Ovarian carcinoma-associated mesenchymal stem cells (CA-MSC) produce not only high levels of IL6 but also the related cytokine leukemia inhibitory factor (LIF). Interleukin 6 (IL6) mediated activation of STAT3 is implicated as a critical therapeutic target for cancer therapy. Less is known about the role of LIF, which can similarly activate STAT3, in ovarian cancer. We therefore sought to evaluate the tumorigenic effects of CA-MSC paracrine LIF signaling and the redundancy of IL6 and LIF in activating ovarian cancer STAT3 mediated cancer growth. As expected, we found that both IL6 and LIF induce STAT3 phosphorylation in tumor cells. In addition, both IL6 and LIF increased the percentage of ALDH+ ovarian cancer stem-like cells (CSC). Supporting redundancy of function by the two cytokines, CA-MSC induced STAT3 phosphorylation and increased cancer cell ‘stemness’. This effect was not inhibited by LIF or IL6 blocking antibodies alone, but was prevented by dual IL6/LIF blockade or JAK2 inhibition. Similarly, small hairpin RNA (shRNA)-mediated reduction of IL6 or LIF in CA-MSC partially decreased but could not completely abrograte the ability of CA-MSC to induce STAT3 phosphorylation and stemness. Importantly, the in vivo pro-tumorigenic effect of CA-MSC is abrogated by dual blockade with the JAK2 inhibitor ruxolitinib to a much greater extent than treatment with anti-IL6 or anti-LIF antibody alone. Ruxolitinib treatment also improves survival in the immunocompetent ovarian cancer mouse model system with ID8 tumor cells plus MSC. Ruxolitinib-treated tumors in both the immunocompromised and immunocompetent animal models demonstrate decreased phospho-STAT3, indicating on-target activity. In conclusion, CA-MSC activate ovarian cancer cell STAT3 signaling via IL6 and LIF and increase tumorigenesis cancer stemness. This functional redundancy suggests that therapeutic targeting of a single cytokine may be less effective than strategies such as dual inhibitor therapy or targeting shared downstream factors of the JAK/STAT pathway.

The dopamine transporter (DAT) is a plasma membrane protein that mediates the reuptake of extracellular dopamine (DA) and controls the spatiotemporal dynamics of dopaminergic neurotransmission. The transporter is subject to fine control that tailors clearance of transmitter to physiological demands, and dysregulation of reuptake induced by psychostimulant drugs, transporter polymorphisms, and signaling defects may impact transmitter tone in disease states. We previously demonstrated that DAT undergoes complex regulation by palmitoylation, with acute inhibition of the modification leading to rapid reduction of transport activity, and sustained inhibition of the modification leading to transporter degradation and reduced expression. Here, to examine mechanisms and outcomes related to increased modification, we co-expressed DAT with palmitoyl acyltransferases (PATs), also known as DHHC enzymes, which catalyze palmitate addition to proteins. Of twelve PATs tested, DAT palmitoylation was stimulated by DHHC2, DHHC3, DHHC8, DHHC15, and DHHC17, with others having no effect. Increased modification was localized to previously identified palmitoylation site Cys580 and resulted in upregulation of transport kinetics and elevated transporter expression mediated by reduced degradation. These findings confirm palmitoylation as a regulator of multiple DAT properties crucial for appropriate DA homeostasis and identify several potential PAT pathways linked to these effects. Defects in palmitoylation processes thus represent possible mechanisms of transport imbalances in DA disorders.

DNA damage repair alterations play a critical role in ovarian cancer tumorigenesis. Mechanistic drivers of the DNA damage response consequently present opportunities for therapeutic targeting. The chromatin-binding DEK oncoprotein functions in DNA double-strand break repair. We therefore sought to determine the role of DEK in epithelial ovarian cancer. DEK is overexpressed in both primary epithelial ovarian cancers and ovarian cancer cell lines. To assess the impact of DEK expression levels on cell growth, small interfering RNA and short hairpin RNA approaches were utilized. Decreasing DEK expression in ovarian cancer cell lines slows cell growth and induces apoptosis and DNA damage. The biologic effects of DEK depletion are enhanced with concurrent chemotherapy treatment. The in vitro effects of DEK knockdown are reproduced in vivo, as DEK depletion in a mouse xenograft model results in slower tumor growth and smaller tumors compared to tumors expressing DEK. These findings provide a compelling rationale to target the DEK oncoprotein and its pathways as a therapeutic strategy for treating epithelial ovarian cancer.

Purpose: Current immunotherapy response rates in high-grade serous carcinoma (HGSC) of the ovary, fallopian tube, and peritoneum are 10-15%. We sought to determine if decreasing levels of the DNA damage repair protein DEK or inhibiting its downstream effector aurora kinase A (AURKA) induces type I interferon (IFN-I) signaling to improve the immune sensing of HGSC. Experimental Procedures: RNA-Seq analysis was performed on HGSC cell lines with stable expression of shRNA targeting DEK (shDEK) or control. Gene expression patterns were analyzed by gene set enrichment analysis and confirmed by RT-PCR. As AURKA/B were identified to be downregulated by shDEK, cell lines were treated with aurora kinase inhibitors and analyzed for DNA damage and apoptosis. IFN-I signature transcripts were analyzed by RT-PCR following shDEK therapy or aurora kinase inhibitor therapy compared to controls. Tumor-infiltrating lymphocyte (TIL) profiles in HGSC from The Cancer Genome Atlas (TCGA) were characterized. Using the ID8 immunocompetent ovarian cancer mouse model, IFN-I signature transcripts were quantified after transfection with siRNA targeting Dek or control without or with overexpression of the DNA damage sensing protein Sting. In vivo studies were performed by injecting ID8 cells intraperitoneally and then treating with the AURKA inhibitor alisertib, anti-PD-L1 antibody, combination therapy or control. TILs were analyzed by flow cytometry. Results: RNA-Seq analysis identified interferon-alpha response as an upregulated pathway in the setting of DEK deficiency. In vitro validation revealed that decreasing DEK levels increases IFN-I signaling. RNA-Seq analysis also showed decreased AURKA/B following shDEK treatment. A positive correlation between DEK and AURKA/B transcript levels was also found in primary patient samples. AURKA/B inhibitor therapy resulted in increased DNA damage and apoptosis, and increased IFN-I signature gene transcripts including IFNB1, IFNA4, ISG15, and MX1. TCGA analysis showed that elevated levels of IFN-I genes including chemokines CXCL9 and CXCL10 are correlated with TIL subsets essential for antitumor immunity. In ID8 cells, Dek-deficiency enhanced Sting-mediated induction of Ifnb1, Cxcl9, and Cxcl10. In the ID8 in vivo studies, AURKA inhibitor therapy resulted in increased TCRβ and TCRγδ TIL subpopulations. Combinatorial therapy animal studies are ongoing. Conclusions: Decreasing levels of the DNA damage repair protein DEK or inhibiting its effector AURKA/B induces DNA damage and increases IFN-I signaling in both HGSC cells lines and primary patient samples. Our TCGA analysis supports the hypothesis that IFN-I signaling is pivotal for the HGSC immunogenicity. Inhibition of AURKA in the ID8 mouse model system results in a shift in the immune phenotype, and further preclinical combinatorial studies are under way. Our results identify a new synthetic immune toxicity combination by priming HGSC with AURKA-targeted therapy with the goal of increasing immunotherapy responses. Citation Format: Danielle E. Bolland, Yuning Hao, Yee Sun Tan, Jake Reske, Lijun Tan, Ronald L. Chandler, Yuying Xie, Yu L. Lei, Karen McLean. Induction of DNA damage in high-grade serous carcinoma induces type I interferon signaling [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 B05.

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Antiestrogen therapy (AET) is an alternative to cytotoxic chemotherapy for recurrent ovarian cancer, yet the often short duration of response suggests mechanisms of resistance. We previously demonstrated that tumor microenvironment interleukin-6/leukemia inhibitory factor (IL6/LIF) cytokines induce tumor cell JAK-STAT signaling to promote cancer growth. Crosstalk between estrogen signaling and cytokine signaling has been reported. Therefore, we sought to characterize the impact of IL6/LIF signaling on estrogen signaling in epithelial ovarian cancer and investigate the efficacy of combination therapy. We first assessed patient tumors for cytokine expression and compared it with response to AET to determine clinical relevance. In vitro, we determined the effect of IL6/LIF on estrogen receptor expression and signaling. Cell viability assays were used to determine the efficacy and potential synergy of cytokine blockade and AET. We then extended studies to animal models, incorporating patient-derived stromal cells. Our results demonstrated shorter progression-free interval on AET in patients with stromal IL6/LIF expression. In vitro, IL6/LIF increased tumor cell estrogen receptor expression and signaling, and combination cytokine blockade and AET resulted in synergistic inhibition of tumor cell growth. The anticancer effect was verified in a mouse model. In conclusion, due to crosstalk between IL6/LIF cytokine signaling and estrogen signaling, dual blockade is a potential new treatment approach for ovarian cancer.

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