Alternative splicing of its 5'-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies.

Abstract

Aberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5' untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. 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 contained upstream open reading frames 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, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor 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 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.

• Authors: Zhiwei Ang, Luca Paruzzo, Katharina E Hayer, Carolin Schmidt, Manuel Torres Diz, Feng Xu, Urvi Zankharia, Yunlin Zhang, Samantha Soldan, Sisi Zheng, Catherine D Falkenstein, Joseph P Loftus, Scarlett Y Yang, Mukta Asnani, Patricia King Sainos, Vinodh Pillai, Emeline Chong, Marilyn M Li, Sarah K Tasian, Yoseph Barash, Paul M Lieberman, Marco Ruella, Stephen J Schuster, Andrei Thomas-Tikhonenko
• Journal: Blood (Blood) Vol. 142 Issue 20 Pg. 1724-1739 (11 16 2023) ISSN: 1528-0020 [Electronic] United States
• PMID: 37683180 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, N.I.H., Extramural)
• Copyright: © 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

Chemical References

• RNA, Messenger
• 5' Untranslated Regions
• Antigens, CD20
• Protein Isoforms

Topics

• Humans
• Alternative Splicing
• RNA, Messenger (genetics)
• 5' Untranslated Regions
• Epstein-Barr Virus Infections (genetics)
• Herpesvirus 4, Human (genetics)
• Antigens, CD20 (genetics)
• Protein Isoforms (genetics)
• Immunotherapy
• Protein Biosynthesis
• Neoplasms (genetics)