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Introduction: Vascular endothelial growth factor (VEGF) is a key promoter of angiogenesis and disease progression in epithelial ovarian cancer (OC). Bevacizumab, a humanized anti-VEGF monoclonal antibody, is approved for treatment of patients with ovarian and other cancers. Despite the wide use of anti-VEGF therapy for cancer treatment, most patients develop resistant disease. Therefore, new combinations/strategies are needed to reduce adaptive resistance to anti-VEGF antibody (AVA) therapy. Our aim is to determine the potential role of epigenetic changes in AVA-adaptive resistance. We identified and characterized epigenetic changes in BARD1, a tumor suppressor, at the emergence of AVA resistance. Methods: To define epigenetic changes during AVA therapy induced resistance, we performed series of in vitro (methylation array, RNA sequencing) and in vivo (orthotopic OC (PDX-MDA-OVCA1, immunocompetent-IG10, and immunocompromised-SKOV3) and fibrosarcoma-HT1080, an inherently VEGF resistant model) experiments. The biological effects of DNA methylation agent (5`-Azacytidine; 5`-Aza) with B20 (murine anti-VEGF antibody) treatment were subsequently tested using in vivo models. Results: To determine whether 5`-Aza can enhance the efficacy of AVA therapy and overcome resistance to AVA in OC, we established AVA-resistant SKOV3 model. In AVA-resistant mice treated with B20 + 5`-Aza, there was a statistically significant reduction in tumor weight (p=0.03). Next, we performed a Human 450k methylation and mRNA arrays from xenografts of SKOV3 tumors. Based on both analyses, we demonstrated that BARD1 was one of the top candidate which was highly expressed (p=0.02) and less methylated (p4-fold, p=0.006). Tumor weight and BARD1 expression were inversely correlated. To determine the biological significance of BARD1, we knockdown BARD1 using Tet-inducible shRNA system. Our data showed a significant reduction in tumor weight in the BARD1 knockdown group in SKOV3 model (p=0.021). RNA-sequencing of BARD1 knockdown showed an enrichment of the angiogenesis pathway. Moreover, BARD1 expression levels were decreased in hypoxic conditions both in vitro and in vivo. Conclusion: Our findings indicate a previously unrecognized role for BARD1 in mediating resistance to AVA therapy. Use of epigenetic drugs to control the expression of tumor suppressor genes may enhance the efficacy of AVA therapy in OC. Citation Format: Emine Bayraktar, Cristian Rodriguez-Aguayo, Sudhir Kumar, Elaine Stur, Lingegowda S. Mangala, Stephen Baylin, Gabriel Lopez-Berestein, Sunila Pradeep, Anil K. Sood. Epigenetic regulation of BARD1 confers resistance to anti-VEGF therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6001.

Introduction: Ovarian cancer is primarily driven by copy number variations, mutations in p53, BRCA1 or BRCA2 genes. Using the single nucleotide polymorphism (SNP) array data of The Cancer Genome Atlas (TCGA), we identified that FXR1 (Fragile X-Related protein 1), which is in the 3q26.3 chromosomal locus is either amplified or copy-gained in ~40% of ovarian cancer patients. As FXR1 plays a critical role in the pathogenesis of ovarian cancer, it is thought to be an attractive target for anticancer drugs. Nevertheless, till date, no specific chemical inhibitors are available that can target FXR1. Therefore, the goal of this study is to develop an agent that can knockdown the expression of FXR1. Methods: We performed Surface sensing of translation (SUnSET) assay to demonstrate that FXR1 enhances the overall translation in cancer cells. RNA electromobility shift and proximity ligation assays were done to show that FXR1 binds to the AU Rich Elements (ARE) present within the 3'UTR of cMYC. Immunoprecipitation assay was performed to show interaction of FXR1 with translation initiation factors (eIFs). Western blotting was performed to evaluate the target specificity of customized FXR1-specific siRNAs FXR1. Flow cytometry analysis was then performed to see cell death and cell cycle arrest in ovarian cancer cells after siFXR1s (1-5) treatment. Live cell immunofluorescence was done to measure cellular uptake of siFXR1 labelled with Texas Red in cells. FXR1-specific siRNA incorporated DOPC was injected as 5 μg (200 μL) intraperitoneally twice a week starting 1 week after inoculation of cancer cells. The IVIS Lumina II Bioluminescence and Fluorescence Imaging System was used for in vivo bioluminescent imaging. Results: Our previous data suggest that FXR1 is an important oncoprotein and has critical roles in the pathophysiology of ovarian cancer. We also found that FXR1 promoted the overall translation in cancer cells, which is a critical feature for oncogenesis. Our mechanistic investigation provided evidence that FXR1 upregulated cMYC protein levels by stabilizing cMYC mRNA via binding to AREs within its 3′UTR. We also found that FXR1 recruits eIFs to the translation initiation site. In our therapeutic approaches, we found that custom designed siRNAs against FXR1 shows maximum potency in reducing the FXR1 protein levels, induces G1-phase of cell cycle and apoptosis in ovarian cancer cells. Importantly, intraperitoneally delivery of siFXR1 incorporated in DOPC nanoliposomes inhibited ovarian tumor growth and prolonged survival of ovarian cancer bearing mice. Conclusion: Our studies have identified that FXR1 is a critical molecule that is important for ovarian cancer progression. We discovered that cMYC is an important target of FXR1 required for FXR1-mediated oncogenesis. Moreover, DOPC encapsulation enhances the siFXR1 uptake, stability, efficiency of cellular delivery, biodistribution, and target-specific knockdown in vivo. Citation Format: Jasmine George, Ishaque P. Kadamberi, Anjali Geethadevi, Sudhir Kumar, Sonam Mittal, Cristian Rodriguez-Aguayo, Lingegowda S. Mangala, Anil K. Sood, Sunila Pradeep, Pradeep Chaluvally-Raghavan. Fxr1 is an oncogenic driver in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1463.

Background: Despite advances in cancer diagnosis and therapy, high-grade serous ovarian cancer (HGSC) is often diagnosed when spread to multiple intraperitoneal areas; it is prone to metastasize to adipose-rich tissues such as the omentum. To gain a deeper understanding of the molecular determinants of the HGSC milieu, we carried out a single-cell analysis of primary and metastatic tumors of HGSC. Thus, our aim is to identify molecular mechanisms that lead to immunosuppressive mechanisms of HGSC in primary and metastatic tumors of HGSC. Methods: Fresh HGSC surgical samples from 19 patients were collected right after surgery, dissociated, and then frozen. For single-cells analysis, cells were sorted by a viability dye and CD45+/- populations followed by Single cell 10 × 3’v3 protocol (10X Genomics) and sequenced using the NovaSeq6000 S2 sequencer. Cell Ranger toolkit v3.1.0 (10x Genomics) was applied for data processing, followed by further downstream analysis using multiple packages from R Package Seurat. Results: To elucidate the cellular heterogeneity of HGSC, we analyzed 100,480 cells, including epithelial, lymphoid, and myeloid populations; these were identified and represented across all patients. We explored the epithelial compartment further, including a total of 21,144 cells. Given the importance of metastatic lesions for the treatment and outcome of patients with HGSC, we examined expression programs among primary and omentum tumors as well treated and untreated tumors. The major differences between primary and omental metastatic tumors included enrichment of EMT pathways and angiogenesis, as well as a decrease in the IFNα and IFNγ response in the omentum. IFNα and IFNγ response pathways were also upregulated in primary treated (P-NACT) tumors, when compared with primary untreated (P-UT) tumors. To explore the immune compartment, we clustered the immune cells across patients in 28 sub-clusters, including 19 sub-clusters of T-cells and Natural Killer cells. Altogether, the immune infiltration on P-UT indicated an immune infiltrate environment and the composition of P-UT was highly enriched in lymphoid cells with a late stage of differentiation. This finding was also demonstrated by the analysis of T cell trajectories, with a clear definition that CD8 and CD4 cells from P-UT tumors are in a late/final and exhausted stage of differentiation, while post-NACT tumors (independent of tissue type) are in early stages (naïve/central memory). The measurement of a dysfunctional score showed that P-UT tumors have the highest scores, indicating a potential increase in tumor reactivity in P-UT tumors. Conclusions: Collectively, these data indicate that HGSC primary and omentum tumors are very distinct niches for immune cells, with primary being much more dysfunctional than omentum tissue, which could indicate that the application of immunotherapies would have different impacts in variable niches of HGSC. Citation Format: Elaine Stur, Fuduan Peng, Pang-ning Teng, Emine Bayraktar, Min Hu, Sara Corvigno, David J. Brown, Sanghoon Lee, Kathleen N. Moore, Nicholas W. Bateman, Kathleen M. Darcy, George L. Maxwell, Thomas Conrads, Nicole Fleming, Nicholas Navin, Linghua Wang, Anil K. Sood. The dynamic immune behavior of primary and metastatic tumors of ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5782.

Plant-derived vesicles (PDVs) were attractive for therapeutic applications, including as potential nanocarriers. However, a concern with oral delivery of PDVs is whether they would remain intact in the gastrointestinal tract. We found that 82% of cabbage PDVs were destroyed under conditions mimicking the upper digestive tract. To overcome this limitation, we developed a delivery method whereby lyophilized Eudragit S-100-coated cabbage PDVs were packaged into a capsule (Cap-cPDVs). Lyophilization and suspension of PDVs in phosphate-buffered saline did not have an appreciable effect on PDV structure, number of PDVs, or therapeutic effect. However, packaging the lyophilized Eudragit S-100-coated PDVs into capsules allowed them to pass through the upper gastrointestinal tract for delivery into the colon better than did suspension of PDVs in phosphate-buffered saline. Cap-cPDVs demonstrated robust therapeutic effect in a dextran sulfate sodium-induced colitis mouse model. These findings could have broad implications for oral delivery of PDVs. Citation Format: Yuan Liu, Adrian Lankenau-Ahumada, Emine Bayraktar, Paul Schwartz, Mamur Chowdhury, Sixiang Shi, Manu M. Sebastian, Htet Khant, Natalia De Val, Zuliang Jie, Nicholas B. Jennings, Cristian Rodriguez-Aguayo, Jody Swain, Elaine Stur, Lingegowda S. Mangala, Yutuan Wu, Supriya Nagaraju, Brooke Erimas, Chun Li, Gabriel Lopez-Berestein, Janet Braam, Anil K. Sood. Enhancing oral delivery of plant-derived vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2720.

Chromosomal instability (CIN) is one of the major forms of genomic instability in various human cancers and is recognized as a common hallmark of tumorigenesis and heterogeneity. However, some malignant tumors show a paucity of chromosomal alterations, suggesting that tumor progression and evolution can occur in the absence of CIN. It is unclear whether CIN is stable between precursor lesions, primary tumor, and metastases or if it evolves during these steps. In this review, we describe the influence of CIN on the various steps in tumor initiation and development. Given the recognized significant effects of CIN in cancer, CIN-targeted therapeutics could have a major impact on improving clinical outcomes.

Background: Clinical response to therapy of patients diagnosed with high-grade serous ovarian carcinoma (HGSC) is highly variable. Mechanisms underlying response to treatment are not well understood. We previously performed bulk multi-omics analysis aimed at identifying biological differences between poor (PR) and excellent responders (ER) to neoadjuvant chemotherapy (NACT). Although differences were found, these were not as extensive as we had anticipated, suggesting that important molecular differences between such tumors may not be fully evident by bulk analyses. Thus, we carried out in-depth spatial analysis of HGSC to explore heterogeneity of disease and determine how the dynamic behavior between the tumor and the tumor microenvironment varies between PR and ER groups. Methods: We selected a cohort of patients with highly annotated HGSC samples categorized by response to NACT (PR and ER) and analyzed pre-treatment tumor tissues using bulk RNA sequencing (bRNASeq) and Visium Spatial Gene Expression. The definition of PR was stable or progressive disease after 3-4 cycles and/or suboptimal interval cytoreduction after NACT. ER was complete response or only microscopic disease left at the time of interval surgery. Results: To generate expression profiles, we used an unsupervised approach and identified nine distinct clusters, with at least 20 cell types. A comparison of the cell populations identified more stroma-dominated cell groups in the PR group. ER tumors contained more immune-related areas, with a high proportion of T, B and natural killer cells. This finding supports the established role of cytotoxic immune cells in strengthening chemotherapy response and better overall survival. In comparison with bRNASeq, we observed a blending phenomenon that hides deeper heterogeneity of the tumor. Some pathways, such as epithelial-mesenchymal transition (EMT), were statistically enriched in specific clusters of PR compared with the ER group, proving that deep characterization of tumor heterogeneity can be achieved only when exploring specific clusters of cells. The spatial distribution of the clusters showed that in the PR group, the clusters tended to be physically larger and distributed throughout the whole tissue. The ligand-receptor analysis revealed that co-expression of specific ligand-receptor pairing exists depending on their geographical localization, with closer interactions leading to stronger co-expression patterns. Conclusion: Two important findings emerged: importance of the stromal component as a potential driver of poor response to NACT; and identification of differential clusters (distribution and composition) in ER compared with PR tissues. Comparison of the in situ technique with bRNASeq allowed characterization of specific cell subpopulations that might be important determinants of lack of response to therapy, representing candidate therapeutic targets. Citation Format: Elaine Stur, Sara Corvigno, Mingchu Xu, Ken Chen, Sanghoon Lee, Jinsong Liu, Emilly Ricco, Nicole Fleming, Emine Bayraktar, Daniel Kraushaar, Jianhua Zhang, Anil K. Sood. Spatially resolved transcriptomics to understand mechanisms of response to neoadjuvant chemotherapy [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 1175.

Background: High-grade serous ovarian cancer (HGSC) is the most aggressive epithelial ovarian cancer, exhibiting tumor heterogeneity, molecular abnormality, and variable clinical outcomes. The heterogeneity and apparent adaptability of the HGSC genome under selective pressure by chemotherapy likely explain the high rates of drug resistance in HGSC. However, the underlying mechanisms of drug resistance are not well understood. Following our previous findings of significant distinct molecular and cellular characteristics in pre-treatment tumor samples from highly clinically annotated HGSC patients who received neoadjuvant chemotherapy (NACT), we examined post-treatment transcriptomic changes associated with excellent or poor responders to NACT in HGSC patients. Methods: Thirty tumor tissues (pre- and post-treatment) after 3 to 4 cycles of NACT were collected from HGSC patients at interval cytoreductive debulking surgery from the following groups: excellent response to NACT with carboplatin/paclitaxel (post-ER, n=8); poor response to NACT (post-PR, n=8). Primary or metastatic sites for each patient were obtained (n=13, post-ER; n=17, post-PR) and subjected to RNA sequencing. The downstream bioinformatics analysis was then performed and compared with the data from the matched pre-NACT samples from the same patients (Lee et al., Cell Reports, 2020). Results: We identified a total of 9,764 upregulated and 2,762 downregulated differentially expressed genes (DEGs, absolute log2FC >=2, adj-p Conclusions: Our transcriptomic analysis revealed distinct transcripts changes in post-NACT vs pre-NACT tumor tissues from HGSC patients with excellent or poor response to the NACT. These findings could enhance treatment decision-making and rationale for developing alternative therapies for patients with HGSC. Citation Format: Sanghoon Lee, Li Zhao, Nicole D. Fleming, Joseph Celestino, Richard A. Hajek, Margaret B. Morgan, Yan Liu, Shannon N. Westin, Amir A. Jazaeri, Jinsong Liu, Jianhua Zhang, P Andrew Futreal, Anil K. Sood. Transcriptomic profiles of response to neoadjuvant chemotherapy in patients with high-grade serous ovarian cancer [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 5738.

Introduction: Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system despite advances in targeted drugs such as anti-VEGF antibody (AVA). Additionally, resistance to AVA therapy is nearly ubiquitous given the occurrence of metabolic adaptations such as a dependence on glutamine metabolism via the enzyme glutaminase. Objectives: To examine the metabolic vulnerability of AVA-resistant cancer with glutaminase inhibitor (GLSi) therapy and to test the utility of hyperpolarized magnetic resonance spectroscopy (HP-MRS) for assessing changes in lactate metabolism associated with GLSi treatment following injection of hyperpolarized [1-13C] pyruvate. Methods: We used a well-characterized SKOV3ip1 orthotopic mouse model of high-grade serous ovarian cancer with adaptive resistance to AVA (bevacizumab) treatment. Following three weeks of GLSi and bevacizumab treatment, we performed HP-MRS imaging to directly and non-invasively compare metabolic changes between the treatment groups of bevacizumab monotherapy versus a combined treatment of bevacizumab plus GLSi therapy. Upon injection of hyperpolarized [1-13C] pyruvate, we quantitatively assessed a normalized lactate ratio (nLac), defined as the 13C resonance signal of lactate divided by the sum of the pyruvate and lactate signals, sixty seconds after injection. GLSi treatment effect was subsequently compared between GLSi monotherapy, bevacizumab monotherapy and control mice receiving no therapy. This was performed via gross necropsy to assess disease burden and quantification of tumor weight and nodule number. Results: Significance analysis microarrays (SAM) of bevacizumab treated tumors from the mouse models revealed greater glutamine and glutamate abundance in all tissues suggestive of a glutamine dependence in AVA resistant tumors that could be susceptible to glutaminase inhibition. The in vivo HP-MRS provided a direct and non-invasive investigation of pyruvate metabolism of tumors in situ. Our analysis indicated that the pyruvate-to-lactate conversion was significantly reduced in vivo by GLSi treatment (0.337 vs 0.178, p=0.0002), suggestive of therapeutic efficacy of GLSi in the setting of AVA-resistant tumors. Consistent with this, we observed a statistically significant reduction in tumor weight (0.05g vs 0.62g and 0.64g, p Conclusions: Using HP-MRS in vivo, we directly monitored metabolic changes occurring after GLSi treatment. These findings are consistent with the notion of glutamine dependence being an important resistance mechanism to AVA therapy and imply potential future use of HP-MRS in monitoring therapeutic efficacy in a clinical realm. Citation Format: Deanna Glassman, Mark S. Kim, Prasanta Dutta, Charles V. Kingsley, Emine Bayraktar, Yutuan Wu, Elaine Stur, Lingegowda S. Mangala, Katherine Foster, Sanghoon Lee, Timothy A. Yap, Shannon N. Westin, Pratip Bhattacharya, Anil K. Sood. Hyperpolarized magnetic resonance spectroscopy assessment of the metabolic effects of glutaminase inhibitor therapy in an ovarian cancer model [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 2484.

Chemotherapy resistance represents a major problem in ovarian cancer prognosis and reliable indicators of chemotherapy sensitivity remain an unmet need. Here, we examined metabolic profiles of pre- and post-neoadjuvant chemotherapy (NACT) tumor samples from patients with high-grade serous ovarian cancer (HGSC). We performed spatially resolved, desorption electrospray ionization-mass spectrometry (DESI-MS) on sections from clinically annotated HGSC samples collected according to a laparoscopic triage algorithm from patients pre- and post-NACT. Patients were considered to be either excellent (ER) if they presented complete response or only microscopic disease left at interval surgery, or poor responders (PR) if they presented stable or progressive disease after NACT. Tumor tissues from 50 patients (29 ER and 21 PR, 13 ER matched pre and post NACT, 13 PR matched pre and post-NACT) were eligible for the analyses Data relative to the detected metabolites, were collected from epithelium and stroma separately. We first identified differentially upregulated metabolites in ER and PR groups and performed pathway analysis. The over-represented pathways in the chemo-naïve PR tissues included the nucleotide metabolism pathway in epithelial areas, and degradation of cysteine and homocysteine, metabolism of amino acids and derivatives, as well as sulfur amino acids metabolism in the stromal areas (p values FDR adjusted 0.045). In general, both the epithelium and stroma of PR tissues of chemo-naïve tumors, contained a higher number and a higher variety of lipid species compared with ER tissues. In post-NACT tumor epithelial areas, there was global downregulation of metabolites across all patient samples, compared to the pre-chemo specimens; this was more evident in ER patients. In post-NACT tumors from PR patients, glutathione synthesis and recycling pathway, and sulfite oxidation to sulfate pathway and many glycerolipids species were upregulated in the epithelium, while phosphatidic acids were upregulated in the stroma. Lastly, we evaluated the predictive power of the metabolic data extracted by the epithelial areas of chemo-naïve tissues, in classifying patients as ER or PR. We used 10-fold cross-validation to create a ridge regression model for the classification of ER and PR samples from pre-NACT tumor tissues. This model, based on 78 differentially expressed metabolites (small molecules and lipids), correctly classified patients as ER or PR with an accuracy of 76.2% (sensitivity 84.6% and specificity 62.5%). Our findings indicate altered metabolic pathways in ovarian cancer tissues based on response to NACT. The identification of differentially activated pathways, such as sulfate metabolism and glutathione metabolism in tumors with poor response, suggest novel actionable targets to overcome chemotherapy resistance in patients with HGSC. Citation Format: Sara Corvigno, Meredith Spraldin, Michael Keating, Sunil Badal, Igor Pereira, Elaine Stur, Nicholas Bateman, Waleed Barakat, Thomas Conrads, Sanghoon Lee, George Maxwell, Susan K. Lutgendorf, Premal Thaker, Jinsong Liu, Nicole Fleming, Katheleen Darcy, Livia S. Eberlin, Anil K. Sood. Metabolic profiling of pre and post treatment ovarian cancer tissues using in situ mass spectrometry imaging [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 2331.

PARP inhibitors (PARPi) offer major clinical benefit for patients with ovarian or other cancers, especially those with deficiencies in the homologous recombination DNA repair pathway. However, most patients with advanced cancer eventually acquire resistance to PARPi, and the mechanisms by which tumors escape PARPi therapy are still not fully understood. Here, we report that treatment with the PARPi olaparib induces the formation of polyploid giant cancer cells (PGCCs) in ovarian and breast cancer cell lines (Hey, SKOV3, MCF-7), three human high-grade serous cancer (HGSC)-derived organoids, and HGSC patient-derived xenografts. Live-cell fluorescence time-lapse tracking of ovarian cancer cells labeled with FUCCI (fluorescent ubiquitination cell-cycle indicator) or histone H2B-mCherry/α-tubulin-EGFP revealed that PGCCs primarily developed from the endoreplication of diploid cancer cells after exposure to sublethal concentrations of olaparib. PGCCs exhibited markers of senescent cells; however, they were able to escape senescence to generate mitotically competent daughter cells via budding, multipolar mitosis, and acytokinetic mitosis following olaparib withdrawal. PGCCs and derived daughter cells conferred resistance to olaparib-induced cytotoxicity, which could be blocked by the anti-progesterone drug mifepristone. Whole transcriptome analysis by RNA sequencing of ovarian cancer cell lines and ovarian cancer-derived organoids showed activation of a senescence-associated secretory phenotype with upregulated cytokines and chemokines and downregulation of MYC signaling in PGCCs. Mifepristone/olaparib combination therapy significantly mitigated tumor growth in patient-derived xenograft models with acquired resistance to olaparib. Thus, targeting of PGCCs may represent a promising approach to overcome PARPi resistance in recurrent ovarian cancer. Citation Format: Xudong Zhang, Jun Yao, Xiaoran Li, Na Niu, Yan Liu, Anil K. Sood, Jinsong Liu. Targeting polyploid giant cancer cells to overcome resistance to PARP inhibitors in ovarian cancer [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 3249.

Introduction: Long non-coding RNAs (lncRNAs) are important regulators of gene expression and are involved in cancer progression and metastasis. Although studies have shown that aberrant expression of lncRNAs mediates disease progression in ovarian cancer (OC), the role of lncRNA ADAMTS9-AS2 has not been well studied. Objective: To determine the role of ADAMTS9-AS2 on OC pathogenesis. Methods: We examined ADAMTS9-AS2 expression in human OC cells and tumor tissues using in situ hybridization (ISH). ADAMTS9-AS2 was overexpressed in A2780cp20 and IGROV1cp20 cells and evaluated its effects on cell proliferation, viability and apoptosis. To evaluate the role of ADAMTS9-AS2 in tumor suppression, nude mice were injected with ADAMTS9-AS2 over expressing cells. Reverse phase protein array (RPPA) was performed to identify the downstream regulators. Luciferase reporter assay was performed to confirm the interaction of targeted protein with ADAMTS9-AS2. In addition, epigenetic regulation of ADAMTS9-AS2 was studied using 5-Azacytidine. Results: A2780cp20 and IGROV1cp20 cells expressed lower amounts of ADAMTS9-AS2, while SKOV3 and PEO4 cells showed higher expression levels. ISH analysis showed dense nuclear staining of ADAMTS9-AS2 in normal fallopian tube sections, whereas OC sections showed reduced expression. Kaplan-Meier survival analysis showed that patients with high expression of ADAMTS9-AS2 had significantly better overall survival (p=0.0079) and progression-free survival (p=0.00018) than those with low ADAMTS9-AS2 expression. Overexpression of ADAMTS9-AS2 in A2780cp20 cells resulted in decreased proliferation (p Conclusion: ADAMTS9-AS2 acts as a tumor suppressor, by enhancing the expression of ANNEXIN A1, and thereby regulating tumor growth. Thus, ADAMTS9-AS2-ANNEXIN A1 axis may represent a novel therapeutic target in OC. Citation Format: Emine Bayraktar, Guy Katz, Dahai Jiang, Cristian Rodriguez-Aguayo, Lingegowda S. Mangala, Santosh K. Dasari, Elaine Stur, Cristina Ivan, Gabriel Lopez-Berestein, Anil K. Sood. Elucidating the role of ADAMTS9AS2 lncRNA in ovarian cancer [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 1550.

Background: Despite high rates of remission after frontline management, most patients with advanced stage ovarian cancer recur. Second look laparoscopy (SLL) can provide more sensitive detection of minimal residual disease (MRD) allowing for more individualized prognostication and possible therapeutic intervention. It may also assess tumor biology and microenvironment associated with undetectable chemoresistant MRD phase of ovarian cancer. The objective of this study was to determine SLL feasibility, and clinicopathologic and molecular characteristics of MRD after frontline therapy in ovarian cancer. Methods: SLL was offered to patients with stage III-IV high grade epithelial ovarian cancer who achieved complete response after frontline surgery and 6 cycles of carboplatin and paclitaxel chemotherapy. Patients were offered standard of care or investigational management options based on homologous recombination deficiency (HRD) and MRD status at SLL. Preliminary translational studies included RNAseq for comparison of serial, matched tumor from primary or interval TRS and SLL derived MRD+ biopsies at the time of SLL. In addition, Nanostring analyses of SLL surgical biopsies both with and without residual tumor were performed. Molecular profiling employed hierarchical clustering, principle component analysis, non-parametric testing, and pathway analyses. Results: Between 4/2017- 7/2021, 39 patients underwent SLL. The majority had stage III disease (74%), high grade serous histology (90%) and underwent neoadjuvant chemotherapy with interval tumor reductive surgery (TRS; 54%). MRD was present in 49% (n=19) of patients and was more frequent among neoadjuvant chemotherapy recipients (73% vs 26%, P=0.015) and patients with homologous recombination proficient tumors (81% vs 25%, P=0.01). Most patients without MRD were dispositioned to observation; 4 patients with HRD received a PARP inhibitor per standard of care. All other patients with MRD received bevacizumab as part of a clinical trial, except for one patient who received a PARP inhibitor. Median follow-up was 6.74 months (range 0.66 - 28.62). Presence of MRD was associated with worse PFS (HR 3.7, 95% CI 1.3- 10.9; 5.5 vs 24.6 months; P= 0.02). Based on transcriptional signature, MRD are immunologically distinct from untreated tumors or those collected at interval TRS (after 3 cycles of chemo) but also displayed heterogeneity with two distinct subclusters. Conclusions: SLL to assess for MRD has potential to further individualize post-frontline therapy, and identify patients at high risk for progression for early intervention clinical trials. Transcriptional profiling suggests that MRD phase of ovarian cancer is characterized by a distinct, heterogeneous, and evolving tumor and immune microenvironment. Citation Format: Roni Nitecki, Minghao Dang, Sanghoon Lee, Bryan Fellman, J Alejandro Rauh-hain, Jolyn Taylor, Lois Ramondetta, Michaela Grinsfelder, Lauren Cobb, David M. Boruta, Pamela T. Soliman, Aaron Shafer, Nicole D. Fleming, Shannon N. Westin, Anil K. Sood, Chris Tanguma, Pedro T. Ramrez, Karen H. Lu, Linghua Wang, Amir A. Jazaeri. Initial clinicopathologic and molecular characterization of minimal residual disease detected by second look laparoscopy after completion of frontline surgery and chemotherapy in patients with advanced stage ovarian cancer [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 4135.

Background: While high-grade serous ovarian cancers (HGSC) look alike at a histological level, there is marked variation in clinical outcomes; however, the underlying mechanisms are not well understood. We previously highlighted significant distinct molecular and cellular characteristics in highly clinically annotated HGSC tumor samples from patients who underwent complete gross resection (R0) or received neoadjuvant chemotherapy (NACT). Neurofibromatosis type 1 (NF1) is known to negatively regulate the Ras/MAPK signaling pathways and tumorigenesis. Here, we specifically validate the higher loss of NF1 copy number exhibited in the R0 compared to the NACT groups in patients with HGSC. Methods: Fresh-frozen tumor biopsies were collected from patients with HGSC treated under a systematic surgical algorithm: complete gross resection after primary surgery (R0, n=40); poor tumor response to NACT (NACT-PR, n=26); excellent tumor response to NACT with carboplatin and paclitaxel (NACT-ER, n=14). Primary or metastatic tumor sites for each patient were obtained pre-treatment and subjected to high-depth targeted DNA sequencing. Results: The average coverage of 568X for tumors and 291X for germline controls (matched blood samples) was obtained by targeted sequencing for each sample from the entire cohort. The median numbers of non-synonymous somatic mutations identified in the panel sequencing were 5 in R0, 3 in NACT-ER, and 5 in NACT-PR. Sixty-nine of the 80 samples (86.25%) had alterations in at least one of the ovarian cancer-related genes. The most frequently altered genes including somatic mutations and copy number variations were TP53 (68%), NF1 (65%) and ERBB2 (62%) in the R0 group whereas TP53 (50%), AKT1 (48%) and TERT (42%) in the NACT group. Gene alteration enrichment was evaluated by comparing between two groups. 17 genes were found to be enriched for alterations in the R0 group comparing to the NACT group; MLH3 (OR= 15.03, p Conclusions: Our findings, using an independent validation cohort of patients, verified the higher loss of NF1 copy number in the R0 versus the NACT group. These findings could have major implications for clinical decision making and developing novel therapies for patients with HGSC. Citation Format: Sanghoon Lee, Li Zhao, Joseph Celestino, Richard A. Hajek, Margaret B. Morgan, Mark S. Kim, Shannon N. Westin, Amir A. Jazaeri, Nicole D. Fleming, Jinsong Liu, Jianhua Zhang, P Andrew Futreal, Anil K. Sood. NF1 copy number alterations in clinically-defined subsets of high-grade serous ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2200.

The emergence of tumor cells with certain stem-like characteristics, such as high aldehyde dehydrogenase (ALDH) activity due to ALDH1A1 expression, contributes to chemotherapy resistance and tumor relapse. However, clinically applicable inhibitors of ALDH activity have not been reported. There is evidence to suggest that epigenetic regulation of stem-related genes contributes to chemotherapy efficacy. Here, we show that bromodomain and extraterminal (BET) inhibitors suppress ALDH activity by abrogating BRD4-mediated ALDH1A1 expression through a super-enhancer element and its associated enhancer RNA. The clinically applicable small-molecule BET inhibitor JQ1 suppressed the outgrowth of cisplatin-treated ovarian cancer cells both in vitro and in vivo. Combination of JQ1 and cisplatin improved the survival of ovarian cancer–bearing mice in an orthotopic model. These phenotypes correlate with inhibition of ALDH1A1 expression through a super-enhancer element and other stem-related genes in promoter regions bound by BRD4. Thus, targeting the BET protein BRD4 using clinically applicable small-molecule inhibitors, such as JQ1, is a promising strategy for targeting ALDH activity in epithelial ovarian cancer. Cancer Res; 76(21); 6320–30. ©2016 AACR.

The risk for venous thromboembolism (VTE) is increased in cancer and particularly with chemotherapy, and it portends poorer survival among patients with cancer. However, many fundamental questions about cancer-associated VTE, or Trousseau syndrome, remain unanswered. This report summarizes the proceedings of a working group assembled by the NCI and NHLBI in August 2014 to explore the state of the science in cancer-associated VTE, identify clinically important research gaps, and develop consensus on priorities for future research. Representing a convergence of research priorities between the two NIH Institutes, the workshop addressed epidemiologic, basic science, clinical, and translational issues in cancer-associated VTE. Cancer Res; 76(13); 3671–5. ©2016 AACR.

Objectives: To evaluate safety and efficacy of durvalumab (D) in combination with paclitaxel (P) and carboplatin (C) in newly diagnosed, advanced-stage, high grade, non-mucinous ovarian cancer (OC). To investigate informative pharmacodynamic changes including proteome changes and immune-related pathways by treatment with immuno-oncology therapy in combination with chemotherapy. Methods: In this single arm phase II study, patients (pts) with untreated, advanced stage (III/IV) OC dispositioned to neoadjuvant chemotherapy (NACT) received D (750mg) with weekly P (80mg/m2) and C (AUC6) after baseline tissue biopsy. Matched biopsies were obtained at tumor reductive surgery (TRS) after 3 cycles. Progression-free survival (PFS) and overall survival (OS) were estimated using Kaplan-Meier method. Reverse-phase protein arrays (RPPA) were performed on pre- and post-treatment tissues. Protein expression alteration and an enriched Reactome pathway analysis using differentially expressed proteins between pre- and post-treatments were assessed. Results: 19 pts were enrolled and 18 treated. Median age was 63 and 50% had stage III disease. No dose-limiting toxicities were observed. 94% of pts had an adverse event (AE). Most common AEs were anemia (94%), neutropenia (83%), fatigue (72%), hyperglycemia (73%), and thrombocytopenia (66%). Immune-related AEs observed included hyperthyroidism (22%), hypothyroidism (22%), diarrhea (6%), hypophysitis (6%). Eighty-three percent had optimal interval TRS, 70% had a pre-operative response by RECIST (PR=11, CR=1). At 13 months (mo) median follow up, 9 pts (50%) had disease progression and median PFS was 14.5 mo (95% CI: 9.2 - NA). Median OS was not reached; OS rate at 18 mo was 69% (95% CI 0.21 - 0.91). Fifty-five tumor specimens were available from 9 pts (31 pre- and 24 post-treatment). Thirteen significantly differentially expressed proteins among 211 proteins (adj. p Citation Format: Shannon N. Westin, Sanghoon Lee, Li Zhao, Ying Yuan, Jimin Wu, Richard Hajek, Sara Sharafi, Larissa A. Meyer, Nicole D. Fleming, Amir Jazaeri, Robert L. Coleman, Karen H. Lu, Gordon B. Mills, Jianhua Zhang, Andrew Futreal, Anil K. Sood. Pharmacodynamic changes by durvalumab in combination with chemotherapy in women with untreated, advanced stage ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT188.

Background: Prior molecular characterization efforts for high-grade serous ovarian cancer (HGSOC) were restricted to those who had upfront surgical debulking with variable treatment paradigms. Thus, we sought to examine molecular and cellular differences between clinically defined groups (tumor tissues from patients who had complete gross resection (CGR) versus those who were triaged to neoadjuvant chemotherapy (NACT) and experienced either excellent or poor response. Methods: Tumor biopsies were collected from three patient groups managed under a systematic surgical algorithm: CGR after primary surgery (R0, n=10); poor tumor response to NACT (NACT-PR, n=10); excellent tumor response to NACT (NACT-ER, n=10). Primary and multiple metastatic tumor sites per each patient were obtained pre-treatment and subjected to comprehensive omics analyses including high-pass whole-genome (WGS) and targeted deep DNA sequencing. Results: An average of 71 nonsynonymous somatic mutations from each sample for 75 samples with high-purity tumors (≥75%) by WGS were identified from each sample for the entire cohort. Fourteen ovarian-cancer-associated genes were found mutated in our patient cohort and, as expected, the most frequently mutated gene was TP53 in both primary and metastatic sites in all three groups. TP53 nonsense mutations were exclusively identified in the NACT-ER (36.0%) and NACT-PR groups (15.4%), while in the R0 group most TP53 mutations were missense mutations (62.5%). Nonsense mutations in CSMD3 and PIK3CA were exclusively identified in both primary and metastatic sites in the NACT-PR group. The most frequent copy number variations (CNVs) in the R0 were copy number gain/loss of CSMD3 (67%) and copy number loss of NF1 (54%) and CDK12 (50%) in both primary and metastatic sites. Interestingly, copy number losses of NF1 were significantly lower in the NACT groups (18%, p=0.002), especially in the NACT-PR (8%, p=0.0004), when compared to the R0 group (54%). We also identified significant less observation of chromothripsis-like patterns, and a significantly higher level of strong-binding neoantigens in the R0 than in the NACT groups. Conclusions: Our findings using HGSOC samples obtained from patients treated on a prospective algorithm identified distinct molecular abnormalities, and could have prognostic and therapeutic implications for patients with HGSOC. Citation Format: Sanghoon Lee, Li Zhao, Shannon N. Westin, Nicholas W. Bateman, Amir A. Jazaeri, Nicole D. Fleming, Karen H. Lu, Robert L. Coleman, Gordon B. Mills, Jianhua Zhang, Thomas P. Conrads, George L. Maxwell, P. Andrew Futreal, Anil K. Sood. High-depth whole genome sequencing of clinically-annotated high-grade serous ovarian cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2510.

Introduction: EphB4 is frequently overexpressed in ovarian and other solid tumors and involved in interactions between tumor cells and tumor microenvironment, contributing to metastasis. EphB4 and its membrane-bound ligand ephrin B2 (EFNB2) mediates bi-directional signaling. Currently, no small-molecule-based, peptides with dual functional binding to EphB4 are available. Therefore, we developed an EphB4 agonist that is capable of activating the forward EphB4 signaling and interfering with the reverse EFNB2 signaling. Methods: A high-affinity bi-directional ephrin agonist peptide (BIDEN-AP) that selectively activated EphB4 was discovered through structural modifications to the known EphB4 antagonist TNYLFSPNGPIARAW (TNYL-RAW) by surface plasma resonance and cell based assays. To improve pharmacokinetics and enhance peptide stability, BINDEN-AP peptide was conjugated to Core-Crosslinked Polymeric Micelles (CCPM). Anti-tumor and anti-angiogenic effects of BINDEN-AP peptide and its conjugate with CCPM were evaluated using orthotopic human A2789cp20-Luc ovarian cancer xenograft model, and PDX model of high grade serous ovarian carcinoma (HGSC). Results: We found that BIDEN-AP was selectively internalized via receptor-mediated endocytosis, mediated phosphorylation of EphB4 and its downstream adaptor protein Crk1 (MAPK14), and suppressed invasion and epithelial-to-mesenchymal transition in ovarian cancer cells. BIDEN-AP also inhibited endothelial migration and tube formation by 50% (p Conclusion: Given the robust anti-tumor effects of BIDEN-AP, these data could have direct relevance for further therapeutic development. Citation Format: Chiyi Xiong, Yunfei Wen, Jun Zhao, Dengke Yin, Lingyun Xu, Anca Chelariu-Raicu, Cody Yao, Xiaohong Leng, Jinsong Liu, Rajan R Chaudhari, Shuxing Zhang, Anil K Sood, Chun Li. Targeting forward and reverse EphB4/EFNB2 signaling by a peptide with dual functions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 122.

Objective: Autophagic cell death is an important mechanism induced by blocking folate receptor (FRα) in inhibiting tumor growth. Here, our objective is to examine the biological effects of IMGN853 (mirvetuximab soravtansine) in preclinical models of ovarian cancer and to identify potential combination therapies. Methods: In vitro, immunoblotting, assays for cell viability, proliferation and cell cycle by fluorescence-activated cell sorting (FACS) were performed in an array of high grade serous carcinoma (HGSC) cell lines. Autophagy flux and activities of autophagy factors were also determined. In vivo, metastasis mouse models were used for testing the therapeutic efficacy of IMGN853 monotherapy or in combination with olaparib or cisplatin. Potential synergy with these drugs was assessed using in vitro assays. The resulting tumors were subjected to immunohistochemistry and reverse phase protein arrays (RPPA). Results: Data from the Cancer Genome Atlas Data (TCGA) revealed that FRα copy-number alterations was significantly correlated with shorter disease-free survival in patients with HGSC. Autophagy marker LC3 was upregulated following monotherapy with IMGN853 as well as in combination of IMGN853 with olaparib or cisplatin in HGSC cell lines such as OVCA8 and OVCA432, which express high-level of FRα. Experiments using metastatic HGSC models showed that IMGN853 monotherapy reduced tumor growth (30% change); the combination of IMGN853 and olaparib was superior to olaparib monotherapy (24% change; p 0.0033). Integrated analysis on RPPA revealed increased expression of ABL1 and PEA15 in the groups treated with IMGN853 monotherapy or in combination with olaparib. We further found reduced FRα expression and increased caspase 3 expression in IMGN853 groups compared with control. Conclusion: Our results point to sustained autophagy in FRα positive cells as a mechanism of effect of IMGN853. These findings could have implications for clinical development of anti-FRα therapies, especially in combination with other drugs such as olaparib. Citation Format: Anca Chelariu-Raicu, Elaine Stur, Cristina Ivan, Robert L. Coleman, Anil K. Sood, Wen Yunfei. Biological effects of a FRα-targeting antibody-drug conjugate, IMGN853 (mirvetuximab soravtansine) in high-grade serous ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1237.

Background: High-grade serous ovarian cancer (HGSOC) remains the leading cause of death from gynecologic malignancies. Here, we examined tumoral proteome changes following neoadjuvant chemotherapy (NACT) to identify potential predictive and prognostic biomarkers of response to primary chemotherapy. Methods: A total of 65 tissue specimens from 10 patients with advanced-stage HGSOC were collected from matched pre- and post-NACT (3 cycles of dose-dense paclitaxel and carboplatin). Protein expression was assessed using reverse-phase protein arrays (RPPA). All relative protein levels using 297 antibodies were normalized by the expression level of pre-treatment samples, and then protein expression alterations and functional analyses were performed by Reactome pathway analysis followed by statistical analysis. Results: The protein expression patterns of samples tended to cluster according to the time point (pre- and post-treatment) by a non-supervised clustering analysis using all the proteins assessed in the RPPA panel. Five differentially expressed proteins among 239 proteins (adj. p Conclusion: Our findings identified significant proteomic alterations following NACT, and could provide insights into interval proteomic alterations following induction chemotherapy in advanced-stage ovarian cancer patients. These data present information to optimize future clinical trial designs for patients with ovarian cancer. Citation Format: Sanghoon Lee, Li Zhao, Joseph Celestino, Kelly M. Rangel, Richard A. Hajek, Mark S. Kim, Sara E. Sharafi, Jinsong Liu, Nicole D. Fleming, Karen H. Lu, Jianhua Zhang, P. Andrew Futreal, Gordon B. Mills, Shannon N. Westin, Anil K. Sood, Amir A. Jazaeri, Robert L. Coleman. Functional proteomic aberrations post-chemotherapy with paclitaxel and carboplatin in patients with advanced ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2941.

Purpose: There is limited success with chemotherapeutic agents in women with advanced or recurrent endometrial cancer. Dysregulation of the PI3K/RAS signaling pathways in endometrial cancer have been well documented. However, responses with mTOR inhibitor such as temsirolimus have been modest. Previously we have shown that metformin prevents temsirolimus-induced AKT activation. Therefore, we enrolled patients with advanced endometrial carcinoma in the expansion cohort of a phase I study of temsirolimus in combination with metformin. Methods: Patients with advanced endometrial cancer refractory to or relapse after standard therapies, ECOG performance status 0 or 1, and with significant organ function reserve were enrolled. Patients were administered intravenous temsirolimus 25mg weekly and oral metformin 2000 mg daily in 28-day cycles. Response was assessed every 2 cycles by clinical evaluation, tumor markers, and imaging per RECIST 1.1. All toxicities were graded using NCI CTCAE, version 4.0. Results: Forty patients were treated. Median age is 67 years (range, 33-78). Drug-related adverse events of any grade were reported in 34 patients. The most common toxicities were mucositis (n=13), AST increase (n=13), anorexia (n=12), diarrhea (n=12) and anemia (n=10). Eleven grade 3 drug-related adverse events were reported. They were anemia (n=2), thrombocytopenia (n=2), mucositis, fatigue, weight loss, hypokalemia, hypophosphatemia, AST increase and ALT increase (n=1 each). Of the 33 patients evaluable for response, objective response was seen in 2 (6%) patients. Both had partial response (PR) and were on the study for 8.7 and 18.2 months respectively. In addition, 13 (39%) patients had stable disease (SD), including 11 with SD ≥4 months, representing a clinical benefit rate of 39%. Molecular characterization of tumor was available for 35 patients. Thirty of 35 patients had molecular alterations in the PI3K and/or RAS pathway. Of the 30 patients, 1 had benign PI3K mutation and 4 were not evaluable for response. Of the remaining 25 patients with PI3K and/or KRAS pathway, 11 (44%) had either objective response or SD ≥4 months. Eighteen of them had molecular alteration only in the PI3K pathway. Seven of 18 (39%) had objective response or SD ≥4 months. Importantly, all 3 patients who had molecular alterations in both the PI3K and RAS pathway achieved SD ≥4 months, while 3 of the 4 patients with exclusive resistant KRAS mutation had progressive disease. Collectively, of the 13 patients with either objective response or SD ≥4 months, 11 (85%) patients had molecular alteration in the PI3K and/or RAS pathway. Conclusion: Temsirolimus in combination with metformin was well tolerated. Anti-tumor activity was seen in patients with advanced/refractory endometrial cancer, particularly in patients with molecular alterations in the PI3K and/or RAS pathway that warrants further study. Citation Format: Aung Naing, Daniel Karp, Sarina A. Piha-Paul, Shubham Pant, Vivek Subbiah, Diane C. Bodurka, Siqing Fu, Amir A. Jazaeri, Shumei Kato, Kathleen Schmeler, Alpa Nick, Yali Yang, Fechukwu O. Akhmedzhanov, Senait Fessahaye, Jing Gong, Bettzy Stephen, Amber M. Johnson, Pamela T. Soliman, Anil K. Sood, Funda Meric-Bernstam, Karen H. Lu. Temsirolimus in combinaton with metformin in patients with advanced or refractory endometrial cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT163.

Background: Psychological stress can promote progression of gynecological malignancies by increasing secretion of sympathetic nervous system (SNS) mediators, namely catecholamines. However, the mechanisms underlying these effects are poorly explored. Here, we examined the effect of stress hormones on small extracellular vesicles (EVs) release in gynecological malignancies and the biological impact of these stress-conditioned EVs on the tumor microenvironment. Methods: Supernatants were collected from epithelial ovarian cancer cell lines treated with norepinephrine, epinephrine or hydrocortisone. Small EVs were isolated from the supernatants and assessed for number and particle size. Auto re-uptake studies, invasion assays and tube formation assays were performed on ovarian cancer cells and RF24 endothelial cells exposed to control and stress-conditioned EVs. Tipifarnib was used to inhibit small EV secretion to study dependence of outcomes of previously performed functional assays on EV number. Mass spectrometry (MS) was performed on control and stress-conditioned small EVs to compare protein content. Reverse-phase protein array (RPPA) was performed on RF24 cells exposed to control and stress-conditioned EVs to observe differences in active pathways. Results: Addition of 1uM and 10uM of catecholamines and glucocorticoids increased small EV secretion by ~ 4-fold in OVCAR5 and HeyA8 cell lines. Maximum increase in secretion was seen between 8 hr and 24 hr after treatment with stress hormones, after which secretion decreased. EV uptake experiments with ovarian cancer cells showed that the percentage of uptake of stress-conditioned EVs at all timepoints was the same, around 65%, indicating that the capacity of the cell to produce EVs might be decreasing after 24 hours. Stress-conditioned EVs increased invasion of ovarian cancer cells and tube formation capacity of RF24 cells by ~ 2-fold. MS analysis of EV contents from control and stress-conditioned EVs showed a differential expression of angiogenesis and cell growth-related proteins in the stress-conditioned EVs alone (p=0.007). Conclusions: Our results demonstrate that addition of stress-hormones increases the angiogenic potential of cancer cell-derived small EVs. Further exploration of the effects of stress-conditioned small EVs on cancer cells and other cell types in the tumor microenvironment might uncover other mechanisms of stress-induced cancer progression. AACR Category: TB06 (Tumor Microenvironment) Key words: ovarian cancer, EVs, norepinephrine, angiogenesis Citation Format: Sujanitha Umamaheswaran, Santosh K. Dasari, Akira Yokoi, Elaine Stur, Anil K. Sood. Effect of sympathetic nervous system mediators on the tumor microenvironment via small extracellular vesicles in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6202.

Despite the wide use of anti-vascular endothelial growth factor (VEGF) therapy (AVT) for many solid cancers, most of them become resistant to such therapy, resulting in progressive disease. Therefore, new biomarkers and strategies for blocking adaptive resistance of cancer to AVT are needed. Small extracellular vesicles (small EVs, with a size of Citation Format: Shaolin Ma, Lingegowda S. Mangala, Wen Hu, Emine Bayaktar, Wei Hu, Nicholas B. Jennings, Robert L. Coleman, Gabriel Lopez-Berestein, Anil K. Sood. Cloaking VEGF in small extracellular vesicles as a cause of adaptive resistance to anti-VEGF therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6331.

Background: After the frontline treatment, the majority of patients diagnosed with ovarian cancer at an advanced stage will develop recurrence (80%); thus, there is an urgent need for novel therapeutic approaches. Purpose: Selinexor is a CRM1 inhibitor, which is a selective inhibitor of nuclear export and has demonstrated antitumor effects in ovarian cancer. Here, we examined the efficacy of selinexor alone and in combination with PARP inhibitors in ovarian cancer. As Selinexor reduces the expression of DNA damage repair proteins, we hypothesized that this combination should reduce the number of DNA damage repair proteins, resulting in synergistic tumor cell death. Methods: Western blot was performed to assess baseline CRM1 expression in eleven ovarian cancer cell lines. We tested the efficacy of selinexor alone and in combination with olaparib in ovarian cancer cells (A2780ip2, OVCAR5). Combination Indices (CI) were calculated with CompuSyn and were considered synergistic when CI less than 1 for classic MTT arrays. We used the annexin V-FITC/PI assay to assess apoptosis as a result of selinexor and olaparib alone or in combination. Results: Baseline CRM1 expression was positive in eight of eleven ovarian cancer cell lines tested. The average IC50 in A2780ip2 for selinexor and olaparib was 0.09uM and 0.97uM, respectively. The median combination index was 0.81 in A2780ip2 and 0.92 in OVCAR5 cells. Flow cytometry demonstrated an additive effect of selinexor and olaparib in A2780ip2 cell apoptosis (7.34% and 7.93%, respectively, v. 17.86%). Conclusions: Selinexor demonstrates synergistic activity with olaparib, and this combination warrants further investigation. Citation Format: Katelyn F. Handley, Cristian Rodriguez-Aguayo, Shaolin Ma, Mark S. Kim, Prahlad T. Ram, Anil K. Sood. Rational combination approaches with CRM-1 inhibitor in ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 569.

Whole genomic duplications (WGD) have been reported in 37% to 56% of high-grade cancers. WGD leads to formation of polyploid giant cancer cells (PGCCs) with salient and atypical nuclear morphology noted by light microscopy. However, PGCCs are generally considered nonviable due to their inability to execute mitosis. We have recently reported that PGCCs are capable of generating mitosis-competent progeny cells via primitive amitotic division including budding, splitting, and burst. We have further demonstrated that PGCCs are part of the process of somatic reprogramming named the giant cell life cycle composed of four distinct but overlapping phases including initiation, self-renewal, termination, and stability. The giant cell life cycle resets the somatic cell from the mitotic cycle to stress-induced endoreplication cell cycle via WGD to form PGCCs. The PGCCs activate endogenous embryonic factors for the reprogramming, which leads to the birth of drug-resistant cells by clonal selection from newly generated heterogeneous cancer stem cells. However, the underlying genetic and epigenetic mechanisms during WGD remain unclear. By mapping DNA methylation and whole-genome sequencing, monitoring the telomere dynamics, and tracking the cellular and nuclear morphology using microscopic time-lapse imaging observations during the time course of giant cell life cycle, we hereby provide new evidence that the daughters cells derived from PGCCs have massive alterations at the whole-genome and epigenome levels. The reprogrammed resistant daughter cells are achieved, at least in part, by genomic instability via shortened telomeres. Targeting PGCCs and promoting their differentiation during the course of giant cell life cycle may represent a novel approach to intercept drug resistance. Citation Format: Na Niu, Xiaoran Li, Jun Yao, Anil Sood, Jinsong Liu. Polyploid giant cancer cells lead to evolution of drug resistance in high-grade ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A17.

Introduction: Approximately 70% of people living with metastatic breast cancer have metastases to their bones. Osteolytic bone metastasis is a common, debilitating and essentially incurable skeletal complication of breast cancer, in which tumor cells migrate to and destroy bones, causing extreme pain, fractures, life-threatening hypercalcemia, limited mobility and eventually motality. The bone resorbing osteoclasts significantly contribute to this process. MicroRNAs (miRNAs) play important roles in physiology and disease, and present tremendous therapeutic potential. Nonetheless, how miRNAs regulate skeletal biology is underexplored. We hypothesize that miRNAs that can suppress osteoclast function may ameliorate breast cancer bone metastasis. Methods: A strategy of ex vivo osteoclast differentiation from mouse bone marrow cells was used to examine the levels of several cancer-related miRNAs during a time course of osteoclastogenesis, and test the effects of miR-34a mimic or inhibitor. Micro-Computed Tomography was conducted to quantify bone mass. ELISA analyses were performed to measure serum bone resorption and bone formation markers. Ovariectomy was employed as a model for postmenopausal osteoporosis. As a model for bone metastases, luciferase labelled bone-metastasis prone MDA-MB-231 human breast cancer cell subline were injected into the left cardiac ventrical of nu/nu mice. Bone metastases were detected and quantified weekly post injection by bioluminescence imaging. Results: MiR-34a is down-regulated during osteoclast differentiation. Osteoclastic miR-34a over-expressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast cancer are diminished in osteoclastic miR-34a transgenic mice. Pharmacologically, systemic delivery of miR-34a mimics via a chitosan nanoparticle vehicle (miR-34a-CH) can target multiple tissues. Our bio-distribution analysis shows that miR-34a-CH delivery to the bone marrow is among the highest compared to other tissues. Administration of miR-34a nanoparticles not only diminished ovariectomy induced bone loss but also attenuated breast cancer bone metastasis. Systemic miR-34a-CH delivery affected neither tumor growth when cancer cells were injected subcutaneously nor tumor metastasis to other organs such as lung when cancer cells were injected intravenously. These results further support the notion that the suppression of bone metastases by miR-34a-CH was mediated by its inhibition of the bone metastatic niche rather than the cancer cells. Mechanistically, we identify the homeodomain transcription factor Tgif2 as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Conclusion: Using mouse genetic, pharmacological and disease models, we have identified mir-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche, revealing miR-34a as a potential new therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of breast cancer. Citation Format: Jing Y Krzeszinski, Wei Wei, HoangDinh Huynh, Zixue Jin, Xunde Wang, Tsung-Cheng Chang, Xian-jin Xie, Lin He, Lingegowda S Mangala, Gabriel Lopez-Berestein, Anil K Sood, Joshua T Mendell, Yihong Wan. MicroRNA-34a suppresses breast cancer bone metastasis by inhibiting osteoclastogenesis and targeting tgif2 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-17-01.

Chemotherapy is commonly used in the treatment of ovarian cancer, yet most ovarian cancers harbor inherent resistance or develop acquired resistance. Therefore, novel therapeutic approaches to overcome chemoresistance are required. In this study, we developed a hyaluronic acid-labeled poly(d,l-lactide-co-glycolide) nanoparticle (HA-PLGA-NP) encapsulating both paclitaxel (PTX) and focal adhesion kinase (FAK) siRNA as a selective delivery system against chemoresistant ovarian cancer. The mean size and zeta potential of the HA-PLGA-NP were 220 nm and -7.3 mV, respectively. Incorporation efficiencies for PTX and FAK siRNA in the HA-PLGA-NPs were 77% and 85%, respectively. HA-PLGA-NP showed higher binding efficiency for CD44-positive tumor cells as compared with CD44-negative cells. HA-PLGA (PTX+FAK siRNA)-NP caused increased cytotoxicity and apoptosis in drug-resistant tumor cells. Treatment of human epithelial ovarian cancer tumor models HeyA8-MDR (P < 0.001) and SKOV3-TR (P < 0.001) with HA-PLGA (PTX+FAK siRNA)-NP resulted in significant inhibition of tumor growth. Moreover, in a drug-resistant, patient-derived xenograft (PDX) model, HA-PLGA (PTX+FAK siRNA)-NP significantly inhibited tumor growth compared with PTX alone (P < 0.002). Taken together, HA-PLGA-NP acts as an effective and selective delivery system for both the chemotherapeutic and the siRNA in order to overcome chemoresistance in ovarian carcinoma. Significance: These findings demonstrate the efficacy of a novel, selective, two-in-one delivery system to overcome chemoresistance in epithelial ovarian cancer. Cancer Res; 78(21); 6247–56. ©2018 AACR.

The MYC oncogene broadly promotes transcription mediated by all nuclear RNA polymerases, thereby acting as a positive modifier of global gene expression. Here, we report that MYC stimulates the transcription of DANCR, a long noncoding RNA (lncRNA) that is widely overexpressed in human cancer. We identified DANCR through its overexpression in a transgenic model of MYC-induced lymphoma, but found that it was broadly upregulated in many human cancer cell lines and cancers, including most notably in prostate and ovarian cancers. Mechanistic investigations indicated that DANCR limited the expression of cell-cycle inhibitor p21 (CDKN1A) and that the inhibitory effects of DANCR loss on cell proliferation could be partially rescued by p21 silencing. In a xenograft model of human ovarian cancer, a nanoparticle-mediated siRNA strategy to target DANCR in vivo was sufficient to strongly inhibit tumor growth. Our observations expand knowledge of how MYC drives cancer cell proliferation by identifying DANCR as a critical lncRNA widely overexpressed in human cancers. Significance: These findings expand knowledge of how MYC drives cancer cell proliferation by identifying an oncogenic long noncoding RNA that is widely overexpressed in human cancers. Cancer Res; 78(1); 64–74. ©2017 AACR.

Triple-negative breast cancer (TNBC) is a highly heterogeneous and recurrent subtype of breast cancer that lacks an effective targeted therapy. To identify candidate therapeutic targets, we profiled global gene expression in TNBC and breast tumor-initiating cells with a patient survival dataset. Eight TNBC-related kinases were found to be overexpressed in TNBC cells with stem-like properties. Among them, expression of PKC-α, MET, and CDK6 correlated with poorer survival outcomes. In cases coexpressing two of these three kinases, survival rates were lower than in cases where only one of these kinases was expressed. In functional tests, two-drug combinations targeting these three kinases inhibited TNBC cell proliferation and tumorigenic potential in a cooperative manner. A combination of PKC-α-MET inhibitors also attenuated tumor growth in a cooperative manner in vivo. Our findings define three kinases critical for TNBC growth and offer a preclinical rationale for their candidacy as effective therapeutic targets in treating TNBC. Cancer Res; 74(17); 4822–35. ©2014 AACR.

Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific T cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5low cells conferred immune resistance. Mechanistic investigations revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCδ/ERK effector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCδ, or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies. Cancer Res; 74(13); 3556–66. ©2014 AACR.

The Notch pathway plays an important role in the growth of high-grade serous ovarian (HGS-OvCa) and other cancers, but its clinical and biologic mechanisms are not well understood. Here, we found that the Notch pathway alterations are prevalent and significantly related to poor clinical outcome in patients with ovarian cancer. Particularly, Notch3 alterations, including amplification and upregulation, were highly associated with poor patient survival. Targeting Notch3 inhibited ovarian cancer growth and induced apoptosis. Importantly, we found that dynamin-mediated endocytosis was required for selectively activating Jagged-1–mediated Notch3 signaling. Cleaved Notch3 expression was the critical determinant of response to Notch-targeted therapy. Collectively, these data identify previously unknown mechanisms underlying Notch3 signaling and identify new, biomarker-driven approaches for therapy. Cancer Res; 74(12); 3282–93. ©2014 AACR.

Background: Preclinical data from our team demonstrated that MEK inhibition increases antitumor efficacy of PARP inhibitors in tumors with Ras pathway alterations. We sought to identify dose limiting toxicities (DLTs) and maximum tolerated dose (MTD) of olaparib and selumetinib combination in solid tumors with Ras pathway alterations. Methods: Olaparib and selumetinib were given orally twice daily. Dose escalation with three planned dose levels (DL) was performed using the modified toxicity probability interval 2 design with toxicity assessed by CTCAE v4.03. RECIST criteria (v1.1) were used to assess response. Clinical benefit was defined as partial (PR) or complete response, or stable disease for 4 months or longer. Results: 14 patients are evaluable for toxicity in the dose escalation phase, and 12 patients are evaluable for response. Median age was 56.5 years, and 10/14 (71%) patients were female. Median number of prior treatments was 4 (1-9). Of the 14 patients enrolled, 11 had KRAS mutations, 1 amplified KRAS, 1 NRAS mutation and 1 amplified NRAS. The majority of patients had gynecologic cancers. 4 and 3 patients were treated at DL1 and DL2, respectively, and 7 patients at DL3. No DLTs were observed (Table 1). MTD was not reached. DL3 (olaparib 300mg; selumetinib 75mg) was confirmed as the recommended Phase II dose (RP2D). Of 12 evaluable patients, ORR was 17%, and CBR 33%. 2 patients had a PR: 1 patient with KRAS mutant primary peritoneal cancer, and 1 with NRAS mutant ovarian cancer. 2 patients remained on treatment for more than 15 months: 1 with KRAS mutant primary peritoneal cancer (on DL-1), and 1 with KRAS mutant non-small cell lung cancer (DL1). All patients with clinical benefit were BRCA wildtype. Conclusions: The combination of olaparib and selumetinib is well-tolerated at the identified RP2D. This combination also shows promising preliminary anti-tumor activity in patients with mutant RAS. Enrollment to expansion cohorts is ongoing. Table 1Grade 3/4 and Most Common Adverse Events. NOTE: No Grade 4 Events Occurred.Adverse EventGrade 3Any GradeAbdominal pain7%7%Acneiform Rash7%71%Anemia79%Anorexia29%Constipation29%Decreased ejection fraction7%14%Decreased white blood cell count7%36%Diarrhea50%Dizziness29%Dry mouth43%Dry skin29%Dysgeusia36%Edema29%Elevated aspartate aminotransferase7%50%Elevated bilirubin7%7%Elevated CPK7%36%Elevated creatinine29%Fatigue7%64%Hepatic pain7%7%Hypophosphatemia79%Nausea57%Neutropenia7%21%Oral mucositis50%Other skin effects36%Thromboembolic event7%7% Citation Format: Katherine C. Kurnit, Funda Meric-Bernstam, Kenneth Hess, Robert L. Coleman, Priya Bhosale, Katerina Savelieva, Filip Janku, David Hong, Aung Naing, Shubham Pant, Jordi Rodon, Timothy A. Yap, Anil K. Sood, Pamela T. Soliman, David M. Gershenson, Gordon B. Mills, Shannon N. Westin. Phase I dose escalation of olaparib (PARP inhibitor) and selumetinib (MEK Inhibitor) combination in solid tumors with Ras pathway alterations [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 CT020.

Non-coding RNAs (ncRNA), which account for more than 75% genome transcripts, have unique functions in regulating cancer pathogenesis. Long non-coding RNAs (lncRNAs) are key regulators of gene expression and are involved in cancer progression and metastasis. However, the role of long non-coding RNAs (lncRNAs) in high-grade serous ovarian cancer (HGSC) is not well understood. We used the NCode™ non-coding RNA array to screen downregulated lncRNAs in HGSCs. After analyzing the array data of lncRNAs in HGSCs (n=29) and normal ovarian (n=11) samples, the probes mapping to the top 25 most down-regulated non-coding RNAs were analyzed. Two lncRNAs (ADAMTS9-AS1 and ADAMTS9-AS2) were identified as the antisense of gene ADAMTS9, and we validate by in vitro translation assay, q-RT-PCR, in-situ hybridization and the biological functions of ADAMTS9-AS2 in ovarian cancer cells. Moreover, using A2780-CP20 orthotopic mouse models of ovarian cancer, we further demonstrated restoring ADAMTS9-AS2 resulted in profound reduction of tumor growth and metastasis. Here, we demonstrate that ADAMTS9-AS2 is highly down-regulated (5.01x10-13 and 1.6x10-19) in ovarian cancers. Overexpression of ADAMTS9-AS2 leads to significantly increased apoptosis (7.5% control and 19.5% AS2OE) and decreased proliferation (49.5% control and 37.0% AS2OE) compared with controls. Consistently, increased expression of ADAMTS9-AS2 led to significantly reduced tumor burden in A2780-CP20 orthotopic ovarian cancer mouse models. We identified the promoter region of Annexin A1 as the major target of ADAMTS9-AS2. The binding of lncRNA ADAMTS9-AS2 to the promoter region can facilitate the expression of tumor suppressor Annexin A1 thereby decrease the proliferation and increase apoptosis of cancer cells. Collectively, our data point to an important role for ADAMTS9-AS2 as a tumor suppressor in ovarian cancer. Note: This abstract was not presented at the meeting. Citation Format: Cristian Rodriguez-Aguayo, Emine Bayraktar, Lingegowda S. Mangala, Cristina Ivan, Gabriel Lopez-Berestein, Anil K. Sood. Long non-coding RNA ADAMTS9-AS2 inhibits ovarian tumor growth via Annexin A1 [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 3579.

TP53 is the most commonly mutated gene in cancer. Certain mutations can produce gain-of-function (GOF) phenotypes, resulting in pathogenic effects beyond the loss of p53 function. Recent study suggested that tumor cells could affect the p53 status of stromal cells while the mechanisms remains unclear. Exosomes have emerged as an important mode of extracellular communication and a major player in cancer pathogenesis, by promoting pro-tumor microenvironment. We hypothesized that packaging of mutated p53 protein into exosomes and dissemination via exosomes could impact a variety of stromal cell processes, producing a pro-tumorigenic microenvironment. In this study, we carried out a series of in vitro (immunoblot analysis, Q-PCR, Gene array, Co-Immunoprecipitation and lentiviral transfection) and in vivo (subcutaneous mouse models) experiments to determine the presence of mutant p53 in exosomes and its biological significance in tumor microenvironment. We demonstrated that gain-of-function (GOF) p53 protein is packaged into p53 mutant cancer cell-derived exosomes and transferred to fibroblasts. Further studies showed that this process is facilitated by the enhanced stability and consequent intracellular concentration of GOF p53. The packaging of GOF p53 into exosomes is selectively bound by HSP90 chaperone proteins. Inhibition of HSP90 blocks the packaging of GOF p53, but not wild-type into exosomes. Uptake of GOF p53-containing exosomes by fibroblasts results in conversion to a cancer-associated phenotype. In vivo study have shown that GOF p53 exosomes-induced cancer-associated fibroblasts promote tumor growth in a colon cancer cells (HT29 cells) xenograft mouse model. These findings reveal a crucial role of exosomes in modifying tumor microenvironment and hold great consequence in broadening our understanding of tumor biology. Citation Format: Shaolin Ma, Michael H. McGuire, Elaine Stur, Emine Bayraktar, Alejandro Villar-Prados, Sherry Y. Wu, Akira Yokoi, Santosh K. Dasari, Cristian Rodriguez-Aguayo, Jinsong Liu, Gabriel Lopez-Berestein, Anil K. Sood. Exosomal gain-of-function p53 protein promotes cancer-associated phenotype [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 LB-311.

Aberrations in Gas6/AXL signaling is associated with many human diseases including ovarian cancer, where in patients expressing high levels of AXL have shorter overall survival than patients expressing low levels. Aptamers are short synthetic oligonucleotides (DNA or RNA) that can bind with highest specificity to various other macromolecules such as peptides, proteins and carbohydrates, based on their tertiary structures. Aptamers are similar in their functionality to antibodies in their specificity; easier to manufacture than antibodies; have minimal side effects; and thus hold a high promise as therapeutics. The typical issues with the use of aptamers as therapeutic agents are their selectivity, stability, and bioavailability. We present the development of the next generation DNA aptamers, targeting p-AXL specifically to as a therapeutic in ovarian cancer (OC) by chemical modifications to their primary sequence. Improved bioavailability and stability, based on the comparative analysis the library of 17 DNA aptamers that differed in the position and number of fluoro- and thio- modifications on their nucleotide sequence, were used as the criteria for the selection and characterization of the best aptamer candidates to target p-AXL. Two of the best candidates, GLB-G25 and GLB-A04 thus chosen with the highest bioavailability and stability, when tested in ovarian cancer cell lines, decreased the migration and invasion of cells in vitro. Treatment of ovarian cancer orthotopic murine model animals with modified DNA aptamer candidates chosen above significantly reduced tumor growth and the number of metastases. The candidates, GLB-G25 and GLB-A04 show highest potential to be developed into therapeutics to target p-AXL that is elevated in several cancers including ovarian cancer. Note: This abstract was not presented at the meeting. Citation Format: Paola AMERO, Cristian Rodriguez-Aguayo, Rajan R. Chaudhari, Shuxing Zhang, Anil K. Sood, Gabriel Lopez-Berestein. Next generation DNA aptamers with prolonged stability and antitumor activity [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 368.

Purpose: Extra-pituitary prolactin (PRL) is involved in development and progression of uterine carcinoma. However, the underlying mechanisms of PRL/PRLR axis in uterine cancer are not well understood, and current means of targeting this axis are limited. Here, we used a potent PRL antagonist (G129R) to interfere with oncogenic activities of uterine cancer cells and further inhibit tumor growth. Experimental Design: We performed integrated analyses by comparing expression of PRLR isoforms in a broad panel of tumors and normal countparts using TCGA and GTex databases. Therapeutic effects of G129R treatment in uterine cancers were assessed in both orthotopic PTENwt and PTENmut models. Additionally, we carried out reverse phase protein assay (RPPA) at in vivo condition from the resulting tumors and at in vitro conditions to interrogate downstream targets and mechanism of action for G129R. Further functional analyses including cell viability, cell cycle and nascent protein synthesis were accessed in two types of uterine cancer cells under serum-depleted condition. Results: We found that PRLR_S1a, a short form of PRLR that is generated from alternative splicing during transcription, is expressed in human uterine cancers to a greater extent than other isoforms. Blocking activities of the PRL/PRLR_S1a axis with the antagonist G129R significantly reduced growth and progression of both orthotopic PTENwt and PTENmut uterine tumors. Data from reverse-phase protein arrays (RPPA) by in the resulting tumors showed that pathways involved in epithelial-mesenchymal transition such as PI3K/mTOR activities were significantly downregulated by G129R treatment. In addition, based on the RPPA data from the in vitro uterine cancer cells treated with G129R as monotherapy or in combination with PRL, we discovered a novel function of G129R in initiating cell death mediated by nuclear FOXO3a and inducing the G0/G1 arrest in both PTENwt and PTENmut uterine cancer cells. Furthermore, G129R decreased the nascent protein synthesis in these uterine cancer cells mediated by transcriptional suppressor EIF-4EBP1. Collectively, our findings reveal a new role of the PRLR_S1a isoform in uterine cancer and identify G129R as a potential therapeutic in uterine cancer. Citation Format: Yun-Fei Wen, Ying Wang, Shaolin Ma, Anca Chelari Raicu, Keith A. Baggerly, Anil K. Sood. Blockade of the short-form of prolactin receptor induces FOXO3a/EIF-4EBP1-mediated cell death in uterine 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 3720.

Background: While immune checkpoint inhibitors have revolutionized the treatment of multiple cancers, such therapies have had limited efficacy in ovarian cancer. We hypothesized that a lytic peptide could stimulate local immune response and enhance efficacy of checkpoint blockade. Here, we tested the effects of a synthetic lytic peptide targeting the luteinizing hormone receptor (LHRH-R), EP-100, on immune therapy in ovarian cancer models. Methods: We carried out series of in vitro (MTT assay, immunoblot analysis, cytokine array, and immune profiling assay) and in vivo (orthotopic mouse model) experiments to determine the biological effects of EP-100 monotherapy and its combination with checkpoint blockade, α-PD-L1. Results: We found that LHRH-R positive murine ovarian cancer cells (ID8, IG10, IF5, and 2C12) were sensitive to EP-100 and were specifically killed at low micromolar levels through LHRH-R. The in vivo syngeneic mouse models (ID8 and IG10) demonstrated that EP-100 reduced tumor volume, tumor weight, and ascites volume as a single agent. The greatest effects on tumor volume reduction and ascites volume were observed with the combination of EP-100 and α-PD-L1 antibody, indicating a potentially synergistic effect in vivo. Immune profiling of tumors showed that the population of CD8+ T cells, NK cells, dendritic cells, and macrophages were significantly increased in tumor and ascites treated with α-PD-L1, EP-100 and in the combination group. However, monocytic myeloid suppressor cells, B cells, and regulatory T cells were decreased in tumors treated with α-PD-L1, EP-100 and in the combination group. We found similar effects of EP-100 combination with α-PD-L1 antibody in ascites from each group. These data indicate that EP-100 promotes a tumor microenvironment that is favorable for immune response. In vitro cytokine arrays revealed that EP-100 induced IL-1α, IL-33, CCL20, VEGF, and LDLR secretion. Among them, we validated increasing Il-33 level following EP-100 treatment in vivo and in vitro and determined the specific biological role of CD8+ T cell activation with IL-33 gene silencing using siRNA and Cas9-CRISPR approaches. In addition, we found that CD8+ T cell expressed a low level of LHRH-R and had minimal direct effects from EP-100. Conclusion: These data demonstrate strong efficacy of EP-100 and checkpoint blockade therapy. Our results provide new directions to enhance the efficacy of immune therapies for ovarian cancer. Citation Format: Mark S. Kim, Shaolin Ma, Carola Leuschner, Sanghoon Lee, Robert L. Coleman, Anil K. Sood. Targeting LHRH-R to improve immune response 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 3199.

Metastatic breast cancer is the most dreadful malignant disease that accounts for the majority of cancer-related deaths worldwide among women. A number of studies have shown that the tumor microenvironment (TME) plays a crucial role in regulating metastasis. It is therefore imperative to understand the dynamic interactions between cancer cells and their microenvironment to examine the molecular interaction and to effectively target cancer cells. TME comprises a variety of cells including immune cells which can influence tumor survival, growth and metastasis. Tumor-infiltrating lymphocytes (TILs), in particular, the CD8 T lymphocytes, has emerged as a promising prognostic marker for immunotherapy in a variety of cancers. However, the key molecular factors that regulate the cross-talk between tumor cells and CD8 T lymphocytes and its impact on metastatic traits in breast cancer is still inconclusive. Platelets are crucial components of the tumor microenvironment that are known to modulate tumor promotion and metastasis. The contribution of platelets and platelet secreted molecules are also carefully examined in metastasis of various cancers. The primary objective of this study is to investigate the role of CD8 T lymphocytes and platelets in breast tumor progression using isogenic tumor lines that form identical primary tumors but differ in their ability to develop metastasis. Citation Format: Robiya Joseph, Rama Soundararajan, Suhas Vasaikar, Fei Yang, Sevinj Isgandarova, Lin Tian, Monika Haemmerle, Barbara Mino, Tieling Zhou, Geraldine Vidhya Raja, Esmeralda Ramirez Pena, Petra Den Hollander, Neeraja Bhangre, Crystal Shin, Melisa Martinez, Jaime Rodriguez Canales, Jeffrey Chang, Anil Sood, Ignacio Ivan Wistuba, Don L. Gibbons, Jeffrey M. Rosen, Ghanashyam Acharya, Navin Varadarajan, Xiang H. Zhang, Sendurai A. Mani. Regulation of metastasis by CD8 T lymphocytes [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 3761.

Objective: Several factors have been proposed to contribute to racial disparities in gynecologic cancers, however there are no comprehensive studies that have investigated inherent molecular features that may be racially unique. Our objective was to perform a pan-cancer clinical and molecular analysis of African-Americans (AA) and European American (EA) patients. Methods: We performed cross-platform analyses utilizing cancer registry and molecular databases [Surveillance, Epidemiology and End Results (SEER) and The Cancer Genome Atlas (TCGA)] to evaluate differences in AA vs EA patients. To characterize genetic ancestry we utilized the recently established The Cancer Genetic Ancestry Atlas (TCGAA). We further evaluated epigenetic modifications through non-coding RNA expression in these tumors. Results: A total of 2,045,500 patients (87.5% EA, 12.5% AA) with 64 primary tumor types were evaluated using SEER to evaluate clinical outcome differences. Compared to white race, AA race had higher rate of death in 45 cancer types (HR>1.0, CI 1.00-5.23). 19 cancer types were then identified to have the largest differences in overall survival (HR>1.2). Utilizing TCGA, we analyzed molecular data of 7028 tumors (88.9% EA, 10.9% AA) having miRSeq data and genetic ancestry data. We identified differentially expressed miRNA’s in five cancer types: breast invasive (300 miRs, p Conclusions: Our results identify molecular and genetic features unique to African Americans that may contribute to worse survival outcomes. While the cause of cancer disparities is multifactorial, a focus on molecular signatures unique to AA may identify actionable molecular abnormalities. Note: This abstract was not presented at the meeting. Citation Format: Olivia D. Lara, Ying Wang, Wei Hu, Lin Zhang, Alejandro Rauh-Hain, Anil Sood. Pan-cancer molecular study of disparities among African Americans [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 4205.

Exosomes are small extracellular vesicles secreted from all living cells; its contents are highly varied, including proteins, small RNAs and recently discovered, genomic DNA (gDNA). The presence of nuclear content in exosomes suggests that different cell compartments can contribute to exosome cargo; however, little is known about this sub population of nuclear derived exosomes (nExo). Here, we analyzed nExo isolated from ovarian cancer cells and characterized them to explore potential clinical relevance such as cancer detection and therapeutic response. Exosomes were isolated from ovarian cancer cell lines, normal human fallopian epithelial tube cells and bio-fluids from mouse and human samples. To determine the purity of exosomes, nanoparticle tracking system, immunoblotting assay and cryo-transmission electron microscopy were used. To analyze the content of exosomes, protein mass spectrometry and imaging flow cytometry analysis was performed. First, we determined that mass spectrometry analyses for ovarian cancer cell exosomes revealed that 12.5% of proteins are derived from the cell nucleus sub-population. Additionally, using a novel imaging flow cytometry technique which enables us to identify single exosome and to quantify them, we determined that 10% of exosomes carried gDNA. Conversely, normal cells secreted only 0.2% of nExo. Then we hypothesized that the prevalence of gDNA is affected by genotoxic drugs. Treating cells with either the PARP inhibitor olaparib or topoisomerase inhibitor topotecan increased the proportion of nExo secreted from ovarian cancer cells. In addition, PARP proteins exist in ovarian cancer cell exosomes and 80.4 % of the exosome co-localized with gDNA. Using in vivo preclinical models, serum from ovarian cancer bearing mice contained a higher number of nExo than non-tumor controls, and this population was increased in response to treatment of genotoxic drugs. Furthermore, plasma and ascites from ovarian cancer patients contain around 0.3 % of nExo, which include nuclear proteins and gDNA. We demonstrate that nExo are predominantly secreted from cancer cells and could serve as an important biomarker. Although the subpopulation is relatively rare, their presence in bio-fluids isolated from cancer patients holds the potential of serving as novel biomarkers for early detection and therapeutic response to genotoxic agents. Citation Format: Akira Yokoi, Alejandro Villar-Prados, Paul Allen Oliphint, Theresa J. O'Halloran, Jason Roszik, Anil K. Sood. A novel approach for liquid biopsy by using nExo [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 LB-232.

Purpose: To identify mechanisms responsible for the development of adaptive resistance to anti-vascular endothelial growth factor (VEGF) therapy, and to develop novel agents to reverse its occurrence. Methods: Gene expression profiling was performed on endothelial cells isolated from tumors sensitive or resistant to anti-VEGF therapy in a syngeneic ovarian cancer mouse model. A series of in vitro (lentiviral transfection, tube formation, siRNA knockdown, luciferase promoter assays) and in vivo (including Tie2; PPARγ KO mice) experiments were then used to elucidate the upstream and downstream effectors of CD5L, as well as the efficacy of CD5L inhibition in pre-clinical orthotopic mouse models. Results: We identified substantially increased CD5L expression in endothelial cells isolated from anti-VEGF therapy resistant vs. sensitive tumors. Ectopic expression of CD5L in RF24 endothelial cells resulted in increased tube formation, cell migration, and cell proliferation, whereas siCD5L treatment resulted in an overall decrease. Observing that a PPARγ binding site was present in the CD5L promoter, we found that ectopic expression of PPARγ into RF24 cells results in increased CD5L expression, whereas siPPARγ treatment decreased it. Ectopic PPARγ expression increased luciferase activity but decreased activity with siPPARγ treatment when using a reporter assay for CD5L promoter. Conversely, mutation of the PPARγ binding site in the promoter construct lacked any increase in activity after ectopic PPARγ expression. IPA analysis identified HIF1α as playing an integral role in the PPARγ /CD5L activation pathway. Indeed, hypoxic conditions resulted in an increase of both PPARγ and CD5L in RF24 cells. HIF1α stabilizers (cobalt chloride) and inhibitors (YC-1 and topotecan) also showed an increase and decrease in PPARγ and CD5L expression, respectively. RPPA analysis of anti-VEGF resistant tumor endothelial cells implicated an upregulation of the PI3K pathway. While exogenous CD5L treatment led to increased p-AKT, the addition of the PI3K inhibitor LY294002 negated this effect and also decreased tube formation and cell migration. Next, we injected both WT B6 and Tie2:PPARγ KO mice with the ID8 mouse ovarian cancer cell line and treated with B20 until moribund. Mice harboring the PPARγ KO had significantly increased survival as well as decreased tumor weight, number of nodules, Ki67 expression, and blood vessel density. Our non-syngeneic model was then treated with B20 and either CD5L RNA aptamer or vehicle. The aptamer group treated with CD5L aptamer displayed significantly decreased tumor weight, number of nodules, Ki67 expression, and blood vessel density. Conclusions: This study identifies a pathway centered on CD5L as an important driver of adaptive resistance to anti-angiogenic therapy. Citation Format: Christopher J. LaFargue, Paola Amero, Kyunghee Noh, Selanare Mangala, Chunhua Lu, Minsoon Cho, Sunila Pradeep, Yihong Wan, Wei Hu, Rajesha Rupaimoole, Robert L. Coleman, Ningyan Zhang, Zhiqiang An, Gabriel Lopez-Bernstein, Vittorio de Franciscis, Anil K. Sood. Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L [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 194.

Introduction: The objective was to identify molecular mechanisms and protein networks involved in synthetic lethality as well as mechanisms of adaptive changes to the combination of olaparib (O) combined with inhibitors of the PI3K-AKT axis. Methods: 110 patients with recurrent ovarian, endometrial, and triple negative breast cancer were treated with a combination of O and vistusertib (V) or capivasertib (C) (mTORC1/2 and AKT inhibitors, respectively). We have previously reported durable clinical efficacy of these combinations, regardless of BRCA status. Tumor samples were collected pre-treatment and 30 days post-treatment and reverse-phase protein array (RPPA) was performed. Baseline expression was assessed and change in expression was calculated. Bioinformatics and statistical methods were developed to assess activity of signaling pathways. Pathway scores were determined based on the expression of sets of proteins known to be involved in a specific pathway, including PI3K, RAS/RAF, and DNA damage repair. Association between protein expression and treatment efficacy was determined. Results: We analyzed a total of 55 paired samples from patients treated with O/V (n=25) and O/C (n=30). These included 20 endometrial (37% PR, 31.5% SD, 31.5% PD), 21 ovarian (10% PR, 55% SD, 35% PD) and 14 triple negative breast (15% PR, 23% SD, 62% PD) tumors. Response to O/V was associated with low pre-treatment mTOR pathway activity and led to altered immune activity based on decreased CD4 and PD1 expression. Resistance to O/V was associated with high pre-treatment cell proliferation (high CCNB1, CDK1 and PLK1 protein expression). In contrast, response to O/C was associated with adaptive responses indicated by decreased mTOR activity and induction of the DNA damage checkpoint (high phospho-CHK1, -WEE1 and -CDC2). Resistance was associated with high pre-treatment RTK activity levels (high phospho-HER3, -EGFR, -IGFR, -MET) and AKT-independent activation of mTOR in the on-treatment samples. Conclusion: Analysis of pre- and on-treatment samples from responders and non-responders to PARP and PI3K pathway combination therapy identified biomarkers of patients likely to benefit from therapy and helps to understand molecular mechanisms involved in response and resistance to these drug combinations. Citation Format: Marilyne Labrie, Zhenlin Ju, Jennifer K. Litton, Tae-Beom Kim, Sanghoon Lee, Ken Chen, Pamela T. Soliman, Michael Frumovitz, Larissa A. Meyer, Stacy Moulder, Amir A. Jazaeri, Karen H. Lu, Anil K. Sood, Robert L. Coleman, Gordon B. Mills, Shannon N. Westin. Exploration of markers of synergistic lethality of PARP and PI3K-Akt-mTOR inhibitors in women's cancers [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 2070.

Cancer cells actively promote their tumorigenic behavior by reprogramming gene expression. Loading intraluminal vesicles with specific miRNAs and releasing them into the tumor microenvironment as exosomes is one mechanism of reprogramming whose regulation remains to be elucidated. Here, we report that miR-6126 is ubiquitously released in high abundance from both chemosensitive and chemoresistant ovarian cancer cells via exosomes. Overexpression of miR-6126 was confirmed in healthy ovarian tissue compared with ovarian cancer patient samples and correlated with better overall survival in patients with high-grade serous ovarian cancer. miR-6126 acted as a tumor suppressor by directly targeting integrin-β1, a key regulator of cancer cell metastasis. miR-6126 mimic treatment of cancer cells resulted in increased miR-6126 and decreased integrin-β1 mRNA levels in the exosome. Functional analysis showed that treatment of endothelial cells with miR-6126 mimic significantly reduced tube formation as well as invasion and migration capacities of ovarian cancer cells in vitro. Administration of miR-6126 mimic in an orthotopic mouse model of ovarian cancer elicited a relative reduction in tumor growth, proliferating cells, and microvessel density. miR-6126 inhibition promoted oncogenic behavior by leading ovarian cancer cells to release more exosomes. Our findings provide new insights into the role of exosomal miRNA-mediated tumor progression and suggest a new therapeutic approach to disrupt oncogenic phenotypes in tumors. Cancer Res; 76(24); 7194–207. ©2016 AACR.

Photothermal ablation (PTA) is an emerging technique that uses near-infrared (NIR) laser light–generated heat to destroy tumor cells. However, complete tumor eradication by PTA therapy alone is difficult because heterogeneous heat distribution can lead to sublethal thermal dose in some areas of the tumor. Successful PTA therapy requires selective delivery of photothermal conducting nanoparticles to mediate effective PTA of tumor cells, and the ability to combine PTA with other therapy modalities. Here, we synthesized multifunctional doxorubicin (DOX)-loaded hollow gold nanospheres (DOX@HAuNS) that target EphB4, a member of the Eph family of receptor tyrosine kinases overexpressed on the cell membrane of multiple tumors and angiogenic blood vessels. Increased uptake of targeted nanoparticles T-DOX@HAuNS was observed in three EphB4-positive tumors both in vitro and in vivo. In vivo release of DOX from DOX@HAuNS, triggered by NIR laser, was confirmed by dual-radiotracer technique. Treatment with T-DOX@HAuNS followed by NIR laser irradiation resulted in significantly decreased tumor growth when compared with treatments with nontargeted DOX@HAuNS plus laser or HAuNS plus laser. The tumors in 6 of the 8 mice treated with T-DOX@HAuNS plus laser regressed completely with only residual scar tissue by 22 days following injection, and none of the treatment groups experienced a loss in body weight. Together, our findings show that concerted chemo-photothermal therapy with a single nanodevice capable of mediating simultaneous PTA and local drug release may have promise as a new anticancer therapy. Cancer Res; 72(18); 4777–86. ©2012 AACR.

Emerging evidence suggests that the Notch/Delta-like ligand 4 (Dll4) pathway may offer important new targets for antiangiogenesis approaches. In this study, we investigated the clinical and biological significance of Dll4 in ovarian cancer. Dll4 was overexpressed in 72% of tumors examined in which it was an independent predictor of poor survival. Patients with tumors responding to anti-VEGF therapy had lower levels of Dll4 than patients with stable or progressive disease. Under hypoxic conditions, VEGF increased Dll4 expression in the tumor vasculature. Immobilized Dll4 also downregulated VEGFR2 expression in endothelial cells directly through methylation of the VEGFR2 promoter. RNAi-mediated silencing of Dll4 in ovarian tumor cells and tumor-associated endothelial cells inhibited cell growth and angiogenesis, accompanied by induction of hypoxia in the tumor microenvironment. Combining Dll4-targeted siRNA with bevacizumab resulted in greater inhibition of tumor growth, compared with control or treatment with bevacizumab alone. Together, our findings establish that Dll4 plays a functionally important role in both the tumor and endothelial compartments of ovarian cancer and that targeting Dll4 in combination with anti-VEGF treatment might improve outcomes of ovarian cancer treatment. Cancer Res; 71(18); 6030–9. ©2011 AACR.

Prexigebersen (liposome-incorporated nuclease resistant antisense oligonucleotides specific for GRB2, L-GRB2) has proven highly effective in the treatment of leukemia, however its application in solid malignancies including gynecologic cancer has yet to be investigated. Given the biological significance of GRB2, we aim to assess the effect of prexigebersen in pre-clinical models. We tested the clinical significance of GRB2 using publicly available high-throughput data. We investigated the expression of GRB2 in a series of in vitro (Western Blotting) experiments in high-grade serous (HGSC) and uterine (UC) carcinoma models. We also tested in vivo (orthotopic mouse model) the biological effects of prexigebersen in HGSC models (OVCAR 5), first in a dose-defining experiment then in combination with standard dose paclitaxel. We first examined GRB2 using TCGA and GISTIC data and found alterations (amplifications, deletions and mutations) in 6% (33 of 594) of HGSC cases and 5% (25 of 547) of UC cases. Amplifications of GRB2 are associated with a decreased patient survival (p Citation Format: Olivia D. Lara, Cristian Rodriquez-Aguayo, Emine Bayrakter, Paola Amero, Selanere Mangala, Chris LaFargue, Ana Tari-Ashizawa, Gabriel Lopez-Berestein, Anil Sood. Grabbing GRB2: The use of liposome-incorporated Grb2 antisense oligonucleotides as a novel therapy in gynecologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5786.

We have previously demonstrated that an oncogenic microRNA miR551b-3p (also known as miR551b) regulates STAT3 transcription thereby enhancing tumor growth and metastasis in ovarian cancer. Herein, we have identified that Oncostatin M (OSM) is upregulated through STAT3-driven transcription, which plays an important role in promoting migration and invasion of breast cancer cells. The purpose of this study was to evaluate the role of miR551b mediated STAT3 activation and the precise mechanism that regulates OSM-Receptor (OSMR) signaling for breast cancer growth and metastasis. MDA-MB231 cells were either overexpressed or silenced with miR551b and anti-miR551b respectively and the effects on proliferation, migration/invasion (Matrigel transwell assay), wound healing and expression of OSMR and its downstream targets (Western blotting, Quantitative RT-PCR) were assessed. Our data demonstrate that miR551b promoted survival, proliferation, invasion and migration and epithelial to mesenchymal transition (EMT) and spheroid formation of breast cancer cells. Our results also show that miR551b-mediated STAT3 activation upregulated the components of OSMR signaling through elevated expression of OSMR, Interleukin 31 receptor-alpha (IL31RA) and their ligands Oncostatin (OSM) and IL31 thereby inducing phosphorylation of STAT3 for tumor cell proliferation, migration and invasion. Conversely, anti-miR551b reduced the expression of OSMR, IL31R and their ligands OSM and IL31 as well as the phosphorylation of STAT3. Briefly, our results provide the first report indicating that miR551b regulates OSMR expression by activating STAT3 and thereby promoting tumor cell growth and metastasis in breast cancer cells through autocrine feed-forward signaling loop. A.G, D.P., contributed equally. Citation Format: Anjali Geethadevi, Deepak Parashar, Miriam Ragle-Aure, Bindu Nair, Yiling Lu, Vessela N. Kristensen, Ming You, Anil K. Sood, Gordon B. Mills, Sunila Pradeep, Pradeep Chaluvally Raghavan. MiR551b-mediated Oncostatin M signaling in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4423.

Background: Both experimental and clinical studies suggest that beta-blockers, a common antihypertensive agent, may reduce ovarian tumor aggressiveness, inhibit angiogenesis and metastasis, and improve survival. However, no study has considered the potential role of other antihypertensive medications in conjunction with beta-blockers in ovarian cancer survival. Methods: We evaluated the associations of pre-diagnostic (n=899) and post-diagnostic (n=683) use of individual antihypertensive medications with ovarian cancer survival in the Nurses' Health Study (NHS; diagnosed between 1994-2012) and NHSII (diagnosed between 2001-2013), with at least 4 years of follow-up for death. Cox proportional hazards models were used to estimate hazard ratios (HR) for ovarian cancer mortality according to antihypertensive medication use. Results: In analyses of individual drugs adjusted for age, BMI and tumor characteristics, pre-diagnostic use of calcium-channel blockers (CCB) was associated with higher ovarian cancer mortality (HR: 1.52; 95% CI: 1.15, 2.00), whereas lower mortality was observed for post-diagnostic use of beta-blockers (HR: 0.76; 95% CI: 0.58, 1.01) and angiotensin-converting enzyme (ACE) inhibitors (HR: 0.64; 95% CI: 0.44, 0.93). While hypertension itself was not associated with ovarian cancer mortality (HR: 0.99; 95% CI: 0.83, 1.17), the associations between antihypertensive medications and ovarian cancer mortality were suggestively stronger among women with history of hypertension. Conclusion: Our findings suggest that post-diagnosis use of beta-blockers and ACE inhibitors is associated with lower ovarian cancer mortality and pre-diagnosis use of CCBs may be associated with higher mortality. These results warrant further investigation, and ongoing work is exploring whether the observed associations may be explained by changes in antihypertensive medication prescription after ovarian cancer diagnosis or changes in overall prescription patterns over time. Citation Format: Tianyi Huang, Anil K. Sood, Shelley S. Tworoger. Antihypertensive medication use and ovarian cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4252.

EP-100 is a synthetic lytic peptide specific for targeting LHRH receptors on cancer cells that is being clinically tested for treatment of ovarian cancer. In this study, we aimed to identify combination approaches with EP-100 in ovarian cancer models. We carried out a series of in vitro (MTT assay, immunoblot analysis, Q-PCR, reverse phase protein array (RPPA), comet assay and immunofluorescence staining) and in vivo (orthotopic mouse model) experiments in ovarian cancer models to determine the biological effects of EP-100 alone and in combination with standard of care drugs. We first examined the cytotoxic effects of EP-100 alone on 8 ovarian cancer cell lines with variable expression level of LHRH receptors and the IC50 values ranged from 0.5 to 2.0 µM. We then tested the effect of combining EP-100 with standard drugs (e.g., cisplatin, paclitaxel, doxorubicin, topotecan, and olaparib) on ovarian cancer cells. Of those drugs, we found that combination of EP-100 and olaparib was highly synergistic in drug sensitive and multi-drug resistant ovarian cancer cell lines (HeyA8, HeyA8-MDR, A2780, and A2780CP20). The combination indices (CI) were < 0.9 at the ED50 level. Furthermore, the nuclear foci of γH2AX were significantly increased in combination group of EP100 and olaparib after 24 h incubation compared with control, EP-100 alone, or olaparib alone groups (p < 0.0001, ANOVA). In addition, there was increased DNA accumulation in tails using comet assay after treating cells with EP-100 and olaparib for 24 h (p < 0.001, ANOVA). RPPA results identified that PI3K/AKT pathway can be blocked significantly by the combination treatment. The study in HeyA8 xenograft mouse models showed that mice treated with EP-100 and olaparib had the lowest tumor weight (0.06 ± 0.05 g) compared with those treated with vehicle (1.19 ± 0.39 g, p < 0.008), EP-100 alone (0.62 ± 0.28 g, p < 0.008), and olaparib alone (0.50 ± 0.22 g, p < 0.01). EP-100 has efficacy in preclinical models of ovarian cancer, especially in combination with olaparib. Our findings suggested that combining EP-100 with olaparib could be a promising therapeutic strategy for ovarian cancer. Citation Format: Shaolin Ma, Sunila Pradeep, Sherry Wu, Mark Seungwook Kim, Wei Hu, Selanere Mangala, Robert L. Coleman, Anil K. Sood. EP-100 sensitizes BRCA wild-type ovarian cancer cells to PARP inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4817.

The dynamic properties of RNA interference (RNAi) in cancer biology have led investigators to pursue with significant interest its role in tumorigenesis and cancer therapy. We recently reported that decreased expression of key RNAi enzymes, Dicer and Drosha, in epithelial ovarian cancers was associated with poor clinical outcome in patients. Dicer expression was also functionally relevant in that targeted silencing was limited with RNAi fragments that require Dicer function compared with those that do not. Together, this and other studies suggest that RNAi machinery expression may affect key pathways in tumorigenesis and cancer biology. Understanding alterations in the functional RNAi machinery is of fundamental importance as we strive to develop novel therapies using RNAi strategies. Cancer Res; 70(7); 2571–4