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

基因突变决定了许多靶向治疗的治疗反应。DNMT3A突变是AML中最常见的突变之一。在最近的一项临床试验中，我们发现密码子882(最常见的突变)存在DNMT3A突变的老年AML患者对DNMT抑制剂5-azacytidine和化疗的组合非常敏感。体外实验进一步支持DNMT3A-R882突变对5-氮胞苷治疗敏感的假设。因此，DNMT抑制剂似乎可以作为DNMT3A突变AML的靶向表观遗传疗法。本项目的目的是分析这种致敏的机制，并为进一步开发AML的表观遗传疗法提供知识。为此，我们将分析我们为该项目开发的DNMT3A-R882H敲入小鼠以及来自DNMT3A突变/不突变患者的原代AML样本。为了确定R882H中的DNMT3A突变在哪些情况下对DNMT抑制剂治疗敏感，我们将分析来自DNMT3A突变敲入小鼠和野生型小鼠的白血病前期和白血病干细胞。将在体外和体内进行分析，以确定白血病的启动和对5-氮胞苷的治疗反应(以及化疗反应)的差异。其次，我们将研究DNMT3A-R882突变体对DNMT抑制剂敏感性的机制。我们将通过质谱仪在小鼠和人类样本中鉴定DNMT3A突变体和野生型蛋白的相互作用伙伴。5-氮胞苷对RNA转录和受影响基因的影响将在带有/不带有DNMT3A突变的小鼠和人类细胞中进行探索。相关基因和可能的机制将与临床试验的数据(甲基化和RNA-Seq)以及先前shRNA文库筛选的结果进行比较。5-氮胞苷敏化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-氮胞苷单一治疗以及与阿糖胞苷联合治疗对携带免疫缺陷小鼠候选基因DNMT3A突变/野生型或特定shRNA的人类白血病母细胞的影响。主要的焦点是看看哪些已确定的机制可以用来使5-氮胞苷在DNMT3A野生型患者中更有效。总体而言，本项目将确定DNMT3A突变诱导5-氮胞苷增敏的机制，并将这些机制用于DNMT3A WT的AML的靶向表观遗传治疗。

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.