Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice

www.nature.com/scientificreports OPEN Received: 21 January 2019 Accepted: 19 March 2019 Published: xx xx xxxx Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice Keith R. Loeb1,2, Bridget T. Hughes1,3,4, Brian M. Fissel5, Nyka J. Osteen1, Sue E. Knoblaugh6, Jonathan E. Grim1,7, Luke J. Drury8, Aaron Sarver9, Adam J. Dupuy8 & Bruce E. Clurman1,3 Insertional mutagenesis is a powerful means of identifying cancer drivers in animal models. We used the Sleeping Beauty (SB) transposon/transposase system to identify activated oncogenes in hematologic cancers in wild-type mice and mice that express a stabilized cyclin E protein (termed cyclin ET74AT393A). Cyclin E governs cell division and is misregulated in human cancers. Cyclin ET74AT393A mice develop ineffective erythropoiesis that resembles early-stage human myelodysplastic syndrome, and we sought to identify oncogenes that might cooperate with cyclin E hyperactivity in leukemogenesis. SB activation in hematopoietic precursors caused T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL). Analysis of >12,000 SB integration sites revealed markedly different oncogene activations in EL and T-ALL: Notch1 and Ikaros were most common in T-ALL, whereas ETS transcription factors (Erg and Ets1) were targeted in most ELs. Cyclin E status did not impact leukemogenesis or oncogene activations. Whereas most SB insertions were lost during culture of EL cell lines, Erg insertions were retained, indicating Erg’s key role in these neoplasms. Surprisingly, cyclin ET74AT393A conferred growth factor independence and altered Erg-dependent differentiation in EL cell lines. These studies provide new molecular insights into erythroid leukemia and suggest potential therapeutic targets for human leukemia. Insertional mutagenesis is a powerful means of identifying the molecular drivers of cancer initiation and progression in animal models. Sleeping Beauty (SB) is a transposon/transposase insertional mutagenesis system that is designed to either overexpress nearby genes or inactivate genes, depending on the transposon’s integration site and orientation1,2. By combining conditional expression of the SB transposase with the T2Onc transposon in various genetic backgrounds, SB screens have been used extensively to identify\cancer genes and how they cooperate with one another in wild type and cancer-sensitizing backgrounds, and across many cancer types3–6. In this study, we employed SB to identify oncogenes that might promote multi-step carcinogenesis in a mouse model engineered to express a stabilized version of the cyclin E protein. Cyclin E, in conjunction with its catalytic partner CDK2, has crucial roles in cell division, and cyclin E-CDK2 deregulation causes genome instability and contributes to cancer development and progression7. One important means of cyclin E regulation is phosphorylation-dependent degradation by the SCFFbw7 ubiquitin ligase8–12. To study the physiologic consequences of abnormal cyclin E degradation, we previously created a knock-in mouse model that ablated two cyclin E phosphorylation sites (T74 and T393) that trigger its degradation13,14. The cyclin ET74AT393A mutation caused increased cyclin E abundance and epithelial cell hyperproliferation. However, these mice did not spontaneously develop epithelial dysplasia or tumors, suggesting that compensatory mechanisms maintain 1 Divisions of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA. 2Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA. 3Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA. 4Present address: University of Utah, Salt Lake City, UT, USA. 5 Present address: Boston University School of Medicine, Boston, MA, USA. 6Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, 43210, USA. 7VA Puget Sound Health Care System, Seattle, WA, 98108, USA. 8Department of Anatomy & Cell Biology, University of Iowa, Iowa City, IA, 52242, USA. 9Institute for Health Informatics, University of Minnesota, Minneapolis, MN, 55455, USA. Correspondence and requests for materials should be addressed to B.E.C. (email: ) Scientific Reports | (2019) 9:5488 | https://doi.org/10.1038/s41598-019-41805-x 1 www.nature.com/scientificreports/ www.nature.com/scientificreports tissue architecture and suppressed tumorigenesis. Cyclin ET74AT393A expression also caused ineffective erythropoiesis with marked expansion of immature erythroid precursors in the spleen and bone marrow, impaired erythroid differentiation, and mild anemia. These features resemble the early stages of human refractory anemia/ myelodysplastic syndrome (MDS). Because MDS can evolve to leukemia in humans, we speculated that cyclin ET74AT393A mice may provide a sensitized background to identify genetic events that cooperate with abnormal cyclin E regulation to promote leukemia. We thus used interferon-inducible Mx-Cre to activate the SB transposase in hematopoietic precursors to identify genes that might cooperate with abnormal cyclin E regulation to promote leukemia. The stabilized cyclin E allele neither predisposed mice to hematologic cancers nor altered gene activations by SB. Strikingly however, Mx-Cre-induced SB activation caused highly penetrant hematologic cancers within 8–13 weeks after Cre induction. To control for biases in transposon integrations that frequently occur proximal to the T2Onc array15,16, we used two different T2/Onc2 strains that contained the transposon array on different chromosomes. The most common malignancies were immature T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL), and there was a non-significant trend towards more EL in the cyclin ET74AT393A mice. To identify activated oncogenes in these neoplasms, we determined the transposon insertion sites in all ELs and T-ALLs. Transposon insertions that are shared by multiple independent tumors, termed common insertion sites (CIS), often occur in the vicinity of cancer-associated genes, which provides the selective pressure for these shared insertions. We identified CIS using two different statistical methods and found that the CIS profile of ELs and T-ALLs differed markedly. Whereas Notch and Ikaros insertions were most common in T-ALL, ETS family transcription factors (Erg and Ets1) were the most commonly activated genes in ELs and were activated in the almost all of these tumors. While T-ALL is common in SB screens performed in blood cells, EL has not, and we thus examined EL in more detail by developing 5 transplantable EL cell lines. Transposon analyses indicated that the vast majority of CISs found in primary ELs were lost during culture of EL cell lines, suggesting that they were not required for their proliferation and maintenance in vitro. However, all EL lines retained their (...truncated)