Tumours with genomic instability display enhanced immunogenicity and potential for response to immune checkpoint blockade (ICB). Interestingly, chemotherapy mediated DNA damage can also stimulate the immune system through activation of the cGAS/STING innate immune pathway and therefore, improve clinical outcomes in combination with ICB. Here we set out to compare the ability of classically used chemotherapies to activate innate immunity, as well as characterise the mechanism(s) behind it. We identified topoisomerase 2 (TOP2) poisons, such as anthracyclines, as the most effective activators of cGAS/STING, leading to a potent type I interferon response. Mechanistically, we demonstrate that abortive TOP2cc repair intermediates (dsDNA fragments containing 5’-phosphotyrosyl residues) get encapsulated within micronuclei and activate cGAS, which can be blocked with antimitotic agents (e.g. taxanes). Crucially, we discovered that these 5’-phosphotyrosyl linked fragments are resistant to nuclease degradation by the cytoplasmic nuclease TREX1, which stabilises them, resulting in robust cGAS/STING mediated inflammation. Finally, using in vivo modelling, we confirmed that Top2 poisons enhance anti-tumour responses to ICB in comparison to taxanes. In line with this, using pre and on-treatment breast tumour biopsies, we have shown that anthracycline-based chemotherapy induces a robust type I interferon response in breast tumours, driving tumour lymphocytic infiltration. Moreover, following anthracycline treatment with taxane treatment results in supression of lymphocytic infiltration in these same tumours. Collectively, our findings reveal that TOP2 poison mediated DNA lesions enhance cGAS substrate availability by impairing cytoplasmic dsDNA degradation, highlighting their ability to drive anti-tumour immune responses and enhance the therapeutic efficacy of ICB. Citation Format: Kienan Savage, Eliana M. Barros, Richard D.A. Wilkinson, Guido Zagnoli-Viera, Stuart A. McIntosh, Katrina M. Lappin, Oliver Barker, Ieuan L. Morgan, Eileen E. Parkes, Marc A. Fuchs, Nuala McCabe, Roger A. Greenberg, Tim Harrison, Keith W. Caldecott, Richard D. Kennedy. CHEMOTHERAPEUTIC TOPOISOMERASE 2 POISONS GENERATE TREX1 RESISTANT DNA FRAGMENTS THAT INDUCE A POTENT cGAS/STING RESPONSE [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-02-02.

Supplementary Tables S1 & S2 and Figures S1a & S1b from Deficiency in the Repair of DNA Damage by Homologous Recombination and Sensitivity to Poly(ADP-Ribose) Polymerase Inhibition

Supplementary Figures 1-4. Figure 1: ATM is a potential drug target for PTEN-deficient cells Figure 2: The synthetic lethality with PTEN loss and ATM inhibition is independent of AKT function Figure 3: The synthetic lethality with PTEN loss and ATM inhibition is independent of RAD51 function Supplementary Figure 4: In vivo efficacy of ATM inhibition with PTEN loss

<p>Supplementary Methods and Table 1. Supplementary Table 1: siRNA target genes</p>

Background: HER2+ breast cancer is treated with anti-HER2 IgG monoclonal antibody therapies such as trastuzumab and pertuzumab. These antibodies inhibit HER2-driven intracellular signaling and can also be described as a form of passive immunotherapy, engaging effector immune cells in antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). Tumor-associated autoantibodies (AAbs) to tumor neo-antigens can be generated by the adaptive immune response before, or in response to, therapy. There is growing evidence of a functional role for tumor-associated AAbs in the immune response to tumors, with IgG and IgA AAbs capable of mediating ADCC and ADCP. Using the innovative Nucleic Acid-Programmable Protein Array (NAPPA) platform we examined the levels and targets of circulating AAbs to ~1700 human proteins before, during and after treatment in HER2+ breast cancer treated in the neo-adjuvant setting. . Methods: Pre-treatment, On-treatment (pre- Cycle 2) and Post-treatment (post-Cycle 6) serum samples were obtained from HER2+ breast cancer patients (n=19) treated on the ICORG 10-05 clinical trial. Patients were treated with neo-adjuvant chemotherapy (docetaxel (T)/carboplatin (C)) +/- trastuzumab (H), lapatinib (L) or HL, TCH n=9, TCHL n= 7, TCL n=3. Patients were classified as having a pathological complete response (pCR, n=6), a partial response (PR, n=6) or a non-response (NR, n=7) to treatment. IgG and IgA autoantibody levels were determined using the HD-NAPPA system, focussing on a subset of ~ 1700 human protein targets. A normalised intensity score of > 5 was utilised as a cut-off for detection. Results: 92 IgG AAbs and 29 IgA AAbs were detected in all samples, across all timepoints (n=57). When IgG AAbs were divided by patient response, 26 AAbs unique to pCR, 26 AAbs unique to PR and 16 AAbs unique to NR were detected. 5 IgG AAbs (BCL11B, MAP6, PRDM8, SYTL2, TP53BP2) were common to all HER2+ breast cancer patients. When IgA AAbs were divided by patient response, 11 AAbs unique to pCR, 6 AAbs unique to PR and 7 AAbs unique to NR were detected. 2 IgA AAbs (TDP1 and MAP6) were common to all HER2+ breast cancer patients. Treatment-induced AAbs were detected in On-treatment and Post- treatment samples in 16/19 patients examined. Conclusions: Our results suggest HER2+ BC patients have unique circulating AAbs associated with treatment response and AAb levels change in response to treatment. Further investigation of AAb levels as biomarkers of response in HER2+ BC is warranted in a larger cohort of patients. Citation Format: Denis Martin Collins, Ji Qiu, Jaine Blayney, Nuala McCabe, Richard Kennedy, Joshua LaBaer, John Crown. Investigation of autoantibodies (AAbs) in HER2+ breast cancer (BC) patients treated in the neo-adjuvant setting [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-09.

Abstract Tumor mutational burden (TMB) is emerging as a promising predictive biomarker to select cancer patients who will benefit from immunotherapy. However, accurate TMB estimation from sequencing data remains hampered by the ability to correctly distinguish somatic variants from both germline variants and spurious base changes introduced by sample preparation and sequencing processes. The latter is particularly prevalent in poor quality FFPE material where degradation and chemical modifications of the DNA manifest as variant artefacts leading to over-estimation of TMB. One method of removing germline variants and artefacts is to compare tumor variant calls to those obtained from matched normal tissue, however inclusion of a matched normal adds significant logistical and financial challenges to clinical trial assays. Therefore, there remains a critical need to establish alternative approaches enabling accurate TMB estimation from FFPE tumor-only samples. To address these challenges, we have developed a tumor-only somatic variant calling pipeline for accurate determination of TMB from FFPE material profiled by whole exome sequencing (WES). The process is underpinned by two key features (a) enhanced germline variant filtering that uses a systematic rule-based strategy, (b) FFPE artefact removal using an ensemble machine learning model incorporating multiple variant call QC parameters. The germline filtering approach was developed on a matched fresh frozen (FF)/FFPE/blood dataset of 23 cancer patients. The FFPE artefact filtering model was built on a dataset of 59 FF and FFPE matched pairs, where FFPE artefacts were defined as variants present in FFPE but absent in the FF sample. Benchmarked against internal and TCGA datasets of somatic variant calls from >30 FF samples with a matched normal, our germline and FFPE filtering steps decreased false discovery rates by over 20%. This translated into improved TMB scoring performance with tumor-only pipeline estimates from three SeraCare reference standards closely matching expected TMB scores. Critically, we establish the clinical relevance of our approach on a published cohort of 98 patients treated with an immune checkpoint inhibitor. Here we observed increased association between patient response and TMB estimated from our pipeline (p=0.042) compared to a standard pipeline with no enhanced germline or FFPE artefact filters (p=0.56). Overall, our combined germline and FFPE artefact filtering pipeline results in significantly improved somatic variant detection and TMB scoring performance in FFPE samples where a matched normal is unavailable. It is applicable across multiple disease types and adaptable to other platforms. Therefore, the pipeline has utility in clinical laboratories where accurate somatic variant detection is crucial to successful deployment of predictive biomarkers such as TMB. Citation Format: Hui Sun Leong, Emma O'Connor, Nuala McCabe, Sinead Donegan, Steven M. Walker, Jayna Mistry, Karen Keating, Denis P. Harkin, Richard D. Kennedy, Laura A. Knight, James R. Bradford. A machine learning approach to detect FFPE artefacts leads to accurate estimation of tumor mutational burden from sequencing data without a matched normal [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 152.

Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8–18 months. The aim of this research was to determine if loss of PTEN (highly prevalent in advanced prostate cancer) is a novel therapeutic target in the treatment of advanced prostate cancer. Previous work has demonstrated PTEN-deficient cells are sensitised to inhibitors of ATM, a key regulator in the response to DSBs. Here, we have shown the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, we have confirmed ATM inhibition in PTEN-depleted cell models, enhances ionising radiation-induced cell killing with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. We have further shown PTEN loss is accompanied by increased endogenous levels of ROS and DNA damage. Taken together, these findings provide pre-clinical data for future clinical evaluation of ATM inhibitors as a neoadjuvant/adjuvant in combination with radiation therapy in prostate cancer patients harbouring PTEN mutations.

Abstract Introduction: Multiple biomarkers and molecular subtypes have been proposed in prostate cancer (PCa) with limited overlap, in part due to the heterogeneity of the disease and issues with data scaling. By applying Almac Diagnostic's unique software driven tool, claraT, cancer gene expression datasets can be characterized according to six of the well-established Hallmarks of Cancer using published signatures, proprietary assays and single gene targets. This retrospective study aimed to identify robust molecular subtypes of PCa that are prognostic and validated by applying claraT to multiple gene expression datasets. Experimental procedures: claraT was applied to three primary PCa gene expression datasets, TCGA (n = 331, Illumina HiSeq), Walker et al Discovery (n = 91, Almac Prostate DSA) and Walker et al Validation (n = 322, Almac Prostate DSA). Six sets of signature scores were generated, each representing a different hallmark and consensus clustering was performed using the signature scores. Clinical relevance was assessed using metastatic-free survival (MFS) and disease-free survival (DFS) as end-points. Using statistical tests, association of the identified subtypes with variables was investigated. Results: Of the six hallmarks investigated, robust and stable molecular subtypes were established using the Genome-Instability (GI) signature set. Four subtypes, two with good prognosis and two with poor prognosis, were characterized in the Walker Validation (MFS log-rank, p=6.798e-10), TCGA (DFS log-rank, p=0.0012) and Walker Discovery (chi-square - metastases, p=0.004). Subtypes were significantly associated with Gleason, biochemical recurrence, and Almac's Prostate Dx assay (chi-square, p<0.00001). Clustering analysis revealed the poor prognosis subtypes to have high GI signature expression e.g. chromosome instability and homologous recombination deficiency signatures. The median number of copy number alterations (CNAs) amongst the subtypes was higher in the poor prognosis subtypes (Wilcoxin, p=1.0363e-18) thus validating the genome-unstable phenotype association with the poor prognosis subtypes. Conclusion: Of the six hallmarks investigated, Genome-Instability was the most robust and prognostic. Molecular subtypes with distinct prognoses have been identified and validated. These poor prognosis subtypes are characterized by an active genome instability biology and high levels of CNAs. Therefore, these poor prognosis subtypes of PCa, with a dominant genome instability biology, may represent a larger target subgroup of PCa's which may benefit from DNA repair targeted therapies such as PARP inhibitors. Citation Format: Cathal McKinney, Nuala McCabe, Paul Harkin, Richard Kennedy, Jaine Blayney. Characterization of robust and prognostic molecular subtypes in prostate 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 2135.

Introduction: Previous work from our lab has identified a molecularly distinct subgroup of primary prostate cancers with biology similar to metastatic prostate cancers. These “met-like” cancers can be identified by a 70 gene classifier and are statistically more likely to result in distant metastases than their “non-met like” counterparts. This study aimed to identify exploitable biology common to both “met-like” primary tumours and metastatic prostate cancer. Experimental procedures: Differential gene expression and Ingenuity pathway analysis (IPA) was used to identify nodal genes upregulated in both “met-like” primary prostate cancers and metastatic prostate cancers. siRNA screening was performed on 40 nodal genes in a panel of normal, primary and metastatic prostate cancers. Cell viability was assessed using cell titer-glo luminescent Assay. Relative levels of apoptosis were assessed using PARP, caspase-8 and caspase-3 cleavage Western Blots. siRNA mediated knockdowns were confirmed by Western Blot and rtPCR. Results: We have identified upregulation of the homeobox transcription factor DLX1 in “met-like” primary prostate cancers and metastatic prostate cancers. High levels of DLX1 expression were found to be independently predicative of biochemical recurrence and metastatic disease in primary prostate cancers. DLX1 knockdown resulted in significantly decreased cell viability in primary and metastatic prostate cancer cell lines with minimal reduction of viability in normal prostate cells. Additionally DLX1 knockdown was shown to result in increased PARP, caspase-8 and caspase-3 cleavage providing evidence of apoptotic cell death. Furthermore knockdown of DLX1 resulted in significantly increased transcript levels of the pro-apoptotic BH3 protein BIK, suggesting its involvement in the observed apoptotic cell death. Conclusion: DLX1 has previously been identified as a marker of prostate cancer metastasis with increased expression being observed in both primary and metastatic prostate cancers. However we have observed increased DLX1 expression in a subset of primary prostate cancers that developed metastatic diseased compared to primary tumors that did not metastasise. The elucidation of apoptotic mechanisms following DLX1 knockdown may uncover potential exploitable biology that may be used for the treatment of prostate cancer. Citation Format: Nicholas Forsythe, Cathal McKinney, Nuala McCabe, Richard D. Kennedy. The role of DLX1 as a biomarker and novel target in poor prognostic prostate 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 370.

Recent trials of the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib, rucaparib, and niraparib have resulted in approval of these agents in ovarian cancer, initially in patients with germline BRCA1 or BRCA2 mutations, now extended to the maintenance setting for all platinum-sensitive ovarian cancer. PARP inhibitors were first developed as chemosensitizing agents and early clinical trials focused on combination treatment with DNA-damaging chemotherapy. However, the identification of synthetic lethality of PARP inhibitor treatment in BRCA1 and BRCA2-mutant cancer led to trials in BRCA1/2-mutant ovarian cancer with clinical responses to single agent treatment. These DNA-repair targeted agents have been a success story of translational research. However, questions remain over their optimal use in ovarian cancer and the effectiveness of currently available predictive biomarkers.

Historically the development of anticancer treatments has been focused on their effect on tumor cells alone. However, newer treatments have shifted attention to targets on immune cells, resulting in dramatic responses. The effect of DNA repair deficiency on the microenvironment remains an area of key interest. Moreover, established therapies such as DNA damaging treatments such as chemotherapy and PARP inhibitors further modify the tumor microenvironment. Here we describe DNA repair pathways in breast cancer and activation of innate immune pathways in DNA repair deficiency, in particular, the STING (STimulator of INterferon Genes) pathway. Breast tumors with DNA repair deficiency are associated with upregulation of immune checkpoints including PD-L1 (Programmed Death Ligand-1) and may represent a target population for single agent or combination immunotherapy treatment.

Introduction: WEE1 kinase is a key component in maintaining the G2/M cell cycle checkpoint for pre-mitotic DNA repair, and is overexpressed in several cancer types. Novel therapeutics are currently being developed to target WEE1 kinase in cancer, however, to date no predictive biomarkers have been approved to aid patient stratification and clinical trial design. To address this, we employed a siRNA screening to identify tumour suppressor genes (TSGs) whose loss mediates sensitivity to WEE1 inhibition. Experimental procedures: U2OS cells were reverse transfected with a customised siRNA library containing 3 independent siRNAs targeting 178 tumour suppressor genes and 24 hours later treated with either DMSO control or MK-1775 (Wee1 Kinase Inhibitor). Cell viability was measured using a cell titer-glo luminescent Cell Viability Assay 72 hours post-treatment. Hits were selected based on robust z-score analysis. Those genes with 2 or more targeted siRNAs demonstrating a robust z-score of ±1 median absolute deviation (MAD) were taken forward for validation studies. Sensitive hits were selected on a z-score of <-1 and resistant hits were selected on a z-score of >1. siRNA knockdown of WEE1 was performed in multiple human cancer cell lines and confirmed by western blotting and RT-q-PCR. Basal expression levels of phosphorylated WEE1, total WEE1, FOS and JUNB were assessed by western blotting. Results: Consistent with previously published findings, the siRNA screen demonstrated that loss of BRCA2 conferred increased sensitivity to WEE1 inhibition (Aarts et al. 2015). The siRNA screen also identified an additional 12 TSGs whose loss mediated sensitivity and 14 TSGs whose loss mediated resistance to WEE1 kinase inhibition. Interestingly, we found that loss of two early response genes, FOS and JUNB conferred resistance to WEE1 inhibition. FOS and JUNB interact to form the AP1 heterodimer, and previous published work has demonstrated the presence of an AP1 binding motif on the WEE1 promoter (Kawasaki et al. 2003). Using publically available gene expression data (TCGA) we have shown a significant correlation between expression of WEE1 with FOS and JUNB in multiple cancer types. Conclusions: Using a TSG siRNA screen, we have identified that loss of JUNB and FOS confers resistance to the WEE1 inhibitor MK1775. Future studies will investigate the mechanisms by which the loss of these genes affects response to WEE1 inhibition, and will also investigate the utility of these genes as predictive biomarkers for response to WEE1 inhibition in clinical samples, thereby aiding patient stratification. Citation Format: Victoria L. Dunne, Niamh McGivern, Kienan I. Savage, Nuala McCabe, Richard Kennedy. The role of early response genes (ERG’s) as a biomarker of response to Wee1 targeted therapies [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 3163.

e21045 Background: Traditionally gene expression signatures (GES) are used individually to classify patients into subgroups. Signatures targeting the same biology are often developed independently and may not classify identically. We developed the claraT software tool that uses consensus between multiple published GES categorised by the Hallmarks of Cancer (Hanahan & Weinberg, 2011) to classify cancers. As metastatic melanoma represents poor prognostic disease (5-yr survival 15-20%), we applied claraT to the TCGA melanoma dataset to identify targetable biologies, validated in a cohort of melanoma patients treated with Ipilimumab. Methods: TCGA RNA-seq data ( n= 472) was analysed using the claraT platform including GES for immune ( n= 14), angiogenesis ( n= 9) and epithelial-mesenchymal transition (EMT) ( n= 12) Hallmarks. Samples were clustered for the combined and individual Hallmarks. Median progression-free (PFS) and overall-survival (OS) differences were analysed across identified subgroups. Analysis was validated in an Ipilimumab treated melanoma dataset ( n= 42) (Van Allen, 2015). Results: Clustering the combined Hallmarks identified 4 subgroups in the TCGA cohort: 1) Immune active, 2) Immune-EMT active, 3) EMT-Angiogenesis active, 4) All inactive. Groups 1&2 had significantly improved OS compared to Groups 3&4 (HR = 0.50, p< 0.0001). Clustering using single Hallmarks revealed that immune-positive tumours had significantly improved OS (HR = 0.53, p< 0.0001) compared to immune-negative tumours. Angiogenesis-negative tumours displayed improved PFS (HR = 0.73, p= 0.03) and OS (HR = 0.53, p <0.0001) compared to angiogenesis-negative tumours. Interestingly the EMT Hallmark was not found to be individually prognostic. When validated in the Ipilimumab treated dataset, patients classified as immune-positive had improved OS (HR = 0.357, p= 0.010) when compared to immune-negative. Similar trends were also observed for angiogenesis and EMT Hallmarks. Conclusions: This study demonstrates how simultaneous analysis of multiple GES ( n= 35 in this study) can identify robust biologies through consensus expression. This platform may have value in the identification of reliable biomarkers for clinical trials and could inform how combination therapies targeting key biologies may be used in cancer treatment.

OBJECTIVE:High grade serous carcinoma (HGSC) is the most common and most aggressive, subtype of epithelial ovarian cancer. It presents as advanced stage disease with poor prognosis. Recent pathological evidence strongly suggests HGSC arises from the fallopian tube via the precursor lesion; serous tubal intraepithelial carcinoma (STIC). However, further definition of the molecular evolution of HGSC has major implications for both clinical management and research. This study aims to more clearly define the molecular pathogenesis of HGSC.METHODS:Six cases of HGSC were identified at the Northern Ireland Gynaecological Cancer Centre (NIGCC) that each contained ovarian HGSC (HGSC), omental HGSC (OMT), STIC, normal fallopian tube epithelium (FTE) and normal ovarian surface epithelium (OSE). The relevant formalin-fixed paraffin embedded (FFPE) tissue samples were retrieved from the pathology archive via the Northern Ireland Biobank following attaining ethical approval (NIB11:005). Full microarray-based gene expression profiling was performed on the cohort. The resulting data was analysed bioinformatically and the results were validated in a HGSC-specific in-vitro model.RESULTS:The carcinogenesis of HGSC was investigated and showed the molecular profile of HGSC to be more closely related to normal FTE than OSE. STIC lesions also clustered closely with HGSC, indicating a common molecular origin.CONCLUSION:This study provides strong evidence suggesting that extrauterine HGSC arises from the fimbria of the distal fallopian tube. Furthermore, several potential pathways were identified which could be targeted by novel therapies for HGSC. These findings have significant translational relevance for both primary prevention and clinical management of the disease.

Cathepsin S (CTSS) has previously been implicated in a number of cancer types, where it is associated with poor clinical features and outcome. To date, patient outcome in breast cancer has not been examined with respect to this protease. Here, we carried out immunohistochemical (IHC) staining of CTSS using a breast cancer tissue microarray in patients who received adjuvant therapy. We scored CTSS expression in the epithelial and stromal compartments and evaluated the association of CTSS expression with matched clinical outcome data. We observed differences in outcome based on CTSS expression, with stromal-derived CTSS expression correlating with a poor outcome and epithelial CTSS expression associated with an improved outcome. Further subtype characterisation revealed high epithelial CTSS expression in TNBC patients with improved outcome, which remained consistent across two independent TMA cohorts. Further in silico gene expression analysis, using both in-house and publicly available datasets, confirmed these observations and suggested high CTSS expression may also be beneficial to outcome in ER-/HER2+ cancer. Furthermore, high CTSS expression was associated with the BL1 Lehmann subgroup, which is characterised by defects in DNA damage repair pathways and correlates with improved outcome. Finally, analysis of matching IHC analysis reveals an increased M1 (tumour destructive) polarisation in macrophage in patients exhibiting high epithelial CTSS expression. In conclusion, our observations suggest epithelial CTSS expression may be prognostic of improved outcome in TNBC. Improved outcome observed with HER2+ at the gene expression level furthermore suggests CTSS may be prognostic of improved outcome in ER- cancers as a whole. Lastly, from the context of these patients receiving adjuvant therapy and as a result of its association with BL1 subgroup CTSS may be elevated in patients with defects in DNA damage repair pathways, indicating it may be predictive of tumour sensitivity to DNA damaging 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.