Abstract 3592: Evaluation of OT-82, a nicotinamide phosphoribosyltransferase inhibitor (NAMPTi), as a potential therapeutic option for Ewing sarcoma

Targeting NAMPT, the rate-limited enzyme in the NAD salvage pathway, is a potentially attractive anticancer strategy, since cancer cells preferentially rely on this pathway to produce NAD. We explored the use of NAMPTi in Ewing sarcoma (ES), an aggressive pediatric malignancy for which novel therapeutic options are critically needed. We previously showed that ES cells are exceptionally sensitive to NAMPTi compared to other cancer types and have single agent activity in vitro and in vivo. We also demonstrated that combining NAMPTi with PARP inhibitors results in enhanced efficacy in vitro and in vivo. In this study, we further characterized the mechanisms of action of OT-82, a NAMPTi expected to enter early phase clinical trials, in ES cells and evaluated various dosing schedules for OT-82 combinations with cytotoxic therapy. On-target activity of OT-82 was assessed using NAD quantification assays and rescue experiments with multiple NAD intermediates. Characterization of NAD salvage pathway enzyme expression was performed by western blot. Mechanistic studies of cell cycle analysis, DNA damage, and cell death were performed using flow cytometry, western blotting, and comet assays. Glycolytic profiles of ES cell lines were analyzed using the Agilent Extracellular Flux Analyzer. Orthotopic ES cell line and PDX models in immunodeficient mice were used to evaluate in vivo combinations. Tumor measurements were performed with calipers twice weekly and toxicity was assessed with weekly body weights and general observations. Tumors were harvested at intermediate time points and at endpoints for biology and pharmacodynamic studies. Treatment with OT-82 revealed dose-dependent reduction of NAD both in vitro and in vivo. Addition of NMN, the product of NAMPT, rescued ES cell viability, indicating on-target activity of OT-82. While delayed NMN administration up to 48 hours after treatment with OT-82 rescued viability, a 72-hour delay rendered the effects irreversible in all cell lines. Treatment with OT-82 resulted in DNA damage, G2/M arrest and induction of apoptosis at 72 hours. Extracellular flux analysis indicated that OT-82 treatments resulted in a decrease in both oxidative and glycolytic activity. While NAMPT expression did not correlate with sensitivity to OT-82, low expression of NAPRT, an enzyme in a parallel NAD salvage pathway, appeared to be predictive of greater sensitivity to OT-82. Further, ES cells that expressed NAPRT could be fully rescued with addit