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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).

#4743

•  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    

TVB Sensitive TVB Insensitive0

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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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CKIR

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TGCTMESO

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Tumor Type Abbreviation

Num

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Lipogenic-Glyclotic Stratification of TCGA Tumor Data - Sample Number

Q1 - LipogenicQ2 - UndeterminedQ3 - UndeterminedQ4 - Glycolytic

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40

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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

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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.

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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

H460   H1975  

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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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

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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

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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.