Abstract 1798: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor-resistant rhabdomyosarcoma (RMS) models exhibit alterations in metabolic and genomic profiles

Abstract RMS is a common pediatric soft tissue sarcoma for which new therapies are critically needed. We previously demonstrated that RMS is highly sensitive to inhibitors of NAMPT, which catalyzes the rate-limiting step of the NAD+ salvage pathway and is the only pharmacologically targetable NAD+ production enzyme. Treatment with the NAMPT inhibitor OT-82 results in complete tumor regressions in vivo, however, upon intermittent treatment, acquired resistance develops in some models. As acquired drug resistance is a known impediment to the clinical efficacy of targeted agents, we sought to elucidate potential mechanisms of OT-82 resistance 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. After stopping treatment, mice were observed for recurrence and retreated when tumors regrew to 900 mm3. Tumors that progressed on treatment were harvested and converted to cell lines. Two resistant cell lines (1 FP - Rh30-mRes and 1 FN - RD-mRes) were selected for further study. Incucyte live cell analysis confirmed retention of OT-82 resistance in vitro with resistant cells maintaining proliferation at doses of OT-82 up to 30X above the IC50 of parental cells. Resistant cells exposed to OT-82 maintained ATP levels consistent with that of untreated controls. After 24h of OT-82 treatment, NAD+ loss was observed in both parental and resistant cells, however resistant cells recovered NAD+ levels within 48-96h. Effects on glucose metabolism, measured using extracellular flux and metabolomic analyses demonstrated that in the presence of OT-82, only resistant cells maintained glycolytic function. Specifically, metabolites downstream of the NAD+-dependent enzyme glyceraldehyde-3-phosphase dehydrogenase were reduced in parental cells but maintained in resistant cells. Analysis of protein expression of NAD+ synthesis enzymes NAMPT, NAPRT, and QPRT revealed that Rh30-mRes expresses more QPRT, however, genetic silencing of QPRT did not reverse resistance, suggesting upregulation of compensatory NAD production enzymes is not a primary mechanism of resistance. Whole exome sequencing revealed that each resistant cell line has a distinct, previously unreported mutation in NAMPT. Protein modeling suggests each mutation affects the drug binding pocket of NAMPT, with the S17F variant in Rh30-mRes resulting in a collapse of the pocket and the S241C variant in RD-mRes reducing binding affinity of OT-82. These findings are consistent with functional studies demonstrating that Rh30-mRes is resistant to multiple other NAMPT inhibitors whereas RD-mRes is resistant only to OT-82. Together, these data suggest that acquired resistance to NAMPT inhibitors in RMS models involves the development of mutations in the target protein affecting drug binding and affinity. Citation Format: Ariana Elizabeth Nelson, Abantika Chakraborty, David Bell, Victor J. Collins, Ali Mokhtar Mahmoud, Ying Wu, SOPHIA VARRIANO, Arnulfo Mendoza, Sameer Issaq, Parthav Jailwala, Jack F. Shern, Ernesto Suárez, Joseph Ivanic, Christine M. Heske. Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor-resistant rhabdomyosarcoma (RMS) models exhibit alterations in metabolic and genomic profiles abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1798.