Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.

◥Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine avail-ability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNAdamage response pathways.

The pathogenesis of systemic lupus erythematosus is believed to involve defects in regulatory T cell (Treg) activity and abnormal activation of B and T lymphocytes. The purpose of this study was to test the therapeutic potential of rabbit anti-mouse thymocyte globulin (ATG), a lymphocyte-depleting agent, in conjunction with transforming growth factor (TGF)-beta1, a factor involved in the induction and expansion of Tregs. MRL/lpr mice with active disease were treated with ATG followed by a 12-day course of latent TGF-beta1 during the period of lymphocyte repopulation. Treatment with ATG + latent TGF-beta1 synergistically inhibited the progression of proteinuria and albuminuria and provided a significant improvement in long-term survival. This therapeutic benefit correlated histologically with reduced glomerular pathology and protein cast formation. The mechanism of action did not involve suppression of autoantibody formation but may involve the activity of CD4+CD25+FoxP3+ Tregs, which were found to be induced by ATG + TGF-beta1 treatment in vitro.

Fatty acid synthase (FASN) is a key enzyme responsible for fatty acids synthesis de novo in mammals. Overexpression of FASN is common in many cancers including prostate, breast and colon cancer and elevated expression of FASN has been linked with poor prognosis and reduced disease-free survival. Experiments with RNAi and small molecule inhibitors suggest that FASN is a metabolic oncogene with an important role in tumor growth and survival and an appealing target for cancer therapy. However, studies utilizing small molecule FASN inhibitors like orlistat and C75 have been confounded by the lack of potency and selectivity, as well as the poor pharmacological properties of these inhibitors. Herein we report pharmacological target validation studies of FASN using a potent, selective and orally bioavailable FASN inhibitor IPI-9119. Building on previous experience with serine hydrolase inhibitors, a series of novel mechanism-based FASN inhibitors were designed based on a tetrazolone carboxamide scaffold. Like orlistat, these analogs are irreversible inhibitors that specifically target the FASN thioesterase domain. Tetrazolone carboxamide analogs were shown to potently inhibit cellular FASN using an occupancy assay and to completely block de novo palmitate synthesis in HCT-116 colon cancer cells using a 13C-glucose incorporation assay. Lead optimization of the tetrazolone carboxamide series resulted in the identification of IPI-9119 as a tool for in vivo proof-of-concept studies. IPI-9119 is a potent FASN inhibitor in both biochemical (IC50∼1nM) and cellular occupancy assays (IC50∼10nM), and shows more than 400-fold selectivity against several additional serine hydrolases. Importantly, IPI-9119 is orally bioavailable and has pharmacokinetic (PK) properties suitable for in vivo pharmacology studies. IPI-9119 was tested for growth inhibition in cancer cell lines in vitro and tumor xenograft models in vivo. Unexpectedly, in contrast to the knock-down studies and to data reported for orlistat and C75, IPI-9119 failed to elicit anti-proliferative effects in multiple cancer cell lines in vitro. Similarly, PK/PD experiments demonstrated that a single oral dose of IPI-9119 at 200 mg/kg leads to complete and sustained blockade of FASN in HCT-116 tumor xenografts, but IPI-9119 failed to show any anti-tumor activity when dosed as a single agent at 200 mg/kg BID for 10 days. In summary, we identified IPI-9119 as a potent, selective and orally bioavailable FASN inhibitor. Preliminary target validation studies with IPI-9119 in cancer cell lines and an HCT-116 xenograft model suggest that FASN inhibition alone is not sufficient to affect cancer cell proliferation and tumor growth. Further studies exploring combination treatments with IPI-9119 are warranted. Citation Format: Erin Brophy, James Conley, Patrick O'Hearn, Mark Douglas, Culver Cheung, John Coco, Laura D'Anello, Andrew Wylie, Thomas Tibbitts, Gregg Keaney, Lawrence Chan, Adilah Bahadoor, Dan Snyder, Marta Nevalainen, Alfredo Castro, Vito Palombella, Massimo Loda, Stephane Peluso. Pharmacological target validation studies of fatty acid synthase in carcinoma using the potent, selective and orally bioavailable inhibitor IPI-9119. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1891. doi:10.1158/1538-7445.AM2013-1891