High-throughput screen for specific small-molecule inhibitors of HIF-2A activity

描述(申请人提供)：大多数实体肿瘤及其转移都会经历缺氧区，这会促进肿瘤的进展和对治疗的抵抗。低氧对肿瘤干细胞(CSC)的增殖和维持也很重要，CSC是异质肿瘤中的少数群体，能够产生不同的肿瘤细胞群体，并与治疗后的肿瘤复发有关。低氧反应的关键介质是低氧诱导因子HIF-1和HIF-2，它们是调节重叠和独特下游靶基因的非冗余转录因子。HIF-2是肾细胞癌(RCC)、神经母细胞瘤、胶质母细胞瘤(GBM)和非小细胞肺癌(NSCLC)肿瘤发病的重要驱动因素。这与HIF-2(但不是HIF-1)在这些肿瘤类型中驱动增殖、侵袭和促进癌症干细胞的维持和生长的能力有关。在这种情况下，高HIF-1预示着更好的患者预后。因此，我们认为，HIF-2的特异性抑制将是治疗HIF-2驱动的肿瘤和靶向CSC群体的有用策略。我们的目标是确定HIF-2的小分子特异性抑制剂，这将导致NE抗癌疗法的发展。我们已经产生了稳定表达缺氧反应元件(HRE)-荧光素酶结构的786-0肾癌细胞。786-0 HRE细胞不表达HIF-1，我们将使用这些细胞来筛选NIH MLSMSR收集的HIF-2抑制剂。我们对384孔板中的786-0 HRE细胞进行了必要的优化分析，发现这些细胞适合HTS，并使用我们的PAN-HIF抑制剂PX478作为阳性对照。最优检测条件的Z‘因子为0.67，最大耐受DMSO浓度为0.75%(v/v)，从细胞接种到荧光素酶读数的总检测时间为48小时。作为计数器筛选，我们已经生成了只表达HIF-1的MIAPACA-2 HRE细胞，我们将使用这些细胞来消除也抑制HIF-1的HITS。作为验证屏幕，我们将使用HRE荧光素酶活性依赖于HIF-2的PANC HRE细胞来确认来自主屏幕的点击对HIF-2的抑制。作为潜在抗肿瘤活性的指标，HIT化合物将通过二次检测来确定它们抑制HIF-2下游靶基因的能力，以及在相关细胞模型中的功能结果，如上皮细胞到间充质细胞的转变和3D克隆形成，如肾细胞癌和GBM。拟议的筛选将导致新的HIF-2特异性抑制剂的鉴定，这将为新的抗癌疗法提供结构线索。此外，这一筛选还将有助于识别PAN HIF-1/HIF-2抑制剂 可能对HIF-1/2具有治疗应用价值，并为HIF-1/2的特异性调控机制提供新的见解。

DESCRIPTION (provided by applicant): Most solid tumors and their metastases experience regions of hypoxia, which promotes both tumor progression and resistance to therapy. Hypoxia is also important for the proliferation and maintenance of cancer stem cells (CSC), a minority population within the heterogenous tumor mass capable of generating the diverse tumor cell population and implicated in tumor recurrence following therapy. Critical mediators of the hypoxic response are the hypoxia-inducible factors HIF-1¿ and HIF-2¿, non-redundant transcription factors regulating both overlapping and unique downstream target genes. HIF-2¿ is an important driver of tumor morbidity in renal cell carcinoma (RCC), neuroblastoma, glioblastoma (GBM) and non small cell lung cancer (NSCLC). This has been linked with the ability of HIF-2¿ (but not HIF-1¿) to drive proliferation, invasion and to promote the maintenance and growth of cancer stem cells within these tumor types. In this setting, high HIF-1¿ predicts for better patient prognosis. Hence, we propose that the specific inhibition of HIF-2¿ will be a useful strategy for treating HIF-2¿ driven tumors and for targeting the CSC population. Our goal is to identify small molecule specific inhibitors of HIF-2¿ that will lead to the development of ne anti-cancer therapies. We have generated 786-0 RCC cells stably expressing a hypoxia responsive element (HRE)-luciferase construct. The 786-0 HRE cells do not express HIF-1¿ and we will use these cells to screen the NIH MLSMSR collection for inhibitors of HIF-2¿. We have performed the necessary optimization assays for the 786-0 HRE cells in 384-well plates and find that these cells are suitable for HTS using our pan-HIF inhibitor PX478 as a positive control. Signal to background ratios were ~20, Z' factor for optimized assay conditions was 0.67, maximum DMSO concentration tolerated was 0.75% (v/v) and total assay time from cell seeding to luciferase reading was 48 hours. As a counter screen, we have generated the MIAPACA-2 HRE cells that only express HIF-1¿ and we will use these cells to eliminate hits that also inhibit HIF-1¿. As a validation screen, we will use the PANC HRE cells in which HRE luciferase activity is HIF-2¿ dependent to confirm HIF-2¿ inhibition by hits from the primary screen. As an indicator of potential anti-tumor activity, hit compounds will be evaluated using secondary assays to determine their ability to inhibit HIF-2¿ downstream target genes and functional outcomes such as epithelial to meseenchymal transition and 3D colony formation in relevant cell models such has RCC and GBM. The proposed screen will lead to the identification of novel HIF-2¿ specific inhibitors that will provide structural leads for new anti-cancer therapies. Additionally, this screen will also facilitate the identification pan HIF-1¿/ HIF-2¿ inhibitor that may have therapeutic application and provide new insights into novel mechanisms of HIF-1/2¿ is form specific regulation.