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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).