The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model
Thomas Mercher, Glen D. Raffel, Sandra A. Moore, Melanie G. Cornejo, Dominique Baudry-Bluteau, Nicolas Cagnard, Jonathan L. Jesneck, Yana Pikman, Dana Cullen, Ifor R. Williams, Koichi Akashi, Hirokazu Shigematsu, Jean-Pierre Bourquin, Marco Giovannini, William Vainchenker, Ross L. Levine, Benjamin H. Lee, Olivier A. Bernard, D. Gary Gilliland
Thomas Mercher, Glen D. Raffel, Sandra A. Moore, Melanie G. Cornejo, Dominique Baudry-Bluteau, Nicolas Cagnard, Jonathan L. Jesneck, Yana Pikman, Dana Cullen, Ifor R. Williams, Koichi Akashi, Hirokazu Shigematsu, Jean-Pierre Bourquin, Marco Giovannini, William Vainchenker, Ross L. Levine, Benjamin H. Lee, Olivier A. Bernard, D. Gary Gilliland
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Research Article Oncology

The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model

Abstract

Acute megakaryoblastic leukemia (AMKL) is a form of acute myeloid leukemia (AML) associated with a poor prognosis. The genetics and pathophysiology of AMKL are not well understood. We generated a knockin mouse model of the one twenty-two–megakaryocytic acute leukemia (OTT-MAL) fusion oncogene that results from the t(1;22)(p13;q13) translocation specifically associated with a subtype of pediatric AMKL. We report here that OTT-MAL expression deregulated transcriptional activity of the canonical Notch signaling pathway transcription factor recombination signal binding protein for immunoglobulin κ J region (RBPJ) and caused abnormal fetal megakaryopoiesis. Furthermore, cooperation between OTT-MAL and an activating mutation of the thrombopoietin receptor myeloproliferative leukemia virus oncogene (MPL) efficiently induced a short-latency AMKL that recapitulated all the features of human AMKL, including megakaryoblast hyperproliferation and maturation block, thrombocytopenia, organomegaly, and extensive fibrosis. Our results establish that concomitant activation of RBPJ (Notch signaling) and MPL (cytokine signaling) transforms cells of the megakaryocytic lineage and suggest that specific targeting of these pathways could be of therapeutic value for human AMKL.

Authors

Thomas Mercher, Glen D. Raffel, Sandra A. Moore, Melanie G. Cornejo, Dominique Baudry-Bluteau, Nicolas Cagnard, Jonathan L. Jesneck, Yana Pikman, Dana Cullen, Ifor R. Williams, Koichi Akashi, Hirokazu Shigematsu, Jean-Pierre Bourquin, Marco Giovannini, William Vainchenker, Ross L. Levine, Benjamin H. Lee, Olivier A. Bernard, D. Gary Gilliland

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Figure 5

MPL mutant transforms 6133 AMKL cells.

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MPL mutant transforms 6133 AMKL cells. (A) Parental 6133 cells were tran...
(A) Parental 6133 cells were transduced with empty vector (MIG) or JAK2WT, MPLWT, JAK2V617F, JAK2T875N, MPLW515L, and FLT3ITDN51 retroviruses. 24 hours after infection, cells were transferred to SCF-free medium, and viable cells were counted daily using a trypan blue exclusion assay. (B) 6133 cells growing with SCF stimulation or transduced with MPLW515L were cytospun on glass slides and stained for AchE. Representative pictures are shown. Original magnification, ×100. (C) Flow cytometric analysis of phospho-ERK1/2 in 6133 cells, 48 hours after transduction with the different mutants and controls. Mean fluorescence intensity is indicated on the histograms. (D) Growth inhibition of 6133+MplW515L cells by MAPK inhibitor. The MAPK inhibitor PD98059 was used to treat 6133+MplW515L cells. Growth at 48 hours compared with that of untreated cells is shown. Mean ± SD of 3 independent experiments is shown. (E) ERK1/2 phosphorylation in 6133+MplW515L cells in presence of MAPK inhibitor. 6133+MplW515L cells were treated with PD98059, and cell lysates were used in direct Western blot for phospho-ERK1/2, followed by total ERK1/2. (F) 6133 cells transduced with WT MPL were maintained under SCF culture and then stimulated with TPO (10 ng/ml). RNA was extracted before TPO stimulation (control) and 2 hours after (+TPO). Expression of Hes1 and Gata1 transcripts was compared with Gapdh by quantitative RT-PCR. Mean ± SD of 3 independent experiments is shown. (G) 6133 cells were analyzed as in F and treated with MAPK inhibitor (PD98059: 5 μM) during TPO stimulation. Mean ± SD of 3 independent experiments is shown.