Richard Ventura

A complementary transposon tool kit for Drosophila melanogaster using P and piggyBac

With the availability of complete genome sequence for Drosophila melanogaster, one of the next strategic goals for fly researchers is a complete gene knockout collection. The P-element transposon, the workhorse of D. melanogaster molecular genetics, has a pronounced nonrandom insertion spectrum. It has been estimated that 87% saturation of the ∼13,500-gene complement of D. melanogaster might require generating and analyzing up to 150,000 insertions. We describe specific improvements to the lepidopteran transposon piggyBac and the P element that enabled us to tag and disrupt genes in D. melanogaster more efficiently. We generated over 29,000 inserts resulting in 53% gene saturation and a more diverse collection of phenotypically stronger insertional alleles. We found that piggyBac has distinct global and local gene-tagging behavior from that of P elements. Notably, piggyBac excisions from the germ line are nearly always precise, piggyBac does not share chromosomal hotspots associated with P and piggyBac is more effective at gene disruption because it lacks the P bias for insertion in 5′ regulatory sequences.

Inhibition of de novo Palmitate Synthesis by Fatty Acid Synthase Induces Apoptosis in Tumor Cells by Remodeling Cell Membranes, Inhibiting Signaling Pathways, and Reprogramming Gene Expression

Inhibition of de novo palmitate synthesis via fatty acid synthase (FASN) inhibition provides an unproven approach to cancer therapy with a strong biological rationale. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types. TVB-3166, an orally-available, reversible, potent, and selective FASN inhibitor induces apoptosis, inhibits anchorage-independent cell growth under lipid-rich conditions, and inhibits in-vivo xenograft tumor growth. Dose-dependent effects are observed between 20–200 nM TVB-3166, which agrees with the IC50 in biochemical FASN and cellular palmitate synthesis assays. Mechanistic studies show that FASN inhibition disrupts lipid raft architecture, inhibits biological pathways such as lipid biosynthesis, PI3K–AKT–mTOR and β-catenin signal transduction, and inhibits expression of oncogenic effectors such as c-Myc; effects that are tumor-cell specific. Our results demonstrate that FASN inhibition has anti-tumor activities in biologically diverse preclinical tumor models and provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN. The reported findings inform ongoing studies to link mechanisms of action with defined tumor types and advance the discovery of biomarkers supporting development of FASN inhibitors as cancer therapeutics. Research in context Fatty acid synthase (FASN) is a vital enzyme in tumor cell biology; the over-expression of FASN is associated with diminished patient prognosis and resistance to many cancer therapies. Our data demonstrate that selective and potent FASN inhibition with TVB-3166 leads to selective death of tumor cells, without significant effect on normal cells, and inhibits in vivo xenograft tumor growth at well-tolerated doses. Candidate biomarkers for selecting tumors highly sensitive to FASN inhibition are identified. These preclinical data provide mechanistic and pharmacologic evidence that FASN inhibition presents a promising therapeutic strategy for treating a variety of cancers.

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

Palmitate, the enzymatic product of FASN, and palmitate-derived lipids support cell metabolism, membrane architecture, protein localization, and intracellular signaling. Tubulins are among many proteins that are modified post-translationally by acylation with palmitate. We show that FASN inhibition with TVB-3166 or TVB-3664 significantly reduces tubulin palmitoylation and mRNA expression. Disrupted microtubule organization in tumor cells is an additional consequence of FASN inhibition. FASN inhibition combined with taxane treatment enhances inhibition of in vitro tumor cell growth compared to treatment with either agent alone. In lung, ovarian, prostate, and pancreatic tumor xenograft studies, FASN inhibition and paclitaxel or docetaxel combine to inhibit xenograft tumor growth with significantly enhanced anti-tumor activity. Tumor regression was observed in 3 of 6 tumor xenograft models. FASN inhibition does not affect cellular taxane concentration in vitro. Our data suggest a mechanism of enhanced anti-tumor activity of the FASN and taxane drug combination that includes inhibition of tubulin palmitoylation and disruption of microtubule organization in tumor cells, as well as a sensitization of tumor cells to FASN inhibition-mediated effects that include gene expression changes and inhibition of β-catenin. Together, the results strongly support investigation of combined FASN inhibition and taxane treatment as a therapy for a variety of human cancers.

Abstract LB-214: FASN inhibitor TVB-2640 shows pharmacodynamic effect and evidence of clinical activity in KRAS-mutant NSCLC patients in a phase I study

TVB-2640 is an oral, first-in-class, selective and reversible inhibitor of fatty acid synthase (FASN) in Phase 1 testing in solid tumor patients (study 3V2640-CLIN-002). FASN is a central mediator of neoplastic lipogenesis and uniquely catalyzes the production of palmitate, a key signaling molecule and the building block for long chain fatty acids. Tumor cells frequently increase FASN expression compared to normal cells to support an increased dependence on de novo lipogenesis to produce palmitate, phospholipids, lipid second messengers, membranes and lipid rafts for oncogenic signaling. High FASN correlates with poor prognosis in several tumor types including NSCLC. Palmitoylation, the reversible attachment of palmitate to proteins regulates membrane interactions and dynamically alters protein trafficking and function. RAS proteins require palmitoylation for plasma membrane localization and signaling, and KRAS4A mutated at G12 specifically requires palmitoylation for oncogenic activity. FASN inhibitors may therefore provide a novel approach to target KRAS which to date has eluded many drug development approaches. In vitro viability assays showed that KRAS mutant NSCLC cell lines show greater sensitivity to FASN inhibitors than KRAS WT. Furthermore gene expression analysis of TCGA NSCLC tumors and PDX models showed that KRAS mutant cases are associated with a higher incidence of lipogenic features than KRAS WT. The ongoing Phase 1 study includes 2 NSCLC expansion cohorts; TVB-2640 as monotherapy or in combination with paclitaxel. Several biomarker approaches are ongoing to investigate pharmacodynamic activity and patient enrichment strategies. Metabolomic profiling of serum from 12 NSCLC patients showed increased serum malonyl carnitine and decreased palmitate- derived lipids after 8 or 15 days of TVB-2640 treatment. These changes are consistent with FASN inhibition, and also observed in preclinical models. A novel non-invasive approach using Sebutape patches to collect forehead sebum showed inhibition of de novo lipogenesis in 7/7 patients including 2 NSCLC. Decreases in wax esters, saturated and monounsaturated triglycerides were observed, indicating that these lipids are not restored by diet. To date, 16 NSCLC patients are considered evaluable for clinical activity; 6 have KRAS mutant tumors, 2 have wild type KRAS and 8 are unknown. Of the 6 known KRAS mutant patients, 3 are in the monotherapy cohort and all 3 have stable disease for 20, >25 and >37 weeks respectively. The remaining 3 KRAS mutant patients are on combination therapy; one achieved a partial response and remains on therapy (>22 weeks), 1 had stable disease (24 weeks), and 1 progressed. This study shows that TVB-2640 1) inhibits palmitate production and lipogenesis, and 2) shows evidence of clinical activity in KRAS mutant NSCLC patients. Additional biomarker and clinical analyses are ongoing in NSCLC and other tumor types. Citation Format: Marie O’Farrell, Tim Heuer, Katharine Grimmer, Richard Crowley, Joanna Waszczuk, Marina Fridlib, Richard Ventura, Claudia Rubio, Julie Lai, Doug Buckley, William McCulloch, George Kemble. FASN inhibitor TVB-2640 shows pharmacodynamic effect and evidence of clinical activity in KRAS-mutant NSCLC patients in a phase I study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-214.

Abstract 4743: Preclinical studies characterize tumor type sensitivity to FASN inhibition and the mechanism and efficacy of novel drug combinations with TVB-2640

Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. Previously we showed that FASN inhibition results in tumor cell apoptosis in vitro and xenograft tumor growth inhibition in vivo. Our studies demonstrated that diverse tumor types exhibit sensitivity to FASN inhibition and characterized mechanisms of action that associate with the antitumor activity of highly selective small molecule FASN inhibitors. In vitro studies with diverse tumor cell types elucidated a mechanism of action that includes plasma membrane remodeling, signal transduction pathway inhibition, and gene expression reprogramming. TVB-2640, TVB-3166, and TVB-3664 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences. Analysis of gene expression data from tumor cell lines and human tumors, both primary and patient-derived xenografts, has allowed for the classification of FASN sensitivity by tumor type, histology, and molecular genetic markers. Discoveries from these analyses are being characterized further using in vitro and in vivo studies. Combined inhibition of FASN and microtubule function with taxane treatment, e.g. paclitaxel, results in synergistic inhibition of tumor growth. Indeed, in Phase I clinical investigation, TVB-2640 combined with paclitaxel has shown promising early signs of clinical activity. Previous in vitro studies revealed that FASN inhibition causes changes in beta-tubulin expression and disrupts the organization of cellular microtubule structures in varied tumor cell types such as CALU-6 non-small-cell lung and 22Rv1 prostate tumor cell lines. Extending our investigation of the mechanism of FASN/taxane synergy, we now show that FASN inhibition prevents beta-tubulin palmitoylation. This likely plays a significant role in the observed effects on beta-tubulin expression and microtubule architecture. As disruption of protein palmitoylation is believed to contribute significantly to the anti-tumor activity of FASN inhibition in general, we expanded the analysis of protein palmitoylation following inhibition of FASN with TVB-3166 and TVB-3664 to include key oncogenic drivers of cell growth, proliferation, and survival such as K-Ras and EGFR. Additionally, the efficacy of FASN inhibition in combination with additional, non-taxane approved cancer therapies, including immunomodulatory agents and bevacizumab, is being investigated. Citation Format: Timothy S. Heuer, Richard Ventura, Julie Lai, Joanna Waszczuk, Claudia Rubio, Glenn Hammonds, Marie O’ Farrell, Douglas Buckley, George Kemble. Preclinical studies characterize tumor type sensitivity to FASN inhibition and the mechanism and efficacy of novel drug combinations with TVB-2640. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4743.

Abstract C175: FASN inhibition studies in preclinical tumor models identify biomarkers that align with in vitro and in vivo sensitivity to TVB-2640

Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. Previously we showed that FASN inhibition results in tumor cell apoptosis in vitro and xenograft tumor growth inhibition in vivo. Our studies demonstrated that diverse tumor types exhibit sensitivity to FASN inhibition and characterized mechanisms of action that associate with the antitumor activity of highly selective small molecule FASN inhibitors. In vitro studies with diverse tumor cell types elucidated a mechanism of action that includes plasma membrane remodeling, signal transduction pathway inhibition, and gene expression reprogramming. TVB-2640 and TVB-3166 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences. Tumor xenograft studies were conducted in rats and mice to examine the in vitro and in vivo relationship of tumor cell sensitivity to FASN inhibition. Pharmacodynamic analyses of tumor and serum samples from these studies characterized the mechanism of action and biomarkers of sensitivity to in vivo FASN inhibition. Once daily oral dosing of TVB-2640 or TVB-3166 caused inhibition of xenograft tumor growth for varied tumor models that included COLO-205 and HCT-116 colon adenocarcinoma cell lines. In vivo sensitivity to FASN inhibition was in agreement with in vitro data. Analysis of lipid, metabolite, protein, and RNA expression in tumor, blood or serum samples showed drug-induced modulation that was consistent with independent in vitro or in vivo studies. Lipid and metabolite changes included decreased palmitate and palmitate-associated lipids as well as increased expression of acylcarnitine species. Decreased expression of pAkt (S473), β-catenin, pβ-catenin (S675), and Myc proteins were found to associate quantitatively with xenograft tumor growth inhibition. Additionally, mRNA expression was modulated in a manner that revealed coordinated changes in the mRNAs from fatty acid, metabolism, cell survival, and cell growth-associated pathways. Expression changes in lipids, metabolites, proteins, and RNA species are leading to the development of a biomarker panel that describes FASN inhibitor target engagement and tumor sensitivity in both in vitro and in vivo studies. These mechanism-based marker panels will be evaluated in current and upcoming clinical studies of TVB-2640. Citation Format: Timothy S. Heuer, Richard Ventura, Kasia Mordec, Julie Lai, Joanna Waszczuk, Claudia Rubio, Marie O9 Farrell, Douglas Buckley, George Kemble. FASN inhibition studies in preclinical tumor models identify biomarkers that align with in vitro and in vivo sensitivity to TVB-2640. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C175.

Abstract 4446: Discovery of tumor types highly susceptible to FASN inhibition and biomarker candidates for clinical analysis

Inhibition of de novo palmitate synthesis by fatty acid synthase (FASN) is a novel cancer therapeutic approach with strong biological rationale. Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. 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. TVB-2640 and TVB-3166 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences that exhibit anti-tumor activity in diverse preclinical tumor models. FASN inhibition induces tumor cell apoptosis by remodeling tumor cell membranes, blocking signal transduction, and reprogramming gene expression. These effects lead to inhibition of anchorage-independent tumor cell growth under lipid-rich conditions and inhibition of in vivo xenograft tumor growth in mice and rats. Studies to understand the mechanisms of action and biological consequences of FASN inhibition are guiding the discovery of tumors highly dependent on FASN activity and biomarkers for assessment of pharmacodynamic activity and patient selection. Using quantitative genomics and a variety of directed analytical approaches, we identified lipid, mRNA, and protein profiles in tumor cells that change in response to FASN inhibition. Subsets of the changes show correlation with FASN inhibitor sensitivity. Marker candidates, such as gene expression signatures that classify in vitro sensitivity to FASN inhibition, are being investigated for detection in clinical human tumor data sets. Our studies also identified FASN inhibitor-mediated mechanisms of action that function in specific tumor types and biomarkers with potential utility for selecting responsive patients and measuring tumor response. Inhibition of the Wnt/b-catenin pathway and expression of TCF-promoter-regulated genes such as c-Myc are examples. Tumors dependent on Wnt/b-catenin activity or with lower intracellular palmitate stores prior to drug treatment may be susceptible to FASN inhibitor treatment. Pairing MYC expression with additional markers improves the performance of MYC as a marker to select tumor cell lines highly sensitive to FASN inhibition. Biomarker candidates from in vitro studies have been examined in vivo using xenograft tumor studies. These preclinical data support 3-V Biosciences’ ongoing Phase I clinical study of a first-in-class FASN inhibitor TVB-2640, and help guide the next steps in its development. Citation Format: Timothy S. Heuer, Richard Ventura, Kasia Mordec, Julie Lai, Joanna Waszczuk, Glenn Hammonds, Marina Fridlib, Russell Johnson, Lily Hu, Allan Wagman, Marie O9 Farrell, Douglas Buckley, George Kemble. Discovery of tumor types highly susceptible to FASN inhibition and biomarker candidates for clinical analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4446. doi:10.1158/1538-7445.AM2015-4446

Abstract 1815: Efficacy of FASN-selective small molecule inhibitors in preclinical tumor models

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnFatty acid synthase (FASN) expression increases with tumor progression and is associated with impaired response to chemotherapy and diminished patient survival in many solid and hematopoietic tumor types. Palmitate, the product of FASN enzymatic activity, functions in vital cellular processes including energy metabolism and cell growth, cellular membrane biosynthesis and architecture, and protein localization and function. 3-V Biosciences has discovered and developed a series of potent, selective, orally available, and reversible FASN inhibitors with excellent pharmaceutical properties. Single agent FASN inhibition has been shown to cause anti-tumor activities in preclinical models of human cancer, including inhibition of AKT and mTOR signal transduction, induction of tumor cell apoptosis, and tumor growth inhibition and regression in pancreatic, ovarian, and lung cancer xenograft tumor models of human cancer. Current studies are supporting the active clinical development of TVB-2640, 3-V Biosciences first-in-class FASN inhibitor, as an oncology therapeutic. These studies have multiple objectives: (1) characterizing a wide range of tumor types for responsiveness to FASN inhibition using tumor cell line and Champions TumorGraftTM efficacy models; (2) investigating the benefit of FASN combination therapy with chemotherapeutic or targeted anti-cancer agents; (3) developing biomarkers that characterize efficacy response; and (4) further elucidating the mechanisms of action that emanate from the inhibition of palmitate synthesis and lead to tumor cell apoptosis and in vivo anti-tumor efficacy. The results of these investigations show that FASN inhibition causes tumor growth inhibition or regression in diverse tumor types; and moreover, that tumor growth inhibition additivity or synergy is observed when FASN inhibition is combined with chemotherapy agents. Genomics and directed analysis of mRNA, proteins, and lipids following FASN inhibition have identified biomarker candidates and provided insights into the tumor growth inhibition mechanisms of action. For example, genome-wide gene expression analysis shows FASN inhibition to significantly increase the expression of many genes in biosynthetic (sterol, lipid, etc.) and apoptosis pathways while significantly reducing the expression of many genes in cell growth and proliferation pathways (DNA replication, cell cycle progression, mitosis, etc.). These data strongly support the clinical development of TVB-2640 as a first-in-class single and combination oncology therapeutic and are advancing the discovery and validation of biomarkers to inform clinical utility and response.nnCitation Format: Timothy S. Heuer, Richard Ventura, Joanna Waszczuk, Kasia Mordec, Julie Lai, Russell Johnson, Lilly Hu, Haiying Cai, Allan Wagman, Douglas Buckley, Stanley T. Parish, Elizabeth Bruckheimer, George Kemble. Efficacy of FASN-selective small molecule inhibitors in preclinical tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1815. doi:10.1158/1538-7445.AM2014-1815

Abstract B261: Characterization of FASN-selective small-molecule inhibitors in preclinical tumor models.

The purpose of this study is to characterize the activity of small-molecule FASN inhibitors in preclinical models of human cancer to evaluate their potential utility as novel cancer therapeutics. FASN-synthesized palmitate is required for vital cellular processes that include energy metabolism, cellular membrane biosynthesis, and protein localization and function. Many solid and hematopoietic tumors overexpress FASN. Moreover, tumor expression of FASN is increased in a stage-dependent manner that is correlated with patient survival. FASN activity can promote the tumorigenic capacity of cells by multiple mechanisms that include: activating cell growth and survival signal transduction pathways such as PI3K-AKT and enhancing cellular processes such as macromolecular biosynthesis, cellular stress response, and energy metabolism. 3-V Biosciences has discovered and developed a series of selective, reversible FASN inhibitors that have been optimized and characterized using in vitro and in vivo assays. These molecules are highly active in both in vitro and in vivo studies, and selected FASN inhibitors demonstrate excellent biochemical (< 10 nM) and cell-based (<100 nM) IC50 values. In vitro, inhibitory activity of cell-based palmitate synthesis aligns with activity in cell phenotypic (e.g. growth and viability) and mechanistic (e.g. AKT phosphorylation and PARP cleavage) assays in cell lines derived from pancreas, breast, lung, colon, and other tumor tissues. Moreover, pharmacologic supplementation of palmitate to cell culture medium rescues cells from cell death in FASN-inhibition cell viability assays. Together, these data show that in vitro FASN inhibition is associated with growth-inhibitory and apoptosis-inducing effects. These novel FASN inhibitors show oral bioavailability, dose dependent plasma exposure, and demonstrate excellent in vivo pharmacodynamic activity. Tumor xenograft studies in mice have been conducted with the FASN inhibitor TVB-3166, and the results demonstrate in vivo tumor growth inhibition both as a single agent and in combination with standard of care chemotherapy agents. TVB-3166 in combination with different chemotherapy agents shows synergistic or additive anti-tumor efficacy in these xenograft studies. In conclusion, these data support continued investigation of FASN inhibition as an approach for the development of a novel cancer therapeutic with the potential for anti-tumor activity in multiple tumor types. Further characterization of FASN inhibitor activity as a single agent and in combination with chemotherapeutics is ongoing using cell-based mechanistic and phenotypic assays as well as tumor xenograft studies.nnCitation Information: Mol Cancer Ther 2013;12(11 Suppl):B261.nnCitation Format: Timothy S. Heuer, Minchao Chen, Richard Ventura, Joanna Waszczuk, Satya Yendluri, Julie Lai, Samnang Tep, Shirley Feng, Russell Johnson, Cristiana Zaharia, Douglas Buckley, George Kemble. Characterization of FASN-selective small-molecule inhibitors in preclinical tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B261.