Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-19-2558 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 Murine Oviductal High-grade Serous Carcinomas Mirror the Genomic Alterations, Gene Expression Profiles and Immune Microenvironment of Their Human Counterparts Kevin McCool*1, Zachary T. Freeman*2,8, Yali Zhai*3, Rong Wu3, Kevin Hu4, Chia-Jen Liu3, Scott A. Tomlins3,8, Eric R. Fearon3,5,6,8, Brian Magnuson7,8, Rork Kuick7,8, and Kathleen R. Cho3,6,8 *Equal contribution Departments of Obstetrics and Gynecology1, Unit for Laboratory Animal Medicine2, Pathology3, Computational Medicine and Bioinformatics4, Human Genetics5, and Internal Medicine6, University of Michigan Medical School, and Department of Biostatistics7, University of Michigan School of Public Health, and the Rogel Cancer Center8, University of Michigan, United States Running Title: Integrated Molecular Analysis of Murine Serous Carcinomas Corresponding Author: Kathleen R. Cho, 1504 BSRB, 109 Zina Pitcher Place, University of Michigan, Ann Arbor, MI 48109-2200; email: [email protected]; phone: 734-615-0323. Conflict of Interest Disclosure: S.A. Tomlins is a prior consultant and current employee at Strata Oncology and reports receiving other commercial research support from Compendia Bioscience/Life Technologies/Thermo Fisher Scientific. The other authors have no potential conflicts of interest to disclose. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-19-2558 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 2 Abstract Robust pre-clinical models of ovarian high-grade serous carcinoma (HGSC) are needed to advance our understanding of HGSC pathogenesis and to test novel strategies aimed at improving clinical outcomes for women with the disease. Genetically engineered mouse models of HGSC recapitulating the likely cell of origin (fallopian tube), underlying genetic defects, histology, and biologic behavior of human HGSCs have been developed. However, the degree to which the mouse tumors acquire the somatic genomic changes, gene expression profiles, and immune microenvironment that characterize human HGSCs remains unclear. We used integrated molecular characterization of oviductal HGSCs arising in the context of Brca1, Trp53, Rb1, and Nf1 (BPRN) inactivation to determine whether the mouse tumors recapitulate human HGSCs across multiple domains of molecular features. Targeted DNA sequencing showed the mouse BPRN tumors, but not endometrioid carcinoma-like tumors based on different genetic defects (e.g., Apc and Pten), acquire somatic mutations and widespread copy number alterations similar to those observed in human HGSCs. RNA sequencing showed the mouse HGSCs most closely resemble the so-called immunoreactive and mesenchymal subsets of human HGSCs. A combined immuno-genomic analysis demonstrated the immune microenvironment of BPRN tumors models key aspects of tumor-immune dynamics in the immunoreactive and mesenchymal subtypes of human HGSC, with enrichment of immunosuppressive cell subsets such as myeloid derived suppressor cells and regulatory T cells. The findings further validate the BPRN model as a robust pre-clinical experimental platform to address current barriers to improved prevention, diagnosis, and treatment of this often lethal cancer. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-19-2558 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 3 Statement of Significance: The acquired gene mutations, broad genomic alterations, and gene expression and immune cell-tumor axis changes in a mouse model of oviductal serous carcinoma closely mirror those of human tubo-ovarian high-grade serous carcinoma. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-19-2558 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 4 Introduction Ovarian high-grade serous carcinoma (HGSC) is the most lethal gynecologic malignancy, and fifth most common cause of cancer death of women in the United States (1). Most women with HGSC present with advanced stage disease, and despite maximal surgical effort combined with chemotherapy, over 75% will experience recurrence and die of their disease (2). These challenges highlight the need for high-fidelity pre-clinical models to advance our understanding of HGSC pathogenesis and to test novel strategies aimed at improving prevention, early diagnosis, and treatment of HGSC. Initial genetically engineered mouse models (GEMMs) of HGSC were based on the assumption that the tumors arise from the ovarian surface epithelium (3,4). However, an expanding body of literature strongly supports the current view that many, if not most HGSCs arise from epithelial cells in the fallopian tube, preferentially in the tubal fimbriae (5-7). As a consequence, more recent HGSC GEMMs have been developed based on transformation of fallopian tube (oviductal) epithelial cells in the mouse (8-10). While tumors arising in these models recapitulate many histopathologic features of human HGSCs, the extent to which the HGSC GEMMs manifest the molecular features of their human tumor counterparts beyond the specific driver genetic lesions instigating tumorigenesis in the models, is unclear. Data from The Cancer Genome Atlas (TCGA) revealed human HGSCs harbor nearly universal mutations in the TP53 tumor suppressor gene, but show recurrent somatic mutations in only a limited collection of additional key tumor suppressor genes (TSGs) such as BRCA1, BRCA2, RB1, NF1, and CDK12 (11). We have previously established a GEMM of HGSC that uses the Ovgp1 promoter to drive expression of a tamoxifen-inducible Cre recombinase (iCre-ERT2) to inactivate various combinations of the Brca1, Trp53, Rb1, and Nf1 TSGs selectively in epithelial cells of the murine oviduct (9). Tumors arising in this so-called BPRN model recapitulate the histopathology of human HGSC and other key features of the human disease, including progression from intraepithelial precursor lesions that closely mimic human serous tubal Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-19-2558 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 5 intraepithelial carcinomas (STICs). While tumor development in BPRN mice is highly penetrant, tumors typically develop after a lengthy latency period of several months. The latency period likely allows each individual tumor to select for the additional genetic alterations that are needed for tumor progression and development, beyond the instigating lesions. Integrated genomic and transcriptomic analysis of TCGA datasets has identified other key molecular features of human ovarian HGSC beyond ubiquitous TP53 mutations. Notably, HGSCs are also characterized by structural genomic instability and a diverse range of resultant widespread copy number alterations, amplifications, deletions, and gene breakage events that may play key roles in the phenotypes of advanced cancers (11-13). Further, based on their gene expression profiles, human HGSCs can be classified into four molecular subtypes: differentiated, immunoreactive, mesenchymal, and proliferative (11). The mesenchymal subtype in particular has been associated with worse clinical outcomes, including lower rates of optimal surgical cytoreduction, higher rates of chemotherapy-resistant disease, and shorter progression- free and overall survival (14,15). In human HGSCs, a high degree of tumor-infiltrating lymphocytes (TILs) has been associated with improved prognosis (16,17), but there are immunosuppressive factors in the tumor microenvironment that can impair TIL infiltration and activity (18). A recent study of human HGSC samples identified significant tumor-immune microenvironment heterogeneity in tumors before adjuvant chemotherapy. Post-treatment samples showed augmentation of pre-existing TIL responses, but no treatment-associated relief of major immune-suppressive mechanisms (19). These findings, together with the rather disappointing clinical response of HGSC patients to currently available immune checkpoint therapies, highlights the complexity of tumor-immune dynamics in HGSC and the need for better in vivo models to clarify the key factors and mechanisms regulating the immune microenvironment in HGSC (20). There is optimism that Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 5, 2019; DOI: 10.1158/0008-5472.CAN-19-2558 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 6 GEMMs can provide tractable and immunologically relevant model systems with which to better understand the immune response to human HGSC before and after therapy (21). Most extant pre-clinical