Discovery of tumor types highly susceptible to FASN ......pAKT (S473) 100 60 42 66 75 78 70 70 70...

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c-Myc

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Discovery of tumor types highly susceptible to FASN inhibition and biomarker candidates for clinical analysis Timothy S. Heuer, Richard Ventura, Kasia Mordec, Julie Lai, Joanna Waszczuk, Marie O’Farrell, Douglas Buckley, and George Kemble

3-V Biosciences, Menlo Park, CA

Introduction

FASN Inhibition of Tumor Cell Viability is an On-Target Effect

•   3-V Biosciences’ lead, oral FASN inhibitor is in Phase I clinical trials for the treatment of solid tumors

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

•   Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells

•   FASN expression increases with tumor progression in human tumors and associates with chemoresistance, metastasis, and diminished patient survival in many tumor types.

•   Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival

•   Studies to understand the mechanisms of action and biological consequences of FASN inhibition are guiding the discovery of tumors highly dependent on FASN and biomarkers for assessment of pharmacodynamic activity and patient selection

•   Inhibition of the AKT and Wnt/β-catenin pathways, including TCF-promoter-regulated genes such as c-Myc, provide examples of mechanistic responses to FASN inhibition that identify biomarker candidates

Additive and Synergistic Inhibition of Xenograft Tumor Growth by FASN Inhibition in Combination with Taxanes

TVB-2640 Inhibits COLO-205 Rat Xenograft Tumor Growth in Association with β-Catenin, c-Myc and pAKT Inhibition

Figure 4. Combination treatment of patient-derived and cell-line-derived xenograft tumors with TVB-3166 and paclitaxel (or docetaxel) shows synergistic tumor growth inhibition and tumor regression. 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.

Conclusions and Status

Mechanism of Taxane Synergy Insights: FASN Inhibition Affects β-tubulin Expression and Microtubule Organization

•   TVB-2640 a first-in-class oral FASN inhibitor, is in Phase I clinical development for the treatment of solid tumors.

•   TVB-2640 demonstrates dose-dependent single agent tumor growth inhibition in the COLO-205 rat xenograft tumor model.

•   Tumor growth inhibition by TVB-2640 is associated with inhibition of β-catenin, c-Myc and AKT and modulation of tumor gene expression.

•   FASN inhibition combined with paclitaxel or docetaxel shows synergistic tumor growth inhibition, including tumor regression in many different xenograft tumor models that include NSCLC, ovarian, and prostate tumors types.

•   The mechanism of FASN-taxane synergy likely includes: (1) decreased expression of β-tubulin and disrupted microtubule organization as a result of FASN inhibition; (2) taxane-mediated sensitization of tumor cells to modulation of gene expression by FASN inhibition.

•   In-vitro and in-vivo evaluation of additional, potent drug combinations is ongoing

Figure 6. CTG-0165 NSCLC PDX xenograft tumors (Champions Oncology) were harvested after 20 days of once-daily, oral treatment as indicated (6 hours post final dose). RNA isolation and data analysis was performed at 3-V Biosciences. RNA sequencing (RNASeq-25, Illumina, Inc.) was performed by Expression Analysis (Durham, NC). Differential gene expression data analysis was performed at 3-V Biosciences using Partek Genomics Suite software (St. Louis, MO).

Figure 1. Cell-based assays for palmitate synthesis and cell viability show alignment of IC50 values in CALU-6 tumor cells. MRC5 lung fibroblasts show inhibition of palmitate synthesis but minimal effect on viability. Palmitate assay measures incorporation of 13C into palmitate from 13C sodium acetate. Cell viability is measured using the Cell Titer Glo assay following treatment with TVB-3166 for 7 days in Advanced MEM media with 1% charcoal-stripped FBS. Absolute fatty acid levels were measure using Metabolon’s FAME analysis.

Results NSCLC Cell Line - Inhibition of palmitate synthesis and cell viability

Lung Fibroblast Cell Line - Inhibition of palmitate synthesis, not cell viability

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Fig.2. Antitumor Activity of TVB-0001 and TVB3166 as a Single Agent or in Combination with Irinotecan in the Treatment of Colo205 Colon cancer xenograft model in nude rats

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TVB-2640 Induces Gene Expression Changes in COLO-205 Rat Xenograft Tumors In Association with Treatment Efficacy

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B-catenin 100 82 68 62 86 81 72 76 78 99pB-catenin 100 63 50 49 76 94 87 51 59 115

cMyc 100 21 20 19 48 56 38 32 37 126pAKT (S473) 100 60 42 66 75 78 70 70 70 113

!-tubulin 100 96 85 80 100 96 105 97 97 105% Tumor Growth 467 190 103 183 188 269 600 179 467

Tumor Size (mm3) 356 288 538 749 436 972 681

Figure 2. Tumor growth and pharmacodynamic analysis of of COLO-205 tumors. Female Rowett nude rats (NIH-Foxn1rnu ), 10-12 weeks of age, were inoculated subcutaneously at the right flank with COLO-205 tumor cells (2 x 107) in 0.2 mL of PBS with matrigel (1:1). Tumor growth inhibition (TGI) was calculated as the percentage of tumor growth, relative to tumor size at the start of treatment in drug-treated compared to vehicle-treated groups. The Mann-Whitney U test was used to assess statistical significance. In-life phase of the efficacy studies were performed by Crown Biosciences (Santa Clara, CA; Beijing, China). Western blot analysis of tumor lysates was performed at 3V Biosciences.

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Combined FASN-Paclitaxel Inhibition Induces PDX mRNA Changes in Apoptosis, Metabolism, and Tubulin-Associated Genes

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7 days Treatment TVB-3166 + Paclitaxel vs Vehicle •   p<0.01 •   1.5 fold change •   1143 genes •   Counter selection for

paclitaxel-induced genes (n=151)

Figure 5. Immunofluorescence of β-tubulin in 22Rv1 cells shows decreased expression and disrupted cellular organization following treatment with TVB-3166 for 72 hours. Imaging was performed using a Zeiss LSM510 confocal microscope (100X objective). β-tubulin mRNA expression was measured by RNA sequencing (RNASeq-25, Illumina, Inc.) following 48 hours of TVB-3166 treatment. RNA sequencing was performed by Expression Analysis (Durham, NC).

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Figure 3. COLO-205 rat xenograft tumors were harvested after 17 days of once-daily, oral treatment with TVB-2640 or vehicle (2 hours post final dose). RNA isolation and data analysis was performed at 3-V Biosciences. RNA sequencing (RNASeq-25, Illumina, Inc.) was performed by Expression Analysis (Durham, NC). Differential gene expression data analysis was performed at 3-V Biosciences using Partek Genomics Suite software (St. Louis, MO).

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CALU-6 NSCLC Cell Line

22Rv1 CRPC Tumor Cell Line

OVCAR-8 Ovarian Tumor Cell Line

A549 NSCLC Cell Line

CTG-0165 NSCLC PDX