CB-839, a selective glutaminase inhibitor,
synergizes with signaling pathway inhibitors
to produce an anti-tumor effect in cell lines
and tumor xenografts
Francesco Parlati, Ph.D.
Calithera Biosciences
South San Francisco, California
Disclosure Information
AACR Annual Meeting 2015
Francesco Parlati
• I have the following financial relationships to disclose:
Stockholder in Calithera Biosciences
Employee of Calithera Biosciences
• I will not discuss off label use and/or investigational use in my
presentation.
Tumor Cells Have Increased Glutamine and
Glucose Consumption
Normal Cell
Lactate
Tumor Cell
GLUCOSE
Pyruvate
TCA
Cycle
Lactate
GLUCOSE
Pyruvate
Energy
Building
Blocks
for Cell
Growth
Energy
Building
Blocks
for Cell
Growth
TCA
Cycle
a-KG
a-KG
Glutamate
Glutamate
Glutaminase
Glutaminase
GLUTAMINE
GLUTAMINE
CB-839
CB-839 Suppresses Metabolic Pathways
Downstream of Glutamate
NSCLC cell lines
CB-839 (1 µM)
Timepoint: 24 h
glutamine
fumarate
Glutaminase
CB-839
malate
TCA
cycle
glutamate
a-ketoglutarate
(a-KG)
glutathione
G lu t a m a t e
F o ld C h a n g e in M e t a b o lit e
G lu t a m in e
oxaloacetate
aspartate
glutamate
a-KG
citrate
GSH
M a la te
C it r a t e
A s p a rta te
16
16
16
16
16
16
8
8
8
8
8
8
4
4
4
4
4
4
2
2
2
2
2
2
1
1
1
1
1
1
0 .5
0 .5
0 .5
0 .5
0 .5
0 .5
0 .2 5
0 .2 5
0 .2 5
0 .2 5
0 .2 5
0 .2 5
0 .1 2 5
0 .1 2 5
0 .1 2 5
0 .1 2 5
0 .1 2 5
0 .1 2 5
0 .0 6 2 5
0 .0 6 2 5
0 .0 6 2 5
0 .0 6 2 5
0 .0 6 2 5
0 .0 6 2 5
C e ll G r o w t h
( % p la t e d )
-1 0 0
Hs 578T
SU M1 4 9 P T
HCC38
M X -1
HCC1806
M C F -1 0 A
HCC70
M D A -M B -2 3 1
B T-2 0
B T-5 4 9
M D A -M B -4 3 6
H M C -1 -8
SU M1 5 9 P T
HCC1395
HCC1187
H s 6 0 6 .T
M D A -M B -4 6 8
H s 7 3 9 .T
HCC1937
M D A -M B -4 5 3
H s 3 4 3 .T
DU4475
J I M T-1
HCC1954
M C F -7
HCC1428
A U -5 6 5
T-4 7 D
S K -B R -3
M D A -M B -1 7 5 -V I I
TU H R 1 0 TK B
A704
7 8 6 -0
C a k i -1
A498
7 6 9 -P
RCC GH
R C C F G -1
C a k i -2
ACHN
RCC ER
W T-C L S 1
J M U -R TK 2
S K -N E P -1
G402
G401
B F TC -9 0 9
A549
H2023
H1568
H358
H2030
H2122
H2347
H23
H1703
HCC827
H441
H661
H1437
H647
H1650
H1975
H226
H2073
H1563
H1299
H2085
H838
H1693
C h a G o -K 1
H727
211H
H28
H2452
H2052
R P M I8 2 2 6
M O L P -2
P C M6
E JM
K M S -2 0
K M S -1 1
MM1 R
S K -M M -2
MM1 S
K M S -2 7
K M S -2 1 B M
IM 9
U266
K M S -3 4
K M M -1
H929
M O L P -8
K M S -2 8 B M
d e l ta -4 7
K M S -1 2 -B M
L P -1
K M S -1 2 P E
K M S -2 8 P E
H uns1
O P M -2
A M O -1
K M S -2 6
L -3 6 3
JJN3
S U -D H L -6
DOHH2
N U -D U L -1
S U -D H L -1 0
M IN O
S U -D H L -4
G r a n ta -5 1 9
J V M -2
HUT 102
DB
RL
W S U -D L C L 2
U937
S U P -B 1 5
S U P -T1
BDCM
REH
CB-839 Has Anti-Proliferative Activity in Multiple
Cancer Types
B re a s t
T r ip le - n e g a t iv e
K id n e y
ER+ or
H e r2 +
non-
M e s o t h e lio m a
RCC
RCC
NSCLC
Lym phom a
M y e lo m a
ALL
100
( % c o n t r o l)
50
0
C e ll D e a t h
-5 0
C e ll L in e
1 mM CB-839
72 h Treatment
Renal Clear Cell Carcinoma Cells are Sensitive to
Glutaminase Inhibition
100
C e ll G r o w t h
50
( % c o n t r o l)
0
C e ll D e a th
-5 0
B F T C -9 0 9
G 401
G 402
R C C ER
ACHN
S K -N E P -1
J M U -R T K 2
C a k i -2
W T -C L S 1
R C C F G -1
RCC GH
7 6 9 -P
A498
C a k i -1
7 8 6 -0
A704
-1 0 0
T UHR10T KB
( % p la t e d )
R e n a l C le a r C e ll C a r c in o m a
K id n e y T u m o r (n o n -R C C )
1 mM CB-839
72 h Treatment
Cross Talk Between Signal Transduction
Pathways and Cancer Metabolism
Growth Factor
Receptor
CB-839
Ras/Raf
Pathway
PI3K/mTOR
Pathway
↑ Glutamine
Utilization
↑ Glucose
Utilization
MTOR Signaling is Downregulated by CB-839 in
Sensitive RCC Cells
Sensitive Cells
-
+ -
+ -
+ -
Resistant Cells
+ -
+ -
+ -
+ -
+ -
+ -
( - ) C B -8 3 9
3
(+ ) C B -8 3 9
2
1
R e n a l C e ll C a rc in o m a
K id n e y T u m o r (n o n -R C C )
 phospho S6
0
( n o r m a liz e d A .U .)
(n o r m a liz e d to to ta l S 6 )
p h o s p h o S 6 ( S e r 2 4 0 /2 4 4 )
phospho S6
(Ser240/244)
total S6
0 .0
p = 0.026*
- 0 .5
- 1 .0
- 1 .5
- 2 .0
S e n s it iv e R e s is t a n t
+ -
+ -
+
CB-839
(1 mM; 24h)
MTOR Signaling is Downregulated by CB-839 in
Sensitive RCC Cells
Sensitive Cells
-
+ -
+ -
+ -
Resistant Cells
+ -
+ -
+ -
+ -
+ -
+ -
phospho 4E-BP1
(Ser65)
1 .5
( - ) C B -8 3 9
(+ ) C B -8 3 9
1 .0
0 .5
R e n a l C e ll C a rc in o m a
K id n e y T u m o r (n o n -R C C )
( n o r m a liz e d A .U .)
0 .0
 p h o s p h o 4 E -B P 1 1
( t n o r m a l iz e d t o t o t a l 4 E - B P 1 )
p h o s p h o 4 E -B P 1 (S e r 6 5 )
total 4E-BP1
0 .0
p = 0.04*
- 0 .2
- 0 .4
- 0 .6
- 0 .8
S e n s it iv e R e s is t a n t
+ -
+ -
+
CB-839
(1 mM; 24h)
Cross Talk Between Signal Transduction
Pathways and Cancer Metabolism
Growth Factor
Receptor
CB-839
Ras/Raf
Pathway
PI3K/mTOR
Pathway
↑ Glutamine
Utilization
↑ Glucose
Utilization
Everolimus
(mTOR inhibitor)
Synergistic Anti-Proliferative Activity of CB-839
and Everolimus in Renal Clear Cell Carcinoma Cells
1 .0
72 h
Treatment
0 .8
phospho 4E-BP1
(Ser65)
0 .6
0 .4
300
100
0.38
150
50
0.33
G lu c o s e
75
25
0.20
37.5
3.1
0.36
18.8
1.6
0.19
CB-839 (nM)
Everolimus (nM)
Mixture (Comb. Index)
G lu t a m in e
C o n s u m p t io n C o n s u m p t io n
E x t r a c e ll u la r
O xygen
A c id if ic a t io n R a te
C o n s u m p tio n R a te
1 .0
C B -8 3 9
0 .5
E v e ro lim u s
C om bo
0 .0
1 .0
B a s e lin e O C R
DMSO
B a s lin e E C A R
1 .0
(r e la t iv e t o D M S O )
( r e l a t iv e t o D M S O )
N u t r ie n t C o n s u m p t io n
4h Treatment
Plating Density
0 .5
0 .0
24 h Treatment
(r e la t iv e t o D M S O )
C e ll S u r v iv a l
( r e a l t iv e t o D M S O )
ACHN
0 .5
0 .0
24 h Treatment
24 h Treatment
( % c o n t r o l)
( % p la t e d )
A5 4 9
H2 0 2 3
H1 5 6 8
H3 5 8
H2 0 3 0
H2 1 2 2
H2 3 4 7
H2 3
H1 7 0 3
HCC8 2 7
H4 4 1
H6 6 1
H1 4 3 7
H6 4 7
H1 6 5 0
H1 9 7 5
H2 2 6
H2 0 7 3
H1 5 6 3
H1 2 9 9
H2 0 8 5
H8 3 8
H1 6 9 3
C h a Go -K 1
Anti-Proliferative Activity of CB-839 in NSCLC
sensitive
resistant
100
C e ll G r o w t h
50
0
C e ll D e a th
-5 0
1 mM CB-839
72 h Treatment
KRAS and EGFR Mutated Tumor Cells are More
Sensitive to Glutaminase Inhibition with CB-839
sensitive
resistant
100
KRAS
KRAS
C e ll G r o w t h
( % c o n t r o l)
EG FR
50
m ut
am p
m ut
W ild T y p e
0
C e ll D e a th
-5 0
A5 4 9
H2 0 2 3
H1 5 6 8
H3 5 8
H2 0 3 0
H2 1 2 2
H2 3 4 7
H2 3
H1 7 0 3
HCC8 2 7
H4 4 1
H6 6 1
H1 4 3 7
H6 4 7
H1 6 5 0
H1 9 7 5
H2 2 6
H2 0 7 3
H1 5 6 3
H1 2 9 9
H2 0 8 5
H8 3 8
H1 6 9 3
C h a Go -K 1
( % p la t e d )
Correlation Between CB-839
Sensitivity & KRAS/EGFR Status
Chi-square
P=0.005**
t test
P=0.026*
1 mM CB-839
72 h Treatment
Cross Talk Between Signal Transduction
Pathways and Cancer Metabolism
Erlotinib
(EGFR inhibitor)
Selumetinib
(Mek inhibitor)
CB-839
Growth Factor
Receptor
Ras/Raf
Pathway
PI3K/mTOR
Pathway
↑ Glutamine
Utilization
↑ Glucose
Utilization
CB-839 is Synergistic with Selumetinib in
KRASmut Cells
H 2 1 2 2 X e n o g ra ft
72 h
Treatment
1500
V e h ic le
C B - 8 3 9 T G I= 4 6 %
3
T u m o r V o lu m e ( m m )
H2122
0 .8
0 .6
0 .4
Plating Density
0 .2
0 .0
100
1000
0.30
50
500
0.25
25
250
0.40
12.5
125
0.56
6.25
62.5
0.41
CB-839 (nM)
Selumetinib (nM)
Mixture (Comb. Index)
S e lu m e tin ib T G I= 4 9 %
C o m b o T G I= 7 8 %
1000
***
G lu c o s e
**
S ta rt
*
0
5
C o n s u m p t io n C o n s u m p t io n
10
15
20
25
S e lu m e tin ib
C om bo
0 .5
0 .0
24 h Treatment
( r e l a t iv e t o D M S O )
C B -8 3 9
GSH
1 .0
C e llu la r M e ta b o lite
( r e l a t iv e t o D M S O )
M a la te
DMSO
v s . S e lu m e t in ib
D o s in g
500
G lu t a m in e
1 .0
v s . C B -8 3 9
****
D a y s P o s t - Im p la n t
N u t r ie n t C o n s u m p t io n
C e ll S u r v iv a l
( r e la t iv e t o D M S O )
1 .0
0 .5
0 .0
BQL
4 h Treatment
v s . C B -8 3 9
v s . S e lu m e t in ib
CB-839 is Synergistic with Erlotinib in
EGFRmut Cells
1 .0
72 h
Treatment
0 .8
0 .6
0 .4
Plating Density
0 .2
0 .0
500
25
0.67
500
12.5
0.45
G lu c o s e
300
12.5
0.43
100
6.25
0.44
100
3.125
0.81
CB-839 (nM)
Erlotinib (nM)
Mixture (Comb. Index)
G lu t a m in e
C o n s u m p t io n C o n s u m p t io n
M a la te
E r lo tin ib
C om bo
0 .5
0 .0
24 h Treatment
( r e l a t iv e t o D M S O )
C B -8 3 9
C e llu la r M e ta b o lite
1 .0
GSH
1 .0
DMSO
( r e l a t iv e t o D M S O )
N u t r ie n t C o n s u m p t io n
C e ll S u r v iv a l
(r e la t iv e t o D M S O )
HCC827
0 .5
0 .0
24 h Treatment
CB-839 Enhaces the Anti-Tumor Effect of
Erlotinib in an Erlotinib Resistant Model
M a la te
72 h
Treatment
0 .6
Plating Density
0 .4
62.5
2,500
0.27
62.5
1000
0.43
62.5
500
0.39
62.5
250
0.46
( r e l a t iv e t o D M S O )
0 .8
CB-839 (nM)
Erlotinib (nM)
Mixture (Comb. Index)
0 .5
0 .0
24 h Treatment
H 1 6 5 0 X e n o g ra ft
V e h ic le
3
62.5
5,000
0.23
1000
C B -8 3 9
E r lo tin ib
T G I= 2 6 %
C om bo
750
n .s .
D o s in g
500
S ta r t
T G I= 6 6 %
***
***
P < 0 .0 0 1
250
TR = 11%
0
0
5
10
15
20
GSH
1 .0
C e llu la r M e ta b o lite
H1650
T u m o r V o lu m e ( m m )
C e ll S u r v iv a l
( r e a l t iv e t o D M S O )
1 .0
25
****
Conclusions
• CB-839 is anti-proliferative in multiple tumor types and suppresses mTOR
pathway signaling
• Clear cell RCC lines are sensitive to CB-839
• KRAS and EGFR mutant NSCLC lines show enhanced sensitivity to CB-839
• CB-839 in combination with signal transduction inhibition offers a novel
therapeutic strategy for the treatment of cancer:
– Everolimus in RCC
– Mek inhibitor in KRAS mutant NSCLC
– EGFR inhibitor in EGFR mutant NSCLC
Acknowledgements
Biology
Francesco Parlati
Mirna Rodriguez
Melissa Works
Andy MacKinnon
Winter Zhang
Ethan Emberley
Susanne Steggerda
Alison Pan
Susan Demo
Chemistry
DMPK
Matthew Gross
Julie Janes
Jim Li
Guy Laidig
Lijing Chen
Tim Stanton
Weiqun Le
Tracy Wang
Jing Zhang
Frances Zhou
Clinical
Pharm. Dev.
Keith Orford
Evan Lewis
Peter Shwoenek
Scientific
Management Team
Pharmacology
Susan Molineaux
Mark Bennett
Eric Sjogren
Chris Molineaux