Abstract 6415: NAPRT expression and epigenetic regulation in pediatric rhabdomyosarcoma as a potential biomarker for NAMPT inhibition

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, with an overall survival rate of less than 20% for patients with advanced disease. Therefore, exploring novel therapeutic strategies is crucial for improving prognosis. One potential approach involves targeting nicotinamide adenine dinucleotide (NAD) biosynthesis pathways, primarily driven by NAPRT and NAMPT, key enzymes of the Preiss-Handler and Salvage pathways, respectively. Due to increased metabolic demands, tumors have a high NAD+ turnover. NAD+ depletion with small molecule NAMPT inhibitors (NAMPTi) has demonstrated significant anti-tumor activity, but success in clinical trials has been limited due to dose-limiting toxicities and the lack of a predictive biomarker. Probing data extrapolated from DepMap, we identified a high frequency of NAPRT gene promoter methylation in RMS cell line models. Therefore, we hypothesized that loss of NAPRT expression due to tumor-specific promoter CpG island methylation in RMS may serve as a biomarker for synthetic lethal interaction with NAMPTi. To extend our findings from cell lines, we analyzed methylation array data from 99 RMS patient samples and identified a subset of tumors with hypermethylation of the NAPRT promoter. NAPRT protein expression was assessed via immunohistochemistry (IHC) using a newly developed and validated NAPRT monoclonal antibody (4A5D7) on tissue microarrays (TMAs) from the Children's Oncology Group (COG), comprising 278 cores from 130 patients aged 0-18 years. Loss of NAPRT expression, defined as < 1% of tumor cells with positive staining, was observed in approximately 40% of tumors. There was no correlation between FOXO1 fusion status and positive staining. Corresponding methylation data was available for 23 of the samples in the TMA and was used to assess correlation between NAPRT promoter methylation and loss of NAPRT protein expression. To investigate the role of NAPRT in mediating sensitivity to NAMPTi, we conducted in vitro cell proliferation assays using isogenic RMS models. NAPRT-silenced cells exhibited pronounced sensitivity to NAMPTi, which was driven by NAD depletion and not reversible with nicotinic acid (NA) supplementation. In vivo, NAMPTi induced significant tumor regression in NAPRT-silenced orthotopic RMS models and this anti-tumor effect was not abrogated with NA administration. Future research is also needed to elucidate mechanisms of NAPRT regulation beyond methylation and to determine the optimal biomarker assay modality. Overall, our results suggest that NAPRT expression is lost in a subset of pediatric RMS and warrants evaluation as a potential biomarker in future clinical trials using NAMPTi. Citation Format: Angela Kim, Prateek Bhardwaj, Sophia J. Zhao, Karlie N. Lucas, Katelyn J. Noronha, Sam Friedman, Collin D. Heer, Raffaella Morotti, Ranjini K. Sundaram, Wenyue Sun, Frederic Barr, Josh Spurrier, Mitch Raponi, Juan C. Vasquez. NAPRT expression and epigenetic regulation in pediatric rhabdomyosarcoma as a potential biomarker for NAMPT inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6415.