Elucidating function of disease-related SAMD9L mutations in hematopoiesis

阐明疾病相关 SAMD9L 突变在造血中的功能

• 批准号: 10644725
• 负责人: Sushree S Sahoo
• 金额: $ 9万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10644725
• 关键词: Accounting; Advisory Committees; Affect; Amino Acids; Anemia; Area; B-Lymphocytes; Biological; Biological Models; Biology; Bone marrow failure; CRISPR/Cas technology; Cell Death; Cell physiology; Cells; Child; Childhood; Chromosome Mapping; Confocal Microscopy; Constitution; Constitutional; Crista ampullaris; Defect; Disease; Disease model; Dysmyelopoietic Syndromes; Electron Microscopy; Engineering; Engraftment; Ensure; Environment; Erythro; Etiology; Exhibits; Experimental Models; Failure; Foundations; Gene Expression; Genes; Genotype; Genus Hippocampus; Germ-Line Mutation; Growth; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterozygote; Homeostasis; Homing; Human; Immunology; Impairment; In Vitro; Inflammatory; Inherited; Institution; Interferons; Investigation; Knowledge; Link; Lymphopenia; Maps; Marrow; Mediating; Membrane Potentials; Mesenchymal Stem Cells; Mitochondria; Modeling; Molecular; Monosomy 7; Mus; Mutant Strains Mice; Mutate; Mutation; Myelopoiesis; Natural Immunity; Nature; Orthologous Gene; Oxidative Phosphorylation; Pathway interactions; Patients; Phase; Phenotype; Physiological; Play; Point Mutation; Positioning Attribute; Predisposition; Proteins; Proteome; Reactive Oxygen Species; Recurrence; Reporter; Research; Resources; Role; Severities; Stress; Structure; Swelling; Syndrome; System; Testing; Toxic effect; Translations; Transmission Electron Microscopy; Up-Regulation; Valine; Virus; Wild Type Mouse; Work; career; cell growth; comparative; cytokine; cytopenia; experimental study; fatty acid oxidation; gain of function; gain of function mutation; hematopoietic stem cell differentiation; in vivo; induced pluripotent stem cell; insight; interest; mitochondrial dysfunction; mitochondrial membrane; mouse model; mutant; novel; novel therapeutics; overexpression; segregation; stem cell biology; stem cell homeostasis; transcriptome

Project Summary Hereditary predisposition is the major etiological contributor to diseases leading to bone marrow failure (BMF) in children. Recently, germline mutations in two interferon responsive genes, SAMD9 and SAMD9L (SAMD9/9L) were shown to cause a group of multisystemic disorders with the common denominator of BMF with cytopenias and a propensity for myelodysplasia. Overexpression of wildtype SAMD9/9L results in translation block and cellular growth inhibition, and these phenotypes are exacerbated by patients’ gain-of-function (GOF) mutations. The molecular mechanisms by which both wildtype and mutant SAMD9/9L proteins exert these activities are largely unknown. Moreover, how the mutants impair hematopoiesis is limited by their cell toxic effect and lack of experimental models. Towards this, by using CRISPR/Cas9 engineering, we have modelled two patient GOF mutations (V1512M, V1512L) at a recurrently mutated SAMD9L amino acid residue, into the endogenous loci of human-induced pluripotent stem cells (hiPSCs) and mouse (mouse V1507M/L). My preliminary analysis exhibited decreased erythro- and myelopoiesis upon differentiation of mutant hiPSC-derived hematopoietic progenitors, and significant anemia with B-cell lymphopenia in mutant mice, with increased severity of phenotypes in V1512M genotype. Pilot gene expression studies showed a mitochondrial stress signature depicted by upregulation of oxidative phosphorylation and reactive oxygen species pathways in mutants. Further exploration into the mitochondrial phenotype revealed mutant-specific alteration of mitochondria network and structure with swollen cristae by electron microscopy, with prominent defect in V1512M. This prompted the investigation of a physiological link between SAMD9L and mitochondria, which I established by demonstrating a strong co-localization of Samd9l with mitochondria in mesenchymal and hematopoietic stem cells (HSC) of wildtype mice. Altogether, my work suggests SAMD9L to play a role in mitochondria biology. Since mitochondria are critical for HSC homeostasis, differentiation, and commitment, I hypothesize that SAMD9L mutant-induced hematotoxicity is mediated by the underlying mitochondrial dysfunction. I will pursue this hypothesis through two specific aims: 1) interrogate the the variable effect of GOF SAMD9L V1512M and V1512L point mutations on hematopoiesis in a in vivo murine model and 2) determine if SAMD9L V1512M and V1512L affect mitochondrial structure, function, and dynamics. I will carry out the K99 phase of these aims in an exceptional research environment under the guidance of Drs. Marcin Wlodarski and John Crispino, and my advisory committees composed of experts in hematopoiesis, mitochondria biology, and immunology. In the independent phase, I will extend my studies of the molecular link between SAMD9L and mitochondria in hematopoiesis and exploit molecular reporters to refine this link. The institutional resources, academic environment and the planned courses outlined in my proposal will ensure my successful transition to independence.

项目摘要 遗传易感性是导致骨髓衰竭（BMF）的疾病的主要病因， 孩子最近，两个干扰素应答基因SAMD 9和SAMD 9 L（SAMD 9/9 L）的种系突变 被证明会导致一组多系统疾病，其共同特征是BMF伴血细胞减少 和骨髓增生异常的倾向野生型SAMD 9/9 L的过表达导致翻译阻断， 细胞生长抑制，并且这些表型因患者的功能获得性（GOF）突变而加剧。 野生型和突变型SAMD 9/9 L蛋白发挥这些活性的分子机制是 大部分未知。此外，突变体如何损害造血受到其细胞毒性作用和缺乏免疫抑制的限制。 实验模型为此，通过使用CRISPR/Cas9工程，我们模拟了两名患者的GOF， 将反复突变的SAMD 9 L氨基酸残基处的突变（V1512 M，V1512 L）导入内源性基因座中， 人诱导的多能干细胞（hiPSC）和小鼠（小鼠V1507 M/L）。我的初步分析 在突变hiPSC衍生的造血干细胞分化后表现出减少的造血和骨髓生成。 在突变小鼠中，祖细胞和显著贫血伴B细胞淋巴细胞减少， V1512 M基因型的表型。先导基因表达研究显示线粒体应激信号 通过突变体中氧化磷酸化和活性氧途径的上调来描述。进一步 对线粒体表型的探索揭示了线粒体网络的突变体特异性改变， 电子显微镜下观察到隆起嵴结构，V1512 M存在明显缺陷。这促使 研究SAMD 9 L和线粒体之间的生理联系，我通过证明 Samd 91与线粒体在骨髓间充质和造血干细胞（HSC）中强共定位 野生型小鼠总之，我的工作表明SAMD 9 L在线粒体生物学中发挥作用。由于线粒体 对于HSC的稳态、分化和定型至关重要，我推测SAMD 9 L突变体诱导的 血液毒性由潜在的线粒体功能障碍介导。我将通过两个方面来探讨这个假设。 具体目的：1）询问GOF SAMD 9 L V1512 M和V1512 L点突变对 和2）确定SAMD 9 LV 1512 M和V1512 L是否影响线粒体 结构、功能和动力学。我将在K99阶段进行一项特殊的研究， 在Marcin Wlodarski博士和John Crispino博士以及我的咨询委员会的指导下， 由造血、线粒体生物学和免疫学专家组成。在独立阶段，我将 扩展了我对SAMD 9 L和线粒体在造血中的分子联系的研究， 分子报告者来完善这种联系。机构资源、学术环境和计划 我的建议中概述的各项方针将确保我成功地过渡到独立。