The von Hippel-Lindau Tumor Suppressor Gene and Kidney Cancer: Insights into Oxygen Sensing and Treating Cancers Caused by Undruggable Mutations

VHL肿瘤抑制蛋白(PVHL)失活是最常见的常见的启动(“躯干”)事件 肾癌的形式，肾透明细胞癌(CcRCC)。PVHL形成一种泛素连接酶，靶向 HIF转录因子用于降解。HIF2，而不是HIF1，促进ccRCC。绑定到pVHL要求 HIFAlpha亚基被3种氧敏感的EglN Pro羟基酶中的一种进行Pro羟基化，这3种酶 是依赖于2-氧戊二酸(2-OG)的双加氧酶(如Tet DNA去甲基酶和KDM组蛋白 去甲基酶)。IDH突变的癌症积累了2-OG竞争对手2-羟基戊二酸(2-HG)。我们的工作 参与了治疗慢性肾细胞癌的血管内皮生长因子抑制剂/HIF2抑制剂和治疗慢性肾细胞癌的2-HG抑制剂的开发 突变白血病。我们发现沙利度胺重新编程小脑以摧毁IKZF1/3骨髓瘤 癌蛋白也激发了人们对小分子降解物的兴趣。 并不是所有的ccRCC对血管内皮生长因子抑制剂/HIF2抑制剂都有反应，而2-HG抑制剂对 IDH突变型胶质瘤。合成致命性(SL)应该是此类肿瘤替代药物靶点的来源，以及 更广泛地说，是与“无法用药”的突变有关的癌症。我们已经确定了新的潜在的SL交互因素 VHL(CDK4/6和ITGAV)和突变体IDH(DHODH和GSK3b)，现在建议进一步验证和 机械学研究。有趣的是，CDK4/6的合作伙伴Cyclin D1是ccRCC的HIF2靶标，但SL VHL和CDK4/6之间的关系不依赖于HIF2。我们还开始在果蝇细胞中进行SL筛选 因为Paralog补偿可能会在人类细胞的基因筛查中导致许多假阴性。 我们为化学物质和基因敲除创建了一个“向上”屏幕，这些化学物质和基因敲除可以降解感兴趣的蛋白质。我们 利用它发现Spautin-1是一种不依赖于Brablon的IKZF1降解剂，并正在追求潜在的 机制。我们还将其应用于各种不可药物的癌蛋白(例如，c-Myc、K-RAS、b-catenin)。 未能下调Cyclin D1导致pRB-VHL-/-ccRCC对HIF2抑制剂的耐药性 以独立的方式。我们正在使用生化方法来鉴定相关的Cyclin D1底物(S)。 我们意外地发现，HIF1和HIF2结合到大致相同的基因组位置，但招募 不同的蛋白质。我们正在生化和功能分析中验证这些相关蛋白质。我们是 还使用底物捕获条件回收非HIF EglN底物和非组蛋白KDM底物。 我们正在继续努力利用CRISPR的体细胞基因编辑来制作HIF2的小鼠模型- 依赖VHL-/-ccRCC。我们还鉴定了新的依赖pVHL的分泌蛋白，包括PTH-1H， 这可能会导致某些慢性肾细胞癌患者出现恶病质和高钙血症。 CcRCC具有很强的免疫原性，但其原因尚不清楚。我们发现HIF2驱动了HIF2的表达 许多内源性逆转录病毒，其中一些可以翻译和呈现为与人类白细胞抗原结合的多肽。我们 正在检查其他ccRCC细胞株和肿瘤中是否有这种多肽，以及它们是否具有免疫原性。

VHL tumor suppressor protein (pVHL) inactivation is the usual initiating (“truncal”) event in the most common form of kidney cancer, clear cell renal cell carcinoma (ccRCC). pVHL forms a ubiquitin ligase that targets the HIF transcription factor for degradation. HIF2, but not HIF1, promotes ccRCC. Binding to pVHL requires that the HIFalpha subunit be prolyl hydroxylated by one of the 3 oxygen-sensitive EglN prolyl hydroxylases, which are 2-oxoglutarate (2-OG)-dependent dioxygenases (as are the TET DNA demethylases and KDM histone demethylases). IDH mutant cancers accumulate the 2-OG competitor 2-hydroxyglutarate (2-HG). Our work contributed to the development of VEGF inhibitors/HIF2 inhibitors for ccRCC and 2-HG inhibitors for IDH mutant leukemia. Our discovery that thalidomide reprograms cereblon to destroy the IKZF1/3 myeloma oncoproteins has also galvanized interest in small molecule degraders. Not all ccRCCs respond to VEGF inhibitors/HIF2 inhibitors and 2-HG inhibitors are fairly inactive against IDH mutant gliomas. Synthetic lethality (SL) should be a source of alternative drug targets for such tumors, and more generally, for cancers linked to “undruggable” mutations. We have identified new potential SL interactors for VHL (CDK4/6 and ITGAV) and mutant IDH (DHODH and GSK3b) and now propose further validation and mechanistic studies. Intriguingly, Cyclin D1, the partner for CDK4/6, is a HIF2 target in ccRCC, but the SL between VHL and CDK4/6 is not HIF2-dependent. We also embarked on SL screens in Drosophila cells because paralog compensation likely causes many false-negatives in genetic screens with human cells. We created an “up” screen for chemicals and gene knockouts that can degrade a protein of interest. We used it to discover that Spautin-1 is a cereblon-independent IKZF1 degrader and are pursuing the underlying mechanism. We are also applying it to various undruggable oncoproteins (e.g., c-Myc, K-Ras, b-Catenin). Failure to downregulate Cyclin D1 causes resistance of VHL-/- ccRCC to HIF2 inhibitors in a pRB- independent manner. We are using biochemical approaches to identify the relevant Cyclin D1 substrate(s). We unexpectedly found that HIF1 and HIF2 bind to approximately the same genomic sites, yet recruit different proteins. We are validating these associated proteins in biochemical and functional assays. We are also using substrate-trapping conditions to recover non-HIF EglN substrates and non-histone KDM substrates. We are continuing our efforts to use somatic gene editing with CRISPR to make a murine model of HIF2- dependent VHL-/- ccRCC. We have also identified new pVHL-dependent secreted proteins including PTH-LH, which might cause the cachexia and hypercalcemia seen in some ccRCC patients. ccRCC is highly immunogenic, but the reason is unknown. We found that HIF2 drives the expression of many endogenous retroviruses, some of which can be translated and presented as HLA-bound peptides. We are examining additional ccRCC cell lines and tumors for such peptides and whether they are immunogenic.