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.

项目总结