CD41 and Mpl Positive Fraction Shows an Efficient Leukemia-Initiating Capacity in Evi1-Induced Leukemia

Abstract 1295

Ecotropic viral integration site 1 (Evi1) is a transcription factor which is highly expressed in hematopoietic stem cells and crucial for their self-renewal capacity. Aberrant expression of Evi1 is observed in several types of acute myeloid leukemia (AML), of which the prognosis is generally poor, mainly due to therapeutic resistance. Effective Evi1-targeted therapies, however, have not yet been developed. Therefore, cell surface molecules or their downstream signaling pathways specific to Evi1 high-expressing leukemia cells would be good candidates for novel targeted therapy. Here in this study, we first revealed by gene expression data of AML patients that the expression of several megakaryocytic differentiation markers including CD41, CD61 and thrombopoietin (Thpo) receptor, Mpl was positively correlated to that of Evi1. To validate the association between these surface markers and Evi1, murine c-kit(+) hematopoietic progenitors were transduced with Evi1 or other several leukemia oncogenes and serially replated in methylcellulose semisolid medium. FACS analysis showed that the CD41(+) fraction emerged after third or fourth replating in Evi1-transduced progenitors, but not in progenitors immortalized by MLL-ENL or AML1-ETO. Similarly, the CD41(+) fraction was clearly observed in the bone marrow and spleen mononuclear cells of Evi1-induced leukemia mice. These results indicated the association between Evi1 and CD41 in AML. We next investigated the functional significance of the CD41(+) population in the Evi1-induced leukemia mouse model. The CD41(+) leukemia cells were morphologically more immature than the CD41(-) cells. Interestingly, the CD41(+) cells were highly positive for c-kit, and showed an enhanced colony-forming capacity in semisolid medium than the CD41(-) cells. Moreover, the CD41(+) cells developed AML with shorter latency than the CD41(-) cells in a murine bone marrow transplantation model (p<0.05, log-rank test). These results demonstrated that leukemia-initiating cells are more enriched in the CD41(+) fraction compared with the CD41(-) counterpart. Although the CD41(+) fraction was also observed in hematopoietic progenitors transduced with another leukemia oncogene, PML-RARA, these CD41(+) cells had differentiated and lost self-renewal capacity, which was in stark contrast to the case of Evi1. Remarkably, we found that Mpl was predominantly expressed in the CD41(+) fraction of Evi1 leukemia cells. Consistent with this finding, when Evi1 leukemia cells were cocultured with OP9 stromal cells, the addition of Thpo stimulated the proliferation and suppressed the apoptosis of the CD41(+) cells whereas the CD41(-) cells were not affected. Furthermore, stimulation of Evi1 leukemia cells with Thpo resulted in a marked increase in phosphorylation of Stat3, Stat5 and Erk1/2, while the addition of stem cell factor instead of Thpo failed to exert those effects. Therefore, Thpo/Mpl pathway is critical for proliferation and survival of the CD41(+) cells of Evi1 leukemia mice. To test whether the CD41(+) cells are resistant to anticancer treatment, Evi1 leukemia cells maintained on OP9 layers were treated with Ara-C for 48 hours, and the CD41 positive rate was measured by FACS. Notably, the CD41(-) cells were decreased in number at a lower concentration of Ara-C than the CD41(+) cells. While both fractions had similar cell cycle status, the CD41(+) cells showed a higher expression of pro-survival protein, Bcl-xl than the CD41(-) cells. Given that Bcl-xl could be activated by Stat family or Erk1/2, the CD41(+) fraction of Evi1 leukemia cells possibly acquire antiapoptotic potential through Thpo/Mpl signaling. In conclusion, we revealed that the CD41(+) fraction of Evi1 leukemia mice has more efficient leukemia-initiating capacity than the CD41(-) fraction. Importantly, the CD41(+) cells express Mpl, and Thpo/Mpl signaling participates in growth and survival of the CD41(+) cells in vitro. In addition, upregulation of Bcl-xl could confer antiapoptotic properties and chemo-resistance on the CD41(+) Evi1 leukemia cells. Our data would provide a rationale for targeting CD41 or Mpl in the novel treatment of Evi1 high-expressing leukemia.