Integrated Genomic and Proteomic Analysis of Murine CLL-like Cells Reveals SF3B1 Mutation to Impact DNA Damage Response and BCR Signaling

Collective large-scale sequencing efforts have unexpectedly revealed the high frequency of mutations in the splicing factor genes (SF3B1, U2AF1, SRSF2, ZRSR2) in various solid and hematological cancers, suggesting the association of splicing dysregulation with tumorigenesis. Mutations in SF3B1 occur in 5-20% of patients with chronic lymphocytic leukemia (CLL) and are associated with poorer overall survival and chemotherapy resistance. These mutations are restricted to hotspots (>50% at K700E site) and strongly co-occur with ATM mutations (loss-of-function) and deletion of 11q (ATM minimal deleted region). Numerous studies including ours have demonstrated that somatic alterations in this gene cause RNA splicing dysregulation, however, how this splicing factor mutation alone and in combination with ATM deletion impacts cellular processes and contributes to CLL remains to be fully defined. To this end, we modeled the effects of these combined alterations by crossing mice with conditional knockout of Atm and mice with a conditional knock-in allele of SF3B1 mutation (Sf3b1-K700E). We achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion by breeding these mice with CD19-Cre homozygous transgenic mice. Conditional expression of heterozygous Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters B-cell development, and induces a state of cellular senescence. Combined with Atm deletion in B cells led to the overcoming of cellular senescence and the development of clonal CLL cells in elderly mice at low penetrance (6%). These malignant cells could be propagated by in vivo passaging, with detectable disease within 4 weeks following transfer, thus making this mouse line amenable to further drug discovery and biologic investigations. To fully understand the underlying mechanisms of how the combined alterations led to CLL, we performed integrated genome, transcriptome, and proteome analysis using mouse CLL (DM-CLL) cells and B cells with either Sf3b1 mutation or Atm deletion, or with double genetic lesions (DM). Whole-genome sequencing of paired DNA from B cells (or DM-CLL) and non-B cell tissue (kidney) revealed the somatic mutation rate in the CLL cells to be ~0.5 mutations/Mb. Few recurrent mutations were identified among the samples. However, copy number variation analysis of DM-CLL cells revealed recurrent amplifications of chromosomes 15 and 17. RNA-seq analysis revealed that these amplifications were associated wi