The Oncogenic functions of YAP/TAZ in Renal Cell Carcinoma

项目摘要 肾细胞癌是美国第八种最常见的成人癌症。尽管最近 治疗方案的改进，大多数转移性肾癌患者继续屈从于这一点 这种疾病每年在全世界造成100,000多人死亡。因此，确定新的治疗方法是很重要的 更有效地治疗这种致命疾病的战略。我的初步研究表明YAP/TAZ功能 作为核心致癌转录网络的主要调节者，是维持 肾癌肿瘤的转录动态平衡。我的建议的第一个主要目的是确定 YAP/TAZ对癌基因转录和超级增强子维持的药理和遗传抑制作用 在患者来源的原代肾细胞癌细胞中。我将使用尖端的表观基因组学技术，包括Cut&Tag和 QuantSeq以探索YAP/TAZ的遗传和药物抑制如何影响成分和 YAP/TAZ转录复合体的活性与全球肾癌的表观遗传和转录 风景。鉴于已报道的YAP/TAZ在协调阶段分离转录中的功能 在超级增强剂的缩合物，我将结合免疫荧光分析内源性YAP/TAZ和 YAP/TAZ转录复合体的其他成分以及DNA荧光原位杂交 (DNA-FISH)针对YAP/TAZ结合的增强子区域，以研究遗传或药理学 抑制YAP/TAZ在这些特定的基因上影响活性转录缩合物的形成 YAP/TAZ靶基因调控的要素及其必要性。通过这些分析，我将能够 对YAP/TAZ在肾癌肿瘤中的分子功能有重要的了解，但也重建了核心 肾细胞癌致癌转录网络的集合。这项提案的第二个主要目标是确定 YAP/TAZ与缺氧诱导因子2α(HIF2A)的功能相互作用以及YAP/TAZ是否共同作用于HIF2α。 在体内模型中，抑制可以克服对HIF2A抑制剂的耐药性。而HIF2A抑制剂出现的时间较晚 阶段性临床试验表明，对这种小分子抑制剂的前期和获得性耐药很普遍。我的初选 研究结果提示，YAP/TAZ信号通路可能参与了对HIF2A抑制剂的耐药性。vbl.使用 YAP/TAZ和HIF2A的药理抑制剂，我打算用Cut&Tag和QuantSeq来研究 HIF2A和YAP/TAZ在维持透明细胞RCC癌基因转录中的功能相互作用 YAP/TAZ调控的基因可能介导对HIF2A抑制剂的耐药性。此外，我会 研究YAP/TAZ抑制剂与HIF2A抑制剂联合应用如何影响HIF2A-I的体内生长 耐药患者来源的ccRCC异种移植，对收获的肿瘤执行QuantSeq以在体外验证My 调查结果。总之，这些研究将大大增强我们对串扰和冗余的理解 在控制肾癌细胞生长和存活的致癌途径之间，潜在地产生更多 有效的联合策略以克服肾癌患者的治疗耐药性。

PROJECT ABSTRACT Renal cell carcinoma (RCC) is the 8th most common form of adult cancer in the US. Despite recent improvement in treatment options, the majority of patients with metastatic RCC continue to succumb to this disease, resulting in over 100,000 deaths per year worldwide. Thus, it is important to identify novel therapeutic strategies to more effectively treat this deadly disease. My preliminary studies suggest that YAP/TAZ function as master regulators of the core oncogenic transcriptional network and are required for maintaining the transcriptional homeostasis of RCC tumors. The first major aim of my proposal is to determine the effects of pharmacologic and genetic inhibition of YAP/TAZ on oncogenic transcription and super enhancer maintenance in patient-derived primary RCC cells. I will employ cutting-edge epigenomic techniques including Cut&Tag and QuantSeq to probe how genetic and pharmacological inhibition of YAP/TAZ affects the compositions and activities of the YAP/TAZ transcriptional complexes and the global RCC epigenetic and transcriptional landscape. Given the reported functions of YAP/TAZ in orchestrating phase-separated transcriptional condensates at super enhancers, I will combine immunofluorescence analysis of endogenous YAP/TAZ and other components of the YAP/TAZ transcriptional complexes, along with DNA fluorescent in situ hybridization (DNA-FISH) against YAP/TAZ-bound enhancer regions, to investigate how genetic or pharmacological inhibition of YAP/TAZ affects the formation of active transcriptional condensates at these particular genetic elements and their necessity in regulating YAP/TAZ target genes. Through these analyses, I will be able to gain critical insights into the molecular functions of YAP/TAZ in RCC tumors, but also reconstruct the core ensemble of the RCC oncogenic transcriptional network. The second major aim of this proposal is to determine how YAP/TAZ functionally interacts with hypoxia-inducible factor 2 alpha (HIF2A) and whether YAP/TAZ co- inhibition can overcome resistance to HIF2A inhibitors in an in vivo model. While HIF2A inhibitors are in late stage clinical trials, upfront and acquired resistance to this small molecule inhibitor is prevalent. My preliminary findings suggest that YAP/TAZ signaling may contribute to resistance to HIF2A inhibitors. Using pharmacological inhibitors of YAP/TAZ and HIF2A, I intend to use Cut&Tag and QuantSeq to investigate the functional interactions between HIF2A and YAP/TAZ in maintaining clear cell RCC oncogenic transcription, as well as possible YAP/TAZ regulated genes mediating resistance to HIF2A inhibitors. Furthermore, I will investigate how YAP/TAZ inhibitors, in combination with HIF2A inhibitors, impact the in vivo growth of HIF2A-i- resistant patient-derived ccRCC xenografts, performing QuantSeq on harvested tumors to validate my in vitro findings. Together, these studies will greatly enhance our understanding of the crosstalk and redundancies between the oncogenic pathways that govern the growth and survival of RCC cells, potentially yielding more effective combination strategies to overcome treatment resistance in RCC patients.