supplemental figure 1 - dm5migu4zj3pb.cloudfront.net · 35.9 24.0 18.8 11.6 33.9 21.4 45.2 11.2...

Supplemental Figure 1. Screening of epigenetic targets that affect the differentiation status of CD8+ T cells. Flow cytometry

plots of CD45RA, CD62L and CCR7 expression in CD8+ T cells treated with each chemical probe 14 days following initial

stimulation with aAPC/mOKT3.

Supplemental Figure 1

UNC0638 UNC0642 A366 SGC0946 UNC1999 GSK343 GSK-J4 OICR-9429

CD45RA

CD

62L

CCR7

34.4

22.4

39.3

23.7

48.2

30.8

25.6

11.1

23.1

11.1

27.9

16.3

43.1

19.3

42.6

20.5

62.1

2.97

64.8

16.5

45.9

8.06

41.7

4.51

41.1

7.86

42.5

8.34

43.5

6.54

44.1

10.3

PFI-2 GSK-LSD1 UNC1215 SGC-CBP30 PFI-1I-CBP112 C646 JQ1

CD45RA

CD

62L

CCR7

51.8

28.4

63.1

32.0

60.0

38.8

58.1

39.6

52.9

27.8

27.7

27.3

54.7

35.8

65.2

57.3

76.1

8.27

68.0

18.7

60.1

31.4

58.3

15.9

73.2

16.0

52.2

12.5

62.8

13.6

57.2

6.62

Bromosporine GSK2801 BAZ2-ICR OF-1 NI-57 PFI-4 PFI-3 CI-994

CD45RA

CD

62L

CCR7

69.1

57.9

41.4

29.5

50.1

30.9

62.4

40.5

48.7

28.9

42.8

26.7

40.1

24.7

55.7

46.3

51.3

33.8

62.7

9.03

59.2

8.99

57.7

7.01

63.0

4.13

50.0

6.67

53.5

6.01

78.3

6.83

VPA Olaparib IOX2 LLY-507 GSK-484 Decitabine DMSOLAQ824

CD45RA

CD

62L

CCR7

66.7

60.9

73.8

52.5

41.9

28.7

72.7

44.1

27.4

21.1

35.9

24.0

18.8

11.6

33.9

21.4

45.2

11.2

26.8

7.79

40.1

10.4

42.5

8.74

66.4

3.52

61.6

9.83

79.3

15.0

60.7

26.7


Supplemental Figure 2. Effects of epigenetic chemical probes on CD8+ T cell differentiation. (A, B) Frequency of

CD45RA+ CD62L+ CCR7+ (A) and CD45RA- CD62L+ CCR7+ cells (B) within CD8+ T cell population cultured for 14 days in

the presence of each chemical probe. Inhibitors for p300 and BET proteins are indicated in bold. The error bars indicate the S.

D. of three technical replicates. The dotted lines indicate the mean values in DMSO control wells.

A

CD

45R

A+

CD

62L+

CC

R7+

cel

ls

with

in C

D8

+ T

cel

ls (

%)

B

UNC0638

UNC0642

A-366

SGC0946

UNC1999

GSK343

GSK-J4

OICR-9429

PFI-2

GSK-LSD1

UNC1215

SGC-CB

P30

I-CBP112C646 JQ

1PFI-1

Brom

osporine

GSK2801

BAZ2-ICROF-1

Ni-57PFI-4

PFI-3

CI-994

LAQ824

Valproic acid

OlaparibIO

X2

LLY-507

GSK-484

Decitabine

DMSO

0

20

40

60


Supplemental Figure 3. Effects of epigenetic targets on differentiation of CD45RO+ memory T cells. (A, B) Peripheral

blood CD3+ CD45RO+ T cells were stimulated with aAPC/mOKT3 and subsequently treated with each epigenetic chemical

probe. The frequency (A) and absolute fold expansion (B) of CD45RA- CD62L+ CCR7+ cells within the CD8+ T cell population

14 days following initial stimulation are shown. Inhibitors for p300 and BET proteins are indicated in bold. The error bars

indicate the S. D. of three technical replicates. The dotted lines indicate the mean values in DMSO control wells.

UNC0638

UNC0642

A-366

SGC0946

UNC1999

GSK343

GSK-J4

OICR-9

429PFI-2

GSK-LSD1

UNC1215

SGC-CBP30

I-CBP112C646 JQ

1PFI-1

Bromosporine

GSK2801

BAZ2-IC

ROF-1

Ni-57PFI-4

PFI-3

CI-994

LAQ82

4

Valpro

ic ac

id

Olapar

ibIO

X2

LLY-5

07

GSK-484

Decita

bine

DMSO

0

10

20

30

CD

45R

A-C

D62

L+ C

CR

7+ c

ells

with

in C

D8+

T c

ells

(%

)A

UNC0638

UNC0642

A-366

SGC0946

UNC1999

GSK343

GSK-J4

OICR-9

429PFI-2

GSK-LSD1

UNC1215

SGC-CBP30

I-CBP112C646 JQ

1PFI-1

Bromosporine

GSK2801

BAZ2-IC

ROF-1

Ni-57PFI-4

PFI-3

CI-994

LAQ82

4

Valpro

ic ac

id

Olapar

ibIO

X2

LLY-5

07

GSK-484

Decita

bine

DMSO

Fol

d ex

pans

ion

of

CD

8+ C

D45

RA

-C

D62

L+ C

CR

7+ c

ells

B

JQ1

(-)-J

Q1JQ

1

(-)-J

Q1

0

200

400

600

A

CD4+ CD8+

B

JQ1

(-)-J

Q1

0

2

4

6

8

10

Dea

d ce

lls (

%)

n.s.n.s.

n.s.


Supplemental Figure 4. Effects of JQ1 treatment on T cell division rate and viability. (A, B) CD3+ T cells labeled

with CFSE were stimulated with aAPC/mOKT3 and cultured in the presence of JQ1 or (-)-JQ1 for 3 days. The average

mean fluorescence intensity of CFSE in CD4+ and CD8+ T cells (A) as well as the frequency of dead cells (B) were

evaluated by flow cytometry (n=5). Error bars depict the S. D.

C

JQ1

Contro

l0

20

40

60

80 P<0.05

Supplemental Figure 5. Differentiation status of CD4+ T cells treated with JQ1. (A, B) CD45RA+ CD62L+ CCR7+ T

cells were stimulated weekly with aAPC/mOKT3 and cultured in the presence or absence of JQ1. Representative FACS plots

showing expression of CD45RA, CD62L and CCR7 (A), and the frequency of CD45RA+ CD62L+ CCR7+ and CD45RA-

CD62L+ CCR7+ cells within the CD4+ T cell population 21 days following initial stimulation is shown (B; n=5, paired t test).

(C) IL-2 secretion upon restimulation with aAPC/mOKT3 in CD4+ T cells cultured for 14 days with or without JQ1 (n=7,

paired t test).


B

P<0.01

JQ1

(-)-J

Q1CD

45R

A- C

D62

L+ C

CR

7+ c

ells

in C

D4+

Tce

lls (

%)

P<0.01

JQ1

(-)-J

Q1

0

2

4

6

8

10

CD

45R

A+ C

D62

L+ C

CR

7+

in C

D4+

T c

ells

(%

)

CD45RA

CD

62L

CCR7

49.6 11.3

82.8

69.1

50.4 5.54

60.9

47.5

JQ1

(-)-JQ1

A

CM CM CM CM EM EM EM EM

Nor

mal

ized

inte

nsity

val

ues


Nor

mal

ized

inte

nsity

val

ues


Nor

mal

ized

inte

nsity

val

ues


Nor

mal

ized

inte

nsity

val

ues


Nor

mal

ized

inte

nsity

val

ues


Nor

mal

ized

inte

nsity

val

ues

SCMSCM

SCM CM CM CM EM EM EM

Nor

mal

ized

inte

nsity

val

ues

SCMSCM


Nor

mal

ized

inte

nsity

val

ues

SCMSCM


Nor

mal

ized

inte

nsity

val

ues

SCMSCM


Nor

mal

ized

inte

nsity

val

ues

SCMSCM


Nor

mal

ized

inte

nsity

val

ues

SCMSCM


Nor

mal

ized

inte

nsity

val

ues

GSE11057 GSE23321

P<0.01

P<0.01

P<0.05

P<0.01

P<0.05

P<0.01

P<0.01

P<0.01

P<0.01

P<0.01

P<0.01

P<0.05

ACTN1

SELL

TCF7

GZMA

KLRG1

PLEK

Supplemental Figure 6. Differentially expressed genes between the TSCM/TCM and TEM phenotypes. Normalized intensity values of the indicated genes from the published microarray data (GSE11057 and GSE23321) are shown.


JQ1

(-)-J

Q1

0

10

20

30

40

B

JQ1

Contro

l0

20

40

60

80

100

JQ1

DMSO

0

20

40

60P<0.05 P<0.01n.s.

ACTN1SELL

TCF7

GZMA

KLRG1PLEK

Rel

ativ

e ex

pres

sion

(JQ

1-tr

eate

d / c

ontr

ol)

D

IL-2

+ IF

N-

+ T

NF-

+ c

ells

with

in C

D8

+ T

cel

ls (

%)

C

P<0.05

Supplemental Figure 7. Comparison of the functional properties of CD45RO+ memory T cells treated with JQ1 or (-)-JQ1.

(A) CD3+ CD45RO+ cells were stimulated with aAPC/mOKT3 and cultured with JQ1 or (-)-JQ1 for 14 days. The frequency of

CD45RA- CD62L+ CCR7+ cells within the CD8+ T cell population is shown (n=8, paired t test). (B, C) CD45RO+ T cells treated

with JQ1 or (-)-JQ1 for 14 days were restimulated with aAPC/mOKT3, and the production of IL-2, IFN- and TNF- in CD8+ T

cells was assessed with intracellular flow cytometry. The frequency of individual cytokine-secreting cells (B) and those

producing all three cytokines (C) is shown (n=4, paired t test). (D) Expression profiles of representative genes with differential

expression between the TCM and TEM phenotypes. The average expression levels in the JQ1-treated CD8+ T cells relative to those

in (-)-JQ1-treated CD8+ T cells are shown (n=4). Error bars indicate the S. D.


JQ1

(-)-J

Q1

Fre

quen

cy o

f CD

45R

A-

CD

62L+

CC

R7+

cel

ls (

%)

AP<0.01


Supplemental Figure 8. Effects of JQ1 on CD8+ T cell differentiation in the absence of costimulatory signals. (A, B)

CD3+ T cells were stimulated every week with plate-coated anti-CD3 mAb (clone OKT3) and cultured in the presence or

absence of JQ1. Representative FACS plots (A) and the frequencies of CD45RA+ CD62L+ CCR7+ and CD45RA- CD62L+

CCR7+ cells (B) within the CD8+ T cell population 14 days following the initial stimulation (n=7, paired t test). (C, D)

Secretion levels of IL-2, IFN-, and TNF- were evaluated by intracellular flow cytometry in JQ1- and (-)-JQ1-treated

CD8+ T cells. The frequencies of each type of cytokine-secreting cells (C) and cells producing all three cytokines (D) are

shown (n=5, paired t test).

JQ1

(-)-J

Q1

0

2

4

6

8

IL-2

sec

retio

n (%

)

JQ1

(-)-J

Q1

20

30

40

50

60

JQ1

(-)-J

Q1

0

10

20

30

C P<0.01n.s.

n.s.

JQ1

(-)-J

Q1IL-2

+ IF

N-

+ T

NF-

+ c

ells

(%

)

P<0.05

D

JQ1

(-)-J

Q1CD

45R

A+ C

D62

L+ C

CR

7+ c

ells

(%

)

JQ1

(-)-J

Q1CD

45R

A- C

D62

L+ C

CR

7+ c

ells

(%

)

P<0.01 P<0.01

BA JQ1 (-)-JQ1

27.6 11.651.7 14.6

35.3

34.2

23.9

22.6

CD45RA

CD

62L

CCR7

Supplemental Figure 9. Differentiation of CD8+ T cells upon antigen-specific T cell stimulation. CD3+ T cells were

stimulated weekly with aAPC/A2 loaded with mutant MART127–35 peptide in the presence of JQ1 or (-)-JQ1. The CD8+

A2/MART1 multimer+ cells were analyzed for CD45RA, CD62L and CCR7 expression 21 days after initial stimulation.

Representative FACS plots of four independent experiments are shown.


JQ1

(-)-JQ1

CD8

MA

RT

1 m

ultim

er

6.01

8.64

CD45RA

CD

62L

10.532.4

5.7221.6

CCR7

8.2

5.3

17.1

9.97


Supplemental Figure 10. Cytokine secretion by CAR-transduced T cells following restimulation with K562-CD19. JQ1- or

(-)-JQ1-treated CAR-T cells were stimulated with K562-CD19 in the absence of drugs. Five days later, they were restimulated

with K562-CD19, and cytokine secretion was evaluated by intracellular flow cytometry. Frequencies of each type of cytokine-

producing cell and cells secreting all three cytokines are shown (n=5, paired t test).

JQ1

(-)-J

Q1

0

10

20

30

IL-2

sec

retio

n (%

)

JQ1

(-)-J

Q1

0

20

40

60

80

IFN

- s

ecre

tion

(%

)

JQ1

(-)-J

Q1

0

10

20

30

40

50

TNF-

sec

retio

n (

%)

JQ1

(-)-J

Q1

IL-2

+ IF

N-

+ T

NF-

+ce

lls (

%)P<0.05 P<0.05

n.s.

P<0.05


Supplemental Figure 11. Treatment of CD19+ acute lymphoblastic cell line NALM-6 with anti-CD19 CAR-transduced T cells. (A) NSG mice were intravenously injected with NALM6-GL and, 14 days later, treated with CAR-transduced T cells treated with JQ1 or (-)-JQ1. In vivo bioluminescent imaging of luciferase activity at the indicated time points following T cell infusion is shown. (B) Phenotypic analysis for persistent CD8+ CAR-T cells in the peripheral blood. Representative FACS plots at day 7 following T cell infusion are shown.

Protein L / Streptavidin-APC

CD8

FS

C

CD45RA

CD

62L

CCR7

18.7

(-)-JQ1

74.440.1 14.3

37.1

32.4

35.9

JQ1

51.840.2 15.2

31.6

24.0

B

CAR-negative control T cells

Gated CD8+ T cells

A(-)-JQ1

Days after T cell infusion

0

7

14

21

28

35

42

No T cells

Days after T cell infusion

0

7

JQ1

0

7

14

21

28

35

42

49

56

63

70

77


Supplemental Figure 12. Autopsy analysis of mice transplanted with JQ1- or (-)-JQ1-CAR-T cells. (A) Representative

FACS plots evaluating the persistence of NALM6-GL and CD19 expression in the spleen and bone marrow. (B)

Representative FACS plots showing the persistence of CAR-T cells in the spleen.

A

In vitro cultured NALM6-GL

CD19

JQ1-CAR-T cell transplanted mice

Spleen Bone marrow

(-)-JQ1-CAR-T cell transplanted mice

Spleen Bone marrow

GFP

61.8 66.0 38.4 55.8

GFP+ cells

K562

B

CD45

FS

C

CD8

CD

4

3.2475.6

Protein L / Streptavidin-PE

0.65(-)-JQ1

0.5760.5

5.76JQ1 Residual CD8+ T cells

Control T cells


Supplemental Figure 13. Cytokine secretion by A2/MART1-T cells following restimulation with aAPC/A2. JQ1- or (-)-

JQ1-treated A2/MART1-T cells were stimulated with aAPC/A2 loaded with MART127-35 peptide in the absence of drugs.

Five days later, the T cells were restimulated with aAPC/A2 with MART127-35, and cytokine secretion was evaluated by

intracellular flow cytometry. Frequencies of each type of cytokine-producing cell and cells secreting all three cytokines are

shown (n=4; paired t test).

JQ1

(-)-J

Q1

0

10

20

30

40

IL-2

sec

retio

n (%

)

JQ1

(-)-J

Q1

50

55

60

65

70

75

IFN

- s

ecre

tion

(%

)

JQ1

(-)-J

Q1

36

38

40

42

44

46

TNF-

sec

retio

n (

%)

P<0.01 n.s.

n.s.

P<0.05

JQ1

(-)-J

Q1

IL-2

+ IF

N-

+ T

NF-

+ce

lls (

%)


Supplemental Figure 14. Surface marker phenotypes of CD8+ T cells with BATF knockdown. Surface expression of CD27,

CD28, and CD127 in the CD8+ NGFR+ T cells transduced with control or siBATF 14 days following initial stimulation with

aAPC/mOKT3. Representative plots of the samples in Figure 7C are shown.

CD27

CD28

Control siBATF-1 siBATF-2

CD127

CD45RA+ CD62L+ CCR7+

CD45RA- CD62L+ CCR7+

CD45RA- CD62L- CCR7-


Supplemental Figure 15. BATF knockdown by lentiviral shRNA. (A, B) CD3+ T cells were transduced with lentiviral

shRNAs against BATF and stimulated with aAPC/mOKT3. Protein levels of BATF in the CD8+ ZsGreen+ T cell population

were analyzed by intracellular flow cytometry three days following stimulation. Representative FACS plots (A) and average

relative mean fluorescence intensity normalized to the control (B) (n=4; one-way ANOVA). Error bars indicate the S.D.

(C) Naïve T cells were purified by magnetic selection, pre-treated with IL-7, and transduced with lentiviral shRNA targeting

BATF at day 5-8. Representative FACS plots of CD45RA, CD62L and CCR7 expression in the purified naïve T cells and

after shRNA transduction are shown.

Contrl

shBATF

-1

shBATF

-2

0.0

0.5

1.0

1.5

P<0.01

P<0.01B

BATF

Controlsh-BATF

shBATF-1 shBATF-2A

C

Purified naïve T

Cultured naïve T

CD45RA

CD

62L

CCR7

99.7

GFP

48.455.751.3

96.6 96.2 96.1

99.6 99.6

Control shBATF-1 shBATF-2

99.8

shRNA-transduced T cells

Untransduced control T cells

100.0


Supplemental Figure 16. Memory T cell formation from naïve T cells transduced with lentiviral shRNA against BATF. (A)

Naïve T cells were transduced with shRNA against BATF following pretreatment with IL-7. They were subsequently stimulated

with aAPC/mOKT3, and the expression profiles of CD45RA, CD62L and CCR7 within the CD8+ ZsGreen+ T cell population were

analyzed 10 days following stimulation. Representative FACS plots of the five experiments are shown. (B) Naïve T cells

transduced with shRNA against BATF were transplanted into irradiated NSG mice. Mice were sacrificed 11 days following T cell

infusion, and the CD8+ ZsGreen+ cells engrafted in the spleen were analyzed. Representative FACS plots analyzing expression of

CD45RA, CD62L and CCR7 within the CD45+ CD8+ ZsGreen+ population are shown. (C) Naïve T cells with shRNA against

BATF were transplanted into irradiated NSG mice, as shown in Figure 7H. Mice were sacrificed 16 days following T cell

infusion, and the frequency of CD45RA+/- CD62L+ CCR7+ cells within the ZsGreen+ CD8+ T cell population in the spleen was

analyzed (n=6, one-way ANOVA).

B

29.445.816.3

CD45RA

CD

62L


67.0 31.3 34.0

96.8

81.0

93.4

61.3

91.8

66.6

CCR7

CD8

ZsG

reen

3.736.324.68

C

P<0.01P<0.01

n.s.

Control

shBATF-1

shBATF-2C

D45

RA

+ C

D62

L+ C

CR

7+ c

ells

with

in C

D8+

ZsG

reen

+ T

cel

ls (

%)

Control

shBATF-1

shBATF-2

0

5

10

15

20

CD

45R

A- C

D62

L+ C

CR

7+ c

ells

with

in C

D8

+ Z

sGre

en+ T

cel

ls (

%)

CD45RA

CD

62L


CCR7

35.0 25.737.2 23.740.8 15.1

51.6

19.6

75.6

37.8

79.7

44.5

A


Supplemental Figure 17. Knockdown effects of genes suppressed by JQ1 on memory T cell differentiation. (A) Expression

of the candidate genes targeted by JQ1 was assessed by quantitative real-time PCR. Relative expression levels of the indicated

genes in JQ1-treated T cells relative to (-)-JQ1-treated cells (normalized to UBC) three days after stimulation with aAPC/mOKT3

are shown (n=4, one-sample test compared to one). Error bars indicate the S.D. ND, not detected within 45 cycles of qPCR. *

P<0.05, ** P<0.01. (B) CD3+ T cells were retrovirally transduced with the control, siFOSL2, siID2, or siPRDM1 and NGFR.

Expression of each target gene compared with the control was assessed by quantitative PCR (n=4, one sample t test compared to

one). Error bars indicate the S.D. * P<0.05, ** P<0.01. (C) CD3+ T cells were stimulated with aAPC/mOKT3 and transduced

with control, siFOSL2, siID2, or siPRDM1, and NGFR. The frequency of CD45RA+/- CD62L+ CCR7+ cells within the NGFR+

CD8+ T cell population 14 days after initial stimulation is shown (n=5, paired ANOVA).

Contro

l

siFOSL2

siID2

siPRDM1

0

5

10

15

CD

45R

A+ C

D62

L+ C

CR

7+ c

ells

with

in C

D8

+ T

cel

ls (

%) n.s.

Contro

l

siFOSL2

siID2

siPRDM1

0

2

4

6

8

10

CD

45R

A- C

D62

L+ C

CR

7+ c

ells

with

in C

D8

+ T

cel

ls (

%)

n.s.C

siFOSL2

siID2

siPRDM1

Rel

ativ

e ex

pres

sion

B

*

**

**

AHR

CREMEGR2

FOSL1

FOSL2ID2

NFIL3

PLAGL2

STAT3

TRIP13

UHRF

PRDM1

RUNX3

STAT4

TBX21

0.0

0.5

1.0

1.5

2.0

2.5

A

**

**

ND


Supplemental Figure 18. Phosphorylation of ribosomal protein S6 kinase (S6K) is decreased in JQ1-treated T cells. (A,

B) CD3+ T cells were stimulated with aAPC/mOKT3 at an E:T ratio of 3:1 and treated with JQ1 or (-)-JQ1 in the presence of

IL-2 and IL-15. Phosphorylation of the indicated proteins in CD8+ T cell population was analyzed 5 days after stimulation.

Representative FACS plots (A) and relative mean fluorescence intensity normalized to freshly isolated CD8+ T cells (B) (n=4;

unpaired t test). (C, D) CD3+ T cells were stimulated with aAPC/mOKT3 and transduced with lentiviral shRNA against

BATF. The shRNA-transduced T cells were rested in cytokine-free media and then restimulated with aAPC/mOKT3.

Phosphorylation of S6K in the ZsGreen+ CD8+ T cell population was quantified by intracellular flow cytometry.

Representative FACS plots (C) and relative mean fluorescence intensity normalized to the control plasmid-transduced cells

(D) (n=4; one-way ANOVA).

n.s.

Day 0

JQ

1

(-)-J

Q1

Rel

ativ

e m

ean

fluor

esce

nce

inte

nsity

Day 0

JQ

1

(-)-J

Q1

0

5

10

15n.s.

Day 0

JQ

1

(-)-J

Q1

0

1

2

3n.s.

Day 0

JQ

1

(-)-J

Q1

0

5

10

15 n.s.P<0.01

pSTAT5 p-p38 MAPK pERK pAKT pS6KB

Day 0

JQ1

(-)-J

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10

20

30

40

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nsity

CControl

shBATF-1

shBATF-2

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pSTAT5 p-p38 MAPK pERK pAKT pS6K

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(-)-JQ1

Day 0 CD8+ T cells

A


Supplemental Figure 19. Phenotypic analysis of CAR-T cells after adoptive transfer. C646- or DMSO-treated CAR-T cells were infused into NSG mice transplanted with NALM6-GL. The frequency of CD45RA+/- CD62L+ CCR7+ T cells within the CD8+ CAR+ T cell population was analyzed (n=10, unpaired t test).

Fre

qunc

y w

ithin

CD

8+ C

AR

+ T

cel

ls (

%)

CD45RA+

CD62L+ CCR7+CD45RA-

CD62L+ CCR7+

n.s.

n.s. n.s.

n.s.n.s.

n.s.

Supplemental Table 1. Epigenetic chemical probes with defined targets.

Probe Target Tested dose (M) References Probe Target Tested dose (M) References

Histone-modifying enzymes Histone readers

UNC0638 G9a/GLP 0.1 1 JQ1 BET Bromodomain 0.15 15

UNC0642 G9a/GLP 0.1 2 PFI-1 BET Bromodomain 1 16

A-366 G9a/GLP 0.5 3 Bromosporine pan-Bromodomain 1

SGC0946 DOT1L 0.5 4 GSK2801 BAZ2A/B 1 17

UNC1999 EZH2 0.2 5 BAZ2-ICR BAZ2B/A 1 18

GSK343 EZH2 0.5 6 OF-1 BRPF1-3 1

GSK-J4 JMJD3/UTX 1 7 Ni-57 BRPF1-3 0.5

OICR-9429 WDR5 1 8 PFI-4 BRPF1B 0.25

PFI-2 SETD7 1 9 PFI-3 SMARCA4 1 19

GSK-LSD1 LSD1 1 10 CI-994 HDAC 1

UNC1215 L3MBTL3 1 11 LAQ824 HDAC 0.01 20

SGC-CBP30 CREBBP/EP300 0.2 12 VPA HDAC 400

I-CBP112 CREBBP/EP300 1 13 Other targets

C646 EP300 10 14 Olaparib PARP 1

IOX2 HIF1a 50 21

GSK484 PAD-4 0.5 22

LLY-507 SMYD2 1 23

Decitabine DNMT 0.05

Supplemental Table 2. Detailed information of the mice transplanted with CAR-T cells.

Probe Overall survival

NALM6-GL at autopsy (%)

CAR-T cell at autopsy (%)

Signs of GVHD Bone

marrow Spleen

T cells in the bone marrow T cells in the spleen

CD4+ CD8+ CAR+ in

CD4+ T cells CAR+ in

CD8+ T cells CD4+ CD8+

CAR+ in CD4+ T cells

CAR+ in CD8+ T cells

1

JQ1

85 35.6 25.2 2.02 0.46 0.53 1.56 7.25 0.93 0.41 2 fur loss and

red skin

2 64 67.8 42.8 0.56 0.08 3.60 6.56 0.38 0.08 1.23 6.92 none

3 65 66 61.8 0.06 0.06 2.60 7.55 0.11 0.35 2.34 5.76 none

4 59 24.6 14.1 1.05 0.06 3.07 8.7 2.35 0.03 0.00 2.13 none

5 91

(alive) NA

fur loss and red skin

6 91

(alive) NA none

7 45 66.1 21.6 0.40 0.02 1.36 4 0.35 0.02 0.76 0 none

8 91

(alive) NA none

9 33 73.2 35.2 0.05 0.02 6.12 0.00 0.06 0.05 2.72 5.17 none

10 46 45.1 33 0.01 0.06 0.00 1.79 0.00 0.01 0.00 0 none

1

(-)-JQ1

40 82.3 22.4 0.02 0.05 0.00 0 0.53 1.78 0.89 0.484 none

2 41 84.2 48.5 0.06 0.23 2.33 6.44 0.30 2.45 0.86 0.649 none

3 37 83.4 48.6 0.04 0.23 4.00 2.76 0.10 1.00 0.00 0.813 none

4 54 55.8 38.4 0.00 0.00 NA NA 0.00 0.02 NA 0 none

5 61 28.4 16.8 0.07 0.02 2.71 12.3 0.79 0.21 0.89 3.39 none

6 46 82.6 45.3 0.21 0.27 1.32 2.11 0.28 1.54 0.63 0.794 none

7 36 72.9 22.2 0.02 0.00 5.88 NA 0.13 0.01 7.32 0 none

8 33 68.3 21.5 0.01 0.02 0.00 8.7 0.13 0.12 2.20 6.71 none

9 40 60.2 21.1 0.03 0.04 0.00 0 0.59 0.49 1.26 2.76 none

10 34 84.5 37.7 0.07 0.19 2.94 2.25 0.13 1.08 0.00 1.21 none

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