NAMPT抑制剂联合嘧啶类似物抗代谢药氟尿苷和5-FU损害横纹肌肉瘤的增殖和存活

Pediatric rhabdomyosarcoma (RMS) is a soft tissue sarcoma of high unmet need that requires novel treatment approaches. Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate limiting step of the nicotinamide adenine dinucleotide (NAD + ) salvage pathway. Previously, we showed that RMS is highly sensitive to NAMPT inhibition, with in vivo RMS models undergoing tumor regression upon treatment with the NAMPT inhibitor OT-82. However, we observed recurrence after cessation of treatment, suggesting single agent therapy is not durable. The aim of this project is to identify and evaluate drug combinations that synergize with NAMPT inhibitors in RMS in an effort to improve treatments for patients with RMS. To identify synergistic drug combinations, we used a matrix drug screen of 2 RMS cell lines (Rh30 (fusion-positive (FP)) and Rh36 (fusion-negative (FN)) testing 2 NAMPT inhibitors (daporinad and GNE-618) in combination with 62 other anticancer agents. We identified that floxuridine, a pyrimidine analog antimetabolite, was the most highly synergistic agent with both NAMPT inhibitors. Validation of the screen results using longitudinal proliferation assays confirmed synergy between floxuridine and an additional NAMPT inhibitor, OT-82, across additional FP and FN RMS cell lines. Doses of each drug that had minimal effect on proliferation as single agents resulted in durable cell death when combined. Notably, when this combination was tested in proliferating non-cancer cell lines, there was a minimal effect on proliferation, suggesting a therapeutic window exists. Rescue experiments using nicotinamide mononucleotide (NMN), the product of NAMPT, reversed the antiproliferative effect of the combination, indicating that the synergy is NAD + dependent. Protein analysis revealed that floxuridine-related ternary complex formation of thymidylate synthase is blocked by NAD + loss mediated by NAMPT inhibition. To evaluate this combination for potential translation into the clinic, we extended our testing to include clinical agents, including RPT1G (Remedy Plan Therapeutics), a NAMPT inhibitor currently under early phase evaluation, and 5-FU, a prodrug of floxuridine. Validation studies of these agents using longitudinal proliferation assays again demonstrated that single agent doses had minimal effect on proliferation but when combined, resulted in persistent cell death. Preliminary toxicity testing in Rh30 tumor-bearing NSG mice demonstrated tolerability of OT-82 plus 5-FU up to the highest doses tested (15 mg/kg and 25 mg/kg respectively) and reduced tumor volume. Efficacy experiments are ongoing and will be reported. These findings demonstrate that combining NAMPT inhibitors with floxuridine or 5-FU results in significant synergy and preliminarily suggest that this is a promising and feasible combination regimen for patients with RMS.