Abstract
BOB.1, encoded by POU2AF1, is one of many factors regulating physiological B-cell maturation in the germinal center. Recently, several studies have described recurrent mutations in a three-nucleotide region in the POU2AF1 splice site in the two most common B-cell non-Hodgkin lymphomas: diffuse large B-cell lymphoma and, more frequently, follicular lymphoma. In this study, we introduced a C→G mutation at the + 1 position of the POU2AF1 splice site in two B-cell lymphoma cell lines (WSU-NHL and SUDHL4) using CRISPR/Cas9 gene editing. Our results demonstrate how point mutations in the POU2AF1 splice site decreased BOB.1 expression levels. The mutation did not produce significant changes in cell proliferation, migration, or invasiveness, but did affect cell morphology, aggregation, and cell survival in a cell-line-dependent manner. Lastly, we found that the POU2AF1 mutation c.16 + 1G > C increased BCR activation, especially in SUDHL4 cells, downregulated oxidative phosphorylation (OxPhos) metabolism, and modified therapy sensitivities in both cell lines. Mutated B-cells were more sensitive to the BTK inhibitor ibrutinib. In conclusion, mutations in the POU2AF1 splice site impact B-cell lymphomagenesis at multiple levels and represent a potential therapeutic target for patients with tumors harboring this mutation.
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Data availability
The datasets generated and/or analysed during the current study are available in the Sequence Read Archive (SRA) repository, BioProject code PRJNA1289409, [https://dataview.ncbi.nlm.nih.gov/object/PRJNA1289409?reviewer=37slsedlut6pfr1rbgp8bkd2os](https:/dataview.ncbi.nlm.nih.gov/object/PRJNA1289409?reviewer=37slsedlut6pfr1rbgp8bkd2os) .
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Acknowledgements
We want to thank Dr. Giovanna Roncador, Head of the Monoclonal Antibodies Unit at the Spanish National Cancer Research Center (CNIO, Madrid, Spain), and Dr. Patricia Pérez-Galán (Institut d’Investigacions Biomèdiques August Pi i Sunyer, IDIBAPS, Spain), for donating some cell lines. This work was supported by: Spanish Ministry of Economy and Competence (MINECO) and Instituto de Salud Carlos III (ISCIII), ISCIII-MINECO AES-FEDER (PI17/00272, PI20/00591, PI23/01587); Dirección General de Universidades e Investigación de la Consejería de Educación e Investigación de la Comunidad de Madrid (CM) (B2017/BMD-3778); and Fundación de Investigación Biomédica H. U. Puerta de Hierro-Majadahonda (FIB HUPHM), Madrid. L.P. received an iPFIS predoctoral fellowship (IFI18/0004) ISCIII-MINECO AES-FEDER, Plan Estatal I+D+I 2014-2020). I.F.M. was supported by B2017/BMD-3778 and the FIB HUPHM. N.Y.C. is supported by the Fundación Científica de la Asociación Española Contra el Cáncer (POSTD18029SANC). B.H. and M.P.A. are supported by the Plan de Empleo Juvenil de la CM (PEJ-2020-TL/BMD-19530, and PEJ-2023-AI/SAL-GL-28806 respectively).
Funding
This work was supported by: Spanish Ministry of Economy and Competence (MINECO) and Instituto de Salud Carlos III (ISCIII), ISCIII-MINECO AES-FEDER (PI17/00272, PI20/00591, PI23/01587); Dirección General de Universidades e Investigación de la Consejería de Educación e Investigación de la Comunidad de Madrid (CM) (B2017/BMD-3778); and Fundación de Investigación Biomédica H. U. Puerta de Hierro-Majadahonda (FIB HUPHM), Madrid. L.P. received an iPFIS predoctoral fellowship (IFI18/0004) ISCIII-MINECO AES-FEDER, Plan Estatal I + D+I 2014–2020). I.F.M. was supported by B2017/BMD-3778 and the FIB HUPHM. N.Y.C. is supported by the Fundación Científica de la Asociación Española Contra el Cáncer (POSTD18029SANC). B.H. and M.P.A. are supported by the Plan de Empleo Juvenil de la CM (PEJ-2020-TL/BMD-19530, and PEJ-2023-AI/SAL-GL-28806 respectively).
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N.Y.C. designed the study, performed experimental procedures and analyzed data, performed the statistical analysis, and wrote the paper; L.P., B.H. and S.G. performed experimental procedures; A.G.G. was involved in the flow cytometry assays; R.M.V., I.F.M. and M.P.A. performed the bioinformatics analysis and data processing; R.T.R and S.R.P were involved in the design of genetic edition procedures; M.S.B. designed the study, discussed and analyzed results and wrote the paper. All the authors reviewed and approved the manuscript.
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Yanguas-Casás, N., Pedrosa, L., Horcajo, B. et al. Splice-site mutations in POU2AF1 are associated with B-cell lymphomagenesis and therapeutic response. Sci Rep (2026). https://doi.org/10.1038/s41598-026-43710-6
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DOI: https://doi.org/10.1038/s41598-026-43710-6
