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不可少的基因进行了基因组规模的调查，并对卵巢癌基因组进行了全面的表征，发现PAX8转录因子的表达是大多数卵巢肿瘤的关键易损性。Pax8是转录因子配对盒家族中的一员，在女性Müllerian导管和输卵管中表达，被认为是卵巢癌的起源。Pax8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选择了PAX8转录程序来维持其生存和增殖。我们发现，PAX8是卵巢癌细胞系存活所需的最高等级的基因，但不是正常卵巢细胞或其他各种血统的细胞。要确定卵巢肿瘤这种新的依赖性背后的潜在生物学机制需要PAX8特异性的化学探针，然而到目前为止还没有报道过这样的探针。本项目致力于鉴定能特异性抑制表达PAX8的卵巢癌细胞存活的PAX8活性的小分子抑制剂。这种抑制剂不仅将有助于调查 卵巢癌对PAX8成瘾的生物学机制，但也可能作为未来治疗途径开发的有用起点。为了确定PAX8活性的抑制物，我们开发并验证了一种基于细胞的LUC报告基因分析方法，该方法以384孔的形式测量卵巢癌细胞中的PAX8活性。我们选择了一个卵巢癌细胞系(从25个特征良好的卵巢癌细胞系中)，它表达高水平的PAX8，并强烈依赖于PAX8的表达，因此与PAX8生物学高度相关。抑制PAX8活性的化合物将经过两个计数器筛选以去除：i)非特异性转录抑制物；ii)Luc抑制剂；以及iii)一般的细胞毒性化合物。然后，我们将对优先降低依赖PAX8的癌细胞与独立癌细胞的存活率的化合物进行表征。然后将对得分最高的化合物进行优化，以实现所需的选择性、有效性和效力。作为一个短期目标，我们将使用这些化合物作为探针来阐明PAX8在卵巢肿瘤中的重要性的分子基础。同时，将使用基于质谱学的蛋白质组学来确定直接结合靶标，从而提供有关PAX8上游调控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，PAX8的表达代表了一个明确的生物标记物，可以预测卵巢肿瘤对PAX8抑制的敏感性，我们设想在卵巢癌的异种移植模型中使用这些PAX8调节剂，以实现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.