Biologic and therapeutic relevance of DNMT3A mutations in acute myeloid leukemia

描述(申请人提供)：急性髓系白血病DNMT3A突变的生物学和治疗相关性尽管在癌症研究和治疗方面取得了许多新进展，但治疗耐药性仍然是临床肿瘤学的核心挑战。基因组测序工作使临床上识别与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个谜。在25%-30%的急性髓系白血病(AML)患者中检测到复发的DNMT3A突变，并与不良结局和对一线化疗的耐药性有关。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降低和异常结合特性。此外，先前的研究表明，DNMT3A表达的增加在基因毒性应激反应中起到促凋亡开关的作用。我们假设，DNMT3A突变保护细胞免受癌基因激活、电离辐射(IR)或化疗引起的DNA损伤，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了髓系分化，导致干/祖细胞的聚集，减少了细胞凋亡，并增加了体外的自我更新。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DNA损伤反应受损，包括P53活性减弱。这项研究计划概述了一种三层方法，以全面表征DNMT3A突变在正常和白血病造血和治疗耐药中的作用。首先，我们将采用体内移植和体外研究的方法，评估白血病相关DNMT3A突变在不同干细胞/祖细胞群体分化、凋亡和自我更新变化中的贡献。接下来，我们将探讨突变型DNMT3A在AML发病机制中的分子机制。我们将通过全基因组甲基化图谱和芯片序列研究来解决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突变的细胞的DNA损伤感知、反应和修复进行详细的分析。这些研究将在指导阶段启动，并将持续到奖项的独立阶段，届时主要重点将转移到机械性知情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遗传小鼠模型中进行，该模型表达与白血病相关的DNMT3A突变。总体而言，我们认为DNMT3A基因的遗传改变在许多患者的AML发展中起着重要作用，对这一现象的更彻底了解可能最终具有诊断、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.