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.

FBXW7ß isoform drives transcriptional activation of the proinflammatory TNF cluster in human pro-B cells.

Noncanonical exon usage plays many important roles in cellular phenotypes, but its contribution to human B-cell development remains sketchily understood. To fill this gap, we collected various B-cell fractions from bone marrow (BM) and tonsil donors, performed RNA sequencing, and examined transcript variants. We identified 150 genes that harbor local splicing variations in all pairwise comparisons. One of them encodes FBXW7, an E3 ubiquitin ligase implicated as a driver in several blood cancers. Surprisingly, we discovered that in normal human pro-B cells, the predominant transcript used an alternative first exon to produce the poorly characterized FBXW7ß isoform, previously thought to be restricted to neural tissues. The FBXW7ß transcript was also abundant in cell lines and primary samples of pediatric B-cell acute lymphoblastic leukemia (B-ALL), which originates in the BM. When overexpressed in a heterologous cell system, this transcript yielded the expected protein product, as judged by anti-FLAG immunoblotting and mass spectrometry. Furthermore, in REH B-ALL cells, FBXW7ß mRNA was the only FBXW7 isoform enriched in the polyribosome fraction. To shed light on possible functions of FBXW7ß, we used gain- and loss-of-function approaches and identified an FBXW7-dependent inflammatory gene signature, apparent in a subset of B-ALL with high FBXW7ß expression. This signature contained several members of the tumor necrosis factor superfamily, including those comprising the HLA Class III cluster (LTB, LST1, NCR3, LTA, and NFKBIL1). Our findings suggest that FBXW7ß expression drives proinflammatory responses, which could contribute to normal B-cell development, leukemogenesis, and responses to anticancer therapies.

miR-17-92 cluster components analysis in Burkitt lymphoma: overexpression of miR-17 is associated with poor prognosis.

Burkitt lymphoma (BL) is an aggressive B cell lymphoma characterized by the reciprocal translocation of the c-Myc gene with immunoglobulin genes. Recently, MYC has been shown to maintain the neoplastic state via the miR-17-92 microRNA cluster that suppresses chromatin regulatory genes and the apoptosis regulator Bim. However, the expression and prognostic impact of miR-17-92 members in pediatric BL (pBL) are unknown. Therefore, we investigated miR-17, miR-19a, miR-19b, miR-20, and miR-92a expression and prognostic impact in a series of 41 pBL samples. In addition, Bim protein expression was evaluated and compared to miR-17, miR-19a, miR-19b, miR-20, and miR-92a levels and patient outcomes. The expression of miR-17-92 members was evaluated by qPCR and Bim protein by immunohistochemistry. Log-rank test was employed to assess prognostic impact. We found that upregulated expression of miR-17 and miR-20a correlates with lack of pro-apoptotic Bim expression. Patients bearing tumors with upregulated miR-17 displayed decreased overall survival (OS), and multivariate analysis revealed that miR-17 was a significant predictor of shortened OS. Using hairpin inhibitors, we showed that inhibition of miR-17 resulted in enhanced Bim expression in a BL cell line overexpressing the miR-17-92 cluster. Our results describe for the first time miR-17, miR-19a, miR-19b, miR-20a, and miR-92a expression profiles in pBL. The prognostic impact of miR-17 should be validated in a larger series, and may provide new therapeutic avenues in the era of anti-miRNA therapy research. Additional functional studies are further required to understand the specific role of miR-17-92 cluster members in BL.