Abstract
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 with overexpression of 835 of 987 Chr15 and Chr17 genes detected in DM-CLL vs. DM cells. Of note, 146 genes were overexpressed in human CLLs with SF3B1 mutations (DFCI cohort), compared to normal B-cells (p<0.05).
Integrated transcriptome and proteome analysis of the DM-CLL cells showed coordinated dysregulation of multiple CLL-associated cellular processes with B-cell receptor (BCR) signaling as the most dramatically downregulated compared to DM cells. Since BCR signaling is a therapeutic target in CLL and has critical roles in B cell biology, we asked how SF3B1 mutation contributes to gene expression of BCR signaling. Through RNA-seq data analysis derived from two independent patient cohorts (DFCI and ICGC), we identified downregulation of BCR gene expression in SF3B1 mutant CLL cells. In line with this, human CLLs harboring SF3B1 mutations exhibit greater sensitivity to in vitro treatment with ibrutinib, and altered response kinetics in vivo to ibrutinib, per analysis of patients with SF3B1 mutations treated with ibrutinib. These studies together highlight a role of SF3B1 mutation in BCR signaling.
In summary, we have generated a genetically-engineered murine model that recapitulates human CLL genetics, and presents an informative model to functionally dissect the effects of mutant SF3B1 in a B cell context. Starting from computation-based identification of recurrent co-occurring events in CLL, our study employs murine lines that express genetic alterations in an lineage-specific fashion, utilizes integrated genomics and proteomics approaches to dissect pathways that are fundamental to CLL phenotype, and more importantly, links the dysregulated pathways back to human CLL gene expression data and clinical trials to reveal novel mechanisms underlying therapeutic response.
Disclosures
Wiestner: Pharmacyclics LLC, an AbbVie Company: Research Funding. Wu:Neon Therapeutics: Equity Ownership.
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