CDX2 expression in the hematopoietic lineage promotes leukemogenesis via TGFβ inhibition

Abstract

The intestine-specific caudal-related homeobox gene-2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane-bound inhibitor (Bambi), an inhibitor of transforming growth factor-β (TGF-β) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2-expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF-β-dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF-β signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment.

Keywords: BAMBI; acute monoblastic leukemia; ectopic expression; oncogene; tumor suppressor.

Fig. 1

Leukemia development by ectopic expression of CDX2 in the hematopoietic lineage. (A) Overall survival (in days) of control (n = 13, black line) and MxCDX2 mice (n = 18, red line) after poly(I:C) injections, and of NSG mice after transplantation of bone marrow cells from MxCDX2 mice (n = 8, red dotted line). (B) Bone marrow cellularity of control (n = 3) and MxCDX2 mice (n = 3). Boxes extend from 25 to 75th percentile, and whiskers represent mean to max. *P < 0.05. (C) Representative cytology (MGG staining) of smears of bone marrow cells of control and MxCDX2 mice. Bars: 25 µm. (D) Relative CDX2 mRNA expression analyzed by RT‐qPCR in the bone marrow of control (n = 3) and MxCDX2 mice (n = 6). Data are given means with SEM. *P < 0.02. (E) Representative western blot of CDX2 protein in the bone marrow of control and MxCDX2 mice. (F) Histology (HE) and immunohistochemical staining of the CDX2 protein in the bone marrow of control and MxCDX2 mice. Bars: 100 µm. (G) Weight (mg) and cellularity of the thymus and spleen of control (n = 3, open boxes) and MxCDX2 mice (n = 3, gray boxes). Boxes extend from 25th to 75th percentile, and whiskers represent mean to max. *P < 0.05; **P < 0.01. (H) Histology (HE) and immunohistochemical staining of the CDX2 protein in the liver, spleen, and thymus of control and MxCDX2 mice. Bars: 100 µm.

Fig. 2

Ex vivo replating activity of MxCDX2 bone marrow cells. (A) Number of colonies grown ex vivo from bone marrow cells of control (white boxes) and MxCDX2 mice (gray boxes) during five consecutive passages (P1–P5). Data are given means with SEM. n = 4. (B) Representative pictures of colonies grown from MxCDX2 bone marrow cells at the 5th replating step. Bars: 500 µm.

Fig. 3

Cellular consequences of the ectopic expression of CDX2. (A) Example of cytometry pattern in one control and one MxCDX2 mouse. Monocytes–macrophages are Cd11b⁺ Gr1^low Ly6C^+and− SSC^low. Immature neutrophils are Cd11b⁺ Gr1^low; mature neutrophils are Cd11b⁺ Gr1^high. Percentages for neutrophils are given compared with single cells, and for CLP compared with Lin⁻ Il7R⁻ Sca1^low cKitl^ow cells. In the illustrated examples, CLP represented 0.0243% and 0.000339% of the bone marrow single cells of control and MxCDX2 mice, respectively. (B) Percent of the indicated cell types in the bone marrow, spleen, and thymus of control and MxCDX2 mice. CLPs: common lymphoid progenitors; CMPs, common myeloid progenitors; LSK, Lin⁻ Sca⁺ cKit⁺ progenitors; MEPs, megakaryocyte–erythroid progenitors; MPPs, multipotential progenitors. Bars represent means, *P < 0.05; **P < 0.01.

Fig. 4

Molecular consequences of the ectopic expression of CDX2. (A) Heat map of the differentially expressed genes in the bone marrow of three control and three MxCDX2 mice. The position of the CDX2 gene and of major genes involved in hematopoiesis is indicated. (B) Gene Ontology term enrichment of the differentially expressed genes. (C) Circos plot of the relationship between the CDX2 protein or several transcription factors involved in hematopoiesis, and the presence of consensus DNA‐binding sites for these factors in their −2000 to +50 bp gene promoter. Green boxes and red boxes represent transcription factors whose genes are, respectively, upregulated and downregulated by ectopic expression of CDX2. Arrows connect a given transcription factor to the presence of consensus DNA‐binding site(s) for this factor in the promoters of transcription factor genes involved in hematopoiesis. Arrows are, respectively, in blue or orange when the factor and its target change in the same way or in opposite ways by ectopic expression of CDX2. A table version of this graph is given in Table S5.

Fig. 5

Involvement of BAMBI in the oncogenic effect of CDX2. (A) Volcano plot of the differentially expressed genes in the bone marrow of MxCDX2 compared with control littermates. Red: upregulated genes; green downregulated genes. The position of Bambi is indicated. (B) Comparative expression of the Bambi gene by RT‐qPCR in MxCDX2 (n = 7) and control mice (n = 6). Bars represent means, ***P < 0.001. (C) Expression of the BAMBI gene in human K562 cells transfected with the pFlag‐CDX2 plasmid compared to cells transfected with the control pFlag vector. n = 3; data are given means with SEM. *P < 0.02. (D) Luciferase activity produced by pBAMBI‐Luc in human K562 cells transfected with pFlag‐CDX2 or with pFlag. n = 9; data are given means with SEM. **P < 0.01. (E) CDX2 chromatin immunoprecipitation of BAMBI promoter fragments overlapping the CDX‐type DNA‐binding sites 1 and 2 (a) in human K562 cells transfected with pFlag‐CDX2 vs pFlag (n = 5), (b) in bone marrow cells of MxCDX2 vs control mice (n = 2), and (c) in mouse AGK463 nuclear extracts immunoprecipitated with anti‐CDX2 antibodies or with control IgG (n = 5). Data are given means with SEM. *P < 0.05. (F) Expression of the CDX2 and BAMBI proteins by western blot in AGK463 cells (lane 1) in comparison with the bone marrow of control and MxCDX2 mice (respectively, lanes 2 and 3). (G) Karyotype analysis revealing supernumerary chromosomes (n = 41) in the AGK463 cell line. (H) Relative Cd11b mRNA expression in AGK463 cells transfected with siRNA@CDX2 or siRNA@Bambi compared with control siRNA, in the absence or presence of TGF‐β (n = 9). Boxes extend from 25th to 75th percentile, and whiskers represent mean to max. *P < 0.05; ***P < 0.001. (I) Proportion of CD11b⁺ cells among siGLO⁺ AGK463 cells transfected with siGLO and the indicated siRNA, and treated or not with TGF‐β.