The Effects of Down-Regulation of MZB1 Expression on Apoptosis in Chronic Lymphocytic Leukemia B Cells With Mutations in the Splicing Factor 3b-1 Protein

Abstract

The goal of this work was to explore the role of the B cell specific Marginal Zone B and B1 cell (MZB1) protein in B cell viability and apoptosis with relation to mutations Splicing Factor 3B subunit 1 (SF3B1) protein, one of the core members of the spliceosomal complex, in Chronic Lymphocytic Leukemia (CLL). CLL arises from clonal B cell populations harboring genetic mutations and is the most common leukemia in adults in the western world (Hallek, 2015; Puiggros, Blanco, & Espinet, 2014). In CLL, SF3B1 mutation has been reported to be found at incidence rates near 3-9% in newly diagnosed patients and 17-18% in chemo-refractory patients (Wan & Wu, 2013) and modulation of SF3B1 function is thought to offer a means of treating this disease (Xargay-Torrent, et al., 2015; Kashyap, et al., 2015). Mutations in SF3B1 lead to an increase in aberrant splicing events (DeBoever, et al., 2015; Darman, et al., 2015) among the genes affected is MZB1 – a B cell specific gene encoding two protein isoforms found in the ER. SF3B1 mutations in CLL B cells induce a preferential splicing of the mRNA responsible for the PACAP isoform of MZB1, a protein reported to participate in apoptosis by binding Caspases 2 and 9 (Bonfoco, Li, Kolbinger, & Cooper, 2001). Characterization of CLL patient B cells obtained from Weill-Cornell Medical College verified that patients with SF3B1-mutated CLL trend toward higher expression of the PACAP isoform mRNA and a decrease in full length MZB1 protein. It was also verified that the most frequent K700E mutation was effective at decreasing MZB1 protein expression to nearly undetectable levels with variant allele fractions as low as 17-19% and up to 50%. CLL patient B cells stimulated with sCD40L and IL-4 cytokines were analyzed by fluorescence assisted cell sorting (FACS) for Annexin V and Propidium Iodide (PI) staining did not show any differential in apoptotic response among the SF3B1-wildtype and SF3B1-mutant samples indicating that the downregulation of MZB1 in SF3B1-mutant CLL patient B cells may not provide a protective effect to apoptosis. Both MZB1 and PACAP mRNA were knocked down using shRNA in the SF3B1-wildtype CLL B cell line MEC-1 and tested for effects on viability after treatment with cytotoxic drugs. Knockdown of both isoforms did not result in any detectable effects on viability of MEC-1 cells after drug treatment. Although the data presented suggest that downregulation of MZB1 in CLL B cells does not mediate effects through apoptosis via the PACAP isoform, it does not negate the likelihood that loss of canonical MZB1 function in the ER confers some tumorigenic advantage to the cell. In fact, the data support the bioinformatic prediction that loss of canonical MZB1 expression in SF3B1-mutated CLL B cells is mediated via aberrant splicing to the PACAP isoform mRNA that is degraded by Nonsense Mediated mRNA Decay (NMD).