Poster Board I-447
AML1-ETO (AE) fusion protein is generated as a result of translocation (8;21), accounting for 40% of the FAB M2 subtype of acute myeloid leukemia (AML). This group of patients has a 5-year survival rate of approximately 50% due to disease relapse. Retroviral gene transfer of AE in CD34+ human umbilical cord blood (hUBC) cells enhances self-renewal phenotypes, making this model a powerful tool to study cellular pathophysiology in response to fusion protein expression during the early stage of disease formation. Based on the current understanding of t(8;21) AML as a stem cell disease, these AE-expressing hUBC cells may also represent the chemotherapy-non-targetable leukemic stem cells that are responsible for the relapsing disease. In the current study, we identified thrombopoietin (TPO) as an essential cytokine for the growth of AE cells. Whereas normal CD34+ hUBC cells cultured in the absence or presence of TPO showed similar growth rates, withdrawing TPO in AE cell cultures resulted in a gradual proliferative defect that was associated with loss of colony forming ability and long-term culture-initiating cells (LTC-IC) frequency. Adding TPO to methylcellulose media leads to an increase in colony numbers and an enhanced replating ability, further confirming that TPO is particularly critical for the growth of the clonogenic AE progenitor cells. Moreover, TPO withdrawal caused a decrease in the percentage of CD34+ cells, and an increase in the percentages of both CD11b+ and CD14+ cells. AE cells incubated in media without TPO adapted a more mature morphology, including nuclear segmentation and cytoplasmic granule formation. At the molecular level, various genes involved in hematopoietic cell self-renewal are downregulated in AE cells cultured without TPO. Taken together, these results indicate that TPO signaling is a critical mediator of the self-renewal phenotypes of the AE cells. Interestingly, AE cells displayed addiction to TPO-mediated signaling as early as one week after AE expression. Therefore, we further hypothesized that AE is directly involved in this process. Indeed, we found that the c-Mpl gene, which encodes the Janus Kinase 2 (JAK2)-activating TPO receptor, is upregulated in AE cells. Additionally, a chromatin immunoprecipitation assay provided evidence of direct AE binding to the promoter region of c-Mpl. These data indicate that AE directly upregulates c-Mpl gene expression in hematopoietic stem and progenitor cells. In the study of the downstream effectors of the TPO signaling, we found that Bcl-xL, an essential anti-apoptotic protein in AE cells, is maintained at high levels by TPO, mainly in a post-translational manner, suggesting that an important component of TPO signaling is to promote cell survival. In fact, recent evidence on the opposing effects of DNA damage and JAK2 activation on Bcl-xL post-translational modifications (deamidation) led us to speculate that the negative effects from increased DNA damage associated with AE expression may be counterbalanced by TPO signaling. In conclusion, our data provide a novel therapeutic approach for t(8;21) AML treatment by targeting the TPO signaling pathway, thereby augmenting conventional chemotherapy in the prevention of disease relapse.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.