Molecular determinants of fetal hemoglobin induction by hydroxyurea to treat sickle cell disease

项目摘要 高度增殖的细胞需要协调它们的基因表达程序与细胞代谢， 养分有效性然而，我们对转录反应的理解很差， 细胞中的核苷酸是有限的，核苷酸是新陈代谢的产物，而启动这些代谢的机制是有限的。 程序.这是一个关键的需求，因为核苷酸合成抑制剂被用作许多疾病的治疗。 一个条件是治疗与核苷酸合成抑制剂是镰状细胞病（SCD），遗传血液 由于β-珠蛋白的突变导致全世界数百万人的显著发病率和死亡率的疾病， 导致红细胞呈镰刀状。几十年前，核糖核苷酸还原酶抑制剂羟基脲（HU）， 阻断核苷酸合成，被发现可以诱导γ-珠蛋白的表达，γ-珠蛋白通常在 胎儿作为胎儿血红蛋白（HbF）的组成部分。由于HbF可以在功能上替代成人血红蛋白， 如果在红细胞中存在足够水平的β-珠蛋白，则可以掩盖β-珠蛋白突变的影响并防止镰状化。 不幸的是，HU诱导HbF的能力是异质的;许多患者仅具有部分应答， 有些人根本没有反应。改善SCD治疗的进展受到我们缺乏 对HU如何诱导HbF的机械理解。 我的建议的目的是解开核苷酸限制和HbF诱导之间的联系， 利用遗传学和代谢学中的现代工具，确定调节这种转录反应的基因。在 目的1，研究HU引起的核苷酸限制和复制应激如何诱导红系细胞中的HbF 祖细胞，以及这是否通过已知的γ-珠蛋白转录抑制因子发生。在目标2中，我 将决定在生理环境中核苷酸消耗和复制应激的程度，如原发性 红系细胞和骨髓，其中诱导HbF。最后，在目标3中，我将使用谱系条形码策略 为了检测决定个体细胞是否响应于HU诱导HbF的基因表达差异， 目的是鉴定其遗传或药理学调节可以提高HU功效的基因 in vivo.我预期这些实验将揭示HU重新激活HbF的机制 并为改善SCD患者的治疗提供新的策略。更广泛地说，它将提供洞察力， 高度增殖的细胞如何协调临床有效的转录程序， 核苷酸限制。 我的培训计划充分利用了麻省理工学院、波士顿儿童医院和哈佛医学院的丰富资源 学校进行尖端研究，并获得临床培训。我还概述了旨在 促进实质性的科学职业发展，培养作为医生科学家的导师。总的来说， 我的研究计划和职业发展计划的重点是为我提供培训， 作为一名医生科学家，我需要进一步发展。

Project Summary Highly proliferative cells need to coordinate their gene expression programs with cellular metabolism and nutrient availability. However, we have a poor understanding of the transcriptional responses that arise when cells are limited in nucleotides, which are products of metabolism, and the machinery that initiates these programs. This is a critical need because nucleotide synthesis inhibitors are used as treatment for many diseases. One condition that is treated with a nucleotide synthesis inhibitor is sickle cell disease (SCD), a genetic blood disorder that causes significant morbidity and mortality in millions worldwide due to a mutation in beta-globin that causes red blood cells to sickle. Decades ago, the ribonucleotide reductase inhibitor hydroxyurea (HU), which blocks nucleotide synthesis, was found to induce expression of gamma-globin, which is normally expressed in the fetus as a component of fetal hemoglobin (HbF). Since HbF can functionally replace adult hemoglobin, it masks the effect of the beta-globin mutation and prevents sickling if present in red blood cells at sufficient levels. Unfortunately, the ability of HU to induce HbF is heterogeneous; many patients only have a partial response, and some do not respond at all. Progress in improving SCD therapy has been hampered by our lack of mechanistic understanding of how HU induces HbF. The objective of my proposal is to unravel the link between nucleotide limitation and HbF induction and identify genes that modulate this transcriptional response, using modern tools in genetics and metabolism. In Aim 1, I will determine how nucleotide limitation and replication stress caused by HU induces HbF in erythroid progenitors, and whether this occurs through the known transcriptional repressors of gamma-globin. In Aim 2, I will determine the degree of nucleotide depletion and replication stress in physiological contexts, such as primary erythroid cells and bone marrow, where HbF is induced. Finally, in Aim 3, I will use lineage barcoding strategies to detect gene expression differences that determine whether individual cells induce HbF in response to HU, with the goal of identifying genes whose genetic or pharmacologic modulation could improve the efficacy of HU in vivo. I anticipate that these proposed experiments will reveal the mechanism by which HU reactivates HbF and inform novel strategies to improve treatment of patients with SCD. More broadly, it will provide insight into how highly proliferative cells can coordinate a clinically efficacious transcriptional program in response to nucleotide limitation. My training plan leverages the rich resources of MIT, Boston Children’s Hospital, and Harvard Medical School to perform cutting-edge research and obtain clinical training. I have also outlined activities designed to facilitate substantial scientific career development and cultivate mentors who are physician scientists. Overall, my research proposal and career development plan are focused on providing me with the training and proficiencies needed to further my development as a physician scientist.