Abstract B016: Novel mutations in nicotinamide phosphoribosyltransferase (NAMPT) arising from OT-82 exposure drive resistance in rhabdomyosarcoma (RMS) models

Abstract

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma, and new treatments are urgently needed. Our prior work showed RMS has enhanced sensitivity to pharmacological inhibitors of NAMPT, the rate-limiting enzyme of the NAD+ salvage pathway and the only clinically targetable enzyme of NAD+ synthesis. In vivo treatment with the NAMPT inhibitor OT-82 produced complete tumor regressions, but acquired resistance developed in a subset of models. Given that acquired resistance remains a major limitation to clinical activity of targeted agents, the purpose of this study was to characterize mechanisms underlying OT-82 resistance in RMS. Resistant cell lines (Rh30-mRes and RD-mRes) were derived from orthotopic xenograft models of fusion-positive and fusion-negative RMS treated intermittently with OT-82 on the clinical schedule until tumors progressed on therapy. Live cell imaging confirmed resistance to OT-82 was retained in resistant cell lines at doses up to 30-fold higher than the IC50 of corresponding parental cells. Both parental and resistant cells underwent NAD+ depletion within 24h of OT-82 treatment, however resistant cells subsequently restored NAD+ levels. Expression analysis of NAD+ biosynthetic enzymes NAMPT, NAPRT, and QPRT revealed increased QPRT in Rh30-mRes, however QPRT knockout did not restore OT-82 sensitivity, indicating that increased expression of compensatory NAD+ production enzymes is not the key driver of resistance. In the presence of OT-82, resistant cells maintained ATP levels comparable to untreated controls. Quantification of effects on glucose metabolism via extracellular flux and metabolomic analyses showed that resistant cells, but not parental cells, preserved glycolytic function during OT-82 treatment. Consistent with this finding, metabolites downstream of glyceraldehyde-3-phosphase dehydrogenase, an NAD+-dependent enzyme, were reduced in parental cells but sustained in resistant cells. Whole exome sequencing identified the presence of novel, previously unreported mutations in NAMPT in each resistant cell line. Structural modeling predicted that both mutations alter the NAMPT drug-binding pocket; the S17F variant in Rh30-mRes was predicted to collapse the binding pocket and the S241C variant in RD-mRes was predicted to reduce OT-82 binding affinity. These predictions were validated by functional data showing that Rh30-mRes cells are resistant to several NAMPT inhibitors, while RD-mRes cells display selective resistance to OT-82. The functional significance of these mutations was further evaluated by introducing the mutations into the sensitive parental cell lines. Cells expressing the mutant NAMPT variants continued to proliferate in the presence of OT-82, whereas cells transfected with either empty vector or wild-type NAMPT overexpression constructs retained sensitivity to OT-82, consistent with untransfected cells. Collectively, these findings indicate acquired resistance to NAMPT inhibitors in RMS models is associated with the emergence of target protein mutations which disrupt drug binding.