The ParaHox gene Cdx4 induces acute erythroid leukemia in mice

Abstract

Acute erythroid leukemia (AEL) is a rare and aggressive form of acute leukemia, the biology of which remains poorly understood. Here we demonstrate that the ParaHox gene CDX4 is expressed in patients with acute erythroid leukemia, and that aberrant expression of Cdx4 induced homogenously a transplantable acute erythroid leukemia in mice. Gene expression analyses demonstrated upregulation of genes involved in stemness and leukemogenesis, with parallel downregulation of target genes of Gata1 and Gata2 responsible for erythroid differentiation. Cdx4 induced a proteomic profile that overlapped with a cluster of proteins previously defined to represent the most primitive human erythroid progenitors. Whole-exome sequencing of diseased mice identified recurrent mutations significantly enriched for transcription factors involved in erythroid lineage specification, as well as TP53 target genes partly identical to the ones reported in patients with AEL. In summary, our data indicate that Cdx4 is able to induce stemness and inhibit terminal erythroid differentiation, leading to the development of AEL in association with co-occurring mutations.

Graphical abstract

Figure 1.

CDX4 is expressed in patients with AML M6 and induces an expansion of erythroid cells in vitro. (A) Microarray analyses showing expression of CDX4 in samples from patients with FAB M6 compared with non-M6 AML. (B) Fold expression measured by qRT-PCR of CDX4 in embryonic stem cells as a positive control, CD34⁺ human bone marrow cells, CB-derived hematopoietic stem cells (HSC), common lymphoid progenitors (CLP), common myeloid progenitor (CMP), granulocyte-macrophage progenitors (GMP), myeloid-erythroid progenitors compared with and human AEL patient samples (n = 8). Fold values were obtained through normalization to expression of TATA binding protein/β-actin. (C) Total cell number in liquid culture expansion assay of HSPCs transduced with Cdx4 (n = 6) or vector control (n = 7). Fluorescence-activated cell sorter-purified cells were plated 72 hours after retroviral infection, and viable cells were counted every 7 days. After day 14, all cells in the control group showed mast cell characteristics and were counted as 0. Total cell number difference was significant on day 7 and day 14; *P < .05 (Wilcoxon test, P = .0313). (D) Total number of Ter119⁺ cells at day 7 and day 21 of proliferation assay (n = 3-7). (E) CFC assay for HSPCs transduced with vector control and Cdx4 plated 72 hours after retroviral infection. The primary CFCs were euthanized and replated 4 times every 7 to 9 days. Total number of colonies generated from 500 cells plated in primary CFCs (n = 7) derived from BM cells transduced with the vector control (ctrl) or Cdx4 (*P < .05; Wilcoxon test, P = .027). (F) Total number of GFP⁺ Ter119⁺ cells generated in primary and tertiary CFCs (n = 3). Total GFP⁺ Ter119⁺ cell number in Cdx4 primary CFC compared with vector ctrl, *P < .05 (Wilcoxon test, P = .0313). Total increase in GFP⁺ Ter119⁺ cell number in Cdx4 primary CFC compared with tertiary CFC is indicated. *P < .05 (Mann-Whitney U test, P = .017).

Figure 2.

Cdx4 induces an expansion of erythroid cells in vivo and an AEL in transplanted mice. (A) Spleen colony formation (CFU-S) was compared between Cdx4 (n = 4) and control vector (n = 5) transduced HSPCs. Transduced cells were sorted 72 hours after retroviral infection and injected in the tail vein of lethally irradiated mice 12 days before euthanasia. CFU-S frequency was calculated per 10⁴ cells. Data are represented in a Log10 scale. Values are shown as mean ± standard error of the mean. Significance was calculated by the Mann-Whitney U test (**P < .01). (B) Spleen cells from panel A were stained and analyzed by fluorescence-activated cell sorting for the expression of myeloid and erythroid markers. The bar graph shows the mean percentage (± standard error of the mean) myeloid marker (Gr-1⁺/Mac-1⁺, Gr-1⁺/Mac-1⁻, Gr-1⁻/Mac-1⁺) and erythroid marker (CD71⁺/Ter119⁻, CD71⁺/Ter119⁺, and CD71⁻/Ter119⁺)-expressing cells and refers to the GFP⁺ compartment. Significance was calculated by the Mann-Whitney U test (*P = .0159). (C) Kaplan-Meier survival curves of primary (n = 12) and secondary (n = 22) mice transplanted with 5FU-stimulated BM cells expressing Cdx4 or the vector control. Mantel-Cox log-rank test was performed on mice injected with control vector vs Cdx4 (primary recipient mice; **P = .0045) and Cdx4 (secondary recipient mice; **P = .0019), respectively. Mantel-Cox log-rank test on all 3 survival curves was also found significant (****P < .0001).

Figure 3.

The histopathological analysis of a representative Cdx4 mouse showing the presence of erythroblasts in different mouse organs. (A) Wright-Giemsa staining of peripheral blood. (B) Ter119 staining of the spleen. Hematoxylin and eosin (H&E) staining of the liver (C), testis (D), and pancreas (E). (F) Myeloperoxidase (MPO) staining of the lung.

Figure 4.

The erythroid phenotype is shaped by differential Hox gene expression. (A) qRT-PCR analysis of members of HoxA and B cluster genes along with Cdx genes in Cdx2 and Cdx4 transduced cells of primary CFC. ΔC_t values were obtained by normalizing to Gapdh, and fold expression compared with empty vector was calculated. The diagram shows average expression levels of 3 independent experiments ± SD. Vector control transduced cells negative for Cdx2 expression; *significant difference between vector control, Cdx2 and Cdx4, respectively. (B) Expression of Hox cluster genes in diseased Cdx4 transplanted mice. Mice that died of an acute leukemia after transplantation with Cdx2 or Cdx4 transduced BM were analyzed for their expression of Hox genes by TaqMan qRT-PCR. Multiple tests using Holm-Sidak method were performed on each gene. Cdx2, Cdx4, Hoxb3 and Hoxb4 were significantly differentially expressed. *Significant difference between Cdx2 and Cdx4, respectively.

Figure 5.

Cdx4 promotes expression of genes involved in oncogenic pathways and suppresses genes associated with erythroid differentiation. (A) Volcano plot showing all genes in the RNA sequencing analysis >P < .05. Each green dot represents a gene that is not differentially regulated. Upregulated DEGs in HSPCs, fold change >1.5 are indicated in red, downregulated DEGs in blue. Cumulative data of 3 biological independent experiments are shown for the vector control and Cdx4. (B) Heat map showing selected DEGs determined by RNA-Seq of Cdx4 and vector control-transduced HSPCs. Heat map representing unsupervised hierarchical clustering of all 6 samples for DEGs (fold change >1.5; P < .05). (C) Enrichr, ChEA, and ENCODE databases consensus transcription factor (TFs) enrichment analysis of downregulated (DN) DEGs, demonstrating enrichment for known targets of Gata1 and Gata2. (D) ENCODE and ChEA consensus TFs from ChIP-X analysis of downregulated DEGs (upper) and upregulated DEGs (lower). (E) The Molecular Signatures Database (MSigDB) oncogenic signature analysis of Cdx4 induced DEGs. (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) 2019 pathway analysis of Cdx4 induced DEGs. The analysis in panels D-F was performed on Enrichr, web-based analysis platform.

Figure 6.

Genes upregulated in the RNA-Seq analysis of Cdx4 transduced murine HSPCs show an enrichment of AML LSC signature genes and overlap with protein coding genes in human AEL. (A) GSEA analysis based on RNA-Seq of Cdx4 transduced HSPCs, showing enrichment of human LSC signature. Right, upregulated genes significantly enriched for the human LSC signature are shown as heat map. (B) Venn diagram representing the overlap of the DEGs on Cdx4 overexpression (P < .05) with published RNA sequencing data (protein coding genes) in human patients with AEL.  NES, normalized enrichment score.

Figure 7.

The protein profile of Cdx4 overexpressing HSPCs resembles the protein signature of immature erythroid stages. (A) Venn diagram representing the overlap of liquid chromatography-MS–based proteomics data (expressed proteins) to the published protein clusters, cluster 1 and cluster 2, derived from primitive stages of erythroid development. (B) Gene variants represented in a matrix plot found in 3 mice indicating 34 genes that were also found in patients with AEL. The 3 genes marked on top of the list were recurrently mutated (>2 patients) in patients with AEL.