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Eltrombopag inhibition of acute myeloid leukemia cell survival does not depend on c-Mpl expression

Leukemia volume 27, pages 1207–1210 (2013)Cite this article

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We recently reported that a proto-type thrombopoietin (TPO) receptor agonist, SB-559457, is toxic to acute myeloid leukemia (AML) cell lines and primary AML cells.1 SB-559457 is a nonpeptidyl, hydrazone class, TPO receptor agonist similar to the Food and Drug Administration approved TPO mimetic, Eltrombopag (E). Here, we confirm that E itself is toxic to AML cell lines and primary AML cells, and initiate studies to understand the drug’s mechanism of action in the inhibition of AML cell survival. Myeloproliferative leukemia virus proto-oncogene c-Mpl is the gene encoding TPO-receptor, a member of the hematopoietic growth factor receptor superfamily. TPO and c-Mpl are both necessary for the proliferation and differentiation of megakaryocytes and production of platelets. E also binds to c-Mpl through a defined transmembrane domain, and activates c-Mpl-dependent signaling in cell lines and primary normal CD34+ stem and progenitor cells.2 It is hypothesized that these effects are necessary for the clinical activity of E in stimulating platelet production. c-Mpl is expressed in some myeloid leukemia cells, but its function in AML is not defined. Some reports have stated that TPO activates signaling pathways in primary AML cells, but this is not consistently observed.3, 4 In this paper, we studied the role of c-Mpl in the toxic effects of the hydrazone class of TPO-receptor agonists on AML cells.

To investigate whether E inhibits the proliferation of myeloid leukemia cells without a correlation to c-Mpl expression, we initially focused on AML cell lines. We studied several myeloid and lymphoid leukemic cell lines, which are not known to express c-Mpl (Jurkat, Ramos, KCL22, KG1a, HL-60, Molm-14 and K562 cells), and one c-Mpl highly expressing megakaryocytic leukemia cell line, Mo7e (Figure 1c, d). First, we analyzed c-Mpl expression using quantitative real-time PCR (qRT–PCR) and western blot methods. We confirmed that the Mo7e cell line expresses high levels of c-Mpl. To validate relative expression analysis, qRT–PCR was performed using two different reference genes. The qRT–PCR results showed that relative c-Mpl mRNA expression levels in all tested cell lines were less than 1% when compared with that in Mo7e cells (Figure 1c). Second, we performed western blot analyses to evaluate c-Mpl protein expression (Figure 1d). A very strong c-Mpl signal was detected in Mo7e cells, however, no signal was detected in lysates from the other cell lines. Taken together, these data confirm the c-Mpl expression in Mo7e cells, but show no evidence of c-Mpl expression in other cell lines. Of note, inhibition of cell growth by E in Mo7e cells, which express c-Mpl, is similar to E-mediated cell growth inhibition in other cell lines that do not have detectable c-Mpl expression (Figure 1a; data not shown), which suggests that E inhibition of leukemic growth may be independent of c-Mpl expression.

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Acknowledgements

We thank Connie Erickson-Miller for helpful comments and Dr Susan Shetzline for critical reading of the manuscript.

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  1. Division of Hematology and Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia,, PA, USA

    M Sugita, A Kalota, A M Gewirtz & M Carroll

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  1. M Sugita
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Correspondence to M Carroll.

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This work was supported by the National Cancer Institute grant 1R21CA153018-01, and by support from GlaxoSmithKline.

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Sugita, M., Kalota, A., Gewirtz, A. et al. Eltrombopag inhibition of acute myeloid leukemia cell survival does not depend on c-Mpl expression. Leukemia 27, 1207–1210 (2013). https://doi.org/10.1038/leu.2012.310

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