Downregulation of SOX7 by DNA Methylation and Its Tumor Suppressing Role In Acute Myeloid Leukemia

The SOX (Sry-related HMG box) gene family is a group of transcription factors containing in common a High-Mobility-Group (HMG) box domain which shares more than 60% homology to that in Sry. SOX proteins are involved in diverse embryonic processes and recently Sox7 was shown to regulate hematopoietic stem and progenitor cells during mouse development. In this study, we examined the expression, regulation and function of SOX7 in human acute myeloid leukemia with a view to understand its link to leukemogenesis.

Bone marrow (BM) or blood samples from patients with primary hematological malignancies, as well as cord blood obtained from normal Caesarian Sections were prospectively collected and mononuclear cells (MNC) fractions were obtained. Screening for SOX gene expression was performed by reverse-transcription polymer chain reaction (RT-PCR) and SOX7 expression in different experiments was further evaluated by quantitative real-time RT-PCR. Methylation of CpG islands around the sox7 transcription start site was studied by bisulphate DNA sequencing and methylation-specific PCR. Leukemic cell-lines (KG1, ML2, K562) and primary AML samples were treated with demethylating agent 5-aza-2′-deoxycytidine (5AdC). Cell proliferation of GFP or GFP-SOX7 expressing K562 cells was evaluated by SNARF-1 staining, cell-cycle analysis, ³H-thymidine incorporation and clonogenic assays. Apoptosis were evaluated by Annexin V/7-AAD assay. Canonical wnt activity of K562 cells expressing GFP or GFP-SOX7 was measured by TOP-FLASH dual luciferase assay.

The expression of 19 SOX genes was tested by RT-PCR in normal umbilical cord blood (UCB) as well as bone marrow or blood samples from patients with hematological malignancies. SOX7 was uniquely expressed in CD34⁺ cells from UCB (N=11) and most case of precursor B-cell acute lymphoblastic leukemia (ALL) (17 out of 20 tested) and a ALL derived cell line Nalm-20, but not any case of acute myeloid leukemia (N=22), myelodysplastic syndrome (N=16) or chronic myelogenous leukemia (N=13). In myeloid leukemia cell lines (KG1, ML2, K562) and primary AML samples, but not Nalm-20, the transcription start site of SOX7 contained CpG islands which were heavily methylated. Treating myeloid leukemic cell lines with 5AdC induced SOX7 expression. Enforced expression of SOX7 in K562 cells by lentiviral transduction significantly reduced cell proliferation as shown by cell growth in cultures (SOX7: 6.5-fold increase; GFP: 21.7-fold increase on Day 9, N=2), SNARF-1 staining (SOX7: 57.5%; GFP: 78.0% of total cells divided twice, N=2), ³H-thymidine incorporation assay (SOX7: 3987 cpm; GFP: 5767 cpm, N=2) and colony-forming unit (SOX7: 262±99 per 1000 input cells; GFP: 464±145 per 100 input cells, p=0.055). It also induced cell cycle delay in S/G₂/M phases (SOX7: 53.4±0.35%; GFP: 44.4±2.28%, p=0.029). Apoptosis was not affected. SOX7 expression in K562 cells significantly reduced canonical-wnt activity as measured by TOP-FLASH dual luciferase assay (SOX7: 30.0±7.1-fold to FOP-FLASH; GFP: 130±18.8-fold to FOP-FLASH, p=0.0081).

SOX7 expression in AML was regulated by promoter hypermethylation and its forced expression in K562 cells reduced cell proliferation and inhibited the canonical wnt signaling pathway. The pathogenetic link between SOX7 gene silencing and AML leukemogenesis is being investigated in our laboratory.

The project was supported by a grant from the strategic Research Theme of cancer stem cells in the HKU.

No relevant conflicts of interest to declare.