PBRM1 bromodomain missense mutations in ccRCC vascular signaling

项目摘要 透明细胞肾细胞癌（ccRCC）是肾细胞癌的最常见亚型。虽然 早期发现和手术的结果是有利的，大约30%的患者存在 转移性疾病这突出表明，需要提高对生物化学的理解， ccRCC的治疗选择。ccRCC是一种独特的血管癌，由于von Hippel-Lindau基因突变， (VHL)基因，其编码降解缺氧的泛素连接酶的底物识别亚基- 诱导因子-α（HIF-α）转录因子在常氧条件下。VHL缺失导致细胞核HIF-α积聚， 下游促血管生成基因的异常表达可能以HIF-2α偏向的方式靶向。因此，在本发明中， ccRCC肿瘤对抗血管生成疗法特别敏感，虽然有效，但与肿瘤的生长相关。 对患者具有显著的不良毒性。最近的临床试验表明， 多溴-1（PBRM 1）是一种在ccRCC中常见突变的表观遗传乙酰赖氨酸阅读蛋白，与 改善抗血管生成治疗的结果。然而，这些研究假设所有PBRM 1突变 功能丧失，忽略了约15%的ccRCC相关PBRM 1突变是错义突变 突变，产生全长蛋白质，具有一系列不确定的结构和功能影响。 ccRCC相关的PBRM 1错义突变聚集在溴结构域（BD）中，溴结构域是 PBRM 1结合组蛋白和其他核蛋白及核酸中的乙酰赖氨酸残基。因此 更好地了解PBRM 1 BD错义突变在ccRCC中的生化影响对于促进 PBRM 1作为患者对抗血管生成治疗反应的预测生物标志物， 病人可避免不必要的治疗毒性，并寻求其他治疗方法。该奖学金将 阐明ccRCC相关PBRM 1 BD错义突变对蛋白质稳定性的生物化学影响 和乙酰化组蛋白结合功能，并确定PBRM 1在调节HIF-2α和 血管生成途径。目的1将确定哪些PBRM 1 BD错义突变是生物化学良性的， 通过将计算预测与生物物理分析相结合， 验证。目的2：探讨PBRM 1 BD错义突变对HIF-2α和血管生成的影响 通过评估HIF-2α转录输出、HIF-2α启动子可及性和血管生成， 在ccRCC细胞测定中的潜力。该奖学金解决知识差距的生化基础， 以ccRCC发病机制为总体目标，促进ccRCC标准治疗的最有效使用 方式。这项研究的成功完成将进一步推动精准医学的方法， 通过将PBRM 1突变状态作为一种微妙的预测性生物标志物来治疗ccRCC， 不必要的患者药物暴露。

PROJECT SUMMARY Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma. Although outcomes are favorable with early detection and surgery, approximately 30% of patients present with metastatic disease. This highlights the need for an improved biochemical understanding of and targeted therapeutic options for ccRCC. ccRCC is a uniquely vascular cancer due to mutations in the von Hippel-Lindau (VHL) gene, which encodes the substrate recognition subunit of a ubiquitin ligase that degrades hypoxia- inducible factor-α (HIF-α) transcription factors in normoxia. VHL loss results in nuclear HIF-α accumulation and aberrant expression of downstream pro-angiogenic gene targets in a likely HIF-2α-biased manner. Thus, ccRCC tumors are uniquely susceptible to anti-angiogenic therapies, which though effective, are associated with substantial undesirable toxicities for patients. Recent clinical trials have demonstrated that mutations in Polybromo-1 (PBRM1), an epigenetic acetyl-lysine reader protein commonly mutated in ccRCC, correlate with improved outcomes for anti-angiogenic therapies. However, these studies presume that all PBRM1 mutations are loss-of-function and neglect to consider that ~15% of ccRCC-related PBRM1 mutations are missense mutations, generating full-length proteins with a spectrum of undetermined structural and functional impacts. ccRCC-associated PBRM1 missense mutations cluster in bromodomains (BDs), the functional domains of PBRM1 that bind acetyl-lysine residues in histones and other nuclear proteins and nucleic acids. Therefore, a better knowledge of the biochemical impacts of PBRM1 BD missense mutations in ccRCC is critical to promote PBRM1 as a predictive biomarker for patient response to anti-antiangiogenic therapies, so that non-responding patients can avoid unnecessary therapeutic toxicity and seek alternative treatments. This fellowship will elucidate the biochemical impacts of ccRCC-associated PBRM1 BD missense mutations on protein stability and acetylated histone binding function and determine the role of PBRM1 in the regulation of the HIF-2α and angiogenesis pathways. Aim 1 will identify which PBRM1 BD missense mutations are biochemically benign or deleterious for protein structure and function by integrating computational prediction with biophysical assay validation. Aim 2 will determine the effects of PBRM1 BD missense mutations on HIF-2α and angiogenesis pathway regulation by assessing HIF-2α transcriptional output, HIF-2α promoter accessibility, and angiogenic potential in ccRCC cellular assays. This fellowship addresses knowledge gaps in the biochemical basis of ccRCC pathogenesis with the overall goal to promote the most effective use of standard ccRCC treatment modalities. Successful completion of this fellowship will further precision medicine approaches for the treatment of ccRCC by advancing PBRM1 mutational status as a nuanced and predictive biomarker to prevent unnecessary patient drug exposures.