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.

F.N. received honoraria from Janssen, AbbVie, AstraZeneca, and SOPHiA GENETICS for speaking in educational activities, and research funding from Gilead. E.C. has been a consultant for Takeda; has received honoraria from Janssen, EUSA Pharma and Roche for speaking at educational activities and research funding from AstraZeneca and is an inventor on 2 patents filed by the National Institutes of Health, National Cancer Institute: Methods for selecting and treating lymphoma types, licensed to NanoString Technologies, and Evaluation of mantle cell lymphoma and methods related thereof, not related to this project. F.N. and E.C. licensed the use of the protected IgCaller algorithm for Diagnostica Longwood. The remaining authors declare no competing financial interests.

This study was supported by Ministerio de Ciencia e Innovacion PID2020-117185RB-I00 and PID2023-148997OB-I00 to X.S.P., PID2021-123054OB-I00 to E.C. and PID2021-123165OB-I00 to D.C.; the Spanish Association Against Cancer (AECC PRYGN211258SUAR) to X.S.P.; La Caixa Foundation CLLSYSTEMS (HR22-00172); European Union NextGenerationEU/Mecanismo para la Recuperacion y la Resilencia (MRR)/PRTR and the Instituto de Salud Carlos III (ISCIII) (PMP21/00015); Centro de Investigacion Biomedica en Red Cancer (CIBERONC). E.C. acknowledges the Generalitat de Catalunya Suport Grups de Recerca AGAUR (2021-SGR-01172). F.N. acknowledges research support from the American Association for Cancer Research (2021 AACR-Amgen Fellowship in Clinical/Translational Cancer Research, 21-40-11-NADE), European Hematology Association (EHA Junior Research Grant 2021, RG-202012-00245), and Lady Tata Memorial Trust (International Award for Research in Leukaemia 2021-2022, LADY_TATA_21_3223). E.C. is an Academia Researcher of the Institucio Catalana de Recerca i Estudis Avancats (ICREA) of the Generalitat de Catalunya. S.L-T. is supported by a Severo Ochoa fellowship from the Asturian government. We also acknowledge support from the Institute of Oncology of Asturias (IUOPA, supported by Obra Social Cajastur, Spain).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The Comite de Etica de la Investigacion con Medicamentos del Principado de Asturias has granted premision to carray out the research "Characterization of functional mutations in cancer driver genes involved in RNA maturation" with Ethics Committee code CEImPA 2020.043.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced are available online at European Genome-phenome Archive (EGA) repository, under the accession number EGA50000000572. Additionally, the anndata, NMF, and TCA outputs can be accessed via Zenodo (DOI: 10.5281/zenodo.13835696). The code used in this paper is publicly accessible in a GitHub repository (https://github.com/xa-lab) for reproducibility.