Adenylate Kinase 2 Deficiency and the Failure of Myelopoiesis

腺苷酸激酶 2 缺乏和骨髓生成失败

• 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
• 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

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.

项目总结/摘要 细胞ATP需求和线粒体ATP合成密切相关。ATP的合成，反过来，取决于 (1)TCA循环中的NADH生成和（2）腺嘌呤核苷酸库的再循环。当ATP 耗尽和AMP上升，细胞增加能量产生并减少能量消耗， 体内平衡得以恢复在线粒体病理学的背景下，系统变得混乱... 网状细胞发育不全（RD）是一种罕见的血液病，是由造血干细胞中的双等位基因功能丧失突变引起的。 线粒体酶腺苷酸激酶2（AK 2）。AK 2催化AMP磷酸化为ADP， 产生ATP合成底物1. RD患者患有严重的先天性 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5。 我们开发了一种新的原代人造血干细胞中AK 2的双等位基因CRISPR敲除模型， 和祖细胞，以精确地模拟培养中和移植后的人骨髓生成的失败 变成老鼠使用广泛的代谢组学分析，我们的初步研究表明，AK 2缺陷的髓系细胞 祖细胞表现出高的NADH/NAD+比率和NAD+消耗，与还原应激一致。在 此外，AK 2缺陷型髓系祖细胞显示线粒体代谢物减少，包括TCA 循环中间体和天冬氨酸，而脂质肉毒碱增加，脂滴中发现， 细胞质我们还检测到嘌呤中间体肌苷一磷酸的水平高度升高 (IMP)以及核糖体和核糖体亚基的减少。有趣的是，我们的研究表明，高IMP股骨柄 从AMP的脱氨基，而不是在嘌呤从头合成的块。综上所述，这些观察 提高了AK 2缺乏导致线粒体还原应激，减少TCA循环活性， 将碳和电子池转移到脂质合成中，同时抵消AMP的积累。 这些发现使我们假设AK 2缺乏导致两种相互关联但不同的病理： I.还原应激将能量代谢重定向到脂质储存而不是OXPHOS; 二. AMP和IMP蓄积，导致核苷酸代谢缺陷。我们建议的研究将 测试骨髓生成失败是否主要是还原性应激和能量利用受损的结果， 嘌呤代谢受损，或两者兼有。我们将确定是否可以通过纠正 NADH/NAD+比率或核苷酸库。最后，我们将在RD的体内模型中验证我们的发现， 研究不同血液谱系中的不同代偿机制是否导致RD表型。 我们以RD为模型，剖析能量代谢、氧化还原应激、 和核苷酸稳态。这些见解将推动减轻还原性压力的治疗， 嘌呤代谢的细胞类型特异性操纵作为免疫抑制和癌症治疗的策略。