efficacy of fasn-selective small molecule inhibitors in ......tumor volume tumor growth vehicle...

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TGI (%): 14 10

•  PIK3CA Q546R mutant •  Adenocarcinoma

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TGI (%): 87

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

Tumor Volume Tumor Growth

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•  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.

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- 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

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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

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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

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•  KRAS G12S mutant •  Stage IV •  Adenocarcinoma


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TVB-3166 is well tolerated

CTG-0165 Body Weight Change CTG-0743 NSCLC PDX


PANC-1 Tumor Cell Line

57% Tumor Growth Inhibitionp=.0431

22Rv1 Tumor Cell Line


•  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

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Inhibition of Deuterium-Labeled De Novo Lipogenesis

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efficacy of fasn-selective small molecule inhibitors in ......tumor volume tumor growth vehicle...

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