Decoding the Paradox of DDX41-mutant MDS

项目摘要 RNA解旋酶基因DDX41的遗传突变导致成人发病的骨髓增生异常 综合征和相关的髓系恶性肿瘤，占MDS病例的5%。这些突变是 总是杂合的，通常是蛋白质N-末端区域的移码，这表明它们会导致 蛋白质功能丧失。DDX41蛋白已经描述了在mRNA剪接、先天免疫信号等方面的功能 和核糖体生物发生，但DDX41突变对髓系细胞贡献的确切机制 恶性并不为人所知。在大约70%带有种系DDX41突变的MDS患者中，一种突变 DDX41的另一个等位基因是在患病的骨髓细胞中获得的。这种体细胞突变几乎 总是导致特定的氨基酸取代精氨酸-525为组氨酸(R525H)。在最近的一份出版物中， 我们证明了小鼠造血干细胞和祖细胞(HSPC)具有两种功能丧失 Ddx41突变(Ddx41-/-)或一个功能丧失突变和一个Ddx41R525H突变(Ddx41R525H/-) 细胞周期停滞和细胞凋亡。这表明造血需要一个拷贝的野生型DDX41 R525H突变体缺乏所需的功能。在我们的小鼠模型中，我们发现 干细胞(HSC)大多处于静止状态，可以在双等位基因Ddx41突变中存活，但不会在 活着。这些发现提出了一个明显的悖论，即DDX41突变的准确组合 在许多患者中存在，是在增殖期HSPC池中选择的，因此克隆携带这些 突变不太可能存活和扩大到足以导致疾病的程度。我们建议的研究将 从种系DDX41-突变体中筛选获得的DDX41R525H突变的机理 病人。我们的总体假设是，具有必需的低蛋白翻译活动的HSC是细胞 由于DDX41蛋白功能降低而导致的DDX41R525H突变产生、持续和扩展的类型 导致核糖体合成减少。DDX41R525H突变体HSC的选育 其他共突变，由于其有限的增殖能力而导致造血效率低下 分化祖细胞，导致MDS。Ddx41R525H/-小鼠HSC在骨髓中不能扩增 有必要对携带这种突变组合的患者细胞进行检查，以了解它们是如何存活的 然后扩张。使用单细胞测序方法，我们建议分析MDS患者的BM细胞以 确定细胞中存在的分化状态、转录变化和共突变 获得性R525H突变。为了确定细胞具有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 R525H突变蛋白与野生型的比较。对收购的DDX41的影响有了更好的了解 关于MDS的发病机制，我们随后将检测基因或化学抑制剂的合理选择靶点 携带双等位基因DDX41突变的HSPC的治疗根除或机械抢救。

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.