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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.

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.

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.

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.

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.