Microrna 124 and Its Role in Response to Epigenetic Therapy in Patients with Acute Mylogenous Leukemia and Myelodysplastic Syndrome

Abstract

MicroRNAs (miRs) are small RNA molecules of approximately 1–722 bps in length. They have been shown to control gene expression at the mRNA and protein levels by binding to the 3′ UTR of genes within a RNA Induced Silencing Complex (RISC). Recently miRs have been shown to have globally altered expression in several types of hematological malignances. However there is little data on how these miRs may affect the biology of the disease or their effects on response to chemotherapy. Some miRs have also been shown to be regulated at an epigenetic level, and we speculated that their expression may be involved in response to epigenetic therapy. Here, we focused on miR124 in patients with Acute Mylogenous Leukemia (AML) and Myelodysplastic Syndrome (MDS) who were treated with epigenetic chemotherapy, decitabine or decitabine and valproic acid. This miR is transcribed in three different regions of the genome hsa-miR124a-1 chr.8p, hsa-miR124a-2 chr.8q and hsa-miR-124a-3 chr.20q, however only two have CpG islands: a-1 and a-3. MiR124a-1 and a-3 have recently been shown to be frequently methylated in patients with AML which may lead to altered expression of the miR as well as the miR targets.We began our study by looking at baseline (pretreatment) methylation, by bisulfite pyrosequencing, in 56 patients (36=MDS,20=AML) and found that miR124a-1 was hypermethylated in 78% of MDS and 95% of AML patients compared to the 95% CI of normal controls. We also looked at methylation of miR124a-3 at baseline and found that 47% MDS patients and 65% of AML patients were hypermethylated above the 95% CI of normal controls. In a separate set of 60 MDS patients, we found that methylation of miR124a-1 at the time of diagnosis could significantly predict shorter overall survival in patients who were methylated (p=0.04).Next we studied how the DNA methylation inhibitor decitabine (DAC) may affect the methylation of miR124a-1 and a-3. To do this we looked at miR124a-1 and miR124a-3 methylation levels at day 5, 12 and 30 post DAC treatment in patients who showed pretreatment methylation. We found that patients who responded to DAC (Complete Response CR or Hematolgic Imporvement HI) had a significant decrease in methylation at day 5 (a-1 p=0.020 and a-3 p=0.014) and at day 12 (a-1 p=0.043 and a-3 p=0.029) whereas patients who did not respond to therapy had no significant de-methylation during these times. Additionally we found no significant differences in the patients treated with DAC(n=40) vs. the patients treated with DAC + valproic acid(n=16).Since promoter methylation has been well correlated with gene silencing we looked for altered expression levels of the mature miR by real time PCR. We first found that miR124 was expressed in CD34+ cells from cord blood and normal bone marrow CD34+ cells. Additionally we found that out of 30 (15 CR/HI, 15 Non Responders) patients with available RNA; patients who responded to DAC had a significant increase in expression at day 5 (p=0.03) whereas patients who did not respond to therapy had no significant change in expression. Since miR124 has been shown to down regulate CDK6, a proto-oncogene, we proceeded to see how its expression was effected in these patients at the mRNA and protein level. We found that again patients who responded to therapy had a significant decrease in CDK6 mRNA expression at day 5 (p=0.002) whereas patients with no response had no significant change in expression. These data were confirmed by western blot where we found that 7/9 responders had a decrease in expression and 4/4 non-responders had no changes in expression. Also we found that miR124a-1 methylation was positively correlated with CDK6 expression (p=0.007), while there was no correlation with miR124a-3.In summary we have found that miR124 hypomethylation and subsequent re-expression is a good marker of response to decitabine, possibly also mediating these responses by reducing the levels of CDK6.