Biologic and therapeutic relevance of DNMT3A mutations in acute myeloid leukemia

描述（由申请人提供）：急性髓系白血病中DNMT3A突变的生物学和治疗相关性尽管癌症研究和治疗取得了许多新进展，但治疗耐药性仍然是临床肿瘤学的核心挑战。基因组测序的努力使得鉴定与临床治疗失败相关的基因和特定突变成为可能。然而，治疗耐药的分子基础在很大程度上仍然是谜。在25-30%的急性髓性白血病（AML）患者中检测到复发性DNMT3A突变，并与不良结局和一线化疗耐药相关。DNMT3A突变最常影响氨基酸残基R882，最近的研究表明，这些突变体表现出酶活性下降和异常结合特性。此外，先前的研究表明，DNMT3A的表达增加在基因毒性应激反应中起促凋亡开关的作用。我们假设DNMT3A突变保护细胞免于因癌基因激活或电离照射（IR）或化疗引起的DNA损伤而凋亡，从而允许恶性造血细胞存活。我们的初步研究表明，aml相关的DNMT3A突变体在原代造血细胞中的表达抑制髓系分化，随着干细胞/祖细胞的积累，减少细胞凋亡并增加体外自我更新。此外，携带突变DNMT3A的细胞表现出基因组不稳定的特征。我们的机制研究指出受损的DNA损伤反应包括p53激活的衰减。本研究计划概述了一种三层方法来全面表征DNMT3A突变在正常和白血病造血和治疗耐药性中的作用。首先，我们将采用过继性转移方法进行体内研究和离体研究，以评估白血病相关DNMT3A突变体对不同干细胞/祖细胞群分化、凋亡和自我更新改变的贡献。接下来，我们将探讨突变体DNMT3A在AML发病机制中致病作用的分子机制。我们将通过全基因组甲基化分析和ChIP-seq研究来解决DNMT3A突变细胞中DNA甲基化模式和DNA结合的变化，并对有和没有DNMT3A突变的细胞中的DNA损伤感知、反应和修复进行详细分析。这些研究将在指导阶段启动，并将继续进入该奖项的独立阶段，届时主要重点将转移到机械信息抗白血病治疗的临床前开发。这些功能、机制和临床前研究将在一种新的基因小鼠模型中进行，该模型表达白血病相关的Dnmt3a突变体，来自其原生位点。总的来说，我们假设DNMT3A基因的遗传改变在许多患者的AML发展中起着重要作用，并且更彻底地了解这一现象可能具有最终的诊断，预后和治疗意义。

DESCRIPTION (provided by applicant): Biologic and therapeutic relevance of DNMT3A mutations in acute myeloid leukemia Despite the many new advances in cancer research and treatment, therapeutic resistance remains the core challenge in clinical oncology. Genome sequencing efforts have made possible the identification of genes and specific mutations associated with therapeutic failure in the clinic. However, the molecular basis of therapeutic resistance remains largely enigmatic. Recurrent DNMT3A mutations are detected in 25-30% of acute myeloid leukemia (AML) patients and are associated with adverse outcome and resistance to frontline chemotherapy. DNMT3A mutations most often affect amino acid residue R882, and recent work has shown that these mutants display decreased enzymatic activity and aberrant binding properties. In addition, previous studies have shown that increased expression of DNMT3A functions as a pro-apoptotic switch in response to genotoxic stress. We hypothesized that DNMT3A mutations protect cells from apoptosis in response to DNA damage caused by oncogene activation, or by ionizing irradiation (IR) or chemotherapeutics, allowing for the survival of malignant hematopoietic cells. Our preliminary studies show that expression of the AML-associated DNMT3A mutants in primary hematopoietic cells inhibits myeloid differentiation, with accumulation of stem/progenitor cells, reduces apoptosis and increases self-renewal in vitro. In addition, cells harboring mutant DNMT3A exhibited hallmarks of genomic instability. Our mechanistic studies point at impaired DNA damage response including attenuation of p53 activation. This research proposal outlines a three-tiered approach to the comprehensive characterization of the role for DNMT3A mutations in normal and leukemic hematopoiesis and therapeutic resistance. First, we will use adoptive transfer approach in vivo studies and ex vivo studies to assess the contribution of leukemia-associated DNMT3A mutants to alterations in differentiation, apoptosis, and self-renewal in different stem/progenitor cell populations. Next, the molecular mechanisms underlying etiologic role of mutant DNMT3A in AML pathogenesis will be explored. We will address the changes in DNA methylation patterns and DNA binding in DNMT3A-mutant cells through genome-wide methylation profiling and ChIP-seq studies, and perform detailed analysis of DNA damage sensing, response and repair in cells with and without DNMT3A mutations. These studies will be initiated during the mentored phase, and will continue into the independent stage of the award, when the main focus will shift to pre-clinical development of mechanistically-informed anti-leukemic treatments. These functional, mechanistic and pre-clinical studies will be carried out in a novel genetic mouse model expressing leukemia-associated Dnmt3a mutant from its native locus. Overall we posit that genetic alterations in the DNMT3A gene play an important role in development of AML in many patients, and a more thorough understanding of this phenomenon is likely to have eventual diagnostic, prognostic and therapeutic implications.