Mechanisms underlying USP1-mediated bypass of EWS-FLI1 oncogene-induced replication stress in Ewing sarcoma

项目摘要 导致基因组不稳定性的复制应激是肿瘤发生的早期驱动因素， 与癌基因的过度表达有关。在正常细胞中，DNA损伤反应（DDR）的激活 途径作为肿瘤发生的屏障，导致细胞周期停滞，诱导细胞衰老或细胞凋亡。 基因组不稳定性的高负荷导致的死亡。然而，在癌细胞中， 复制压力DDR的保护屏障，细胞死亡和衰老被绕过，导致不受控制的 细胞增殖因此，破译绕过癌基因诱导的复制应激的机制， 衰老将有助于理解疾病进展的基础科学，并将确定新的靶点 治疗 尤文肉瘤（EWS）是由染色体易位和染色体间的框内基因融合驱动的。 EWSR 1和ETS转录因子家族。在大多数EWS病例中，染色体易位 导致EWS-FLI 1癌基因的产生。EWS-FLI 1作为异常转录因子发挥功能， 推动EWS的发展和进步。EWS-FLI 1癌基因的表达导致癌基因诱导的 复制压力和基因组不稳定性。然而，EWS-FLI 1旁路的分子机制 癌基因诱导的复制应激反应途径在很大程度上是未知的。我们的初步数据显示， USP 1去泛素化酶在EWS细胞系和肿瘤中过表达。USP 1规范DDR，并要求 基因组稳定性和干细胞维持。我们发现USP 1的表达受EWS-FLI 1的调控。 重要的是，使用小分子USP 1抑制剂抑制USP 1活性导致EWS生长停滞 表明USP 1表达和活性对于EWS细胞增殖和进展是重要的。 值得注意的是，USP 1缺失导致HELLS染色质重塑蛋白水平降低。的功能 USP 1或HELLS在EWS发病机制中的作用尚未研究。在本研究中，我们将研究 通过USP 1去泛素化酶（Aim 1）对HELS的作用，确定USP 1促进EWS细胞增殖的机制 增殖（目的2），并确定USP 1敲低对体内EWS肿瘤形成的影响以及 USP 1抑制剂与化疗药物联合抑制EWS细胞增殖的功效 (Aim 3）。 本研究的成功完成将揭示USP 1介导的旁路的新机制 EWS-FLI 1癌基因诱导的复制应激，并帮助评估USP 1靶向治疗策略， EWS。

PROJECT SUMMARY Replication stress leading to genome instability is an early driver of tumorigenesis and has been associated with overexpression of oncogenes. In normal cells, activation of the DNA damage response (DDR) pathway serves as a barrier to tumorigenesis leading to cell cycle arrest inducing cellular senescence or cell death in response to high burden of genome instability. However, in cancer cells upon oncogene-induced replication stress the protective barrier of DDR, cell death and senescence is bypassed leading to uncontrolled cell proliferation. Therefore, deciphering the mechanisms that bypass oncogene-induced replication stress and senescence will help understand the basic science underlying disease progression and will identify new targets for therapy. Ewing sarcoma (EWS) is driven by a chromosomal translocation and in-frame gene fusion between EWSR1 and ETS family of transcription factors. In majority of the EWS cases, the chromosomal translocation results in the generation of EWS-FLI1 oncogene. EWS-FLI1 functions as an aberrant transcription factor that drives the development and progression of EWS. Expression of EWS-FLI1 oncogene leads to oncogene-induced replication stress and genome instability. However, the molecular mechanism underlying bypass of EWS-FLI1 oncogene-induced replication stress response pathways is largely unknown. Our preliminary data shows that USP1 deubiquitinase is overexpressed in EWS cell lines and tumors. USP1 regulates DDR and is required for genome stability and stem cell maintenance. We find that USP1 expression is regulated by EWS-FLI1 in EWS. Importantly, inhibition of USP1 activity using small molecule USP1 inhibitors resulted in growth arrest of EWS cell lines indicating that USP1 expression and activity is important for EWS cell proliferation and progression. Notably, USP1 depletion led to a decrease in the levels of HELLS chromatin remodeling protein. The function of USP1 or HELLS in EWS pathogenesis has not been investigated. In this study, we will examine the regulation of HELLS by USP1 deubiquitinase (Aim 1), determine the mechanism by which USP1 promotes EWS cell proliferation (Aim 2), and determine the effect of USP1 knockdown on EWS tumor formation in vivo and the efficacy of USP1 inhibition in combination with chemotherapeutic drugs at suppressing EWS cell proliferation (Aim 3). Successful completion of this study will unravel novel mechanistic insights into USP1 mediated bypass of EWS-FLI1 oncogene-induced replication stress and help evaluate USP1 targeted treatment strategies for EWS.