PLAGL2 Cooperates in Leukemia Development by Direcly Inducing c-Mpl Expression.

Inversion of chromosome 16 is found in 12% of human acute myeloid leukemia (AML). This inversion creates the CBFΒ-SMMHC fusion protein, which hinders myeloid differentiation and triggers AML in collaboration with additional mutations. We have previously shown that the zinc finger transcription factor PLAGL2 efficiently cooperates with CbfΒ-SMMHC in murine AML, and that PLAGL2 transcript levels are significantly increased in inversion 16 human AML. The mechanism of PLAGL2 action in AML, however, is not understood.

In order to identify candidate PLAGL2 target genes in AML, we performed gene expression profiling of primary hematopoietic progenitors expressing CbfΒ-SMMHC and PLAGL2, as well as in AML samples induced by CbfΒ-SMMHC and PLAGL2. The expression of c-Mpl transcript was increased 8 to 32 fold in progenitors and AML samples when compared to controls not expressing PLAGL2. This increase was confirmed by quantitative PCR analysis and correlated with increased c-Mpl receptor in membrane by flow cytometry. Analysis of the c-Mpl proximal promoter identified three conserved Plag binding sites. PLAGL2 binding to these sites was confirmed by chromatin immunoprecipitation and luciferase reporter assays. Furthermore, PLAGL2/CbfΒ-SMMHC AML cells show a significant increase in sensitivity to the c-Mpl-ligand thrombopoietin when compared to non-PLAGL2 CbfΒ-SMMHC-AML samples as measured by Jak2 phosphorylation.

To test whether PLAGL2 induces AML by increasing c-Mpl levels, AML development was analyzed in bone-marrow transplantation assays using CbfΒ-SMMHC-expressing donor cells transduced with c-Mpl expressing retrovirus. All transplanted mice (8/8) succumbed to leukemia with a median latency of 8 weeks, while control groups remained unaffected for 16 weeks. The pathology of Mpl/CbfΒ-SMMHC AML was immuno-phenotypically and morphologically identical to that of PLAGL2/CbfΒ-SMMHC AML.

This study demonstrates that PLAGL2 directly induces c-Mpl receptor expression, and that such increase cooperates with CbfΒ-SMMHC in AML development. These results suggest that AML can result from the alteration of upstream regulators of Mpl/Jak2 signaling.