TY - JOUR
T1 - Alternative splicing of its 5′-UTR limits CD20 mRNA translation and enables resistance to CD20-directed immunotherapies
AU - Ang, Zhiwei
AU - Paruzzo, Luca
AU - Hayer, Katharina E.
AU - Schmidt, Carolin
AU - Torres Diz, Manuel
AU - Xu, Feng
AU - Zankharia, Urvi
AU - Zhang, Yunlin
AU - Soldan, Samantha
AU - Zheng, Sisi
AU - Falkenstein, Catherine D.
AU - Loftus, Joseph P.
AU - Yang, Scarlett Y.
AU - Asnani, Mukta
AU - King Sainos, Patricia
AU - Pillai, Vinodh
AU - Chong, Emeline
AU - Li, Marilyn M.
AU - Tasian, Sarah K.
AU - Barash, Yoseph
AU - Lieberman, Paul M.
AU - Ruella, Marco
AU - Schuster, Stephen J.
AU - Thomas-Tikhonenko, Andrei
N1 - Publisher Copyright:
© 2023 The American Society of Hematology
PY - 2023/11/16
Y1 - 2023/11/16
N2 - 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.
AB - 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.
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U2 - 10.1182/blood.2023020400
DO - 10.1182/blood.2023020400
M3 - Article
C2 - 37683180
AN - SCOPUS:85173897526
SN - 0006-4971
VL - 142
SP - 1724
EP - 1739
JO - Blood
JF - Blood
IS - 20
ER -