TLS-ERG Fusion Protein Blocks Terminal Differentiation of Myeloid Progenitor Cells through Deregulation of Cyclin-Dependent Kinase 1 (CDK1).

In a subset of acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) in blast crisis, the TLS (translocation liposarcoma) gene is fused to the ERG (ets-related gene) gene through a recurrent t(16;21) translocation. This chromosomal translocation results in the generation of TLS-ERG fusion protein. More recently, the same TLS-ERG fusion protein has also been identified in Ewing’s sarcoma in children. To investigate the oncogenic mechanism of TLS-ERG, we have stably expressed TLS-ERG in mouse L-G myeloid progenitor cells through retroviral transduction. L-G cells are dependent on IL-3 for proliferation and undergo terminal differentiation into mature neutrophils when treated with G-CSF. Retroviral expression of TLS-ERG fusion protein in L-G cells blocks G-CSF induced terminal differentiation and enables these cells to proliferate in the absence of IL-3. The ability to transform L-G myeloid progenitor cells appears to be an acquired function of the TLS-ERG fusion protein since neither wild-type TLS nor wild-type ERG is able to transform cells. The evolutionarily conserved arginine at position 367 within the ets domain of TLS-ERG is known to be critical to DNA binding, we therefore have replaced arginine at this position with a leucine (R367L). When stably expressed in L-G cells, this TLS-ERG mutant no longer possesses the ability to transform cells. As cellular transformation often reflects a robust cell cycle machinery, we have examined the effects of TLS-ERG on Cdk1, a major protein kinase involved in all four phases of the cell cycle. While the level of Cdk1 protein decreases to an undetectable level during G-CSF-induced terminal differentiation or after IL-3 withdrawal in control L-G cells harboring the empty retroviral vector, the level of Cdk1 protein remains unchanged in TLS-ERG cells following the same treatments. Through RT-PCR analysis, we have found that the steady state level of Cdk1 mRNA is not directly affected by TLS-ERG. Instead, our results show that TLS-ERG increases the stability of Cdk1 protein through repressing genes involved in protein degradation. Using a luciferase reporter construct, we have found that transcriptional repression by TLS-ERG can be reversed by 5-aza-2′-deoxycytidine (also called Decitabine, a DNA methyltransferase inhibitor) or by trichostatin A (a histone acetyltransferase inhibitor). Interestingly, treatment of TLS-ERG-expressing L-G cells with these two epigenetic drugs can also destabilize Cdk1 protein and facilitate terminal differentiation in these TLS-ERG cells. To investigate whether deregulation of Cdk1 protein is indeed correlated with oncogenic transformation by the TLS-ERG fusion protein, we have constructed a lentiviral vector for delivery of a dominant-negative form of Cdk1. In L-G cells harboring TLS-ERG, lentiviral expression of the dominant-negative Cdk1 is found to restore the ability of cells to undergo G-CSF-induced terminal differentiation. In addition, stable siRNA knockdown of Cdk1 is also able to release cells from TLS-ERG blockage of terminal differentiation. Together, these findings suggest that deregulation of Cdk1 activity by TLS-ERG fusion protein plays a critical role in cellular transformation in vitro, and the role of Cdk1 deregulation in leukemogenesis is currently being examined in vivo in a TLS-ERG mouse model.