Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9 (1), 5488

Insertional Mutagenesis Using the Sleeping Beauty Transposon System Identifies Drivers of Erythroleukemia in Mice

Affiliations

Insertional Mutagenesis Using the Sleeping Beauty Transposon System Identifies Drivers of Erythroleukemia in Mice

Keith R Loeb et al. Sci Rep.

Abstract

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Breeding scheme, survival, and tumor spectrum of Mx-1 sleeping beauty mice. (a) Breeding scheme for transposon mutagenesis in cyclin ET74AT393A and wild-type mice. Cyclin ET74AT393A or wild-type mice homozygous for both a T2/Onc transposon array and Cre recombinase-inducible SB transposase allele (T2/Onc2/T2/Onc2; RosaSBLSL/RosaSBLSL) were mated with cyclin ET74AT393A mice heterozygous for Mx-1 Cre to generate cyclin ET74AT393A or wild-type mice with T2/Onc transposon array and Mx-1 Cre recombinase. (b) Kaplan Myer survival curve of SB-Cyclin ET74AT393A and SB-wild-type mice following induction of Mx-1 Cre and transpose expression by poly I/C injection. Time indicates intervention due to illness related to hematologic malignancy. Induction of malignancy was completely penetrant in both backgrounds with a median survival of 11.9 weeks post-injection for wild-type and 12.9 weeks for cyclin ET74AT393A mice (p = 0.08). (c) Spectrum of hematologic neoplasms that arise in Mx-1 Sleeping beauty mice. Diagnosis was made by a combination of histology and flow cytometry (see text).
Figure 2
Figure 2
Morphology and immunophenotype of hematopoietic neoplasms in Mx-1 sleeping beauty mice. (a,b) Histologic sections (H&E) of erythroleukemia involving spleen (A: 20X objective) and liver (B: 40X objective). (c) Bone marrow cytospin preparations (Wright Giemsa) of erythroleukemia with block-like clumped chromatin and dark blue basophilic cytoplasm characteristic of erythroid precursor cells (63X objective). (d) Peripheral blood smear (Wright Giemsa) with circulating erythroblasts (40X objective). (e) Histologic section (H&E) of erythroleukemia with prominent megakaryocytic differentiation (large atypical cells) involving the liver (20X objective). (f) Representative immunophenotype of immature T-cell leukemia in blue (top row) and erythroleukemia in red (bottom row). (g) Summary of T-cell leukemia and erythroleukemia immunophenotype.
Figure 3
Figure 3
Immunophenotypic change and loss of insertion sites of erythroleukemia following transplantation and cell culture models. (a) Flow cytometry results from primary leukemia (top), transplanted leukemia (middle), and cell culture (bottom). The abnormal erythroblasts are CD45 dim/low side scatter/high CD71/variable CD117/variable Ter119 (red). (b) Loss of insertion sites following cell culture. Number of total insertion sites and common insertion site (CIS) identified in primary tumor (blue and green) and cell lines (red and purple) in five separate tumors and derived cell lines. (c) Quantification of inserts and CIS in tumor-derived cell lines.
Figure 4
Figure 4
Erg over-expression inhibits erythroid differentiation. (a) Transposon insertion sites within Erg are predicted to promote the expression of a truncated transcript containing the full ETS domain. All of the insertions are in the same transcriptional orientation as the Erg gene. (b) Immunoblot showing partial knockdown of ERG with mErg-specific shRNA in EL cell lines. (c) ERG knockdown in erythroleukemia cell lines induces immunophenotypic maturation (decreased CD117 and increased Ter119 expression) in wt EL cell lines (4230 and 4460) but not in cyclin ET74AT393A EL cell lines (3945 and 4489). (d) Growth factor dependence of the erythroleukemia-derived cell lines. Two cell lines (3945 and 4489) from cyclin ET74AT393A mice are growth factor independent, and three cell lines (4230, 4456, 4460) from wt mice are growth factor dependent (EPO and c-Kit ligand).
Figure 5
Figure 5
(a) Transposon insertion site within the gene encoding Flt3. All of the insertions are in the same orientation of the gene and are predicted to result in overexpression of a truncated protein containing the tyrosine kinase domain of Flt3. (b) Immunoblot showing over-expression of truncated FLT3 protein (65 kDa) in the 4489 cell line that contained an insertion in the Flt3 gene. Endogenous FLT3 was not detected (113 KDa). (ce) Over-expression of the truncated FLT3 in 4489 imparts increased sensitivity to a panel of FLT3 tyrosine kinase inhibitors including Lestaurtinib (c), PKC412 (d), and Sorafenib (e).

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Largaespada DA. Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification. Methods. 2009;49:282–286. doi: 10.1016/j.ymeth.2009.07.002. - DOI - PMC - PubMed
    1. Dupuy AJ. Transposon-based screens for cancer gene discovery in mouse models. Semin Cancer Biol. 2010;20:261–268. doi: 10.1016/j.semcancer.2010.05.003. - DOI - PMC - PubMed
    1. Abbott KL, et al. The Candidate Cancer Gene Database: a database of cancer driver genes from forward genetic screens in mice. Nucleic Acids Res. 2015;43:D844–848. doi: 10.1093/nar/gku770. - DOI - PMC - PubMed
    1. Copeland NG, Jenkins NA. Harnessing transposons for cancer gene discovery. Nat Rev Cancer. 2010;10:696–706. doi: 10.1038/nrc2916. - DOI - PubMed
    1. Moriarity BS, Largaespada DA. Sleeping Beauty transposon insertional mutagenesis based mouse models for cancer gene discovery. Curr Opin Genet Dev. 2015;30:66–72. doi: 10.1016/j.gde.2015.04.007. - DOI - PMC - PubMed

Publication types

Feedback