Overexpression Of DNMT3a and DNMT3b Is Related To Downregulation Of Mir-29a In Juvenile Myelomonocytic Leukemia (JMML)

Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD). It occurs in infancy and young children with a progressive course leading to death within one year after diagnosis. This disease is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. Mutations or other abnormalities in RAS , NF1 , PTPN11 , and CBL have been linked to be responsible for the pathogenesis of JMML in up to 85% of cases. Treatment is very difficult in JMML, and only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28-55%), with 5-year disease-free survival rates of 25-40%. JMML occurs in an age-range when genes are actively being turned on or off in children in adaption to the oxygenized environment after birth. Epigenetics plays a key role in this developmental plasticity. We previously reported hypermethylation on the promoter of PTEN in 77% of JMML patients, and decitabine, a DNA-hypomethylating reagent, significantly inhibited colony formation (CFU-GM) in JMML cells in vitro. In addition, other groups found that aberrant DNA methylation on promoters of BMP4, CALCA, CDKN2B, and RARB is significantly associated with poor prognosis in JMML. Taking together, these data suggest that epigenetic mechanisms may contribute to the pathogenesis of JMML. MicroRNAs (miRNAs) have been reported to play an important role in myeloid differentiation and activation. miRNA function is highly dependent on the cell type. Recently, we reported that miR-183 is overexpressed in JMML. Other groups have reported aberrant expression of miR-29a in acute myeloid leukemia and other cancers. Both miR-183 and miR-29a are located on chromosome 7q32 in humans, which is frequently disrupted in JMML. We hypothesized that miR-29a may be deregulated in JMML, and contribute to the aberrant epigenetic regulation in JMML. In order to test our hypothesis, we collected peripheral blood or bone marrow from 41 JMML patients and 14 normal individuals. Total RNAs were extracted from mononuclear cells (MNCs) using Trizol. We first evaluated the expression levels of miR-29a by using relative-quantitative real-time RT-PCR (qRT-PCR). We found that the expression levels (RQ) of miR-29a in patients are significantly lower than that in normal individuals (median 0.45 vs. 1.11, p< 0.001). By analysis of the expression levels of miR-29a together with previous data on miR-183 in these JMML patients and normal controls, we found that the RQ of miR-29a is inversely correlated with RQ of miR-183 (Spearman’s r=-51, p<0.001). This suggests that the expression of miR-29a is segregated from that of miR-183 in MNCs, although they are located only 1.1 million base-pairs apart on chromosome 7q32. When further evaluating the mRNA expression levels of miR-29a targeting genes by using qRT-PCR, we found that RQ of DNA methyltransferases 3A and 3B (DNMT3a and DNMT3b) were significantly increased in JMML patients’ samples in comparison with normal controls (median 5.16 vs. 1.00, p=0.001 for DNMT3a; median 3.45 vs. 1.08, p=0.001). Strikingly, the RQ of miR-29a was inversely correlated with the RQs of both DNMTs (Spearman’s r=-0.62 for DNMT3a; Spearman’s r=-0.79 for DNMT3b, p<0.001), which suggests that miR-29a plays a significant role in the regulation of DNMT 3a and DNMT3b in MNCs. In conclusion, we found that overexpression of DNMT3a and DNMT3b, the two key molecules in DNA methylation of epigenetic modification in developmental plasticity, is related to downregulation of miR-29a in JMML. In addition to our previous report on overexpression of miR-183 in JMML, this finding provides new insights into the role of miRNAs in the aberrant epigenetic regulation in JMML, and it may apply to other pediatric malignancies. Further investigation is ongoing to uncover the mechanism of down-regulated miR-29a in JMML. If the underexpression of miR-29a in JMML is due to the hypermethylation on miR-29a promoter, as reported in prostate cancer cells, demethylating agents, such as decitabine, or soybean products, isoflavone, could be potential drugs to treat JMML. Disclosures: No relevant conflicts of interest to declare.