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bioRxiv · 10.64898/2026.03.17.712388

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

Ferrao Blanco, M. N.·Kazybay, B.·Perzolli, A.·Kester, L.·Heidenreich, O.·Vormoor, H. J.
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Abstract

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.

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Ferrao Blanco, M. N., Kazybay, B., Perzolli, A., Kester, L., Heidenreich, O., Vormoor, H. J.. 2026-03-18. The bone marrow microenvironment of RAS pathway mutant B-ALL is enriched for immunosuppressive regulatory T cells. https://doi.org/10.64898/2026.03.17.712388

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