Using The Genome Editing Techniques TALENs To Study The Molecular Mechanism Of DNMT3A Mutation In Acute Leukemia

Objective:To detect the incidence of DNMT3A mutation, its relevance with other clinical characteristics and impact on prognosis in adult patients with acute lymphoblastic leukemia (ALL).Methods:Genomic DNA was extracted from bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNCs), which were isolated from57patients with ALL by Ficoll density gradient centrifugation. Then the exon23of DNMT3A gene, where the hotspot mutation was located, was amplified by using polymerase chain reaction (PCR) method. After the DNA sequencing of purified products was applied, DNMT3A mutations were identified by comparing the results with the normal genome sequence. The clinical characteristics of the57patients were obtained from our own biological specimens’library.Results:In the57patients with ALL, a total of3(5.3%) T-ALL cases had the DNMT3A R882H mutation, which was significant greater than in B-ALL subtype (P=0.048). The patients aged between40and60years old had higher mutation rate than other age groups (P=0.042). Patients with DNMT3A mutation had worse overall survival (OS) than wild-type patients (P=0.02and P=0.014)Conclusion:DNMT3A mutation was found in adult ALL patients with a low frequency and exerts a negative impact on prognosis, suggesting that it is of great significance in the risk stratification and treatment instruction for Chinese ALL patients. Objective:To study the incidence of DNMT3A mutation, its relevance with other clinical characteristics and impact on prognosis in adult patients with acute myeloid leukemia (AML).Methods:Genomic DNA was extracted from bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNCs), which were isolated from392patients with AML by Ficoll density gradient centrifugation. Then the exon23of DNMT3A gene, where the hotspot mutation was located, was amplified by using polymerase chain reaction (PCR) method. After the DNA sequencing of purified products was applied, DNMT3A mutation were identified by comparing the results with the normal genome sequence. The clinical characteristics of the392patients were obtained from our own biological specimens’ library.Results:In the392patients with AML, a total of37(9.44%) cases harboring the DNMT3A R882mutation.29(78.38%) were type R882H,8(21.62%) were type R882C. DNMT3A mutations were more frequently associated with older age but there was no difference in gender, WBC counts, platelets counts, hemoglobin and blasts between the patients with and without DNMT3A mutation. The patients with DNMT3A mutation were significantly associated with FAB groups M4and M5and DNMT3A mutation more frequently had a mutation in NPM1and FLT3-ITD (P<0.001). In addition, DNMT3A mutated more often focused on CN-AML (83.78%,31/37, P<0.001) and had intermediate risk cytogenetics (91.89%,34/37, P<0.001). In the whole cohort and CN-AML, DNMT3A mutated patients had a shorter overall survival(P=0.0218and P=0.0006) and event-free survival(P=0.0203and P=0.0006) compared with DNMT3A wild-type patients. In CN-AML, DNMT3A mutation independently predicted a shorter overall survival (HR=1.986,95%CI1.207to3.269, P=0.007) by multivariate analysis. Conclusion:DNMT3A mutation was found in adult AML patients with a high frequency and exerts a negative impact on prognosis, suggesting that it is of great significance in the risk stratification and treatment instruction for AML patients. Objective:Our study aimed to establish one K562cell line carried with DNMT3A R882H mutation by using transcription activator-like effector nucleases (TALENs) and to investigate the biological effect of DNMT3A R882H mutation on cells.Methods:DNMT3A-TALENs plasmid were designed and constructed by using modified rapid assembly method. TALENs plasmid and donor plasmid were electroporated into K562cells and subcloned to PCR screening for DNMT3A R882H mutation clones. Whole-exome sequencing was performed to access the potential off-target effect of DNMT3A-TALEN plasmids. Western blot was used to analysis the expression of DNMT3A and SLC7A11. Colony forming assay and CSFE staining were applied to evaluate the proliferation ability of K562cells with DNMT3A R882H mutation. The apoptosis of cells was quantified using Annexin V and PI staining by flow cytometry.Results:In this study, we first established one K562cell line carried with DNMT3A R882H mutation by using transcription activator-like effector nucleases (TALENs) and found that the mutant gene can promote proliferation of K562cells. Further RNA microarray analysis revealed that some genes crucial for glutathione (GSH) synthesis, including CTH, PSPH, PS AT1and especially SLC7A11(the cysteine/glutamate transporter), were significantly upregulated, which result in significant elevation of the intracellular GSH levels. In addition, the mutant clones showed chemo-resistance and combined utilization of Sulfasalazine (SSZ), a specific inhibitor of SLC7A11, could sensitize the cells to chemotherapeutics.Conclusion:Our results provided novel insight of the role of DNMT3A R882H mutation played in AML pathogenesis. The GSH level of mutant clones increased significantly and showed chemo-resistance. Combined utilization of SSZ could sensitize the cells to chemotherapeutics, which provided a new thought for the future treatment of AML patients with DNMT3A R882H mutation.