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Preclinical studies characterize tumor type sensitivity to FASN inhibition and the mechanism and efficacy of novel drug combinations with TVB-2640
Timothy S. Heuer, Richard Ventura, Julie Lai, Joanna Waszczuk, Kasia Mordec, Claudia Rubio, Glenn Hammonds, Marie O’ Farrell, Douglas Buckley and George Kemble 3-V Biosciences, Menlo Park, CA
Introduction
Classification of Tumor Lipogenic and Epithelial Gene Expression Aligns with FASN-Inhibitor Sensitivity In Vitro
• 3-V Biosciences’ lead, oral FASN inhibitor is in Phase I clinical development 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 Inhibition decreases cellular levels of palmitate and saturated fatty acids and induces tumor cell apoptosis
• In vitro and in vivo studies have identified multiple mechanisms of action for FASN inhibition efficacy that include: (1) Membrane and lipid raft architecture remodeling; (2) Tumor cell gene expression reprogramming; (3) Signal transduction pathway inhibition
• 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
Figure 7. CALU-6 NSCLC cells respond to FASN inhibition in the presence of 1% glutamine but not glutamine-free media. Similar results observed in PANC-1 tumor cells. CASP1 and ELOVL6 gene expression by qPCR are representative of more global gene expression effects.
Conclusions
Combined FASN Inhibition with PD1 Immunotherapy Enhances Xenograft Efficacy
• Lipogenic gene expression signatures classify FASNi sensi7vity • Lipogenic gene expression is increased in Kras-‐mutant NSCL tumors; Kras-‐
mutant NSCLC cell lines have increased FASNi sensi7vity • Palmitoyla7on of Ras-‐associated signaling proteins is increased in Kras-‐
mutant NSCLC cell lines and is inhibited by FASN inhibi7on with TVB-‐3664 • FASN inhibi7on combines with PD1 immunotherapy or VEGF inhibi7on
(bevacizumab) to increase significantly in vivo tumor xenograR efficacy • TVB-‐2640 CLIN-‐002 tumor mRNA shows b-‐catenin, mito7c cell cycle, VEGFR
and Ras pathway dysregula7on in responsive, not unresponsive, pa7ents
Figure 9. TVB-3664 combined with bevacizumab shows increased tumor growth inhibition compared to single agent treatment. In vitro assays measuring endothelial cell tube formation (Incucyte analysis, Essen biosciences) shows that media conditioned with COLO-205 cells treated with TVB-3644 inhibits tube length in combination with axitinib better that axitinib alone or COLO-205 cells treated with vehicle (DMSO).
Results
Figure 1. Tumor cell line classification by gene expression analysis. Methods described in the Collisson et al and Daemen et al reports were followed using in vitro viability data (cell titer glo) with TVB-3166. Gene expression data were from the CCLE (Broad Institute, Cambridge, MA).
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• Published literature reports gene expression signatures of lipogenic, glycoly7c, epithelial, and mesenchymal phenotypes • Lipogenic and epithelial phenotypes co-‐segregate • Glycoly7c and mesenchymal phenotypes co-‐segregate
• Applica7on to 3-‐V In vitro data shows associa7on with FASN inhibitor sensi7vity
• Applica7on to TCGA tumor data (8588 samples) to iden7fy candidate tumor types and biomarkers of FASN inhibitor sensi7vity • Stra7fica7on and analysis of molecular gene7c features of tumors
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Epithelial/Mesenchymal
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Lipogenic/Glycolytic
LipogenicGlycolytic
References
1. Collisson et al. Nat Med. 2011 Apr;17(4):500-‐3. Epub 2011 Apr 3. PMID: 21460848
2. Daemen et al. Proc Natl Acad Sci U S A. 2015 Aug 11;112(32):E4410-‐7. Epub 2015 Jul 27. PMID: 26216984
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Tumor Type Abbreviation
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Lipogenic-Glyclotic Stratification of TCGA Tumor Data - Sample Number
Q1 - LipogenicQ2 - UndeterminedQ3 - UndeterminedQ4 - Glycolytic
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Tumor Type Abbreviation
Perc
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Lipogenic-Glyclotic Stratification of TCGA Tumor Data
Q1 - LipogenicQ2 - UndeterminedQ3 - UndeterminedQ4 - Glycolytic
Ø Normalized gene expression scores
§ W-‐Score: Weighted sum of 4 gene sets plus VIM and FASN. Weights determined by mul7-‐variable linear least squares regression for best match to FASNi sensi7vity.
§ VIM: Vimen7n, high mesenchymal expression, low epithelial expression.
§ Alpha: FASN sensi7vity-‐derived gene set. Difference between mean z-‐score of 17 posi7ve correla7on genes and mean z-‐score of 20 nega7ve correla7on genes.
§ MES: PDAC-‐derived mesenchymal/epithelial gene set. Difference between mean z-‐score of 20 mesenchymal genes and mean z-‐score of 22 epithelial genes.
§ FASN: TVB-‐2640 target. Synthesizes palmitate that provides a substrate for complex lipids that func7on in cell signaling and metabolism.
KRAS-Mutant NSCLC Tumors are More Lipogenic
Classification of Lipogenicity in TCGA Tumor mRNA Data
RAS Mutation Associated with FASN Sensitivity in NSCLC Cell Lines
FASN Inhibition
Remodeled Membrane Architecture
Disrupted Signal Transduction
Altered Gene Expression
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• Increased Glutamine metabolism – ReducFve glutamine metabolism can promote
lipid synthesis via pyruvate and NADPH producFon • Increased Glucose metabolism
– GlycolyFc pyruvate anabolism can promote lipid synthesis
– Increased non-‐oxidaFve PPP biosynthesis and NADPH producFon
Oncogenic KRAS Reprograms Tumor Cell Metabolism
Combined FASN and Angiogenesis Inhibition Enhances Efficacy
Figure 5. In vitro acyl-biotinyl exchange and Western blot analysis of protein palmitoylation and inhibition by treatment with 2-bromopalmitate or the FASN inhibitor TVB-3664. TVB-3664 treatment for 72 hours. 2-BP treatment for 16 hours.
+ 1% L-Glutamine - 1% L-Glutamine
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Figure 8. FASN inhibition sensitizes the LLC/LL NSCLC mouse tumor model to anti-PD immunotherapy. In vitro, FASN inhibition induces PD-L1 expression in NSCLC tumor cell lines
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Glutamine Sensitizes KRAS-Mutant Tumor Cells to FASN Inhibition
TVB-3664 Inhibits Increased Palmitoylation of RAS-Associated Signaling Proteins in KRAS-Mutant NSCLC Lines
COLO-‐205 XenograV TVB-‐3664
DMSO
FASN Inhibition Blocks RAS-Associated Signaling Pathways
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RMP1-14 10 mg/kg
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A549 (LUAD2): Undefined Lipogenic Score
Figure 3. In vivo mouse tumor xenograft efficacy analysis of the HEPG2 liver tumor and A549 NSCLC tumor model. Liver tumors classify as the most highly lipogenic by gene expression analysis (GEA); lung adenocarcinomas are moderately lipogenic by GEA. HEPG2 and A549 tumor cell lines classify as undetermined; thus, not as highly lipogenic as other liver or lung adenocarcinoma tumor cell lines. In-life analysis of tumor growth efficacy was conducted by Crown Biosciences (Santa Clara, CA)
Ventura et al. EBioMedicine. 2015 Jul 2;2(8):806-22 PMID: 26425687; 3-V Biosciences.
Figure 6. Activated KRAS reprograms tumor cell metabolism to increase utilization of glutamine and glucose. One of the consequences is high levels of pyruvate production. Son et al. 2013 Nature; Ying et al. 2012 Cell. A possible fate of pyruvate is metabolism to citrate and acetyl CoA, the substrate for palmitate synthesis by FASN.
LLC/LL2 NSCLC Mouse Syngeneic XenograV Mean Tumor Growth
In Vitro Tube Formation
In vitro induction of PD-L1 expression
Classifier Gene Set
Z-score normalization across samples per gene
Classifier score calculation
Top Half Bottom Half0
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G12 MutantWild Type
Low score = increased lipogenicity
p=0.001 p=0.002 p=0.002 p=0.046 p=0.074
Tumor Cell Line Xenograft Sensitivity Aligns with TCGA Tumor Classification
A427 NCI-H520 KRAS G12D KRAS WT
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TVB-2640 Biomarker Analysis in CLIN-002 Tumor RNA Tumor mRNA expression of genes in the Wnt/b-‐catenin, mitoFc cell cycle, VEGFR and Ras signaling pathways segment responding and non-‐responding paFents treated with TVB-‐2640 Further invesFgaFon in larger set of paFents Ayasdi (Menlo Park, CA) Core Topological Data Analysis
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Figure 2. Classification of 8558 TCGA tumor sample data using the Daemen et al 2015 methods. Data were downloaded, processed, and analyzed at 3-V Biosciences. In figure 3 below, tumor cell line xenografts representing (1) liver tumors (LIHC) and (2) lung adenocarcinoma tumors (LUAD) were assessed for tumor growth inhibition efficacy using TVB-3166.
Figure 4. Cell Titer Glo in vitro assay was used to asses cell viability in response to treatment with the FASN inhibitor TVB-3166. Cell were treated for 7 days with TVB-3166.