Targeting BRPF3 moderately reverses olaparib resistance in high grade serous ovarian carcinoma.

PARP inhibitors (PARPi) kill cancer cells by stalling DNA replication and preventing DNA repair, resulting in a critical accumulation of DNA damage. Resistance to PARPi is a growing clinical problem in the treatment of high grade serous ovarian carcinoma (HGSOC). Acetylation of histone H3 lysine 14 (H3K14ac) and associated histone acetyltransferases (HATs) and epigenetic readers have known functions in DNA repair and replication. Our objectives are to examine their expression and activities in the context of PARPi-resistant HGSOC, and to determine if targeting H3K14ac or associated proteins has therapeutic potential. Using mass spectrometry profiling of histone modifications, we observed increased H3K14ac enrichment in PARPi-resistant HGSOC cells relative to isogenic PARPi-sensitive lines. By reverse-transcriptase quantitative PCR and RNA-seq, we also observed altered expression of numerous HATs in PARPi-resistant HGSOC cells and a PARPi-resistant PDX model. Knockdown of HATs only modestly altered PARPi response, although knockdown and inhibition of PCAF significantly increased resistance. Pharmacologic inhibition of HBO1 depleted H3K14ac but did not affect PARPi response. However, knockdown and inhibition of BRPF3, a bromodomain and PHD-finger containing protein that is known to interact in a complex with HBO1, did reduce PARPi resistance. This study demonstrates that depletion of H3K14ac does not affect PARPi response in HGSOC. Our data suggest that the bromodomain function of HAT proteins, such as PCAF, or accessory proteins, such as BRPF3, may play a more direct role compared to direct HATs function in PARPi response.

Wnt Signaling in Gynecologic Malignancies.

Gynecologic malignancies, including ovarian cancer, endometrial cancer, and cervical cancer, affect hundreds of thousands of women worldwide every year. Wnt signaling, specifically Wnt/ß-catenin signaling, has been found to play an essential role in many oncogenic processes in gynecologic malignancies, including tumorigenesis, metastasis, recurrence, and chemotherapy resistance. As such, the Wnt/ß-catenin signaling pathway has the potential to be a target for effective treatment, improving patient outcomes. In this review, we discuss the evidence supporting the importance of the Wnt signaling pathways in the development, progression, and treatment of gynecologic malignancies.

Activation of Wnt signaling promotes olaparib resistant ovarian cancer.

Epithelial ovarian cancer (EOC) has one of the highest death to incidence ratios among all cancers. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype due to the lack of therapeutic options following debulking surgery and platinum/taxane-based chemotherapies. For recurrent chemosensitive HGSOC, poly(ADP)-ribose polymerase inhibitors (PARPi; olaparib, rucaparib, or niraparib) represent an emerging treatment strategy. While PARPi are most effective in homologous recombination DNA repair-deficient (HRD) HGSOCs, recent studies have observed a significant benefit in non-HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance. Therefore, there is an urgent clinical need to understand PARPi resistance and to introduce novel combinatorial therapies to manage PARPi resistance and extend HGSOC disease-free intervals. In a panel of HGSOC cell lines, we established matched olaparib sensitive and resistant cells. Transcriptome analysis of the matched olaparib-sensitive vs -resistant cells revealed activation of the Wnt signaling pathway and consequently increased TCF transcriptional activity in PARPi-resistant cells. Forced activation of canonical Wnt signaling in several PARPi-sensitive cells via WNT3A reduced olaparib and rucaparib sensitivity. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA-approved compound, pyrvinium pamoate, which has been shown to promote downregulation of ß-catenin. In both an HGSOC cell line and a patient-derived xenograft model, we observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARP and Wnt inhibitors.

Neuropilin-1 promotes Hedgehog signaling through a novel cytoplasmic motif.

Hedgehog (HH) signaling critically regulates embryonic and postnatal development as well as adult tissue homeostasis, and its perturbation can lead to developmental disorders, birth defects, and cancers. Neuropilins (NRPs), which have well-defined roles in Semaphorin and VEGF signaling, positively regulate HH pathway function, although their mechanism of action in HH signaling remains unclear. Here, using luciferase-based reporter assays, we provide evidence that NRP1 regulates HH signaling specifically at the level of GLI transcriptional activator function. Moreover, we show that NRP1 localization to the primary cilium, a key platform for HH signal transduction, does not correlate with HH signal promotion. Rather, a structure-function analysis suggests that the NRP1 cytoplasmic and transmembrane domains are necessary and sufficient to regulate HH pathway activity. Furthermore, we identify a previously uncharacterized, 12-amino acid region within the NRP1 cytoplasmic domain that mediates HH signal promotion. Overall, our results provide mechanistic insight into NRP1 function within and potentially beyond the HH signaling pathway. These insights have implications for the development of novel modulators of HH-driven developmental disorders and diseases.

Naturally occurring canine sarcomas: Bridging the gap from mouse models to human patients through cross-disciplinary research partnerships.

Fueled by support from the National Cancer Institute's "Cancer Moonshot" program, the past few years have witnessed a renewed interest in the canine spontaneous cancer model as an invaluable resource in translational oncology research. Increasingly, there is awareness that pet dogs with cancer provide an accessible bridge to improving the efficiency of cancer drug discovery and clinical therapeutic development. Canine tumors share many biological, genetic, and histologic features with their human tumor counterparts, and most importantly, retain the complexities of naturally occurring drug resistance, metastasis, and tumor-host immune interactions, all of which are difficult to recapitulate in induced or genetically engineered murine tumor models. The utility of canine models has been particularly apparent in sarcoma research, where the increased incidence of sarcomas in dogs as compared to people has facilitated comparative research resulting in treatment advances benefitting both species. Although there is an increasing awareness of the advantages in using spontaneous canine sarcoma models for research, these models remain underutilized, in part due to a lack of more permanent institutional and cross-institutional infrastructure to support partnerships between veterinary and human clinician-scientists. In this review, we provide an updated overview of historical and current applications of spontaneously occurring canine tumor models in sarcoma research, with particular attention to knowledge gaps, limitations, and growth opportunities within these applications. Furthermore, we propose considerations for working within existing veterinary translational and comparative oncology research infrastructures to maximize the benefit of partnerships between veterinary and human biomedical researchers within and across institutions to improve the utility and application of spontaneous canine sarcomas in translational oncology research.

ATF6-Mediated Signaling Contributes to PARP Inhibitor Resistance in Ovarian Cancer.

High-grade serous ovarian cancer (HGSOC) is the deadliest ovarian cancer histotype due in-part to the lack of therapeutic options for chemotherapy-resistant disease. PARP inhibitors (PARPi) represent a targeted treatment. However, PARPi resistance is becoming a significant clinical challenge. There is an urgent need to overcome resistance mechanisms to extend disease-free intervals. We established isogeneic PARPi-sensitive and -resistant HGSOC cell lines. In three PARPi-resistant models, there is a significant increase in AP-1 transcriptional activity and DNA repair capacity. Using RNA-sequencing and an shRNA screen, we identified activating transcription factor 6 (ATF6) as a mediator of AP-1 activity, DNA damage response, and PARPi resistance. In publicly available datasets, ATF6 expression is elevated in HGSOC and portends a poorer recurrence-free survival. In a cohort of primary HGSOC tumors, higher ATF6 expression significantly correlated to PARPi resistance. In PARPi-resistant cell lines and a PDX model, inhibition of a known ATF6 regulator, p38, attenuated AP-1 activity and RAD51 foci formation, enhanced DNA damage, significantly inhibited tumor burden, and reduced accumulation of nuclear ATF6. IMPLICATIONS: This study highlights that a novel p38-ATF6-mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors.

Targeting DUSP Activity as a Treatment for High-Grade Serous Ovarian Carcinoma.

Identifying novel, durable treatments for high-grade serous ovarian cancer (HGSOC) is paramount to extend both progression-free survival (PFS) and overall survival (OS) in patients afflicted with this disease. Dual-specificity phosphatase 1 (DUSP1) was identified as one of seven genes that may significantly affect prognosis in patients with HGSOC; however, the role of DUSP inhibition (DUSPi) in the treatment of HGSOC remains largely unknown. In this study, we show that DUSP1 is highly expressed in HGSOC and confers worse PFS and OS. Further, we corroborate data that show DUSP1 expression is directly associated with therapy resistance. Using a tissue microarray of 137 different serous ovarian carcinomas, we demonstrate the high expression of DUSP1 in primary and recurrent serous ovarian cancer. In both acquired and de novo therapy HGSOC-resistant models, DUSPi both inhibited cellular proliferation and promoted cell death. RPPA analysis of HGSOC cells revealed DUSPi led to the differential regulation of several pathways, including AMPK and mTORC. Further, in a patient-derived xenograft HGSOC model, DUSPi significantly inhibited tumor progression.

Plexins promote Hedgehog signaling through their cytoplasmic GAP activity.

Hedgehog signaling controls tissue patterning during embryonic and postnatal development and continues to play important roles throughout life. Characterizing the full complement of Hedgehog pathway components is essential to understanding its wide-ranging functions. Previous work has identified neuropilins, established semaphorin receptors, as positive regulators of Hedgehog signaling. Neuropilins require plexin co-receptors to mediate semaphorin signaling, but the role of plexins in Hedgehog signaling has not yet been explored. Here, we provide evidence that multiple plexins promote Hedgehog signaling in NIH/3T3 mouse fibroblasts and that plexin loss of function in these cells results in significantly reduced Hedgehog pathway activity. Catalytic activity of the plexin GTPase-activating protein (GAP) domain is required for Hedgehog signal promotion, and constitutive activation of the GAP domain further amplifies Hedgehog signaling. Additionally, we demonstrate that plexins promote Hedgehog signaling at the level of GLI transcription factors and that this promotion requires intact primary cilia. Finally, we find that plexin loss of function significantly reduces the response to Hedgehog pathway activation in the mouse dentate gyrus. Together, these data identify plexins as novel components of the Hedgehog pathway and provide insight into their mechanism of action.

Targeting Fatty Acid Oxidation to Promote Anoikis and Inhibit Ovarian Cancer Progression.

Epithelial-derived high-grade serous ovarian cancer (HGSOC) is the deadliest gynecologic malignancy. Roughly 80% of patients are diagnosed with late-stage disease, which is defined by wide-spread cancer dissemination throughout the pelvic and peritoneal cavities. HGSOC dissemination is dependent on tumor cells acquiring the ability to resist anoikis (apoptosis triggered by cell detachment). Epithelial cell detachment from the underlying basement membrane or extracellular matrix leads to cellular stress, including nutrient deprivation. In this report, we examined the contribution of fatty acid oxidation (FAO) in supporting anoikis resistance. We examined expression Carnitine Palmitoyltransferase 1A (CPT1A) in a panel of HGSOC cell lines cultured in adherent and suspension conditions. With CPT1A knockdown cells, we evaluated anoikis by caspase 3/7 activity, cleaved caspase 3 immunofluorescence, flow cytometry, and colony formation. We assessed CPT1A-dependent mitochondrial activity and tested the effect of exogenous oleic acid on anoikis and mitochondrial activity. In a patient-derived xenograft model, we administered etomoxir, an FAO inhibitor, and/or platinum-based chemotherapy. CPT1A is overexpressed in HGSOC, correlates with poor overall survival, and is upregulated in HGSOC cells cultured in suspension. CPT1A knockdown promoted anoikis and reduced viability of cells cultured in suspension. HGSOC cells in suspension culture are dependent on CPT1A for mitochondrial activity. In a patient-derived xenograft model of HGSOC, etomoxir significantly inhibited tumor progression. IMPLICATIONS: Targeting FAO in HGSOC to promote anoikis and attenuate dissemination is a potential approach to promote a more durable antitumor response and improve patient outcomes.

Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma.

BACKGROUND: Euchromatic histone-lysine-N-methyltransferases 1 and 2 (EHMT1/2, aka GLP/G9A) catalyze dimethylation of histone H3 lysine 9 (H3K9me2) and have roles in epigenetic silencing of gene expression. EHMT1/2 also have direct roles in DNA repair and are implicated in chemoresistance in several cancers. Resistance to chemotherapy and PARP inhibitors (PARPi) is a major cause of mortality in high-grade serous ovarian carcinoma (HGSOC), but the contribution of the epigenetic landscape is unknown. RESULTS: To identify epigenetic mechanisms of PARPi resistance in HGSOC, we utilized unbiased exploratory techniques, including RNA-Seq and mass spectrometry profiling of histone modifications. Compared to sensitive cells, PARPi-resistant HGSOC cells display a global increase of H3K9me2 accompanied by overexpression of EHMT1/2. EHMT1/2 overexpression was also observed in a PARPi-resistant in vivo patient-derived xenograft (PDX) model. Genetic or pharmacologic disruption of EHMT1/2 sensitizes HGSOC cells to PARPi. Cell death assays demonstrate that EHMT1/2 disruption does not increase PARPi-induced apoptosis. Functional DNA repair assays show that disruption of EHMT1/2 ablates homologous recombination (HR) and non-homologous end joining (NHEJ), while immunofluorescent staining of phosphorylated histone H2AX shows large increases in DNA damage. Propidium iodide staining and flow cytometry analysis of cell cycle show that PARPi treatment increases the proportion of PARPi-resistant cells in S and G2 phases, while cells treated with an EHMT1/2 inhibitor remain in G1. Co-treatment with PARPi and EHMT1/2 inhibitor produces an intermediate phenotype. Immunoblot of cell cycle regulators shows that combined EHMT1/2 and PARP inhibition reduces expression of specific cyclins and phosphorylation of mitotic markers. These data suggest DNA damage and altered cell cycle regulation as mechanisms of sensitization. RNA-Seq of PARPi-resistant cells treated with EHMT1/2 inhibitor showed significant gene expression changes enriched in pro-survival pathways that remain unexplored in the context of PARPi resistance, including PI3K, AKT, and mTOR. CONCLUSIONS: This study demonstrates that disrupting EHMT1/2 sensitizes HGSOC cells to PARPi, and suggests a potential mechanism through DNA damage and cell cycle dysregulation. RNA-Seq identifies several unexplored pathways that may alter PARPi resistance. Further study of EHMT1/2 and regulated genes will facilitate development of novel therapeutic strategies to successfully treat HGSOC.