Abstract A69: The PAX8 cistrome in benign and malignant Mullerian epithelia.

Background: PAX8 is a highly evolutionarily conserved transcription factor with essential roles in both Mullerian development as well as high grade serous ovarian cancer. While PAX8 has emerged as a common clinical tool to identify Mullerian primary tumors, the transcriptional network directed by PAX8 in Mullerian epithelia is unknown. In this study, we sought to understand how PAX8 interacts with chromatin in the context of both benign fallopian tube secretory epithelial cells as well as in high grade serous carcinomas. Methods: This study examined PAX8 using six cell lines: three immortalized fallopian tube secretory cell lines (FT33TAg, FT194, FT246) generated by our laboratory and three high grade serous ovarian cancer cell lines (KURAMOCHI, OVSAHO, and JHOS4) with high genomic fidelity to human tumors according to The Cancer Genome Atlas. First, we used chromatin immunoprecipitation combined with DNA sequencing (ChIP-Seq) to map PAX8 binding sites along the genome. Overlapping peaks among the cell lines were aligned to define a minimum consensus binding motif for PAX8. Unsupervised hierarchical clustering then segregated the peaks into groups. The nearest gene within 50000 base pairs to each peak was identified. Next, we used a series of RNAi experiments to knockdown PAX8 in all six cell lines using three independent siRNA sequences plus a pool and examined the effect on the transcriptome in each cell line using RNA-seq. Finally, we used pathway analysis software and gene ontology tools to map the signaling networks directed by PAX8. Results: Among the six cell lines, there were 11,361 unique PAX8 peaks by ChIP-seq. Approximately half of all PAX8 binding sites were located in intergenic sequences and about a 40% were in intronic sequences. The consensus DNA binding motif was almost identical between benign and malignant cell lines and was consistent with a predicted PAX homology motif. Unsupervised clustering identified 3 sets of peaks: 1) a highly conserved set of peaks among all six cell lines comprised of 558 peaks; 2) a common set of peaks common to benign cell lines but absent in cancer cell lines comprised of 3013 peaks; and 3) a very heterogeneous pattern of DNA binding unique to each cancer cell line ranging from few PAX8 peaks (JHOS4) to more than 3000 PAX8 peaks (both KURAMOCHI and OVSAHO). By gene ontology analysis, the common set of peaks for all six cell lines clustered around apoptosis and programmed cell death. Using the combination of RNAi with RNA-seq, few genes (ranging from 1 – 32 genes) were affected by loss of PAX8 in the fallopian tube cell lines or JHOS4, while more than 200 genes were altered in KURAMOCHI and OVSAHO. While in KURAMOCHI these genes were more likely to involve metabolic processes, in OVSAHO these genes were more likely to involve cell cycle regulation. Conclusions: PAX8 expression is shared among benign fallopian tube secretory cells and high grade serous carcinoma, but PAX8 function appears to be context dependent. While there is a shared set of PAX8 binding sites in benign and malignant Mullerian epithelia, PAX8 appears to be essential for the expression of very few targets in benign cells compared to cancer. In addition, PAX8 appears to have greater access to chromatin in the setting of cancer as evidenced by an increase in the number of PAX8 binding sites, while at the same time losing affinity for a set of binding sites seen only in benign fallopian tube cells. Together, these data suggest a dynamic reorganization of chromatin in the process of malignant transformation. Citation Format: Kevin M. Elias, Megan Emori, Myles Brown, Ronny Drapkin. The PAX8 cistrome in benign and malignant Mullerian epithelia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A69.