| Objective:Acute myeloid leukemia (AML) is a heterogeneous hematology disease, which caused by the maturation disorders, differentiation and malignant clonal proliferation of myeloid leukemia cells. t(8;21)(q22;q22) is the most common chromosomal translocation of AML, generating AML1-ETO chimeric gene that can transcript and translate AML1-ETO chimeric protein. Although t(8;21) AML have the same genetics background, but this type of AML have greater heterogeneity. And the reason for this heterogeneity is unclear. Epigenetics participate in leukemogenicity: ETO recruits transcript depress complex N-CoR/SMRT/Sin3/HDAC, which leading to depress express of downstream genes. AML 1-ETO chimeric protein interacts with other transcription factors (for example, CEBPa, PU.1 and MEF) depress or re-depress express of downstream genes. PRMT1 methylates arginine 142 of AMLl-ETO9a, which is mediated transcriptional activation in Kasumi-1 cells. The AML1-ETO at lysine 43 acetylation promotes leukemogenicity self-renewal-promoting. The bioinformatics TCGA expression profile data show that EZH1 (Enhancer of zeste homolog 1) is higher expression in t(8;21)AML than normal karyotype AML. Ezhl is an important histone methyltransferase for hematopoietic stem cell maintenance and BM Failure upon Loss of Ezhl in hematopoietic Cell, indicating the important relation between Ezhl and hematopoietic system. The role of Ezhl in leukemia cell is largely unknown. The aims of our study are to investigate the relation between Ezhl and AML 1-ETO, the biological function of Ezhl in the t(8;21)AML.Methods:The mRNA and protein expression level of Ezhl were detected using real-time quantitative PCR and western blot, respectively. CoIP and Western blot experiments were conducted to verify the interacting proteins. Transfect siRNA to slience the express of EZH1, use flow cytometer method to detect transfection efficiency, the cell proliferation was determined by colony-forming. To address if Ezhl determines AML1-ETO cell fate in vivo, SKNO-1 cells were transfected with Ezhl control vehicle or siEzhl. At 48th hour after transfection, cells were subcutaneously injected into the flanks of nude mice. The tumor volume, tumor weight and aggressive infiltration of leukemic cells into bone marrow, lung, liver and spleen were observed.Results:The mRNA and protein expression level of Ezhl were high expression. Ezhl and AML1-ETO are binded to complex. Ezhl knockdown greatly disrupted the colony-forming capabilities of Kasumi-1 and SKNO-1 compared to the vehicle-siRNA group. To address if Ezhl determines AML1-ETO cell fate in vivo, SKNO-1 cells were transfected with siEzhl or control vehicle. After 48 hours after transfection, alive cells were subcutaneously injected into the flanks of nude mice (n= 6 mice/group). We observed that the tumor latency was greatly different, with tumor detectable at 24 days in vehicle group, but at 45 days in siEzhl group after injection. Tumors in the vehicle group grew faster and had larger final tumor volume than that in siEzhl group. Furthermore, in contrast to vehicle group, the mice engrafted with siRNA-transfected cells had much lower tumor weight (68±23 vs. 262±64 mg, P< 0.05). H&E/Giemsa-stained BM cells and sections of spleen, liver and lung showed a less infiltration of myeloid blasts in siEzhl group than vehicle group.Conclusion:Ezhl is highly expression in AML1-ETO positive cell lines. Ezhl is binded to AML1-ETO. Ezhl induces proliferation of AML1-ETO positive cells, and plays the role of an oncogene. It could be a potential novel target for treating t(8;21) AML.Objective:The second generation exome sequencing technology sets up a milestone. The mutation gene found by it have a great impact on the diagnosis and treatment stratification of acute myeloid leukemia (AML). t(8;21)(q22;q22) is the most common chromosomal translocation of AML, generating AML1-ETO chimeric gene that can transcript and translate AML1-ETO chimeric protein. Only the expression of AML1-ETO single express cannot lead to AML, other mutations are required for leukemogenicity. More recurring mutations can be found in t(8;21)AML than the other types of AML. Therefore, it is very important to sequence and find out new mutations. According to the risk stratification of NCCN-2015-AML-Version 1, t(8;21)AML classified as favorable-risk. About 1/3 patients with KIT mutations are considered as intermediate-risk. But from the perspective of clinical epidemiology, this type of AML has greater heterogeneity, and the reason for this heterogeneity is unclear. So screening of mutations in t(8;21)AML is of great significance for the future treatment stratification. The aim of our study is the application of second generation exome sequencing technology in AML, explore the mutations of de novo and relapse t(8;21)AML, the clone evolving of t(8;21)AML during the period of de novo and relapse.Methods:Our NimbleGen exome chip covered the following 111 genes exon region: the AML related gene mutation that WHO-2008 guideline and NCCN-2014 guideline reported, the drive mutations that AML related research reported, and the mutation genes that clinical chip covered. Part of genes covered all exon regions. We evaluated the sensitivity of second generation exome sequencing chip using the mixture of Kasumi-1 and K.562 cell line. Tirty-three t(8;21)AML bone marrow samples were tested using Illumina Hiseq of NimbleGen exome chip. Varscan 2 method was to compare the sequences of the following two pairs:de novo and complete remission, relapse and complete remission. The de novo gene mutations and relapse gene mutations were screened. Then refer the 79 normal database and other methods to screen harm mutations. At last, the recurring mutations were screened.Results:We evaluated the sensitivity of second generation exome sequencing chip using the mixture of Kasumi-1 and K562 cell line. Under the condition of 1600× sequencing depth,3% dilution condition,100% homozygous mutation, the heterozygous mutation occupied 95%. We analyzed the mutations of eighteen t(8;21)AML samples, the result showed that the highest frequency of mutation is KIT, following by ASXL1, FLT3, KDM6A, NRAS, PHF6 and RAD21-AS1. The mutations of de novo AML are as follows:PHF6, KDM6A, FLT3, SH2B3, RAD21, MEF2B, IDH2, IDH1, CBL and ETV6. The mutations of relapse AML are as follows:SETD2, IKZF1 and FLT3-ITD. The de novo and relapse AML share mutations are as follow:KIT, RAD21-AS1, NRAS and ASXL1. Among the eighteen t(8;21)AML samples, there are three samples are serials. No.#1-de novo patient has NRAS and FLT3 mutation, No.#1-relapse patient has FLT3-ITD. No. #2-de novo patient has FLT-3 mutation, No.#2-relapse patient has NRAS and IKZF1. We speculated that patient#1 and patient#2 had clonal evolution. Both No. #7-de novo and No.#7-relaspe have KIT mutation, and we speculated that the relapse of patient#7 have the same leukemia clone with the de novo.Conclusion:The sensitivity of second generation leukemia chip is high. In t(8;21)AML patient, we found other mutations other than KIT mutation, and we explored the de novo and relapse mutation of t(8;21)AML preliminary, which help us to analyze the clonal evolution. |
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