Leukemias arise from genetic alterations in precursor cells in the bone marrow that prevent normal blood cell development and differentiation. A mutated cell will divide, expand and progressively replace normal functional blood cells. One of the predispositions for leukemia is the myelodysplastic syndrome (MDS), a condition in which bone marrow stem cells are dysfunctional, leading to anemia, leukopenia, and thrombocytpenia. In 2005, Drs. Loeb and Clurman (Human Biology and Clinical Research Divisions) established a new mouse model preventing degradation of Cyclin E (T74AT393A mutation) and demonstrated that this alteration caused anomalies resembling early stages of MDS, including expansion of immature erythroid precursors, impaired erythroid differentiation and anemia. Understanding how MDS can predispose to leukemia motivated the author to identify additional genetic aberrations that would cooperate with cyclin E stabilization in the tumorigenic process. The Clurman lab published these results in the Scientific Reports journal last month.
In the study, the researchers used a powerful strategy as explained by the first author Dr. Loeb: “We performed a genetic screen using Sleeping Beauty transposon insertion to identify oncogenic drivers of leukemia”. The Sleeping Beauty system consists of a mouse system conditionally expressing a transposase. Transposases are enzymes catalyzing the random insertion of DNA sequences called transposons in the genome. Genetically engineered transposons can either silence or enhance the expression of the affected gene depending on the site and orientation of the insertion. This model has been extensively used to screen for new oncogenes and tumor suppressors based on the characterization of the transposon insertion site in animals presenting increased or decreased tumorigenesis respectively. Researchers reasoned that activating the transposon system in erythroid precursors in animals accumulating Cyclin E (Cyclin ET74AT393A), which develop MDS-like anomalies, would allow the identification of new drivers of tumorigenesis. They used the Mx1-Cre allele that drives expression of the Cre recombinase in hematopoietic precursors under interferon gamma stimulation (conditional and inducible model). After interferon gamma stimulation, the Cre recombinase removes the STOP cassette in front of the SB transposase sequence (LSL-SB). The transposase is expressed and randomly inserts genetically engineered transposons (T2/Onc2) derived from an array of 300 transposons, throughout the genome of the host cell. Animals bearing the three alleles Mx1-Cre; LSL-SB; T2/Onc2 were considered as control or wild-type animals whereas the animals bearing all four alleles Mx1-Cre; LSL-SB; T2/Onc2; Cyclin ET74AT393A were referred to as Cyclin ET74AT393A.
Loeb commented on the results they obtained: “We had previously shown that expression of the stabilizing Cyclin E mutation (T74AT393A) inhibits erythroid maturation and results in erythroid hyperplasia in the marrow and spleen and peripheral anemia. Following transposon expression, wild-type and cyclin ET74AT393A mice rapidly develop leukemia (T-cell lymphoblastic leukemia and erythroleukemia) and share similar sets of insertion sites suggesting that Cyclin E status does not impact leukemogenesis or oncogene activation in this study.” When comparing the insertion sites in T-cell leukemia versus erythroleukemia (EL), the authors found that Erg and Ets1 were the most common insertion sites leading to EL whereas T-cell leukemia essentially arose from insertion affecting the expression of Notch1, Ikzf1 and Erg. “To further identify functional insertions that promote leukemia, we then passaged the EL in cell culture”, says Loeb. “Since the transposase is constitutively active, we predicted the cell culture would further select for functional oncogenic inserts. Whereas Sleeping Beauty insertions in many loci were lost during culture of EL cell lines, Erg insertions were retained, indicating ERG’s key role in these neoplasms”.
Loeb commented on the results they obtained: “We had previously shown that expression of the stabilizing Cyclin E mutation (T74AT393A) inhibits erythroid maturation and results in erythroid hyperplasia in the marrow and spleen and peripheral anemia. Following transposon expression, wild-type and cyclin ET74AT393A mice rapidly develop leukemia (T-cell lymphoblastic leukemia and erythroleukemia) and share similar sets of insertion sites suggesting that Cyclin E status does not impact leukemogenesis or oncogene activation in this study.” When comparing the insertion sites in T-cell leukemia versus erythroleukemia (EL), the authors found that Erg and Ets1 were the most common insertion sites leading to EL whereas T-cell leukemia essentially arose from insertion affecting the expression of Notch1, Ikzf1 and Erg. “To further identify functional insertions that promote leukemia, we then passaged the EL in cell culture”, says Loeb. “Since the transposase is constitutively active, we predicted the cell culture would further select for functional oncogenic inserts. Whereas Sleeping Beauty insertions in many loci were lost during culture of EL cell lines, Erg insertions were retained, indicating ERG’s key role in these neoplasms”.
Although Cyclin ET74AT393A animals developed leukemia with the same penetrance as the wild-type animals, Loeb explains that “in primary cultured erythroleukemia cells, Cyclin ET74AT393A conferred growth factor independence and altered Erg-dependent differentiation”, thus clarifying the role of Cyclin E in erythroleukemia.
This study paves the path to elucidating the function and targetability of ERG and ETS1 in leukemia. Loeb comments: “Based on these studies, we believe that ERG and ETS1 will also be important in human acute myeloid leukemia (AML). ERG knockdown induces differentiation and decreases proliferation in erythroleukemias arising in wild type mice. Therefore, ERG may be a good therapeutic target in leukemias with high levels of ERG expression by its ability to block cell maturation. In support of this possibility, several recent studies have shown that overexpression of ERG is a poor prognostic factor for AML. Small molecule inhibitors against ERG are currently being developed and some of these are in clinical trials for prostate cancer with a TMPRSS/ERG (T/E) gene fusions.”
This work was supported by the National Institute of Health, the American Cancer Society and the Core Center of Excellence in Hematology.
Fred Hutch/UW Cancer Consortium member Drs. Loeb, Grim and Clurman contributed to this research.
Loeb KR, Hughes BT, Fissel BM, Osteen NJ, Knoblaugh SE, Grim JE, Drury LJ, Sarver A, Dupuy AJ, Clurman BE. Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice. Sci Rep 9(1):5488. doi: 10.1038/s41598-019-41805-x.