Implications of PARP1 in myelodysplastic syndromes and targeted therapy

项目摘要/摘要 骨髓增生异常综合征(MDS)是一种克隆性造血干细胞疾病的异质性组，是一种 获得性骨髓衰竭综合征。MDS的特征是无效的造血导致 外周血细胞减少和祖细胞扩增。编码RNA剪接因子(U2AF1、SF3B1、 SRSF2和ZRSR2)频繁突变，并出现在MDS的创始克隆中，代表着一种独特的 靶向治疗的遗传易损性类别。然而，尽管剪接体突变很普遍， 这些突变如何影响不同的细胞机制在很大程度上尚不清楚。我们和其他人最近的研究 提示R-环，一组含有RNA：DNA杂交体和移位的单链的转录中间产物- 链状DNA是不同剪接体突变体引起的基因组不稳定的一个来源。在预赛中 导致这一应用的研究，我们发现PARP1是由R环激活的，它在 抑制与R环相关的DNA损伤。此外，我们还发现MDS相关的RNA剪接因子 突变促进了R环的积累，使细胞对PARP抑制变得敏感。这些令人兴奋的发现 推测PARP1是R环的关键感受器，是R环相关的关键抑制因子 DNA损伤。此外，异常的R循环累积代表了MDS中的一个新的靶向漏洞- 相关剪接因子突变细胞，使抑制PARP成为靶向R环脆弱性的一种有吸引力的方法 MDS。最后，由于PARP抑制剂在不同疾病中获得的FDA批准有限，因此重新调整PARP的用途 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 我们的调查结果会反馈给诊所。为了检验这些假设，在目标1中，我们将阐明PARP1通过哪些机制 由R-环激活。在目标2中，我们将确定全球PARP1底物和R-环在 U2AF1-突变细胞，提供了PARP1如何调节R-环的蛋白质组和基因组学观点。在目标3中，我们 将评估PARP抑制剂olaparib是否可以选择性地消除MDS相关剪接突变细胞 体外和体内。总之，这些研究将从机械上解释PARP1是如何在 拼接突变细胞，揭示PARP1如何保护细胞免受R环相关基因组的不稳定性，并解决 PARP抑制剂能否利用剪接体突变MDS细胞中的R环相关漏洞 作为MDS的靶向治疗。R-LOOP、PARP1、DNA损伤反应和剪接体的综合专业知识 MDS突变(阮氏实验室)，蛋白质组学方法调节PARP(梁实验室，co-I)，以及 MDS GEMM小鼠模型的U2AF1和SRSF2突变(Lee实验室，co-I)为我们提供了独特的 在表达MDS相关突变的细胞中表征PARP1功能的机会。这些研究将 不仅极大地促进了我们对R环生物学和PARP1信号的理解，而且还重新调整了 FDA批准的PARP抑制剂在靶向治疗携带RNA剪接因子的MDS患者中的应用 通过利用与R环路相关的漏洞进行突变。

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.