Dissecting stress granule dependencies in leukemia stem cells

项目总结 急性髓系白血病(AML)每年在2万多名美国公民中确诊，其5年存活率仅为 20%-30%。尽管经过了几十年的研究，AML的有效治疗仍然是一个重要的临床需求， 由于坚持治疗，复发率和死亡率很高，这突显了护理标准- 导致这种疾病的耐药白血病干细胞(LSC)。我们和其他人的工作，以及一个巨大的 以RNA剪接基因突变为代表的基因组亚群，结合起来提供了令人信服的证据 建议在RNA新陈代谢水平上解除管制是定义和调节各方面的特征 疾病病理学的。RNA结合蛋白(RBPs)是转录后调控的核心效应物， 通过调节RNA的性质来精确控制基因表达，包括剪接、多聚腺苷酸化、 本地化、降级和翻译，作为一个类别，可以强制实施极其多样化的监管网络 小路。一类特殊的RBPs在应激颗粒、无膜细胞质结构中起作用 由RBPs和mRNAs组成，协调转录后程序，介导适应性细胞 回应。通过基因筛查和体内研究的结合，我们最近发现LSCs 选择性地依赖于形成压力颗粒的能力来维持其生存和增殖。因此，我们 假设应激颗粒在AML发病机制中起关键作用，以及应激颗粒蛋白 代表了一种可在新的靶向治疗策略中利用的脆弱性。在这个项目中，我们的目标是 明确高度治疗的白血病干细胞中应激颗粒的动力学和功能要求 难治性MLL重排(MLL-r)AML。我们将首先进行一系列的体外应力颗粒形成和在 用急性分离的MLL-r AML和正常HSCs进行体内细胞植入试验以识别应激 从原始AML到更稳定的AML和正常造血细胞的颗粒动力学和特征。 我们将发现选择性结合在应激颗粒中的mRNAs，定义并功能验证可能的关键LSC- 特定应激颗粒相互作用蛋白，包括我们已经确定的一个候选蛋白，并绘制其作用图 新的LSC特异应激颗粒蛋白和隔离在其中的RNA在LSC功能中的作用。最后，我们将 系统地筛选新的与应激颗粒相关的限制性商业惯例，这些限制性商业惯例是复发性喉鳞状细胞功能的基础。我们的 因此，研究将确定应激颗粒RNA和蛋白质成分以及基因表达网络的作用 他们控制促进白血病的发病机制，从而确定新的潜在的治疗AML的靶点。

PROJECT SUMMARY Acute myeloid leukemia (AML), diagnosed in over 20,000 US citizens each year, has a 5-year survival of only 20-30%. Despite decades of research, the effective treatment of AML remains a significant unmet clinical need, with standard of care underscored by high relapse and mortality rates due to the persistence of treatment- resistant leukemic stem cells (LSC) that drive the disease. Work by us and others, and the existence of a large genomic subgroup represented by mutations in RNA splicing genes, in combination present compelling evidence to suggest that deregulated control at the level of RNA metabolism is a feature that defines and mediates aspects of disease pathology. RNA binding proteins (RBPs) are core effectors of post-transcriptional regulation that execute precise control of gene expression by modulating RNA properties that include splicing, polyadenylation, localization, degradation and translation, and as a class can enforce an extremely diverse network of regulatory pathways. One specific class of RBPs functions within stress granules, membraneless cytoplasmic structures that are composed of RBPs and mRNAs to orchestrate post-transcriptional programs mediating adaptive cellular responses. Through a combination of genetic screens and in vivo studies, we recently discovered that LSCs selectively depend on the ability to form stress granules for their survival and proliferation. We therefore hypothesize that stress granules play a critical role in AML pathogenesis and that stress granule proteins represent a vulnerability that can be exploited in novel targeted therapeutic strategies. In this project, we aim to define the dynamics and functional requirements for stress granules in the leukemic stem cells of highly treatment refractory MLL-rearranged (MLL-r) AML. We will first perform a series of in vitro stress granule formation and in vivo cell engraftment studies assays using acutely isolated MLL-r AML and normal HSCs to identify stress granule dynamics and characteristics across primitive to more committed AML and normal hematopoietic cells. We will uncover mRNAs bound selectively in stress granules, define and functionally validate putatively key LSC- specific stress granule-interacting proteins, including one candidate we have already identified, and map the role of novel LSC-specific stress granule proteins and RNAs sequestered in them, in LSC function. Lastly, we will systematically screen for novel stress granule-relevant RBPs that underlie the function of relapsed LSCs. Our study will thus define the role of stress granule RNA and protein components and the gene expression networks they control to promote leukemia pathogenesis, thereby identifying novel potential therapeutic targets in AML.