Combining EHMT and PARP Inhibition: A Strategy to Diminish Therapy-Resistant Ovarian Cancer Tumor Growth while Stimulating Immune Activation.

Despite the success of Poly-ADP-ribose polymerase inhibitors (PARPi) in the clinic, high rates of resistance to PARPi presents a challenge in the treatment of ovarian cancer, thus it is imperative to find therapeutic strategies to combat PARPi resistance. Here, we demonstrate that inhibition of epigenetic modifiers Euchromatic histone lysine methyltransferases 1/2 (EHMT1/2) reduces the growth of multiple PARPi-resistant ovarian cancer cell lines and tumor growth in a PARPi-resistant mouse model of ovarian cancer. We found that combinatory EHMT and PARP inhibition increases immunostimulatory dsRNA formation and elicits several immune signaling pathways in vitro. Using epigenomic profiling and transcriptomics, we found that EHMT2 is bound to transposable elements, and that EHMT inhibition leads to genome-wide epigenetic and transcriptional derepression of transposable elements. We validated EHMT-mediated activation of immune signaling and upregulation of transposable element transcripts in patient-derived, therapy-naïve, primary ovarian tumors, suggesting potential efficacy in PARPi-sensitive disease as well. Importantly, using multispectral immunohistochemistry, we discovered that combinatory therapy increased CD8 T cell activity in the tumor microenvironment of the same patient-derived tissues. In a PARPi-resistant syngeneic murine model, EHMT and PARP inhibition combination inhibited tumor progression and increased Granzyme B+ cells in the tumor. Together, our results provide evidence that combinatory EHMT and PARP inhibition stimulates a cell autologous immune response in vitro, is an effective therapy to reduce PARPi resistant ovarian tumor growth in vivo, and promotes anti-tumor immunity activity in the tumor microenvironment of patient-derived ex vivo tissues of ovarian cancer.

VDX-111, a novel small molecule, induces necroptosis to inhibit ovarian cancer progression.

Epithelial ovarian cancers that are nonhomologous recombination deficient, as well as those that are recurrent and in a platinum-resistant state, have limited therapeutic options. The objectives of this study were to characterize the mechanism of action and investigate the therapeutic potential of a small molecule, VDX-111, against ovarian cancer. We examined the ability of VDX-111 to inhibit the growth of a panel of ovarian cancer cell lines, focusing on BRCA wild-type lines. We found that VDX-111 causes a dose-dependent loss of cell viability across ovarian cancer cell lines. Reverse phase protein array (RPPA) analysis was used to identify changes in cell signaling in response to VDX-111 treatment. An RPPA analysis performed on cells treated with VDX-111 detected changes in cell signaling related to autophagy and necroptosis. Immunoblots of OVCAR3 and SNU8 cells confirmed a dose-dependent increase in LC3A/B and RIPK1. Incucyte live cell imaging was used to measure cell proliferation and death in response to VDX-111 alone and with inhibitors of apoptosis, necroptosis, and autophagy. Annexin/PI assays suggested predominantly nonapoptotic cell death, while real-time kinetic imaging of cell growth indicated the necroptosis inhibitor, necrostatin-1, attenuates VDX-111-induced loss of cell viability, suggesting a necroptosis-dependent mechanism. Furthermore, VDX-111 inhibited tumor growth in patient-derived xenograft and syngeneic murine models. In conclusion, the cytotoxic effects of VDX-111 seen in vitro and in vivo appear to occur in a necroptosis-dependent manner and may promote an antitumor immune response.

CASC4/GOLM2 drives high grade serous carcinoma anoikis resistance through the recycling of EGFR.

Ovarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.

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.

Novel chromobox 2 inhibitory peptide decreases tumor progression.

BACKGROUND: The Polycomb Repressor Complex 1 (PRC1) is an epigenetic regulator of differentiation and development, consisting of multiple subunits including RING1, BMI1, and Chromobox. The composition of PRC1 dictates its function and aberrant expression of specific subunits contributes to several diseases including cancer. Specifically, the reader protein Chromobox2 (CBX2) recognizes the repressive modifications including histone H3 lysine 27 tri-methylation (H3K27me3) and H3 lysine 9 dimethylation (H3K9me2). CBX2 is overexpressed in several cancers compared to the non-transformed cell counterparts, it promotes both cancer progression and chemotherapy resistance. Thus, inhibiting the reader function of CBX2 is an attractive and unique anti-cancer approach. RESEARCH DESIGN & METHODS: Compared with other CBX family members, CBX2 has a unique A/T-hook DNA binding domain that is juxtaposed to the chromodomain (CD). Using a computational approach, we constructed a homology model of CBX2 encompassing the CD and A/T hook domain. We used the model as a basis for peptide design and identified blocking peptides that are predicted to directly bind the CD and A/T-hook regions of CBX2. These peptides were tested in vitro and in vivo models. CONCLUSION: The CBX2 blocking peptide significantly inhibited both 2D and 3D growth of ovarian cancer cells, downregulated a CBX2 target gene, and blunted tumor growth in vivo.

Combinatory EHMT and PARP inhibition induces an interferon response and a CD8 T cell-dependent tumor regression in PARP inhibitor-resistant models.

Euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/2), which catalyze demethylation of histone H3 lysine 9 (H3K9me2), contribute to tumorigenesis and therapy resistance through unknown mechanisms of action. In ovarian cancer, EHMT1/2 and H3K9me2 are directly linked to acquired resistance to poly-ADP-ribose polymerase (PARP) inhibitors and are correlated with poor clinical outcomes. Using a combination of experimental and bioinformatic analyses in several PARP inhibitor resistant ovarian cancer models, we demonstrate that combinatory inhibition of EHMT and PARP is effective in treating PARP inhibitor resistant ovarian cancers. Our in vitro studies show that combinatory therapy reactivates transposable elements, increases immunostimulatory dsRNA formation, and elicits several immune signaling pathways. Our in vivo studies show that both single inhibition of EHMT and combinatory inhibition of EHMT and PARP reduces tumor burden, and that this reduction is dependent on CD8 T cells. Together, our results uncover a direct mechanism by which EHMT inhibition helps to overcome PARP inhibitor resistance and shows how an epigenetic therapy can be used to enhance anti-tumor immunity and address therapy resistance.

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.

Distinct roles of treatment schemes and BRCA2 on the restoration of homologous recombination DNA repair and PARP inhibitor resistance in ovarian cancer.

Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a major advance in ovarian cancer, now as a treatment and as a maintenance therapy in the upfront and recurrent settings. However, patients often develop resistance to PARPis, underlining the importance of dissecting resistance mechanisms. Here, we report different dosing/timing schemes of PARPi treatment in BRCA2-mutant PEO1 cells, resulting in the simultaneous development of distinct resistance mechanisms. PARPi-resistant variants PEO1/OlaJR, established by higher initial doses and short-term PARPi treatment, develops PARPi resistance by rapidly restoring functional BRCA2 and promoting drug efflux activity. In contrast, PEO1/OlaR, developed by lower initial doses with long-term PARPi exposure, shows no regained BRCA2 function but a mesenchymal-like phenotype with greater invasion ability, and exhibits activated ATR/CHK1 and suppressed EZH2/MUS81 signaling cascades to regain HR repair and fork stabilization, respectively. Our study suggests that PARPi resistance mechanisms can be governed by treatment strategies and have a molecular basis on BRCA2 functionality. Further, we define different mechanisms that may serve as useful biomarkers to assess subsequent treatment strategies in PARPi-resistant ovarian cancer.

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.

Loss of Claudin-4 Reduces DNA Damage Repair and Increases Sensitivity to PARP Inhibitors.

High-grade serous ovarian cancer is the deadliest gynecologic malignancy due to progression to resistant disease. Claudin-4 is classically defined as a tight junction protein and is often associated with epithelial cancers. Claudin-4 is aberrantly expressed in nearly 70% of all ovarian cancer tumors and conveys a worse overall prognosis. Elevated claudin-4 expression correlates to increased DNA repair activity and resistance to DNA damaging agents. PARP inhibitors are emerging as an effective therapeutic option for patients with ovarian cancer and function by promoting DNA damage. The study examines the relationship between claudin-4 expression and the response to PARP inhibitors using both genetic and pharmacologic inhibition of claudin-4 in in vitro and ex vivo models of ovarian cancer to examine DNA repair markers and functional activity. Genetic inhibition of claudin-4 results in the downregulation of several DNA damage repair effectors, including 53BP1 and XRCC1. Claudin-4 knockdown did not change homology-directed repair but inhibited nonhomologous end-joining and reduced 53BP1 foci formation. In 15 primary ovarian cancer tumors, higher claudin-4 expression significantly correlated to a dampened PARP inhibitor-mediated antiproliferation response. Further, claudin-4 inhibition in high claudin-4 tumors sensitized tumor sections to PARP inhibition. These data highlight that claudin-4 expression in ovarian cancer tumors could serve as both a marker of PARP inhibitor response and a therapeutic target to improve PARP inhibitor response.

The Capacity of the Ovarian Cancer Tumor Microenvironment to Integrate Inflammation Signaling Conveys a Shorter Disease-free Interval.

PURPOSE: Ovarian cancer has one of the highest deaths to incidence ratios across all cancers. Initial chemotherapy is effective, but most patients develop chemoresistant disease. Mechanisms driving clinical chemo-response or -resistance are not well-understood. However, achieving optimal surgical cytoreduction improves survival, and cytoreduction is improved by neoadjuvant chemotherapy (NACT). NACT offers a window to profile pre- versus post-NACT tumors, which we used to identify chemotherapy-induced changes to the tumor microenvironment. EXPERIMENTAL DESIGN: We obtained matched pre- and post-NACT archival tumor tissues from patients with high-grade serous ovarian cancer (patient, n = 6). We measured mRNA levels of 770 genes (756 genes/14 housekeeping genes, NanoString Technologies), and performed reverse phase protein array (RPPA) on a subset of matched tumors. We examined cytokine levels in pre-NACT ascites samples (n = 39) by ELISAs. A tissue microarray with 128 annotated ovarian tumors expanded the transcriptional, RPPA, and cytokine data by multispectral IHC. RESULTS: The most upregulated gene post-NACT was IL6 (16.79-fold). RPPA data were concordant with mRNA, consistent with elevated immune infiltration. Elevated IL6 in pre-NACT ascites specimens correlated with a shorter time to recurrence. Integrating NanoString (n = 12), RPPA (n = 4), and cytokine (n = 39) studies identified an activated inflammatory signaling network and induced IL6 and IER3 (immediate early response 3) post-NACT, associated with poor chemo-response and time to recurrence. CONCLUSIONS: Multiomics profiling of ovarian tumor samples pre- and post-NACT provides unique insight into chemo-induced changes to the tumor microenvironment. We identified a novel IL6/IER3 signaling axis that may drive chemoresistance and disease recurrence.

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.

Amplification of the Mutation-Carrying BRCA2 Allele Promotes RAD51 Loading and PARP Inhibitor Resistance in the Absence of Reversion Mutations.

Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in homologous recombination (HR) DNA repair and deleterious mutations in BRCA2 confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the BRCA2 gene. In this study, we generated olaparib- and rucaparib-resistant BRCA2-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant BRCA2 allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of BRCA2 or DOT1L was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a BRCA2 reversion, mutation amplification of a mutant-carrying BRCA2 contributes to PARP inhibitor resistance.

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.

Wnt family member 4 (WNT4) and WNT3A activate cell-autonomous Wnt signaling independent of porcupine O-acyltransferase or Wnt secretion.

Porcupine O-acyltransferase (PORCN) is considered essential for Wnt secretion and signaling. However, we observed that PORCN inhibition does not phenocopy the effects of WNT4 knockdown in WNT4-dependent breast cancer cells. This suggests a unique relationship between PORCN and WNT4 signaling. To examine the role of PORCN in WNT4 signaling, here we overexpressed WNT4 or WNT3A in breast cancer, ovarian cancer, and fibrosarcoma cell lines. Conditioned media from these lines and co-culture systems were used to assess the dependence of Wnt secretion and activity on the critical Wnt secretion proteins PORCN and Wnt ligand secretion (WLS) mediator. We observed that WLS is universally required for Wnt secretion and paracrine signaling. In contrast, the dependence of WNT3A secretion and activity on PORCN varied across the cell lines, and WNT4 secretion was PORCN-independent in all models. Surprisingly, WNT4 did not exhibit paracrine activity in any tested context. Absent the expected paracrine activity of secreted WNT4, we identified cell-autonomous Wnt signaling activation by WNT4 and WNT3A, independent of PORCN or Wnt secretion. The PORCN-independent, cell-autonomous Wnt signaling demonstrated here may be critical in WNT4-driven cellular contexts or in those that are considered to have dysfunctional Wnt signaling.

Multi-Omic Approaches Identify Metabolic and Autophagy Regulators Important in Ovarian Cancer Dissemination.

High-grade serous ovarian cancers (HGSOCs) arise from exfoliation of transformed cells from the fallopian tube, indicating that survival in suspension, and potentially escape from anoikis, is required for dissemination. We report here the results of a multi-omic study to identify drivers of anoikis escape, including transcriptomic analysis, global non-targeted metabolomics, and a genome-wide CRISPR/Cas9 knockout (GeCKO) screen of HGSOC cells cultured in adherent and suspension settings. Our combined approach identified known pathways, including NOTCH signaling, as well as novel regulators of anoikis escape. Newly identified genes include effectors of fatty acid metabolism, ACADVL and ECHDC2, and an autophagy regulator, ULK1. Knockdown of these genes significantly inhibited suspension growth of HGSOC cells, and the metabolic profile confirmed the role of fatty acid metabolism in survival in suspension. Integration of our datasets identified an anoikis-escape gene signature that predicts overall survival in many carcinomas.

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.

CBX2 identified as driver of anoikis escape and dissemination in high grade serous ovarian cancer.

High grade serous ovarian carcinoma (HGSOC) is often diagnosed at an advanced stage. Chromobox 2 (CBX2), a polycomb repressor complex subunit, plays an oncogenic role in other cancers, but little is known about its role in HGSOC. We hypothesize that CBX2 upregulation promotes HGSOC via induction of a stem-like transcriptional profile and inhibition of anoikis. Examination of Gene Expression Omnibus (GEO) datasets and The Cancer Genome Atlas (TCGA) established that increased CBX2 expression conveyed chemoresistance and worse disease-free and overall survival. In primary HGSOC tumors, we observed CBX2 expression was significantly elevated compared to benign counterparts. In HGSOC cell lines, forced suspension promoted CBX2 expression. Subsequently, CBX2 knockdown inhibited anchorage-independent proliferation and potentiated anoikis-dependent apoptosis. Furthermore, CBX2 knockdown re-sensitized cells to platinum-based chemotherapy. Forced suspension promoted increased ALDH activity and ALDH3A1 expression and CBX2 knockdown led to a decrease in both ALDH activity and ALDH3A1 expression. Investigation of CBX2 expression on a HGSOC tissue microarray revealed CBX2 expression was apparent in both primary and metastatic tissues. CBX2 is an important regulator of stem-ness, anoikis escape, HGSOC dissemination, and chemoresistance and potentially serves as a novel therapeutic target.

Alternative Polyadenylation of PRELID1 Regulates Mitochondrial ROS Signaling and Cancer Outcomes.

Disruption of posttranscriptional gene regulation is a critical step in oncogenesis that can be difficult to observe using traditional molecular techniques. To overcome this limitation, a modified polyadenylation site sequencing (PAS-seq) protocol was used to generate a genome-wide map of alternative polyadenylation (APA) events in human primary breast tumor specimens and matched normal tissue. This approach identified an APA event in the PRELID1 mRNA that enhances its steady-state level and translational efficiency, and is a strong breast cancer subtype-dependent predictor of patient clinical outcomes. Furthermore, it has been demonstrated that PRELID1 regulates stress response and mitochondrial reactive oxygen species (ROS) production in a cell type-specific manner. Modulation of PRELID1 expression, including its posttranscriptional control, appears to be a common stress response across different cancer types. These data reveal that PRELID1 mRNA processing is an important regulator of cell type-specific responses to stress used by multiple cancers and is associated with patient outcomes.Implications: This study suggests that the regulation of PRELID1 expression, by APA and other mechanisms, plays a role in mitochondrial ROS signaling and represents a novel prognostic factor and therapeutic target in cancer. Mol Cancer Res; 15(12); 1741-51. ©2017 AACR.

Fibroblast Subtypes Regulate Responsiveness of Luminal Breast Cancer to Estrogen.

Purpose: Antiendocrine therapy remains the most effective treatment for estrogen receptor-positive (ER+) breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer-associated fibroblasts (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance.Experimental Design: Tissues from patients with ER+ breast cancer were analyzed for the presence of CD146-positive (CD146pos) and CD146-negative (CD146neg) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evaluated in ER+ tumor cells cocultured with CD146pos or CD146neg fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrence in tamoxifen-treated patients with ER+ breast cancer.Results: We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer cells are sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes.Conclusions: Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER+ breast cancer and should be considered a target for drug development. Clin Cancer Res; 23(7); 1710-21. ©2016 AACR.