Diverse Role of DNA Hypermethylation in Reduced Expression of Tumor Suppressor NR4A3 in Acute Myeloid Leukemia

The expression of NR4A3, which is a member of the gene encoding NR4A orphan nuclear receptor subfamily, has been reported to be commonly silenced in blasts of patients with acute myeloid leukemia (AML), irrespective of karyotype. In line with this finding, Nr4a1^-/-/Nr4a3^-/- mice rapidly develop AML within one month following birth (Mullican et al., 2007). In addition, Nr4a1^+/-/Nr4a3^-/- and Nr4a1^-/-/Nr4a3^+/- mice show myelodysplastic/myeloproliferative neoplasms (Ramirez-Herrick et al., 2011), suggesting that NR4A3 functions as a tumor suppressor gene in myeloid malignancies. The extremely short latency of AML development in Nr4a1^-/-/Nr4a3^+/- mice indicates that silencing these tumor suppressors is sufficient to induce AML and that NR4A3 has a crucial role in the pathogenesis of AML. Thus, unveiling the molecular mechanism that regulates NR4A3 expression in AML would facilitate the development of novel therapies, including transcriptional reactivation of the gene. However, the therapeutic modalities targeting NR4A3 have been hindered by our minimal understanding of the mechanism underlying reduced NR4A3 expression, particularly in human AML cells. Abnormal epigenetic regulation is a common mechanism in the pathogenesis of several types of cancers. For instance, the expression of several tumor suppressor genes, such as p16 and MLH1, is repressed due to DNA hypermethylation at their promoter regions. Given that loss-of-function mutations in NR4A3 have not been reported in AML to date, we hypothesized that DNA hypermethylation contributes to a reduction in NR4A3 expression in AML. To test our hypothesis, we analyzed DNA methylation status of NR4A3 in human AML cells.

We first compared the level of NR4A3 expression in eight human AML cell lines and two human primary AML samples, with that in CD34+ mononuclear bone marrow (BM) cells from healthy human controls. As expected, the expression of NR4A3 was markedly reduced in all of the AML cell lines and primary AML cells compared with that in the cells of the healthy controls. To evaluate the function of NR4A3 in human AML cells, we ectopically overexpressed NR4A3 in a human AML cell line (NB4 cells). The growth of NR4A3 -overexpressing NB4 cells was remarkably compromised compared with that of the controls, suggesting a tumor suppressive function of NR4A3 in both human AML and murine cells. To investigate the DNA methylation status of NR4A3, we performed bisulfite sequencing assays using eight human AML cell lines (HL60, NB4, Kasumi, TF1, U937, K562, MOLM13, and THP1) as well as CD34+ BM cells from healthy controls. Unexpectedly, a hypermethylated CpG site in the promoter region was not detected in any of the cell lines. However, the drastically or mildly methylated region including twenty eight CpGs was identified approximately 3 kb downstream of the transcription start site in six AML cell lines (97.5%, 78.3%, 77.1%, 89.9%, 95.2%, and 86.9% in HL60, NB4, Kasumi, TF1, U937, and K562, respectively) and two mixed lineage leukemia-related cell lines (31.0% and 53.6% in MOLM13 and THP1, respectively), whereas this site was hypomethylated in the controls (n = 2; mean, 12.7%; range, 7.1%-18.2%). To evaluate the contribution of this hypermethylated region to reduced NR4A3 expression, the six AML cell lines with heavily hypermethylated CpGs at NR4A3 and two human primary AML cell samples were treated with a DNA methyltransferase inhibitor (decitabine; DAC) for three or five days. DAC exposure inhibited cell growth and restored the expression of NR4A3 in all AML cell lines and primary cells in a dose- and time-dependent manner. Next, we examined the status of DNA methylation at the CpG site following DAC treatment with bisulfite sequencing assays. The frequencies of methylated CpG in HL60, NB4, and K562 cells was reduced from 97.5% to 53.6%, 78.3% to 68.5%, and 86.9% to 67.5% after DAC treatment, respectively. In contrast, the methylation status in Kasumi, TF1, and U937 cells did not significantly changed after DAC treatment.

Our findings in the present study suggest that DNA hypermethylation may partially account for the transcriptional inactivation of NR4A3 in AML. However, the mechanism of reduced NR4A3 expression is complex and variable depending on the genetic background. We are currently working on a more detailed analysis of DNA methylation using human primary cells, by extending the regions for investigation, such as enhancer regions.