Transcriptomes of normal human mature B cells and REH B-cell acute lymphoblastic leukemia cells: effect of FBXW7 isoform knockdown or knockout followed by reconstitution

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. For normal bone marrow cell subset data, please go to GEO Series GSE115655. Overall design: The sample groups are as follows. 1. LN: We performed fluorescence activated cell sorting to isolate primary normal B-cell subsets from human tonsil donors. 2. SYY53 + 54: To investigate functions of individual FBXW7 isoforms, we performed transient knockdown with Morpholinos in REH cell line. SYY53 and SYY54 are biological replicates from two independent experiments. 3. SYY123 + 141: We established REH cell lines where all FBXW7 isoforms were knocked out (pan-FBXW7 CRISPR KO clone 5 = cl5) and stably reconstituted with each FBXW7 isoform via the MIGR1 retroviral vector. SYY123 and SYY141 are biological replicates from two independent retroviral transduction experiments. 4. REH_ctrl: REH parental cell line.