Decoding the Paradox of DDX41-mutant MDS

解读 DDX41 突变型 MDS 的悖论

• 批准号: 10905168
• 负责人: Timothy Michael Chlon
• 金额: $ 43.58万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10905168
• 关键词: ATP Hydrolysis; Accounting; Adult; Alleles; Amino Acid Substitution; Animal Model; Apoptosis; Arginine; Binding Sites; Biogenesis; Biological Assay; Bone Marrow; Bone Marrow Cells; Bone Marrow Diseases; Cell Cycle Arrest; Cell model; Cell surface; Cells; Chemicals; DNA; Defect; Development; Disease; Dysmyelopoietic Syndromes; Essential Genes; Flow Cytometry; Genes; Genetic; Genetic Predisposition to Disease; Genotype; Helicase Gene; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterozygote; Histidine; Human; Immune signaling; Inherited; Manuscripts; Messenger RNA; Molecular; Mus; Mutation; Myeloproliferative disease; N-terminal; Pathogenesis; Pathway interactions; Patients; Predisposition; Prevention strategy; Process; Production; Proliferating; Protein Region; Proteins; Proteomics; Publications; RNA Binding; RNA Helicase; RNA Splicing; Reporter; Resolution; Ribosomes; Role; Sampling; Small Nucleolar RNA; Somatic Mutation; Testing; Therapeutic; Translations; cell type; disorder prevention; genetic testing; genetically modified cells; improved; in vivo; inhibitor; loss of function; loss of function mutation; mouse model; mutant; mutant mouse model; pharmacologic; protein function; rational design; single cell sequencing; stem cells; targeted treatment; transcriptome; transcriptomics; treatment strategy

Project Summary Inherited mutations in the RNA helicase gene DDX41 cause predisposition to adult-onset Myelodysplastic Syndrome and related myeloid malignancies, accounting for up to 5% of MDS cases. These mutations are always heterozygous and are often frameshifts in the N-terminal region of the protein, suggesting that they cause loss of protein function. The DDX41 protein has described functions in mRNA splicing, innate immune signaling and ribosome biogenesis, but the precise mechanism by which DDX41 mutations contribute to myeloid malignancy is not understood. In approximately 70% of MDS patients with germline DDX41 mutations, a mutation in the other allele of DDX41 is acquired in the diseased bone marrow (BM) cells. This somatic mutation almost always causes the specific amino acid substitution Arginine-525 to Histidine (R525H). In a recent publication, we demonstrated that murine hematopoietic stem and progenitor cells (HSPC) bearing two loss-of-function Ddx41 mutations (Ddx41-/-) or one loss-of-function mutation and one Ddx41R525H mutation (Ddx41R525H/-) undergo cell cycle arrest and apoptosis. This indicates that one copy of wild-type DDX41 is required for hematopoiesis and that the R525H mutant is deficient for the required function. In our mouse model, we found that hematopoietic stem cells (HSC), which are mostly quiescent, can survive with biallelic Ddx41 mutations but do not expand in vivo. These findings raise an apparent paradox in that the precise combination of DDX41 mutations that is present in many patients is selected against in the proliferative HSPC pool and thus clones bearing these mutations would be unlikely to survive and expand sufficiently to contribute to disease. Our proposed studies will elucidate the mechanistic basis of selection for the acquired DDX41R525H mutation in germline DDX41-mutant patients. Our overall hypothesis is that HSC, which have obligatory low protein translation activity, are the cell type in which DDX41R525H mutations arise, persist, and expand due to decreased function of the DDX41 protein causing reduced ribosome synthesis. The selection for DDX41R525H mutant HSC, possibly with the assistance of other co-mutations, causes hematopoietic inefficiency due to the limited proliferative capacity of their differentiating progenitor cells, leading to MDS. The inability of Ddx41R525H/- mouse HSC to expand in BM necessitates examination of patient cells bearing this combination of mutations to understand how they survive and expand. Using single-cell sequencing approaches, we propose to analyze MDS patient BM cells to determine the differentiation state, transcriptomic changes, and co-mutations present in cells bearing the acquired R525H mutation. To determine the molecular basis for the selective advantage of cells bearing the R525H mutation, we will use genetically-engineered cell models to quantitatively assess the function of the R525H mutant protein compared to wild-type. With an improved understanding of the effect of acquired DDX41 mutations on MDS pathogenesis, we will then test genetic or chemical inhibitors of rationally-chosen targets for therapeutic eradication or mechanistic rescue of HSPC bearing biallelic DDX41 mutations.

项目摘要 RNA解旋酶基因DDX41中的遗传突变导致成人发作的骨髓倍增加性 综合征和相关的髓样恶性肿瘤，最多占MDS病例的5％。这些突变是 始终在蛋白质的N末端区域中杂合，通常是移料器，这表明它们引起 蛋白质功能的丧失。 DDX41蛋白已经描述了MRNA剪接，先天免疫信号的功能 和核糖体生物发生，但DDX41突变有助于髓样的精确机制 不了解恶性肿瘤。在大约70％的MDS种系DDX41突变的MDS患者中 在DDX41的另一个等位基因中，在患病的骨髓（BM）细胞中获得。这个躯体突变几乎 始终导致特异性氨基酸取代精氨酸525对组氨酸（R525H）。在最近的出版物中 我们证明了两个功能丧失的鼠造血茎和祖细胞（HSPC） DDX41突变（DDX41 - / - ）或一个功能丧失突变和一个DDX41R525H突变（DDX41R525H/ - ）发生 细胞周期停滞和凋亡。这表明造血需要一份野生型DDX41的副本 R525H突变体缺乏所需功能。在我们的鼠标模型中，我们发现造血 干细胞（HSC）大多是静止的，可以通过双质DDX41突变生存，但不会扩展 体内。这些发现引起了明显的悖论，因为DDX41突变的精确组合是 许多患者存在于增殖的HSPC池中，因此在带有这些的克隆中选择了这些。 突变不太可能生存并足够扩展以促进疾病。我们提出的研究将 阐明在种系DDX41突变中获得的DDX41R525H突变的选择机械基础 患者。我们的总体假设是，具有强制性低蛋白质翻译活性的HSC是细胞 DDX41R525H突变的类型由于DDX41蛋白的功能降低而产生，持久和扩展 导致核糖体合成降低。 DDX41R525H突变体HSC的选择，可能在 其他联合杂交导致造血效率低下，因为它们的增殖能力有限 区分祖细胞，导致MDS。 DDX41R525H/ - 鼠标HSC无法在BM中扩展 需要检查具有这种突变组合的患者细胞，以了解它们如何生存 并扩展。使用单细胞测序方法，我们建议分析MDS患者BM细胞 确定带有的细胞中存在的分化状态，转录组变化和共突变 获得的R525H突变。确定带有细胞的选择性优势的分子基础 R525H突变，我们将使用基因工程细胞模型来定量评估 与野生型相比，R525H突变蛋白。随着对获得的DDX41的影响有了改进的了解 然后，我们将在MDS发病机理上进行突变，然后测试合理选择目标的遗传或化学抑制剂 消除治疗性的或机械拯救双重双重DDX41突变的HSPC。