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 遗传性是导致骨髓衰竭(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)的种系突变 被证明引起了一组多系统疾病，其共同特征是BMF伴细胞减少症 并有骨髓发育不良的倾向。过表达通配类型SAMD9/9L会导致转换块和 细胞生长抑制，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 很大程度上是未知的。此外，突变体如何损害造血功能受到它们的细胞毒性作用和缺乏 实验模型。为此，通过使用CRISPR/CAS9工程，我们已经对两个患者GOF进行了建模 反复突变的SAMD9L氨基酸残基的突变(V1512M，V1512L)进入内源基因座 人诱导多能干细胞和小鼠(小鼠V1507M/L)。我的初步分析 突变型HiPSC来源的造血细胞分化后红系和骨髓生成减少 在突变小鼠中，祖细胞和伴有B细胞淋巴细胞减少的显著贫血，随着疾病严重程度的增加 V1512M基因的表型。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物种途径的上调来描述。进一步 对线粒体表型的探索揭示了线粒体网络和突变体特异性的改变 电子显微镜下有明显的隆起结构，V1512M有明显缺陷。这促使 研究SAMD9L和线粒体之间的生理联系，这是我通过展示一种 Samd9l与线粒体在小鼠骨髓间充质干细胞和造血干细胞中的强共定位 野生型老鼠。综上所述，我的工作表明SAMD9L在线粒体生物学中发挥了作用。因为线粒体 对HSC的动态平衡、分化和承诺至关重要，我假设SAMD9L突变诱导 血液毒性是由潜在的线粒体功能障碍所介导的。我将通过两个例子来探讨这一假设 具体目的：1)探讨GOF SAMD9L V1512M和V1512L点突变对人类免疫功能的影响 体内小鼠模型的造血功能和2)确定SAMD9L V1512M和V1512L是否影响线粒体 结构、功能和动力。我将在一项特殊的研究中执行这些目标的K99阶段 在Marin Wlodarski博士和John Crispino博士以及我的顾问委员会的指导下 由造血学、线粒体生物学和免疫学专家组成。在独立阶段，我将 扩大我对SAMD9L与线粒体在造血中的分子联系的研究和开发 分子记者来完善这一联系。机构资源、学术环境和规划 我的提案中列出的课程将确保我成功地过渡到独立。