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

项目总结/摘要 2016年，近63，000人将被诊断患有肾细胞癌。七成 这些患者将患有透明细胞肾细胞癌（ccRCC），其中90%具有失活的 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，以便被蛋白质降解。 蛋白酶体VHL的失活导致这些蛋白质的积累，并导致细胞内蛋白质的变化。 细胞通路这些细胞变化中最具特征的是细胞内的 HIF 2a转录因子。HIF 2a诱导许多基因的转录，包括几个转录因子。 参与血管生成和肿瘤发生。最近，HIF 2a抑制被证明可以减少 原位植入缺乏功能性pVHL的ccRCC细胞的小鼠的肿瘤生长。这些 结果强调了利用VHL失活引起的细胞变化来治疗 ccRCC。然而，并非所有的ccRCC细胞系都对HIF 2a抑制有应答，也并非所有的患者来源的细胞系都对HIF 2a抑制有应答。 异种移植肿瘤在用HIF 2 α拮抗剂治疗后显示生长减少。因此，我们认为， 需要治疗VHL-/- ccRCC的替代靶点。这项研究旨在确定基因 与VHL失活有合成致死关系。一种合成的基因， VHL失活，当操纵时，将优先降低细胞中的适应性，其中VHL活性 在具有完整VHL的细胞中没有类似的损害。这些基因将通过以下方法鉴定： 在具有或缺乏pVHL途径的细胞中筛选编码CRISPR sgRNA的慢病毒文库 功能以这种方式，可以开发用于治疗VHL-/- ccRCC的疗法的新靶标。 发现了VHL合成致死率筛选之间的跨种属比较 将在果蝇细胞和人类细胞中进行失活以鉴定靶点 其是稳健的并且可能耐受VHL-/- ccRCC细胞系之间的差异，并且最终， 患者将在体外和体内验证筛选“命中”。对于验证的命中，生化 与VHL失活的合成致死关系的基础功能开始是 通过遗传学和（如果可能的话）药物学操纵体内的特定结构域来探测 目标.这项研究的结果可能最终有助于开发新的治疗方法 用于治疗ccRCC。

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.