cells with leukemia-associated dnmt3amutations are …
TRANSCRIPT
CELLSWITHLEUKEMIA-ASSOCIATEDDNMT3A MUTATIONSAREMORESENSITIVETOCYTARABINE-INDUCEDDNADAMAGE
OlgaGuryanova |AssistantProfessorDepartmentofPharmacologyandTherapeutics
Acutemyeloidleukemia(AML)islargelyincurable
NCISEERCancerStatistics
6.8% 24.0%5-yearsurvival
intensivechemo,HSCT,bettersupportivecare
5-10monthsmediansurvival
inpatients60+yearsoldunfitforhigh-dosechemo
Döhner,Weisdorf &Bloomfield,NEJM 2015
Two-hitmodelofAMLpathogenesis
ClassIproliferation(JAK2,FLT3)
Myeloproliferativeneoplasms(MPN)
Acutemyeloidleukemia
(AML)
Othergenes–epigeneticandchromatinmodifiergenes(DNMT3A,TET2,IDH1/2,EZH2,ASXL1),
etc.
ClassIIdifferentiation(RUNX1,CEBPA)
Myelodysplasticsyndromes(MDS)
me
mod
RecurrentDNAmethyltransferase3A(DNMT3A)mutationsinAML
Brunettietal.2017TCGA,2013
Acutemyeloidleukemia
MDSClonalhematopoiesis
modestDNAhypomethylationnotassociatedwithgenedysregulation
OGlabresearchinterests
Dnmt3amut
RELA
PSEDnmt3a
mut
REMISSION
NORM
AL
Dnmt3amut
CLONAL
HEMATOPO
IESIS
Dnmt3amut
DIAG
NOSIS
2.pre-leukemichematopoiesisandclonalevolution
3.therapeuticresponse
1.modelingandmechanism
DNMT3Amutationsare associatedwithadvancedageandunfavorableoutcomeinAML
Leyetal.2010,Yanetal. 2011
DNMT3AWT DNMT3AMUTAgeatdiagnosis* 60yrs 67yrs *p <0.001
Balasubramanian etal.2018
low-dosecytarabine
Low-dosecytarabine – atreatmentoptionforelderlyAMLpatients
60+yearsold,poorperformancestatus
investigationaltherapy(clinicaltrial)
supportivecare
Chaintermination
ATR
Chk1Checkpointactivation
gH2A.X
Forkcollapse
ATM
Chk2p53
G2àMcheckpoint
originfiring
Apoptosis
Negativeenrichmentofthecellcycle-relatedsignaturesincellsexpressingDNMT3AR882
DNMT3A R882 vs WT(TCGA)
NES-1.70FDR0.004
+/m vsWT stem&prog cells
NES-2.05FDR0.001
TCGA(2013)NEJMGlassetal. (2017)CancerDiscovGuryanova etal.(2016)NatMed
DNMT3A directstherapeuticresponsetoreplicationstress-inducingagents
LeukemiacelllineswithDNMT3Amutationsaremoresensitivetoreplicationstressinducers
Transform of Trial 2: Cladribine
1 2 3 40.0
0.5
1.0
1.5
Via
bilit
y, r
elat
ive
to u
ntre
ated
Ara-C, log(uM)
K-562
KU-812
SET-2
KO-52
Transform of Ara-C - 4 cell lines
1 2 3 40.0
0.5
1.0
1.5
Ara-C, log(uM)
Via
bilit
y, r
elat
ive
to u
ntre
ated
K-562KU-812
SET-2KO-52
IC50297.6465.246.1165.8
Transform of Fludarabine -4 cell lines
-1 0 1 20.0
0.5
1.0
1.5
Fludarabine, log(ug/ml)
Via
bilit
y, r
elat
ive
to u
ntre
ated
K-562KU-812SET-2KO-52
Transform of Ara-C - 4 cell lines
1 2 3 40.0
0.5
1.0
1.5
Ara-C, log(uM)
Via
bilit
y, r
elat
ive
to u
ntre
ated
K-562KU-812
SET-2KO-52
IC50ND16.121.1221.303
Cellnu
mbe
r
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
AMLcelllines
K562 SET2 KU812 KO52
AraC,5μMcleavedPARP
γH2AX
GAPDH
0 4 8 12 24 0 4 8 12 24 0 4 8 12 24 0 4 8 12 24
DNMT3A-mutantcelllinestreatedwithAra-CaccumulatemarkersofDNAdamageandapoptosis
DNMT3Amut
DNMT3Awt
AMLcelllines
IncreasedapoptosisinDNMT3A-mutantcelllinesafterAra-Ctreatment
K-562
KU-812
SET-2KO-52
0
5
10
15
20
Transpose of AnnexinV+DAPI-
% A
nnex
inV+
DA
PI- c
ells 0h
4h
24h
0 4 24 0 4 24 0 4 24 0 4 24
K-562 KU-812 SET-2 KO-52
Annexin V
K-562
KU-812
KO-52SET-2
0
5
10
15
20
25
Data 2
% c
ells
in s
ub-G
1
untreated
0.5 uM
5 uM
0 0.5 5 0 0.5 5 0 0.5 5 0 0.5 5
K-562 KU-812 SET-2 KO-52
Sub-G1
DNMT3Amut
DNMT3Awt
AMLcelllines
Ara-Csensitivity correlateswithnuclearstructurechangesincellsexpressingDNMT3A variants
pPICH
D3A W
T
D3A R
C0
500
1000
1500
2000
2500
Nuc
lear
are
a (p
ixel
s)
U2OS untr nuclei - batch 1
Vector DNMT3A(wt)
DNMT3A(mut)
**
3.5 4.0 4.5 5.0 5.50.0
0.5
1.0
1.5
Ara-C, log(uM)
Via
bilit
y, r
elat
ive
to u
ntre
ated
Transform of AraC 48h 3
MiGRi
WT
44
Vector
DNMT3A wt
DNMT3A mut
Cellnu
mbe
r
Dose
wt
mut
DNMT3Amut
Vector
U2OSDNMT3AWT
nuclearsize
Ara-C
IncreasedapoptosisafterAra-CexposureincellsexpressingDNMT3Amutants
AraC,50µM
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
Untre
ated
24
h0
10
20
30
40
50
Time after treatment(hours)
%ce
lls in
su
b-G
1
EV
DNMT3A(wt)
DNMT3A(mut)
ns
**
**ns
****
***
24hUnt
reat
ed
24h
0
10
20
30
40
50
Time after treatment(hours)
%ce
lls in
su
b-G
1
EV
DNMT3A(wt)
DNMT3A(mut)
ns
**
**ns
****
***
Untre
ated
24
h0
10
20
30
40
Time after treatment(hours)
% A
nn
exin
V +
cel
ls EV
DNMT3A(wt)
DNMT3A(mut)
ns
****
****
ns**
24hUnt
reat
ed
24h
0
10
20
30
40
Time after treatment(hours)
% A
nn
exin
V +
cel
ls EV
DNMT3A(wt)
DNMT3A(mut)
ns
****
****
ns**
CellsexpressingmutantDNMT3A showpersistentDNAdamagesignalingafterAra-C
0 2 8 12 0 2 8 12 0 2 8 12 AraC,10µM(h)
p-Chk1
GAPDH
totalChk1
DNMT3A
p-p53
totalp53
Vector DNMT3Awt DNMT3Amut
0
20
40
60
80
100
120
140
160
0 2 4 6 8 10 12 14
Relativ
eDe
nsity
Timeaftertreatment
EV
WT
mut
DNMT3Amut
DNMT3AwtVector
p-Chk1
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
U2OS
AccumulationofDNAdamagemarkergH2A.XafterAra-CexposureincellswithDNMT3Amutations
MiGRi 1
2h
WT 12h
RH 12h
0
2×1010
4×1010
6×1010
8×1010
Nor
mal
ized
fluo
resc
ence MiGRi 12h
WT 12h
RH 12h
Vector DNMT3A(wt)
DNMT3A(mut)
NS*
****12h
Vector
DNMT3A
(wt)
DNMT3A
(mut)
U2OS
MiGRi 2
4h
WT 24h
RH 24h
0
2×1010
4×1010
6×1010
8×1010
Nor
mal
ized
fluo
resc
ence MiGRi 24h
WT 24hRH 24h
NS***
***24h
MiGRi 3
6h
WT 36h
RH 36h
0
2×1010
4×1010
6×1010
8×1010
Nor
mal
ized
fluo
resc
ence MiGRi 36h
WT 36h
RH 36h*****
****36h
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
Untreat
ed 12h
24h
36h
0
1×1010
2×1010
3×1010
4×1010
Data 1
Nor
mal
ized
fluo
resc
ence MiGRi
WT
RH
AccumulationofDNAdamagemarkergH2A.XafterAra-CexposureincellswithDNMT3Amutations
*
****
Vector
DNMT3A(wt)
DNMT3A(mut)
U2OS
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
MiGRi UTWT UT RH UT0
20
40
60
80
100
Untreated
%Ta
il D
NA
MiGRi UT
WT UT
RH UT
Vector DNMT3A(wt)
DNMT3A(mut)
*NSNS
AccumulationofDNAdamageafterAra-CexposureincellswithDNMT3Amutations
U2OS
0h
MiGRi 12hWT 12h RH 12h0
20
40
60
80
100
Time after treatment(hours)
%Ta
il D
NA
MiGRi 12h
WT 12hRH 12h
Vector DNMT3A(wt)
DNMT3A(mut)
***NS***
12h
MiGRi 24hWT 24h RH 24h0
20
40
60
80
100
Time after treatment(hours)
%Ta
il D
NA
MiGRi 24h
WT 24h
RH 24h
Vector DNMT3A(wt)
DNMT3A(mut)
****NS****
24h
Vector
DNMT3A
(wt)
DNMT3A
(mut)
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
0h 12h
24h
36h
0
20
40
60
80
100
Time after treatment(hours)
%Ta
il D
NA
MiGRi
WT
RH
*
*******
Vector
DNMT3A(wt)
DNMT3A(mut)
U2OS
AccumulationofDNAdamageincellswithDNMT3AmutationsaftercontinuousAra-Cexposure
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
Ara-C
AlteredcellcycleprofileafterAra-CtreatmentincellswithDNMT3Amutations
Untreate
d 4h 8h0
5
10
15
20
Time after treatment(hours)
% c
ells
in G
2 ph
ase
MIG
WT
RH
0h 4h 8 h
Vector
DNMT3A(wt)
DNMT3A(mut)
G1
Sub-G1
S G2
G1
Sub-G1
SG2
G1Sub-G1
S
G2
Vector
DNMT3A
(wt)
DNMT3A
(mut)
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
U2OS
Ara-C,10µM
8 h
WhydocellswithDNMT3AmutationsaccumulateDNAdamageafterAra-C?
impairedDNArepair
susceptibilitytoDNAdamage
NodefectinHRorNHEJincellswithDNMT3AmutationstreatedwithAra-C
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
Vector DNMT3A(wt) DNMT3A(mut)
RAD5
1(HR)
53BP
1(NHE
J)
0h 12h
24h
36h
0
20
40
60
80
100
Time after treatment(hours)
%Ta
il D
NA
MiGRi
WT
RH
Vector
DNMT3A(wt)
DNMT3A(mut)
DNMT3AmutantcellsefficientlyresolveAra-CinducedDNAdamage
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
Untreate
d Ara-
C30
min
60 m
in0
10
20
30
40
50
Repair kinetics%
Tai
l DN
A
Vector
WT
MUT
washout
Ara-Crepair
U2OS
MethoCult (colony forming) assay schematic
Dnmt3amut cKI
wt control MethoCult
7-10 days:count colonies( = progenitors)
Col
ony
nu
mb
er
Dose
mut
wt
treat
Cell
num
ber
Dose
mut
wttreat
DNMT3Amut
DNMT3Awt
WT mut +/–DNMT3AmutDNMT3Awt
DNMT3Amut
VectorDNMT3AWT
Dnmt3awt:Flt3ITD:Npm1c
Dnmt3amut:Flt3ITD:Npm1c
1 2 3 4 50.0
0.5
1.0
1.5
AraC, log(nM)
Via
bilit
y, r
elat
ive
to u
ntre
ated
Dnmt3a wt:Flt3ITD:Npm1c(193.8)
Dnmt3a mut:Flt3ITD:Npm1c(52.38)
MouseleukemiawithaDnmt3amutationismoresensitivetocytarabine exvivo
DNMT3AmutDNMT3Awt
Ara-C
Apoptosis
DNAdamageDNAdamage
gH2A.X gH2A.X
Ara-C
Summary
DNArepairDNArepair ✓✓
Continuousexposure=continuousintravenousinfusion
pPICH
D3A W
T
D3A R
C0
500
1000
1500
2000
2500
Nuc
lear
are
a (p
ixel
s)
U2OS untr nuclei - batch 1
Vector DNMT3A(wt)
DNMT3A(mut)
**
DNMT3Amut
Vector
U2OSDNMT3AWT
nuclearsizeDnmt3amut
SPT-16|
SPT-16
Guryanova etal.(2016)NatMed
Ara-CsensitivityinDNMT3Amut setting:Nextsteps
DNMT3AWT
Dungrawala etal.(2015)Mol Cell
293T (HU)
DNMT3ApromotesrecruitmentofSPT-16toDNAaftertorsionalstress,throughdirectinteraction;attenuatedbyDNMT3Amutations
DNMT3Adetectedatstalledreplicationforks30’afterHU
Proteomeatreplicationforks– iPOND(isolationofproteinsonnascentDNA)
Mass-spec
Differentialsensitivitytocytarabine invivo
Dnmt3amut :Flt3ITD :Npm1c
Dnmt3awt :Flt3ITD :Npm1c treat
%su
rvival
Time
mutwt
Chromatinaccessibility– ATAC-seq
Ara-CsensitivityinDNMT3Amut setting:Nextsteps
Replicationforkdynamics– CldU/IdU labeling
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Undergraduates:
UFHealthCancerCenterCollaborativePilotGrant
DNMT3A R882mutationsdisrupttetramerizationandattenuatecooperativeDNAbinding
denovo DNAmethyltransferase
DNMT3Awt
DNMT3AR882mut2x processivity
Holz-Schietingeretal.2012
Homotetramer- processive catalysis
Homodimer- distributivecatalysis