Abstract 288: Metabolic profiling of MEKi-treated fusion-negative rhabdomyosarcoma cell lines suggests a role for glutamine antagonism as a preclinical therapeutic approach

Abstract Improved genetic characterization of rhabdomyosarcoma (RMS) subtypes, including PAX3/PAX7 fusion-negative RMS (FN-RMS) with RAS pathway mutations [1], has led to hope for individualized treatments with fewer side effects than conventional chemotherapy. Understanding the metabolic vulnerabilities of RMS remains an area with room for improvement. We hypothesized that determining the metabolic effects of RAS pathway inhibition in RAS-active RMS could inform new therapeutic combinations. We performed polar metabolomic profiling comparing trametinib and DMSO-treated FN-RMS cell lines, including two cell types with RAS mutations (SMS-CTR and RD) and one with an FGFR1 amplification (RMS-YM). Although inhibition of phospho-ERK was observed after 6 hours of treatment with trametinib in cells, limited effects on polar metabolites were observed at this time point. Following 24h of trametinib treatment, we observed alterations in purine and pyrimidine metabolism, amino acid metabolism, and central carbon metabolism. Based on these findings and our prior research on metabolic vulnerabilities other RAS-active sarcomas [2], we investigated RMS cell dependence on glutamine, the most abundant amino acid in serum and a crucial metabolic contributor of nitrogen and carbon substrates to these pathways [3]. FN-RMS cell lines demonstrated reduced colony formation in media with low glutamine. The clinical glutamine antagonist, DRP-104 [4], inhibited the growth of RAS-RMS cells with IC50 values in the low micromolar range. We investigated the effects of DRP-104 on markers of tumor growth, DNA damage, and cell death by western blotting. As our own and other data support a role for MEKi and glutamine antagonism in RAS-active tumor models [5], future investigations will seek to combine DRP-104 with trametinib in cell-based and in vivo RAS-RMS models. References [1]Shern, J. F., et al. (2015). "Pediatric Rhabdomyosarcoma." Crit Rev Oncog 20(3-4): 227-243.[2]Lemberg, K. M., et al. (2023). "Pro-905, a Novel Purine Antimetabolite, Combines with Glutamine Amidotransferase Inhibition to Suppress Growth of Malignant Peripheral Nerve Sheath Tumor." Mol Cancer Ther 22(12): 1390-1403.[3]Yoo, H. C., et al. (2020). "Glutamine reliance in cell metabolism." Exp Mol Med 52(9): 1496-1516.[4]Rais, R., et al. (2022). "Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug." Sci Adv 8(46): eabq5925.[5]Encarnacion-Rosado, J., et al. (2024). "Targeting pancreatic cancer metabolic dependencies through glutamine antagonism." Nat Cancer 5(1): 85-99. Citation Format: Konstantinos Flamourakis, Lior Levy, Jingdong Tan, Kathryn M. Lemberg. Metabolic profiling of MEKi-treated fusion-negative rhabdomyosarcoma cell lines suggests a role for glutamine antagonism as a preclinical therapeutic approach [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 288.