PDF(Pubmed)
Abstract：
The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.
摘要：
5-氮杂胞苷（AZA）和地西他滨（DEC）是非细胞毒性的，分化诱导疗法被批准用于治疗骨髓增生异常综合征，急性髓性白血病（AML），并被评估为AML异基因造血干细胞移植后的维持疗法和治疗血红蛋白二联疗法。恶性细胞细胞减少被认为是通过关键表观遗传调节因子的S期特异性消耗而发生的。DNA甲基转移酶1(DNMT1)，在癌症的情况下，从而发布终端差异化程序。DNMT1靶向还可以提高免疫功能基因的表达（HLA-DR，MICA,MICB）刺激移植物抗白血病作用。在体内,由于药物遗传因素，DEC和5-AZA活性存在很大的个体间差异，和定量DNMT1耗竭的分子药效学效应的测定是迈向个体化或个性化治疗的合理步骤。我们开发并分析验证了由免疫表型和细胞周期状态定义的血液和骨髓细胞亚群中DNMT1表位水平的流式细胞术测定。使用野生型(WT)和DNMT1敲除(DKO)HC116细胞来选择和优化高度特异性DNMT1单克隆抗体。该方法的方法学验证包括HC116-WT和-DKO细胞和外周血单核细胞的细胞计数和匹配免疫印迹；用DEC处理的H116-WT的流式细胞术，和在用5-AZA治疗之前和之后的患者样品。对患者样本的分析证明了测定的可重复性，治疗前患者DNMT1水平的变化，和DNMT1耗竭治疗后。已经开发了流式细胞术测定，在临床试验的研究环境中，可以为DEC或5-AZA治疗的研究提供信息，以实现靶向分子药效学作用并更好地了解治疗抵抗/失败。
