Abstract 2449: Transcriptome and whole-genome sequencing analysis of a novel murine model of chronic lymphocytic leukemia (2017)

Abstract Large-scale cancer sequencing of primary chronic lymphocytic leukemia (CLL) has identified SF3B1, an RNA splicing factor, as one of the most frequently mutated CLL genes. SF3B1 mutations localize to a hotspot (&amp;gt;50% at K700E site) and highly co-occur with mutations in ATM or deletion of chromosome 11q (minimally deleted region contains ATM). How this splicing factor mutation alone or in cooperation with ATM deletion contributes to CLL remains elusive. Genetically engineered mice are powerful tools in understanding genetic lesions and cancer phenotypes. We therefore generated a mouse line that conditionally expresses heterozygous Sf3b1-K700E mutation. We modeled the effects of the combined alterations by crossing mice with conditional knockout of Atm and mice with Sf3b1-K700E. By breeding these mice with homozygous CD19-Cre transgenic mice, we achieved B cell-restricted expression of heterozygous Sf3b1 mutation and Atm deletion. B cell co-expression of these two mutations in vivo led to clonal expansion of CD19+CD5+ B cells in blood, marrow and spleen in aged mice (18 to 24-month old) at low penetrance. No leukemia cells were found in the Sf3b1-K700E mice (up to 24-month old). The CLL cells from the double mutant mice could be engrafted in both immunocompetent and immunodeficient mice, with detectable disease within 2-4 weeks following transfer, thus making this mouse line amenable to drug discovery and biologic investigations. To investigate how Sf3b1 mutation and Atm deletion synergistically contribute to CLL, we asked if there are RNA and DNA level changes in the double mutant mice with CLL. First, we performed transcriptome sequencing of splenic B cell RNA collected from age-matched mice that either express wild-type, singly mutant alleles of Sf3b1 or Atm, or doubly mutant alleles with or without CLL-like disease. Using the tool JuncBASE, we classified and quantified splice variants associated with the different genetic alterations. Consistent with prior findings in human CLL, we observed that splice variants in Sf3b1-mutant mice alone were highly enriched at 3' splice sites. Sf3b1 and Atm doubly mutant B cells displayed a splicing pattern similar to that in Sf3b1 singly mutant cells. Moreover, we identified unique CLL splice variants in genes (Setdb2, Phf11c) previously shown to be associated with CLL. Next, we examined the mutation rate in DNA derived from splenic B cells collected from mice with a singly mutated allele of Sf3b1 or Atm, or with doubly mutated alleles with and without CLL-like disease through comparison against matched germline DNA from kidney by whole-genome sequencing. We have observed that co-expression of Sf3b1 mutation and deletion of Atm results in a higher mutation rate compared to singly mutant cells. Our analysis has revealed that altered RNA splicing and genomic instability all contribute to CLL leukemogenesis. We will further dissect how the two lesions contribute to CLL functionally using this model. Citation Format: Lili Wang, Jing Sun, Amaro Taylor-Weiner, Jaegil Kim, Zachary J. Cartun, Angela N. Brooks, Donna Neuberg, Mark D. Fleming, Benjamin L. Ebert, Gad Getz, Ruben Carrasco, Catherine J. Wu. Transcriptome and whole-genome sequencing analysis of a novel murine model of chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2449. doi:10.1158/1538-7445.AM2017-2449