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

Abstract Purpose New treatments are needed to improve survival in children with rhabdomyosarcoma (RMS). NAD⁺ biosynthesis, regulated by the enzymes NAPRT and NAMPT, represents a metabolic vulnerability due to high NAD⁺ turnover in cancers. Although NAMPT inhibitors (NAMPTi) show preclinical promise, clinical translation has been limited by toxicity and the lack of predictive biomarkers. Here, we evaluated NAPRT expression in RMS and its potential as an actionable biomarker to guide NAMPTi therapy. Experimental Design NAPRT promoter methylation, transcript levels, and protein expression were assessed in RMS cells, PDXs, and primary tumors (n=109) from the Children’s Oncology Group. In vitro sensitivity to NAMPTi was tested in molecularly diverse and isogenic RMS cell lines, examining the role of NAPRT expression in mediating cytotoxicity and the ability of nicotinic acid (NA) to rescue viability. In vivo efficacy was assessed using NAPRT-isogenic orthotopic xenograft models. Results NAPRT promoter hypermethylation was found in a subset of RMS models and patient samples. Immunohistochemistry showed loss of NAPRT protein in 30–40% of tumors, defined as <1% tumor cell staining. Methylation modestly correlated with protein expression. NAPRT-silenced cells were highly sensitive to NAMPTi, driven by NAD⁺ depletion and not reversible with NA. In vivo, NAMPTi induced significant tumor regression, which was not abrogated with NA administration in NAPRT-silenced models. Conclusions NAPRT loss occurs in a subset of RMS, offering a potential strategy to expand the therapeutic window of NAMPTi. Further research is needed to understand NAPRT regulation and optimize biomarker assay strategies for use in future clinical trials.