Supp1

Cell free binding toBRD2/3/4 EC50 = 10 – 19 nM

Cell free inhibition ofBRD2/3/4 binding

to AcH4IC50 = 92 – 112 nM

Cellular proliferation(72 hour MTT assay)

IC50 ≥17 nM

IC50=0.49 IC50=0.23

IC50=0.28 IC50=0.19

Cl

S N

NN

NH

OH

ON

A

D

B C

F

CFSE

CFSE

101 102 103 104 105

101 102 103 104 105

Untreated Control

Live/Dead-BV510

SSC

-H (x

100

0)

-10

42.5

95

147.5

200

92.24%

SSC

-H (x

100

0)

Untreated Control

Live/Dead-BV510

SSC

-H (x

100

0)

-10

42.5

95

147.587.07%

SSC

-H (x

100

0)

-10

42.5

95

147.5

200200

CD4+ CD4+

CD8+ CD8+

Live/Dead-BV510

MK-8628 0.19µM

Live/Dead-BV510

101 102 103 104 105 101 102 103 104 105

101 102 103 104 105 101 102 103 104 105

MK-8628 0.19µM

-10

42.5

95

147.5

200

75.31%

70.60%

0

13

25

38

50

0

11

22

32

43

Cou

ntC

ount

0.19nM MK-8628Untreated ControlUnstimulated Control

0.19nM MK-8628Untreated ControlUnstimulated Control

CD4+

CD8+

5.00

1.67

0.56

0.19

0.06

0.02

0.00

0

20

40

60

80

100

5.00

1.67

0.56

0.19

0.06

0.02

0.00

0

20

40

60

80

100

CD

4+ T

Cel

l Via

bilit

y (%

)

CD

8+ T

Cel

l Via

bilit

y (%

)

Prol

ifera

ting

CD

4+ (%

)

Prol

ifera

ting

CD

8+ (%

)

0

20

40

60

80

100

0

20

40

60

80

100

MK-8628 (µM) MK-8628 (µM)

E

G

************

****n.s**

********

****

n.sn.s*

** n.s

******** ********

n.s

************ ****

****

5.00

1.67

0.56

0.19

0.06

0.02

0.00

5.00

1.67

0.56

0.19

0.06

0.02

0.00

MK-8628 (µM) MK-8628 (µM)

Supplemental Figure 1. Chemical structure and anti-proliferative activity of MK-8628. (A) 2D and (B) 3D chemical structure of MK-8628. (C) MK-8628 potency and toxicity summary table. (D) Flow cytometry contour plots of viability dye staining in anti-CD3/CD28 mAb stimulated CD4+ and CD8+ T lymphocytes treated with 0.19µM MK-8628 or vehicle treated controls after 96 hours. (E) Dose response depicting frequency of viable CD4+ and CD8+ T lymphocytes as shown in D. (F) Flow cytometry histograms analyzing CFSE cell dye dilution in anti-CD3/CD28 mAb stimulated daughter CD4+ and CD8+ T lymphocytes treated with 0.19µM MK-8628 or vehicle treated controls after 96 hours. (G) Frequency of CFSE+CD4+ and CD8+ T lymphocytes as shown in F. Data are from one experimental representative of at least three independent experiments and represent triplicate wells (E & G); small horizontal red lines indicate the mean. *P<0.05, **P<0.01 ***P<0.001, ****P<0.0001, one-way ANOVA with post-test analysis.

Supp2+

SSC

-H (x

100

0)

0

50

100

150

200

73.69%

SSC

-H (x

100

0)0

50

100

150

200

33.24%

SSC

-H (x

100

0)

0

100

150

200

68.51%

SSC

-H (x

100

0)

0

50

100

150

200

11.77%

SSC

-H (x

100

0)

0

50

100

150

200

63.28%

SSC

-H (x

100

0)

0

100

150

200

41.12%

SSC

-H (x

100

0)0

50 50

100

150

200

38.71%

SSC

-H (x

100

0)

0

100

150

200

18.43%

50

102 103 104 105 101101 100 102 103 104 105 102 103 104 105

101100 102 103 104 105 101100 102 103 104 102101 101103 104 105 102 103 104 105

102 103 104 105

CD4+ CD4+ CD4+ CD4+

CD8+ CD8+ CD8+ CD8+

MK-8628 1µM

CD71-PerCP-Cy5.5

MK-8628 1µM

CD71-PerCP-Cy5.5

A C

B D

50

0

20

40

60

80

100

%C

D25

+ am

ong

CD

8+

0

20

40

60

80

%C

D71

+ am

ong

CD

4+

0

20

40

60

MK-8628 (µM)

+ C

D 7

1+ a

mon

g C

D8+

01 0.1 0.01 0 1 0.1 0.01 0 1 0.1 0.01 0 1 0.1 0.01 0

20

40

60

80

100

%C

D25

+ am

ong

CD

4+

********

n.s

********

n.s

********

n.s

********

n.s

Untreated Control

CD25-PE-Cy7

Untreated Control

CD25-PE-Cy7

MK-8628 (µM)

CD25-PE-Cy7

MK-8628 1µM

MK-8628 1µM

CD25-PE-Cy7

MK-8628 (µM)

Untreated Control

CD71-PerCP-Cy5.5

Untreated Control

CD71-PerCP-Cy5.5

MK-8628 (µM)

Supplemental Figure 2. MK-8628 impairs bulk T lymphocyte activation. (A) Flow cytometry contour plots of IL-2R alpha chain (CD25) expression in anti-CD3/CD28 mAb stimulated CD4+ and CD8+ T lymphocytes treated with 1µM MK-8628 or untreated controls after 24 hours. (B) Dose response depicting CD25 expression in CD4+ and CD8+

T lymphocytes as shown in A. (C) Flow cytometry contour plots of transferrin receptor (CD71) expression in anti-CD3/CD28 mAb stimulated CD4+ and CD8+ T lymphocytes treated with 1µM MK-8628 or untreated controls after 24 hours. (D) Dose response depicting CD71 expression in CD4+ and CD8+ T lymphocytes as shown in C. Data are from one experimental representative of at least three independent experiments and represent triplicate wells; small horizontal red lines indicate the mean. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA with post-test analysis.

Supp3

SS

C-H

(x 1

000)

0

50

100

150

200

SSC

-H (x

100

0)

0

50

100

150

200

MK-8628 1µM

SSC

-H (x

100

0)

0

SS

C-H

(x 1

000)

0

50

100

150

200

SSC

-H (x

100

0)

0

100

150

200

0

SS

C-H

(x 1

000)

0

50

100

150

200

SSC

-H (x

100

0)

0

50

100

150

200

SS

C-H

(x 1

000)

0

50

100

150

200

SSC

-H (x

100

0)

0

50

100

150

200

Untreated Control

CD25-PE-Cy7

MK-8628 1µM

CD25-PE-Cy7

Untreated Control

CD25-PE-Cy7 CD25-PE-Cy7

Untreated Control

CD25-PE-Cy7

MK-8628 1µM

CD25-PE-Cy7

Untreated Control

CD25-PE-Cy7

MK-8628 1µM

CD25-PE-Cy7

Untreated Control

CD71-PerCP-Cy5.5

MK-8628 1µM

CD71-PerCP-Cy5.5

Untreated Control

CD71-PerCP-Cy5.5

MK-8628 1µM

CD71-PerCP-Cy5.5

Untreated Control

CD71-PerCP-Cy5.5

MK-8628 1µM

CD71-PerCP-Cy5.5

Untreated Control

CD71-PerCP-Cy5.5

MK-8628 1µM

CD71-PerCP-Cy5.5

SSC

-H (x

100

0)

100

150

200

SSC

-H (x

100

0)

50

100

150

200

SSC

-H (x

100

0)

0

50

100

150

200

SSC

-H (x

100

0)

0

50

100

150

200

SSC

-H (x

100

0)

0

50

100

150

200

SSC

-H (x

100

0)

0

50

100

150

200

SSC

-H (x

100

0)

0

50

100

150

200

Naïve CD4+ Naïve CD4+ Naïve CD8+ Naïve CD8+

Naïve CD4+ Naïve CD4+ Naïve CD8+ Naïve CD8+

Memory CD4+ Memory CD4+ Memory CD8+ Memory CD8+

Memory CD4+ Memory CD4+ Memory CD8+ Memory CD8+

101 102 103 104 105 101 102 103 104

101 102 103 104 105

102 103 104 105

101 102 103 104 105 101100 102 103 104 105

101 102 103 104 105

102 103 104

102 103 104 105

101100 102 103 104 105101 102 103 104 105

102 103 104 105

102 103 104 105 102 103 104 105

50

0

50 50

100

150

200

102 103 104 105

102 103 104 105

8.51% 72.91%

45.92% 84.29%

63.55%

78.12%

1.08%

30.87%

84.07% 44.81% 86.36% 35.29%

62.87% 23.26% 47.26% 10.92%

MK-8628 (µM)

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

0

25

50

75

100

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

%C

D25

+ am

ong

Naï

ve C

D4+

%C

D25

+ am

ong

Mem

ory

CD

4+

%C

D25

+ am

ong

Mem

ory

CD

8+%

CD

71+

amon

gN

aïve

CD

8+

%C

D71

+ am

ong

Naï

ve C

D4+

%C

D71

+ am

ong

Mem

ory

CD

4+

%C

D71

+ am

ong

Mem

ory

CD

8+%

CD

25+

amon

gN

aïve

CD

8+

1 0.1 0.0

1 0 1 0.1 0.0

1 0

1 0.1 0.0

1 0 1 0.1 0.0

1 0

MK-8628 (µM)

MK-8628 (µM)

1 0.1 0.0

1 0 1 0.1 0.0

1 0

10.1

0.0

1 0 10.1

0.0

1 0

MK-8628 (µM)

MK-8628 (µM)

MK-8628 (µM)

A B

C D

MK-8628 (µM) MK-8628 (µM)

********n.s.

********

*

****** n.s.

********n.s.

****n.s.n.s.

****** n.s.

********n.s.

******

*

Supplemental Figure 3. MK-8628 suppresses activation in naïve and memory T lymphocyte subsets. (A) Flow cytometry contour plots of IL-2R alpha chain (CD25) expression in anti-CD3/CD28 mAb stimulated naïve (CD3+CD45RA+CCR7+) and memory (CD3+CD45RO+CD45RA-CCR7-) T lymphocyte subsets treated with 1µM, 0.1µM or untreated controls after 24 hours. (B) Dose response depicting CD25 expression in naïve and memory T lymphocyte subsets as shown in A. (C) Flow cytometry contour plots of transferrin receptor (CD71) expression in anti-CD3/CD28 mAb stimulated naïve and memory T lymphocyte subsets treated with 1µM, 0.1µM or untreated controls after 24 hours. (D) Dose response depicting CD71 expression in naïve and memory T lymphocyte subsets as shown in C. Data are from one experimental representative of at least three independent experiments and represent triplicate wells; small horizontal red lines indicate the mean. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA with post-test analysis.

Supp4

0

50

100

150

MK-8628 (µM)

0

5000

10000

15000

0

500

1000

1500

2000

010203040506070

0

5

10

15

20

0

5

10

15

20

0

20

40

60

80

0

1

2

3

4

5

0

6

12

18

0

2000

4000

6000

0

7000

14000

21000

28000

35000

10 .1

0 .01 010 .1

0 .01 00

2000

4000

6000

8000

10000

0

4

8

12

0

10

20

30

0

10

20

30

40

0

1

2

3

4

5

0

2

4

6

8

0

5

10

15

20

25

IFN

γ (p

g/m

L)%

IFN

γ+ a

mon

gN

aïve

CD

4+%

IFN

γ+ a

mon

gN

aïve

CD

8+

%IF

Nγ+

am

ong

Mem

ory

CD

4+%

IFN

γ+ a

mon

gM

emor

y C

D8+

%IL

-2+

amon

gN

aïve

CD

4+%

IL-2

+ am

ong

Naï

ve C

D8+

%IL

-2+

amon

gM

emor

y C

D4+

%IL

-2+

amon

gM

emor

y C

D8+

%TN

Fα+

amon

gN

aïve

CD

4+%

TNFα

+ am

ong

Naï

ve C

D8+

%TN

Fα+

amon

gM

emor

y C

D4+

%TN

Fα+

amon

gM

emor

y C

D8+

IFN

γ (p

g/m

L)

IL-2

(pg/

mL)

IL-2

(pg/

mL)

TNFα

(pg/

mL)

TNFα

(pg/

mL)

10 .1

0 .01 010 .1

0 .01 010 .1

0 .01 010 .1

0 .01 0

10 .1

0 .01 010 .1

0 .01 010 .1

0 .01 010 .1

0 .01 010 .1

0 .01 010 .1

0 .01 0

10 .1

0 .01 010 .1

0 .01 010 .1

0 .01 010 .1

0 .01 010 .1

0 .01 010 .1

0 .01 0

MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM)

MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM)

MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM)

A B

C D

E Fn.s. n.s. n.s.

********

*****

n.s.*

******** n.s.

********

********

********

********

*******

******

********

****

****n.s.n.s.

n.s.n.s.

********

*****

*******

********

********

******

****

****n.s.

n.s. ********

* ********

****

****

******

****

CD

4-BU

V395

69.94%

2-NBDG

CD

4-BU

V395

Untreated Control

2-NBDG

CD

8-BU

V395

63.40%MK-8628 0.19µM

2-NBDG

CD

8-BU

V395

58.47%

5.00

1.67

0.56

0.19

0.06

0.02 0

20

40

60

80

100

1000

2000

3000

4000

5000

2-N

BDG

MFI

20

40

60

80

1000

2000

3000

4000

5000

2-N

BDG

MFI

CD4_MK-8628

CD4_Control

CD8_MK-8628

CD8_Control

10

10.5

11

11.5

12

Mea

n Ex

pres

sion

(Log

2)

SLC2A1 - GLUT1

11.5

12

12.5

13

Mea

n Ex

pres

sion

(Log

2)

HIF1A

5.00

1.67

0.56

0.19

0.06

0.02 0

5.00

1.67

0.56

0.19

0.06

0.02 0

5.00

1.67

0.56

0.19

0.06

0.02 0

MK-8628 (µM) MK-8628 (µM) MK-8628 (µM) MK-8628 (µM)

% 2

NB

DG

+am

ong

ng C

D4+

%2-

NBD

G+

amon

g ng

CD

8+

105

104

103

102

101 102 103 104 105 101 102 103 104 105 101 102 103 104 105 101 102 103 104 105

105

104

103

102

105

104

103

102

105

104

103

102

Untreated ControlG H

I J

M N

K L

2-NBDG

CD4_MK-8628

CD4_Control

CD8_MK-8628

CD8_Control

********

*****

n.s.n.s.

********

********

*****

********

********

n.s.n.s.

********

********

********

IC50=0.67 IC50=0.10 IC50=0.49 IC50=0.20

58.38%MK-8628 0.19µM

Supplemental Figure 4. MK-8628 suppresses cytokine production in naïve and memory T lymphocyte subsets and dysregulates naïve T cell glycolysis. (A-B) Meso scale discovery (MSD) analysis of IFN-γ, IL-2, and TNF-α levels in supernatants of anti-CD3/CD28 mAb stimulated naïve (CD3+CD45RA+ CCR7+) and memory (CD3+CD45RO+CD45RA-CCR7-) CD4+ (A) or CD8+ T lymphocytes (B) treated with 1µM, 0.1µM or untreated controls after 24 hours. (C-F) Intracellular flow cytometry assessment of IFN-γ, IL-2, and TNF-α expression in anti-CD3/CD28 mAb stimulated naïve and memory CD4+ (C-D) and CD8+ T lymphocytes (E-F) treated with 1µM, 0.1µM or untreated controls after 24 hours. (G-H) Flow cytometry contour plots of 2-NBDG expression in anti-CD3/CD28 mAb stimulated naïve CD4+ and CD8+ T lymphocytes treated with 0.19µM MK-8628 or untreated controls after 24 hours. (I-L) Dose response depicting frequency and geometric mean fluorescent intensity (MFI) of 2-NBDG in naïve CD4+ and CD8+ T lymphocytes as shown in G-H. (M-N) Quantitative PCR analysis of Hif1a and Glut1 transcripts in anti-CD3/CD28 mAb stimulated naïve CD4+ and CD8+ T lymphocytes treated with 200nM MK-8628 for 24 hours, presented relative to expression in untreated controls. Data are from one experimental representative of at least three independent experiments and represent triplicate wells (A-H) or from three independent experiments where each symbol represents one of four donors (M-L); small horizontal red lines indicate the mean. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA with post-test analysis (A-F, I-L) or unpaired two-tailed Student t-test (M-N).