The leukemias with core-binding factor inv(16)(p13;q22) frequently present oncogenic mutations in receptor tyrosine-kinase pathway proteins KIT, FLT3 and RAS, which provide proliferation and survival capacity to hematopoietic blasts (8
). We have previously shown that the related transcription factors PLAG1 and PLAGL2 cooperate with CBFβ-SMMHC in leukemia development (10
). This study shows that the transcription factor PLAGL2 activates Mpl receptor, inducing activation of the Erk, Stat, Akt pathways.
The members of the PLAG zinc finger transcription factors PLAG1 and PLAGL2 have been shown to activate growth factor associated genes, including IGFII and CLF1, in epithelial cells (24
). We found that the PLAG factors promote proliferation of hematopoietic progenitors and participate in AML development (9
). Here we designed a gene expression profiling approach to identify genes consistently deregulated by PLAGL2 in hematopoietic progenitors as an “early” readout of PLAG function, and in leukemic cells expressing PLAGL2 as a “leukemic” readout. In this analysis, 23 genes were upregulated over 2 fold while no repressed genes were found, highlighting the transcription activation role of PLAG proteins. The sustained upregulation of Mpl levels in membrane by PLAGL2 strongly suggests that Mpl is a major player in PLAG mediated oncogenicity. Consistent with these findings, mutations in the MPL
genes that promote translation and protein stability have been found in the inherited myeloproliferative syndrome familial essential thrombocythemia (25
). In addition, somatic activating mutations in MPL
are present in a fraction of patients with myelofibrosis with myeloid metaplasia and essential thrombocythemia (27
). Future studies should evaluate the levels of PLAGL2 and MPL in myeloproliferative disorders that lack oncogenic mutations in MPL
Mpl expression is regulated by the ETS, RUNX and GATA factors in megakaryocytes (29
). The Tpo/Mpl pathway is a major regulator of hematopoietic stem cells (32
). However, the mechanism of regulation of Mpl expression in HSCs is not well understood. We identified two PLAG consensus sites within a 180 base pair evolutionary conserved region upstream of the Mpl
translation initiation site. These sites can be bound by PLAGL2 which upregulates transcription, strongly implicating PlagL2 as regulator of Mpl
transcription. Importantly, RUNX and ETS binding sites are also located within the Mpl proximal promoter, suggesting that Mpl expression could be regulated by the combinatorial role of multiple transcription factors in megakaryocytes, stem cells and leukemic cells. Future studies should unravel the specific PLAG, RUNX and ETS factors that interact to regulate Mpl expression in different cell types. In addition to the oncogenic role of PLAG1 and PLAGL2, PLAGL1 (the third member of the PLAG family) has been implicated as a tumor suppressor in multiple types of cancer, including breast and ovarian cancer and neck squamous cell carcinomas (reviewed in (35
)), and has recently been found repressed in diffuse large B-cell lymphoma (36
). Since PLAGL1 binds to the first GC-block, it is also possible that MPL expression in hematopoietic progenitors and leukemia may be affected by PLAGL1-loss of expression. Coexpression analysis of available gene expression datasets of human AML (www.oncomine.org
) suggest no correlation between PlagL2 and Mpl transcript levels. However, further study of the three PLAG proteins and other factors as modulators of Mpl signaling in human AML is warranted.
The present study demonstrates that PLAGL2 activates expression of Mpl, using two PLAG-consensus binding sites in its proximal promoter, and activates its downstream signaling in hematopoietic progenitors and leukemic cells. This activation is at least one of PLAGL2 induced oncogenic signals that promotes leukemia development in cooperation with CBFβ-SMMHC in mice.