Cross-species identification and validation of synthetic lethal relationships with VHL inactivation

项目摘要/摘要 2016年，将有近6.3万人被诊断为肾癌。70%的 这些患者将患有肾透明细胞癌(Ccrcc)，其中90%处于失活状态。 VHL肿瘤抑制基因突变。VHL肿瘤抑制因子的蛋白产物 PVHL基因是一种E3泛素连接酶，它标记某些蛋白质被 蛋白酶体。VHL的失活会导致这些蛋白的积聚，并导致 细胞通路。这些细胞变化中最有特点的是放松对 HIF2A转录因子。HIF2A诱导许多基因的转录，其中包括几个 参与血管生成和肿瘤的发生。最近，对HIF2A的抑制被证明可以减少 原位移植缺乏功能性pVHL的ccRCC细胞的小鼠肿瘤生长。这些 结果强调了利用VHL失活产生的细胞变化来治疗的前景 中国铁路总公司。然而，并不是所有的ccRCC细胞系都对HIF2A抑制有反应，也不是所有患者来源的细胞株都对HIF2A抑制有反应 异种移植瘤在用HIF2A拮抗剂治疗后生长减少。因此， VHL-/-ccRCC的治疗需要替代靶点。这项研究旨在识别基因 与VHL失活有合成致命关系的病毒。一种人工合成致死的基因 当VHL失活时，VHL失活将优先降低VHL活动所在细胞的适合度 在具有完整VHL的细胞中没有类似的伤害。这些基因将通过以下方式进行识别 在有或无pVHL途径的细胞中筛选编码CRISPR sgRNAs的慢病毒文库 功能。通过这种方式，可以开发治疗VHL-/-ccRCC的新靶点 被发现了。VHL合成致死率筛查间的跨物种比较 将在果蝇黑腹果蝇细胞和人类细胞中进行灭活，以确定目标 它们是健壮的，并可能经受住VHL-/-ccRCC细胞系之间的差异，最终， 病人。屏幕“命中”将在体外和体内进行验证。对于经过验证的命中率，生化 VHL失活与Be合成致死关系的基础功能(S) 通过遗传和(如果可能的话)对特定结构域的药理学操作来探索 目标。这项研究的结果可能最终有助于开发新的治疗方法 根据已确定的靶点治疗慢性肾细胞癌。

PROJECT SUMMARY/ABSTRACT In 2016, nearly 63,000 people will be diagnosed with renal cell carcinoma. Seventy percent of these patients will have clear cell renal cell carcinoma (ccRCC), 90% of which have inactivating mutations in the VHL tumor suppressor gene. The protein product of the VHL tumor suppressor gene, pVHL, is an E3 ubiquitin ligase that marks certain proteins for degradation by the proteasome. Inactivation of VHL leads to buildup of these proteins and results in changes in cellular pathways. The most well-characterized of these cellular changes is deregulation of the HIF2a transcription factor. HIF2a induces transcription of many genes including several that are involved in angiogenesis and tumorigenesis. Recently, HIF2a inhibition was shown to reduce tumor growth in mice orthotopically implanted with ccRCC cells lacking functional pVHL. These results highlight the promise of exploiting cellular changes resultant from VHL inactivation to treat ccRCC. However, not all ccRCC cell lines respond to HIF2a inhibition, nor do all patient-derived xenograft tumors show a reduction in growth upon treatment with a HIF2a antagonist. Therefore, alternative targets for treatment of VHL-/- ccRCC are needed. This study aims to identify genes that have a synthetic lethal relationship with VHL inactivation. A gene that is synthetic lethal to VHL inactivation will, when manipulated, preferentially reduce fitness in cells in which VHL activity is lost, without comparable harm in cells with intact VHL. Such genes will be identified by screening a lentiviral library encoding CRISPR sgRNAs in cells that have or lack pVHL pathway function. In this way, novel targets for the development of therapy to treat VHL-/- ccRCC can be discovered. Cross-species comparisons between screens for synthetic lethality with VHL inactivation in Drosophila melanogaster cells and human cells will be conducted to identify targets that are robust and likely to withstand differences between VHL-/- ccRCC cell lines and, eventually, patients. Screen “hits” will be validated in vitro and in vivo. For validated hits, the biochemical function(s) that underlies the synthetic lethal relationship with VHL inactivation with begin to be probed by genetic and, when possible, pharmacological manipulation of specific domains within the target. The results of this study may eventually contribute to the development of novel therapy for the treatment of ccRCC based on the targets identified.