Roles for the AML1/RUNX1 Point Mutation in the Pathogenesis of MDS/AML.

Myelodysplastic syndermes (MDS) are clonal hematological disorders of hematopoietic stem cells (HSC), which are characterized by ineffective hematopoiesis, dysplastic morphology of blood cells, and high possibility to transit to acute myeloid leukemias (AML). Until now, a number of genetic or epigenetic alterations involved in the pathogenesis of MDS have been identified. Among them, point mutations of AML1/RUNX1 were found in 15 to 17% of MDS/AML (high risk MDS and AML following MDS) patients. Although it has been reported that loss-of-function mutations of AML1 inhibited hematopoietic differentiation and they led to MDS/AML, the direct role for AML1 mutations in the onset of MDS/AML has not been elucidated. To clarify the influence of mutated AML1 on the biology of HSCs, we transduced C-terminus-truncated form of AML1 (AML1dC: dominant-negative form of wild-type AML1), which were derived from high-risk MDS patient, into murine Lineage^-Sca1⁺c-Kit⁺(LSK) cells. In lethally irradiated murine bone marrow transplantation (BMT) experiment, AML1dC-transduced LSK cells showed higher peripheral blood contribution than mock-transduced cells at 4 weeks after BMT. In mice that received transplants of AML1dC-transduced cell, the ratio of donor-derived hematopoietic cells gradually increased over several months after the transplantation, but not in mice that received transplants of mock-transduced cells. AML1dC-transduced cells more strongly contributed to all hematopietic lineages than mock-transduced cells, especially granulo-monocytic lineage. However, AML1dC-transduced LSK cells did not develop MDS/AML in transplanted mice during over 7 months of observation. Methylcellose colony assay revealed that AML1dC-transduced cells formed CFU-Mix more efficiently than mock-transduced cells in the presence of thrombopoietin (TPO). To further explore the effect of AML1dC on the HSCs differentiation into myeloid cells, we utilized the Tet-off system in the OP9 system. In this system, after deprivation of LIF from the culture medium, Flk-1⁺ hemangioblasts that have both potential to develop into hematopoietic cells and endothelial cells develop from ES cells after 4.5 days and definitive hematopoietic stem/progenitor cells appear after 8.5 days. After collection of hemangioblasts on day 4.5, we inducibly expressed AML1dC by depriving Tet from the culture medium, cultured for 7 days, and performed FACS analysis on day 11.5. After the culture with Tet, 37.8% of AML1dC-transduced ES cells were positive for Mac-1 in the GFP-negative (AML1dC-negative) fraction. In contrast, after the culture without Tet, 53.3 % of AML1dC-transduced ES cells were positive for Mac-1 in the GFP-positive (AML1dC-expressed) fraction. Because in vivo and in vitro assays suggested that mutated AML1 had some role in the proliferation of MDS clone, we analyzed the activation of signal transduction molecules in AML1dC-transduced LSK cells. As the results, TPO-induced STAT5 phosphorylation was enhanced in AML1dC-transduced LSK cells compared to mock-transduced cells. Moreover, TPO-induced STAT3 activation was detected in AML1dC-transduced LSK cells, but not in mock-transduced LSK cells. There was no significant difference in the phosphorylation of ERK1/2 between AML1dC- and mock-transduced LSK cells. In the realtime PCR analysis, the mRNA expression of CIS, which is one of the STAT5-target genes, was increased 2 to 4-fold in AML1dC-transduced LSK cells compared to mock-transduced cells. Next, to elucidate the effect of mutated AML1 on the determination of HSC fate, we performed colony assay using AML1dC-transduced common lymphoid progenitor (CLP) cells. As the results, lymphoid colony-forming ability was attenuated in AML1dC-transduced CLPs compared to mock-transduced CLPs and only AML1dC-transduced CLPs could form CFU-Mix in the methylcellose culture medium including stem cell factor, IL-3, IL-6, erythropoietin and TPO. These results suggest that AML1dC transduction reprograms CLP to multipotent progenitor or converts CLP to common myeloid progenitor. Together, we show that C-terminus-deletion mutant of AML1 enhances hematopietic stem cell activity and promotes HSC commitment into myeloid progenitors. These results indicate that some types of mutated AML1 give rise to the proliferation of malignant clone via cytokine-induced STATs activation.