Anti-Tumor Activity of Glutaminase Inhibitor CB-839 in ... · Anti-Tumor Activity of Glutaminase...

Anti-Tumor Activity of Glutaminase Inhibitor CB-839 in Solid Tumor Malignancies

Francesco Parlati, Ph.D.Calithera Biosciences

Keystone Tumor Metabolism January 25, 2018

Disclosure

• I am an employee and shareholder at Calithera Biosciences

Agenda

• Introduction to CB-839

• CB-839 combination with signal transduction inhibitors

• CB-839 combination with taxol in TNBC

• Novel combination strategies with CB-839

– CDK4/6 and PARP inhibitors

Agenda




• Novel combination strategies


Cancer vs. Normal Cell Metabolism

GLUCOSE

Pyruvate

α-KG

Glutamate

GLUTAMINE

Lactate

TCACycle

Mitochondrion

Normal Cells Cancer Cells

Intermediates for biosynthesis

Glutathione

Glutaminase

GLUCOSE

Pyruvate

α-KG

Glutamate

GLUTAMINE

Lactate

TCACycle

Glutaminase

Mitochondrion

CB-839

aspartate

a-ketoglutarate(a-KG)

citrate

fumaratemalate

oxaloacetateTCAcycleglutamate

a-KGglutamate

glutamine

Glutaminase

CB-839 Suppresses Metabolic Pathways Downstream of Glutamate in vitro

glutathione

0 . 0 6 2 5

0 . 1 2 5

0 . 2 5

0 . 5

1

2

4

8

1 6

G l u t a m i n e

Fo

ld C

ha

ng

e i

n M

eta

bo

lite

0 . 0 6 2 5

0 . 1 2 5

0 . 2 5

0 . 5

1

2

4

8

1 6

G l u t a m a t e

0 . 0 6 2 5

0 . 1 2 5

0 . 2 5

0 . 5

1

2

4

8

1 6

G S H

0 . 0 6 2 5

0 . 1 2 5

0 . 2 5

0 . 5

1

2

4

8

1 6

M a l a t e

0 . 0 6 2 5

0 . 1 2 5

0 . 2 5

0 . 5

1

2

4

8

1 6

C i t r a t e

0 . 0 6 2 5

0 . 1 2 5

0 . 2 5

0 . 5

1

2

4

8

1 6

A s p a r t a t e

6

CB-839

NSCLC*24 h treatment 1 mM CB-839

* Average of 4 cell lines

- 3

- 2

- 1

0

1

2

3

4

5

6

mR

NA

ex

pr

es

sio

n (

log

2)

p = 0 . 0 7 * * * * * * * * * *

T N B C c c R C C N S C L C m e l a n o m a

no

rm

al

tum

or

no

rm

al

tum

or

no

rm

al

tum

or

no

rm

al

tum

or

Glutaminase is Overexpressed in Solid Tumors

Expression of GAC isoform in Clinical Samples

mRNA levels were obtained from Compendia Bioscience™ Translational Bioinformatics Services (Life Technologies, Ann Arbor, MI)

CB-839 Has Broad Anti-tumor Activity Across Multiple Tumor Cell Lines

Panel of Tumor Cell Lines Treated with CB-839 (1 mM) for 72 Hours• Effects of CB-839 on the growth/death of a panel of cancer cell lines

– % cell growth compared to untreated cells– % cell death compared to starting cell number

Incr

easi

ng

effe

ct o

f C

B-8

39

0

25

50

75

100

no drugeffect

completegrowthsuppression

completekilling

% c

ell g

row

th%

cel

l dea

th

Triple Negative Breast Cancer Non-Small Cell Lung CancerRenal Cell Carcinoma Melanoma

High Levels of GLS Expression in PatientsOn-study or Archival Tumor Samples

Non-Small Cell Lung Cancer

Triple Negative Breast Cancer

Renal CellCarcinoma

Melanoma

G l u t a m i n a s e E x p r e s s i o n ( I H C )H

Sc

or

e

TN

BC

RC

C

NS

CL C

me

l an

om

a

0

5 0

1 0 0

1 5 0

2 0 0

2 5 0

3 0 0h i g h e s t

e x p r e s s i o n

l o w e s t

e x p r e s s i o n

Tumor FFPE stained with a-GLS antibody

[ C B - 8 3 9 @ 4 h ] ( n g / m L )

% G

lut

am

ina

se

Ac

tiv

ity

1 1 0 1 0 0 1 0 0 0

0

2 0

4 0

6 0

8 0

1 0 01 0 0 T I D

1 5 0 T I D

2 5 0 T I D

4 0 0 T I D

6 0 0 T I D

8 0 0 T I D

e x v i v o p l a t e l e t

d o s e r e s p o n s e

8 0 0 B I D

6 0 0 B I D

L L O Q

CB-839 Monotherapy in Patients

• Well tolerated at active doses

– MTD not reached

– 800 mg PO BID selected as RP2D

• Clear PK/PD relationship

– Glutaminase inhibition tested in patients with solid tumors (n=88)

– Sustained and near-complete inhibition of glutaminase in platelets and tumors

• CB-839 monotherapy was active in RCC pts (n=21)

– 1 PR; on study 356 days

– 52% SD, 2 longest ongoing at 25 mo and 15 mo

Tumor GIST NSCLC colon meso RCC

662 1384 1945 2352 11530

C1D15 AUC (0-8h) (ng*hr/mL)

Approximately 3 weeks on study drug

Glu

ta

min

as

e A

ct

ivit

y

(n

mo

l/m

in/

mg

pr

ot

ein

)

5

1 0

1 5

2 0

2 5

3 0

3 5

- 8 6 %

- 7 5 %

- 8 4 %- 5 7 %

U n i n h i b i t e d

I n h i b i t e d

- 9 6 %

C1D1 4 hours post dose

Platelets

Pharmacodynamic Glutaminase Inhibition

Tumors

Meric-Bernstam F. et. al, European Journal of Cancer, Vol. 69, S12–S13

Agenda






CB-839 is Synergistic with Growth Factor Signaling Pathway Inhibitors• Signal Transduction inhibitors indirectly decrease glucose and glutamine utilization

• CB-839 is synergistic with multiple growth factor signaling pathway inhibitors

CB-839

Erlotinib(EGFRi)

Everolimus(mTORi)

= Inhibition

Growth Factor Receptor

Ras/RafPathway

PI3K/mTOR Pathway

↑ Glutamine Utilization

↑ Glucose Utilization

Metabolic Rewiring Supporting Tumor Growth

Crizotinib(ALKi/ROSi)

12

Cabozantinib(VEGFRi/METi)

Synergistic Anti-Proliferative Activity of CB-839 and Everolimus in Renal Clear Cell Carcinoma

0 .4

0 .6

0 .8

1 .0

Ce

ll S

urv

iva

l

(re

alt

ive

to

DM

SO

)

A C H N

CB-839 (nM)Everolimus (nM)Mixture (Comb. Index)

3001000.38

18.81.6

0.19

15050

0.33

7525

0.20

37.53.1

0.36

Plating Density

72 hTreatment

0 .0

0 .5

1 .0

G lu c o s e

C o n s u m p tio n

Nu

trie

nt

Co

ns

um

pti

on

(re

lati

ve

to

DM

SO

)

G lu ta m in e

C o n s u m p tio n

D M S O

C B -8 3 9

E v e ro lim u s

C o m b o

24 h Treatment

CB-839 Enhances the Anti-tumor Activity of Signal Transduction Inhibitors

D a y P o s t I m p l a n t

Tu

mo

r V

olu

me

(m

m3

)

8 1 6 2 4 3 2

3 0 0

6 0 0

9 0 0

1 2 0 0

1 5 0 0

V e h i c l e

C B - 8 3 9

C o m b o

E v e r o l i m u s

D o s i n g

s t a r t

* * *


Tu

mo

r V

olu

me

(m

m3

)

1 0 2 0 3 0 4 0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

1 2 0 0

1 4 0 0

V e h i c l e

C B - 8 3 9

C o m b o

C a b o z a n t in ib

*D o s i n g

s t a r t

D a y s P o s t I m p l a n t

Tu

mo

r V

olu

me

(m

m3

)

2 0 3 0 4 0 5 0

2 5 0

5 0 0

7 5 0

1 0 0 0V e h i c l e

C B - 8 3 9

C r i z o t in ib

C o m b o

* *

D o s i n g

s t a r t


Tu

mo

r V

olu

me

(m

m3

)

0 2 5 5 0 7 5 1 0 0 1 2 5

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

V e h i c l e

C B - 8 3 9

E r lo t i n ib

C o m b o

* * * *

D o s i n g

s t a r t

Everolimus Combination Cabozantinib Combination

Crizotinib Combination Erlotinib Combination

RCCCaki-1 xenograft

RCCCaki-1 xenograft

NSCLCH2228 xenograft

NSCLCHCC827 xenograft

CB-839 + Everolimus in RCC

Phase 1b Dose escalation and expansion

15

Escalation in Advanced RCC

Expansion in clear cell and papillary

RCC

CB-839 + Everolimus in RCC Clinical Outcomes in Phase 1b

N=17 patients (14 ccRCC and 3 papillary RCC)

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

Median PFS is 8.5 moSu

rviv

al P

rob

abili

ty (

%)

Months

Number at risk: 16 10 8 5 4 2 1 0

Median PFS (95% C.I.)8.5 mo (5.3 - 11 mo)

^*

Ever

olim

us

med

ian

PFS

^ Motzer et al, NEJM 2015* Choueiri et al, NEJM 2015

Meric-Bernstam F. et. al, European Journal of Cancer, Vol. 69, S12–S13

Clinical Studies in RCC with CB-839

• Phase 2: CB-839 + Everolimus vs. Everolimus

– Placebo control double blind study in RCC patients

• Phase 1b: CB-839 + Cabozantinib in RCC patients

• Phase 1/2: CB-839 + Nivolumab in RCC patients

Combination Partner

Nivolumab

Ph 2: Renal Cell Carcinoma Everolimus

Ph 1b: Renal Cell Carcinoma Cabozantinib

Ph 1/2: Renal Cell Carcinoma

Agenda






CB-839 Has Broad Anti-tumor Activity Across Multiple Tumor Cell Lines

Panel of Tumor Cell Lines Treated with CB-839 (1 mM) for 72 Hours• Effects of CB-839 on the growth/death of a panel of cancer cell lines

– % cell growth compared to untreated cells– % cell death compared to starting cell number

Incr

easi

ng

effe

ct o

f C

B-8

39

0

2 5

5 0

7 5

1 0 0

n o d r u g

e f f e c t

c o m p l e t e

g r o w t h

s u p p r e s s i o n

c o m p l e t e

k i l l i n g

% c

ell

gr

ow

th

% c

ell

de

at

h

T N B C N S C L CR C C M e l a n o m a

CB-839 in TNBC PatientsMonotherapy

• CB-839 was well tolerated in TNBC patients

• 19 patients were treated

– 3 patients had stable disease

• 2 patients with stable disease greater than 8 months

CB-839 plus Paclitaxel Exacerbates Cell Cycle Defect in TNBC cells

21

• Paclitaxel targets tubulin and blocks cell cycle progression at mitosis (G2/M-phase) • CB-839 treatment causes cells to accumulate in G1 or S-phase • The combination of CB-839 plus paclitaxel causes cells to accumulate in G2/M or S-phase• Synergy between CB-839 and Paclitaxel in 8 out of 11 TNBC cell lines

DM

SO

CB

- 83

9

Pa

cl i

t ax

el

Co

mb

o

0

2 5

5 0

7 5

1 0 0

J I M T - 1

% C

ell

s

% G 1

% S

% G 2 / M

0 . 0 0

0 . 2 5

0 . 5 0

0 . 7 5

1 . 0 0

J I M T - 1

Re

lativ

e C

ell

Gr

ow

th

CB-839 (nM)Paclitaxel (nM)Combination Index

50050.10

25025.16

12512.5.15

62.56.25.07

31.33.13.11

15.61.56.16

7.80.78.19

00-

CB-839 plus Paclitaxel Shows Enhanced Anti-tumor Activity In Vivo

22

T N B C

J I M T - 1 X e n o g r a f t

S t u d y D a y

Tu

mo

r V

olu

me

(m

m3

)

0 5 1 0 1 5 2 0 2 5

5 0 0

1 0 0 0

1 5 0 0 V e h i c l e

C B - 8 3 9 , B I D

P a c l i t a x e l , I P Q O D x 5

C o m b o

*

v s p a c l i t a x e l

T N B C

J I M T - 1 X e n o g r a f t

S t u d y d a y

Tu

mo

r V

olu

me

(m

m3

)

0 7 1 4 2 1 2 8 3 5

0

2 0 0

4 0 0

6 0 0

8 0 0

P a c l i t a x e l , I V Q O D x 5

C o m b o

* *

* *v s p a c l i t a x e l

v s v e h i c l e

C B - 8 3 9 , B I D

V e h i c l e

CB-839 + Paclitaxel in TNBCStudy Design

23

Advanced TNBC

CB-839 400 – 800 mg BIDPaclitaxel 80 mg/m2 IV D1,8,15 Q28

Expansion in TNBC pts with and without prior taxane in

the metastatic setting

CB-839 + Paclitaxel in TNBCKey Demographics

• Median of 3 prior lines of therapy for metastatic disease

– 35% ≥ 5L therapy

• Prior taxane exposure

– 88% any line of therapy• 51% advanced/metastatic

• 37% adjuvant/neoadjuvant

24

Kalinsky et al. SABCS 2017

CB-839 + Paclitaxel in TNBCBest Change in Target Lesions

25

% c

ha

ng

e i

n t

ar

ge

t t

um

or

s

60

0

80

0

60

0

40

0

80

0

80

0

80

0

80

0

40

0

80

0

80

0

80

0

80

0

80

0

60

0

80

0

40

0

60

0

80

0

80

0

80

0

80

0

60

0

80

0

80

0

40

0

60

0

40

0

80

0

60

0

80

0

60

0

80

0

80

0

60

0

80

0

60

0

80

0

80

0

- 1 0 0

- 8 0

- 6 0

- 4 0

- 2 0

0

2 0

4 0

6 0

8 0

1 0 0

P r i o r T a x a n e

[ A d v / M e t ]

P r i o r T a x a n e

[ N e o / A d j o n l y ]

N o P r i o r

T a x a n e

D o s e ( m g )


Patients were dosed with CB-839 400-800 mg BID and Paclitaxel 80 mg/m2 IV D1,8,15 Q28

CB-839 + Paclitaxel in TNBCResponse Table CB-839 ≥ 600 mg dose

26

Adv/Met^

Neo/Adj only

None

22 16 3

20 14 3

4 (20) 3 (21) 1 (33)

7 (35) 5 (36) 2 (67)

9 (45) 6 (43) 0

11 (55) 8 (57) 3 (100)

4 2 6

Patients:

Total Enrolled (N)

RECIST Evaluable [N (%)]*

PR

SD

PD

DCR (CR + PR + SD)

Lines of Prior Adv/Met Therapy

By Prior Taxane (≥600 mg)

400mg

≥600mg

7 42

7 37

0 8 (22)

3 (43) 14 (38)

4 (57) 15 (41)

3 (43) 22 (59)

3 4

By CB-839 Dose

*pts receiving a post-baseline tumor assessment, discontinued due to drug-related AE, or died due to diseasehaving received 16 days of treatment

^Includes 3 pts that progressed on neo-adjuvant taxane therapy


Molecular Subtyping of TNBC Patients on CB-839 + Pacitaxel Combination

• RNA expression analysis typically used to subtype TNBC tumors:

– TNBC-type:

• Tumor-intrinsic and tumor-extrinsic gene signature

• 6 subtypes identified (LAR, BL1, BL2, M, MSL, IM)

– Stroma-Axis:

• Tumor-extrinsic gene signature

• 4 subtypes identified (T, B, E, D)

3 6 9 1 2 1 8 2 4

T i m e o n S t u d y ( M o n t h s )

P R

S D

P D

Trend between Clinical Benefit and LAR Subtype in TNBC Patients on CB-839 + Pacitaxel

28

Trend between clinical benefit (PR or SD> 4 mo):• LAR subtype (3 of 6 patients w/benefit; 0 of 7 w/o benefit)

• Expression of androgen receptor and several metabolic enzymes

• e.g. TCA cycle, GSH biosynthesis, amino acid metabolism

LAR

LAR

MSL

IM

LAR

M

IM

M

M

BL2

IM

BL2

BL1

BL1

BL1

IM

Clin

ical

Ben

efit

No

Cli

nic

al

Ben

efi

t


TNBC-Type

3 6 9 1 2 1 8 2 4

T i m e o n S t u d y ( M o n t h s )

P R

S D

P D

Trend between Clinical Benefit and Stroma Axis D in TNBC Patients on CB-839 + Paclitaxel

29

Stromal axis D “high” subtype • 5 of 6 pts with “high” stromal D had clinical benefit

– Characterized by expression of collagens and collagen modifying enzymes associated with desmoplasia

– Recent publication linking collagen production by fibroblasts to glutaminolysis

High

High

High

Intermed

High

High

Intermed

Low

Low

Intermed

Intermed

High

Low

Low

Low

Low

Clin

ical

Ben

efit

No

Clin

ical

B

enef

it

Stroma-D Score


SummaryCombination of CB-839 with Paclitaxel

• CB-839 is well tolerated in combination with paclitaxel in TNBC patients

• Clinical benefit demonstrated in heavily pre-treated TNBC population

• Strongest clinical benefit in pts with LAR and/or Desmoplastic Stromal gene expression biomarker signatures

• Phase 2 Study initiated to further evaluate CB-839 + Paclitaxel combination in patients with TNBC

Agenda






Cancer vs. Normal Cell Metabolism

GLUCOSE

Pyruvate

α-KG

Glutamate

GLUTAMINE

Lactate

TCACycle

Mitochondrion

Normal Cells Cancer Cells

Intermediates for biosynthesis

(e.g. nucleotides)

Glutaminase

GLUCOSE

Pyruvate

α-KG

Glutamate

GLUTAMINE

Lactate

TCACycle

Glutaminase

Mitochondrion

CB-839

CB-839 Decreases Nucleotide Pools In Vivo

D a y s P o s t - I m p l a n t

Tu

mo

r V

olu

me

(m

m3

)

1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

V e h i c l e

C B - 8 3 9

D o s i n g

S t a r t

* * * *

A M P

Sc

ale

d I

nte

ns

ity

Ve

hi c

l e

CB

- 83

9

0

2

4

6

* *

G M P

Ve

hi c

l e

CB

- 8

39

0 . 0

0 . 5

1 . 0

1 . 5* *

U M P

Ve

hi c

l e

CB

- 83

9

0

1

2

* *

C M P

Ve

hi c

l e

CB

- 83

9

0

1

2

*

T M P

Ve

hi c

l e

CB

- 83

9

0

1

2

* *

PDn

RPMI-8226 Xenograft Model

aspartate

a-KG citrate

fumaratemalate

oxaloacetateTCAcycleglutamate

glutamine

glutathione

CB-839succinate

nucleotides

DMSO

EdU

Inco

rpo

rati

on

CB-839

DNA Content

CB-839 Induces G1/S Cell Cycle Defect in TNBC cells

34

SUM159PT

Hs 587T

• CB-839 treatment causes cells to accumulate in G1 or S-phase (9 out of 11 cell lines)

Cells were treated with 1 mM CB-839 for 24 hours and labelled with EdU for 2 hours

DM

SO

CB

- 83

9

0

2 5

5 0

7 5

1 0 0

H s 5 8 7 T

G 1 - p h a s e

G 2 / M - p h a s e

S - p h a s e

% p

op

ula

tio

n

DM

SO

CB

83

9

0

2 5

5 0

7 5

1 0 0

S U M 1 5 9 P T

G 1 - P h a s e

G 2 / M - P h a s e

S - p h a s e

% p

op

ula

tio

n

S%

29

20.6

Rationale for Combining CB-839 with CDK4/6 and PARP Inhibitors

• CB-839 decreases in glutamate and aspartate

• Lower aspartate decreases the pool of nucleotides in tumors

• Decreased nucleotides lead to decreased rate of DNA synthesis and increased DNA damage

• We hypothesized that this effect by CB-839 on DNA synthesis and DNA damage may enhance the activity of CDK4/6 and PARP inhibitors

35

CB-839 Potentiates the Activity of CDK 4/6 Inhibitor in TNBC

36

0 . 0

0 . 5

1 . 0

Ce

ll S

ur

viv

al

(r

ela

tiv

e t

o

DM

SO

)

C B - 8 3 9

C o m b o

P a l b o c i c l i b

CB-839 (mM)

CDK4/6 inh. (mM)

plating density Day 0

1

10

.5

5

.25

2.5

.13

1.3

.063

.63

.031

.31

0

0

.0156

.16

HCC1569

DMSO CB-839 (1 mM) Palbo (2.5 mM) Combination

DNA Content

Edu

Inco

rpo

rati

on

M C F 7 ( E R+

b r e a s t c a n c e r )

C D K 4 / 6 i

C B - 8 3 9

C o m b o

1

10

37

CB-839 Enhances the Activity of CDK4/6 Inhibitor in ER+ Breast Cancer


0

.5

5

.25

2.5

.13

1.3

.063

.63

.031

.31

.016

.16

0CB-839 (mM)

Palbociclib (mM)

D a y s P o s t I m p l a n t

Tu

mo

r V

olu

me

(m

m3

)

5 1 0 1 5 2 0 2 5 3 0 3 5 4 0

4 0 0

6 0 0

8 0 0 V e h i c l e

C B - 8 3 9

P a l b o c i c l i b

C o m b o

* * * *

M C F - 7 X e n o g r a f t

No calculated CI values due to lack of dose response

CB-839 Synergizes with PARP Inhibitor in Ovarian and Breast Cancer Cell Lines

38

0 . 0

0 . 5

1 . 0

U W B 1 . 2 8 9

O v a r i a n C a n c e r

Re

lativ

e C

ell

Gr

ow

th

C B - 8 3 9

P A R P i

C o m b o

CB-839 (µM)PARP inh. (µM)Combination Index

.252.5.03

.06

.63

.02

.131.3.04

.03

.31

.04

00-

.02

.16

.02

UMB1.2896 d CellTiterGlo assayPAPR inh.=Niraparib

.01

.08

.03

.004.04.02

0 . 0

0 . 5

1 . 0

H C C 1 3 9 5

T N B C

.252.5

.06

.63.131.3

.03

.3100


.55

110

No calculated CI values due to lack of dose response

HCC13953 d CellTiterGlo assayPAPR inh.=Talazoparib

Summary

• CB-839 is an oral, highly selective inhibitor of GLS with in vitro and in vivo anti-tumor activity

• Glutaminase inhibition has several downstream effects rationalizing novel combinations – Signal transduction inhibitors: everolimus, cabozantinib, crizotinib,

erlotinib

– Cell cycle inhibitors: paclitaxel and CDK4/6 inhibitors

– DNA repair inhibitors: PARP inhibitors

• Phase 1 clinical studies have shown promising clinical activity– Renal cell carcinoma in combination with everolimus

– Triple-negative breast cancer in combination with paclitaxel

Acknowledgements

BiologyEthan Emberley

Clarissa LeeAndy MacKinnonGisele Marguier

Silinda NeouSusanne Steggerda

Sandra Spurlock

PharmacologyMatthew Gross

Jason ChenTony Huang

Amani Makkouk

DMPKWeiqun LiYong Ma

Jing ZhangWinter Zhang

ChemistryEric Sjogren

Jim LiRoland Billedeau

Lijing ChenGuy Laidig

Tim Stanton

Pharm. Dev.Peter Shwonek

Natalija Sotirovska

Clinical TeamKeith OrfordSam Whiting Mark BennettSacha Holland

Yu LiangAlison Pan

Anti-Tumor Activity of Glutaminase Inhibitor CB-839 in ... · Anti-Tumor Activity of Glutaminase...

Documents

Transcript of Anti-Tumor Activity of Glutaminase Inhibitor CB-839 in ... · Anti-Tumor Activity of Glutaminase...