Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both in vitro and in vivo by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis.Significance: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.

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.

EWS is an aggressive malignancy affecting adolescents and young adults, for which novel therapies are critically needed. Recent insights have revealed that NAMPT is a potential therapeutic target, mediating the rate limiting step of the NAD salvage pathway which converts nicotinamide (NAM) to nicotinamide mononucleotide (NMN). This pathway is preferentially utilized in tumor cells where it is essential for maintaining cellular bioenergetics and provides the necessary substrate for NAD-dependent proteins. OT-82 (OncoTartis) is a novel NAMPT inhibitor expected to enter early phase clinical trials in the near future. The purpose of this study was to evaluate the efficacy and mechanism of action of OT-82 as a single agent and in rational combinations in EWS. Proliferation of EWS cells lines was assessed after treatment with OT-82 as a single agent and in combinations with PARP inhibitors and DNA damaging agents using IncuCyte live cell analysis and MTS assays. Targeted activity of OT-82 was evaluated via NAD/NADH-Glo assays and rescue experiments with metabolic intermediates. Protein expression of targets was assessed with Western blot. For in vivo studies, SCID mice underwent orthotopic injection of EWS cells from established cell lines and PDX cells. When tumors reached a desired size, mice were randomized and treated with vehicle, OT-82, irinotecan, niraparib or combinations of OT-82 plus niraparib or irinotecan. Tumors were measured semiweekly with calipers and harvested at midpoints and at study endpoint for biology studies. EWS cell lines were sensitive to OT-82 in a dose-dependent manner, with IC50 values in the low nanomolar range for all cell lines tested, consistent with the known activity of NAMPT inhibitors in EWS cell lines. While NAMPT expression in EWS cell lines was high, the level of expression did not correlate with sensitivity to OT-82. OT-82 activity was on-target, demonstrated by dose-dependent reduction in NAD/NADH levels in EWS cells after treatment. This effect could be rescued through the addition of NMN in the majority of cell lines tested, supporting the proposed mechanism of OT-82 as a NAMPT inhibitor. When combined with either niraparib or SN-38, anti-proliferative activity was synergistic in vitro. In vivo, treatment with OT-82 resulted in tumor regression or growth inhibition in multiple xenograft models of EWS treated at doses between 25 and 50 mg/kg three days per week. When combined with low dose niraparib (50 mg/kg five days per week), tumor growth was suppressed, and survival time was significantly increased as compared to either single agent. When OT-82 was combined with low-dose irinotecan (2-5 mg/kg two days per week), the combination exhibited enhanced efficacy in multiple models. Biology studies of tumor tissue are ongoing and will be reported. OT-82 may be a novel targeted approach for the treatment of EWS and the addition of niraparib or irinotecan may enhance its efficacy. Citation Format: Anna E. Gibson, Arnulfo Mendoza, Lioubov Korotchkina, Olga Chernova, Christine M. Heske. Targeting nicotinamide phosphoribosyltransferase (NAMPT) with OT-82 in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5477.