Decoding the Paradox of DDX41-mutant MDS

项目摘要 RNA解旋酶基因DDX 41的遗传突变导致成人型骨髓增生异常的易感性 综合征和相关的骨髓恶性肿瘤，占高达5%的MDS病例。这些突变 总是杂合的，并且在蛋白质的N-末端区域经常是移码的，这表明它们导致 蛋白质功能丧失。DDX 41蛋白已被描述在mRNA剪接、先天免疫信号传导和免疫调节中的功能。 和核糖体生物发生，但DDX 41突变有助于髓样细胞的确切机制， 恶性肿瘤还不清楚。在大约70%的具有生殖系DDX 41突变的MDS患者中，突变 DDX 41的另一个等位基因是在患病的骨髓（BM）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组氨酸（R525 H）。在最近的一份出版物中， 我们证明，小鼠造血干细胞和祖细胞（HSPC）具有两种功能丧失， Ddx 41突变（Ddx 41-/-）或一个功能丧失突变和一个Ddx 41 R525 H突变（Ddx 41 R525 H/-）发生 细胞周期停滞和凋亡。这表明造血需要一个野生型DDX 41拷贝 并且R525 H突变体缺乏所需的功能。在我们的小鼠模型中，我们发现造血细胞 干细胞（HSC），大部分是静止的，可以在双等位基因Ddx 41突变的情况下存活，但在Ddx 41突变的情况下不扩增。 vivo.这些发现提出了一个明显的矛盾，即DDX 41突变的精确组合， 在增殖性HSPC库中，选择了存在于许多患者中的这些抗体，因此携带这些抗体的克隆 突变不太可能存活并扩展到足以导致疾病。我们建议的研究将 阐明了在种系DDX 41突变体中获得DDX 41 R525 H突变的选择机制基础 患者我们的总体假设是，具有强制性低蛋白翻译活性的HSC是细胞， 由于DDX 41蛋白功能降低导致DDX 41 R525 H突变出现、持续和扩展的类型 导致核糖体合成减少。DDX 41 R525 H突变HSC的选择，可能在以下辅助下进行： 其他共突变，由于其有限的增殖能力， 分化祖细胞，导致MDS。Ddx 41 R525 H/-小鼠HSC不能在BM中扩增 需要检查携带这种突变组合的患者细胞，以了解它们如何存活 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， 确定分化状态，转录组学变化，和共突变存在于细胞携带 获得性R525 H突变。为了确定携带该基因的细胞的选择优势的分子基础， R525 H突变，我们将使用基因工程细胞模型来定量评估 R525 H突变体蛋白与野生型相比。随着对获得性DDX 41影响的进一步了解， 突变的MDS发病机制，然后我们将测试遗传或化学抑制剂的合理选择的目标， 携带双等位基因DDX 41突变的HSPC的治疗性根除或机械性挽救。

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.