Background Allograft models enable characterisation of genomic drivers and treatment responses by modelling immune and micro-environmental changes more accurately than xenografts. Despite this, few models are available to the prostate cancer (PCa) community. This study presents a novel allograft model of high-risk, localised PCa. In characterising this model we have focused on its response to radiotherapy (RT). Methods The DVL3 cell line was derived from a transgenic trp53 -/- /Pten -/- mouse model and characterised both in vitro and in vivo . The DVL3 cells were allografted and response to RT was investigated and compared to the TRAMP-C1 model. Extensive tumour profiling in the DVL3 model was performed using flow cytometry, immunohistochemistry and RNA-seq. Results In vitro the DVL3 cells expressed basal and luminal markers. DVL3 cells formed tumours with distinct glandular morphology which expressed androgen receptor, similar to human localised PC. DVL3 tumour growth was delayed following administration of fractionated RT, with infiltration of myeloid derived suppressor cells (MDSC). Conclusions The DVL3 allograft model represents substantial progress in PCa modelling, displaying; luminal differentiation, strong AR expression, and an immunosuppressive microenvironment, similar to observations in high-risk PCa patients. This model is ideally suited for development and validation of novel therapeutics, in particular immune-modulatory agents in combination with RT.

The prostate cancer (PCa) field lacks clinically relevant, syngeneic mouse models which retain the tumour microenvironment observed in PCa patients. This study establishes a cell line from prostate tumour tissue derived from the Pten−/−/trp53−/− mouse, termed DVL3 which when subcutaneously implanted in immunocompetent C57BL/6 mice, forms tumours with distinct glandular morphology, strong cytokeratin 8 and androgen receptor expression, recapitulating high-risk localised human PCa. Compared to the commonly used TRAMP C1 model, generated with SV40 large T-antigen, DVL3 tumours are immunologically cold, with a lower proportion of CD8+ T-cells, and high proportion of immunosuppressive myeloid derived suppressor cells (MDSCs), thus resembling high-risk PCa. Furthermore, DVL3 tumours are responsive to fractionated RT, a standard treatment for localised and metastatic PCa, compared to the TRAMP C1 model. RNA-sequencing of irradiated DVL3 tumours identified upregulation of type-1 interferon and STING pathways, as well as transcripts associated with MDSCs. Upregulation of STING expression in tumour epithelium and the recruitment of MDSCs following irradiation was confirmed by immunohistochemistry. The DVL3 syngeneic model represents substantial progress in preclinical PCa modelling, displaying pathological, micro-environmental and treatment responses observed in molecular high-risk disease. Our study supports using this model for development and validation of treatments targeting PCa, especially novel immune therapeutic agents.

Radiation therapy is a common treatment for prostate cancer, however recurrence remains a problem. MicroRNA expression is altered in prostate cancer and may promote therapy resistance. Through bioinformatic analyses of TCGA and CPC-GENE patient cohorts, we identified higher miR-191 expression in tumor versus normal tissue, and increased expression in higher Gleason scores. In vitro and in vivo experiments demonstrated that miR-191 overexpression promotes radiation survival, and contributes to a more aggressive phenotype. Retinoid X receptor alpha, RXRA, was discovered to be a novel target of miR-191, and knockdown recapitulated radioresistance. Furthermore, treatment of prostate cancer cells with the RXRA agonist 9-cis-retinoic acid restored radiosensitivity. Supporting this relationship, patients with high miR-191 and low RXRA abundance experienced quicker biochemical recurrence. Reduced RXRA translated to a higher risk of distant failure after radiotherapy. Notably, this miR-191/RXRA interaction was conserved in a novel primary cell line derived from radiorecurrent prostate cancer. Together, our findings demonstrate that miR-191 promotes prostate cancer survival after radiotherapy, and highlights retinoids as a potential option to improve radiotherapy response.

Abstract Introduction: 40-45% of patients with High Grade Serous Ovarian Cancer (HGSOC) will eventually relapse with platinum resistant disease. Tothill et al and TCGA are two independent gene expression datasets which have demonstrated the presence of a mesenchymal molecular subgroup, characterised by upregulation of angiogenesis regulating genes. Angiogenesis is known to be an integral pathological feature of HGSOC and anti-angiogenics have dominated the field of drug development in EOC. However, despite this, anti-angiogenic agents have failed to demonstrate a significant impact on overall survival (OS) benefit. In this study, we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents and what the underlying biological rationale for this could be. Methods: A meta-analysis of 14 phase II and III clinical trials in EOC were used to investigate the association between platinum resistance and response to anti-angiogenic agents. In addition, we analysed gene expression in 12 matched pre- and post-chemotherapy EOC samples. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the development of an angiogenic phenotype. Further studies were performed in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy. Result: In the clinical trial meta-analysis, an OS benefit for antiangiogenics was observed in platinum-resistant disease (p=0.029), whilst platinum-sensitive EOC only derived progression free survival (PFS) (p=<0.0001) benefit and not OS (p=0.125). In the 12 matched pairs of patient samples, post-platinum samples had a higher micro-vessel density (MVD) relative to their paired treatment-naïve sample (p= 0.0001). Additionally, an in vivo angiogenesis matrigel plug assay demonstrated that cisplatin-resistant EOC cell lines were associated with an increase in MVD (p=<0.0001). MVD was reduced in the platinum-resistant cells following treatment with bevacizumab (p=0.001). Ascites-derived primary cells established from platinum-resistant patients demonstrated overexpression of VEGF-A, consistent with stimulating angiogenesis. Gene expression analysis of pre- and post-platinum paired samples identified that PDGFRα (p=0.007) and PDGFRβ (p=0.005) were differentially expressed in the post-platinum therapy samples. In vitro validation in the platinum-resistant cell lines demonstrated that VEGF-A expression was regulated by PDGFRα. Discussion: We have demonstrated that previous platinum therapy for EOC is associated with an increase in tumor PDGFα and VEGF-A expression, correlating with a response to anti-angiogenic therapies. This data suggests that platinum therapy resistance may inform the selection of EOC patients for novel antiangiogenic therapies in future clinical trials. Citation Format: Aya El Helali, Nuala McCabe, Christopher Steele, Lara Dura Perez, Christina L. O'Neill, Naomi Dickson, Niamh McGivern, Caolan Harkin, Andrena McCavigan, Reinhold J. Medina, Laura A. Knight, Stephen McQuaid, Jacqueline A. James, Caroline O. Michie, Charlie Gourley, W Glenn McCluggage, Denis P. Harkin, Richard H. Wilson, Alan W. Stitt, Richard D. Kennedy. Platinum resistance in epithelial ovarian cancer is dependent on a PDGFR alpha-VEGF-A signalling mechanism that activates downstream angiogenesis pathways [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 7.

5578 Background: Patients with High Grade Serous Ovarian Cancer (HGSOC) initially respond to SOC platinum based treatment but most will eventually relapse with platinum resistant disease. Angiogenesis is known to be an integral pathological feature of HGSOC and anti-angiogenic agents have been trialed in this population, but have failed to demonstrate a significant impact on overall survival (OS). Here, we asked if platinum resistance could be associated with an improved response to anti-angiogenic therapies. Methods: A meta-analysis of 14 phase II and III clinical trials in EOC were used to investigate the association between platinum resistance and response to anti-angiogenic agents. In addition we analysed gene expression in 12 matched pre-and post-chemotherapy samples. Novel cisplatin-resistant HGSOC cell lines and novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy were developed to investigate the relationship between angiogenesis and platinum resistance. Results: The meta-analysis revealed an OS benefit for anti-angiogenics in platinum-resistant disease (p = 0.029), whilst platinum-sensitive disease derived only PFS benefit (p = < 0.0001). In the matched pairs of patient samples, post-platinum samples had a higher micro-vessel density (MVD) relative to their paired treatment-naïve sample (p = 0.0001). Additionally, an in vivo angiogenesis matrigel plug assay demonstrated that cisplatin-resistant EOC cell lines were associated with an increase in MVD (p = < 0.0001). MVD was reduced in the platinum-resistant cells following treatment with bevacizumab (p = 0.001). Ascites-derived cells established from platinum-resistant patients demonstrated overexpression of VEGF-A through increased PDGFRa and PDGFRb expression. Conclusions: We have demonstrated that previous platinum therapy for EOC is associated with an increase in tumour PDGFα and VEGF-A expression, correlating with a response to anti-angiogenic therapies. This data suggests that platinum therapy resistance may inform the selection of EOC patients for novel anti-angiogenic therapies in future clinical trials.

12111 Background: Unsupervised hierarchical clustering of gene expression data from 265 high grade serous ovarian cancer (HGSOC) patients identified 3 major molecular subgroups. One subgroup is driven by activation of the MAPK-pathway and is associated with a mesenchymal phenotype, poor prognosis and resistance to platinum.The MAPK pathway is currently being targeted by novel therapeutics and hence an assay to detect activation of the pathway across cancers would be highly valuable as a clinical trial enrichment tool. Methods: Using TCGA data we show the existence of the mesenchymal subgroup across a range of solid tumours including stomach, bladder colon, lung, melanoma and prostate cancer. Further to this, a common gene list was generated to include only transcripts with high variability and expression across diseases, and used as a starting list for the development of a 15 transcript assay which can be used to prospectively identify the mesenchymal subgroup from archived tissue. The 15 gene expression assay was tested in preclinical model systems to assess its utility at predicting response to MEK inhibitors. Results: The 15 gene expression mesenchymal assay was a poor prognostic marker in 13 different solid tumours: overall HR = 1.78 [95% CI:1.65-1.92]) p < 0.0001. Additionally the assay was associated with a mesenchymal phenotype (migration, invasion) and activated MAPK (phospho-MAPK) signalling in preclinical cell line models. The assay also predicted phospho-MEK expression in clinical samples (p < 0.05). The assay score was reduced by MEK inhibition (p < 0.05) and elevated by KRAS, NRAS and MEK1 overexpression (p < 0.05). The assay predicted response to the MEK inhibitors Trametinib and Selumetinib across cell line models from multiple diseases (p < 0.001) and to Trametinib in mouse xenograft studies of lung cancer cell lines. Conclusions: A 15 gene expression assay has been developed from FFPE samples across multiple diseases to detect a mesenchymal molecular subgroup associated with MAPK signalling. The assay predicted sensitivity to MEK inhibitors in pre-clinical cell line and mouse model systems. Further work aims to validate the assay as a predictive biomarker in clinical samples from patients treated with MEK targeted therapies.

Abstract Introduction: Platinum resistant High Grade Serous Ovarian Cancer (HGSOC) has a poor outcome with limited treatment options. Angiogenesis is a key pathological feature of ovarian cancer and anti-angiogenics have dominated the field of drug development in EOC, particularly in the second-line setting (Marachini et al 2013). In this study we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents. Method: A review of phase III anti-angiogenic clinical trials was used to investigate the association between platinum resistance and response to anti-angiogenic agents. To investigate the effect of chemotherapy on predefined ovarian cancer molecular subgroups (Gourley, et al. J Clin Oncol 32:5s, 2014), we analysed 35 matched pre- and post-chemotherapy samples by gene expression. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the mechanisms of cisplatin section pressure and shift to an angiogenic phenotype. This was further validated in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy. Results: Critical review of 22 phase III anti-angiogenic trials suggested that there was a better response to anti-angiogenics following previous platinum-based chemotherapy. Our analysis demonstrated that 67% of treatment naïve tumours that were initially classified as non-angiogenic shifted to an angiogenic biology, which was associated with platinum resistance. Additionally we found that cisplatin resistant cancer cell lines demonstrated hallmarks of vascular mimicry and an associated increase in vessel density using an angiogenesis Matrigel plug assay in Athymic nude mice (p-value=<0.0001). In addition, cell lines established from platinum-resistant patients as well as cell lines made platinum resistant in-vitro, demonstrated overexpression of VEGFa which would be expected to stimulate angiogenesis. Conclusion: We have demonstrated that platinum-resistance in HGSOC is associated with angiogenic biology supporting the use of anti-angiogenic agents in this setting. Citation Format: Aya El Helali, Nuala McCabe, Naomi Dickson, Lara Dura Perez, Denis P Harkin, Richard Wilson, Richard Kennedy. Acquired platinum resistance enhances tumour angiogenesis through activation of vascular mimicry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 776. doi:10.1158/1538-7445.AM2017-776