Abstract 2893: Sleeping Beauty uncovers cooperating driver genes in a preclinical mouse model of myeloid leukemia
Authors: Mann Karen M. , Newberg Justin , Navin Nicholas , Adams David J. , Jenkins Nancy , Copeland Neal , Mann Michael B. 
Published: August 2015

Abstract
Myeloid leukemia is associated with few predominant mutations and translocations in humans. In order to uncover additional loci that drive the disease, we developed an aggressive, fully-penetrant mouse model of myeloid leukemia using Sleeping Beauty insertional mutagenesis. Animals developed distinct foci of expanded splenic red pulp as early as 30 days of age and had a median survival of 70 days. SB insertions sequenced from 168 spleens of mice with end-stage disease revealed more than 470 statistically defined candidate cancer genes (CCGs), 87 of which have orthologs mutated in human AML. Pathway enrichment analysis using the CCGs confirmed that MAP kinase and Jak-Stat signaling play an important role in the disease. Twenty-five CCGs were defined as trunk drivers based on high sequence read representation in multiple tumors from the population. Among the trunk drivers was a suite of transcription factors that act downstream of MAP kinase signaling, including Ets- family member genes Erg, Ets1 and Fli1.
We sequenced SB insertions from single cells isolated from a single tumor to interrogate cooperating relationships among trunk drivers. Hierarchical clustering of SB insertions revealed intra-tumor heterogeneity and identified three distinct subclonal populations. Importantly, Erg and Ghr anchored one subpopulation that was distinct from a second subpopulation anchored by Ets1 and Notch1. These gene relationships observed in single cells confirmed the population-based statistical analysis of cooperativity between Erg and Ghr and Ets1 and Notch1 from the cohort of 168 tumors. Mutual-exclusivity for Erg and Ets1 in driving myeloid leukemia was supported by microarray expression analysis in a subset of the tumors. RNA-seq analysis confirmed that SB insertions drive expression of many CCGs including trunk divers Erg, Ghr, Ets1 and Notch1. Chimeric transcripts for the four driver genes contained the SB splice donor and downstream wild type exons encoding the functional domains of the translated proteins, suggesting that SB drives the mis-expression of these proteins to induce myeloid leukemia.
We have identified new CCGs that contribute to myeloid disease progression and gained insight into both inter- and intra- tumor heterogeneity of this tumor type. Importantly, SB-driven myeloid leukemia is a robust, transplantable genetic model that offers a unique platform for preclinical testing. Transposon insertion profiles provide a powerful means to stratify tumors for targeted therapies. Finally, SB can be harnessed to uncover loci that confer therapy response or resistance.
Citation Format: Karen M. Mann, Justin Newberg, Nicholas Navin, David J. Adams, Nancy Jenkins, Neal Copeland, Michael B. Mann. Sleeping Beauty uncovers cooperating driver genes in a preclinical mouse model of myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2015-2893