Supplementary Figure 2

Gross et al., “Antitumor Activity of the Glutaminase Inhibitor CB-839 in TNBC”
Supplementary Figure S2
75
50
50
50
25
25
25
0
0
0
55 [39-78]
2000 [1300-3000]
4
10
0
10
00
1
10
0.
1
5
10
0
10
00
10
00
0
1
10
0.5
0.1
0.5
0.0
0
Gluc
0.3
0.2
**
1.0
0.0
0.0
HCC1806 (CB-839)
T47D (DMSO)
0.0
T47D (CB-839)
JIMT-1
100
100
75
75
75
50
50
50
25
25
25
0
0
0
α-
sc
re
o
N
re
sc
N
o
α-
sc
re
o
N
ue
100
ue
αKG
125
KG
125
KG
HCC1806
125
ue
% Cell Growth
MDA-MB-231
B83
9
SO
0
C
M
D
H
0
M
-6
2
D
-6
2
-8
39
-4
4
SO
-4
4
CB
0
-2
6
M
0
-2
SO
Glutamine: EC50 = 8 nM [95% CI: 4-18 nM]
Glutamate: IC50 = 3 nM [95% CI: 1-7 nM]
2
Receptor-positive
Glutamate
6
D
[CB-839] (nM)
2
log2 [Glu]
(nmol/106 cells)
1000
4
TNBC
Glutamate
O
AA
O
G
AA S
H
+
G
SH
0
100
6
4
SO
B83
9
0
6
G
M
10
Receptor-positive
Glutamine
D
5
10
TNBC
Glutamine
C
F
B83
9
10
0
10
00
1
10
1.0
1.5
0.1
***
2.0
10
0
30
[Glutamate]
nmol/106 cells
[Glutamine]
nmol/106 cells
0
******
1.5
[CB-839] (nM)
20
1
**
2.5
***
0.3
Cit
Mal
***
0.4
IC50 (nM) [95% CI]
CB-839
>1000
BPTES
>20000
0.2
1
HCC1806 (DMSO)
10
0.1
2
5
1
0
MDA-MB-231
15
15
2
0
[CB-839] (nM)
20
3
AA
G
O
S
AA
H
+
G
SH
Lactate
0
****
[inhibitor] nM
Fum
****
25
4
C
2
2
***
10
Glut
Asp
5
O
Glucose
***
*
log2 [Gln]
(nmol/106 cells)
4
15
3
(nmol/106 cells)
6
6
E
Metabolite level
8
8
Glu
4
10
10
Gln
T47D
12
12
0.
1
Rate of glucose consumption
or lactate production
(nmol/min/106 cells)
HCC1806
14
1
[inhibitor] nM
IC50 (nM) [95% CI]
CB-839
19 (16-23)
BPTES 2400 [1100-5100]
CB-839
D
0.
[inhibitor] nM
IC50 (nM) [95% CI]
BPTES
C
10
0
10
00
10
00
0
[CB-839] (nM)
Cell count: IC50 = 26 nM [95% CI: 14-45 nM]
CTG: IC50 = 30 nM [95% CI: 24-36 nM]
BLOQ
100
CB-839
BPTES
0
1000
1
10
75
0.
1
0
75
1
5000
0
100
10
2000
100
1
10000
100
0.
4000
125
10
0
10
00
10
00
0
15000
T47D
125
1
20000
6000
MDA-MB-231
125
10
25000
Cell Growth
30000
8000
Cell Count
10000
HCC1806
B
35000
(% of DMSO control)
HCC1806
Cell viability (CTG RFU)
A
Supplementary Figure S2. CB-839 has potent anti-proliferative activity in TNBC cells that is associated
with selective impairment of glutamine utilization. A, anti-proliferative effect of CB-839 on HCC1806
cells treated for 72 hours measured by Cell Titer Glo (CTG; left axis) or by cell counting (right axis). The
mean and SEM of duplicate measurements are plotted; calculated IC50 and 95% CI values are shown.
The dotted line represents the CTG signal or cell number at the time of compound addition. B, cell
proliferation dose-response curves for HCC1806, MDA-MB-231, and T47D cells treated with CB-839 or
BPTES for 72 hours. The mean and SEM of triplicate measurements averaged across at least three
independent experiments (N≥9) are plotted; calculated IC50 and 95% CI values are shown. C, glucose
consumption and lactate production by HCC1806 (left) and T47D cells (right) measured after 1 μM CB839 treatment for 6 hours. Culture medium was analyzed for glucose and lactate with the YSI 2900
Biochemistry Analyzer. The mean and SEM from triplicate measurements are plotted. D, intracellular
metabolite levels measured in HCC1806 and T47D cells treated with DMSO or 1 µM CB-839 for 4 hours.
The level of fumarate in the CB-839 treated HCC1806 cells was below the limit of quantitation (BLOQ) of
~0.01 nmol per 106 cells. The mean and SEM of triplicate measurements are shown and statistical
analysis was done by unpaired t-test: *P ≤ 0.05;**P ≤ 0.01;***P ≤ 0.001;**** P ≤ 0.0001. Gln=glutamine,
Glu=glutamate, Asp=aspartate, Glut=glutathione, Fum=fumarate, Mal=malate, Cit=citrate, Gluc=glucose.
E, dose response curves for intracellular glutamine and glutamate levels measured after treating MDAMB-231 cells with CB-839 for 24 hours. The mean and SEM of duplicate measurements are plotted;
calculated IC50 for glutamate decrease and EC50 for glutamine increase with associated 95% CI values are
shown. F, difference in glutamine concentration between breast cancer cell lines [left, TNBC (N=12);
right, receptor-positive (N=8)] treated with DMSO or CB-839 for 4 hours. G, difference in glutamate
concentration between breast cancer cell lines [left, TNBC (N=12); right, receptor-positive (N=8)]
treated with DMSO or CB-839 for 4 hours. For panels (F) and (G), the mean from duplicate
measurements is plotted. Each cell line is depicted by two symbols (DMSO and CB-839 treated) with a
connecting line showing the magnitude difference between the two treatment groups. The open
symbols in the receptor-positive graphs (right) represent the two basal-like ER-/HER2+ cell lines JIMT-1
and HCC1954. H, anti-proliferative activity of 1 µM CB-839 on MDA-MB-231, HCC1806 and JIMT-1 cell
lines treated for 72 hours in the absence or presence of cell permeable TCA cycle intermediates [alphaketoglutarate (α-KG), oxaloacetate (OAA)] and/or glutathione (GSH). The data is expressed as the
percent proliferation in the presence of CB-839 relative to untreated controls (either DMSO, α-KG alone,
GSH alone, OAA alone, or OAA and GSH). The mean and the SEM of duplicate experiments are plotted.
The dotted line represents the cell signal at the time of compound of addition relative to the DMSO
control at 72 hours.

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