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.