CDX2 regulates Human Leukemogenesis Via Modulation Of The Wnt-Signaling Pathway
Authors: Paczulla Anna , Konantz Martina , Grzywna Sarah , Kanz Lothar , Lengerke Claudia 
Published: October 2019

Abstract

Introduction
The caudal-type homeobox (Cdx) gene family has been mainly studied during early development for its role in axial elongation and antero-posterior patterning. More recently, Cdx genes were shown to regulate embryonic hematopoiesis by interactions with the canonical Wnt pathway and Hox genes. The role of Cdx genes in adult hematopoiesis remains poorly understood. Adult hematopoietic stem and progenitor cells derived from healthy murine bone marrow (BM) express low levels of Cdx1 and Cdx4 but not Cdx2. However, the majority (>80%) of human acute myeloid (AML) and lymphoid leukemias (ALL) were shown to express the human homologue CDX2, and ectopic induction of Cdx2 expression was sufficient to robustly induce myeloid leukemia in murine bone marrow cells. On the molecular level, the leukemogenic activity of Cdx2 was associated with modulation of Hox and Klf4 gene expression (Faber et al, 2013). The current study further explores the role of CDX2 in leukemogenesis by analyzing the effects of CDX2 expression induction or repression on human healthy and malignant hematopoietic cells and its molecular effects on the Wnt signaling pathway known to regulate Cdx genes during embryonic development.

Methods
Human bone marrow or mobilized peripheral blood derived CD34+ cells as well as the human leukemic cell lines SKM-1, NOMO-1, EOL-1 and NALM16 were exposed to lentiviruses containing CDX2 overexpression, shRNAs against CDX2 or control constructs. Efficient modulation of CDX2 expression was verified on gene expression level by qRT-PCR and on protein level by immunoblot analysis. CDX2 modified and control cells were subjected to growth, colony forming (CFU), cell cycle, flow cytometry and qRT-PCR gene expression analysis assays and analyzed in vivo upon xenotransplantation in NOD/SCID/IL2Rγnull (NSG) mice. To explore the effect of Dickkopf-1 (DKK1), recombinant human DKK1 protein or carrier was supplemented to the methylcellulose in CFU assays.

Results
shRNA-mediated knockdown of CDX2 in leukemic cell lines lead to reduced growth (SKM-1, NALM16) and CFU formation (SKM-1 cells). Consistently, CDX2 knockdown SKM-1 cells showed lower ability to repopulate NSG mice and, upon subcutaneous injection in the flank, gave rise to much smaller tumors when compared to control cells, supporting the notion that CDX2 plays roles in human leukemogenesis. In contrast to the data published in mice, healthy human CD34+ cells transduced to overexpress CDX2 were unable to induce leukemia upon transplantation in NSG mice within an observation period of 5 months. On the molecular level, CDX2 modified cells showed differential expression of Klf4 and Hox but also Wnt pathway associated genes. Notably, robust induction of the canonical Wnt-inhibitory molecule DKK1 was observed in both healthy CD34+ stem/progenitor and leukemic cells upon CDX2 induction, while CDX2 suppression showed opposite effects. Analysis of the DKK1 promotor region revealed an interspecies conserved putative binding site for CDX2 as well as multiple HOX gene binding sites, suggesting that CDX2 can modulate DKK1 expression directly but also via its downstream HOX genes. Importantly, CFU assays performed on CDX2-knockdown cells showed a rescue of colony formation upon stimulation with DKK1 protein as compared to treatment with carrier only, demonstrating that the observed molecular interaction is functionally relevant in human leukemic cells. In contrast, control leukemic cells treated with DKK1 showed reduced CFU formation, indicating that CDX2 might act through DKK1 activation to fine-tune Wnt signal activation to the dosage that best promotes leukemogenesis and leukemic cell growth and survival.

Conclusion
Taken together, our data indicate that CDX2 employs DKK1 activation to modulate the Wnt signaling pathway and thereby growth, clonogenic capacity as well as in vivo tumorigenicity of human leukemia cells. In contrast to murine cells, CDX2 activation requires cooperative molecular events in order to induce leukemia in human healthy stem and progenitor cells.

Disclosures:
No relevant conflicts of interest to declare.