Adenylate Kinase 2 Deficiency and the Failure of Myelopoiesis

腺苷酸激酶 2 缺乏和骨髓生成失败

基本信息

批准号：
10446518
负责人：
Katja Gabriele Weinacht
金额：
$ 59.91万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10446518
关键词：
ATP Synthesis Pathway Adenine Nucleotides Adult Affect Aspartate B-Lymphocytes Blood Carbon Carnitine Catabolism Cell Cycle Cells Citric Acid Cycle Clustered Regularly Interspaced Short Palindromic Repeats Cytoplasm Deamination Defect Development Electrons Energy Metabolism Enzymes Erythroid Excretory function Exhibits FRAP1 gene Failure Generations Genetic Transcription Hematological Disease Hematopoietic Hematopoietic stem cells Homeostasis Human Image Immunosuppression Impairment In Vitro Inosine Monophosphate Isotopes Knock-out Life Link Lipids Lymphopenia Malignant Neoplasms Mass Spectrum Analysis Mediating Megakaryocytes Metabolic Metabolic Diseases Mitochondria Mitochondrial Diseases Modeling Mus Myelogenous Myeloid Cells Myelopoiesis NADH NADH oxidase Natural regeneration Neutropenia Nucleoside Hydrolases Nucleotides Oxidation-Reduction Pathology Pathway interactions Patients Phenotype Phosphorylation Purines Pyruvate Recycling Ribosomal RNA Ribosomes Severe Combined Immunodeficiency Signal Transduction Stress System T-Lymphocyte Testing Therapeutic immunosuppression Translations Transplantation Xenograft procedure adenylate kinase cancer therapy cell type improved in vitro Model in vivo in vivo Model insight loss of function mutation metabolomics mitochondrial metabolism novel novel strategies nucleotide metabolism overexpression progenitor purine metabolism reticular dysgenesis stem stress reduction

项目摘要

PROJECT SUMMARY/ABSTRACT Cellular ATP demand and mitochondrial ATP synthesis are tightly linked. ATP synthesis, in turn, depends on (1) NADH generation in the TCA cycle and (2) recycling of the adenine nucleotide pool. When ATP is depleted and AMP rises, the cell increases energy generation and curtails energy expenditure until homeostasis is restored. In the context of mitochondrial pathology, the system becomes unhinged… Reticular Dysgenesis (RD) is a rare hematologic disease, caused by biallelic loss-of-function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the phosphorylation of AMP to ADP in the inter-membrane space to generate substrate for ATP synthesis1. RD patients suffer from severe congenital neutropenia, lymphopenia, and die early in life unless cured by hematopoietic stem cell transplantation5. We have developed a novel biallelic CRISPR-knockout model of AK2 in primary human hematopoietic stem and progenitor cells to precisely mimic the failure of human myelopoiesis in culture and after transplantation into mice. Using broad metabolomic profiling, our preliminary studies revealed that AK2-deficient myeloid progenitors exhibit a high NADH/NAD+ ratio and NAD+ depletion, consistent with reductive stress. In addition, AK2-deficient myeloid progenitors displayed a decrease in mitochondrial metabolites, including TCA cycle intermediaries and aspartate, while lipid carnitines were increased, and lipid droplets were found in the cytoplasm. We also detected highly elevated levels of the purine intermediate inosine monophosphate (IMP) and a decrease in rRNA and ribosome subunits. Interestingly, our studies suggest the high IMP stems from deamination of AMP, rather than a block in purine de novo synthesis. Taken together, these observations raise the possibility that AK2 deficiency causes mitochondrial reductive stress, curtailing TCA cycle activity and diverting carbon and electron pools into lipid synthesis while counteracting the accumulation of AMP. These findings led us to hypothesize that AK2 deficiency causes two interconnected but distinct pathologies: I. Reductive stress redirecting energy metabolism into lipid storage rather than OXPHOS; II. Accumulation of AMP and IMP, leading to defects in nucleotide metabolism. Our proposed studies will test if failure of myelopoiesis is primarily a result of reductive stress and impaired energy utilization, versus impaired purine metabolism, or both. We will determine if myelopoiesis can be rescued by correcting the NADH/NAD+ ratio or nucleotide pools. Lastly, we will validate our findings in an in vivo model of RD and investigate if different compensatory mechanisms in different blood lineages result in the RD phenotype. We use RD as a model to dissect escape mechanisms at the juncture of energy metabolism, redox stress, and nucleotide homeostasis. These insights will advance therapies for mitigating reductive stress and using cell type-specific manipulation of purine metabolism as a strategy for immunosuppression and cancer therapy.

项目概要/摘要细胞 ATP 需求与线粒体 ATP 合成紧密相连，而 ATP 合成又依赖于线粒体。 (1) TCA 循环中的 NADH 生成和 (2) ATP 时腺嘌呤核苷酸库的回收。当 AMP 耗尽且 AMP 升高时，细胞会增加能量产生并减少能量消耗，直到在线粒体病理学的背景下，体内平衡得到恢复，系统变得精神错乱…… 网状发育不全（RD）是一种罕见的血液系统疾病，由双等位基因功能丧失突变引起线粒体酶腺苷酸激酶 2 (AK2) 催化 AMP 磷酸化为 ADP。 RD 患者患有严重的先天性缺陷。中性粒细胞减少症、淋巴细胞减少症，除非通过造血干细胞移植治愈，否则会在生命早期死亡。我们开发了一种新型人类原代造血干细胞中 AK2 的双等位基因 CRISPR 敲除模型和祖细胞精确模拟培养中和移植后人类骨髓生成的失败通过广泛的代谢组学分析，我们的初步研究表明 AK2 缺陷的骨髓细胞。祖细胞表现出高 NADH/NAD+ 比率和 NAD+ 消耗，与还原应激一致。此外，AK2 缺陷的骨髓祖细胞显示线粒体代谢物（包括 TCA）减少循环中间体和天冬氨酸，而脂质肉碱增加，并且在细胞中发现脂滴。我们还检测到嘌呤中间体肌苷单磷酸的水平高度升高。 (IMP) 以及 rRNA 和核糖体亚基的减少。综上所述，这些观察结果是来自 AMP 的脱氨作用，而不是嘌呤从头合成的阻断。提出了 AK2 缺乏导致线粒体还原应激、减少 TCA 循环活性和将碳和电子库转移到脂质合成中，同时抵消 AMP 的积累。这些发现使我们认为 AK2 缺陷会导致两种相互关联但又不同的病理： I. 还原应激将能量代谢重新定向到脂质储存而不是 OXPHOS； II. AMP 和 IMP 的积累，导致核苷酸代谢缺陷。测试骨髓生成失败是否主要是由于应激减少和能量利用受损造成的，而不是嘌呤代谢受损，或两者兼而有之，我们将确定是否可以通过纠正骨髓生成来挽救。最后，我们将在 RD 和 NADH 体内模型中验证我们的发现。研究不同血统的不同补偿机制是否会导致 RD 表型。我们使用 RD 作为模型来剖析能量代谢、氧化还原应激、这些见解将推进减轻还原应激和使用的疗法。嘌呤代谢的细胞类型特异性操作作为免疫抑制和癌症治疗的策略。