Exploring the Therapeutic Potential of OT-82 in Ewing Sarcoma: Targeting the NAD Salvage Pathway

The abstract discusses the exploration of NAMPT inhibitors (NAMPTi) as a potential anticancer strategy, specifically in Ewing sarcoma (ES), a pediatric cancer lacking effective treatments. The study highlights that ES cells are highly sensitive to NAMPTi, showing promising results both in vitro and in vivo. The combination of NAMPTi with PARP inhibitors has shown to enhance effectiveness. The research focuses on OT-82, a NAMPTi under early clinical trial consideration, to understand its impact on ES cells and to assess various treatment schedules in combination with cytotoxic therapy.

OT-82's activity was measured through NAD quantification and rescue experiments, while the expression of NAD salvage pathway enzymes was determined via western blot. The study involved cell cycle analysis, DNA damage assessment, and cell death evaluation using flow cytometry, western blotting, and comet assays. The glycolytic profiles of ES cell lines were examined with an Agilent Extracellular Flux Analyzer. In vivo studies utilized immunodeficient mice with orthotopic ES cell lines and PDX models to test the combination of OT-82 and irinotecan, observing enhanced antitumor effects.

OT-82 treatment led to a dose-dependent reduction in NAD levels both outside and inside the cell. The addition of NMN, a product of NAMPT, rescued cell viability, confirming OT-82's on-target activity. Delayed NMN administration post-treatment could rescue viability up to 48 hours, but not after 72 hours. DNA damage, cell cycle arrest, and apoptosis induction were observed at 72 hours post-treatment. The extracellular flux analysis showed a decrease in oxidative and glycolytic activity due to OT-82 treatment. While NAMPT expression did not predict sensitivity to OT-82, low NAPRT expression, part of a parallel NAD salvage pathway, indicated higher sensitivity. Cells expressing NAPRT could be rescued with NA, its substrate, while low expressers could not. Ongoing studies are examining dosing schedules and tumor biology, with results to be reported. OT-82 is identified as a targeted NAMPTi with potential for treating ES, particularly in strategic combinations.