Role of nuclear profilin-1 in DNA replication fork stability and cancer chemotherapy response

项目总结 DNA复制应激是许多基因毒性化疗的机制基础之一，这些化疗导致 复制分叉停顿。失速的叉子经常倒转形成四向连接结构，作为一种自然的顶盖 在叉子之前修复DNA损伤，避免灾难性的叉子坍塌。然而， 叉子翻转是一把双刃剑，因为倒叉手臂中新生的DNA容易受到过量 核酸酶切除如果没有得到安全的保护，可能会导致严重的基因组不稳定。多种因素在拉锯战中发挥作用 -战火在失速的叉子上加强或削弱它们的稳定性，它们在牢房中的平衡决定着 在压力和化疗后的细胞结果中复制分叉。在本研究中，我们调查了 一种新的复制分叉调节因子Profilin-1(PFN1)的分子机制。作为一种著名的肌动蛋白- PFN1是一种结合蛋白，在肌动蛋白聚合和动力学中起着至关重要的作用。矛盾的是，它还有很好的- 有记录但知之甚少的抗癌活动，包括使癌细胞对 化疗。在最近发表的研究中，我们首次证明了抗癌药物 PFN1茎的影响，至少部分来自其在空间和机械上截然不同的核功能 从它在肌动蛋白调节中的细胞质功能。我们证明了核PFN1直接与ENL相互作用 超级延长复合体(SEC)，并抑制SEC推动各种不同类型的转录延长的能力 包括MYC在内的癌症基因。我们还提出了核PFN1水平经常降低的临床证据。 由于其核出口因子Exportin-6(XPO6)表达上调，其缺失增加了核 降低PFN1水平，减少肿瘤生长。这些发现确立了这样一种概念，即PFN1从根本上说 核内重要的和与癌症相关的功能，并为进一步发现其参与奠定了基础 在额外的核进程中。在这项授权中，我们提出了核PFN1促进正常DNA的新证据 复制，但在压力期间导致叉子不稳定，并增加细胞对复制压力的敏感性- 诱导化疗，包括PARP抑制剂。我们假设核PFN1具有上下文依赖性 对DNA复制分叉的影响。在正常情况下，它通过增加染色质来促进分叉进展 通过Snf2h.放松。在应激条件下，它通过刺激Snf2小时和 通过抑制BOD1L来增加叉子切除，从而导致叉子不稳定。目标1：确定 核PFN1对BOD1L依赖的复制分叉保护。目标2：确定PFN1/Snf2轴在 复制叉子的重塑和稳定性。目的3：了解核型PFN1的化疗增敏能力。 这项拨款中提议的工作有可能产生重要的机械性见解和概念验证 治疗数据使核PFN1作为基因毒性合成致死靶点的进一步研究合理化 化疗。

PROJECT SUMMARY DNA replication stress is one of the mechanistic underpinnings of many genotoxic chemotherapies which cause replication fork stalling. Stalled forks frequently reverse to form four-way junction structures, as a natural coping mechanism, to allow repair of the DNA lesions ahead of the forks and avoid catastrophic fork collapse. However, fork reversal is a double-edged sword because the nascent DNA in the reversed arm is susceptible to excessive nuclease resection if not securely protected which can lead to severe genome instability. Many factors play tug- of-war at stalled forks to strengthen or weaken their stability, and their balance in the cell determines the fate of replication forks during stress and cellular outcome upon chemotherapy treatments. In this study, we investigate the molecular mechanisms of a novel replication fork regulator named profilin-1 (Pfn1). As a well-known actin- binding protein, Pfn1 plays an essential role in actin polymerization and dynamics. Paradoxically, it also has well- documented but poorly understood anticancer activities including the ability to sensitize cancer cells to chemotherapy treatments. In recently published work, we demonstrated for the first time that the anticancer effects of Pfn1 stem, at least partially, from its nuclear functions that are spatially and mechanistically distinct from its cytoplasmic function in actin regulation. We showed that nuclear Pfn1 directly interacts with ENL in the Super Elongation Complex (SEC) and inhibits the ability of SEC to drive transcriptional elongation of various cancer genes including MYC. We also presented clinical evidence that nuclear Pfn1 level is frequently decreased in cancer due to the upregulation of its nuclear exporter exportin-6 (XPO6), whose deletion increases nuclear Pfn1 level and decreases tumor growth. These findings establish the notion that Pfn1 has fundamentally important and cancer-relevant functions in the nucleus, and set the stage for further discovery of its involvement in additional nuclear processes. In this grant, we present novel evidence that nuclear Pfn1 promotes normal DNA replication but causes fork destabilization during stress and increases cellular sensitivity to replication stress- inducing chemotherapies including PARP inhibitors. We hypothesize that nuclear Pfn1 has context-dependent effects on DNA replication forks. Under normal conditions, it promotes fork progression by increasing chromatin relaxation through SNF2H. Under stressed conditions, it promotes fork reversal by stimulating SNF2H and increases fork resection by suppressing BOD1L, leading to fork destabilization. Aim 1: Determine the effect of nuclear Pfn1 on BOD1L-dependent replication fork protection. Aim 2: Define the role of Pfn1/SNF2H axis in replication fork remodeling and stability. Aim 3: Understand the chemotherapy-sensitizing ability of nuclear Pfn1. Work proposed in this grant has the potential to generate important mechanistic insights and proof-of-concept therapeutic data rationalizing further investigation of nuclear Pfn1 as a synthetic lethal target for genotoxic chemotherapies.