(P 178) HIGH FREQUENCY OF NAPRT-SILENCING IN NEUROBLASTOMA AND RHABDOMYSARCOMA CONFERS SENSITIVITY TO TARGETING OF NAD+ METABOLISM USING NAMPT INHIBITORS

Objective: Cells primarily synthesize nicotinamide adenine dinucleotide (NAD+) through the Preiss-Handler and Salvage pathways driven by nicotinic acid phosphoribosyltransferase (NAPRT) and nicotinamide phosphoribosyltransferase (NAMPT), respectively. Loss of NAPRT expression by tumor-specific promoter CpG island methylation may provide a novel predictive biomarker for synthetic lethality following treatment with NAMPT inhibitors (NAMPTi’s). However, there is limited data on NAPRT promoter methylation and expression across cancer types. Therefore, we sought to identify tumor types with loss of NAPRT expression and determine the efficacy of NAMPTi treatment.

Methods: We probed data within the Cancer Dependency Map (DepMap). Bisulfite sequencing data measuring NAPRT methylation was downloaded from DepMap (https://depmap.org/portal/download/) in January of 2021. For in vitro assays, G401, G402, RH18, and RH41 rhabdomyosarcoma (RMS) tumor cell lines, along with SKNAS, SKNDZ, SKNFI, SKNBE, and CHP-212 neuroblastoma (NBL) cell lines, were used. Growth delay assays were performed using the NAMPT inhibitor FK866 with and without nicotinic acid (NA) supplementation. NAD(H) levels were determined using the NAD/NADH-Glo assay kit (Promega). NAPRT protein expression was detected by western blot. For in vivo experiments, RD cells were injected into the flank of athymic nude mice (n = 10 mice/group). Mice were treated with vehicle control or FK866 at 20 mg/kg/dose by intraperitoneal injection twice daily for 4 days per week for a total of 4 cycles.

Results: Based on analysis of bisulfite sequencing data from DepMap, RMS and NBL cells exhibit a high rate of NAPRT promoter methylation. Transcriptomic and proteomic profiling from DepMap demonstrated accompanying loss of NAPRT expression. To validate these findings, we directly tested for NAPRT expression in six NBL cell lines, two of which were predicted to express functional NAPRT enzyme, and four that were predicted to be NAPRT-silenced. We also tested six RMS tumor cell lines predicted to be NAPRT-silenced. NAPRT status as predicted by DepMap analysis aligned with western blot findings. In vitro, NAPRT-silenced cells showed exquisite sensitivity to NAMPTi even with NA supplementation, while NAPRT expressing cells were completely rescued when NA was added to growth media. This NAMPTi-sensitivity was further mirrored by changes in NAD levels. In vivo, NAMPTi treatment resulted in significant tumor regression of NAPRT-silenced RD xenografts.

Conclusion: Overall, this study identified high rates of NAPRT promoter methylation and loss of NAPRT expression in NBL and RMS tumor cell lines, along with significant sensitivity to NAMPTi. These data suggest that NAPRT loss may serve as a biomarker of NAMPTi sensitivity in NBL and RMS. Future studies are needed to validate these findings in isogenic and patient-derived xenograft (PDX) models, and to evaluate the expression of NAPRT in primary patient tumors. Consideration should also be given to the potential of targeting NAD metabolism as a strategy to enhance the efficacy of existing cytotoxic chemotherapy in these difficult-to-treat tumors.