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. 2011 Apr;25(4):655-62.
doi: 10.1038/leu.2010.301. Epub 2011 Jan 25.

The transcription factor PlagL2 activates Mpl transcription and signaling in hematopoietic progenitor and leukemia cells

S F Landrette  1 , D Madera, F He, L H Castilla
Affiliations

The transcription factor PlagL2 activates Mpl transcription and signaling in hematopoietic progenitor and leukemia cells

S F Landrette et al. Leukemia. 2011 Apr.

Abstract

Cytokine signaling pathways are frequent targets of oncogenic mutations in acute myeloid leukemia (AML), promoting proliferation and survival. We have previously shown that the transcription factor PLAGL2 promotes proliferation and cooperates with the leukemia fusion protein Cbfβ-SMMHC in AML development. Here, we show that PLAGL2 upregulates expression of the thrombopoietin receptor Mpl, using two consensus sites in its proximal promoter. We also show that Mpl overexpression efficiently cooperates with Cbfβ-SMMHC in development of leukemia in mice. Finally, we demonstrate that PlagL2-expressing leukemic cells show hyper-activation of Jak2 and downstream STAT5, Akt and Erk1/2 pathways in response to Thpo ligand. These results show that PlagL2 expression activates expression of Mpl in hematopoietic progenitors, and that upregulation of wild-type Mpl provides an oncogenic signal in cooperation with CBFβ-SMMHC in mice.

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Figures

Figure 1
Figure 1. Identification of PLAGL2 target genes in leukemia development using microarray analysis
(A) Cluster of genes specifically up-regulated in PLAGL2+ cells from two-dimensional hierarchical clustering analysis. Hematopoietic progenitor samples infected with MIG (MIG1-HP and MIG2-HP), HPs infected with PLAGL2-MIG (PL2+1HP and PL2+2MIG), AML samples expressing PLAGL2 (PL2+1, PL2+2, and PL2+ AMLs), and AML samples not expressing PLAGL2 (PL2−1, PL2−2, PL2−3 AMLs). All samples express Cbfb-MYH11. (B) Relative Mpl transcript levels of Cbfb-MYH11/hematopoietic progenitor (HP) transduced with MIG (lane 1) or MIG-PLAGL2 (lane 2) retrovirus, and of PLAGL2-negative (PL2−; lanes 3-6) and PLAGL2-positive (PL2+; lanes 7-10) Cbfb-MYH11 AML samples using quantitative RT-PCR analysis. (C) Expression of Mpl receptor in AML cells with non-detectable PLAGL2 expression (left panel) or expressing PLAGL2 (right panel) using FACS analysis. Percentage of cells expressing Mpl is shown relative to negative control.
Figure 2
Figure 2. Mpl expression is upregulated by PLAGL2 in hematopoietic cells
(A) Top: depiction of the Mpl gene structure, including untranslated exon sequences (blank boxes), coding regions (black boxes), translation start (arrow). Middle: zoom-in of the proximal promoter, exon 1 (black box) and section of intron 1 (arrowed line), and 30-way multiz-alignment & mammalian conservation analysis of the mouse genome segment chr4:118,129,933-118,130,333; using the UCSC Genome Browser on Mouse July 2007 (NCBI37/mm9) Assembly. Bottom: ClustalW sequence alignment of the Mpl proximal promoter (−189 to +3 from translation start site) including human (Hsa), monkeys (Rhe), mouse (Mmu), dog (Cfa), horse (Efe), and cow (Bta). The PLAGL2 consensus binding sites GRGGC(6-8)RGGK (P1 and P2) and a third site (P3) with an (N)9 linker are shown in boxes. Arrows indicate orientation of binding site. (B) Analysis of PLAGL2 activation of the Mpl proximal promoter using luciferase reporters in NIH3T3 cells. The luciferase reporters with 300 bp of the Mpl proximal promoter (wt) and respective mutants ablating one or more sites (m1, m2, m3, m1, 2, 3) are illustrated on the left panel. Fold activation relative to wt reporter and no PLAGL2 (MIG + wt) of each construct with (PL2) or without (MIG) PLAGL2 is shown on right panel. (C) Electrophoretic mobility shift assay of PLAGL2 consensus sites P1, P2, and P3 in the Mpl proximal promoter, using labeled wild type oligo (o), unlabeled oligo (c), or labeled oligo with point mutation in the core box of the Consensus PLAGL2 site (m). Arrow indicates PLAGL2:DNA binding.
Figure 2
Figure 2. Mpl expression is upregulated by PLAGL2 in hematopoietic cells
(A) Top: depiction of the Mpl gene structure, including untranslated exon sequences (blank boxes), coding regions (black boxes), translation start (arrow). Middle: zoom-in of the proximal promoter, exon 1 (black box) and section of intron 1 (arrowed line), and 30-way multiz-alignment & mammalian conservation analysis of the mouse genome segment chr4:118,129,933-118,130,333; using the UCSC Genome Browser on Mouse July 2007 (NCBI37/mm9) Assembly. Bottom: ClustalW sequence alignment of the Mpl proximal promoter (−189 to +3 from translation start site) including human (Hsa), monkeys (Rhe), mouse (Mmu), dog (Cfa), horse (Efe), and cow (Bta). The PLAGL2 consensus binding sites GRGGC(6-8)RGGK (P1 and P2) and a third site (P3) with an (N)9 linker are shown in boxes. Arrows indicate orientation of binding site. (B) Analysis of PLAGL2 activation of the Mpl proximal promoter using luciferase reporters in NIH3T3 cells. The luciferase reporters with 300 bp of the Mpl proximal promoter (wt) and respective mutants ablating one or more sites (m1, m2, m3, m1, 2, 3) are illustrated on the left panel. Fold activation relative to wt reporter and no PLAGL2 (MIG + wt) of each construct with (PL2) or without (MIG) PLAGL2 is shown on right panel. (C) Electrophoretic mobility shift assay of PLAGL2 consensus sites P1, P2, and P3 in the Mpl proximal promoter, using labeled wild type oligo (o), unlabeled oligo (c), or labeled oligo with point mutation in the core box of the Consensus PLAGL2 site (m). Arrow indicates PLAGL2:DNA binding.
Figure 3
Figure 3. Mpl expression cooperates with Cbfβ-SMMHC in leukemia development
Schematic representation of viral vectors (A) and experimental strategy (B) used in BM transplantation assay. Hematopoietic progenitors from wild type and Cbfb+/56M;Mx1Cre mice (expressing CBFb-MYH11) were infected with MIG, MIG-Mpl, or MIG-Mpl-Y112F retrovirus and transplanted into sub-lethally irradiated recipient mice. (C) Kaplan Meier survival curves for mice transplanted with wild type (wt) or Cbfb+/56M;Mx1Cre (CM) cells infected with MIG-Mpl (Mpl), MIG-MplY112, or MIG retroviruses. (D) Representative FACS analysis of peripheral blood white blood cells from a leukemic mouse expressing Cbfβ-SMMHC and MIG-Mpl (GFP+cKit+ cells). (E) Representative cell morphology of 2 AML samples from peripheral blood of CM/Mpl leukemic mice, depicting balstlike (red arrow) and monocyticlike (asterisks), with magnification 100X. (F) Spleen weight of leukemic (n=11) and control (n=6) mice.
Figure 3
Figure 3. Mpl expression cooperates with Cbfβ-SMMHC in leukemia development
Schematic representation of viral vectors (A) and experimental strategy (B) used in BM transplantation assay. Hematopoietic progenitors from wild type and Cbfb+/56M;Mx1Cre mice (expressing CBFb-MYH11) were infected with MIG, MIG-Mpl, or MIG-Mpl-Y112F retrovirus and transplanted into sub-lethally irradiated recipient mice. (C) Kaplan Meier survival curves for mice transplanted with wild type (wt) or Cbfb+/56M;Mx1Cre (CM) cells infected with MIG-Mpl (Mpl), MIG-MplY112, or MIG retroviruses. (D) Representative FACS analysis of peripheral blood white blood cells from a leukemic mouse expressing Cbfβ-SMMHC and MIG-Mpl (GFP+cKit+ cells). (E) Representative cell morphology of 2 AML samples from peripheral blood of CM/Mpl leukemic mice, depicting balstlike (red arrow) and monocyticlike (asterisks), with magnification 100X. (F) Spleen weight of leukemic (n=11) and control (n=6) mice.
Figure 3
Figure 3. Mpl expression cooperates with Cbfβ-SMMHC in leukemia development
Schematic representation of viral vectors (A) and experimental strategy (B) used in BM transplantation assay. Hematopoietic progenitors from wild type and Cbfb+/56M;Mx1Cre mice (expressing CBFb-MYH11) were infected with MIG, MIG-Mpl, or MIG-Mpl-Y112F retrovirus and transplanted into sub-lethally irradiated recipient mice. (C) Kaplan Meier survival curves for mice transplanted with wild type (wt) or Cbfb+/56M;Mx1Cre (CM) cells infected with MIG-Mpl (Mpl), MIG-MplY112, or MIG retroviruses. (D) Representative FACS analysis of peripheral blood white blood cells from a leukemic mouse expressing Cbfβ-SMMHC and MIG-Mpl (GFP+cKit+ cells). (E) Representative cell morphology of 2 AML samples from peripheral blood of CM/Mpl leukemic mice, depicting balstlike (red arrow) and monocyticlike (asterisks), with magnification 100X. (F) Spleen weight of leukemic (n=11) and control (n=6) mice.
Figure 4
Figure 4. AML cells expressing Cbfb-MYH11 and PLAGL2 are hypersensitive to Tpo ligand
(A) Phosphorylation analysis of Mpl target Jak2 protein in CBFb-MYH11 AML samples expressing PLAGL2 (PL2+ AML) or not (PL2− AML), after serum starvation and incubation with Tpo ligand. (B) Expression of phospho-Stat5, Stat5, phospho-Erk1/2, Erk1/2, phospho-Akt, Akt, and B-actin by immunoblot analysis after serum starvation and incubation with Tpo ligand. (C) Densitometric quantification of phospho-protein levels shown in B.
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