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Triple-negative breast cancer (TNBC) remains the most lethal breast cancer subtype with poor response rates to the current chemotherapies and a lack of additional effective treatment options. We have identified deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumour DNA from the genotoxic misincorporation of uracil during treatment with standard chemotherapeutic agents commonly used in the FEC regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP), providing dUMP for thymidylate synthase as part of the thymidylate biosynthesis pathway and maintaining low intracellular dUTP concentrations. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporation into DNA. Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by fluoropyrimidines or anthracyclines represents an effective strategy to induce cell lethality. dUTPase inhibition significantly sensitised TNBC cell lines to fluoropyrimidines and anthracyclines through imbalanced nucleotide pools and increased DNA damage leading to decreased proliferation and increased cell death. These results suggest that repair of treatment-mediated DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a promising strategy to enhance the efficacy of TNBC chemotherapy.

An underlying cause of breast cancers has been largely attributed to defects in the DNA damage response (DDR) pathway. In particular, the homologous recombination (HR) pathway repairs double-stranded breaks (DSBs) in DNA, ultimately protecting the cell from genomic instability and thus preventing the accumulation of transforming mutations. In line with this, mutations in a number of genes encoding HR proteins are a well-studied cause of HR deficiency (HRD), and, at the germline level, can confer risk to breast cancer but also occur somatically, contributing to sporadic breast cancer development, progression and response to therapy. Our understanding of the biological processes involved in HR and how these become compromised during breast cancer development has led to a better understanding of how HRD cells can be targeted with specific DNA damaging agents and/or with synthetic lethal targeting approaches such as PARP inhibition. Additionally, in vitro and preclinical modeling has supported the development of clinical trials to assess targeted therapies such as PARP inhibitors (PARPis), ultimately leading to development of therapies with greater clinical benefit. A number of challenges have been encountered, including resistance to therapy; however, addressing these challenges head-on and continually driving scientific research and clinical trials with innovative therapies will contribute to our ability to target HRD in breast cancers. Ongoing research efforts into HRD in breast cancer development are therefore essential, even in the era of targeted therapies, to provide innovative strategies for improved tumor responses.

Introduction: We previously identified an immune-driven gene expression signature recognizing loss of the Fanconi Anemia/BRCA DNA repair pathway - the DNA damage immune response (DDIR) assay. This 44-gene expression-based assay predicted response to DNA damaging chemotherapy in breast cancer, with a 5-year relapse-free survival hazard ratio of 0.37 post adjuvant treatment in assay positive patients. We demonstrated constitutive activation of the cGAS-STING pathway as the underlying mechanism driving immune gene expression in this subgroup, as a result of aberrant cytoplasmic DNA due to deficient DNA repair. We sought to evaluate efficacy of this immune-based signature in predicting response to neoadjuvant DNA-damaging chemotherapy (NAC) in breast cancer. Methods: Patients with early breast cancer were recruited from 2 Northern Ireland centres. All received 3 cycles of 5FU, epirubicin and cyclophosphamide (FEC) chemotherapy initially, with subsequent taxane/HER2 targeted therapies as indicated. Formalin fixed paraffin embedded tumor samples were obtained at baseline, post 3 cycles of FEC and at resection. Chemotherapy response was assessed by residual cancer burden (RCB) scoring. Gene expression data was obtained from all baseline samples and 18 midpoint samples containing sufficient residual tumor. DDIR assay call was determined using a predetermined cut-off. Tumor infiltrating lymphocytes (TILs) were assessed at each time point on H&E stained sections. In addition, gene expression signatures (n=62) encompassing 6 hallmarks of cancer (Immune response, Genome instability, Angiogenesis, EMT, Cell death and Proliferation) were reported using the Almac ClaraT platform. Results: 46 patients were recruited - 26 were assay positive, with high immune gene expression, and 20 assay negative. The DDIR assay predicted NAC response with an odds ratio for RCB 0-1 of 4.67 (95% CI 1.13 - 15.09; p = 0.03). In patients with DDIR positive tumors, there was a strong association with immune response and immune checkpoint response signatures (p < 0.0001), as well as TILs in baseline biopsies (p =0.0262). Matched baseline and mid-point (after 3 cycles of FEC chemotherapy) gene expression analysis was available for 17 non-responders and 4 responders. In responding tumors a trend to reduced immune gene expression was seen, with an associated reduction in TILs (p = 0.0573), probably reflecting eradication of DDIR positive tumor cells (decreased tumor content on midpoint specimens in responders was noted compared to non-responders (p = 0.0257)). Conversely, increased DDIR signature score was observed in non-responders post FEC chemotherapy (p = 0.0332), with a trend to increased TIL infiltration (p = 0.0792). Immune checkpoint gene expression also increased following NAC in these patients. DDIR negative non-responding tumors demonstrated upregulation of angiogenesis signatures at baseline (p < 0.01), along with higher scores for TGFβ driven immune resistance signatures (p < 0.05), suggesting other potential therapeutic approaches for these patients. Conclusions: The cGAS-STING driven DDIR gene expression assay is predictive of response to neoadjuvant DNA damaging chemotherapy and is associated with high baseline immune infiltration. In keeping with the recent TONIC trial, we note anthracycline-based treatment as a potent activator of innate immunity. In non-responders, although TIL infiltration and immune gene expression is induced by DNA damaging chemotherapy, tumors do not respond, presumably due to intact DNA repair pathways and immune checkpoint gene induction. Therefore, there is a rationale to offer this population induction anthracycline-based chemotherapy followed by immune checkpoint targeting treatment to improve outcome. Citation Format: Stuart A McIntosh, Kienan I Savage, Colin James, Tong Lioe, Steven Walker, Keith Lowry, Laura Knight, Andrena McGavigan, Gemma Logan, Jane Hurwitz, Stephen J Kirk, Paul Harkin, Richard D Kennedy, Eileen E Parkes. cGAS-STING driven immune activation predicts response to neoadjuvant chemotherapy and suggests rational IO combination therapies: Results of the Neo-DDIR study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-17.

Modern methods of acquiring molecular data have improved rapidly in recent years, making it easier for researchers to collect large volumes of information. However, this has increased the challenge of recognizing interesting patterns within the data. Atlas Correlation Explorer (ACE) is a user-friendly workbench for seeking associations between attributes in The Cancer Genome Atlas (TCGA) database. It allows any combination of clinical and genomic data streams to be searched using an evolutionary algorithm approach. To showcase ACE, we assessed which RNA sequencing transcripts were associated with estrogen receptor (ESR1) in the TCGA breast cancer cohort. The analysis revealed already well-established associations with XBP1 and FOXA1, but also identified a strong association with CT62, a potential immunotherapeutic target with few previous associations with breast cancer. In conclusion, ACE can produce results for very large searches in a short time and will serve as an increasingly useful tool for biomarker discovery in the big data era. Significance: ACE uses an evolutionary algorithm approach to perform large searches for associations between any combinations of data in the TCGA database.

Background: The DDRD assay was developed to prospectively identify a molecular subtype of cancers defined by DNA damage activated immune signalling. This 44-gene assay has been validated to predict response to anthracycline-cyclophosphamide chemotherapy in both the adjuvant and neoadjuvant settings in breast cancer. The biomarker phase Neo-DDRD trial assessed the clinical utility of this assay prospectively in patients receiving neoadjuvant chemotherapy (NAC). Methods: 48 patients receiving NAC for early (T1-2, N0-1) or locally advanced non-metastatic breast cancer were recruited from March 2014 – Sept 2017. Chemotherapy regimen was selected according to local standard-of-care guidelines (FEC “100” in N0, FEC-D in N1, FEC-DH in HER2 +ve, with the addition of pertuzumab when approved). The DDRD score was applied to RNA expression data obtained from pre-treatment FFPE core biopsies using a custom cDNA microarray (Breast Cancer DSA™). Response to NAC was assessed on resection specimens following definitive surgical treatment by pathologists blinded to the DDRD scores, using the Residual Cancer Burden (RCB) assessment tool. Results: Patients with a positive DDRD assay call had an odds ratio for pathological complete response (pCR) or minimal residual disease (RCB0/RCB1) of 4.64 (95%CI 1.31-16.42; p = 0.0174). Patients with RCB0/RCB1 had significantly higher DDRD assay score than those with class RCB2/RCB3 responses (p =0.0011; unpaired t-test). Tumour subtype and DDRD call are summarised in Table 1. In total, 29.2% of patients had a pathological complete response (pCR) (RCB0). Tumor infiltrating lymphocytes (TILs) were assessed on baseline pre-treatment samples. Increasing DDRD assay score was not significantly associated with increasing stromal TILs (p=0.1403; Pearson correlation). In assessing correlation between stromal TILs and response to chemotherapy, there was no significant difference found across RCB categories (p=0.45; one way ANOVA). Table 1:tumour subtype distribution and DDRD assay resultSubtypeDDRD+DDRD-TotalTriple negative7613HER2+ ER-527HER2+ ER+6612ER+ HER2-8816 Conclusions: Preliminary results from the Neo-DDRD biomarker phase trial support the ability of the DDRD assay to predict response to neoadjuvant chemotherapy. The DDRD assay was confirmed to be a better predictor of neoadjuvant chemotherapy response than the presence of TILs. Translational work from this trial is ongoing: serial tissue and plasma samples collected at treatment mid-point and surgical resection will be used to study changes in RNA expression profiles in both DDRD assay positive and negative during chemotherapy, as well as changes in tumour immune infiltrates, peripheral chemokines and circulating tumour DNA profiles. Based on the results from this pilot study, we propose a biomarker-driven phase II trial in the neoadjuvant setting using the DDRD assay to stratify patients for treatment. Citation Format: Parkes EE, James CR, Lioe T, Walker S, Savage KI, Lowry K, Knight L, McCavigan A, Logan G, Hurwitz J, Kirk SJ, Harkin P, Kennedy RD, McIntosh SA. Biomarker phase neo-DDRD trial: Predicting response to neoadjuvant chemotherapy (NAC) in early breast cancer using the DNA damage repair deficiency (DDRD) assay [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-11-12.

Contralateral breast cancer (CBC) incidence is reported as 0.4-0.7% per year, equating to an approximate 10-15% 20 year risk after primary cancer diagnosis. Young age at primary diagnosis and a strong family history are consistently cited as significant risk factors for CBC development. Requests for contralateral prophylactic mastectomy (CPM) are rising, despite a lack of evidence that CPM significantly improves breast cancer specific survival. Therefore, a method to stratify CBC prognosis and risk at an individual level is required to allow us to personalise surgical decision-making for individual patients. This study aims to: 1. Explore prognostic factors following CBC diagnosis; 2. Investigate metastatic CBC prevalence by determining clonal relationships between primary and CBC tumours; 3. Assess the rate of germline predisposition gene mutations in a large cohort of Northern Irish CBC cases. A cohort of 403 CBC patients and 1:1 matched unilateral breast cancer controls (matched for age, year of diagnosis, nodal positivity and tumour morphology) were identified from the Northern Ireland Cancer Registry. Archival primary, CBC and normal lymph node tissue from these CBC patients were obtained for somatic and germline sequencing of a custom 94-gene panel, comprising genes most commonly mutated in breast cancer and all known predisposition genes. CBC patients had a significantly increased risk of breast cancer specific mortality when compared with 1:1 matched unilateral breast cancer controls (HR 6.45, CI 4.27-9.77). Within the CBC cohort, a shorter time interval between primary and CBC diagnosis is associated with increased breast cancer mortality, whereby women with Targeted next generation sequencing of 42 cases to date has provided evidence of shared somatic mutations suggestive of metastatic clonality between primary and CBC in four cases (9.5%). Germline pathogenic predisposition gene mutations were identified in five (11.9%) patients and a further ten (23.8%) patients were noted to have germline predisposition variants of unknown significance. Breast cancer specific mortality risk was significantly higher in CBC patients compared with matched unilateral cancer controls, in excess of what would be expected from two primary cancer events. This finding suggests that there are as yet unquantified factors contributing to this excess hazard. Poorer outcome with shorter inter-tumour diagnostic intervals, in addition to evidence of primary-CBC clonality in some cases, suggests that metastatic CBC may play a role in excess risk. Additionally, the significant reduction in PR positivity of ER positive CBC tumours after ER+/PR+ primary tumours, could suggest that acquired hormonal resistance following primary anti-oestrogen therapy exposure may contribute to this additional mortality hazard. Citation Format: McIlmunn CG, Bannon FJ, Fitzpatrick D, Ervine A, Boyd C, Cameron I, Harita T, James J, Gonzalez de Castro D, Savage KI, McIntosh SA. Molecular and genetic characterisation of contralateral breast cancers in Northern Ireland: Opportunities for personalised surgery [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-09-10.

Graphical abstract, RNA-sequencing (RNA-seq) is a relatively new technology that lacks standardisation. RNA-seq can be used for Differential Gene Expression (DGE) analysis, however, no consensus exists as to which methodology ensures robust and reproducible results. Indeed, it is broadly acknowledged that DGE methods provide disparate results. Despite obstacles, RNA-seq assays are in advanced development for clinical use but further optimisation will be needed. Herein, five DGE models (DESeq2, voom + limma, edgeR, EBSeq, NOISeq) for gene-level detection were investigated for robustness to sequencing alterations using a controlled analysis of fixed count matrices. Two breast cancer datasets were analysed with full and reduced sample sizes. DGE model robustness was compared between filtering regimes and for different expression levels (high, low) using unbiased metrics. Test sensitivity estimated as relative False Discovery Rate (FDR), concordance between model outputs and comparisons of a ’population’ of slopes of relative FDRs across different library sizes, generated using linear regressions, were examined. Patterns of relative DGE model robustness proved dataset-agnostic and reliable for drawing conclusions when sample sizes were sufficiently large. Overall, the non-parametric method NOISeq was the most robust followed by edgeR, voom, EBSeq and DESeq2. Our rigorous appraisal provides information for method selection for molecular diagnostics. Metrics may prove useful towards improving the standardisation of RNA-seq for precision medicine.

Genomic stability is critical for normal cellular function and its deregulation is a universal hallmark of cancer. Here we outline a previously undescribed role of COMMD4 in maintaining genomic stability, by regulation of chromatin remodelling at sites of DNA double-strand breaks. At break-sites, COMMD4 binds to and protects histone H2B from monoubiquitination by RNF20/RNF40. DNA damage-induced phosphorylation of the H2A-H2B heterodimer disrupts the dimer allowing COMMD4 to preferentially bind H2A. Displacement of COMMD4 from H2B allows RNF20/40 to monoubiquitinate H2B and for remodelling of the break-site. Consistent with this critical function, COMMD4-deficient cells show excessive elongation of remodelled chromatin and failure of both non-homologous-end-joining and homologous recombination. We present peptide-mapping and mutagenesis data for the potential molecular mechanisms governing COMMD4-mediated chromatin regulation at DNA double-strand breaks., Amila Suraweera et al. use a range of biochemical and in vitro cellular assays to examine the role of the COMMD4 in DNA repair. Their results suggest that COMMD4 interacts with the histone H2A-H2B during repair of double-stranded DNA breaks, thereby maintaining genomic stability by regulating chromatin structure.

The cohesin complex plays a major role in folding the human genome into 3D structural domains. Mutations in members of the cohesin complex are known early drivers of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), with STAG2 the most frequently mutated complex member. Here we use functional genomics to investigate the impact of chronic STAG2 loss on three-dimensional genome structure and transcriptional programming in a clinically relevant model of chronic STAG2 loss. The chronic loss of STAG2 led to loss of smaller loop domains and the maintenance/formation of large domains which in turn led to altered genome compartmentalisation. These changes in genome structure were linked with altered gene expression, including deregulation of the HOXA locus and the MAPK signalling pathway, which may contribute to disease development and response to therapy.AUTHOR SUMMARYAcute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) are clonal malignant diseases that affect the myeloid blood cell lineage. Around 40 different mutations have been identified as being associated with MDS and AML; several mutations effect genes in the cohesin complex particularly STAG2. STAG2 is an X-linked gene that is pivotal to the cohesin complex and the mutations result in a truncated gene and loss of function. We have introduced a clinically relevant truncating mutation into an isogeneic AML model. This has shown that changes in the sizes of loop and domains formed in the genome resulting in appropriate gene compartmentalisations. The associated changes in gene transcription has resulted in deregulation of HOX genes, essential for development and differentiation, and in the MAPK signalling pathway that could a therapeutic target.

Background The evaluation of circulating tumour DNA (ctDNA) from clinical blood samples, liquid biopsy, offers several diagnostic advantages compared with traditional tissue biopsy, such as shorter processing time, reduced patient risk and the opportunity to assess tumour heterogeneity. The historically poor sensitivity of ctDNA testing, has restricted its integration into routine clinical practice for non-metastatic disease. The early kinetics of ctDNA during radical radiotherapy for localised NSCLC have not been described with ultra-deep next generation sequencing previously. Materials and methods Patients with CT/PET-staged locally advanced, NSCLC prospectively consented to undergo serial venepuncture during the first week of radical radiotherapy alone. All patients received 55Gy in 20 fractions. Plasma samples were processed using the commercially available Roche AVENIO Expanded kit (Roche Sequencing Solutions, Pleasanton, CA, US) which targets 77 genes. Results Tumour-specific mutations were found in all patients (1 in 3 patients; 2 in 1 patient, and 3 in 1 patient). The variant allele frequency of these mutations ranged from 0.05–3.35%. In 2 patients there was a transient increase in ctDNA levels at the 72 h timepoint compared to baseline. In all patients there was a non-significant decrease in ctDNA levels at the 7-day timepoint in comparison to baseline (p = 0.4627). Conclusion This study demonstrates the feasibility of applying ctDNA-optimised NGS protocols through specified time-points in a small homogenous cohort of patients with localised lung cancer treated with radiotherapy. Studies are required to assess ctDNA kinetics as a predictive biomarker in radiotherapy. Priming tumours for liquid biopsy using radiation warrants further exploration.

Genomic stability is critical for normal cellular function and its deregulation is a universal hallmark of cancer. Here we outline a previously undescribed role of COMMD4 in maintaining genomic stability, by regulating of chromatin remodelling at sites of DNA double strand breaks (DSBs). We demonstrate that COMMD4 is initially recruited to DSBs by a direct-interaction with phosphorylated hSSB1. At break-sites, COMMD4 binds to and protects histone H2B from monoubiquitination by RNF20/RNF40. DNA damage-induced phosphorylation of the H2A-H2B heterodimer disrupts the dimer allowing COMMD4 to preferentially bind H2A. Displacement of COMMD4 from H2B allows RNF20/40 to monoubuquitinate H2B and for remodelling of the break-site. Consistent with this critical function, COMMD4-defficient cells show excessive elongation of remodelled chromatin and failure of both non-homologous-end-joining and homologous recombination. We present a model, developed by molecular dynamic simulation, peptide-mapping and mutagenesis for the potential molecular mechanisms governing COMMD4-mediated chromatin regulation at DSBs.

Tumours with genomic instability demonstrate enhanced immunogenicity and potential for response to immune checkpoint blockade (ICB). We previously demonstrated activation of the cGAS-STING pathway following loss of DNA repair, resulting in cytokine induction, lymphocytic infiltration and immune checkpoint activation. Here we explore the role of chemotherapies in inducing this innate immune response, identifying topoisomerase II (topo-II) inhibitors, particularly doxorubicin and epirubicin, as potent inducers of a cGAS-STING dependent interferon response. Mechanistically, topo-II inhibition resulted in significant induction of cytoplasmic DNA and subsequent micronuclei formation, a requirement for efficient cGAS-STING activation and consequent cytokine and immune checkpoint gene induction. Importantly, increased cytokine and immune checkpoint gene expression, as well as increased immune cell infiltration, was also observed in patient derived breast tumour biopsies following topo-II inhibitor-based treatment. Taken together, this study indicates topo-II inhibitors such as doxorubicin, may be best placed to induce immunogenic inflammation, and thereby increase responses to ICB therapies.SignificanceThis work demonstrates how topo-II inhibitors induce STING-pathway activation, cytokine induction and immune checkpoint protein upregulation in cancer cells and provides a rationale for combining topo-II inhibitors with ICB therapy in early breast cancer.

Background: BRAF mutations (BRAFMT) occur in ∼10-15% of metastatic colorectal cancer (mCRC) and have a poor clinical outcome, in particular those with microsatellite stable (MSS) disease. Anti-BRAF/EGFR combinations have shown some increases in response rate, but this is associated with minor increases in overall survival. There is an unmet need to understand the biology of poor prognostic BRAFMT CRC. Using publicly available gene expression data from 155 CRC cell lines (GSE59857), we recently identified that unfolded protein response (UPR) and DNA repair are dominant pathways deregulated in the consensus molecular subgroup 1/BRAFMT subgroup. The aim of this study was to investigate the role of BOLD-100, an inhibitor of the UPR regulator GRP78, in regulating the survival of CRC cells. Methods: A panel of isogenic paired and non-isogenic V600E BRAFMT and BRAFWT cells were used. BOLD-100, a ruthenium-based small molecule inhibitor, was obtained from BOLD Therapeutics. Cell Titre Glo, Flow Cytometry, Western blotting, Caspase 8, 3/7 activity, RNAi assays were used. RNA seq and IPA bioinformatic analyses were performed on BOLD-100-treated BRAFMT/WT CRC cells. A compound library including small molecules approved by the FDA was used. Results: In vitro CellTitre-Glo® and Annexin V/PI sensitivity studies showed that the BRAFMT, MSS CRC cells were highly sensitive to BOLD-100 with IC50 values between 9.25-31μM. Treatment with BOLD-100 resulted in early decreases in GRP78 levels and increases in expression levels of the endoplasmic reticulum stress protein CHOP, and this was associated with caspase-8 dependent cell death in the BRAFMT CRC cells. Notably, silencing of CHOP did not abrogate BOLD-100-induced cell death in BRAFMT CRC cells, indicating that the UPR pathway played no role in the cell death following BOLD-100. RNA seq and IPA analysis showed that cell cycle regulation and DNA repair were the most significant deregulated pathways following BOLD-100 treatment. Further mechanistic studies revealed that BOLD-100 induced rapid and potent increases in pATRT1989, pChk1S345 and γH2AX expression levels in BRAFMT cells. Using a small molecule compound library, we found that the ATR inhibitors AZD6783 and M4344 resulted in strong synergy and apoptosis when combined with BOLD-100, in particular in BRAFMT CRC cells. Moreover, we found that the ROS scavenger NAC abrogated BOLD-100 induced CHOP, pATRT1989, pChk1S345 and γH2AX levels and rescued cell death following BOLD-100 treatment in BRAFMT CRC cells. Conclusions: Taken together, we have identified a role for BOLD-100 in regulating the survival of BRAFMT CRC cells. Our data support further studies with BOLD-100, in particular in combination with ATRi, for the treatment of BRAFMT CRC tumours. Citation Format: Robbie Carson, Shivaali Karelia, Deborah Lavin, Vijay Tiwari, Richard Kennedy, Kienan Savage, Adam Carie, Jim Pankovich, Mark Bazett, Sandra Van Schaeybroeck. Targeting the DNA repair pathway with BOLD-100 in BRAF mutant colorectal cancer [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 1183.

Background: 5-Fluorouracil (5-FU) is an antimetabolite DNA damaging chemotherapeutics that forms the backbone for treatment of colorectal cancer (CRC) and currently it is the most commonly used anti-cancer drug. Despite the central role played by p53 in regulating DNA-damage response, its role in regulating response to 5-FU remains unclear. Methods: To better understand how p53 status differentially affects response to 5-FU, we conducted a detailed phenotypic analysis of the effects of 5FU alone or in combination with Oxaliplatin on cell cycle, DNA damage, cell death, DNA repair and consequent signaling across a panel of p53 isogenic, null and mutant CRC cell lines. Results: This revealed that while 5-FU induce both p53 dependent and independent cell death, the effects on cell cycle are profoundly different, with p53 deficient cells accumulating in S-phase with prolongated double strand breaks. This is not observed in p53 proficient cells, which are protected from these effects by induction of the p53 target gene p21/CDKN1A limiting cell cycle and enforcing suppression of cell cycle genes. Notably in both p53 or p21 deficient cells 5-FU results in sustained DNA damage-signaling through ATR and ATM in p53 deficient cells which preferentially arrest in S-phase. Importantly, these cells exhibit significant induction of cell surface expression of programmed death ligand-1 (PD-L1), which is enhanced by ATR inhibition concomitant with increased ATM activation. Further analysis indicates that transcriptional induction of PD-L1 is mediated through STAT3 in an interferon independent manner. Conclusion: This work adds significantly to our understanding of how p53 status impacts 5FU induced cell cycle arrest and response in CRC patients and have important implications for understanding 5-FU response, particularly in future combinations with immune checkpoint inhibitors. Citation Format: Tamas Sessler, Fiammetta Falcone, Peter Gallagher, Timothy Wright, Kienan Savage, Daniel B. Longley, Simon S. McDade. 5-FU-induces PD-L1 in the absence of p53 via ATM dependent activation of STAT3 [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 1386.

Modern methods in generating molecular data have dramatically scaled in recent years, allowing researchers to efficiently acquire large volumes of information. However, this has increased the challenge of recognising interesting patterns within the data. Atlas Correlation Explorer (ACE) is a user-friendly workbench for seeking associations between attributes in the cancer genome atlas (TCGA) database. It allows any combination of clinical and genomic data streams to be selected for searching, and highlights significant correlations within the chosen data. It is based on an evolutionary algorithm which is capable of producing results for very large searches in a short time.

Acute Myeloid Leukaemia (AML) is a genetically heterogeneous disease of the blood and bone marrow primarily affecting older populations. Chemotherapy has been the standard of care for four decades, just 10% of elderly patients show a response and it will be curative in only a third of patients aged 18-60. Therefore, it's necessary to identify new therapeutic targets for improved treatment options. The search for novel mutations within the genomes of karyotypically normal AML patients identified Cohesin's STAG2 as a recurrently mutated gene (6%). Cohesin is a ring-shaped multimeric protein complex which has a multitude of roles including maintaining sister chromatid cohesion, DNA replication and repair, gene expression and chromatin architecture. STAG2 remains the most understudied Cohesin gene, how a mutation affects the ability of Cohesin to fulfil certain roles needs elucidation as well as how STAG2 functions as part of Cohesin to repair DNA breaks, promote faithful replication and control gene expression. Therefore, we sought to explore the consequences of loss-of-function STAG2 mutations on DNA repair and replication using a STAG2 knockout (ΔSTAG2) isogenic cell line model created using CRISPR/Cas9. Immunofluorescent staining of DNA damage repair proteins 53BP1 and RAD51 following 2Gy irradiation (IR) suggests ΔSTAG2 cells have a significant DNA repair defect. The average 53BP1 and RAD51 foci counts were significantly higher (p=0.001) in mutant cells which had three times as many foci at 24 hours after IR. In addition, 40% of ΔSTAG2 cells stain positive for 53BP1 and RAD51 respectively at 24 hours post-IR. This damage persists at 48 and 72 hours, whereas wild type (WT) damage returns to basal levels. Given that our data suggests a HR defect, we assessed the response of WT and ΔSTAG2 cells to PARP inhibitors (PARPi). Interestingly, ΔSTAG2 cells showed significantly greater sensitivity than WT cells to highly potent PARPi Talazoparib with an IC50 of 62nM. We next assayed single DNA fibres to evaluate the implications of a STAG2 mutation on the dynamics of individual replication forks. Interestingly, we observed a significantly reduced number of ongoing replication forks compared to WT cells (p=0.01) with a concomitant increase in the number of stalled forks (p=0.01) in the absence of exogeneous DNA damage. Following treatment with hydroxyurea (HU) this phenotype was exacerbated and ΔSTAG2 cells exhibit significantly more stalled replication forks than WTs (p=0.001). This indicates a role for STAG2 in maintaining replication fork stability and promoting fork restart following replication stress. Importantly, our data identifies a potential DNA damage repair deficiency that sensitises ΔSTAG2 cells to PARPi monotherapy as well as unearthing an essential role for STAG2 in replication fork progression and fork stability. This mutation could therefore be exploited therapeutically and represents a novel therapeutic target/approach for AML patients with mutated STAG2. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia (AML) is most commonly seen in people over the age of 65 and has a median age of 63. Globally there is an increasingly elderly population so the rate of incidence of AML is set to increase. The therapy landscape for AML has changed little over the past four decades. Cytarabine, first approved in 1969, is still the standard of care induction therapy for AML. There has been only modest improvements in survival rates during this time and there is currently no method of determining which patients will or will not respond to Cytarabine treatment. An assay, developed in 2014, used microarray data to determine which breast cancer patients had a DNA Damage Repair Deficiency (DDRD) and therefore would be more susceptible to DNA damaging agents. A negative DDRD (DDRD-) score predicts that patients do not to have a DNA Repair Deficiency whilst patients with a positive DDRD (DDRD+) score are predicted to have a DNA Repair Deficiency. This assay has been adapted to different solid cancer types such as ovarian and oesophageal cancer. This project has assessed the potential of using the DDRD assay for AML patients. The assay was applied to publically available microarray data of >600 AML patients (TCGA AML data &GSE6891), who were classed as DDRD- or DDRD+. Excluding patients not treated with Cytarabine, this left 639 patients, 405 DDRD+ and 234 DDRD-. Kaplan Meier analysis showed the DDRD+ patients survived significantly (p=0.00047) worse than the DDRD- cohort. Whole exome sequencing was available for 183 patients (131 DDRD+) and the mutations associated with each group were identified. As the DDRD+ patients had the worst outcome, we focused on group. The list of genes more commonly mutated in the DDRD+ patients (>2 instances and >50% occurring in this group) were subjected to pathway analysis. Deregulated pathways included "leukemogenisis" and "cell proliferation and regulation"; however, the most deregulated pathway was "metabolism of nucleobase containing compounds". As Cytarabine is a nucleobase-containing compound, this is potentially a contributing factor as to why these patients responded poorly to this treatment. The assay was applied to microarray data of a panel of myeloid cell lines, and DDRD-(NB4 & SKM1) and a DDRD+(HL-60) cell line were chosen as experimental models. Clonogenic assays, used to analyse the effect of Cytarabine on these cell lines, showed that the DDRD- cell lines were more sensitive with a lower colony growth rate than the DDRD+cell line. DNA damage induction and repair, following cytarabine treatment or 2gy radiation, were measured using RAD51 foci counts. Whilst foci counts were high in all cell lines 2hrs and 4hrs following radiation, the DDRD+ cell line continued to show high levels after 24hrs whereas the levels in the DDRD- cell lines returned to a basal level. RAD51 response to radiation treatment showed that a repair defect is present in DDRD+ cells as they fail to repair the damage induced by radiation. Following treatment with Cytarabine however, few foci were seen in the DDRD+ cell line 2hrs, 4hrs or 24hrs following treatment whereas the DDRD- cell lines responded in a similar fashion to radiation treatment. That RAD51 foci are not present following Cytarabine treatment indicates that Cytarabine fails to induce damage in these cells. The DDRD assay has shown to be an effective method for determining cellular response to Cytarabine in vivo. The non-response of the DDRD+ cell line to Cytarabine suggests that these cells do not elicit a DNA damage or an apoptotic response. This perhaps contributes to their poorer outcome and suggests that Cytarabine is not an effective treatment plan for patients deemed to be DDRD+. Although alternative induction treatment options are currently unavailable for DDRD+ AML patients, this DDRD assay could be used as a biomarker for Cytarabine response in the future. Disclosures No relevant conflicts of interest to declare.

Available treatments for glioma have limited effectiveness rendering this a disease of poor clinical outcome. Gene expression profiling can uncover the biological mechanisms underlying disease, information that is crucial for drug development or repurposing. RNA sequencing (RNA-seq) is routinely used to assess gene expression but costs remain high. Whilst sample multiplexing reduces RNA-seq costs, the lowered cDNA sequencing depth of multiplexed samples may hinder accurate differential gene expression detection. The impact of sequencing depth alteration on RNA-seq-based downstream analyses such as gene expression connectivity mapping is not known. Connectivity mapping is a method used to identify potential therapeutic compounds for drug repurposing. In this study, published RNA-seq from brain tumour (glioma) patients were analysed and assembled into two gene signature contrasts for astrocytoma disease progression (WHO Grade II-III; III-IV). Gene signatures were subsampled to simulate sequencing depth alterations and analysed in connectivity mapping to investigate target compound robustness. Data loss to gene signatures led to the loss, gain and consistent identification of significant connections to target compounds. The most accurate gene signature contrast with consistent patient gene expression profiles was more resilient to data loss and identified robust target compounds. Target compounds lost included candidate compounds of potential clinical utility in glioma (e.g. Suramin, Dasatinib). Lost connections may have been linked to low abundance genes in the gene signature that closely characterised the disease phenotype. Consistently identified connections may have related to highly expressed abundant genes that were ever-present in gene signatures, despite data reductions. Potential noise surrounding findings included false positive connections that were gained as a result of gene signature modification with data loss. Findings highlight the necessity for gene signature accuracy for connectivity mapping, which should improve the clinical utility of future target compounds identified for drug repurposing.

BACKGROUND: Gene expression profiling can uncover biological mechanisms underlying disease and is important in drug development. RNA-seq is routinely used to assess gene expression but costs remain high. Sample multiplexing reduces RNA-seq costs, however, multiplexed samples have lower cDNA sequencing depth, which can hinder accurate differential gene expression detection. The impact of sequencing depth alteration on RNA-seq-based downstream analyses such as gene expression connectivity mapping is not known, where connectivity mapping can be used to identify potential therapeutic compounds for repurposing. MATERIAL AND METHODS: In this study, published RNA-seq profiles from brain tumour (glioma) patients were assembled into two disease progression gene signature contrasts for astrocytoma. Available treatments for glioma have limited effectiveness rendering this a disease of poor clinical outcome. Gene signatures were subsampled to simulate sequencing alterations and analysed in connectivity mapping to investigate target compound robustness. RESULTS: Data loss to gene signatures led to the loss, gain and consistent identification of significant connections. The most accurate gene signature contrast with consistent patient gene expression profiles was more resilient to data loss and identified robust target compounds. Target compounds lost included candidate compounds of potential clinical utility in glioma (e.g. Suramin, Dasatinib). Lost connections may have been linked to low abundance genes in the gene signature that closely characterised the disease phenotype. Consistently identified connections may have been related to highly expressed abundant genes that were ever-present in gene signatures, despite data reductions. Potential noise surrounding findings included false positive connections that were gained as a result of gene signature modification with data loss. CONCLUSION: Findings highlight the necessity for gene signature accuracy for connectivity mapping, which should improve the clinical utility of future target compound discoveries.

Introduction Currently, risk management options for BRCA1 mutation carriers include risk-reducing mastectomies (RRM), and selective oestrogen receptor modulators (SERMs). However, the majority of BRCA1-associated tumours are oestrogen receptor negative (ER-ve). SERMs do not reduce the incidence of ER-ve tumours, therefore are unlikely to be effective in this group of women. Oestrogen and its metabolites have been shown to cause DNA damage, resulting in genomic instability in BRCA1 deficient breast cells, an early hallmark of BRCA1-related cancers. Based on this, we hypothesise that oestrogen suppression in BRCA1 carriers may reduce DNA damage in breast tissue, and thereby potentially reducing breast cancer risk. Trial Design The study is an interventional, non-randomised, crossover study without masking. The primary purpose is to assess the impact of these drugs on suppression of DNA damage and therefore their potential for use as chemopreventive agents in this setting. Pre-menopausal BRCA1 mutation carriers will be recruited from the Family History Clinic at Belfast City Hospital. The proportion of women who receive information packs who progress to trial entry will be recorded, with target recruitment of twelve women to the trial. Participants will complete quality of life questionnaires, provide blood and urine samples and undergo baseline ultrasound-guided breast biopsy. Half of these women will receive a SERM (tamoxifen) for 3 months, whilst the other half receives oestrogen suppression therapy (goserelin and anastrazole), and then provide repeat questionnaires and samples (blood, urine and breast biopsy). Treatment groups will cross over for a further 3 months treatment, with a final set of questionnaires and samples taken. Compliance with treatment and adverse events will be monitored throughout the study. Eligibility Criteria Inclusion criteria: · Female · Age ≥18 years · Premenopausal · Known pathogenic BRCA1 mutation · Intact ovaries · No previous breast/ovarian/other carcinoma · No previous use of chemoprevention · Willingness to use non-hormonal methods of contraception Exclusion criteria: · BRCA1 mutation of uncertain significance · Contraindications to study drugs or breast biopsies · Pregnancy or breastfeeding · Inability to give informed consent · Patient awaiting risk reducing surgery Specific Aims The primary objective is to assess the feasibility of treatments by measuring successful recruitment rates and compliance. The secondary objective is to establish tolerability of interventions through quality of life measurements and adverse event occurrence. Exploratory objectives will assess the potential of treatments to reduce DNA damage through analysis of breast biopsies using comet assays and immunohistochemistry for 53BP1 and γH2AX (markers of DNA double-strand breaks). Oestrogen and metabolite levels will be measured in blood and urine samples using UPLC-MS/MS. Statistical Methods Not applicable due to nature and size of pilot study Present accrual: 1 participant Target accrual: 12 participants Contact information for people with specific interest in trial: aideen.campbell@qub.ac.uk or s.mcintosh@qub.ac.uk Citation Format: Campbell AM, Savage KI, McIntosh SA. Chemoprevention in BRCA1 mutation carriers (CIBRAC): A proof of concept clinical trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT3-01-01.

Multiple large scale sequencing projects have identified a significant number of mutations in Acute Myeloid Leukaemia (AML) and high-risk Myelodysplastic syndromes (MDS) patients. Emerging from this are those affecting members of the cohesin complex, predominantly STAG2, and it has been highlighted that the poorer overall survival of this chromatin structure group is second only to patients carrying mutations affecting TP53. The Cohesin complex is an evolutionary conserved, multimeric protein complex consisting of SMC1, SMC3, RAD21 and STAG1/ STAG2. The complex plays a pivotal role during mitosis, ensuring sister chromatid cohesion however, substantial data exists highlighting roles in DNA damage repair, homologous recombination and most significantly, contribution to long-range interactions between cis-regulatory elements of the genome. Here, we have used the CRISPR/Cas9 genome editing system to create an isogenic model featuring a premature stop codon within STAG2 at a region which presents as highly mutated in patient data sets. This stop codon generates a loss of function phenotype due to the haplo-insufficient X-linked nature of the STAG2 gene. Following model validation, the impact on genome wide distribution of the cohesin complex was assessed using ChIP-Seq for STAG1, STAG2, SMC3 & the transcriptional insulator CTCF. A modest increase in overall CTCF binding from ~67,000 to ~72,000 high confidence peaks was observed, despite a ~7-fold decrease in enrichment, indicating decreased global insulator activity as a result of the inactivating STAG2 mutation. As expected, compensation between the STAG members was observed at a number of sites within transcriptionally active topologically associated domains, with an increase in STAG1 binding peaks (~17,000 to >25,000 sites) within the mutant model, despite no changes in average global enrichment. This data was integrated with RNA-seq of the isogeneic parental and mutant cells to determine the transcriptional impact of changes in cohesin complex binding on gene expression within active and repressive domains. Assessment of the active and repressive super-enhancer associated marks H3K27ac and H3K27me3 showed increased global levels of both marks without increase in the polycomb repressive complex 2 member, EZH2 total protein levels. These findings indicate that increased H3K27ac and H3K27me3 marks, in this model, are due to altered domain boundaries, associated with local activation of both H3K9ac and H3K9me3, leading to aberrant gene expression and consequently a neomorphic state within these mutant cells. With recent further stratification of de-novo AML, secondary AML and high-risk MDS emerging, this study on the aberrant and non-canonical nature of the cohesin complex provides avenues for an attractive therapeutic target and personalised treatment approach for patients within the chromatin subgroup. Disclosures No relevant conflicts of interest to declare.

We have previously identified a DNA damage response deficient (DDRD) subgroup in breast cancer, associated with loss of the Fanconi Anemia/BRCA DNA repair pathway. The 44-gene DDRD signature developed to prospectively identify this subgroup has been validated as predictive of response to DNA damaging chemotherapy in the treatment of breast cancer. This subgroup is defined by immune signalling, with immune genes, such as the chemokines CXCL10 and CCL5, and immune checkpoints IDO1 and PD-L1 (CD274) overexpressed within the signature and subgroup. Here we report innate immune pathway activation in this subgroup, and association with PD-L1 expression. Methods: We used isogenic cell line models to identify and model pathways constitutively active in DDRD cancer cells. CD4+, CD8+ T-cell infiltration and PD-L1 expression was identified by immunohistochemistry (IHC) on a previously described breast TMA of 191 breast tumor samples, of which 65 were in the DDRD subgroup. DNA microarray data from FFPE samples was used to identify other immune checkpoints associated with this subgroup. Results: CD4+ and CD8+ T-cell infiltration, and PD-L1 expression were significantly associated with DDRD breast tumors on IHC analysis. In addition, expression of immune checkpoints such as IDO1, TIM-3 and LAG-3 were associated with the DDRD subgroup. Increased migration of peripheral blood mononuclear cells was identified into media conditioned by DDRD cells, this was dependent on chemokines secreted by DDRD cells. Endogenous activation of the innate immune pathway STING/TBK1/IRF3 was identified in DDRD cancer cells, and required for expression of chemokines CXCL10 and CCL5. This chemokine expression was associated with cytosolic DNA detected by cGAS and activation of the innate immune pathway STING/TBK1/IRF3. Importantly, this pathway was cell cycle related with upregulation of chemokine expression in S-phase of the cell cycle. Chemokine expression could be induced by S-phase DNA damaging chemotherapy but not by taxanes. Similarly, PD-L1 was induced by treatment with DNA damaging agents in cancer cells dependent on STING. Conclusions: We have identified constitutive activation of the innate immune STING pathway in a DNA damage response deficient subgroup of breast cancer, which is associated with CD4+, CD8+ infiltration and PD-L1 expression. We propose activation of the STING pathway as an important signal for lymphocytic infiltration, independent of tumor-associated neoantigens. The DDRD signature could identify patients with innate immune activation in response to endogenous DNA damage, which may allow stratification for immune checkpoint targeted treatments. Moreover, S-phase DNA damaging agents activate PD-L1 expression, and a combination approach of immune checkpoint inhibitors and S-phase specific chemotherapy may be synergistic in the clinic. Citation Format: Eileen E. Parkes, Steven M. Walker, Nuala McCabe, Laura E. Taggart, Laura Hill, Niamh E. Buckley, Kienan I. Savage, Manuel Salto-Tellez, Stephen McQuaid, Mary T. Harte, Paul B. Mullan, D Paul Harkin, Richard D. Kennedy. A DNA damage response deficiency (DDRD) group in breast cancer is associated with activation of the STING innate immune pathway and PD-L1 expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4000.