Subset Of Ovarian Cancers Research Articles

A comprehensive analysis of germline predisposition to early-onset ovarian cancer

The subset of ovarian cancer (OC) diagnosed ≤ 30yo represents a distinct subgroup exhibiting disparities from late-onset OC in many aspects, including indefinite germline cancer predisposition. We performed DNA/RNA-WES with HLA-typing, PRS assessment and survival analysis in 123 early-onset OC-patients compared to histology/stage-matched late-onset and unselected OC-patients, and population-matched controls. Only 6/123(4.9%) early-onset OC-patients carried a germline pathogenic variant (GPV) in high-penetrance OC-predisposition genes. Nevertheless, our comprehensive germline analysis of early-onset OC-patients revealed two divergent trajectories of potential germline susceptibility. Firstly, overrepresentation analysis highlighted a connection to breast cancer (BC) that was supported by the CHEK2 GPV enrichment in early-onset OC(p = 1.2 × 10–4), and the presumably BC-specific PRS313, which successfully stratified early-onset OC-patients from controls(p = 0.03). The second avenue pointed towards the impaired immune response, indicated by LY75-CD302 GPV(p = 8.3 × 10–4) and diminished HLA diversity compared with controls(p = 3 × 10–7). Furthermore, we found a significantly higher overall GPV burden in early-onset OC-patients compared to controls(p = 3.8 × 10–4). The genetic predisposition to early-onset OC appears to be a heterogeneous and complex process that goes beyond the traditional Mendelian monogenic understanding of hereditary cancer predisposition, with a significant role of the immune system. We speculate that rather a cumulative overall GPV burden than specific GPV may potentially increase OC risk, concomitantly with reduced HLA diversity.

Germline variants in non-BRCA1/2 homologous recombination-related genes and ovarian cancer: Analysis of tumor phenotype and survival.

5565 Background: BRCA1/2-associated ovarian cancer (OC) exhibits homologous recombination (HR)-deficiency (HRD) and a better prognosis than BRCA1/2-wildtype (WT) OC. However, it is unclear if patients with germline pathogenic variants (gPV) in other HR-related genes have a similar tumor phenotype. We sought to define OC molecular features from patients with gPV in other HR genes and analyze survival compared to patients with BRCA1/2 and WT OC. Methods: We identified patients with OC treated at our institution who underwent tumor-normal sequencing using MSK-IMPACT targeting 341-505 cancer-related genes with germline analysis of ≥76 genes from 7/2015-12/2020. Biallelic inactivation was inferred via assessments of loss of heterozygosity (LOH) at gPV in non- BRCA1/2 HR-related genes ( ATM, BARD1, BRIP1, FANCA, FANCC, NBN, PALB2, RAD50, RAD51B, RAD51C, and RAD51D). Clinical characteristics were compared to patients with BRCA1/2-associated and WT OC using non-parametric tests. Progression-free (PFS) and overall-survival (OS) were calculated from date of pathologic diagnosis using the Kaplan-Meier method. Left truncation at date of MSK-IMPACT consent was applied. Whole-exome sequencing (WES) was performed in a subset of OCs. Results: Of 882 patients with OC, 56 (6.3%) had gPV in non- BRCA1/2 HR-related genes compared to 95 (10.8%) patients with BRCA1-associated OC (58 germline, 37 somatic) and 59 (6.7%) patients with BRCA2-associated OC (40 germline, 19 somatic). Patients with a deleterious genetic alteration were diagnosed with OC at younger age and more likely to have high-grade serous histology compared to the WT group (p < 0.01). High rates of biallelic alterations were observed amongst gPV in BRIP1 (11/13), PALB2 (3/4), RAD51B (3/4), RAD51C (3/4), and RAD51D (8/10), and WES was performed in a subset (n = 27) of tumors from these patients with adequate tumor purity (>30%). We observed a higher tumor mutational burden (TMB), median 2.5 (1.1-6.0) vs. 1.2 (0.6-2.6) mut/Mb, and enrichment of HRD mutational signatures in tumors associated with PALB2 and RAD51B/C/D compared with BRIP1 (p < 0.01), although markers of telomeric-allelic imbalance (TAI), large-scale state transitions (LST) and fraction of genome altered (FGA) were similar. PFS and OS varied by gene group with best survival in BRCA1/2-associated OC, even after adjustment for clinical covariates in multivariable models (p < 0.01). Although we observed heterogeneity in PFS and OS for those with gPV in other HR-related genes by biallelic status and HRD phenotype, none had significantly better survival than those with WT OC. Conclusions: OCs associated with gPV in non- BRCA1/2 HR-related genes represent a heterogenous group. OCs in those with gPV in PALB2 and RAD51B/C/D preferentially harbored biallelic alterations and displayed an HRD phenotype.

OR22-6 Ovarian Cancer Cell Glucocorticoid Receptor Activity Is Associated With Cytokine Secretion Promoting Infiltration of Immunosuppressive Cells Into the Tumor Microenvironment

Abstract Immunosuppressive cells abundantly infiltrate tumors during cancer progression. Notably, immunosuppressive myeloid-derived suppressor cells (MDSCs) have recently been identified as a heterogeneous cell population that expand during tumor-associated inflammation and are significantly increased in the tumor, peripheral blood, as well as ascites of ovarian cancer patients. MDSCs contribute to tumor immune tolerance via inhibition of T-cell proliferation and activation. Signals from the tumor microenvironment in the form of soluble cytokines are proposed to lead to MDSC infiltration. It is also known that high tumor cell glucocorticoid receptor (GR) expression is associated with a relatively poor prognosis in ovarian cancer patients. We hypothesized that ovarian cancer cell GR expression and activity may modulate tumor cell cytokine secretion thereby leading to increased MDSC generation and recruitment to the tumor, thereby creating an immunosuppressive environment. Our preliminary data confirm that GR activation in two high-grade ovarian cancer cell lines (HeyA8 and SKOV3) modulates the cytokine secretome. Our results demonstrate GR-mediated secretion of several pro-tumorigenic and immunosuppressive proteins including G-CSF, M-CSF, TGF-b, and MCP-1 in those ovarian cancer models. We also observed downregulation of potent cytotoxic T-cell activating cytokines required for anti-tumor T-cell function including IL-2, IFN-g and MIP1 upon GR activation. Importantly, treatment of cells with a selective GR modulator (SGRM) reverses this immunosuppressive secretome effect. Furthermore, upon culturing healthy peripheral blood mononuclear cells (PBMCs) with tumor conditioned media from GR activated ovarian cancer cells, we show that secreted immunomodulatory factors have the capacity to promote differentiation of human MDSCs from progenitor myeloid cells. The resulting MDSC population was characterized via expression of activation markers including Arginase-1, iNOS, VEGF and TGF-b. Most importantly, the suppressive function of these MDSCs is being evaluated by determining their inhibitory potential on cytotoxic T-cell proliferation and/or activation. Based on the insight provided by these data, we propose to determine whether ovarian tumor cell-intrinsic GR activation and resulting MDSC generation contributes to earlier relapse of GR-positive ovarian cancers, and eventually whether this mechanism may be targeted to improve patient outcomes in this subset of ovarian cancers. Presentation: Monday, June 13, 2022 12:15 p.m. - 12:30 p.m.

Breaking satellite silence: human satellite II RNA expression in ovarian cancer.

Multiple cancer types demonstrate abnormal expression of repetitive RNA sequences as a form of epigenetic instability. There is growing interest in understanding the role of repetitive RNAs in cancer pathogenesis and immunogenicity and in their potential role as diagnostic or therapeutic biomarkers. In this issue of the JCI, Porter and colleagues report on satellite RNA in a subset of ovarian cancers. The authors found that high expression of human satellite (HSAT) repeats — but not other families of repeats — was associated with an immunosuppressive phenotype in ovarian cancer cell lines and tumor samples. Further induction of HSAT RNA levels in vitro, surprisingly, leads to innate immune activation, suggesting a potential therapeutic strategy. This work highlights the expanding role of repetitive RNAs in tumor biology and the need to better define specific classes of repetitive elements expressed in cancer — as well as their role in tumorigenesis, tumor immunity, and the host response to cancer.

Abstract 1893: Deep learning supported high content analysis of primary patient samples identifies ALK inhibition as a novel mechanism of action in a subset of ovarian cancers

Abstract Background: High unmet need of ovarian cancer (OC) suggests the discovery of new targeted therapeutics is crucial to improve patient prognosis. Unlike artificial model systems such as cell lines, primary cancer samples recapitulate the complexity of the original microenvironment consisting of cancer cells as well as stromal and immune cells; this is especially important when evaluating IO targets and signalling pathways. Supported by our previous success predicting therapy for late stage haematological cancer patients in the EXALT-I trial using AI-supported functional single cell quantification of drug action (Kornauth et. al. 2021) we set out to systematically reveal novel targets and pathways in OC using small molecule drugs (SMDs) as tools. Single cell phenotypic screening of OC MPAs (malignant pleural effusion and ascites) was enabled by the quantification of drug effects using an end-to-end scalable deep learning driven image analysis tool chain. This custom state-of-the-art AI software is critical to enable robust primary cell screening given the diversity of cells within each sample. This revealed anaplastic lymphoma kinase (ALK), as well as structurally related targets and pathway associated proteins, as being potential novel targets in a subset of OC patient samples. There is sparse literature evidence for therapeutic utilisation of the ALK pathway in OC, and the diversity of responses indicates a further novel patient selection method. Methods: MPAs from OC patients (n = 20) were collected and the sensitivity of the cancer cells to 85 SMDs was evaluated using high content microscopy. Individual cells were segmented and classified using convolutional neural networks and drug responses were estimated from the resulting cell counts. The integration of these results with whole exome and RNA sequencing guided target and pathway prioritisation. Results: Screening for novel sensitivities using SMDs as tools uncovered inhibitors of ALK and related targets as having strong cancer cell cytotoxic effects, recapitulated in solid tumour biopsies. Transcriptomic profiling revealed pathway correlations to ALK inhibitor sensitivity, however non-annotated polypharmacological effects of each drug cannot yet be excluded. Conclusions: Quantifying SMD sensitivity in a disease relevant model system identified ALK as a promising and overlooked target in OC, providing an upstream and potentially more specific target to the recently suggested MEK, PI3K and STAT3 (Papp et. al. 2018, Izar et al. 2020). While further work to confirm the target is required, this study supports a notion of patient-centric drug development using disease relevant models and deep learning. Our work introduces a novel patient-centric tool to advance understanding of the OC target landscape and provides a resource for the development of novel therapeutic approaches. Citation Format: Irene Gutierrez-Perez, Joost Van Ham, Valentin Aranha, Rin Okumura, Elisabeth Waltenberger, Isabella Alt, Claudia Baumgaertler, Maja Stulic, Edgar Petru, Christoph Minichsdorfer, Lukas Hefler, Judith Lafleur, Nikolaus Krall, Thorsten Füreder, Gregory Ian Vladimer, Robert Sehlke, Bojan Vilagos. Deep learning supported high content analysis of primary patient samples identifies ALK inhibition as a novel mechanism of action in a subset of ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1893.

Abstract 4096: Development of novel anticancer RNA therapeutics with dual-targeting siRNA that silences ubiquitin family members

Abstract Ubiquitination is a metabolic process in eukaryotic cells that modifies proteins with ubiquitin leading to protein degradation via the ubiquitin-proteosome pathway. Cellular levels of ubiquitin are driven by the expression of RPS27A, UBA52, UBB and UBC. In cancer, the dysregulation of ubiquitination can affect various aspects of tumor progression including cell cycle regulation and gene expression. Ubiquitin B (UBB) gene has been shown to be over-expressed in some tumor types, including NSCLC and cervical cancer, and under-expressed in other tumors, in particularly a subset of ovarian cancers. A role for ubiquitin C (UBC) in cancer has not been well established but UBC has been described as a biomarker in renal cancer and CLL. In recent studies, repression of UBB in ovarian cancer cells was associated with sensitivity to UBC knockdown and demonstrated a synthetic lethal relationship. Since both UBB and UBC show substantial sequence homology, we explored the possibility of developing dual targeting siRNA that would silence both UBB and UBC. We identified unique individual siRNA sequences that efficiently silenced both UBB and UBC and led to rapid cytotoxicity in several cancer cell lines including the colon cancer cell line HCT-116, the breast cancer cell line SK-BR3, and the prostate cancer cell line 22Rv1. Treatment with either UBB-specific or UBC-specific siRNA did not show the same lethal phenotype. Dose response assays using the dual targeting siRNA designated U21 indicated EC50 for cytotoxicity between 50–500 pM in several cancer lines. Treatment of cancer cell lines with U21 leads to rapid apoptosis and cell death. Chemical modifications to pharmacologically stabilize the U21 siRNA sequence did not change its activity. The efficient cytotoxic activity of the U21 siRNA sequence has been integrated into our SeekR™ platform to create aptamer-siRNA chimeras for targeted delivery of the lethal U21 payload to specific cancer cell types. Citation Format: David O. Azorsa, David W. Lee, Marvin O'Ketch, Nathalie A. Azorsa, Ioanna Papacharalampous, Lizette A. Castaño, Jeffrey A. Kiefer, Spyro Mousses. Development of novel anticancer RNA therapeutics with dual-targeting siRNA that silences ubiquitin family members [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4096.

Abstract 1335: Ovarian cancer cell glucocorticoid receptor activity is associated with cytokine secretion promoting infiltration of immunosuppressive cells into the tumor microenvironment

Abstract Immunosuppressive cells abundantly infiltrate tumors during cancer progression. Notably, immunosuppressive myeloid-derived suppressor cells (MDSCs) have recently been identified as a heterogeneous cell population that expand during tumor-associated inflammation and are significantly increased in the tumor, peripheral blood, as well as ascites of ovarian cancer patients. MDSCs contribute to tumor immune tolerance via inhibition of T-cell proliferation and activation. Signals from the tumor microenvironment in the form of soluble cytokines are proposed to lead to MDSC infiltration. It is also known that high tumor cell glucocorticoid receptor (GR) expression is associated with a relatively poor prognosis in ovarian cancer patients. We hypothesized that ovarian cancer cell GR expression and activity may modulate tumor cell cytokine secretion thereby leading to increased MDSC generation and recruitment to the tumor, eventually creating an immunosuppressive environment. Our preliminary data confirm that GR activation in two high-grade ovarian cancer cell lines (HEYA8 and SKOV3) modulates the cytokine secretome. Our results demonstrate GR-mediated secretion of several pro-tumorigenic and immunosuppressive proteins including G-CSF, M-CSF, TGF-β, and MCP-1 in those ovarian cancer models. We also observed downregulation of potent cytotoxic T-cell activating cytokines required for anti-tumor T-cell function including IL-2, IFN-γ and MIP1 upon GR activation. Importantly, treatment of cells with a competitive GR antagonist reverses this immunosuppressive secretome effect. Furthermore, upon culturing healthy peripheral blood mononuclear cells (PBMCs) with tumor conditioned media from GR activated ovarian cancer cells, we show that secreted immunomodulatory factors have the capacity to promote differentiation of human MDSCs from PBMCs. The resulting MDSC population was characterized via expression of activation markers including Arginase-1, iNOS, VEGF and TGF-β. Most importantly, the suppressive function of these MDSCs is being evaluated by determining their inhibitory potential on cytotoxic T-cell proliferation and/or activation. Based on the insight provided by these data, we propose to determine whether ovarian tumor cell-intrinsic GR activation and resulting MDSC generation contributes to earlier relapse of GR-positive ovarian cancers, and eventually whether this mechanism may be targeted to improve patient outcomes in this subset of ovarian cancers. Citation Format: Manisha Taya, Janet Gonzalez, Lynda Bennett, Suzanne D. Conzen. Ovarian cancer cell glucocorticoid receptor activity is associated with cytokine secretion promoting infiltration of immunosuppressive cells into the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1335.

Abstract LB125: The role of succinate dehydrogenase in ovarian cancer metabolism

Abstract The reprogramming of cellular metabolism is an important hallmark of cancer allowing tumor cells to survive and proliferate. Recent data revealed that subsets of ovarian cancer with invasive and metastatic phenotype are highly metabolically active with increased mitochondrial respiration. To better understand the metabolic profiles of ovarian cancer, we utilized patient-derived xenografts (PDXs) derived from high-grade serous ovarian carcinoma (HGSOC), and performed comprehensive analysis of metabolic pathways by Reverse Phase Protein Array (RPPA). The RPPA analysis revealed PDX subsets with high or low mitochondrial activity and metabolism, and we identified succinate dehydrogenase (SDHA) to be a top differentially expressed metabolic protein between those PDX subsets. SDHA is a mitochondrial enzyme that participates in the TCA cycle and the electron transport chain, which are both essential for cellular metabolism. Analysis of the TCGA data demonstrated that SDHA gene alterations are highly prevalent in HGSOC patients (19% cases). Despite these findings, the impact of SDHA amplification on cancer biology is poorly understood. To assess the effect of SDHA overexpression on energy metabolism, we used SDHA-overexpressing ovarian cancer cell lines and measured cellular respiration by Seahorse. The results revealed that the overexpression of SDHA leads to a significant increase in basal and maximal respiration shifting cellular metabolism towards increased oxidative phosphorylation in mitochondria. Further, we determined if SDHA overexpression redirects cellular metabolism in response to deprivation of selected nutrients. In the conditions with low glucose or glutamine, SDHA overexpressing cells significantly increased basal respiration when compared with controls. In galactose medium that disrupts glycolysis, the SDHA lost its ability to induce cellular respiration, which indicates that SDHA relays on glycolysis to increase mitochondrial respiration. Together, our data indicate that SDHA requires glycolysis to fuel TCA cycle and and support cellular respiration. Next, we performed in vitro drug screening and found that SDHA overexpressing cells are highly sensitive to glycolysis inhibitor Shikonin. Our pilot in vivo data showed that Shikonin significantly reduces growth of SDHA-amplified tumors. In summary, our studies indicate that SDHA upregulation plays a role in reprogramming of cellular metabolism and pathogenesis of ovarian cancer. Moreover, the unique metabolic state of ovarian cancer associated with SDHA amplification could be successfully targetable offering a potential new treatment strategy for ovarian cancer patients. Citation Format: Magdalena Cybula, Maria Rostworowska, Lin Wang, Summer Wang, Ana Luiza Drumond-Bock, Magdalena Bieniasz. The role of succinate dehydrogenase in ovarian cancer metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB125.

Ovarian cancer cell glucocorticoid receptor activity modulates cytokine secretion promoting infiltration of immunosuppressive cells into the tumor microenvironment

Abstract Myeloid-derived suppressor cells (MDSCs) have been identified as a heterogeneous cell population that expand during tumor progression and infiltrate the tumor, peripheral blood, as well as ascites of ovarian cancer patients. MDSCs contribute to tumor immune tolerance via inhibition of T-cell proliferation/activation. It is also known that high tumor cell glucocorticoid receptor (GR) expression is associated with a relatively poor prognosis in ovarian cancer patients. We hypothesized that ovarian tumor cell GR expression and activity may modulate tumor cell cytokine secretion thereby leading to increased MDSC generation and recruitment, thus creating an immunosuppressive tumor microenvironment. Our preliminary data confirms GR-mediated secretion of pro-tumorigenic cytokines including G-CSF, TGF-b, and MCP-1 in high-grade ovarian cancer models. We also observed GR-mediated downregulation of IL-2 and IFN-g, cytokines required for anti-tumor T-cell function. Importantly, treatment of cells with a competitive GR antagonist reversed this immunosuppressive secretome effect. Moreover, culturing healthy peripheral blood mononuclear cells (PBMCs) with tumor conditioned media from GR activated ovarian cancer cells demonstrated that secreted immunomodulatory factors have the capacity to promote differentiation of human MDSCs from PBMCs. Based on the insight provided by these data, we propose to determine whether ovarian tumor cell-intrinsic GR activation and resulting MDSC generation has an inhibitory effect on cytotoxic T-cell function, contributing to earlier relapse of GR-positive ovarian cancers, and eventually whether this mechanism may be targeted to improve patient outcomes in this subset of ovarian cancers. Supported by grant from CPRIT

Replication catastrophe is responsible for intrinsic PAR glycohydrolase inhibitor-sensitivity in patient-derived ovarian cancer models

BackgroundPatients with ovarian cancer often present at advanced stage and, following initial treatment success, develop recurrent drug-resistant disease. PARP inhibitors (PARPi) are yielding unprecedented survival benefits for women with BRCA-deficient disease. However, options remain limited for disease that is platinum-resistant and/or has inherent or acquired PARPi-resistance. PARG, the PAR glycohydrolase that counterbalances PARP activity, is an emerging target with potential to selectively kill tumour cells harbouring oncogene-induced DNA replication and metabolic vulnerabilities. Clinical development of PARG inhibitors (PARGi) will however require predictive biomarkers, in turn requiring an understanding of their mode of action. Furthermore, differential sensitivity to PARPi is key for expanding treatment options available for patients.MethodsA panel of 10 ovarian cancer cell lines and a living biobank of patient-derived ovarian cancer models (OCMs) were screened for PARGi-sensitivity using short- and long-term growth assays. PARGi-sensitivity was characterized using established markers for DNA replication stress, namely replication fibre asymmetry, RPA foci, KAP1 and Chk1 phosphorylation, and pan-nuclear γH2AX, indicating DNA replication catastrophe. Finally, gene expression in sensitive and resistant cells was also examined using NanoString or RNAseq.ResultsPARGi sensitivity was identified in both ovarian cancer cell lines and patient-derived OCMs, with sensitivity accompanied by markers of persistent replication stress, and a pre-mitotic cell cycle block. Moreover, DNA replication genes are down-regulated in PARGi-sensitive cell lines consistent with an inherent DNA replication vulnerability. However, DNA replication gene expression did not predict PARGi-sensitivity in OCMs. The subset of patient-derived OCMs that are sensitive to single-agent PARG inhibition, includes models that are PARPi- and/or platinum-resistant, indicating that PARG inhibitors may represent an alternative treatment strategy for women with otherwise limited therapeutic options.ConclusionsWe discover that a subset of ovarian cancers are intrinsically sensitive to pharmacological PARG blockade, including drug-resistant disease, underpinned by a common mechanism of replication catastrophe. We explore the use of a transcript-based biomarker, and provide insight into the design of future clinical trials of PARGi in patients with ovarian cancer. However, our results highlight the complexity of developing a predictive biomarker for PARGi sensitivity.

Debulking surgery for malignant tumors: the current status, evidence and future perspectives.

Debulking surgery, also called cytoreductive surgery, is a resection of the tumor as much as possible and an intended incomplete resection for unresectable malignant tumors. Since the most important principle in surgical oncology is complete R0 resection, debulking surgery goes against the basic principle and obscures the concept of operability. However, debulking surgery has been advocated for various types of advanced malignant tumors, including gynecological cancers, urological cancers, gastrointestinal cancers, breast cancers and other malignancies, with or without adjuvant therapy. Positive data from randomized trials have been shown in subsets of ovarian cancer, renal cell carcinoma, colorectal cancer and breast cancer. However, recent trials for renal cell carcinoma, colorectal cancer and breast cancer have tended to show controversial results, mainly according to the survival improvement of nonsurgical systemic therapy alone. On the other hand, debulking surgery still has a therapeutic role for slow-growing and borderline malignant tumors, such as pseudomyxoma peritonei and thymomas. The recent understanding of tumor heterogeneity and clonal evolution responsible for malignancy and drug resistance indicates that select patients may obtain prolonged survival by the synergistic effect of debulking surgery and novel systemic therapy. This review aimed to describe the current status and evidence of debulking surgery in a cross-organ manner and to discuss future perspectives in the current era with advances in systemic therapy.

Development of a DNA damage response (DDR) therapeutics platform for oncology.

e15036 Background: Cancer cells respond to increases in DNA damage by deploying their DNA damage response (DDR) pathways. We are building a platform for the discovery and development of target-specific DDR therapeutics, including small molecule inhibitors and targeted protein degradation warheads, founded on fragment- and structure-based drug discovery. Methods: Our DDR platform, which includes hit-to-lead, lead optimization and candidate selection, starts with hit generation from a new technology that uses high-throughput protein X-ray crystallography to directly screen compound libraries. Our hit generation process produces empirical evidence of direct target engagement. The elucidation of high-quality ligand-bound 3D structures reveals the location and pose of the ligand and details of the protein-ligand interactions. Thus we can predict the structure-function consequences of the hit molecule engagement, which sets the stage for rapid assessment of synthetic tractability and intellectual property. After hit identification, we apply a multi-pronged approach in hit-to-lead conversion and lead optimization using iterative biophysical and biochemical assays, coupled with crystallography. We are applying our approach to several new targets in DDR and will present some early progress in this space. Results: Apurinic/apyrimidinic endonuclease 1 (APE1) is the major repair enzyme for abasic sites in DNA and contributes to DNA strand break processing. Many studies have associated increased APE1 levels with enhanced growth, migration, and drug resistance in human tumor cells, and with decreased patient survival. APE1 has been implicated in over 20 human cancers, including glioblastoma, making the protein an attractive target for the development of anticancer therapeutics. Despite intensive effort, there are no clinical endonuclease inhibitors of APE1. We have identified 25 diverse small molecule fragments that bind to APE1 at two distinct sites, including the endonuclease site. Pol eta (or PolH) is a DNA polymerase implicated, among other things, in the development of cisplatin resistance in a subset of ovarian cancers. In our quest to develop PolH inhibitors, we have identified 5 diverse fragments that bind to two distinct sites in the polymerase including a new potential allosteric site. Our results on APE1 and PolH represent the first known cases of crystal structures of small molecules bound to these proteins. Flap endonuclease (FEN1) is implicated in several cancers including for example ER/tamoxifen-resistant breast cancer. We are developing a targeted protein degradation approach using PROTACs (Proteolysis-Targeting Chimeras) toward the development of novel therapeutics against FEN1. Conclusions: Our results will help us develop small molecule inhibitors and targeted protein degradation against DDR targets that may be effective as single therapies or be used to make existing therapies more effective.

The prognostic significance of GRB7 protein expression and localization in human breast and ovarian cancers.

Objective: To study GRB7 protein expression in normal human tissues and breast and ovarian cancers, and determine its clinical significance. Results: GRB7 protein was expressed in multiple tissues, including myoepithelial cells of normal breast and fibroadenoma. Cytoplasmic GRB7 expression was seen predominantly in HER-2 positive and, to a lesser extent, triple negative breast cancer. Membrane localization of GRB7 was present in a subset of breast cancers with high cytoplasmic GRB7 expression. Univariate and multivariate analysis found that cytoplasmic GRB7 expression was associated with a negative progesterone receptor status, while membrane GRB7 expression was associated with a negative axillary nodal status. Membrane associated GRB7 expression was present in a subset of ovarian cancers with high cytoplasmic GRB7 expression. Membrane GRB7 expression displayed a trend towards improved recurrence free survival (RFS). Landmark analysis suggested an RFS advantage for ovarian cancers that had GRB7 membrane expression and survived beyond 27 months; GRB7 membrane expression in two or more cores (out of three) predicted an improved RFS. Membrane expression of GRB7 protein was observed in breast cancer cell lines with high GRB7 protein expression in vitro. Conclusion: GRB7 protein membrane expression may be associated with a better prognosis in breast and ovarian cancers. The favorable prognostic value of GRB7 protein membrane expression and its underlying mechanism is worthy of further investigation. Methods: Immunohistochemistry of normal human tissues, breast tissues of various pathologies, and clinically annotated ovarian cancers was performed to correlate the patterns of GRB7 expression with biomarkers or clinical outcome.

Opposite Macrophage Polarization in Different Subsets of Ovarian Cancer: Observation from a Pilot Study.

The role of the innate immune system in ovarian cancer is gaining importance. The relevance of tumor-associated macrophages (TAM) is insufficiently understood. In this pilot project, comprising the immunofluorescent staining of 30 biopsies taken from 24 patients with ovarian cancer, we evaluated the presence of total TAM (cluster of differentiation (CD) 68 expression), M1 (major histocompatibility complex (MHC) II expression), and M2 (anti-mannose receptor C type 1 (MRC1) expression), and the blood vessel diameter. We observed a high M1/M2 ratio in low-grade ovarian cancer compared to high-grade tumors, more total TAM and M2 in metastatic biopsies, and a further increase in total TAM and M2 at interval debulking, without beneficial effects of bevacizumab. The blood vessel diameter was indicative for M2 tumor infiltration (Spearman correlation coefficient of 0.65). These data mainly reveal an immune beneficial environment in low-grade ovarian cancer in contrast to high-grade serous ovarian cancer, where immune suppression is not altered by neoadjuvant therapy.

High homogeneity of MMR deficiency in ovarian cancer

ObjectiveMismatch repair (MMR) deficiency and Bethesda panel microsatellite instability (MSI) are increasingly analyzed to identify tumors that might benefit from immune checkpoint inhibitors, but tumor heterogeneity is a potential obstacle for such analyses. In ovarian cancer, data on intratumoral heterogeneity of MMR deficiency/MSI are lacking. MethodsN = 582 ovarian cancers were screened for MMR deficiency by immunohistochemistry (IHC) on a tissue microarray. 10 cases suspect for MMR deficiency were identified among 478 interpretable cancers and repeated IHC on large sections combined with polymerase chain reaction (PCR)-based MSI analysis validated MMR deficiency/MSI in 9 of these tumors. ResultsMMR deficiency/MSI was predominantly seen in endmetrioid cancers (8 of 35, 23%) and also in 1 of 358 serous carcinomas (0.3%), but was absent in 34 mucinous carcinomas, 23 clear cell carcinomas, 17 malignant mixed Mullerian tumors (carcinosarcomas), and 11 mixed carcinomas. MMR deficiency involed protein loss of PMS2/MLH1 in 6 cases and of MSH2 and/or MSH6 in 3 cases. 7 MMR deficient cancers were MSI-high (all endometrioid), one was MSI-low (endometrioid) and one cancer with unequivocal MMR protein loss exhibited microsatellite stability (serous). MLH1 promotor methylation was observed in 4 of 5 endometrioid cancers with MLH1 protein loss. Immunostaining of all available cancer-containing tissue blocks (n = 114) of tumors with confirmed MMR deficiency/MSI revealed uniform MMR status throughout the entire tumor mass. ConclusionsOur data show that MSI is present in a substantial proportion of endometrioid ovarian cancers but can also occur in other tumor subtypes. MMR deficiency/MSI typically involves the entire tumor mass, suggesting that MMR inactivation occurs early in tumorigenesis in a subset of ovarian cancers.

Regulation of human chorionic gonadotropin beta subunit expression in ovarian cancer

PurposeExpression of human chorionic gonadotropin beta subunit by cancers is extensively documented, yet regulation of the multiple genes that can code for this protein is poorly understood. The aim of the study was to examine the mechanisms regulating CGB gene expression in ovarian cancer.MethodsExpression of CGB genes and SP1, SP3, TFAP2A transcription factor genes was evaluated by RT-qPCR. The methylation status of CGB genes promoter regions was examined by methylation-specific PCR.ResultsmRNA arising from multiple CGB genes was detected in both ovarian control and malignant tissues. However, expression of CGB3–9 genes was shown to be significantly higher in malignant than healthy ovarian tissues. CGB1 and CGB2 transcripts were shown to be present in 20% of ovarian cancers, but were not detected in any of the control samples. Malignant tissues were characterized by DNA demethylation of CGB promoter regions. In ovarian cancer CGB expression positively correlated with TFAP2A transcripts level and expression of TFAP2A transcription factor was significantly higher in cancer than in control tissues. In contrast SP3 expression level was significantly lower in ovarian tumours than in control ovarian tissue.ConclusionsIn ovarian cancers increased expression of human chorionic gonadotropin beta subunit is associated with demethylation of CGB promoter regions. CGB3–9 expression level strongly correlates with expression of the TFAP2A transcription factor. Presence of mRNA arising from CGB1 and CGB2 genes appears to be a unique feature of a subset of ovarian cancers.

Abstract 206: miR551b-mediated tumor-specific angiogenesis in ovarian cancer

Abstract Background: Ovarian cancer is the most lethal gynecological cancer. Tumor angiogenesis is essential for the progression and metastasis of ovarian cancer. Our data shows that STAT3, HIF-1α and VEGFA are the key regulators of tumor-specific angiogenesis. Our studies have provided evidence that VEGF is directly regulated by STAT3 in miR551b-3p expressing cells, which indicates that STAT3 is an important regulator of angiogenesis in a subset of ovarian cancers. Recently, we showed that miR551b-3p acts as an upstream regulator of STAT3 in ovarian cancer cells. We identified that anti-miR551b-3p inhibit the expression of miR551b-3p disrupt tumor-specific angiogenesis by preventing the expression of HIF-1α and VEGFA through direct inhibition of STAT3. Methods: In this study, we explored the role of miR551b on angiogenesis in ovarian cancer cells by performing cell proliferation, western blot and qPCR of angiogenic markers, in vitro capillary tube formation, in vivo matrigel plug assay and ex-vivo aortic ring assays. Results: We found that miR551b-3p upregulates STAT3, HIF-1α, VEGFR and VEGF in ovarian cancer cells and increases the abilities of cell proliferation, migration and tube formation of human umbilical vein endothelial (HUVEC) cells. In conjunction, knockdown of miR-551b decreased the level of STAT3, HIF-1α and VEGFR2 proteins as well as the angiogenic activity; also increased cellular apoptosis. Altogether, our data suggest critical roles of miR-551b on STAT-3–mediated angiogenesis in ovarian cancer cells. In complement to our in vitro results, the delivery of anti-miR551b inhibited angiogenesis in the matrigel plug injected in nude mice. Furthermore, the treatment of anti-miR551b reduced the angiogenic sprouting from rat thoracic aorta in our ex vivo culture. Taken together, our findings provide the first evidence that miR-551b is a pivotal regulator of tumor angiogenesis and inhibiting miR551b could be used to inhibit tumor-specific angiogenesis in ovarian cancer. Conclusions: miR551b markedly augmented the pro-angiogenic effects in vitro, in vivo and ex-vivo models. Our data suggest that miR551b is an actionable target to disrupt tumor-specific angiogenesis in ovarian cancer. In our pre-clinical model, we are using anti-miR551b encapsulated in nanoliposomes for in vivo delivery to inhibit and sensitize anti- angiogenic therapy and chemotherapy. We expect that the completion of our studies will identify novel mechanisms regulate tumor-specific angiogenesis and novel targets to treat ovarian cancer patients. Citation Format: Deepak Parashar, Anjali Geethadevi, Jasmine George, Gopakumar Gopinathan Nair, Changliang Chen, Sunila Pradeep, Pradeep Chaluvally-Raghavan. miR551b-mediated tumor-specific angiogenesis in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 206.

Candidate synthetic lethality partners to PARP inhibitors in the treatment of ovarian clear cell cancer.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are new types of personalized treatment of relapsed platinum-sensitive ovarian cancer harboring BRCA1/2 mutations. Ovarian clear cell cancer (CCC), a subset of ovarian cancer, often appears as low-stage disease with a higher incidence among Japanese. Advanced CCC is highly aggressive with poor patient outcome. The aim of the present study was to determine the potential synthetic lethality gene pairs for PARP inhibitions in patients with CCC through virtual and biological screenings as well as clinical studies. We conducted a literature review for putative PARP sensitivity genes that are associated with the CCC pathophysiology. Previous studies identified a variety of putative target genes from several pathways associated with DNA damage repair, chromatin remodeling complex, PI3K-AKT-mTOR signaling, Notch signaling, cell cycle checkpoint signaling, BRCA-associated complex and Fanconi's anemia susceptibility genes that could be used as biomarkers or therapeutic targets for PARP inhibition. BRCA1/2, ATM, ATR, BARD1, CCNE1, CHEK1, CKS1B, DNMT1, ERBB2, FGFR2, MRE11A, MYC, NOTCH1 and PTEN were considered as candidate genes for synthetic lethality gene partners for PARP interactions. When considering the biological background underlying PARP inhibition, we hypothesized that PARP inhibitors would be a novel synthetic lethal therapeutic approach for CCC tumors harboring homologous recombination deficiency and activating oncogene mutations. The results showed that the majority of CCC tumors appear to have indicators of DNA repair dysfunction similar to those in BRCA-mutation carriers, suggesting the possible utility of PARP inhibitors in a subset of CCC.

Analysis of ROR1 Protein Expression in Human Cancer and Normal Tissues.

Purpose: This study examines cell surface ROR1 expression in human tumors and normal tissues. ROR1 is considered a promising target for cancer therapy due to putative tumor-specific expression, and multiple groups are developing antibodies and/or chimeric antigen receptor-modified T cells to target ROR1. On-target, off-tumor toxicity is a challenge for most nonmutated tumor antigens; however, prior studies suggest that ROR1 is absent on most normal tissues.Experimental Design: Our studies show that published antibodies lack sensitivity to detect endogenous levels of cell surface ROR1 by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded tissues. We developed a ROR1-specific monoclonal antibody (mAb) targeting the carboxy-terminus of ROR1 and evaluated its specificity and sensitivity in IHC.Results: The 6D4 mAb is a sensitive and specific reagent to detect cell surface ROR1 by IHC. The data show that ROR1 is homogenously expressed on a subset of ovarian cancer, triple-negative breast cancer, and lung adenocarcinomas. Contrary to previous findings, we found ROR1 is expressed on several normal tissues, including parathyroid; pancreatic islets; and regions of the esophagus, stomach, and duodenum. The 6D4 mAb recognizes rhesus ROR1, and ROR1 expression was similar in human and macaque tissues, suggesting that the macaque is a suitable model to evaluate safety of ROR1-targeted therapies.Conclusions: ROR1 is a promising immunotherapeutic target in many epithelial tumors; however, high cell surface ROR1 expression in multiple normal tissues raises concerns for on-target off-tumor toxicities. Clinical translation of ROR1-targeted therapies warrants careful monitoring of toxicities to normal organs and may require strategies to ensure patient safety. Clin Cancer Res; 23(12); 3061-71. ©2016 AACR.

Nuclear COMMD1 Is Associated with Cisplatin Sensitivity in Ovarian Cancer.

Copper metabolism MURR1 domain 1 (COMMD1) protein is a multifunctional protein, and its expression has been correlated with patients’ survival in different types of cancer. In vitro studies revealed that COMMD1 plays a role in sensitizing cancer cell lines to cisplatin, however, the mechanism and its role in platinum sensitivity in cancer has yet to be established. We evaluated the role of COMMD1 in cisplatin sensitivity in A2780 ovarian cancer cells and the relation between COMMD1 expression and response to platinum-based therapy in advanced stage high-grade serous ovarian cancer (HGSOC) patients. We found that elevation of nuclear COMMD1 expression sensitized A2780 ovarian cancer cells to cisplatin-mediated cytotoxicity. This was accompanied by a more effective G2/M checkpoint, and decreased protein expression of the DNA repair gene BRCA1, and the apoptosis inhibitor BCL2. Furthermore, COMMD1 expression was immunohistochemically analyzed in two tissue micro-arrays (TMAs), representing a historical cohort and a randomized clinical trial-based cohort of advanced stage HGSOC tumor specimens. Expression of COMMD1 was observed in all ovarian cancer samples, however, specifically nuclear expression of COMMD1 was only observed in a subset of ovarian cancers. In our historical cohort, nuclear COMMD1 expression was associated with an improved response to chemotherapy (OR = 0.167; P = 0.038), although this association could not be confirmed in the second cohort, likely due to sample size. Taken together, these results suggest that nuclear expression of COMMD1 sensitize ovarian cancer to cisplatin, possibly by modulating the G2/M checkpoint and through controlling expression of genes involved in DNA repair and apoptosis.