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

Thankyou!

NIH/NCIK99/R00PathwaytoIndependenceCareerDevelopmentAward

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KathrynKrajcik

Undergraduates:

UFHealthCancerCenterCollaborativePilotGrant

DNMT3A R882mutationsdisrupttetramerizationandattenuatecooperativeDNAbinding

denovo DNAmethyltransferase

DNMT3Awt

DNMT3AR882mut2x processivity

Holz-Schietingeretal.2012

Homotetramer- processive catalysis

Homodimer- distributivecatalysis