Discovering modulators of PAX8 for targeting ovarian cancer

描述（由申请人提供）：我们最近对卵巢癌细胞系中必需基因的基因组规模调查以及对卵巢癌基因组的全面表征，明确了PAX 8转录因子的表达是大多数卵巢肿瘤的关键脆弱性。PAX 8是在雌性苗勒管发育期间和输卵管中表达的转录因子的配对盒家族的成员，输卵管被认为是卵巢癌的起源。PAX 8在大多数卵巢肿瘤或细胞系中过表达或扩增，这表明许多卵巢癌细胞已经增选PAX 8转录程序以维持其存活和增殖。我们发现PAX 8是卵巢癌细胞系存活所需的最高排名基因，但不是正常卵巢细胞或来自各种其他谱系的细胞。表征卵巢肿瘤这种新依赖性背后的潜在生物学机制需要PAX 8特异性化学探针，然而迄今为止尚未报道此类探针。该项目的重点是鉴定PAX 8活性的小分子抑制剂，其特异性抑制表达PAX 8的卵巢癌细胞的存活。这种抑制剂不仅有利于研究 卵巢癌对PAX 8成瘾背后的生物学机制，但也可能作为未来治疗发展途径的有用起点。为了鉴定PAX 8活性的抑制剂，我们已经开发并验证了384孔格式的基于细胞的Luc报告基因测定，其测量卵巢癌细胞中的PAX 8活性。我们选择了一种卵巢癌细胞系（从一组25个充分表征的卵巢癌细胞系中），该细胞系表达高水平的PAX 8，并且强烈依赖于PAX 8表达，因此与PAX 8生物学高度相关。抑制PAX 8活性的化合物将经历两次反筛选以去除：i）非特异性转录抑制剂; ii）Luc抑制剂;和iii）通常的细胞毒性化合物。然后，我们将表征优先降低PAX 8依赖性癌细胞相对于独立癌细胞的存活的化合物。然后将优化得分最高的化合物以实现所需的选择性、功效和效力。作为短期目标，我们将使用这些化合物作为探针来阐明PAX 8在卵巢肿瘤中的重要性的分子基础。与此同时，将使用基于质谱的蛋白质组学来鉴定直接结合靶点，从而提供关于PAX 8的上游调节因子和下游效应因子的重要知识。由于我们的初步工作表明PAX 8表达代表了一种明确的生物标志物，可预测卵巢肿瘤对PAX 8抑制的敏感性，因此我们设想在卵巢癌异种移植模型中使用这些PAX 8调节剂，因为我们致力于将这些发现转化为治疗机会的长期目标。

DESCRIPTION (provided by applicant): Our recent genome-scale survey of genes essential in ovarian cancer cell lines coupled with the comprehensive characterization of ovarian cancer genomes pinpointed the expression of the PAX8 transcription factor as a critical vulnerability of the majority of ovarian tumors. PAX8 is a member of the paired- box family of transcription factors expressed during the development of the female M¿llerian ducts and in the fallopian tube, which is considered the origin of ovarian carcinomas. PAX8 is overexpressed or amplified in most ovarian tumors or cell lines indicating that many ovarian cancer cells have co-opted PAX8 transcriptional program to maintain their survival and proliferation. We found that PAX8 was the highest ranked gene required for the survival of ovarian cancer cell lines but not normal ovarian cells or cells from a variety of other lineages. Characterizing the underlying biological mechanisms behind this novel dependency of ovarian tumors requires PAX8-specific chemical probes, however such probes have not been reported to date. This project focuses on the identification of small-molecule inhibitors of PAX8 activity that specifically inhibit the survivalof ovarian cancer cells expressing PAX8. Such inhibitors will not only facilitate the investigation of the biological mechanism behind the addiction of ovarian cancers to PAX8 but may also serve as useful starting points for future therapeutic avenues of development. To identify inhibitors of PAX8 activity we have developed and validated a cell-based Luc reporter gene assay in 384-well format that measures PAX8 activity in ovarian cancer cells. We selected an ovarian cancer cell line (from a panel of 25 well-characterized ovarian cancer cell lines) that expresses high levels of PAX8 and is strongly dependent on PAX8 expression, and thus is highly relevant to PAX8 biology. Compounds that suppress PAX8 activity will be subjected to two counter screens to remove: i) non-specific inhibitors of transcription; ii) Luc inhibitors; and iii) generally cytooxic compounds. We wil then characterize compounds that preferentially reduce the survival of PAX8-dependent versus independent cancer cells. Top scoring compounds will then be optimized to achieve the desired selectivity, efficacy and potency. As a short-term goal, we will use these compounds as probes to elucidate the molecular basis of PAX8 essentiality in ovarian tumors. In parallel, the direct binding targets will be identified using mass-spectrometry- based proteomics, providing important knowledge on upstream regulators and downstream effectors of PAX8. Since our preliminary work suggests that PAX8 expression represents a clear biomarker that predicts sensitivity of ovarian tumors to PAX8 suppression, we envision using these PAX8 modulators in xenograft models of ovarian cancer as we work towards a longer-term goal of translating these findings into therapeutic opportunities.