{"doi":"10.1101/2023.02.19.529123","title":"Alternative splicing of its 5’-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies","abstract":"ABSTRACT Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the MS4A1 gene encoding human CD20 also produces several mRNA isoforms with distinct 5’ untranslated regions (5’-UTR). Four variants (V1-4) were detectable by RNA-seq in distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant by far. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform was found to contain upstream open reading frames (uORFs) and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching Morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, while V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed CAR T cells were able to kill both V3- and V1-expressing cells, but the bispecific T cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on four post-mosunetuzumab follicular lymphoma relapses and discovered that in two of them downregulation of CD20 was accompanied by the V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies. Key Points In normal & malignant human B cells, CD20 mRNA is alternatively spliced into four 5’-UTR isoforms, some of which are translation-deficient. The balance between translation-deficient and -competent isoforms modulates CD20 protein levels & responses to CD20-directed immunotherapies Explanation of Novelty We discovered that in normal and malignant B-cells, CD20 mRNA is alternatively spliced to generate four distinct 5’-UTRs, including the longer translation-deficient V1 variant. Cells predominantly expressing V1 were still sensitive to CD20-targeting chimeric antigen receptor T-cells. However, they were resistant to the bispecific anti-CD3/CD20 antibody mosunetuzumab, and the shift to V1 were observed in CD20-negative post-mosunetuzumab relapses of follicular lymphoma.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":392202,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":5,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.955,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1032169,"name":"Luca Paruzzo","orcid":"0000-0002-6505-0194","position":1,"is_corresponding":false},{"id":244454,"name":"Katharina E. Hayer","orcid":"0000-0002-1463-3111","position":2,"is_corresponding":false},{"id":656015,"name":"Carolin Schmidt","orcid":null,"position":3,"is_corresponding":false},{"id":655021,"name":"Manuel Torres-Diz","orcid":"0000-0002-5487-8113","position":4,"is_corresponding":false},{"id":446028,"name":"Feng Xu","orcid":"0000-0002-7227-7426","position":5,"is_corresponding":false},{"id":1032170,"name":"Urvi Zankharia","orcid":"0000-0002-4980-390X","position":6,"is_corresponding":false},{"id":1032171,"name":"Yunlin Zhang","orcid":"0000-0002-5608-0615","position":7,"is_corresponding":false},{"id":348685,"name":"Samantha S. Soldan","orcid":"0000-0001-6263-519X","position":8,"is_corresponding":false},{"id":656011,"name":"Sisi Zheng","orcid":null,"position":9,"is_corresponding":false},{"id":1032774,"name":"Catherine D. Falkenstein","orcid":null,"position":10,"is_corresponding":false},{"id":324652,"name":"Joseph P. Loftus","orcid":null,"position":11,"is_corresponding":false},{"id":941312,"name":"Scarlett Y. Yang","orcid":"0000-0002-9398-7363","position":12,"is_corresponding":false},{"id":27330,"name":"Mukta Asnani","orcid":"0000-0002-6088-5010","position":13,"is_corresponding":false},{"id":1032775,"name":"Patricia King Sainos","orcid":null,"position":14,"is_corresponding":false},{"id":623398,"name":"Vinodh Pillai","orcid":"0000-0001-7126-6226","position":15,"is_corresponding":false},{"id":1032776,"name":"Emeline Chong","orcid":null,"position":16,"is_corresponding":false},{"id":446032,"name":"Marilyn M. Li","orcid":"0000-0002-4253-2369","position":17,"is_corresponding":false},{"id":273631,"name":"Sarah K. Tasian","orcid":"0000-0003-1327-1662","position":18,"is_corresponding":false},{"id":281155,"name":"Yoseph Barash","orcid":"0000-0003-3005-5048","position":19,"is_corresponding":false},{"id":299552,"name":"Paul M. Lieberman","orcid":"0000-0002-3935-9921","position":20,"is_corresponding":false},{"id":256650,"name":"Marco Ruella","orcid":"0000-0003-4301-5811","position":21,"is_corresponding":false},{"id":273372,"name":"Stephen J. Schuster","orcid":"0000-0002-3376-8978","position":22,"is_corresponding":false},{"id":96147,"name":"Andrei Thomas‐Tikhonenko","orcid":"0000-0002-2739-2206","position":23,"is_corresponding":false},{"id":1032168,"name":"Zhiwei Ang","orcid":"0000-0001-6583-4733","position":0,"is_corresponding":true}],"reference_count":88,"raw_metadata":null,"created_at":"2026-07-19T01:18:52.232318Z","pmid":"37645778","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}