The Effect of the R882H DNMT3a Mutation on Megakaryocyte Cell Lines

Introduction DNA methyltransferase (DNMT) family play an important role in the development and growth of lives, encoding enzymes that catalyze the addition of a methyl group to the cytosine residue of CpG islands. With the increase in methylation, the downstream genes are often associated with reduced expression. In this family, DNMT3a occupies the essential position to implement the de novo methylation. Timothy J. Ley and many other scientists found that in M4 and M5 acute myeloid leukemia (AML), around 20% patients suffered from DNMT3a mutation (most are R882H mutation), always associating with adverse prognosis. But what9s the reason for adverse prognosis? Additionally, our formal Meta analysis showed that the de novo AML patients with DNMT3a mutation have higher platelet counts, WBC and RBC counts. To shed some light on the possible causal relation between the increasing in platelet count and poor prognosis led by DNMT3a mutation, we transduced the MK cell lines with genes null-mCherry (null), DNMT3a wild type-mCherry (DNMT3aWT) and DNMT3a R882H mutation type-mCherry (DNMT3aMT) respectively, trying to figure out the possible role that the mutation plays in the megakaryopoiesis and thrombopoiesis. Also, we tested several drugs that may target the mutation. Methods The SFFV-null-IRES-mCherry, SFFV-DNMT3aWT-IRES-mCherry and SFFV-DNMT3aMT-IRES-mCherry plasmids were constructed by Dr. Qianli Jiang, modified from LEGO-iC plasmids. MK cell lines (chrf-288-11, meg-01) were flow-through transduced with the lentivirus produced by packaging plasmids and those above. All the fluorescence positive cells have been doubly sorted by flow cytometry. Cell ploidy was analyzed by flow cytometry using Propidium Iodide (PI); colony forming unit (CFU-MK) were enumerated 14d after being plated with TPO and IL-3; cell proliferation were tested by CCK-8; apoptosis was measured via flow cytometry with PI and Annexin V-FITC; CD41a and CD61 were tested with flow cytometry. The drug tests including Decitabine, Dasatinib and Rituximab were analyzed using CCK-8 test and cytomorphologic tests. Results With CCK8 test of chrf-288-11 and meg-01, DNMT3aMT proliferates faster than the null and DNMT3aWT (P Conclusion With all the above evidences, we can safely conclude that the megakaryocyte cell lines with DNMT3a mutation are associated with high-differentiation, high-colony formation and low-apoptosis, which could help us to understand the elevation of platelet count in AML patients with DNMT3a mutation. The anti-apoptosis and renewal ability of the cell lines with DNMT3a mutation may lead to a bad prognosis of these AML patients (with DNMT3a mutation). What9s more, according to the drug experiment, we found in the both cell lines, DNMT3aWT and DNMT3aMT cells died significantly at even low concentration of decitabine. Dasatinib also slowed down the proliferation of 3 gene types of chrf-288-11, whether Dasatinib could lead to further treatment of such leukemia with DNMT3A mutation needs more research. Rituximab is helpful in the treatment against refractory thrombocytopenia. However, the mechanism hasn9t been clarified. Interestingly, our results showed that, Rituximab may have a direct effect on MKs, giving a boost to the megakaryopoiesis and thrombopoiesis with certain concentration. Disclosures No relevant conflicts of interest to declare.