT?
SO THERE'S OBVIOUS TURNOVERS
ABOUT IF YOU LOOK FOR A PROTEOME
RESPONSE AND YOU MAKE ASERKZS
ABOUT MECHANISMS OF ACTION BUT
YOU'RE CONSIDERING A SINGLE
ISOGENIC BACKGROUND, WHAT ARE WE
MISSING WITH THAT IN OUR
UNDERSTANDING, SO I WILL MAKE
THE CASE TO YOU, THERE'S A LEVEL
OF UNDERSTANDING WE NEED TO
GLEAN ABOUT CELLULAR STATE, AND
THE IMPORTANCE OF DIFFERENT
FACTORS IN CELL STATE, THEY
COULD BE METABOLIC,
ENVIRONMENTAL, WE WILL FOCUS ON
ESSENTIALLY THE BACKGROUNDS, AS
A MAIN VARIANT AND ALSO, THE
CASE THAT WHILE WE'RE STILL
MISSING FROM THOSE MECHANISMS OF
ACTION STUDY, IS THE IDEA OF
WHAT'S ACTUALLY HAPPENING, WHAT
ARE THE SMALL MOLECULES,
INTERACTING WITH, ARE THEY
ACTUALLY ISHT ACTING WITH THEIR
INTENDED TARGETS.
SO WE WILL DIVE INTO THE FIRST
CELL STATE BASED ANALYSIS
FITTER, SO, CHELSEA IN THE LAB,
ORIGINALLY PUT OUT A PREPRINT IN
THE FIRST PRINT OF THESE STUDIES
WHERE ESSENTIALLY SHE'S BUILT AN
QUANTITATIVE PLATFORM FOR DRUG
PROTERBATION, AND DATA
ACQUISITION TO PULL OUT THE
RESPONSES ON THE PROTEOMIC
LEVEL, SHE ALSO DOES PHOSPHO
PROTEOMICS BUT I WON'T GET INTO
TOO MUCH TODAY.
SHE ESTABLISHED CANCER CELL
LINES, 1 IS LUNG CELL AND 1 IS
HTT116 AND HDT116 WE INCLUDED
BECAUSE IT WAS IN DEEP COVER MOA
ANY IT GIVES US A REFERENCE TO
SEE IF THE CORRELATED PROFILES
WE WILL PULL OUT MATCH TO
CORRELATED PROFILES WITH A STOCK
OF HTCC116 IS IN A DIFFERENT LAB
4 YEARS LATER SO THAT SHOULD BE
A GOOD CONTROL FOR US.
SHE THEN COMPARED THESE
ESSENTIALLY BY TREATING WITH A
SERIES OF IN THIS CASE, HDAC, OR
HISTONE DEACETYLASE, AND 1 EGFR
AND 1 INHIBITOR TO SEE HOW
PROTURBATIONS OF EPIGENETIC
REGULATION WILL AFFECT
ESSENTIALLY THE PROTEOME.
AND WE HAVE 1 GROUP HERE WITH
THE INHIBITOR AND SHE MADE SURE
TO SHOW WHERE EACH 1 IS
ASSOCIATED.
SHE PUT TOGETHER THE LARGE DATA
SET, THE CELL LINE'S SPECIFIC
DRUG EFFECTS TO SEE IF WE'RE
SEEING DIFFERENTIAL RESPONSES
AND OF COURSE WE WERE OR ELSE WE
PROBABLY WOULDN'T PRESENT ON
THIS AND TRY TO PUBLISH IT, JUST
IN VIABILITY DATA.
SO YOU TREAT ALL OF THESE CELL
LINES WITH THE EXACT SAME
CONCENTRATION FOR THE EXACT SAME
TIME RECOLLECT AND THE VIABILITY
AND SMALL MOLECULES VARIES
MARKEDLY AND IN THIS CASE, SOME
OF THEM HAVE VIABILITY, SO THE
ARELATIVE LEVEL OF THIS, SHOULD
BE VIABILITY PRETREATMENT AND
POST TREATMENT SHOULD BE A 1 TO
1 RATIO, ALL THESE NUMBERS
SHOULD BE 1 IF THERE'S NO
EFFECT, WE CAN SEE IN THIS CASE,
RIGHT, FOR PC 9 CELLS EXPOSED TO
CDC101, THEY LOSE 70% OF THEIR
BIODATA.
BUT NONE OF THE OTHER, RIGHT,
CELL LINES HAVE SUCH A DRAMATIC
EFFECT.
WE SEE SIMILAR EFFECTS ACROSS
BASICALLY PC 9 SO THERE'S
CLEARLY A CELL TYPE SPECIFIC
EFFECT BUT THERE'S INTERESTING
FACTORS HERE WHERE THERE'S A
LARGE EFFECT ON 8549S, ONLY 1
HDAC INHIBITOR, IN THIS CASE,
POLINSTAB, AND WE CAN SEE THAT
MORE IN THE PROTEOME SPECIFIC
LEVEL, SO ESSENTIALLY EACH ROW
I'M SHOWING YOU AND THE DATA IS
THE PROTEOME AND THE PROTEOME
RESPONSE COMPARED TO DMSO
CONTROLS, THE LOG 2 PULL CHAINS,
PLURIBU IS HIGHER, RED IS LOWER,
WE SHOULD PROBABLY SWITCH THOSE,
BUT IN THIS CASE, WHAT HOPEFULLY
YOU CAN GREEN FROM THE HEAT MAP
RIGHT IS THAT THE RESPONSES SEEM
TO VARY IN THESE TRAWNCHS GOING
ACROSS BUT WE CAN NICELY SEE IS
THAT THOSE ESSENTIALLY ARE
CONSIST WENT JUST THE CELL
LINES, SO THESE CELL LINES ARE
RESPONDING CONSISTENTLY TO THE
DEACETYLASE INHIBITORS ALMOST OF
WHAT, REALLY ROBUST WAYS, WITH
SPECIFIC CLEVES OR PROTEINS THAT
ARE AFFECTED, IN 1 CELL LINE AND
NOT AFFECTED IN BASICALLY ANY
OTHER CELL LINE.
THAT'S FUNCTIONALLY INTERESTING,
WHAT'S DRIVING THAT EFFECT?
LOOKING BACK THEN TO THE DEEP
COVER MOA STUDY, AND DOING
EVERYTHING IN THE 116S WE CAN
SEE THAT THE HCT 116 RESPONSE IS
QUITE DEFINITE THAN THE 85449
RESPONSE AND INTERESTING CAVEAT
HERE IS BOTH OF THESE ARE DRIVEN
BY KRACE MUTATIONS.
THE SAME DRIVER MUTATION AND
MARKETEDLY DIFFERENT RESPONSES
TO SMALL MOLECULE INHIBITORS,
YEAH, YEAH.
>> [INAUDIBLE QUESTION FROM
AUDIENCE ]
>> YEAH, NORMALLY I TRY TO USE
THE SAME NUMBER OF LIVING CELLS
AND BASICALLY WASH OFF THE DEAD
KIND OF LEVY APOPTOTIC CELLS,
SAME TOTAL PROTEIN INPUT THAT
GOES INTO EACH AFTER GETTING RID
OF THE HOPEFULLY DEAD CELLS.
YEAH.
FREIGHT -- GREAT QUESTION AND A
LEAD IN FOR 2 CLICKS AWAY.
ONE THING BEFORE WE GET
TRUSTEES, WE CAN SEE SOME OF THE
RESPONSES IN THE HEAT MAP, YOU
CAN SEE THESE AFFECTS IN SMALL
CLEVES OF THESE PROEN TOOS WHERE
THERE SEEM TO BE DRUG SPECIFIC
EFFECTS WITHIN THE CELL LINES,
SO WE CAN SEE THIS LINE RIGHT
HERE, OF BLUE AND PC 9s, THAT
IS CONSISTENT WITH PC 1s AND
H292, SO THERE IS SOME
CONSISTENCY ACROSS, THAT IS
LIKELY MORE CLICKS THAN I WILL
JUST SHOW YOU NOW BECAUSE YOU
ASKED, ALL OF THAT, RIGHT THERE
IS ACTUALLY THE P38 KINASE
INHIBITOR.
SO THAT'S A STRIKING THING,
RIGHT?
SO HDAC INHIBITORS ARE ALL OVER
THE PLACE, P38 KINASE INHIBITORS
RESPOND THE SAME, ALL THESE CELL
TYPES RESPOND EXACTLY SAME TO
THE P-KINASE INHIBITION.
SO STEPPING BACK, WE DO SEE SOME
UCIBOLITTOUS RESPONSES NO MATTER
WHAT WHICH WERE ENCOURAGING SO
AGAIN, ON THE LEFT-HAND SIDE
HERE, THIS IS P38 KINASE
INHIBITOR, ESSENTIALLY NO
CONSISTENT CHANGE, THE CLAYED
HERE IS ALL THE CELL LINES
RESPONDING TO RELAMATIN INCREASE
IN BODY, AND EVERYTHING ELSE
HERE IS RESPONSES TO HDAC
INHIBITORS, SO ALL THESE
PROTEINS IN EVERY CELL LINE
CHANGE, THERE'S NICE 1S SHOULD
BE RELATED LIKE THE PSYCHE LYNN,
D3 AND SO THAT WAS ENCOURAGING
TO SEE.
WHAT WAS INTERESTING THOUGH,
RIGHT, WAS THE NOT SO UBIQUITOUS
CHANGES SO DES ACETYLASES DON'T
RESPOND TO THE INHIBITION.
HDAC 7 WAS BY FAR THE MOST
RESPONSIVE GENERALLY, GENERALLY
WENT DOWN IN THIS CASE.
BUT THE OTHERS DON'T SHOW WHAT I
WOULD CALL A CONSISTENT
RESPONSE, YOU SEE MAYBE AN
EFFECT ON HDAC 5, BUT NOTHING WE
CAN POINT TO AND SAY, THIS IS
PROBABLY WHY THERE'S AN EFFECT.
AGAIN, THE RELAMATINIB, YOU CAN
ALSO SEE THESE IN PC SPACE, SO
EVERY 1 OF THESE CLUSTERS OF
POINTS HERE ARE CELL LINES
RESPONDING TO SMALL MOLECULES
AND YOU CAN SEE THE COLORS ARE
ALL THE DIFFERENT SMALL
MOLECULES AND THE SHAPE IS THE
CELL LINES, BASICALLY THE
SHAPE'S ALL CLUSTERED TOGETHER
WITH THE EXCEPTION OF
RELAMATINIB, WHICH RESPONDS
UBIQUITOUSLY.
SO WE CAN SEE DRUG AND CELL
SPECIFIC EFFECTS, THIS AGAIN
COMES BACK TO THE IDEA OF
BUILDING NETWORKS ON THIS TYPE
OF DAT AIT WILL BE PERTINENT TO
THINK ABOUT THE CELL LINE YOU'RE
USING, WE'VE SEEN THE EFFECT
WHERE YOU TRY TO COMPARE IN THE
DEEP COVER MOA STUDY TO THE
DECRYPT E-STUDY, THE CORRELATION
ABOUT .4-.6 FI REMEMBER
CORRECTLY.
SO YOU HAVE TO BE CAREFUL THERE.
OKAY, SO, CHELSEA WAS NOT 1 TO
SIT ON HER HANDS ONCE SHE HAD A
BIG DATA SET AND SO SHE WENT FOR
INCREASING BOTH THE NUMBER OF
CELL LINES AND THE DRUGS USED
FOR TREATMENT, ESSENTIALLY,
ADDED A LOT MORE TO EACH.
SO WHATLY ACTUALLY DID WAS
INCREASE THE NUMBER OF CELL
LINES FROM 5-24.
THEY ENCOMPASS LARGE
HETEROGENEOUS GENETIC POPULATION
OF EUROPEAN, AFRICAN, ASIAN,
MALE AND FEMALE CELL LINES WITH
DIFFERENT DRIVER MUTATIONS EGFR,
KRASE MODEL, P53 AND TREATED
THESE WITH A SERIES OF SMALL
MOLECULE INHIBITORS, TO THINGS
LIKE ORAL KINASE B, P38 KINASE
INHIBITOR, AND SO EXPANDING THE
REPERER TWOAR OF PROTURBATIONS.
AND BUILT UP A NETWORK IN THIS
CASE, TAKING THE TOP HALF OF
THAT HEAT MAP, MAKING IT
CIRCULAR SO CAN YOU SEE
EVERYTHING AT ONCE ON 1 SLIDE,
AND WE CAN SEE THE CORRELATION
ESSENTIALLY, OF THE RESPONSES.
HOPEFULLY WHAT YOU CAN GLEAN BY
LOOKING AT IT ARE CONSISTENT
RESPONSES, THE CELLS CLUSTER,
RTK INHIBITORS AND HDAC
INHIBITORS TOGETHER, THAT'S
INTERESTING, THERE'S A DIVERSITY
OF RESPONSES BUT THESE LARGELY
SEEM TO CLUSTER TOGETHER OR AT
LEAST AWAY FROM THE OTHER
INHIBITORS, SO, THAT IS
INTERESTING TO SEE, AND 1 THING
WE WERE ALSO EXCITED ABOUT FOR
THIS LARGER SET OF DATA AND I
WON'T GO INTO TOO MUCH DETAIL,
CHELSEA WILL BE PRESENTING ABOUT
THIS IN THE ASMS ON THURSDAY,
THIS IDEA THAT ONCE WE HAVE MORE
CELL LINES WE CAN BEGIN TO DO
COMPARISONS AND MOAR MORE
PROTEIN COMPLEXIO GENOMIC LAND
SCAPE, YOU BEE WE DO SEE EFFECTS
DRIVEN BY THE DRIVER MUTATION
BEING PRESENT OR ABNORMALITIES
SEBT.
SO IN THIS CASE, LOOKING AT THE
RESPONSE OF CELLS TO GEFITINIB,
APPARENTLY THAT'S LOW BATTERY,
ALL RIGHT, WITH OR WITHOUT THE
MUTATION, SO WE CAN SEE DCBL,
FD2, ESSENTIALLY IF THERE'S NO
MUTANTS JUST WILD-TYPE EGFR,
GEFITINIB PRODUCES NO EFFECTS,
BUT IF IT HAS A MUSEUM TABT
EFFECT THERE'S A DIG 95 CANT
COUNSEL REGULATION OF THIS
PROTEIN.
IF WE LOOKEDDA THIS, THERE WERE
A SERIES OF INTERESTING PATHWAYS
WITH THE EFFECTS OF EGFR AND WE
HAVE MORE CANDIDATES FOR THIS
BUT THINGS LIKE THE UPICK WITTEN
LIGASE ACTIVITY THAT WE'RE
INTERESTED IN PLAYING IN.
ONE FURTHER STEP SHE WENT AND
LOOKED AT SHE HAS VIABILITY DATA
FOR ALL OF THESE AND IN ADDITION
AND SHE BEGAN TO LOOK AT WHAT
THE EFFECT IS OR THE LIKELY
EFFECT IS OF MUTATIONS ON
VIABILITY, THESE DAT AAND THE
EFFECTS, THAT'S DRIVEN BY SMALL
MOLECULE TREATMENT AND IN THIS
CASE, TREATMENT, AND THE TOP
CANDIDATE HERE, THIS IS JUST THE
LOG 2 VIABILITY RATIO, PRE- AND
POST TREATMENT, THEY ARE
SLIGHTLY MORE VIABLE ODDLY AFTER
GEFITINIB TREATMENT, ON THE SPOT
AND THEN WHAT'S SHOWING UP OVER
HERE ARE EACH 1 OF THESE DOTS IS
A CELL LINE AND WHETHER OR NOT
IT'S MUTATIONAL STATUS IS EGFR,
MUTANT, KITHROORKS CA MUTANT, ET
CETERA, ET CETERA, AND THEN EACH
OF THESE POINTS WOULD BE 3 EGFR
MUTANTS, EGFR MUTATION IS
SEPARATING AWAY FROM THE PACK
OVER HERE IN TERMS OF THE IMPACT
OF THE GEFITINIB, SO IF YOU HAVE
THE MUTANT YOU ARE LIKELY TO
HAVE THE GEFITINIB, IT'S
SLIGHTLY PROTECTIVE IN THIS CASE
AND FEN2 SEEMS TO BE SLIGHTLY
EFFECTIVE AT LEADING THE
GEFITINIB RESPONSE, SO THERE'S
THINGS TO PULL OUT IN THE
LANDSCAPE OF THIS BUT OVERALL I
JUST WANT TO POINT OUT WITH THIS
INITIAL STEP, WE HAD TO DEEP
COVER MOA, YOU HAVE DECRYPT-E,
AND WE'RE MISSING THE KEY OF
WHAT ARE THE CELL LINES SPECIFIC
RESPONSES AND HOW CONSISTENT ARE
THOSE OVERALL?
OKAY, SO THERE'S ANOTHER STEP
HERE, THE FIRST I MENTIONED WAS
THIS IDEA THAT WE WANTED TO LOOK
AT WHAT ARE ESSENTIALLY THE CELL
STATE FACTORS THAT MIGHT IMPINGE
ON WHAT WE'RE LOOKING AT, AND
THE OTHER THING THAT WE'RE
REALLY INTERESTED IN TRYING TO
ASKER TAIN ARE WHAT ARE THE
ACTUAL MECHANISMS OF ACTION, WE
GET AN IDEA OF PUTATIVE
MECHANISMS OF ACTION WHEN WE DO
THE PROTEOME RESPONSIVE DATA,
BUT WE WOULD LIKE TO DIG A
LITTLE BIT FURTHER THAN THAT AND
WE CAN THINK, WE TREAT WITH
DRUGS, SOME STUFF HAPPENS AND
THEN THE PROTEOME SHIFTS, ALL
RIGHT.
WE CAN LOOK AT THIS IN SINGLE
CELL SEQUENCING LAND FOR SOME
FOLKS OUT AT U-W, AND MISSIONARY
AND OTHER LABS WHERE THEY DID
SIMILAR WORK AND SHARE THAD
THERE'S HETEROGENEOUS SINGLE
CELL RESPONSES IN
TRANSCRIPTOMICS AND AGAIN THIS
IS JUST RESPONSES AND SO WE'RE
STILL REALLY INTERESTED IN THIS,
SOME STUFF HAPPENS SO WE OF
COURSE TURNED TO COLLEAGUES' LAB
AND TTP PROTOCOLLINGS AND A
FAVOR OF THEM THAT CAME OUT OF
THE LAB WAS PISA, SO THIS IS THE
IDEA WE'RE TREATING WITH
DENATURING WITH VEHICLE
CONTROLS, PROTEINS WILL DENATURE
AND HAVE SOME SPACE MELTING
TEMPERATURE, YOU ADD DRUG IT'LL
CHANGE THE MELTING TEMPERATURE,
CHANGE THE MELTING TEMPERATURE
IN THE CHANGE IN ABUNDANCE AND
OUR IDEA IS TO INTEGRATE ALL
DATA SETS WE NOW HAVE PROTEOMICS
SINGLE CELL TRANSCRIPTOMICS,
TRANSCRIPTOMICS ON SOME OF THESE
AND ASSESS DRUG SPECIFIC
ENGAGEMENT AND PROTEIN SPECIFIC
ENGAGEMENT AND THIS MULTIOMIC
COMPARISON.
SO WE'VE BEEN ABLE TO DO THIS
RIGHT, AND THE PISA IDEA IS
INSTEAD OF GETTING MELTING
TURNLS YOU GET RELATIVE
COMPARISON TO DMSO CONTROLS AND
SO IN THIS CASE, WHAT THIS MEANS
IS OUR LOG 2 RATIO SPACE, THINGS
THAT ARE ON THE LEFT SIDE HERE,
WOULD BE DESTABILIZED WHEN YOU
ADD INHIBITORS AND THINGS ON THE
RIGHT SIDE THERE, HAD TO BE
STABILIZED AND EITHER WAY,
STABILIZED OR DESTABILIZED IS
INTERESTING TO US BECAUSE IT
MEANINGS SOME CHANGE IN THE
THERMAL STABILITY OF THOSE
PROTEINS.
IT WAS NICE TO SEE HISTONE
DEACETYLASE INHIBITOR AND TREAT.
ALL RIGHT, GREAT, THE ASSAY
WORKS, WE ALSO SEE SOME
DISCRIMINATION RATE, HDAC, 1, 2,
6 ARE ALTERED BUT 3, 4, 7, AITD,
HAVE NO STABILITY CHANGES AT
LEAST IN THE ASSAY THAT WE USED.
AND EVEN IF YOU JUST LOOK AT ALL
THE OTHER POINTS THAT ARE
SIGNIFICANT, TO FIND THE
SIGNIFICANT IN THIS VOLCANO
PLOT, WE SEE SIGNIFICANT CHANGE
FROM ABOUT 40-50 PROTEINS THAT
WE NOW CONSIDER FOR MECHANISM OF
ACTION VERSUS THE 2200 PROTEINS
WE HAD TO ESSENTIA WILY
CONSIDER, SO WE CAN THINK SOME
OF THESE WILL BE ON TARGETS WE
KNOW AND ALL OF THE REST ARE OFF
TARGETS THAT ARE INTERESTING FOR
THE ADVERSE EFFECTS OF THESE.
OKAY, SO, CELL TYPE SPECIFIC
RESPONSES, RIGHT, WE CAN
ACTUALLY GO IN AND USE THESE
ASSAYS, BOTH IN CELLS AND
LIAISON SUITABILITY ZONESS, AND
1 INTERESTING HIT THAT WE PICKED
OUT, THIS IS ANOTHER SMALL
MOLECULE INHIBITOR OF HISTONE
DEACETYLASES, SO AGAIN IN TERMAL
STABILITY LAND, WE SEE
STABILIZATION OF OUR HISTONE
DEACETYLASES, THAT'S GREAT, AND
WE ALSO SAW THIS INTERESTING OFF
TARGET HIT, AND THE ANALYSIS WE
SAW THESE CONSISTENTLY WHETHER
OR NOT WE DID THIS INTACT CELLS
OR IN LIAISON SUITABILITY
ZONESS, AND WE ALSO SEE
PARTICULARLY IN 8549 WHICH WE
DID THIS THERMAL STABILITY ASSAY
AND THEY ARE PARTICULARLY
SUSCEPTIBLE AS I MENTIONED
BEFORE.
SO 1 THING THAT WAS INTERESTING
THERE IS THAT IF WE GO LOOK AT
DEEP COVER MOA, BUT IF WE GO
LOOK IN DEPTH MAP FOR THE
RELATIVE ABUNDANCE OF 8549, AND
H202, IN OUR ANALYSIS, 8549 HAS
THE LOWEST, MEANING THEY'RE
PARTICULARLY SUSCEPTIBLE TO
INHIBITION.
WE CAN ALSO GO FURTHER AND DO
STUDIES ON THE KINASE B, AND
MODELED IN THE CENTER IS THE
INHIBITOR, BX680, AND IF WE PUT
BELINOSTAT IN THERE, IT HAS A
NEAR PERFECT OVERLAP, AND THE
INHIBITOR WILL BE 880 AND WE'RE
TRYING TO FOLLOW THIS UP TO
VERIFY.
SO IF WE CAN DO 1 THERMAL
ANALYSIS, WE CAN DO A HUNDRED
FWE CAN DO A HUNDRED, WE CAN DO
200, SO THAT'S WHAT MEGAN AND
JOHN DID.
RAN THIS LARGE SCALE SMALL
MOLECULE PANEL, FOCUSING ON THE
KINASE INHIBITORS, A FEW
SMATTERING OF OTHERS IN THERE,
SO WE FOCUSED ON CYTOKINASE
INHIBITORS AND DID THIS ON 96
TOTAL, 70 OF THESE ARE KINASE
INHIBITORS, GOT RELATIVELY
MASSIVE DATA SET, WITH THERMAL
STABILITY AND MEASUREMENTS FOR
ALL OF THESE TREATMENTS, RIGHT?
AND NOW, WE HAVE A LARGE SCALE
DATA SET TO TRY AND USE OR
UNDERSTAND NECKANISMS OF ACTION
FOR SMALL MOLECULES.
LARGE SCALE DATA ANALYSIS WE
HAVE LOSS AND LOTS OF POINTS FOR
THE THERMAL STABILITY CHANGES
ABOUT YOU WHAT WAS STRIKING AND
INTERESTING TO US IS THAT WE CAN
USE THE SCALE OF THIS TO
INTERPRET ON AND OFF TARGET
EFFECTS.
AND SO WHAT I'M SHOWING YOU
HERE, AGAIN, ARE THERMAL
STABILITY MEASUREMENTS AND THE
FULL CHANGE THAT WAS THE NEXT
ACCESSOT PLOTS, AND FOCUSING ON
THE K526 CELLS AND OTHER CANCER
CELLS IN THE LINE, LOOKING AT
RIP KINASE 1 STABILITY TREATING
WITH ORAL KINASE INHIBITORS O
CDK9 INHIB THORS IDENTITY ARES
ON AND YOU SEE THAT ACROSS ALL
TREATMENTS THE YELLOW 1S ARE
DMSO, THE OTHERS ARE GRAY SMALL
MOLECULE DRUGS, THERE'S
RELATIVELY LITTLE EFFECT EXCEPT
FOR THESE 4 WHICH HAVE
SIGNIFICANTLY ALTERED AND
INCREASED STABILITY, SO WE CAN
ACTUALLY SEE THESE INHIBITORS
ARE KNOWN TO HAVE THIS AS AN OFF
TARGET EFFECT, RECENTLY, IT WAS
FOUND TO HAVE THIS AND WE HAVE
THIS NEW POTENTIAL CANDIDATE FOR
A RIP KINASE 1 INTERACTIONS WITH
THIS.
AGAINY WE GO BACK TO THE
CORRELATION AND WE LOOK AT
CORRELATION OF THERMAL STABILITY
PROFILES FOR THIS, SO HERE,
WE'RE SHING AGAIN, I SHOWED THE
PROTEOME SCALE PREVIOUSLY, AND
IN THIS CASE, IT'S THE THERMAL
ABILITY SCALE, WE CAN SEE THAT
ESSENTIALLY THE DATA INDICATE
THAT BRD 4 AND 3, AND IF YOU
LOOK REALLY CLOSELY BEHIND THAT,
THERE'S BRD2, AND PLK1 HAVE
SIGNIFICANTLY ALTERED THERMAL
STABILITY, WITH TREATMENT OF
THESE THINGS THAT SHOULD TARGET
THEM.
GREAT.
HOWEVER WHAT IT SAYS TO US IS
THAT INTERESTINGLY STRUCTURAL
THINGS ARE INDUCING THERMAL
ABILITY CHANGES WHICH IS USEFUL
BECAUSE WE CAN LOOK AT SMALL
MOLECULES THAT ARE STRUCTURALLY
SIMILAR AND SEE IF THEY ARE
PRODUCING EFFECTS THAT MIGHT BE
OF INTEREST, IN THIS CASE, I
WILL SHOWING THAT THE BI13536,
AND THE MVP, AND WE CAN LOOK AT
ESSENTIALLY, TITRATIONS OF
THESE, AGAIN, WE'RE STAYING IN
LOG 2 FULL CHAINS BEING THERMAL
STABILITY BASE ANALYSIS AND WE
CAN SEE FOR CDK4 AND 6, WHICH
ARE THE TARGET, AS YOU INCREASE
CONCENTRATION, YOU INCREASE IN
THIS CASE, THE EFFECTIVE CHANGE
IN THERMAL STABILITY, YOU HAVE A
DOSE DEPEBDENT BINDING, WE
CONFIRMED WHAT EVERYONE ALREADY
KNEW.
WHAT WAS MORE FUN IS WE CAN
THINK AND DO THE EXACT SAME
THING AND FIND THIS KNOWN OFF
TARGET INTERACTION WITH PROTEINS
SO BRD4 IN THIS CASE, AND WE
ACTUALLY FOUND STRIKING WAS THAT
BOTH ARE EQUIPPED HERE IN THE
BROWNISH COLORS AND BI2536,
WHICH IS A KNOWN PLK INHIBITOR
SEEMED TO BE ENGAGING PLK 1.
SO THAT'S INTERESTING RIGHT
BECAUSE IT SUGGESTS TO US THAT
PLK1 IS AN OFF TARGET FOR P A
LBOCICLIB, AND IF WE LOOK AT A
MARKER FOR PLK1 IN THESE CELLS,
PHOSPHORYLATION OF CTP, WE CAN
SEE HIGHER CONCENTRATIONS, YOU
CAN INHIB THORS INCREASE IN BODY
THE ACTIVITY OF PLK 1, SO IT'S
NOT ONLY BINDING BUT AFFECTING
THE ACTIVITY OF THIS MITOTIC
REGULATOR, WE CAN DO MY FAVORITE
THING WHICH IS GO SIT IN PIMAL
FOR HOURS AND DO DOCKING AND WE
CAN SEE THAT WHERE PI3526, THE
KINASE ININCREASE IN BODYITOR
CAN'T REALLY BIND TO THIS, AT
THE TOP AND BENEFICIAL OF THE
BINDING POCKET, THE PLK 1
BINDING SITE IS MUCH MORE
PERMISSIVE AND SO, THEREFORE, WE
THINK THAT'S WHY IT CAN DIRECTLY
INHIBIT THE PLK1.
SO GENERATED, I AM A REPETITIVE
PERSON, SO IF I DO SOMETHING
ONCE, I WILL PROBABLY END UP
DOING IT AGAIN, IN THIS CASE WE
LIKE CORRELATION A LOT SO WE
WERE LIKE IT WORKED ON THE
PROTEOME SCALE, LET'S KEEP IT
GOING FOR THE THERMAL STABILITY
SCALE AND WE FOUND IT WAS A BIT
SURPRISING AT THE TIME IS IF YOU
DO ENOUGH OF THESE ANALYSIS AND
LOOK ACROSS ENOUGH TREATMENTS
WITH ENOUGH DRUGS, WE CAN SEE
VERY CONSISTENT RESPONSES, THE
SMALL MOLECULES, WERE
STRUCTURALLY AND COMPLEX RELATED
PROTEINS, SO IN THIS CASE,
LOOKING AT P38 ALPHA AND MAP
KINASE APK2 AND 3.
WE CAN SEE THAT THERE IS A
SIGNIFICANT CORRELATION OF THE
THERMAL STABILITY RESPONSES OF
THESE SMALL MOLECULES.
WHAT WAS GREAT ABOUT THAT IS
THAT WE KNOW THAT THERE'S DIRECT
OR AT LEAST DIRECT PROTEIN
INTERACTIONS BETWEEN THESE
PROTEINS, SO EITHER WE'RE
PULLING DOWN SECONDARY EFFECTS
HERE OR WE'RE SEEING ESSENTIALLY
BINDING TO RELATED COMPLEXES AND
RELATED PROTEINS, BECAUSE THAT
WAS INTERESTING, SO THAT HINTED
THAT THIS CORRELATION OF
CIVILIAN PROFILES MIGHT BE
USEFUL MORE DPLOABALLY SO WE DID
IT MORE GLOBALLY AND WE LOOKED
AT CORRELATION PROFILES OF ALL
PROTEINS AND WE THOSE IT WAS TO
OWNEROUS, SO WE LOOKED AT THE
COMPLETE SET OF KINOAM, WITHIN
THE K5626 CELLS AND YOU CAN SEE
HERE WE HAVE THESE WONDERFUL
CORRELATION PLOTS, SIGNIFICANCE
IS LOW ENOUGH THAT R-HAS TROUBLE
GIVING YOU A REAL ANSWER BECAUSE
THAT'S A GOOD SIGN, WE CAN SEE
THESE KINASES, REALLY STRONGLY
CORRELATED RESPONSES, WE DO THIS
AT LARGE SO THE ENTIRETY OF KINE
OHM, WE CAN BUILD UP A
CORRELATION NETWORK, FOR HOW
CORRELATIVE THE THERMAL CIVILIT
RESPONSES ARE, SO WITHIN THIS,
WE CAN SEE HERE, HERE'S THESE
RPS ON THE PAGE, WE CAN SEE DOWN
HERE ON THE LEFT ARE THE MAP
KINASE 14, AND RELATED AND THOSE
ARE PART OF THIS CORRELATION
NETWORK BUT WE SEE A WHOLE HOST
OF OTHER PROTEIN INTERACTIONS
WITHIN IT.
SO THAT WAS EXCITING AND SO WE
WANTED TO BUILD ON THAT A LITTLE
BIT MORE, BECAUSE WITHIN THAT,
AN ASTUTE OBSERVER MIGHT SEE WE
HAVE KINASE GROUPS UP HERE ON
THE TOP, A BUNCH OF POINTS THAT
ARE NOT CALLING THE WHITE,
THEY'RE NOT COLORED BECAUSE
THEY'RE NOT PROTEIN KINASES,
THEY'RE KINASES, THEY'RE IN THE
KINE OHM, BUT THEY'RE NOT
PROTEIN KINASES.
WE SAW A WHOLE HOST OF THESE
RESPONDING TO SMALL MOLECULES
SPECIFICALLY, CHANGING TO
KINASES CHANGING THERMAL
STABILITY.
WE CAN PULL THOSE OUT, AND WE
CAN THINK ABOUT IN THIS CASE,
PICAIN ICE, AND CHANGING THE
THERMAL PIP-KINASE, AND WITHIN
THE NONKINASES IS A STRONG
CORRELATED PROFILE, THESE SEEM
TO BE HAVING DIRECT ENGAGEMENTS
WITH SMALL MOLECULES AND LARGELY
ARREST THAT IS LARGELY UNBIVED
FROM THESE STUDIES LAST TIDBIT
HERE IS ABOUT THE CONTEXT OF
CELLS AND IT MATTERS SOMETIMES.
WE ACTUALLY SEE THE OPPOSITE
EFFECT FOR SOME KINASES LIKE
BRAF, WHICH AFFECT LIAISON
SUITABILITY ZONESS, THIS IS THE
CONTEXT OF THEM SEMESTERS, THAT
CAN BE IMPORTANT MOVING FORWARD.
OVERALL METHOD OF ACTION WE CAN
INFER THIS FROM THIS, AND WE CAN
WITH POINT CORRELATION AND LARGE
SCALE ANALYSIS AND COMPLEX
INTERACTIONS AND SECONDARY
EFFECTS SO WE SEE HERE, 1 ASPECT
OF THIS AGAIN, JUST SO I DON'T
GO, TOO, TOO, LONG IS THE WHYED
THAT WE'RE INHIBITING KINASES,
KINASES AFFECT OTHER PROTEINS
THROUGH PHOSPHORYLATION WHICH
CAN AFFECT THE THERMAL
STABILITY.
WE CAN ACTUALLY SEE IN THESE
ASSAYS, THAT PHOSPHORYLATION
EFFECT ON THERMAL STABILITY AS A
SECONDARY EFFECT WHICH CAN BE
USEFUL FOR PICKING UP MECHANISM
OF ACTION TOOL.
AND AGAIN THE AFFECT OF CELLULAR
CONTEXT ESPECIALLY ON CHROMATIN
AND COMPLEX BOUNDARIES.
SO WE HAVE PROTEIN COMPLEX TEEN
WIDE DRUG INTERACTIONS AND 1
LAST TIDBIT OF A STORY THAT I
WILL HINT AT IS WE HAVE A HOST
OF THESE SMALL MOLECULES, BIND
PROTEIN ANDS THESE PROTEINS, AND
SMALL MOLECULES AREN'T ALONE,
AND SO WE CAN THINK ABOUT THE
EXAMPLE OF PROTEIN KINASE C, AND
SOME OF THOSE THERE, WHICH IF
YOU TREAT PROTEIN, OR TREAT
CELLS, RIGHT, WHICH INHIBITS
PRCA, AND BINDS, THAT'S ITS
TARGET BUT IF YOU DO THAT IN
LIAISON SUITABILITY ZONESS YOU
GET A STRANG INCREASE IN
STABILITY, IF YOU DO THAT IN
CELLS WITH THE CELLULAR CONTEXT
YOU GET A STRONG DECREASE IN
STABILITY, THERE'S SOMETHING
ABOUT THE CELLULAR CONTEXT THAT
IS CHANGING THE RESPONSE TO THE
SMALL MOLECULE, SO WE WERE
CURIOUS ABOUT THAT AND CURIOUS
OF THINKING ABOUT HOW NATIVE
LIGANDS CHANGE THE PROTEOME, TIM
AND ROSE, AT U-W, AND ROSE WHO
IS NOW GOING TO NORTHWESTERN DID
THIS BY LOOKING AT MUSE LIVER
MITOCHONDRIA, AND HUMAN CELLS
AND YEAST FOR PROTEINS THAT BIND
THE CALCIUM, THE PRKCA EXAMPLE,
WE WERE ABLE TO TEASE OUT A
LARGE NUMBER OF THESE CALCIUM
BINDING PROTEINS THIS WAY USING
THERMAL PROTEOME, AND THERMAL
STABILITY ASSAYS, WHERE WE CAN
SEE THAT SOME OF THESE PROTEINS
ARE DESTABILIZED BY CALCIUM CAN
OR DESTABILIZED BY MAGNESIUM,
AND THE SPECIFIC RESPONSES AND
THAT WAS FUN, AND THEN WE BEGIN
TO SEE PAPERS CONFIRMING, THESE
THINGS THAT WE SPECULATED ON
WERE CALCIUM BINDERS AND WE
THINK YOU HAVE A PREPPER TWOAR
OF NATIVE LIGANDS THERE AND OUR
SMALL MOLECULE NATIVE LIAISON
GAPPEDS WE FOUND THIS
INTERESTING ASPECT OF CAT
IENGAGEMENT WHERE THE SUBSTREAM
EFFECTS CALCIUM BINDING, SO THIS
IS A STRUCTURE OF WE CAN SEE
NADP H THAT SHOULD BIND HERE AND
RIGHT HERE IS THE BINDING POCKET
FOR CALCIUM SO IF YOU HAVE A
NATURAL SUBSTRATE FOR DECR1, YOU
HAVE A KD, OVER HERE IN THE LOW
MICROMOLAR AND ABOUT 10 TOLD
HIGHER AND YOU DON'T HAVE THAT
LIAISON GABBED THERE.
SO WE'RE ABLE TO ACTUALLY USE
THESE PROTEOME PROFILES AND THE
ASSAY TREATMENT OF SUBSTANCE
ABUSE TEASE THESE OUT, SO WE'RE
INTERESTED IN COMBINING THE
NATIVE LIAISON GABBED AND
PROTEIN DRUG INTERACTION.
I COULD TALK MORE ABOUT THE
AGING STUDY, I WILL LEAVE IT
HERE IF SOMEBODY WANTS TO TALK
ABOUT IT, I PUT TOO MUCH IN
THIS, BUT I LEAVE YOU WITH THE
LAST BIG THING WE'RE INTERESTED
IN IS SMALL MOLECULE
PROTERBATIONS DON'T JUST DRIVE
PROTEOME SHIFTS BUTEE CAN USE
THEM TO CHANGE HOW CELLS BEHAVE
IN A MUCH LARGER CONTEXT, THIS
IN CASE, SMALL MOLECULE
INHIBITION WITH CHI RON WITH
INHIBITS GSK3A AND RETINAL
LOCATION NONAPOPTOTICKIC ACID
WHICH INDUCES NEUROMESODERM
FORMATION.
IF WE DO THESE THINGS IN STEM
CELLS, WE CAN ACTUALLY DRIVE THE
FORMATION OF THESE ORGANOIDS AND
THESE REALLY BEAUTIFUL WAYS AND
THEN USE THESE SYSTEMS TO
UNDERSTAND THE PROTERBATIONS
EFFECTS ARE CHANGE NOTHING
DEVELOPMENT AGING AND I AM HAPPY
TO CHAT MORE ABOUT THAT BUT JUST
IN THE INTEREST OF TIME I WILL
BLAZE THROUGH ALL OF THIS JUST
TO MAKE SURE I CAN THANK PEOPLE
AND ANSWER ANY QUESTIONS.
JUST TEASE YOU WITH ALL THE DATA
THAT WAS THERE.
CORRELATION COMES BACK UP, I
PROMISE IT'S EVERYWHERE, THAT'S
PROBABLY, THE OTHER THING JUST
FOR NATHAN WHEN I TALK TO HIM,
SO SHE HAVE A REFERENCE POINT IN
TERMS OF THE SLIDE, SO LED THE
DEVELOPMENT OF THE PROFILING,
CATARINNA IS LEADING THE AGING
PROTEOMES AND WE'RE EVENTUALLY
HOPING TO MARRY THESE 2 WHERE
IT'S LIKE GASTROLLOYDS AND SMALL
ORGANOID BASED PROFILING,
UNDERSTANDING HOW AGING HAPPENS
AND THE CLOSER WE GET THESE THE
MORE COMPREHENSIVE ANALYSIS OF
MORE AGING THERE.
THESE ARE THE FOLKS WHO DID THE
WORK, I HAVE A GREAT LAB, FOLKS
THAT I FEATURED IN THIS WERE
CATARENA, KATHRYN AND CATARINNA
ARE LEADING THE THERMAL
STABILITY WORK, CATARINNA IS
LEADING THE MOUSE AGING WORK AND
COLLABORATORS.
SO I GUESS I WOULD BE REMISS TO
NOT THANK ASMS FOR LETTING MOOY
COME OUT HERE AND PAYING THE
WAY.
THANK YOU ASMS.
APPRECIATE IT.
YEAH.
>> THAT WAS GREAT.
OKAY, SO I HAVE A FEW QUESTIONS,
ACTUALLY, FIRST OF ALL -- SO THE
CELL LINES AND THE DRUGS AND
SEEING THINGS AND SEEING THINGS
THAT CHANGE DIFFERENTLY THAT'S A
VERY NICE DATA SET, I WONDER
FIRST OF ALL IF YOU INCREASE THE
AMOUNT OF THESE DRUGS, WHAT
WOULD BE THE EFFECT ON THE
DIFFERENCES BETWEEN DRUGS AND
BETWEEN -- I MEAN IF YOU REALLY
SLAMMED THE CELLS, WOULD THEY
SORT OF LINE UP WHAT COULD SOME
OF THE DIFFERENCES HAVE BEEN
BECAUSE SOME OF THE, YOU KNOW
PROTEINS WERE MAYBE PARTIALLY
INHIBITED.
>> YEAH, SO IT'S A FUN QUESTION
AND HONESTLY THE REVIEW CRITIQUE
WE GET IS THE OPPOSITE BECAUSE
WE HIT THEM HARD, SO IT'S LIKE
10 MICROMOLAR FOR 24 HOURS.
IF WE HIT THEM MUCH HARDER
THEY'RE BASICALLY ALL DEAD.
SO WE'RE TRYING TO HIT THEM AS
HARD AS WE CAN TO SEE IF WE CAN
INDUCE A SIMILAR RESPONSE AND
DON'T.
AND THERE'S MORE SUBTLE RESPONSE
FIST YOU TUNE DOWN LIKE A
HUNDRED NANO MOLAR, WHAT THE
RESPONSE IS OR PARTIAL RESPONSE
TO IT.
>> SO YOU TOUCH OFFICE OF
DIVERSITY THIS, BUT I WONDER --
SO YOU KNOW YOU SEE CHANGES, YOU
SEE SOME PROTEINS DO THIS AND
SOME CELLS, DO THIS, BUT MAYBE
THESE PROTEINS ARE A LITTLE
DEFINITE BETWEEN CELLS AND DRUGS
BUT SOMETIMES RELATED DRUGS AND
I WONDER IF THEY HAD SIMILAR
GENIAN OITATIONS LIKE SIMILAR
DOMAINS OR THEY'RE BOTH PART OF
THE SAME BIOLOGICAL PATHWAYS AND
YOU WOULD SEE OVERLAP THERE,
HAVE YOU DONE LIKE GENE
ANNOTATION AND THEN, LOOKEDDA
THE IT THAT WAY?
>> YEAH, SOPHISTICATEDY WE
GENERALLY DO -- LIKE GO GSCA, TO
SEE WHAT THE GENERAL THE
PROFILES ARE, I THINK WE CAN GET
SOME, ADDITIONAL INFORMATION, I
GUESS, 1 THING THAT I'M REALLY
CURIOUS ABOUT MOVING FORWARD IS
WE'VE DONE LIMITED AMOUNTS OF
DOMAIN COMPARISONS TO UNDERSTAND
BINARY ASPECTINGS OF THE
INTERACTION, AND THERE'S ALSO 1
THAT WE GOT TUNED INTO FOR A
RECENT MEETING AND JUST TRY TO
THINK OF ESPECIALLY WORKING DOWN
THE HALL FROM MIKE IS THINKING
ABOUT HOW ISOFORMS ARE
DIFFERENT, PROTEIN COMPLEXIO
FORM VARIANTS AND THESE MIGHT BE
AFFECTING AND TUNING RESPONSES
SO PARTICULARLY FOR THE BRD FOR
EXAMPLE, IT HAS 2 ISOFORMS THAT
ARE KNOWN TO CAUSE DIFFERENT
EFFECTS AND YOU CAN INHIBIT THEM
POTENTIALLY SLIGHTLY
DIFFERENTLY.
SO THERE MIGHT BE DOMAIN AND
ISOFORM SPECIFIC EFFECTS THAT
UNDERLIE EVEN THE GENOME
ANNOTATIONS.
>> AND FINALLY, I KNOW YOU DID
LIKE CELL TO CELL LINE
DIFFERENCES BUT I WONDER,
ESPECIALLY IF YOU DID PRIMARY
CELLS OF SOME SORT, IF YOU ARE
THINKING ABOUT SINGLE CELL
PROTEOMICS.
>> YEAH, I LOVE IT, SO WE HAVE
-- I THINK WE'RE TRYING FOR NOW
A TWIST ON SINGLE CELL
PROTEOMICS WHICH IS TRYING TO DO
THINGS IN TISSUES AND LOOK HOW
CELL TYPES RESPOND.
AND SEE IF THAT'S CONSISTENT
ESPECIALLY IN THE CONTEXT OF
TISSUES OR ORGANOIDS, THE SINGLE
CELL ANGLE IS CHALLENGING I
THINK AS WE ALL KNOW, SO I THINK
IT WOULD BE AWESOME TO DO, MY
WORRY IS THAT WE HAVE ENOUGH
CELLS SO IF WE LOOK AT 1 PCA
PLOT FROM THAT SHOWED SINGLE
CELL TRANSCRIPTOMIC RESPONSES TO
HDAC INHIBITION WHICH SHOULD
MASSIVELY REMODEL THE TRANSCRIPT
ORDER OF MICRONS AND THEY STILL
SEE RESPONSES OF POCKETS CELLS
AND A LARGE NUMBER OF CELLS SO
MY WORRY WOULD BE WE HAVE TO DO
SUCH A DEEP DIVE IN SINGLE CELL
LAND TO BE ABLE TO TEASE THAT
OUT THAT THEY WOULD LIKE LIKE
COMPETING WITH PARALENS LENS
LENS LENS TO GET TO 19 OR 50,000
CELLS AND WE DON'T HAVE AS MUCH
MONEY AS THEY DO.
>> SO YOU'RE LOOKING FOR MANY OF
THE SIMILAR CHANGES THAT OCCUR
AND SOME DISCORD ON CHANGES, I
WOULD BE INTERESTED TO SEE IF
YOU SAW 2 DRUGS THAT MAY BE HAD
A SIMILAR CHANGE ON SOMEONE
WOULD SAY EXPECTED TARGET BUT
DISCORDANT CHANGES ON OFF TARGET
EFFECTS AND POTENTIALLY IF YOU
WERE TO TREAT WITH BOTH DRUGS
MAYBE THOSE OFF AGER AT THE TIME
EFFECTS WOULD CANCEL OUT IN THIS
WAY, I DON'T KNOW IF THAT'S AN
ANGLE YOU LOOKED FOR BUT IT'S
SORT OF LIKE LOOKING ON THE
PERIPHERY OF THINGS THAT MIGHT
HELP LET'S SAY CERTAIN DRUGS
WORK TOGETHER.
>> YEAH, I MEAN THE IDEA OF
DOING KIND OF THESE MULTIDRUG
TREATMENTS COCKTAIL TREATMENTS,
THAT'S SUPER APPEALING,
HONESTLY, MITIGATE THE EFFECTS,
NEGATIVE EFFECTS OF 1 BY HAVING
ANOTHER, SO I WILL GET CLOSE TO
THAT IDEA, I MEAN, LET ME KNOW
IF IT ANSWERS, ESSENTIALLY,
THERE'S A EFFECT THAT CHELSEA
SAW IN HER INITIAL DATA ON THE
APOPTOTIC PROTEIN, BUT DIDN'T
SHOW YOU THE PROTEOMICS DATA
THAT WENT ALONG WITH DATA BUT
THERE ARE CELL TYPE SPECIFIC
CHANGES AND BAD PHOSPHORYLATION
WHICH SEEMS TO PARTIALLY
CORRELATE OR POTENTIALLY BE
RELATED TO THE VIABILITY THAT
YOU SEE, BUT WE'RE TREATING
HISTONE DE, SET LACE INHIBITOR
THAT'S CLEARLY NOT
PHOSPHORYLATING BAD, AND IF
THAT'S LEADING THESE CELLS
SURVIVE, HDAC INHIBITION BECAUSE
IT'S BAD, WE TREAT WITH
SOMETHING THAT'S INCONSISTENTLY
EXPRESSED THERE, AND SO, THE
IDEA IS ESSENTIALLY WE FOUND A
MOTIF, IT'S A BASIC MOTIF AND
THERE'S 2 MANY BASED ON THE
KINASES BUT THERE'S ENOUGH THAT
WE HAVE PRETTY DESCENT SMALL
MOLECULE INHIBITORS FOR AKT, AND
SO THE IDEA IS ESSENTIALLY, CAN
WE CHANGE THE VIABILITY OR
CHANGE THE SENSITIVITY, TO HDAC
ININCREASE IN BODY HIGZ BY
PROHIBITING THESE GENERAL
MITOTIC SIGNALS WHICH WOULD BE
REALLY FUN, WE CAN PROVE THAT
AFTER IT HAPPENS.
>> AND MY SECOND QUESTION IS
ABOUT YOUR CORRELATION NETWORK,
SO HOW DO YOU -- HOW DO YOU DO
LIKE QUALITY CONTROL TO ASSIST
OR ASSESS WHETHER OR NOT THESE
CORRELATION EDGES ARE ROBUST
ENOUGH.
>> YEAH, SO THERE'S -- I DON'T
HAVE IT FOR THE SMALL MOLECULE,
I JUST DIDN'T PUT IT IN THERE,
BUT WE DO THESE PERMIAITATION
TESTS TO MAKE SURE -- SO HERE'S
AN EXAMPLE FOR THIS ORGANOID
BASED STUDY SO WE CAN PERMUTE
ALL POSSIBLE COMBINATIONS AND
SEE ESSENTIALLY WHAT IS THE
BACKGROUND CORRELATION, AND SEE
IF IS WHAT WE'RE SEEING
SIGNIFICANTLY DIFFERENT FROM
WHAT'S THERE, WHERE WE KIND OF
BACK TO ASK DEMONSTRATED AT 0 WE
GENERALLY DON'T SEE THAT OUTLIER
AND WE WITH CAN AGAIN TO USE
THAT AND USE SIMILAR CONCEPTS
WHICH CAN WITHIN SETS OF DATA,
GETTING THE IDEA OF ANNOTATIONS
IF WE ANNOTATE NICELY SO IF WE
LOOK AT PROTEIN COMPLEXES, SO A
PROTURBATION LIKE IN THE MAP K
14 EXAMPLE, WE EXPECTED PROBABLY
TO EFFECT THINGS IN A COMPLEX
WITH 814 AND LET SEEEE IF IT'S
HIGHLY CORRELATED RESPONSE TO
THAT, AND IT VALIDATE THAT WE
ALSO APPLY PRETTY STRONG FILTERS
TO USE TO OFFSET, OR GET AN IDEA
OF THE SAWLS DISCOVERY RATE FOR
KIND OF THIS RANDOM PROFILE,
THAT GENERALLY DOES A PRETTY
GOOD JOB OF THAT.
>> THANK YOU.
>> SO, YEAH, VERY NICE TALK.
SO I WAS WONDERING, WHEN YOU
TREAT THE CELLS WITH MULTIPLE
DRUG ANDS IN RESPONSE TO THAT,
DID YOU LOOK AT LIKE SOME PTMs
ALONG WITH LIKE PHOSPHORYLATION,
AND DID YOU TRY TO QUANTIFY?
>> SO THE ONLY AISLE GIVE YOU
THE TECHNICAL ANSWER AFTER THAT,
NOT GENERALLY, SO WE DIDN'T DO
GENERAL ACETYL PROFILING, BUT
THE 1 CAVEAT TO THAT IS WE LOOK
FOR HISTONE METHYLATION
ACETYLATION TO TRY TO VERIFY IF
THE EFFECTS WE WERE SEEING WERE
DIRECTLY AFFECTING HISTONE
MODIFICATIONS, AND WE SAW A
GENERALLY DEACETYLASE INHIBITOR,
THE METHYLATION GOES ON AND
DOWN, AND EXPECT TO BE BUFFERED.
>> THANK YOU.
>> I HAVE A COUPLE OF SMALL
QUESTIONS.
ONE IS YOU USE IN YOUR PANEL OF
DIFFERENT CELLS YOU USE ONLY
CELL LINES NO PRIMARY CELLS, NO?
>> NO PRIMARY CELLS YET.
>> YEAH BECAUSE IT WOULD BE NICE
TO HAVE PRIMARY CELLS FROM THIS
TYPE OF LINEAGE, THIS IS WHAT IS
MOST RELEVANT, RIGHT, AND THE
SAME TYPE OF RELEVANCE, QUESTION
IS ABOUT YOUR COMPAREISON OF
LISATE, OFF OF CELLS SO TREATING
LISATE WITH A DRUG IS KIND OF
INTERESTING BUT THE CELLS ARE
WHAT IS REAL EXISTING
ORGANELLES, SO, YEAH, JUST MAYBE
MORE COMMENT THAN QUESTION.
, YEAH, YEAH, I GUESS YOUR FIRST
POINT IS THAT OUR GOAL IS WE
WOULD LOVE TO DO PRIMARY CELLS
BUT 1 THING WE REALLY LOVE TO DO
IS LOOK ACROSS KIND OF
DEVELOPMENTAL TRAJECTORIES SO
CELLS WITH VERY DIFFERENT
CELLITATES TO TRY TO UNDERSTAND
THE EFFECT THERE AND THEN
OBVIOUSLY IT WOULD BE REALLY FUN
TO BE ABLE TO DO THAT, IN
TESTING.
>> YEAH, ORGANIZATIONS CANCER
CENTERIE, SO KEEP A STEP GOING
OVER PRIMARY CELLS.
>> FOR FOR THE CELL FIRST
LYSATE, YEAH, WE STARTED OFF
WITH THE IDEA THAT THE LISATE
WOULD BE GREAT BECAUSE YOU WILL
GET LIKE REALLY SPECIFIC
INTERACTIONS, RIGHT, AH, GET RID
OF THE MILIEU, ALL THE STUFF
THAT MIGHT INTERFERE WITH
INTERACTIONS, JUST LOOK FOR HARD
CORE BIOCHEMICAL INTERACTIONS
AND LIKE CONTROLLED BUFFERS, WE
WILL GET IT OUT, AND FOR SOME OF
THEM, WORKED BRILLIANTLY, AND
THEN FOR A LOT OF THEM,
ESPECIALLY EPIGENETIC RELATED
PROTEINS WHICH WE'RE MOST
INTERESTED FOR LIKE THE HDAC,
STUFF, WE NEEDED CELLULAR
CONTACTS AND SO, YEAH, WE FOUND
THAT THEY WERE MORE
COMPLIMENTARY I THINK THAN WE
WOULD HAVE EXPECTED TO BEGIN
WITH.
>> SO FOLLOWING UP ON THAT, SO
DID YOU THINK OF GOING THE OTHER
DIRECTION AND DO A SUBCELLULAR
FRACTIONATION AND LOOK AT
DEFINITE SUBCELLULAR COMPONENTS
TO SEE WHERE THE CHANGES ARE
COMING FROM, SO WE DIDN'T DO IT
FOR THE DRUGS BUT FOR THE
CALCIUM BINDING, THE LAB
SPECIFICALLY IS INTERESTED
MITOCHONDRIA RELATIONSHIP TO
CALCIUM SIGNALING SO WE DID IT
IN THAT CASE AND IT WORKED
REMARKABLY WELL, SO THAT WAS IN
WHOLE LIVE RESPIRING
MITOCHONDRIA, ISOLATED FROM MICE
AND YOU CAN STILL DO PSET MAP
AND GET THERMAL STABILITY
MEASUREMENTS SO THE THING I
WOULD LOVE TO DO WHICH I AM
TRYING TO CONVINCE THE GRADUATE
STUDENTS TO DO THIS IS TAKE
THIS, AND TAKE SUBCELLULAR
ENVIRONMENTS AND SEE IF THERE'S
CROSSES BETWEEN THEM.
>> ALL RIGHT, IF THAT'S ALL THE
QUESTIONS, THANKS AGAIN, THIS
WAS A FANTASTIC TALK.
>> THANK YOU.