NAMPT mediates the rate-limiting step of the NAD salvage pathway, which maintains cellular bioenergetics and provides a necessary substrate for functions essential to rapidly proliferating cancer cells. In this study, we evaluated the efficacy and mechanisms of action of OT-82, a novel, high-potency NAMPT inhibitor with a favorable toxicity profile, in preclinical models of Ewing sarcoma (EWS), an aggressive pediatric malignancy with previously reported selective sensitivity to NAMPT inhibition. We show that OT-82 decreased NAD concentration and impaired proliferation of EWS cells in a dose-dependent manner, with IC50 values in the single-digit nanomolar range. Notably, genetic depletion of NAMPT phenocopied pharmacological inhibition. On-target activity of OT-82 was confirmed with the addition of NMN, the product of NAMPT, which rescued NAD concentration and EWS cellular viability. Mechanistically, OT-82 treatment resulted in impaired DNA damage repair through loss of PARP activity, G2 cell-cycle arrest, and apoptosis in EWS cells. Additional consequences of OT-82 treatment included reduction of glycolytic and mitochondrial activity. In vivo, OT-82 impaired tumor growth and prolonged survival in mice bearing EWS xenografts. Importantly, antitumor effect correlated with pharmacodynamic markers of target engagement. Furthermore, combining low-dose OT-82 with low doses of agents augmenting DNA damage demonstrated enhanced antitumor activity in vitro and in vivo. Thus, OT-82 treatment represents a potential novel targeted approach for the clinical treatment of EWS.
Purpose: A hallmark of cancer cells is altered metabolism. Therapeutically, these alterations may be exploited by targeting metabolic vulnerabilities specific to cancer cells. Efficient production of NAD through the NAD salvage pathway is one such potential vulnerability, as some tumor cells demonstrate a high need for rapid NAD turnover. Nicotinamide phosphoribosyltransferase (NAMPT) is the pharmacologically targetable rate-limiting enzyme in this pathway. We report on the effect of targeting NAMPT in models of pediatric rhabdomyosarcoma (RMS), a cancer for which novel therapeutics remain an unmet need. Experimental Procedures: The relative sensitivity of RMS cell lines to NAMPT inhibitors was first compared to NAMPT inhibitor sensitivity of other cancer cell lines using viability assays. A panel of ten molecularly diverse RMS cell lines was used for the remainder of the evaluations. In vitro activity of NAMPT inhibition was evaluated using assays of proliferation and cell death. Measurements of NAD and functional assessment of NAD-dependent processes, such as glucose metabolism, were used to study the mechanistic activity of NAMPT inhibition in these models. In vivo studies included assessments of toxicity, efficacy, and mechanism of action of a clinical NAMPT inhibitor, OT-82, in four orthotopic RMS models. Results: RMS cells showed striking sensitivity to NAMPT inhibition with IC-50 values in the low nanomolar range. In vitro, NAMPT inhibition resulted in NAD depletion and impaired cellular proliferation. Effects on glucose metabolism included decreases in glycolytic activity and glycolytic capacity in all cell lines tested, as well as decreased oxidative phosphorylation in a subset of cell lines. The majority of cell lines exhibited ATP depletion and irreversible necrotic cell death. Apoptotic cell death was not observed. In vivo, the effects of OT-82 treatment delivered on the human clinical schedule replicated those seen in vitro, including loss of glycolytic activity as measured using hyperpolarized 13C MRI spectroscopy. In all four xenograft models, complete tumor regressions were observed at multiple doses and with minimal toxicity. Conclusions: NAMPT inhibition with OT-82 was highly effective in decreasing RMS proliferation and impairing glucose metabolism both in vitro and in vivo. Given these results, there is a critical need for further clinical study of this class of agents for RMS. Citation Format: Grace McKay-Corkum, Victor J. Collins, Choh Yeung, Takeshi Ito, Sameer H. Issaq, Arnulfo Mendoza, Kazutoshi Yamamoto, Murali Cherukuri, Len Neckers, Christine M. Heske. Exploiting metabolic vulnerabilities of pediatric rhabdomyosarcoma with novelnicotinamide phosphoribosyltransferase (NAMPT) inhibitor OT-82 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6718.
e22004 Background: RMS is the most common pediatric cancer of the soft tissues. Patients who present with metastatic disease or experience recurrence have a poor prognosis; survivors often suffer from long-term systemic effects resulting from cytotoxic chemotherapy. Thus, novel therapies are needed. NAMPT inhibitors are a class of drugs targeting a key metabolic enzyme in the production of NAD+, a coenzyme critical to energy generation in some cancer cells. In this study, we evaluated the mechanism and functional outcomes of treatment with the clinical NAMPT inhibitor, OT-82, in preclinical models of RMS. Methods: Live cell analysis via IncuCyte was used to determine the temporal effects of OT-82 on cell growth in a diverse panel of ten fusion positive and negative RMS cell lines. Analysis of the proposed mechanism of action was performed using NAD/NADH detection assays and rescue experiments with nicotinamide mononucleotide (NMN), the product of NAMPT. Markers of cellular apoptosis and necrosis were quantified with flow cytometric assays. Specific metabolic effects of OT-82 were determined with ATP quantification and real-time extracellular flux analysis of oxidative phosphorylation and glycolysis. In vivo studies were performed in orthotopic RMS models. Tumor dimensions were measured with calipers, and toxicity was assessed by observation and body weight measurement. Results: Treatment of RMS cell lines with OT-82 dosed in the low nanomolar range resulted in time- and dose-dependent decreases in NAD+ levels and proliferation in all cell lines tested. Addition of NMN rescued cell growth, confirming the on-target activity and functional effect of OT-82. Flow cytometric assays revealed cell-line dependent differences in cell fates, with a subset of cell lines staining positive for markers of necrosis, and the other subset staining negative for markers of necrosis and apoptosis. Functional investigation verified that necrotic cell lines did not regrow after withdrawal of OT-82 in culture (durable responders), whereas non-necrotic cell lines recovered growth (transient responders). Additionally, ATP levels in durable responders decreased with OT-82 treatment but remained stable in transient responders. Extracellular flux analysis revealed that both durable and transient responders experienced inhibition of glycolysis, but that oxidative phosphorylation was only reduced in the durable responders. In vivo studies using OT-82 on a clinically-relevant schedule demonstrated that all RMS xenografts underwent complete tumor regression, with durable responder models experiencing a longer tumor-free period following discontinuation of treatment. Conclusions: In vitro and in vivo efficacy of OT-82 suggest that targeting NAD+ metabolism through NAMPT inhibition may be a promising approach for the treatment of RMS.
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