Timothy S. Heuer

A genome-wide analysis of immune responses in Drosophila.

Oligonucleotide DNA microarrays were used for a genome-wide analysis of immune-challenged Drosophila infected with Gram-positive or Gram-negative bacteria, or with fungi. Aside from the expression of an established set of immune defense genes, a significant number of previously unseen immune-induced genes were found. Genes of particular interest include corin- and Stubble-like genes, both of which have a type II transmembrane domain; easter- and snake-like genes, which may fulfil the roles of easter and snake in the Toll pathway; and a masquerade-like gene, potentially involved in enzyme regulation. The microarray data has also helped to greatly reduce the number of target genes in large gene groups, such as the proteases, helping to direct the choices for future mutant studies. Many of the up-regulated genes fit into the current conceptual framework of host defense, whereas others, including the substantial number of genes with unknown functions, offer new avenues for research.

EXEL-7647 Inhibits Mutant Forms of ErbB2 Associated with Lapatinib Resistance and Neoplastic Transformation

Purpose: Mutations associated with resistance to kinase inhibition are an important mechanism of intrinsic or acquired loss of clinical efficacy for kinase-targeted therapeutics. We report the prospective discovery of ErbB2 mutations that confer resistance to the small-molecule inhibitor lapatinib. Experimental Design: We did in vitro screening using a randomly mutagenized ErbB2 expression library in Ba/F3 cells, which were dependent on ErbB2 activity for survival and growth. Results: Lapatinib resistance screens identified mutations at 16 different ErbB2 amino acid residues, with 12 mutated amino acids mapping to the kinase domain. Mutations conferring the greatest lapatinib resistance cluster in the NH2-terminal kinase lobe and hinge region. Structural computer modeling studies suggest that lapatinib resistance is caused by multiple mechanisms; including direct steric interference and restriction of conformational flexibility (the inactive state required for lapatinib binding is energetically unfavorable). ErbB2 T798I imparts the strongest lapatinib resistance effect and is analogous to the epidermal growth factor receptor T790M, ABL T315I, and cKIT T670I gatekeeper mutations that are associated with clinical drug resistance. ErbB2 mutants associated with lapatinib resistance transformed NIH-3T3 cells, including L755S and T733I mutations known to occur in human breast and gastric carcinomas, supporting a direct mechanism for lapatinib resistance in ErbB2-driven human cancers. The epidermal growth factor receptor/ErbB2/vascular endothelial growth factor receptor inhibitor EXEL-7647 was found to inhibit almost all lapatinib resistance-associated mutations. Furthermore, no ErbB2 mutations were found to be associated with EXEL-7647 resistance and lapatinib sensitivity. Conclusions: Taken together, these data suggest potential target-based mechanisms of resistance to lapatinib and suggest that EXEL-7647 may be able to circumvent these effects.

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.

Diacylglycerol Metabolism and Signaling Is a Driving Force Underlying FASN Inhibitor Sensitivity in Cancer Cells

Fatty acid synthase (FASN) generates the de novo source of lipids for cell proliferation and is a promising cancer therapy target. Development of FASN inhibitors, however, necessitates a better understanding of sensitive and resistant cancer types to optimize patient treatment. Indeed, testing the cytotoxic effects of FASN inhibition across human cancer cells revealed diverse sensitivities. We show here that metabolic incorporation of glucose into specific complex lipid species strongly predicts FASN inhibitor sensitivity. We also show that the levels of one of these lipid classes, protein kinase C (PKC) stimulator diacylglycerols, are lowered upon FASN inhibitor treatment in sensitive compared to resistant cells and that PKC activators and inhibitors rescue cell death in sensitive cells and sensitize resistant cells, respectively. Our findings not only reveal a biomarker for predicting FASN sensitivity in cancer cells but also a put forth a heretofore unrecognized mechanism underlying the anticancer effects of FASN inhibitors.

Discovery of XL888: A novel tropane-derived small molecule inhibitor of HSP90

With structural guidance, tropane-derived HTS hits were modified to optimize for HSP90 inhibition and a desirable in vivo profile. Through an iterative SAR development process 12i (XL888) was discovered and shown to reduce HSP90 client protein content in PD studies. Furthermore, efficacy experiments performed in a NCI-N87 mouse xenograft model demonstrated tumor regression in some dosing regimens.

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.

Fatty acid synthase – Modern tumor cell biology insights into a classical oncology target

Decades of preclinical and natural history studies have highlighted the potential of fatty acid synthase (FASN) as a bona fide drug target for oncology. This review will highlight the foundational concepts upon which this perspective is built. Published studies have shown that high levels of FASN in patient tumor tissues are present at later stages of disease and this overexpression predicts poor prognosis. Preclinical studies have shown that experimental overexpression of FASN in previously normal cells leads to changes that are critical for establishing a tumor phenotype. Once the tumor phenotype is established, FASN elicits several changes to the tumor cell and becomes intertwined with its survival. The product of FASN, palmitate, changes the biophysical nature of the tumor cell membrane; membrane microdomains enable the efficient assembly of signaling complexes required for continued tumor cell proliferation and survival. Membranes densely packed with phospholipids containing saturated fatty acids become resistant to the action of other chemotherapeutic agents. Inhibiting FASN leads to tumor cell death while sparing normal cells, which do not have the dependence of this enzyme for normal functions, and restores membrane architecture to more normal properties thereby resensitizing tumors to killing by chemotherapies. One compound has recently reached clinical studies in solid tumor patients and highlights the need for continued evaluation of the role of FASN in tumor cell biology. Significant advances have been made and much remains to be done to optimally apply this class of pharmacological agents for the treatment of specific cancers.

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 1010: Evaluation of small-molecule FASN inhibitors in preclinical models of colorectal cancer

Fatty Acid Synthase (FASN), a key enzyme of de novo lipid synthesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN activity is associated with decreased survival and increased disease recurrence. Recently, a first-in-class, oral FASN inhibitor (TVB-2640) entered a Phase I clinical trial (3V2640-CLIN-002) in solid tumor patients demonstrating a favorable tolerability profile with no significant adverse events; however, tumor characteristics that would indicate responsiveness to FASN inhibition are not fully understood. The purpose of our study was: (i) to determine the effect of novel, selective and reversible FASN inhibitors on proliferation of primary CRC cell cultures, established CRC cell lines, and CRC patient-derived xenografts (PDXs); and (ii) to identify potential biomarkers associated with CRC responsiveness to FASN inhibition. METHODS. The effect of TVB-3166, TVB-3664, and TVB-3693 (all developed by 3-V Biosciences) on the proliferation of primary cells (established from 1 st generation PDX tumors) and CRC cell lines was assessed by cell count; apoptosis was assessed by Cell Death ELISA. In addition, tumor growth was assessed in PDX models established in NOD SCID gamma mice using freshly resected CRC specimens (either primary CRC or metastasis) from our patient population. Once the xenografts grew to ∼100 mm 3 , mice were randomized into two groups (n = 5) to receive either vehicle or TVB-3664 (3mg/kg) by gavage daily. Tumor volume and animal weights were measured weekly. Western blot analysis, immunohistochemistry and immunofluorescent staining were used to identify FASN-mediated changes in β-catenin, Akt and AMPK pathways. RESULTS. TVB compounds tested showed similar efficacy in primary and established CRC cells with a wide range of sensitivity to FASN inhibition. The 5 cell lines that were most responsive to FASN inhibition demonstrated a low basal level of pAMPK and pAkt as compared to the 5 least responsive cells. Moreover, we noted that increased FASN protein expression was also associated with increased sensitivity to FASN inhibition. Inhibition of proliferation by TVB compounds was associated with decreased expression of active β-catenin, c-MYC, pAkt, and survivin, while an increase in apoptosis was noted by induction of PARP cleavage. Consistent with our in vitro studies, TVB-3664 treatment significantly reduced tumor volume in vivo with no weight changes or toxicity observed. CONCLUSIONS. Our studies show that the novel FASN inhibitors, as a single agent, significantly inhibit CRC growth both in vitro and in vivo. Importantly, our results suggest that basal activation of AMPK and Akt may be predictive of responsiveness to FASN inhibition and may function as potential biomarkers to allow a more personalized treatment approach. Citation Format: Yekaterina Y. Zaytseva, Piotr G. Rychahou, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, Timothy S. Heuer, George Kemble, B. Mark Evers. Evaluation of small-molecule FASN inhibitors in preclinical models of colorectal cancer. [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 1010.

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.