Abstract 3945: NAMPT inhibitor-resistant rhabdomyosarcoma models exhibit alterations in metabolic and genomic profiles

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and represents a tumor of high unmet clinical need as standard systemic therapy consists of cytotoxic agents only. We have previously demonstrated that RMS models are highly sensitive to inhibition of nicotinamide phosphoribosyltransferase (NAMPT). NAMPT catalyzes the rate-limiting step of the NAD+ salvage pathway and is currently the only pharmacologically targetable NAD+ production enzyme. Treatment with the clinical NAMPT inhibitor OT-82 results in complete tumor regressions in vivo, however, upon intermittent treatment, acquired resistance develops in some in vivo models. As acquired drug resistance is a known impediment to the clinical efficacy of targeted agents, we sought to elucidate potential mechanisms of acquired resistance to OT-82 in RMS. Mice with orthotopic fusion-positive (FP) and fusion-negative (FN) RMS xenograft tumors were treated with OT-82 for 8 weeks on the clinical dosing schedule. Upon cessation of treatment, mice were observed for recurrence and retreated when tumors regrew to a volume of >900 mm3. Tumors that progressed on treatment were converted to cell lines. Two resistant cell lines (1 FP and 1 FN) were selected for further study. Incucyte live cell analysis confirmed retention of OT-82 resistance in vitro with resistant cell lines maintaining proliferation at doses of OT-82 up to 30 times above the IC50 of parental lines. Resistance to multiple other NAMPT inhibitors (daporinad and KPT-9274) was observed in one of the resistant cell lines. Resistant cells exposed to OT-82 maintained ATP levels consistent with that of untreated controls. After 24 hours of OT-82 treatment, NAD+ loss was observed in both parental and resistant cell lines, however resistant cell lines were able to recover NAD+ levels within 48-96 hours. Downstream effects on glucose metabolism, measured using extracellular flux and metabolomic analyses, demonstrated that in the presence of OT-82 resistant cells maintained their glycolytic function, whereas parental cells did not. Specifically, the products downstream of the NAD+-dependent enzyme glyceraldehyde-3-phosphase dehydrogenase were reduced in parental cell lines but maintained in resistant cell lines. Analysis of gene and protein expression of NAD+ synthesis enzymes NAMPT, NAPRT, and QPRT demonstrated changes in NAPRT and QPRT expression at the transcriptional level and at the protein level in resistant cell lines. Whole exome sequencing revealed that each resistant cell line has a distinct, previously unreported mutation in NAMPT. Together, these data suggest that acquired resistance to NAMPT inhibitors in RMS models involves cellular alterations in the biochemical, metabolomic, and genomic profiles of cells. Citation Format: Ariana E. Nelson, Victor J. Collins, Ali Mokhtar Mahmoud, Ying Wu, Sophia Varriano, Arnulfo Mendoza, Sameer Issaq, Parthav Jailwala, Jack F. Shern, Christine M. Heske. NAMPT inhibitor-resistant rhabdomyosarcoma models exhibit alterations in metabolic and genomic profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3945.