SF3B1 mutations are recurrent in chronic lymphocytic leukemia (CLL), particularly enriched in clinically aggressive stereotyped subset #2. To investigate their impact, we conducted RNA-sequencing of 18 SF3B1MUT and 17 SF3B1WT subset #2 cases and identified 80 significant alternative splicing events (ASEs). Notable ASEs concerned exon inclusion in the non-canonical BAF (ncBAF) chromatin remodeling complex subunit, BRD9, and splice variants in eight additional ncBAF complex interactors. Long-read RNA-sequencing confirmed the presence of splice variants, and extended analysis of 139 CLL cases corroborated their association with SF3B1 mutations. Overexpression of SF3B1K700E induced exon inclusion in BRD9, resulting in a novel splice isoform with an alternative C-terminus. Protein interactome analysis of the BRD9 splice isoform revealed augmented ncBAF complex interaction, while exhibiting decreased binding of auxiliary proteins, including SPEN, BRCA2, and CHD9. Additionally, integrative multi-omics analysis identified a ncBAF complex-bound gene quartet on chromosome 1 with higher expression levels and more accessible chromatin in SF3B1MUT CLL. Finally, Cancer Dependency Map analysis and BRD9 inhibition displayed BRD9 dependency and sensitivity in cell lines and primary CLL cells. In conclusion, spliceosome dysregulation caused by SF3B1 mutations leads to multiple ASEs and an altered ncBAF complex interactome, highlighting a novel pathobiological mechanism in SF3B1MUT CLL.

Central European Institute of Technology Masaryk University Brno Czech Republic

Clinical Genetics and Genomics Karolinska University Laboratory Karolinska University Hospital Stockholm Sweden

Department of Biology School of Science National and Kapodistrian University of Athens Athens Greece

Department of Hematology Hospital Pitie Salpetriere Sorbonne University Paris France

Department of Hematology Oncology Institute of Southern Switzerland and Institute of Oncology Research Bellinzona Switzerland

Department of Immunology Erasmus MC University Medical Center Rotterdam Rotterdam Netherlands

Department of Immunology Genetics and Pathology Science for Life Laboratory Uppsala University Uppsala Sweden

Department of Immunology Mayo Clinic Rochester USA

Department of Internal Medicine Hematology and Oncology Medical Faculty Masaryk University and University Hospital Brno Brno Czech Republic

Department of Molecular Medicine and Surgery Karolinska Institutet Stockholm Sweden

Department of Oncology Pathology Science for Life Laboratory Karolinska Institutet Stockholm Sweden

Division of Experimental Oncology IRCCS Ospedale San Raffaele Milan Italy

Division of Hematology Department of Internal Medicine Mayo Clinic Rochester MN USA

Division of Hematology Department of Medicine Stanford University Medical Center Stanford CA USA

Hematology Department and HCT Unit G Papanicolaou Hospital Thessaloniki Greece

Institute of Applied Biosciences Centre for Research and Technology Hellas Thessaloniki Greece

Institute of Medical Genetics and Genomics Medical Faculty Masaryk University and University Hospital Brno Brno Czech Republic

Università Vita Salute San Raffaele Milan Italy

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