Abstract PR06: Dissecting mechanisms of replication fork stabilization in patient-derived high-grade serous organoid cultures and their impact on therapeutic sensitivity and the immune-tumor interaction
Authors: Sarah J. Hill, Patrick Lizotte, Nikolas Kesten, Neil S. Horowitz, Michael G. Muto, Michael J. Worley, Colleen M. Feltmate, Ross S. Berkowitz, Henry Long, Ursula A. Matulonis, Christopher P. Crum, Myles Brown, Alan D. D'Andrea
Journal: Clinical Cancer Research (2020)

Abstract Genomic analyses indicate that 50% of high-grade serous ovarian cancers (HGSC) harbor a genomic alteration in a DNA damage repair gene that may lead to functional defects. Using functional assays on patient-derived HGSC organoid cultures to test the capacity of the tumor cells to repair double-strand DNA breaks and to protect stalled replication forks, we have found that many HGSCs have stalled fork protection defects regardless of the genomic background of the tumor and that these defects correlate with sensitivity to replication stress inducing therapeutic agents. We hypothesized that gaining a better understanding of the mechanisms of replication fork instability and stability in HGSC organoid cultures would help to better understand the mechanisms of therapeutic sensitivity of the tumor cells. The purpose of this work is to understand how replication fork stabilization either in the primary tumor or through selection post-treatment leads to alterations in tumor cell biology, including therapeutic sensitivity and interaction of the tumor cells with the surrounding microenvironment. We utilized bulk RNA sequencing analysis of HGSC organoid cultures with varied replication fork protection capacity, some matched pairs of untreated and post-neoadjuvant tumors, to stratify differences in functional profiles in fork stable versus unstable tumors; we then used basic molecular biology techniques to understand the mechanisms of fork stabilization and how this stabilization affects the therapeutic sensitivity of the cells. We also developed and utilized multiple functional assays to assess the interaction of HGSC organoids of varying fork protection capacity with their immune microenvironment in different drug exposure settings. We identified multiple proteins that through either up- or downregulation lead to stabilization of replication forks in the tumor cells and found that the mechanisms of stabilization can occur at both the level of the replication fork and the overall transcriptional level of the cell and can alter the therapeutic sensitivity of the cells. We have determined that replication fork stability leads to increased mesenchymal characteristics in tumors and to decreased activation of the antitumor immune response within the cultures after treatment with DNA damage repair and immuno-oncologic (IO) agents. Overall, these results indicate that replication fork stabilization in HGSC through multiple different mechanisms can lead to altered interactions of the tumor cells with their microenvironment and altered therapeutic sensitivity. This abstract is also being presented as Poster B10. Citation Format: Sarah J. Hill, Patrick Lizotte, Nikolas Kesten, Neil S. Horowitz, Michael G. Muto, Michael J. Worley, Colleen M. Feltmate, Ross S. Berkowitz, Henry Long, Ursula A. Matulonis, Christopher P. Crum, Myles Brown, Alan D. D'Andrea. Dissecting mechanisms of replication fork stabilization in patient-derived high-grade serous organoid cultures and their impact on therapeutic sensitivity and the immune-tumor interaction [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR06.