The bone marrow microenvironment of RAS pathway mutant B-ALL is enriched for immunosuppressive regulatory T cells

Somatic mutations in the RAS pathway are highly prevalent in B-Cell Acute Lymphoblastic Leukemia (B-ALL), yet their impact on the bone marrow immune microenvironment and response to immunotherapy remains poorly defined. In this study, we integrated bulk RNA-sequencing, single-cell RNA-sequencing (scRNA-seq), and spectral flow cytometry to characterize the immune landscape of RAS-mutant B-ALL. We identified pathogenic mutations in KRAS, NRAS, PTPN11, or BRAF in 42% of the cohort, predominantly as clonal events. Despite similar T-cell frequencies by flow cytometry, bulk transcriptomes from RAS-mutant samples showed suppression of immune-response and T-cell-activation pathways, and T cells from RAS-mutant patients exhibited impaired proliferation ex vivo. Single-cell analysis revealed higher CD8 dysfunction scores and enrichment of regulatory T cells (Tregs) in RAS-mutant bone marrow. These findings were validated by spectral flow cytometry and by CIBERSORTx deconvolution of bulk data. Trajectory analysis supported a higher CD4 to Treg differentiation in the RAS-mutant niche, and CellChat mapping identified contact-dependent and checkpoint interactions (including TIGIT-NECTIN2 and CTLA-4-CD86/ICOSL) enriched in RAS-mutant samples. Functionally, blinatumomab produced limited leukemic-cell killing ex vivo overall, but addition of CTLA-4 blockade (ipilimumab) selectively restored blinatumomab efficacy in RAS-mutant samples. Together, these results indicate that RAS-pathway activation associates with a Treg-enriched, immunosuppressive bone-marrow microenvironment and point to CTLA-4-targeted strategies to enhance T-cell-engager efficacy in this subgroup.