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Mechanisms of DNMT3A-mutation induced sensitization for 5-Azacytidine in AML

DNMT3A 突变诱导 AML 中 5-氮杂胞苷致敏的机制

基本信息

批准号：
280661318
负责人：
Professor Dr. Carsten Müller-Tidow
金额：
--
依托单位：
Klinik für Hämatologie, Onkologie und Rheumatologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/280661318?language=en
关键词：
Mechanisms DNMT3A mutation induced sensitization

项目摘要

Genetic mutations determine therapy response for many targeted therapies. DNMT3A mutations are among the most frequent mutations that occur in AML. In a recent clinical trial we discovered that older AML patients with DNMT3A mutations in codon 882 (most common mutation) are extraordinarily sensitive to the combination of the DNMT inhibitor 5-Azacytidine and chemotherapy. In vitro experiments further support the hypothesis that DNMT3A-R882 mutations sensitize for 5-Azacytidine therapy. Thus, DNMT-inhibitors appear to act as targeted epigenetic therapy for DNMT3A mutant AML. The aim of this project is to analyze the mechanisms of this sensitization and to exploit the knowledge for further epigenetic therapy development in AML. For this purpose we are going to analyze a DNMT3A-R882H Knock-in mouse that we developed for this project as well as primary AML specimens from patients with/without DNMT3A mutation. To identify in which circumstances DNMT3A mutations in R882H sensitize for DNMT-inhibitor therapy we are going to analyse pre-leukemic and leukemic stem cells from DNMT3A-mutant knock-in versus wild-type mice. Analyses will be performed in vitro and in vivo for differences in leukemia initiation and therapy response to 5-Azacytidine (and chemotherapy response). Second, we are going to characterize mechanisms of DNMT-inhibitor sensitivity for DNMT3A-R882 mutants. We are going to identify interaction partners for DNMT3A-mutant and wild-type protein in murine and human samples by Mass-Spec. The effects of 5-Azacytidine onto RNA transcription and affected genes will be explored in murine and human cells with/ without DNMT3A mutation. Relevant genes and potential mechanisms will be compared to data from the clinical trial (methylation and RNA-Seq) as well as to results from a previous shRNA library screen. Relevant mechanisms for 5-Azacytidine sensitization will be functionally verified by shRNA based approaches. We are going to analyze the effects of 5-Azacytidine monotherapy and the combination with cytarabine onto human leukemia blasts that carry DNMT3A-mutations/wildtype or specific shRNAs for candidate genes in immunodeficient mice. Main focus is to see, which of the identified mechanisms can be utilized to render 5-Azacytidine more effective in DNMT3A wild-type patients. Overall, this project will identify mechanisms of DNMT3A mutation induced sensitization for 5-Azacytidine and the utilization of these mechanisms for targeted epigenetic therapy in AML with DNMT3A WT.

基因突变决定了许多靶向治疗的治疗反应。DNMT 3A突变是AML中最常见的突变。在最近的一项临床试验中，我们发现在密码子882（最常见的突变）中具有DNMT 3A突变的老年AML患者对DNMT抑制剂5-氮杂胞苷和化疗的组合非常敏感。体外实验进一步支持DNMT 3A-R882突变对5-氮杂胞苷治疗敏感的假设。因此，DNMT抑制剂似乎作为DNMT 3A突变AML的靶向表观遗传疗法。该项目的目的是分析这种致敏的机制，并利用知识进一步开发AML的表观遗传治疗。为此，我们将分析我们为此项目开发的DNMT 3A-R882 H敲入小鼠以及来自有/无DNMT 3A突变患者的原发性AML标本。为了确定在何种情况下R882 H中的DNMT 3A突变对DNMT抑制剂治疗敏感，我们将分析来自DNMT 3A突变敲入与野生型小鼠的白血病前和白血病干细胞。将在体外和体内分析白血病起始和对5-氮杂胞苷的治疗应答（和化疗应答）的差异。其次，我们将描述DNMT 3A-R882突变体的DNMT抑制剂敏感性机制。我们将通过质谱鉴定小鼠和人样本中DNMT 3A突变体和野生型蛋白的相互作用伴侣。5-氮杂胞苷对RNA转录和受影响基因的影响将在有/无DNMT 3A突变的小鼠和人细胞中进行探索。相关基因和潜在机制将与临床试验数据（甲基化和RNA-Seq）以及先前shRNA文库筛选的结果进行比较。5-氮杂胞苷致敏的相关机制将通过基于shRNA的方法进行功能验证。我们将分析5-氮杂胞苷单药治疗和与阿糖胞苷联合治疗对免疫缺陷小鼠中携带DNMT 3A突变/野生型或候选基因特异性shRNA的人白血病母细胞的影响。主要关注的是，可以利用哪些已鉴定的机制来使5-氮杂胞苷在DNMT 3A野生型患者中更有效。总体而言，该项目将确定DNMT 3A突变诱导5-氮杂胞苷致敏的机制，并利用这些机制对具有DNMT 3A WT的AML进行靶向表观遗传治疗。