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.

项目概要 遗传倾向是导致骨髓衰竭（BMF）疾病的主要病因 孩子们。最近，两个干扰素反应基因 SAMD9 和 SAMD9L (SAMD9/9L) 发生种系突变 被证明会引起一组多系统疾病，其共同点是 BMF 和血细胞减少 以及骨髓增生异常的倾向。野生型 SAMD9/9L 的过度表达导致翻译阻断和 细胞生长抑制，并且这些表型因患者的功能获得（GOF）突变而加剧。 野生型和突变型 SAMD9/9L 蛋白发挥这些活性的分子机制是 很大程度上不为人所知。此外，突变体如何损害造血功能受到其细胞毒性作用和缺乏 实验模型。为此，通过使用 CRISPR/Cas9 工程，我们模拟了两名患者 GOF 反复突变的 SAMD9L 氨基酸残基处的突变（V1512M、V1512L）进入内源基因座 人诱导多能干细胞 (hiPSC) 和小鼠 (小鼠 V1507M/L)。我的初步分析 在突变 hiPSC 衍生的造血细胞分化时表现出红细胞和骨髓细胞生成减少 祖细胞和突变小鼠中伴有 B 细胞淋巴细胞减少的显着贫血，且严重程度增加 V1512M 基因型的表型。试点基因表达研究显示线粒体应激特征 通过突变体中氧化磷酸化和活性氧途径的上调来描述。更远 对线粒体表型的探索揭示了线粒体网络的突变特异性改变 电子显微镜下可见嵴肿胀的结构，V1512M 具有明显的缺陷。这促使 对 SAMD9L 和线粒体之间生理联系的研究，我通过证明 Samd9l 与间充质和造血干细胞 (HSC) 中的线粒体强共定位 野生型小鼠。总而言之，我的工作表明 SAMD9L 在线粒体生物学中发挥作用。由于线粒体 对于 HSC 稳态、分化和定型至关重要，我假设 SAMD9L 突变诱导的 血液毒性是由潜在的线粒体功能障碍介导的。我将通过两个方面来探讨这个假设 具体目标：1) 探讨 GOF SAMD9L V1512M 和 V1512L 点突变对 体内小鼠模型中的造血作用和 2) 确定 SAMD9L V1512M 和 V1512L 是否影响线粒体 结构、功能和动力学。我将在一项特殊的研究中实现这些目标的 K99 阶段 环境在博士的指导下。 Marcin Wlodarski 和 John Crispino，以及我的顾问委员会 由造血、线粒体生物学和免疫学专家组成。在独立阶段，我会 扩展我对 SAMD9L 和线粒体在造血和利用中分子联系的研究 分子记者来完善这一联系。机构资源、学术环境及规划 我的提案中概述的课程将确保我成功过渡到独立。