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将它们的基因表达程序与细胞新陈代谢和 养分可利用性。然而，我们对转录反应的理解很差。 细胞受到核苷酸的限制，核苷酸是新陈代谢的产物，以及启动这些代谢的机制。 程序。这是一种迫切的需求，因为核苷酸合成抑制剂被用来治疗许多疾病。 使用核苷酸合成抑制剂治疗的一种疾病是镰状细胞病(SCD)，一种遗传性血液 由于β-珠蛋白的突变而导致全球数百万人显著患病和死亡的疾病 导致红血球呈镰刀状。几十年前，核苷酸还原酶抑制剂羟基脲(HU)，它 阻断核苷酸合成，被发现诱导γ-珠蛋白的表达，它通常在 胎儿作为胎儿血红蛋白(HBF)的组成部分。由于HBF在功能上可以替代成人的血红蛋白，所以它 掩盖了β-珠蛋白突变的影响，如果红细胞中存在足够的水平，就可以防止镰刀状。 不幸的是，HU诱导HBF的能力是不同的；许多患者只有部分反应， 有些人根本没有回应。在改进SCD治疗方面的进展受到了我们缺乏 对HU如何诱发HBF的机械性理解。 我的建议的目的是解开核苷酸限制和HBF诱导和 利用遗传学和新陈代谢的现代工具，确定调节这种转录反应的基因。在……里面 目的1研究HU引起的核苷酸限制和复制应激是如何诱导红系HbF的。 祖细胞，以及这是否通过已知的伽马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耗尽和复制应激的程度，例如初级 红系细胞和骨髓，在那里诱导HBF。最后，在目标3中，我将使用谱系条形码策略 为了检测决定单个细胞是否对HU产生HBF的基因表达差异， 目的是确定哪些基因或药物调节可以改善HU的疗效 在活体内。我预计这些拟议的实验将揭示HU重新激活HbF的机制。 并为改善SCD患者的治疗提供新的战略。更广泛地说，它将提供对 高度增殖的细胞如何协调临床有效的转录程序以应对 核苷酸限制。 我的培训计划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 促进科学事业的实质性发展，培养内科科学家导师。总的来说， 我的研究建议和职业发展计划的重点是为我提供培训和 作为一名内科科学家，我需要熟练的技能来促进我的发展。

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.