biostudies-literatureParas KIMemorial Sloan-Kettering Cancer Center (MSK)NICHD NIH HHSDamon Runyon Cancer Research Foundation (DRCRF)PaulieStrong FoundationGeoffrey Beene Cancer Research CenterPershing Square Sohn Cancer Research Alliance (PSSCRA)New York Stem Cell Foundation (NYSCF)National Cancer Institute (NCI)NCI NIH HHSEunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)Human Frontier Science Program (HFSP)NIGMS NIH HHSNational Institute of General Medical Sciences (NIGMS)4718-4733https://www.ebi.ac.uk/biostudies/studies/S-EPMC12477665biostudies-literatureUnknown85(23)PAX3-FOXO1, an oncogenic transcription factor, drives a particularly aggressive subtype of rhabdomyosarcoma (RMS) by enforcing gene expression programs that support malignant cell states. In this study, we show that PAX3-FOXO1+ RMS cells exhibit altered pyrimidine metabolism and increased dependence on enzymes involved in de novo pyrimidine synthesis, including dihydrofolate reductase (DHFR). Consequently, PAX3-FOXO1+ cells display increased sensitivity to inhibition of DHFR by the chemotherapeutic drug methotrexate, and this dependence is rescued by provision of pyrimidine nucleotides. Methotrexate treatment mimics the metabolic and transcriptional impact of PAX3-FOXO1 silencing, reducing expression of genes related to PAX3-FOXO1-driven malignant cell states. Accordingly, methotrexate treCancer researchPAX3-FOXO1 Drives Targetable Cell State-Dependent Metabolic Vulnerabilities in Rhabdomyosarcoma.PMC12477665T32 HD060600P30CA08748T32HD060600P30 CA008748L1000200/2021-LF30 HD107943F30CA284711F30HD107943F30 CA284711T32 GM152349T32GM152349Siddiquee ABoyer JARabinowitz JDDela Cruz FSParas KIFinley LWSGuillan KBrunner JSPajuelo Torres LMontero AMYou DChakraborty SJackson BTXie AKung ALfalsePAX3-FOXO1 Drives Targetable Cell State-Dependent Metabolic Vulnerabilities in Rhabdomyosarcoma.PAX3-FOXO1, an oncogenic transcription factor, drives a particularly aggressive subtype of rhabdomyosarcoma (RMS) by enforcing gene expression programs that support malignant cell states. In this study, we show that PAX3-FOXO1+ RMS cells exhibit altered pyrimidine metabolism and increased dependence on enzymes involved in de novo pyrimidine synthesis, including dihydrofolate reductase (DHFR). Consequently, PAX3-FOXO1+ cells display increased sensitivity to inhibition of DHFR by the chemotherapeutic drug methotrexate, and this dependence is rescued by provision of pyrimidine nucleotides. Methotrexate treatment mimics the metabolic and transcriptional impact of PAX3-FOXO1 silencing, reducing expression of genes related to PAX3-FOXO1-driven malignant cell states. Accordingly, methotrexate tre2025-01-01T00:00:00Z2025 Dec2026-06-06T13:41:22.857Z2026-05-31T03:09:23.743ZS-EPMC124776654091178410.1158/0008-5472.CAN-25-031510.1158/0008-5472.can-25-0315