U1 snRNA mutation reshapes tumor microenvironment in chronic lymphocytic leukemia: a role for CD44-mediated signaling

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of monoclonal mature B lymphocytes in peripheral blood, bone marrow, lymphoid tissues, and extranodal sites. Genes involved in RNA splicing such as SF3B1 and U1 are frequently mutated in CLL, leading to altered splicing and generation of tumor neoepitopes. To study the impact of these mutations on the tumor microenvironment (TME), we have developed a comprehensive single-cell atlas of unmutated CLL encompassing 26 bone marrow and lymph node tumor samples from 23 U-CLL patients with mutations in U1 (n=7), SF3B1 (n=8), or without mutations in splicing genes (n=10). We observed high intra-tumor heterogeneity, discerning 12 transcriptional programs, one linked to the U1 g.A3>C mutation and characterized by NFKB hyperactivation. T cell and NK compartments exhibited site- and mutation-specific enrichment, with increased CD4 ⁺ regulatory cells (Treg) and CD8 ⁺ exhausted cells in lymph nodes, while U1 -mutant tumors showed increased CD8 ⁺ cytotoxic activity, with a predominance of effector-like CD8 ⁺ cells. Single-cell T cell receptor sequencing revealed clonotype expansion in U1 -mutated tumors, particularly in CD8 ⁺ effector and exhausted cells, suggesting a neoantigen-driven immune response. Cell-to-cell interaction analysis identified CD44 as a key mediator in U1 -mutated tumors, showing pro-B survival interactions as those involving MIF-CD44-CD74. Furthermore, interactions between CD80 on CLL cells and CTLA4 on Tregs and CD8 ⁺ exhausted were upregulated, reflecting an immunosuppressive phenotype associated with U1 mutated CLL. These findings highlight the complex interplay between mutations in CLL and the TME, offering novel avenues for alternative therapeutic strategies for U-CLL with mutations in U1 .