Implications of PARP1 in myelodysplastic syndromes and targeted therapy

项目总结/摘要 骨髓增生异常综合征（MDS）是一组异质性克隆性造血干细胞疾病， 获得性骨髓衰竭综合征MDS的特征在于无效造血，导致 外周血细胞减少和祖细胞扩增。编码RNA剪接因子（U2 AF 1，SF 3B 1， SRSF 2和ZRSR 2）经常突变，并出现在MDS的建立克隆中，代表了一种独特的 一类遗传脆弱性的靶向治疗。然而，尽管剪接体突变普遍存在， 这些突变如何影响不同的细胞机制在很大程度上还不清楚。我们和其他人最近的研究 表明R环，一组含有RNA：DNA杂合体和置换的单核苷酸的转录中间体， 单链DNA是由不同剪接体突变体诱导的基因组不稳定性的来源。上对初级 研究导致这一应用，我们发现PARP 1是由R环激活，它发挥了关键作用， 抑制R环相关的DNA损伤。此外，我们发现MDS相关的RNA剪接因子 突变促进R环积累并使细胞对PARP抑制敏感。这些令人兴奋的发现 这使我们假设PARP 1是R环关键传感器和R环相关的关键抑制剂 DNA损伤。此外，异常的R环积累代表了MDS中的一个新的可针对性漏洞。 相关剪接因子突变细胞，使PARP抑制成为靶向R-loop脆弱性的有吸引力的方法， MDS最后，由于PARP抑制剂在不同疾病中获得了有限的FDA批准， 治疗携带RNA剪接因子突变的MDS患者的抑制剂可能提供最快的翻译途径。 把我们的发现送到诊所为了验证这些假设，在目标1中，我们将阐明PARP 1是通过什么机制被激活的。 由R环激活在目标2中，我们将确定全球PARP 1底物和R环分布格局， U2 AF 1突变细胞，提供PARP 1如何调节R环的蛋白质组学和基因组学观点。在目标3中， 将评估PARP抑制剂奥拉帕尼是否可以选择性地消除MDS相关的剪接突变细胞， 体外和体内。总之，这些研究将从机制上解释R环是如何被PARP 1感知的。 剪接突变细胞，揭示PARP 1如何保护细胞免受R环相关基因组不稳定性，并解决 PARP抑制剂是否可以利用剪接体突变型MDS细胞中R环相关的脆弱性 作为MDS的靶向治疗。在R环，PARP 1，DNA损伤反应和剪接体方面的综合专业知识 MDS中的突变（Nguyen实验室），通过蛋白质组学方法的PARP调节（Leung实验室，co-I），和 U2 AF 1和SRSF 2突变的MDS GEMM小鼠模型（Lee laboratory，co-I）为我们提供了独特的 有机会在表达MDS相关突变的细胞中表征PARP 1功能。这些研究将 不仅大大推进了我们对R环生物学和PARP 1信号传导的理解， FDA批准的PARP抑制剂在携带RNA剪接因子的MDS患者的靶向治疗中的用途 通过利用R环相关漏洞进行突变。

PROJECT SUMMARY/ABSTRACT Myelodysplastic syndromes (MDS), a heterogenous group of clonal hematopoietic stem cell disorders, are an acquired bone marrow failure syndrome. MDS is characterized by ineffective hematopoiesis resulting in peripheral blood cytopenia and progenitor expansion. Genes encoding for RNA splicing factors (U2AF1, SF3B1, SRSF2, and ZRSR2) are frequently mutated and occur in the founding clones of MDS, representing a unique class of genetic vulnerability for targeted therapy. However, despite the prevalence of spliceosome mutations, how such mutations impact different cellular mechanisms are largely unclear. Recent studies by us and others suggest that R-loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single- stranded DNA, are a source of genomic instability induced by different spliceosome mutants. In the preliminary studies leading to this application, we find that PARP1 is activated by R-loops and it plays a key role in suppressing R-loop-associated DNA damage. Furthermore, we show that MDS-associated RNA splicing factor mutations promote R-loop accumulation and render cells sensitive to PARP inhibition. These exciting findings lead us to hypothesize that PARP1 is a key sensor of R-loops and a critical suppressor of R-loop-associated DNA damage. Furthermore, aberrant R-loop accumulation represents a new targetable vulnerability in MDS- associated splicing factor mutant cells, making PARP inhibition an attractive way to target R-loop vulnerability in MDS. Finally, since PARP inhibitors achieved limited FDA approval in different diseases, repurposing PARP inhibitors to treat MDS patients harboring RNA splicing factor mutations may provide the fastest route to translate our findings to the clinics. To test these hypotheses, in Aim 1, we will elucidate mechanisms by which PARP1 is activated by R-loops. In Aim 2, we will identify global PARP1 substrates and R-loop distribution landscape in U2AF1-mutant cells, providing a proteomic and genomic view of how PARP1 regulates R-loops. In Aim 3, we will evaluate whether PARP inhibitor, olaparib, can selectively eliminate MDS-associated splicing mutant cells in vitro and in vivo. Together, these studies will mechanistically explain how R-loops are sensed by PARP1 in splicing mutant cells, reveal how PARP1 guards cells against R-loop-associated genomic instability, and address whether R-loop-associated vulnerability in spliceosome-mutant MDS cells can be exploited by PARP inhibitors as targeted MDS therapy. The combined expertise in R-loops, PARP1, DNA damage response and spliceosome mutations in MDS (Nguyen laboratory), PARP regulation by proteomic approach (Leung laboratory, co-I), and MDS GEMM mouse models of U2AF1 and SRSF2 mutations (Lee laboratory, co-I) provides us the unique opportunity to characterize PARP1 function in cells expressing MDS-associated mutations. These studies will not only significantly advance our understanding of R-loop biology and PARP1 signaling, but also repurpose the use of FDA-approved PARP inhibitors in targeted therapy for MDS patients harboring RNA splicing factor mutations by exploiting R-loop-associated vulnerability.