Evaluating the effects of critical metabolite depletion in pediatric rhabdomyosarcoma (RMS) using a novel inhibitor of nicotinamide phosphoribosyltransferase (NAMPT).

Abstract

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.