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

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.

An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia.

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B- ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.

An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia.

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B-ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.

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

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.