Abstract B013: Preclinical evaluation of NAMPT inhibitors with pyrimidine agonist antimetabolites floxuridine and 5-FU in rhabdomyosarcoma

Abstract

While therapeutic advances have improved survival across pediatric malignancies, these gains have yet to be fully realized for patients with rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma. Our previous studies have shown that RMS is highly sensitive to targeting of nicotinamide phosphoribosyltransferase (NAMPT), the rate limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway. In in vivo studies using the clinical inhibitor OT-82, we observed complete tumor regressions; however, upon cessation of OT-82 treatment, tumors recurred, indicating that single agent therapy is not sufficient. Therefore, the purpose of this study is to identify and assess feasible combination drug regimens that exhibit synergy with NAMPT inhibitors. Using an unbiased matrix drug screen, we evaluated 62 anticancer agents in combination with 2 NAMPT inhibitors (daporinad and GNE-618) in 2 RMS cell lines (Rh30 (fusion-positive (FP)) and Rh36 (fusion-negative (FN)). Floxuridine, a pyrimidine analog antimetabolite, emerged as a top synergistic hit with both NAMPT inhibitors in both cell lines. Longitudinal proliferation assays in additional FP and FN RMS cell lines testing the combination of floxuridine plus NAMPT inhibitors, including OT-82, validated the screen results, revealing the combination resulted in profound loss of proliferation even when using doses that were ineffective as single agents. Critically, when tested in proliferating non-cancer cell lines at the same doses, the combination minimally impacted proliferation, indicating the existence of a therapeutic window. Flow cytometric analysis established apoptosis as the predominant mechanism of cell death. Addition of nicotinamide mononucleotide (NMN), the product of NAMPT, rescued the effect of the combination, demonstrating that the mechanism of synergy is NAD+-dependent. Interestingly, formation of the floxuridine-induced ternary complex involving thymidylate synthase was impaired by NAMPT inhibition and was reversible with the addition of NMN. To determine whether our findings extended to clinically relevant agents and were of translational significance, we next evaluated the combination of 5-FU, the prodrug of floxuridine which has been established for pediatric clinical use, plus the first-in-class hyperbolic NAMPT inhibitor RPT1G (Remedy Plan Therapeutics), which is currently undergoing early phase investigation. Use of these clinical agents recapitulated the initial findings, confirming that low doses of each single agent that did not impact cell growth resulted in persistent loss of proliferation when combined and that the synergy was NAD+-dependent. Similarly, thymidylate synthase ternary complex formation was blunted with the addition of RPT1G. Pilot testing of the combination of 5-FU plus RPT1G in orthotopic tumor-bearing xenografts is ongoing and will be reported. Together, these results demonstrate that pyrimidine analog antimetabolites plus NAMPT inhibitors may represent both a promising and clinically tractable regimen for children with RMS.