Efficacy of FASN-selective small molecule inhibitors in ......Tumor Volume Tumor Growth Vehicle...

www.postersession.com

0

200

400

600

800

1000

1200

1400

1600

1800

2000

6 8 10 12 14 16 18 20 22 24 26

Tum

or V

olum

e (m

m3 )

Days After Tumor Inoculation

Vehicle

TVB-3166 30 mg/kg

TVB-3166 60mg/kg

Tumor Growth Tumor Volume

TGI (%): 14 10

•  PIK3CA Q546R mutant •  Adenocarcinoma

Vehicl

e

TVB-316

6 (30

)

TVB-316

6 (60

)0

500

1000

1500

2000

Tum

or G

row

th (%

)

Tumor Volume

TGI (%): 57

Tumor Growth

19 *

0

200

400

600

800

14 17 20 23 26 29 32

Tum

or V

olum

e (m

m3 )

Days after Tumor Inoculation

TVB-3166 60 mg/kg p.o.

TVB-3166 100 mg/kg p.o.

Vehicle

*

Vehic

le

TVB-316

6 30 m

pk

TVB-316

6 100

mpk

-100

0

100

200

300

400

500

600

Tum

or G

row

th (%

)

•  KRAS G12D mutant •  Adenocarcinoma

* p<0.05

Vehicl

e

TVB-3166

(60)

0

200

400

600

800

Tum

or G

row

th (%

)

0

100

200

300

400

500

600

700

800

900

1000

0 2 4 6 8 10 12 14 16 18

Cub

ic m

illim

eter

s

Day after Treatment Start

Vehicle

TVB-3166 (60 mg/kg); po/qd

TGI (%): 87

•  KRAS, EGFR wild type •  Stage IV, brain metastasis

Tumor Volume Tumor Growth

Vehicl

e

TVB-3166

(60)

0

200

400

600

800

Tum

or G

row

th (%

)

0

200

400

600

800

1000

1200

1400

0 2 4 6 8 10 12 14 16 18 20

Cub

ic m

illim

eter

s

Day

Vehicle

TVB-3166 (60 mg/kg); po/qd

Tumor Volume For CTG-0160

TGI (%): 50

Tumor Volume Tumor Growth

•  KRAS, EGFR wild type •  MET mutant •  mTOR over-expressing •  PTEN null •  Squamous cell carcinoma

Conclusions and Status

Efficacy of FASN-selective small molecule inhibitors in preclinical tumor models Timothy S. Heuer1, Richard Ventura1, Joanna Waszczuk1, Kasia Mordec1, Julie Lai1, Russell Johnson1, Lily Hu1, Haiying Cai1, Allan Wagman1, Steve Smith1, Douglas Buckley1,

Stanley T. Parish2, Elizabeth Bruckheimer2, and George Kemble1 3-V Biosciences1, Menlo Park, CA, and Champions Oncology2, Baltimore, MD

Introduction Results

Metabolic and Signaling Pathway Interaction

•  3-V Biosciences lead, oral FASN inhibitor has initiated Phase I clinical trails for the treatment of solid tumors

•  Fatty acid synthase (FASN) catalyzes the synthesis of palmitate from acetyl-CoA, malonyl-CoA, and NADPH

•  Palmitate and palmitate-derived lipids function in vital cellular processes such as energy metabolism, and cellular membrane biosynthesis

•  Palmitate is conjugated directly to specific proteins as a mechanism to affect protein localization and activation

•  FASN tumor expression has been found to be increased in a stage-dependent manner with high expression associated with diminished patient survival

•  FASN activity promotes the tumorigenic capacity of cells by multiple mechanisms including enhanced macromolecular biosynthesis and glucose metabolism, cell growth and survival signal transduction, cellular stress response, and resistance to chemotherapeutic agents

•  FASN inhibition can restore sensitivity to chemotherapeutics •  In vitro and in vivo studies in preclinical tumor models demonstrate that FASN

inhibition reduces tumor cell proliferation and induces apoptosis •  Preclinical studies have discovered biomarker candidates and provide insight into

FASN inhibition anti-tumor mechanisms of action

Lipid Regulation of Protein Function •  Membrane-associated lipid rafts localize proteins for cell signaling

–  Protein palmitoylation required for architecture and signal transduction •  Lipid-associated protein activation fuels cancer cell growth and survival

–  Ras, Raf, RTKs (e.g. EGFR, ErbB2), Akt, etc. •  FASN inhibition is a mechanism to inhibit vital tumor-cell-activated pathways regulated

by lipid modification or lipid association

•  Metabolic and signal transduction pathway cross-talk reprograms tumor cell metabolism

•  Tumor cells require increased energy and macromolecular biosynthesis

•  FASN inhibition blocks cellular lipid synthesis and signal transduction

Assay% TVB)3166%IC50%(µM)%

Hu%FASN%Biochem% 0.042%

%CALU)6%Cell%Palmitate% 0.081%

CALU)6%Cell%Viability% 0.061%

FASN Inhibition Disrupts Lipid Rafts and Palmitoylated Protein Localization

•  Discontinuous, lower intensity lipid raft staining following FASN inhibition •  N-Ras localization altered following FASN inhibition

Lipid rafts N-Ras Merge

DMSO 96 H

TVB-3166, 1 µM 96 H

Figure 1. CALU-6 in vitro immunoflouresent staining of lipid rafts (cholera toxin) and N-Ras. CALU-6 cells were treated with TVB-3166 for 96 hours in Advanced MEM media with 1% charcoal-filtered FBS.

K

ErbB3 ErbB2

Cytoplasm p85

Extracellular

NRas PI3K AKT

FASN Inhibition Blocks Signal Transduction and Induces Apoptosis

FASN Inhibition Blocks Anchorage-Independent Tumor Cell Growth

Figure 3. Soft agar colony formation assays show inhibition of anchorage-independent growth by FASN inhibition in lung, prostate, and colon tumor cell lines. Cells were treated with TVB-3166 for 21-28 days in IDEM plus 10% FBS.

Figure 2.FASN inhibition in MDA-MB-468 cells blocks β-catenin and AKT signaling. Annexin V flow cytometry shows induction of apoptosis. Cells were treated with TVB-3166 for 96 hours (Western) or 120 hours (Annexin V) in Advanced MEM media with 1% CF FBS.

MDA-MB-468

- 0 52 77

DM

SO TVB-3166 (µM)

2 0.2

0.02

β-Catenin pS675

β-Catenin

PARP- Cleaved

% Inhibition

% inhibition

% Induction

% Inhibition Akt pS473

- 0 52 77

- 0 49 60

- 39 124 187

FASN Inhibition Induces mRNA Expression Changes in Tumor Cell Growth, Survival, and Metabolism Pathways

•  Lipid, sterol, glucose, metabolism signatures •  Apoptosis, cell cycle, DNA replication signatures •  Concerted modulation of pathway-associated gene expression

DNA Replication, Cell Cycle, Mitosis

Vehi

cle

1.0 µM

0.1 µM

IDH2

DHF

R

CDK2

MCM

6

CCNB

2

PCNA

CDC7

FGFR

3

AURK

A

SKP2

RRM

2 M

CM2

MCM

4 CC

NE2

PLK4

CDK1

CCNB

1

CCNA

1

BUB1

TGFB

2

RCF4

MCM

3

DHF

R

BUB1

ETS2

NFAT

C2

KIF2

C M

KI67

Apoptosis Lipid, Sterol, Glucose Metabolism,

ACLY

AACS

INSI

G1

LIPI

N1

ELO

VL6

ACLY

SCD

ACAC

A AC

SS2

ACSL

1

EIF4

EBP1

EIF5

CASP

4

ACSL

3 EP

B41

EIF1

IDH1

EIF5

ACSL

3

LIPI

N1

FASN

LDLR

CASP

1 AN

XA7

DNA

JA3

CASP

4 CA

SP1

BCL2

A1

BCL2

L13

TP53

BP2

PRKA

CB

PPP1

R15A

EGFR

PRKC

A

DUS

P6

DUS

P5

DUS

P1

SQLE

Figure 4 Affymetrix HU133 Plus 2.0 microarray analysis of gene expression changes induced by FASN inhibition. PANC-1 tumor cells were treated with TVB-3166 for 72 hours in Advanced MEM plus 1% CF FBS and L-glutamine. Unsupervised hierarchical clustering and pathway enrichment analysis of 911 genes with significant TVB-3166 treatment-dependent variance (FDR <0.0001).

TVB-3166 Oral PK and Inhibition of De Novo Lipogenesis

TVB-3166 Inhibits In Vivo Tumor Growth

Figure 5 TVB-3166 plasma exposure in Sprague Dawley rats following oral dosing at 30 mg/kg. Inhibition of de novo lipogenesis in Sprague Dawley rats, measured 8 hours following oral dosing at 10 or 30 mg/kg. Lipogenesis is measured as incorporation of deuterium from D2O into newly synthesized palmitate (Kinemed Inc., Emeryville, CA).

Figure 6 . TVB-3166 inhibits growth of patient-derived and cell-line-derived xenograft tumors. Tumor growth inhibition (TGI) was calculated as the percentage of tumor growth, relative to tumor size at the start of treatment in drug-treated groups compared to vehicle-treated groups. The efficacy studies were performed by Champions Oncology and Crown Biosciences.

Gene Expression Pathway ANOVA Analysis Pathway Name Database Enrichment

p-valueSteroid biosynthesis kegg 5.55E-09

Cell cycle kegg 5.47E-07

Metabolic pathways kegg 0.0001

DNA replication kegg 0.0002

p53 signaling pathway kegg 0.0003

One carbon pool by folate kegg 0.0004

Terpenoid backbone biosynthesis kegg 0.0005

Insulin signaling pathway kegg 0.0009

Viral carcinogenesis kegg 0.0016

Aminoacyl-tRNA biosynthesis kegg 0.0023

HIF-1 signaling pathway kegg 0.0037

NF-kappa B signaling pathway kegg 0.0053

Pyruvate metabolism kegg 0.0085

Glycine, serine and threonine metabolism kegg 0.0138

Biosynthesis of unsaturated fatty acids kegg 0.0155

Folate biosynthesis kegg 0.0156

Glycolysis / Gluconeogenesis kegg 0.0166

Glutathione metabolism kegg 0.0169

2-Oxocarboxylic acid metabolism kegg 0.0200

Mismatch repair kegg 0.0228

Propanoate metabolism kegg 0.0261

Dorso-ventral axis formation kegg 0.0271

Selenocompound metabolism kegg 0.0311

Pyrimidine metabolism kegg 0.0336

Gap junction kegg 0.0506

Mineral absorption kegg 0.0632

Vitamin B6 metabolism kegg 0.0655

Fatty acid biosynthesis kegg 0.0655

Sterol Biosynthesis Up-Regulation Cell Cycle Down-Regulation

AKT

mTOR Raptor

PI3K

FASN

P

RTK

Palmitate

SREBP1C

Glucose

Pyruvate Citrate

Acetyl CoA

FASN Malonyl CoA NADPH

Glutamine

NADPH

KRAS

DMSO TVB-3166 (2µM)

6.9% Apoptosis 26.7% Apoptosis

CTG-0165 NSCLC PDX CTG-0160 NSCLC PDX

87% Tumor Growth Inhibition 2 of 3 tumors: 50% regression

50% Tumor Growth Inhibition

Vehicl

e

TVB-3166

(60)

0

200

400

600

Tum

or G

row

th (%

)

0

200

400

600

800

0 2 4 6 8 10 12 14 16 18

Cub

ic m

illim

eter

s

Day

Vehicle

TVB-3166 (60 mg/kg); po/qd

TGI (%):

•  KRAS G12S mutant •  Stage IV •  Adenocarcinoma

Tumor Volume Tumor Growth

47

-20

-10

0

10

20

0 2 4 6 8 10 12 14 16 18

Bod

y W

eigh

t Cha

nge

(%)

Day

Vehicle

TVB-3166 (60 mg/kg); po/qd

TVB-3166 is well tolerated

CTG-0165 Body Weight Change CTG-0743 NSCLC PDX

47% Tumor Growth Inhibition

PANC-1 Tumor Cell Line

57% Tumor Growth Inhibitionp=.0431

22Rv1 Tumor Cell Line

14% Tumor Growth Inhibition

•  Clinical development of TVB-2640, a first-in-class oral FASN inhibitor, is initiated •  TVB-3166 demonstrates single agent activity in tumor cell line and patient-

derived xenograft tumor models •  FASN inhibition effects on tumor cell biology include: (1) membrane and protein

localization disruption, (2) signal transduction inhibition, (3) concerted gene expression pathway modulation, (4) apoptosis induction, and (5) anchorage-independent tumor cell growth inhibition

•  FASN inhibition-mediated pathway modulation informs selection of drug combinations and discovery of mechanisms of action and biomarker candidates

•  Biomarker discovery for patient and expansion cohort selection is proceeding •  In vitro and in vivo evaluation of multiple, potent drug combinations is ongoing

DMSO

TVB-3166 (µM)

0.1

1.0

CALU-6 LnCAP 22Rv1 HT-29

Inhibition of Deuterium-Labeled De Novo Lipogenesis

Vehicl

e

TVB-316

6 (10

)

TVB-316

6 (30

)0

2

4

6

Deu

teriu

m-la

bele

d Pa

lmita

te (%

)

3.31

1.49 1.01

10#

100#

1000#

10000#

0# 4# 8# 12# 16# 20# 24#

Plasma&Co

ncen

tra.

on&(n

g/mL)&

Time&(H)&

Mean&Rat&Plasma&Concentra.on&Following&Oral&Administra.on&of&TVBA3166&

TVB+3166#PO#(10#mg/kg)#

TVB+3166#PO#(30#mg/kg)#