Julia Stefanie Brunner

Inflammatory responses are associated with recruitment of monocyte-derived cells (Mdcs) into tissues. Although tissue-specific Mdc reprogramming is well established, how Mdc infiltration alters tissue metabolism remains unclear. Here, using a mouse neuroinflammation model coupled with genetic fate mapping, metabolomics and metabolite imaging, we identify that central nervous system (CNS) Mdc infiltration is associated with substantial metabolic changes and assign disease-linked metabolites therein. In particular, we found that increased arginine catabolism driven by lesion-associated arginase 1 ( Arg1 )-expressing Mdcs promoted oxidative damage, lipid accumulation and Mdc dysfunction. Genetic ARG1 deficiency within Mdcs during neuroinflammation increased extracellular arginine and was associated with rewiring of the CNS metabolic landscape, including attenuated disease-linked metabolites. This was accompanied by enhanced Mdc-driven anti-inflammation, regulatory T cell expansion and improved disease outcome. Opposing effects were observed following dietary arginine deficiency. Together, our work highlights key roles for Mdcs in CNS metabolism and reveals the pleiotropic beneficial effects of arginine in neuroinflammation.

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 treatment slows the growth of multiple PAX3-FOXO1+ tumor xenograft models but not their fusion-negative counterparts. Taken together, these data demonstrate that PAX3-FOXO1 induces cell states characterized by altered pyrimidine dependence and nominates methotrexate as an addition to the current therapeutic arsenal for treatment of these malignant pediatric tumors.Significance:PAX3-FOXO1+ rhabdomyosarcoma cells and tumors exhibit increased sensitivity to DHFR inhibition via methotrexate, identifying a potential therapeutic vulnerability that can be exploited to treat this aggressive pediatric sarcoma.

Supplementary Table 1 from PAX3-FOXO1 Drives Targetable Cell State–Dependent Metabolic Vulnerabilities in Rhabdomyosarcoma

Additional data from in vivo studies (related to Figure 5)

Characterization of the PAX3-FOXO1+ RMS cell state (related to Figure 1)

Further validation of DepMap differential essentiality analysis (related to Figure 2)

Metabolic profiling of RMS cell lines (related to Figure 3)

Results of DepMap differential essentiality analysis

Further characterization of the effects of MTX or PAX3-FOXO1 knockdown (related to Figure 4)

Supplementary Table 2 from PAX3-FOXO1 Drives Targetable Cell State–Dependent Metabolic Vulnerabilities in Rhabdomyosarcoma