c-Mpl-del, an Alternative Splicing Isoform of c-Mpl, Promotes Malignancy and Chemoresistance of AMKL by Regulating AKT/DDIT3 Survival Signaling

Background: Acute megakaryocytic leukemia (AMKL) is a clinically heterogeneous subtype of acute myeloid leukemia (AML) characterized by unrestricted megakaryoblast proliferation, myelofibrosis, and poor prognosis. Aberrant c-Mpl signaling has been implicated in a myriad of myeloid proliferative disorders, some of which can lead to AMKL, however the role of c-Mpl in AMKL progression remains largely unexplored. Methods: Quantitative real-time PCR and Western blot were carried out to identify the expression of c-Mpl variants. The binding affinities of c-Mpl to TPO and c-Mpl dimerization were investigated by surface plasmon resonance, microscale thermophoresis, and fluorescence resonance energy transfer. RNA-Seq, Co-IP, and Western blot were performed to examine the functional mechanism of c-Mpl variants in AMKL development and chemotherapy response. Finding: We identified increased expression of a c-Mpl isoform (c-Mpl-del) in the bone marrow of AMKL patients and AMKL cell lines. c-Mpl-del overexpression was correlated with enhanced AMKL cell proliferation and chemotherapeutic resistance, and decreased survival in xenografted mice. Interestingly, c-Mpl-del exhibits decreased receptor internalization resulting in increased surface stability and allowed for sustained TPO signaling. These led to the preferential phosphorylated c-Mpl-del C-terminus Y607 and biased activation of PI3K/AKT pathway, which culminated in downregulation of DDIT3-related apoptotic responses conducive to AMKL survival and proliferation. Interpretation: This study elucidates a novel role of c-Mpl in AMKL progression and drug resistance, which not only improves our understanding of c-Mpl-dependent leukemia development, but also has important implications in prognosis and treatment of AMKL. Funding: A full list of funding can be found in the Acknowledgments section. Declaration of Interest: All authors declare that they have no conflict of interest. Ethical Approval: All clinical samples were obtained with informed consent from Sun Yat-sen University Cancer Center and approved by the Hospital’s Ethical Review Committees. The animal studies were authorized by the Animal Ethic Review Committees of the Sun Yat-sen University.