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

项目总结 导致基因组不稳定的复制应激是肿瘤发生的早期驱动因素，一直以来 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 途径是肿瘤发生的屏障，导致细胞周期停滞，导致细胞衰老或细胞 死亡是对基因组不稳定性高负担的回应。然而，在癌基因诱导的癌细胞中 复制应激DDR的保护屏障，细胞死亡和衰老被绕过导致失控 细胞增殖。因此，破译绕过癌基因诱导的复制应激和 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 接受治疗。 尤文肉瘤(EWS)是由染色体易位和框内基因融合引起的 EWSR1和Ets转录因子家族。在大多数EWS病例中，染色体易位 结果产生了EWS-FLI1癌基因。EWS-FLI1作为一种异常转录因子发挥作用 推动EWS的发展和进步。EWS-FLI1癌基因表达导致癌基因诱导 复制压力和基因组的不稳定性。然而，EWS-FLI1旁路的分子机制 癌基因诱导的复制应激反应通路在很大程度上是未知的。我们的初步数据显示， USP1脱泛素酶在EWS细胞系和肿瘤中高表达。USP1管理DDR，并需要 基因组的稳定性和干细胞的维持。我们发现在EWS中USP1的表达受EWS-FLI1的调控。 重要的是，使用小分子USP1抑制剂抑制USP1活性会导致EWS生长停滞 提示USP1的表达和活性对EWS细胞的增殖和进展是重要的。 值得注意的是，USP1的缺失导致了Hells染色质重塑蛋白水平的下降。的功能 USP1或Hells在EWS发病机制中的作用尚未被研究。在这项研究中，我们将研究这一规定 通过USP1脱泛素酶对地狱的影响(目标1)，确定USP1促进EWS细胞的机制 增殖(目标2)，并确定USP1基因敲除对体内EWS肿瘤形成的影响以及 USP1抑制联合化疗药物对EWS细胞增殖的抑制作用 (目标3)。 这项研究的成功完成将揭开对USP1介导的搭桥的新的机制见解 EWS-FLI1癌基因诱导的复制应激并帮助评估USP1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.