Expressionof Sf3b1- K700Ein Murine B Cells Causes Pre-mRNA Splicing and Altered B Cell Differentiation and Function
Authors: Wang Lili , Gambe Rutendo , Fan Jean , Wan Youzhong , Brooks Angela N , Sun Jing , Obeng Esther A. , Neuberg Donna S , Meyerson Matthew , Fleming Mark D. , Ebert Benjamin L. , Carrasco Ruben D. , Wu Catherine J 
Published: October 2019

Abstract
Mutations in the RNA splicing factor SF3B1 have been identified by large-scale sequencing as putative drivers in chronic lymphocytic leukemia (CLL), but their precise roles in the pathogenesis of CLL remains unknown. Although prior transcriptomic studies using primary CLL samples have led to the appreciation of altered RNA splicing in association with these mutations, understanding of their impact on cellular function has been complicated by their variable mutant allele frequency across samples as well as their common co-occurrence with other heterogeneous gene mutations. We therefore generated a mouse line that conditionally expresses the commonly occurring Sf3b1-K700E mutation at its endogenous murine locus. We obtained B-cell lineage specific expression of the mutant allele by crossing heterozygous floxed Sf3b1-K700E mice with homozygous CD19-Cre knockin mice. We confirmed that expression of the mutant allele was uniquely present as a heterozygous mutation in B cells, but not in other cell lineages.
We sought to characterize the impact of Sf3b1-K700E on RNA splicing, B cell function, and CLL in this in vivo model. By unbiased RNA-sequencing of splenic B cells from wildtype and mutant mice (n=3), we profiled the splice isoform changes that were associated with Sf3b1-K700E. Using the tool JuncBASE, we detected, classified and quantified 54 differentially spliced transcripts (P<0.05, absolute delta percent spliced in >10%). Consistent with the altered splicing pattern reported in human CLL samples, the splice variants in our mouse model were highly enriched with altered selection of 3' splice sites (49 of 54 events, P<0.001). Of these, we validated 3 selected splice variants in independent samples by qPCR. Our murine model of Sf3b1-K700E mutation thus recapitulates altered RNA splicing, as per in human disease.
We therefore next investigated whether Sf3b1-K700E affects B cell development and function. Splenic B cell numbers were significantly lower in the mutant (n=37) compared to control (n=33, P=0.0027) mice while T cell numbers were equivalent. Flow cytometric analysis of various B cell subpopulations from bone marrow and spleen revealed a significant increase in marginal zone B cells in the mutant mice (n=6, P<0.01). Consistent with this finding, we observed evidence of enlarged marginal zone areas on sections of mutant mouse spleens by visual inspection, with a mean reduction by 25% of proliferating germinal centers (per Ki67+ staining, n=6, average, P<0.05). On average, in vitro culture of splenic B cells with LPS and IL4 revealed mutant B cells to undergo 10-15% less proliferation, with reduced survival upon stimulation. Moreover, serum analysis revealed a 30-45% reduction in production of IgG1 and IgG3 from mutant mice (n=8, P<0.05). Altogether, these results suggest that mutant Sf3b1 induces an intrinsic B cell defect leading preferentially to impaired cellular proliferation.
Cellular senescence has been commonly detected in pre-malignant lesions and is related to impaired cell proliferation. Indeed, by quantitative PCR array of 84 genes associated with cellular senescence, we found comparable levels of expression of all genes in T cells from wildtype and mutant splenocytes, but an overall trend of upregulation (range: by 1.5- to 21-fold) in the entire set of 84 genes in mutant B splenocytes, with significant upregulation in 20 genes (n=5, P<0.05). These genes included the critical senescence regulators Cdkn2a (p16) and Cdkn1a (p21), whose elevated expression in Sf3b1 mutated B cells we confirmed at the protein level. Furthermore, we detected increased levels of the cellular senescence mediators Igfbp6 and Igfbp7 in the sera from mutant mice (n=31, P<0.05). Collectively, the data demonstrate that expression of Sf3b1-K700E in B cells leads to the cellular senescent phenotype.
While we observed that expression of Sf3b1-K700E results in RNA splicing changes, B cell developmental dysregulation and cellular senescence, expression of this mutation alone did not lead to expansion of CD5+CD19+ cells in vivo over time, despite observations up to 18-months (n=50). Our ongoing studies are now focused on the combined effects of Sf3b1-K700E and other recurrent CLL mutations on evasion of senescence and CLL disease progression.

Disclosures
No relevant conflicts of interest to declare.