Journal: EBioMedicine

Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

doi: 10.1016/j.ebiom.2016.12.012

Figure Lengend Snippet: FASN inhibition disrupts tubulin palmitoylation, expression and microtubule organization. (A) TVB-3664 inhibits tubulin palmitoylation in NSCLC cell lines. Cells were treated with DMSO, TVB-3664 (48 h), or 2-bromopalimitate (18 h) as a positive control as indicated. Cell lysates were harvested and analyzed for palmitoylation of alpha-or beta-tubulin using acyl-biotin exchange chemistry followed by Western blot analysis. (B) TVB-3166 inhibits b-tubulin mRNA expression. 22Rv1 prostate tumor cells were treated with DMSO, TVB-3166, paclitaxel, or the combination of TVB-3166 and paclitaxel for 48 h in vitro. Three individual biological replicates were treated and used for analysis by RNA sequencing to quantitate mRNA levels. TVB-3166-treated cells showed a significant decrease in b-tubulin mRNA ( p < 0.001) compared to vehicle or paclitaxel-treated cells. Combination-treated cells also showed decreased b-tubulin expression. (C) FASN inhibition disrupts tumor cell microtubule organization. 22Rv1 prostate tumor cells were treated with DMSO, TVB-3166, paclitaxel, or the combination of TVB-3166 and paclitaxel for 96 h. Microtubule organization was visualized by immuno-fluorescence microscopy using an anti-b-tubulin antibody. Similar effects were observed with TVB-3664 in place of TVB-3166 or docetaxel in place of paclitaxel (Fig. S1). Non-tumor cells (MRC-5 lung fibroblasts) were not affected significantly by TVB-3664 and paclitaxel or docetaxel treatment (Fig. S1).

Article Snippet: Paclitaxel and deuterium labeled paclitaxel (Paclitaxel-D5) as internal standard were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of paclitaxel and IS were prepared in acetonitrile at concentrations of 1 mg/ml each.

Techniques: Inhibition, Expressing, Positive Control, Western Blot, In Vitro, RNA Sequencing Assay, Fluorescence, Microscopy

Journal: EBioMedicine

Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

doi: 10.1016/j.ebiom.2016.12.012

Figure Lengend Snippet: Combined treatment of tumor cells with TVB-3166 and paclitaxel in vitro increases soft agar colony growth inhibition and induction of apoptosis. (A) 22Rv1 prostate tumor cells (top panel) or CALU-6 NSCLC cells (bottom panel) were treated with a dose–response matrix of TVB-3166 and paclitaxel. 22Rv1 and CALU-6 tumor cells treated with 0.1 μM TVB-3166 and 0.001 μM paclitaxel showed increased inhibition of colony growth compared to treatment with each agent alone. Colony growth was inhibited completely with 0.1 μM TVB-3166 and 0.003 μM paclitaxel. (B) Intracellular paclitaxel concentration in CALU-6 tumor cells is unaffected by FASN inhibition. CALU-6 tumor cells were treated with DMSO, TVB-3166, or TVB-3664 for 24 (A) or 48 (B) hours. Cell lysates and supernatants were collected and the paclitaxel concentration in each matrix was determined by precipitation, extraction, and LC-MS-MS.

Article Snippet: Paclitaxel and deuterium labeled paclitaxel (Paclitaxel-D5) as internal standard were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of paclitaxel and IS were prepared in acetonitrile at concentrations of 1 mg/ml each.

Techniques: In Vitro, Inhibition, Concentration Assay, Liquid Chromatography with Mass Spectroscopy

Journal: EBioMedicine

Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

doi: 10.1016/j.ebiom.2016.12.012

Figure Lengend Snippet: Paclitaxel quantitation in CALU-6 NSCLC cells before and after FASN inhibition.

Article Snippet: Paclitaxel and deuterium labeled paclitaxel (Paclitaxel-D5) as internal standard were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of paclitaxel and IS were prepared in acetonitrile at concentrations of 1 mg/ml each.

Techniques: Quantitation Assay, Inhibition

Journal: EBioMedicine

Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

doi: 10.1016/j.ebiom.2016.12.012

Figure Lengend Snippet: Tumor growth inhibition and statistical analysis of treatment groups.

Article Snippet: Paclitaxel and deuterium labeled paclitaxel (Paclitaxel-D5) as internal standard were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of paclitaxel and IS were prepared in acetonitrile at concentrations of 1 mg/ml each.

Techniques: Inhibition

Journal: EBioMedicine

Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

doi: 10.1016/j.ebiom.2016.12.012

Figure Lengend Snippet: Combined treatment of NSCLC tumor xenografts with TVB-3166 and a taxane (paclitaxel or docetaxel) inhibits tumor growth synergistically compared to single agent activity. A minimum of 10 mice per treatment group was used in all studies. (A) CALU6 NSCLC adenocarcinoma cell line. (B) A549 NSCLC adenocarcinoma cell line. (C) CTG-0165_P + 6 NSCLC adenocarcinoma patient-derived tumor. TVB-3166 was dosed once daily by oral gavage at 60 mg/kg. Paclitaxel was dosed once every 4 days by intravenous administration at 10 mg/kg. Docetaxel was dosed once every 7 days by intravenous administration at 8 or 6 mg/kg. In groups dosed with both TVB-3166 (60 mg/kg) and paclitaxel (10 mg/kg) or docetaxel (8 or 6 mg/kg), TVB-3166 was administered 2 h before taxane administration. Animals were randomized according to tumor size and drug treatment was started when the mean tumor size was 150–200 mm 3 . Tumors and blood samples were harvested 2 h after the last dose. TVB-3166 and paclitaxel plasma and tumor drug concentrations were determined by mass spectrometry. The in-life phase for the CALU-6 (A) and A549 (B) studies was performed at Crown Biosciences (Santa Clara, CA; Beijing, China). The in-life phase for the CTG-0165_P + 6 study (C) was performed at Champions Oncology (Baltimore, MD).

Article Snippet: Paclitaxel and deuterium labeled paclitaxel (Paclitaxel-D5) as internal standard were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of paclitaxel and IS were prepared in acetonitrile at concentrations of 1 mg/ml each.

Techniques: Activity Assay, Derivative Assay, Mass Spectrometry

Journal: EBioMedicine

Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

doi: 10.1016/j.ebiom.2016.12.012

Figure Lengend Snippet: Combined treatment of ovarian, prostate, and pancreatic tumor xenografts with TVB-3166 and paclitaxel inhibits tumor growth synergistically compared to single agent activity. A minimum of 10 mice per treatment group was used in all studies. (A) OVCAR8 ovarian adenocarcinoma cell line. (B) 22Rv1 castration-resistant prostate tumor cell line. (C) PANC1 pancreatic ductal adenocarcinoma tumor cell line. TVB-3166 was dosed once daily by oral gavage at 60 or 100 mg/kg. Paclitaxel was dosed once every 4 days by intravenous administration at 10 mg/kg. In groups dosed with both TVB-3166 (60 mg/kg) and paclitaxel (10 mg/kg), TVB-3166 was administered 2 h before taxane administration. Animals were randomized according to tumor size and drug treatment was started when the mean tumor size was 150–200 mm 3 . Tumors and blood samples were harvested 2 h after the last dose. TVB-3166 and paclitaxel plasma and tumor drug concentrations were determined by mass spectrometry. The in-life phase for these studies was performed at Crown Biosciences (Santa Clara, CA; Beijing, China).

Article Snippet: Paclitaxel and deuterium labeled paclitaxel (Paclitaxel-D5) as internal standard were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of paclitaxel and IS were prepared in acetonitrile at concentrations of 1 mg/ml each.

Techniques: Activity Assay, Mass Spectrometry

Journal: EBioMedicine

Article Title: FASN Inhibition and Taxane Treatment Combine to Enhance Anti-tumor Efficacy in Diverse Xenograft Tumor Models through Disruption of Tubulin Palmitoylation and Microtubule Organization and FASN Inhibition-Mediated Effects on Oncogenic Signaling and Gene Expression

doi: 10.1016/j.ebiom.2016.12.012

Figure Lengend Snippet: Combined treatment of xenograft tumors with TVB-3166 and paclitaxel enhances pharmacodynamic effects of FASN inhibition to induce (A) gene expression changes similar to that observed in in vitro cell treatments with single-agent FASN inhibition and (B) inhibition of beta-catenin expression and phosphorylation similar to effects observed in in vitro cell treatments with single-agent FASN inhibition. Xenograft tumors were harvested 2 h following the last dose of TVB-3166 and/or taxane. For gene expression analysis, tumor pieces were preserved in RNAlater. RNA isolation was performed at 3-V Biosciences and mRNA changes were determined using RNA sequencing (RNASeq-25, Illumina, Inc). For Western blot analysis, tumor pieces were flash frozen. Tumor lysates were prepared at 3-V Biosciences in a buffer containing protease and phosphatase inhibitors.

Article Snippet: Paclitaxel and deuterium labeled paclitaxel (Paclitaxel-D5) as internal standard were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of paclitaxel and IS were prepared in acetonitrile at concentrations of 1 mg/ml each.

Techniques: Inhibition, Expressing, In Vitro, Isolation, RNA Sequencing Assay, Western Blot