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Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore gamma-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after gamma-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy.

The eleven Fanconi anemia (FA) proteins cooperate in a novel pathway required for the repair of DNA cross-links. Eight of the FA proteins (A, B, C, E, F, G, L, and M) form a core enzyme complex, required for the monoubiquitination of FANCD2 and the assembly of FANCD2 nuclear foci. Here, we show that, in response to DNA damage, Chk1 directly phosphorylates the FANCE subunit of the FA core complex on two conserved sites (threonine 346 and serine 374). Phosphorylated FANCE assembles in nuclear foci and colocalizes with FANCD2. A nonphosphorylated mutant form of FANCE (FANCE-T346A/S374A), when expressed in a FANCE-deficient cell line, allows FANCD2 monoubiquitination, FANCD2 foci assembly, and normal S-phase progression. However, the mutant FANCE protein fails to complement the mitomycin C hypersensitivity of the transfected cells. Taken together, these results elucidate a novel role of Chk1 in the regulation of the FA/BRCA pathway and in DNA cross-link repair. Chk1-mediated phosphorylation of FANCE is required for a function independent of FANCD2 monoubiquitination.

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease–associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIα poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

Two million elderly Americans are abused or neglected each year. Elderly patients visit their physicians approximately five times each year. Yet physicians initiate only about 2% of the reported cases of abuse and neglect. This study's purpose was to assess the experience, knowledge, and attitudes of primary care physicians toward elder mistreatment.A self-report questionnaire was mailed to a random sample of 500 primary care physicians. The population included 250 family physicians and 250 general internists in Ohio, divided equally between large urban, suburban, and rural practice settings.The overall response rate was 78%, with 216 family physicians and 176 internists responding. Nearly 72% of those responding reported no exposure or only minimal exposure to the physical, emotional, or sexual abuse of the elderly. More than half of the respondents in both groups reported that they had never identified a case of elder mistreatment. Both family physicians and general internists estimated its prevalence as roughly less than 25% of the prevalence documented in the medical literature, and both were reluctant to accept the problem as universal. More than 60% of clinicians in both groups indicated they had never asked their elderly patients about abuse. Family physicians tended to have a better knowledge of those elements encompassing elder mistreatment and were more aware of management options.Physicians need more education about elder mistreatment.

BRCA1 has been reported to have roles in DNA damage repair, cell cycle checkpoint control, transcriptional regulation and ubiquitination. We have previously demonstrated that BRCA1 is a potent activator of a subset of interferon (IFN)-regulated genes and that BRCA1 synergistically activated a number of these genes in the presence of IFN-gamma, but not type I IFNs. Here we report that one of these targets, 2,5 oligoadenylate synthetase (2,5 OAS), is a mediator of BRCA1/IFN-gamma-induced apoptosis. We show that the induction of 2,5 OAS in response to IFN-gamma is BRCA1 and STAT1 dependent. Consistent with a role as a negative regulator of proliferation, transient transfection of 2,5 OAS into breast cancer cell lines results in decreased colony growth and apoptosis. Furthermore we show that IFN-gamma-induced apoptosis is dependent on functional BRCA1 and STAT1 and we demonstrate that IFN-gamma-induced apoptosis is dependent on 2,5 OAS induction. 2,5 OAS is the only known upstream regulator of RNaseL, a recently identified hereditary prostate tumour suppressor gene implicated in apoptosis. We propose that BRCA1 may be an upstream regulator of RNaseL, acting in concert with IFN-gamma to transcriptionally activate 2,5 OAS, leading to the downstream activation of RNaseL and apoptosis.

Germline mutations of the BRCA1 gene account for approximately 5% of breast and ovarian cancer cases, and lower than normal BRCA1 expression or function may be an important contributing factor in sporadic cancers. The major role of BRCA1 is to respond to DNA damage by participating in cellular pathways for DNA repair, mRNA transcription, cell cycle regulation, and protein ubiquitination. Because most chemotherapeutic agents function by directly or indirectly damaging DNA, the role of BRCA1 as a regulator of chemotherapy-induced DNA damage has been the subject of an increasing number of investigations. We review published preclinical and clinical evidence that the level of BRCA1 function in an individual patient's tumor can guide the choice of chemotherapeutic agents for breast and ovarian cancer. We conclude that a loss of BRCA1 function is associated with sensitivity to DNA-damaging chemotherapy and may also be associated with resistance to spindle poisons. We recommend that prospective clinical studies investigating the role of BRCA1 in the response to chemotherapy be conducted.

BRCA1 has been implicated in a number of cellular processes, including transcriptional regulation, DNA damage repair, cell cycle arrest, and apoptosis. We identified mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3), an upstream regulator of the c-Jun NH2-terminal kinase/stress-activated protein kinase and p38/MAPK pathways, as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by coimmunoprecipitation in mammalian cells. Deletion mapping demonstrated that amino acids 1611–1863 are required to mediate the interaction with MEKK3 in yeast. BRCA1 disease-associated mutations abrogated the interaction in yeast, and BRCA1 failed to interact with MEKK3 in BRCA1 mutant HCC1937 breast cancer cells. We demonstrate that small interfering RNA-based inhibition of endogenous BRCA1 reduces MEKK3 kinase activity and conversely that inducible expression of BRCA1 activates MEKK3 and p38/MAPK. Finally, we demonstrate using complementary approaches that BRCA1 is required for paclitaxel-induced activation of MEKK3. These data indicate that BRCA1 is a key regulator of the paclitaxel-induced stress response pathway and suggest that the ability of BRCA1 to associate with, and mediate the activation of, MEKK3 represents a potential mechanism through which this pathway is regulated.

We have evaluated the role played by BRCA1 in mediating the phenotypic response to a range of chemotherapeutic agents commonly used in cancer treatment. Here we provide evidence that BRCA1 functions as a differential mediator of chemotherapy-induced apoptosis. Specifically, we demonstrate that BRCA1 mediates sensitivity to apoptosis induced by antimicrotubule agents but conversely induces resistance to DNA-damaging agents. These data are supported by a variety of experimental models including cells with inducible expression of BRCA1, siRNA-mediated inactivation of endogenous BRCA1, and reconstitution of BRCA1-deficient cells with wild-type BRCA1. Most notably we demonstrate that BRCA1 induces a 10-1000-fold increase in resistance to a range of DNA-damaging agents, in particular those that give rise to double-strand breaks such as etoposide or bleomycin. In contrast, BRCA1 induces a1000-fold increase in sensitivity to the spindle poisons, paclitaxel and vinorelbine. Fluorescence-activated cell sorter analysis demonstrated that BRCA1 mediates G(2)/M arrest in response to both antimicrotubule and DNA-damaging agents. However, poly(ADP-ribose) polymerase and caspase-3 cleavage assays indicate that the differential effect mediated by BRCA1 in response to these agents occurs through the inhibition or induction of apoptosis. Therefore, our data suggest that BRCA1 acts as a differential modulator of apoptosis depending on the nature of the cellular insult.

Background: The interaction between a tumor and the surrounding non-malignant stroma impacts on nearly every aspect of malignant progression. The heterogeneity of the tumor microenvironment (TME), however, makes mapping this interaction difficult. Each stromal population contributes to various functions and each tumor type has its own distinct pathway of interaction with different stromal populations. The majority of these interactions are mediated by soluble factors secreted into the TME. The loss of the tumor suppressor PTEN, an early event in prostate tumorigenesis, results in a subsequent increase in secretion of the pro-inflammatory CXCL8 chemokine into the TME (Maxwell et al 2013). The specific role of CXCL8 signaling within the prostate TME has not been fully classified and warrants further investigation. Methods: PTENnull PC3 cells were cultured in the absence and presence of human macrophage-representative THP-1 monocytes. Tumor-derived CXCL8 signaling was attenuated using (i) CXCL8 neutralizing antibodies, (ii) CXCR1 and CXCR2 receptor-targeting small peptide inhibitors (PEPducins), and (iii) a CXCL8-targeted shRNA method to generate stable PC3-120p cells with reduced CXCL8 secretion – a control line termed PC3-NTp was co-developed. PC3 cell motility and invasion was assessed by real-time transwell invasion through MatrigelTM and wound closure assays. Phospho-receptor tyrosine kinase (RTK) arrays were used to identify signaling pathways downstream of CXCL8 signaling which are relevant to prostate tumor cross-talk with infiltrating monocytes. Results: The presence of THP-1 cells more than doubled the rate of PC3 invasion through MatrigelTM (2.4-fold, p=0.0098) and increased wound closure after 6 h (50.63% v 25.65% for PC3 cells alone; p=0.0108). Macrophage-dependent enhancement of invasion and motility was attenuated following inhibition of CXCL8 signaling (i) by neutralizing antibody (90% decrease in normalized invasion and 43% decrease in wound closure, p Conclusions: Tumor associated macrophages (TAMs) are correlated with prostate cancer progression. Secretion of CXCL8 from PTEN-deficient prostate cancer cells activates macrophages, which then sustains cancer cell migration and invasion. Attenuating CXCL8 signaling within the TME and its downstream effects provides potential therapeutic targets to reduce metastasis in PTEN-deficient, CXCL8-high prostate cancer patients. Citation Format: Jessica L. Neisen, Nuala McCabe, Richard D. Kennedy, David JJ Waugh. Cabozantinib attenuates CXCL8-driven macrophage-dependent migration and invasion of PTEN-deficient prostate cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A49. doi:10.1158/1538-7445.CHTME14-A49

As part of ongoing surveillance of infection in the haematology and oncology units at Belfast City Hospital, microbiologically documented bloodstream infections over three 12-month periods 1994/5, 1998/9 and 1999/00 were reviewed. Gram-positive organisms were the most common cause of blood stream infection in the haematology unit causing 66%, 56% and 64% of episodes of monomicrobial bacteraemia in 1994/5, 1998/9 and 1999/00, respectively. In haematology patients, enterococci have emerged as an important cause of bacteraemia, with increasing levels of glycopeptide resistance, and the 'non-fermenting Gram-negative rods other than Pseudomonas aeruginosa' are an increasingly common cause of monomicrobial and polymicrobial bacteraemia. In oncology patients, Gram-negative organisms (predominantly enterobacteriaceae) were more common than Gram-positive organisms, causing 50% and 54% of monomicrobial bacteraemia in 1998/9 and 1999/00, respectively. Changes in patient population, underlying diseases and chemotherapeutic agents may explain these findings. The spectrum of infection seen in haematology and oncology patients changes as management evolves. Ongoing co-operation between haematologists, oncologists and microbiologists is important to detect trends in epidemiology, which can be used to design empirical antibiotic regimens and guide infection control policies.

Breast cancer remains a frequent cause of female cancer death despite the great strides in elucidation of biological subtypes and their reported clinical and prognostic significance. We have defined a general cohort of breast cancers in terms of putative actionable targets, involving growth and proliferative factors, the cell cycle, and apoptotic pathways, both as single biomarkers across a general cohort and within intrinsic molecular subtypes. We identified 293 patients treated with adjuvant chemotherapy. Additional hormonal therapy and trastuzumab was administered depending on hormonal and HER2 status respectively. We performed immunohistochemistry for ER, PR, HER2, MM1, CK5/6, p53, TOP2A, EGFR, IGF1R, PTEN, p-mTOR and e-cadherin. The cohort was classified into luminal (62%) and non-luminal (38%) tumors as well as luminal A (27%), luminal B HER2 negative (22%) and positive (12%), HER2 enriched (14%) and triple negative (25%). Patients with luminal tumors and co-overexpression of TOP2A or IGF1R loss displayed worse overall survival (p=0.0251 and p=0.0008 respectively). Non-luminal tumors had much greater heterogeneous expression profiles with no individual markers of prognostic significance. Non-luminal tumors were characterised by EGFR and TOP2A overexpression, IGF1R, PTEN and p-mTOR negativity and extreme p53 expression. Our results indicate that only a minority of intrinsic subtype tumors purely express single novel actionable targets. This lack of pure biomarker expression is particular prevalent in the triple negative subgroup and may allude to the mechanism of targeted therapy inaction and myriad disappointing trial results. Utilising a combinatorial biomarker approach may enhance studies of targeted therapies providing additional information during design and patient selection while also helping decipher negative trial results. Citation Format: Manuel Salto-Tellez, David P. Boyle, Darragh McArt, Gareth Irwin, Charlotte Charlotte Wilhelm-Benartzi, Tong G. LIoe, Martha Minter, Stephen McQuaid, Paul Mullan, Richard D. Kennedy, Peter Hamilton, D P. Harkin. Defining a therapeutic classification of breast cancer by actionable targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1905. doi:10.1158/1538-7445.AM2014-1905

5502 Background: HGSOC is a histopathological diagnosis and may represent multiple diseases at a molecular level. We investigated whether distinct molecular subgroups may influence treatment choice...

4015 Background: The incidence of esophageal adenocarcinoma (EAC) has increased 6-fold in the last 30 years and in early stage EAC 5-year survival remains poor at 25-35%. Neoadjuvant therapy confer...

63 Background: Accurate prediction of outcome for neo-adjuvant therapy in early stage esophageal adenocarcinoma (EAC) is essential for clinical trial design, patient counseling and treatment decisions. The use of 18F-fluorodeoxyglucose positron emission tomography (PET) has improved the staging of EAC and can predict pathological response and survival. Our aim was to develop a pre-operative prognostic model based on metabolic imaging and clinical parameters. Methods: Between April 2004 and December 2010, 118 patients with early stage adenocarcinoma of the distal esophagus or gastro-esophageal junction were treated with platinum and fluorouracil-based combination chemotherapy prior to surgical resection. Patients were staged by endoscopic ultrasound and PET/CT was performed prior to, and upon completion of, chemotherapy. A metabolic response was defined as a >35% reduction in the maximum standard uptake variable (SUVmax). The final prognostic model was selected by multivariate Cox regression analyses and the concordance-index (c-index) was used to assess the fit of the final multivariate model. Results: In both the univariate and multivariate analysis of patient prognosis post-surgery, five pre-surgery factors were included: clinical T and N staging, PET response, neutrophil-lymphocyte ratio and albumin levels. The majority of the patients were T3 (82.1%) and N1 (73.7%) and 69.8% of patients demonstrated a PET response. In the univariate analysis PET response was the only significant prognostic factor with a median overall survival (OS) of 60.4 months for responders compared to 23.3 months for non-responders (p=0.0241). The final multivariate model of T staging and PET response had a c-index of 0.624 with T3/PET non-response resulting in a poorer prognosis compared to T3/PET response and T2/PET responder or non-responder (median OS 11.5 vs 44.5 vs undefined months; p=0.0284). We seek to validate our model in an independent patient dataset to assess its predictive capability. Conclusions: Metabolic imaging and clinical staging can be combined in a prognostic model in early stage EAC. Our model can inform patient counseling and the development of clinical trial strategies.

455 Background: Only 15-25% of pts with stage II colon cancer (CC) experience recurrence and conventional staging methods neither allow accurate identification of low (L) and high-risk (H) subgroups nor predict benefit of adjuvant chemotherapy. The ColDx assay (Almac Diagnostics) is a 634-probeset gene expression signature shown to be independently prognostic for recurrence-free interval (RFI). The objective of this study was to assess the ability of ColDx to classify stage II CC pts at L- and H-risk of relapse. Methods: This validation study was conducted using formalin fixed paraffin embedded biospecimens and clinical data from CALGB 9581, a phase III trial of edrecolomab v. observation in pts with normal risk, stage II CC. 1,454 CALGB 9,581 pts met eligibility criteria. A case-cohort sampling design was used to randomly select (RS) 514 pts from 901 eligible pts with available tissue; supplemented by 49 non-RS recurrent pts (total 563). Risk status for each pt was based on a positive or negative ColDx score using a pre-specified cutpoint, 0.4377. The Self Prentice method was used to test the association between ColDx categories and RFI (distant recurrence or death due to primary disease). Results: Initial results in 563 pts were erroneous due to a quality failure in a batch of reagent. 524 samples were re-labeled, re-ordered, and re-assayed using reagents that passed quality control (36 samples had insufficient material; 95 failed ColDx QC). Final analysis comprised 393 pts, 360 RS (58 events; 16%); 33 non-RS events. 216 pts (55%) were predicted H (62 events); 177 (45%) pts were predicted L (29 events). H pts exhibited significantly worse RFI (univariable hazard ratio (HR), 2.0; 95% CI, 1.3-3.3; p < 0.01). ColDx remained significant after adjustment for prognostic factors; HR, 2.1 (95% CI, 1.3-3.4; p < 0.01). Conclusions: The ColDx assay result is associated with RFI in the CALGB 9,581 sub-sample and is independent from other prognostic factors, including MSI. Further investigation is needed to establish the role of this classifier in guiding treatment decisions in this patient population.

45 Background: Patients with operable esophageal adenocarcinoma have a poor prognosis (median survival

Background: CXCL8 signaling levels are elevated in localized high grade prostate cancer (CaP). We have shown exposure of CaP cells to chemical DNA-damaging agents potentiates CXCL8 signaling, which underpins an autocrine mode of therapeutic resistance. Data from in vitro and in vivo CaP models has shown loss of the tumor suppressor PTEN also increases expression of the pro-inflammatory chemokine CXCL8. Experiments were designed to determine whether exposure to ionising radiation (IR) selectively induced CXC-chemokine signaling in PTEN-deficient CaP cells, and to determine how CXCL8 signaling may modulate their radiosensitivity. Methods: shRNA-directed strategies were used to establish cell-based models of CaP with differential expression of PTEN. These clonal (DU145) and heterogeneous (22Rv1) populations were subjected to IR under normal or hypoxic culture conditions. PTEN was reconstituted in PC3 cells and was driven by tetracycline-regulated expression of the gene. Changes in chemokine expression were analyzed by RT-PCR, immunoblotting and ELISA. Autocrine CXCL8 signaling was attenuated using siRNA to down-regulate gene expression of its receptors, CXCR1 and CXCR2. The effects of modulating chemokine signaling on cell viability following exposure to clinically-relevant doses of IR were assessed by colony formation assays, cell proliferation assays, and analysis of apoptosis and cellular senescence. The effect of CXCL8 signaling inhibition in vivo was determined using a PC3 xenograft model in SCID mice. The cell penetrating pepducin, x1/2pal-i3, was used to attenuate both CXCR1 and CXCR2 and tumor growth was assessed via caliper measurements. Results: Exposure to IR (3 Gy) increased gene expression of CXCL8, CXCR1 and CXCR2 - with PTEN-deficiency enhancing this response. Loss of PTEN expression differentially modulated the radiosensitivty of CaP cell lines: PTEN-depletion conferred relative radioresistance in DU145 cells but made 22RV1 cells more radiosensitive. However, the inhibition of CXCL8 signaling in all PTEN-depleted models resulted in consistently increased radiosensitivity (SER 1.2, 1.13, and 1.39 in DU145, 22RV1, and PC3 CaP cells, respectively; p Conclusions: Our results demonstrate the role of treatment-induced CXCL8 signaling as a mechanism of resistance to IR in PTEN-depleted CaP cells. Inhibition of IR-induced CXCL8 signaling provides a novel therapeutic opportunity to enhance the radiosensitivity of PTEN-depleted CaP cells. However, mechanistic results indicate that other genetic factors including p53 status dictate the resultant induction of treatment-induced apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C215. Citation Format: Chris WD Armstrong, Jonathan A. Coulter, Steven Walker, Richard D. Kennedy, Kevin M. Prise, Joe M. O'Sullivan, David JJ Waugh. Sensitivity of PTEN-deficient prostate carcinoma xenografts to ionizing radiation through inhibition of treatment-induced CXCL8 signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C215.

In an effort to develop more tailored approaches to managing disease, there has been a surge in biomarker research aimed at providing truly personalized medicine. The increase in interest over the past 20 years suggests great promise that biomarkers will transform patient treatment. However, despite the optimism and intense research, relatively few are in routine clinical use today. Attention has now turned to actively translating actionable biomarkers and biomarker signatures into clinical utility—not an easy task. The major reasons for clinical failure of biomarkers are poor experimental design and inappropriate choice of assay. It is therefore critical to carefully construct biomarker discovery and validation studies using the most appropriate assays, and to develop these into validated analytical methods suitable for clinical practice. This webinar will review the unprecedented range of technology choices, and provide insight into the challenges confronting researchers when selecting the right biomarker to pursue and the best assay to develop for the clinic.

TPS11120 Background: Currently there is no biomarker to predict specific benefit from DNA-damaging anthracycline and cyclophosphamide-based chemotherapy in the clinic. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA-damage response pathway occurs in approximately 25% of breast cancer and results in sensitivity to DNA-damaging agents. We therefore developed an assay to detect loss of the FA/BRCA pathway, for the purpose of predicting benefit from chemotherapy. Methods: 21 FA patient samples were analyzed to identify genetic processes associated with loss of the FA/BRCA pathway. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples and a molecular subgroup was identified that was defined by the molecular processes representing loss of the FA/BRCA pathway. A 44-gene DNA Damage response deficient (DDRD) assay was developed that could identify this subgroup from formalin fixed, paraffin embedded (FFPE) samples in the clinic. Results: In a publicly available independent cohort of 204 patients, the assay predicted response to neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline and cyclophosphamide) with an odds ratio of 4.01, (95% Cl:1.69-9.54). We also analysed samples from an independent cohort of 114 node-negative breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin and cyclophosphamide treatment at the Northern Ireland Cancer Centre. The DDRD assay significantly predicted 5-year relapse free survival with a hazard ratio of 0.27 (95% Cl:0.10-0.83). The assay was not predictive of survival in patients who did not receive chemotherapy. Conclusions: An FFPE tissue-based assay that detects loss of the FA/BRCA pathway has been developed and independently validated as a predictor of response and prognosis following DNA damaging anthracycline/cyclophosphamide-based chemotherapy in the neoadjuvant and adjuvant settings.

The involvement of Wnt signalling in cancers including ovarian, gastric, and breast cancer is well characterised. Wntless (Wls) (also known as GPR177, Evi, and Srt) is a key modulator of Wnt protein secretion, and was recently found to be highly overexpressed in malignant astrocytomas, promoting proliferation, survival, and migration of glioma cells. It was hypothesised that this molecule may be aberrantly expressed in other cancers known to possess aberrant WNT signalling. Using immunohistochemical analysis of ovarian, gastric, and breast cancer tissue microarrays, we found that Wls was overexpressed in a subset of tumours from each cancer subtype. Wls overexpression was associated with poorer clinical outcomes in gastric cancer. Interestingly, a strong correlation was observed between Wls expression and human epidermal growth factor receptor 2 (HER2) expression. Eight of eight (100%) HER2-positive intestinal gastric carcinomas, five of five (100%) HER2-positive serous ovarian carcinomas, and 41 of 64 (64%) HER2-positive breast carcinomas expressed Wls. This finding has clear biological and clinical implications. HER2 is an important predictive biomarker in breast and gastric cancer for selection of patients likely to respond to trastuzimab. However, a substantial proportion of HER2-positive patients do not respond to this therapy. Recent literature suggested a possible pathway in which Wls and HER2 may be involved via the known mediator of trastuzimab and chemotherapy resistance NFkB, which may explain the low response rate of patients with HER2-positive breast, ovarian, and gastric cancers to agents that target HER2. An in-vitro study has been commenced to try to confirm the existence of this pathway. This study should provide significant insights into the mechanistic association between these important signalling molecules. Funding Queens University Belfast.

mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (≈2 x 10-5 of all mRNA). Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development.

10553 Background: Loss of PTEN function has been widely reported to cause up-regulation of the PI3K/AKT signalling pathway resulting in increased cell growth, proliferation and survival. More recently it has been reported that PTEN null cells demonstrate genomic instability and have increased production of reactive oxygen species (ROS) and oxidative stress induced DNA damage. Ataxia Telangiectasia Mutated (ATM) is the primary response kinase, which responds to stalled DNA replication and DNA double strand breaks due to oxidative DNA damage. Methods: A metagene representing ATM activation was generated from cell line data and used to perform hierarchical clustering analysis of public DNA microarray profiling datasets of breast cancer, ovarian cancer and glioblastoma with known PTEN IHC/mutation status. Furthermore, we ask if ATM activation may be therapeutically exploited in PTEN null tumours using ATM specific siRNA and compounds in 2 PTEN isogenic cell line model systems. Results: We show that PTEN null cells have elevated levels of ROS, DNA damage and have endogenous activation of ATM, an enzyme important in responding to DNA damage resulting from oxidative stress. We hypothesised that PTEN deficient tumours may rely on ATM enzyme for survival. To investigate this we generated a 189-gene list representing ATM activation and used this to perform hierarchical clustering analysis of a breast cancer DNA microarray dataset. This list was able to significantly cluster tumours with known loss of PTEN expression (p=0.004). Furthermore, this gene list was able to segregate PTEN null/mutant tumours from PTEN wild-type tumours in 2 independent datasets of glioblastoma and ovarian cancer (p=0.015 and p=0.012). In addition, we found that inhibition of ATM using the selective inhibitor KU-55933 caused DNA damage, cell cycle arrest and apoptosis specifically in PTEN deficient cells when compared to PTEN wild-type cells. Conclusions: These observations suggest that ATM may represent a therapeutic target in PTEN deficient tumours and furthermore ATM activation may be the basis of a biomarker of PTEN status in human cancers.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancies in the Western World. The aim of this study was to identify ovarian cancer subtypes related to biological pathways which are currently being targeted by novel therapeutics. SRC (sarcoma) is a tyrosine kinase which plays critical roles in mediating many cellular pathways such as proliferation, adhesion, survival, differentiation and cell motility. SRC activity is increased in many human cancers including EOC. Furthermore, up-regulation of the SRC pathway has been shown to be associated with cisplatin resistance and patient overall survival in EOC. We therefore hypothesised that up-regulation of the SRC pathway may represent a subtype of EOC which could be therapeutically exploited. We used a semi-supervised analysis approach to perform hierarchical clustering analysis of 198 serous, stage III and IV EOC using a 917 public gene list generated by the differentially expressed genes, after DNA microarray profiling, in MCF10A cells with normal expression and over-expression of the SRC gene. A number of gene clusters were identified upon hierarchical clustering which segregated the patient samples. To examine which of the genes from these clusters represented SRC activation we used a series of in vitro assays including phospho-SRC expression, sensitivity to SRC siRNA, and sensitivity to the SRC inhibitors Saracatinib and Dasatinib in 16 ovarian cancer cell lines. The ovarian cell lines were scored individually with the probesets representing the genes of interest from each of the individual gene clusters and the cell line in vitro data. Two gene clusters were selected for further investigation. A panel of 15 breast cancer cell lines were next investigated for SRC activation using the same in vitro assays described above. Correlation of SRC activation, by the scoring of the in vitro assays and the scoring of probesets from the individual gene clusters, demonstrated one gene cluster which showed good correlation and which potentially represented a metagene of SRC activation. The gene cluster consists of 16 genes, some of which are already known components of the SRC pathway. Furthermore, using the probesets representing the genes from this gene cluster to score public DNA microarray profiling data we were able to predict response to Dasatinib in two independent datasets of prostate and breast cancer cell lines (p= Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4820. doi:1538-7445.AM2012-4820

Background PTEN is frequently lost in cancer cells through genetic mutation or epigenetic silencing. Loss of PTEN function has been widely reported to cause up-regulation of the PI3K/AKT signalling pathway resulting in increased cell growth, proliferation and survival. More recently it has been reported that PTEN null cells demonstrate genomic instability through increased ROS and oxidative stress induced DNA damage. The aim of this study was to identify a biomarker for PTEN status in human breast cancers. Materials and Methods A metagene representing ATM activation was generated from public cell line data of AT fibroblasts treated with gamma-irradiation. This was used to perform hierarchical clustering analysis of a public DNA microarray profiling dataset with known PTEN IHC status. The metagene was validated in PTEN wildtype and null breast cancer cell lines. Results We found that PTEN null cells have elevated levels of ROS and furthermore activation of the DNA damage signalling kinase, ATM. In agreement with this, the ATM metagene signature correlated with PTEN mutation in breast cancer tumours. Scoring of PTEN wildtype and null breast cancer cell lines using the metagene correlated with ATM activation and sensitivity to inhibition of ATM. Furthermore we show that inhibition of ATM caused DNA damage, cell cycle arrest and apoptosis in PTEN deficient cells suggesting a novel therapeutic strategy. Conclusion These observations suggest that ATM may represent a therapeutic target in PTEN deficient tumours and furthermore ATM activation may also be an important biomarker of PTEN mutation or loss in breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-12.

Background: PTEN loss is associated with early prostate cancer progression. We sought to characterize whether PTEN loss regulates pro-inflammatory chemokine signaling and its functional significance to PTEN-depleted prostate cancer cells. Methods: Chemokine/chemokine receptor expression was assessed in vitro in established cell lines, exhibiting different levels of endogenous PTEN expression. Down-regulation of endogenous PTEN expression in DU145 and 22Rv1 cells was effected using transient siRNA or stable shRNA interventions. Chemokine expression was further evaluated by immunohistochemistry in prostate tissue from wild-type and PTEN+/− mice. Results: Loss of PTEN induced transcription-mediated increases in expression of CXCL8 and its receptors, CXCR1 and CXCR1 in DU145 and 22Rv1 cells. Stress-induced expression of CXCL8, CXCR1 and CXCR2 was greater in magnitude and duration in PTEN-null PC3 and LNCaP cells, in contrast to the dampened response observed in PTEN-expressing cells. PTEN loss had no effect on basal or stress-induced expression of CCL2, CXCL12 or their receptors CCR2 or CXCR4 in prostate cancer cells. The efficacy of CXCL8 signaling upon HIF-1 and NF-B transcription was also greater in magnitude and duration in PTEN-null prostate cancer cells or following siRNA-mediated PTEN knockdown. Increased expression of the orthologous murine chemokine KC was elevated in atypical cytological features of PTEN+/− prostate tissue relative to normal epithelium in PTENWT glands. Attenuation of CXCL8 signaling using siRNA oligonucleotides or shRNA vectors in cell-based models of partial or complete PTEN-loss significantly decreased cell viability. Furthermore, attenuation of CXCL8 signaling modulates the response of PTEN-depleted prostate cancer cells to clinically-relevant doses of ionizing radiation. Conclusion: PTEN loss promotes a selective up-regulation of CXCL8 signaling and increases the efficacy of CXCL8 signaling in prostate cancer cells. Functionally, this selective induction of CXCL8 signaling acts to maintain cell viability as an essential adaptation to the genetic instability arising from PTEN loss. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B200.

10511 Background: Loss of a functional DNA-damage response (DDR) sensitizes tumors to DNA-damaging as well as targeted therapeutics such as PARP-1 inhibitors. However, there is no assay to detect D...

5000 Background: Despite histological heterogeneity and evidence of some molecular subtypes (e.g. BRCA1/2-deficient) EOC continues to be treated as a single disease. Using unsupervised hierarchical...

Microarray gene expression profiling has facilitated identification of molecular subtypes which can be associated with disease outcome and treatment response. However current platforms lack disease focus, potentially missing vital information contained in patient tissue samples. Moreover, a wide range of FFPE sample cohorts are available but their use is limited by the mRNA degradation inherent to the sample fixation process. By combining the Breast Cancer DSA® with powerful data analysis methodology, we reproduced the five major molecular subtypes in breast cancer based on a cohort of 107 FFPE samples. Furthermore, we showed that the established subtypes do not extend to BRCA1 and BRCA2 mutant tumours, necessitating expansion of the original breast cancer subtypes definition. Following RMA pre-processing of the 107 raw profiles, the data was transformed to overcome the association between profile quality and expression variability. If left untreated, this technical variability, often observed with FFPE tissues, can impair the study of inherent biological variations. Using the intrinsic list of 277 genes previously identified as differentiating breast cancer subtypes in fresh frozen samples [Sorlie et al., 2003], initially the sporadic samples were examined using hierarchical agglomerative clustering. All five original subtypes were identified, verifying the ability to detect subtypes in FFPE samples using the breast cancer DSA®. When the intrinsic list was applied to all 107 samples including BRCA1 and BRCA2 mutant samples, the subtypes were still distinguishable, although 31% of the mutant tumors were unclassified suggesting that specific biological processes defining BRCA1/BRCA2 mutant tumours are not captured by the original breast cancer molecular classification. We have successfully overcome the limitation imposed by FFPE sample microarray profiling by using the Breast Cancer DSA® and advanced data manipulation and exploration to reproduce molecular subtypes previously identified using fresh frozen samples. Further investigation to extend the previously identifed subtypes will progress the investigation of the heterogeneous biology underlying breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2011-326

Introduction: The tumour suppressor PTEN is frequently lost in multiple cancer types, loss of PTEN function has been linked to activation of the AKT signalling pathway. The regulation of the AKT signalling has long been attributed as the key tumour suppressing function of PTEN. However, it has recently been demonstrated that PTEN null cells demonstrate genomic instability and require base excision repair (BER) for survival. One of the critical functions of BER is the repair of oxidative DNA damage; we postulated that PTEN might have a role in the maintenance of this form of damage. Additionally, we investigated which molecular pathways were involved in the response of this form of DNA damage in PTEN null cells. Results: Using isogenic cell lines we found an increased in baseline ROS levels according to PTEN status. Specifically the increase in ROS levels was correlated to an increase in the levels of double strand breaks as measured by levels of activated γH2AX, formation of γH2AX foci and activation of the DNA damage response kinase ATM. Furthermore, inhibition of ATM function both via siRNA knockdown and small molecule inhibition in PTEN null cells resulted in cell cycle arrest followed by apoptosis. Strikingly this dependence on ATM activity was independent of AKT activity, suggesting that the oxidative DNA damage was the result of a loss in PTEN function independent of its role in the regulation of the AKT activation. Conclusions: These observations suggest that PTEN may play a role in the maintenance of the repair of ROS DNA damage; furthermore it maybe postulated that inhibition of ATM maybe a suitable therapeutic intervention for PTEN null tumours. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 250. doi:10.1158/1538-7445.AM2011-250

Background: Approximately 5-10% of all breast cancers are hereditary and the majority of these arise due to germline mutations in the BRCA1 and BRCA2 tumour suppressor genes. BRCA1 is involved in multiple cellular pathways including DNA damage signalling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. Several distinct pathological features can be used to estimate the likelihood of the presence of a BRCA1 mutation, however, it is not yet possible to fully categorise a BRCA1 mutated tumour. BRCA1-associated breast cancers are generally defined as being ER (Estrogen Receptor) negative and indeed triple negative for ER, PR and HER2. However, approximately 10-36% of BRCA1 mutated breast cancers are, in fact, ER positive. These tumours less frequently demonstrate the characteristics more commonly associated with BRCA 1 -associated breast cancers. Initial molecular evidence also suggests that there is heterogeneity within BRCA1-associated breast tumours and this is dependent on the presence or absence of the estrogen receptor. The aims of this study are to investigate the underlying biology of BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast tumours. Methods: Extensive gene expression profiling and data analysis was performed on a cohort of 70 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA1 mutated breast tumours and matched sporadic controls using the ALMAC Breast Cancer DSA™ research tool. Within this dataset we have generated molecular profiles of: (1) BRCA 1 -mutated ER positive and (2) BRCA1-mutated ER negative breast cancer. Functional analysis was performed using DAVID and METACORE. Validation of gene targets was performed by qRT-PCR and Western blotting. Results: A list of differentially expressed transcripts was derived from the comparison of 35 BRCA1 mutant breast tumours and 35 matched sporadic controls. Further analysis based on the presence and absence of ER identified a set of transcripts defining BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast cancer. Functional analysis of these two datasets has identified the main pathways and processes that are deregulated. Specifically, BRCA1-deficiency in the absence of ER was associated with deregulation of pathways implicated in immune response whereas BRCA 1 deficiency in the presence of ER was associated with pathways implicated in cell adhesion and cytoskeletal remodelling. Validation of the key genes underlying these two BRCA1-deficient breast cancer subtypes has been performed. Discussion: This approach has revealed significant heterogeneity within BRCA1 mutated breast cancer based on the presence or absence of ER. Significant differences in the transcripts and molecular processes underlying BRCA1-mutated (ER positive) and BRCA1-mutated (ER negative) breast tumours have been identified. The ability to identify BRCA 1 -deficiency by gene expression profiling from FFPE derived breast tissue may also have significant clinical application. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-14.

Background The BRCA 1 tumour suppressor gene is mutated in a significant proportion of hereditary breast cancer cases. Additionally, downregulation of BRCA1 mRNA and protein expression is reported in approximately one third of sporadic breast cancers. BRCA1 is strongly implicated in the maintenance of genomic stability by its involvement in multiple cellular pathways including: DNA damage signalling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodelling, transcriptional regulation and apoptosis. To date, gene expression profiling has identified: (1) at least five breast cancer subtypes and (2) that BRCA1 mutant tumours segregate with basal-like breast cancers. These studies also provide evidence that breast cancers with germline mutations in BRCA1 are different from non BRCA1-related tumours. The main aim of this study is to investigate the underlying biology of BRCA1-mutated breast cancer. Methods Extensive gene expression profiling and data analysis were performed on a cohort of 70 FFPE (Formalin Fixed Paraffin Embedded) derived BRCA 1 mutated breast tumours and matched sporadic controls using the Almac Breast Cancer DSATM research tool. Functional analysis was performed with DAVID and METACORE. Validation of gene targets was performed by qRT-PCR and Western blotting. Results A list of differentially expressed transcripts has been derived from the comparison of these BRCA1 mutant breast tumours and matched sporadic controls. Functional analysis of this gene list has identified the key genes and molecular pathways that are deregulated in these tumours. BRCA1 deficiency was associated with deregulation of pathways involved in: (1) immune response, (2) metastasis and invasion, (3) cytoskeletal remodelling, (4) spindle assembly and chromosome separation, (5) apoptosis and survival. Validation of the key genes underlying this BRCA1-deficient breast cancer profile has been performed. Conclusions This approach has revealed a set of transcripts that could potentially be used to identify both hereditary and sporadic breast cancer patients with BRCA1- deficiency. The ability to perform gene expression profiling from FFPE derived breast tissue could also have significant clinical application. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-02.

e21024 Background: There is considerable interest in the use of gene expression data to generate prognostic/predictive biomarkers from NSCLC tissue. Since individual tumors exhibit histological div...

Prostate cancer is the second leading cause of cancer-related deaths in men; specifically one in six men are diagnosed with prostate cancer in their lifetime. The use of serum prostate-specific antigen (PSA) levels has been lauded as a huge step forward in the diagnosis and treatment of prostate cancer, however since it's implemenatation as a biomarker it has become apparent that the numbers of deaths from prostate cancer has only decreased slightly. Therefore the identification of new biomarkers and treatments for highly invasive/metastatic prostate cancers is of a high priority. We sought to identify new biomarkers and drug targets by performing DNA microarray analysis of prostate tumour samples and normal prostate tissue samples. To further these goals we developed a specific array platform, this array was based upon extensive sequencing of prostate tumour samples and contains approximately 90,000 probesets many of which are specific to prostate cancer. We then utilized this technology to profile a series of fresh high Gleason score primary prostate tumour samples and normal prostate samples. We identified approximately 1,600 transcripts and many associated functionally relevant pathways that were significantly differentially expressed in the prostate tumour samples when compared with the normal prostate samples. Encouragingly we also identified several transcripts which are known to be specifically expressed in prostate cancer including prostate cancer antigen 3 (PCA3), α-methylacyl-coA-racemase AKA 2-methylacyl-CoA 2-epimerase (AMACR) and members of the olfactory receptor family 51, thereby demonstrating that this approach was producing reliable information. Additionally to these transcripts 34% of the 1,600 was annotated as being unique to the prostate cancer disease specific array when compared to available generic microarrays, thereby representing novel potential biomarkers and drug targets. Functional analysis of these unique transcripts annotated many to apoptotic processes, DNA repair and cellular proliferation amongst others. This content may be highly relevant to biomarker and target development for advanced prostate cancer. In conclusion we have developed a novel prostate cancer disease specific array, we have utilized this platform to profiled a series of prostate tumour and normal samples identifying several novel transcripts associated with advanced prostate cancer. Functional annotation of these unique transcripts associated many with processes know to be deregulated in cancer. We believe that this approach demonstrates the utility of this novel platform for the discovery of clinical biomarkers and novel drug targets from tumour tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1153.

4041 Background: The discovery and widespread acceptance of molecular subtypes in breast cancer has changed the way that this disease is viewed. Similarly, we have focussed on stage II colorectal cancer where 20–25% of patients are at risk of recurrent disease following surgery. There are currently several types of colorectal cancer recognised by histopathological methods, however, little has been published regarding disease classification at a molecular level. Methods: We have taken an unbiased approach to the molecular classification of colorectal cancer. Using a colorectal cancer disease specific microarray platform we have performed expression profiling and bioinformatic analysis of 500 formalin fixed and paraffin embedded (FFPE) stage II colorectal cancer samples, in order to identify and characterise molecular subtypes. Results: We have identified molecular subtypes for colorectal cancer. One of these subtypes is enriched with tumours with microsatellite instability (MSI) that are predominantly proximal, mucinous and poorly differentiated. It is notable that while MSI is generally accepted to be associated with a good prognosis, we have found both good and poor prognosis patients within this subtype. A second subtype contains tumors with a low CpG island methylator phenotype. Another subtype is defined by an expression pattern consistent with activation of tumor suppressors. The remaining subtypes are newly identified. Conclusions: We have identified molecular subtypes in colorectal cancer. We will present how knowledge about these subtypes may affect patient management and may be used to guide the development of novel therapeutic strategies. [Table: see text]

22086 Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide with low response rates to standard chemotherapy treatment. Identification of reliable genomi...

10509 Loss of the fanconi anemia (FA) pathway function has been described in a number of sporadic tumor types including breast, ovarian, pancreatic, head and neck and hematological malignancies. Functionally, the FA pathway responds to stalled DNA replication following DNA damage. Given the importance of the FA pathway in the response to DNA damage, we hypothesized that cells deficient in this pathway may become hyper-dependent on alternative DNA damage response pathways in order to respond to endogenous genotoxic stress such as occurs during metabolism. Therefore, targeting these alternative pathways could offer therapeutic strategies in FA pathway deficient tumors. To identify new therapeutic targets we treated FA pathway competent and deficient cells with a DNA damage response siRNA library, that individually knocked out 230 genes. We identified a number of gene targets that were specifically toxic to FA pathway deficient cells, amongst which was the DNA damage response kinase Ataxia Telangiectasia Mutated (ATM). To test the requirement for ATM in FA pathway deficient cells, we interbred Fancg ± Atm± mice. Consistent with the siRNA screen result, Fancg-/- Atm-/- mice were non viable and Fancg± Atm-/- and Fancg-/- Atm ± progeny were less frequent that would have been expected. Several human cell lines with FA gene mutations were observed to have constitutive activation of ATM which was markedly reduced on correction with the appropriate wild-type FA gene. Interestingly, FA pathway deficient cells, including the FANCC mutant and FANCG mutant pancreatic cancer cell lines, were selectively sensitive to monotherapy with the ATM inhibitor KU55933, as measured by dose inhibition and colony count assays. FA pathway deficient cells also demonstrated an increased level of chromosomal breakage, cell cycle arrest and apoptosis following KU55933 treatment when compared to FA pathway corrected cells. We conclude that FA pathway deficient cells have an increased requirement for ATM activation in order to respond to sporadic DNA damage. This offers the possibility that monotherapy with ATM inhibitors could be a therapeutic strategy for tumors that are deficient for the FA pathway. No significant financial relationships to disclose.

P53 mutations are frequently detected in high-risk MDS and AML cases with complex aberrant karyotypes (~20% of de novo MDS and AML cases). Pro-apoptotic signaling is a predominant mechanism through which wild-type p53 suppresses malignant transformation, and defects in this process account for the chemo/radio-resistance of many human tumors. The ability to sensitize p53 mutant myeloid cells to undergo apoptosis after drug or radiation therapy could accelerate the development of novel therapies for MDS, AML and other malignancies with p53 mutations. We have engineered a p53 mutant zebrafish line in which the allele, p53e7 (p53M214K), mimics a mutation found in multiple primary human tumors. The line is homozygous viable and fertile with homozygotes developing malignant peripheral nerve sheath tumors with 30% penetrance by 1.5 years of age. We show that p53e7/e7 embryos exhibit fully penetrant radioresistance in both the nervous system and myeloid lineage, as visualized in live embryos stained with acridine orange or carrying a pu.1-EGFP transgene. In a pilot morpholino (MO) screen that targeted 8 checkpoint kinases, we found that chk1 knockdown restores radiosensitivity in neurons and myeloid progenitors of p53e7/e7 mutants, p53 protein-null morphants and p53e6 (p53N168K) homozygotes. Remarkably, chk1 knockdown is compatible with normal zebrafish development and viability in the absence of IR, regardless of p53 status. The radiosensitizing effects of chk1 loss are phenocopied by a recently developed, specific inhibitor of human CHK1, but not by inhibitors of ATM and CHK2. The CHK1 inhibitor shows no apparent toxicity in non-irradiated p53+/+ specimens. Radiosensitization of p53 mutants through Chk1-loss correlates with G2/M checkpoint malfunction and TUNEL-positive DNA fragmentation occurring throughout the cell cycle. Cell death in irradiated p53e7/e7;chk1MO embryos preferentially occurs during mitosis, as evidenced by an increased number of TUNEL/pH3 doubly labeled cells compared to irradiated wild-type embryos. Despite prominent TUNEL staining, p53e7/e7;chk1MO embryos analyzed by whole-mount immunochemistry are devoid of IR-induced activated cellular Caspase-3. Therefore, Chk1-loss in p53 mutants does not restore the usual Casp-3-mediated apoptotic response to IR, a notion supported by the inability of forced Bcl-xL expression to completely block cell-death recovery in p53e7/e7;chk1MO embryos. Rather, Chk1-loss unbuffers a p53-independent pathway that is normally suppressed by Chk1 after DNA damage. Through epistasis analyses, we show that this alternative cell death pathway is activated by ATR and requires Casp-2, but not Casp-8 or -9 activity. Altogether, our data indicate that Chk1 acts as a survival factor in p53 mutants after DNA damage, by blocking an ATR/Casp-2 cell-death program that would otherwise lead to widespread cellular death. As such, our results provide in vivo evidence supporting the use of CHK1 inhibitors in the treatment of MDS and AML cases with p53 mutations, as well as other types of human cancers associated with P53 deficiency.

Fanconi Anemia is a multigenic cancer susceptibility syndrome. The FA pathway controls the monoubiquitination of FANCD2, and its targeting to damage-associated DNA foci. Cells that are deficient in the FA pathway are highly sensitive to bi-functional crosslinking agents such as mitomycin-C (MMC). Although FANCD2 monoubiquitination is activated by DNA damage, how this is coupled to the DNA damage response is unknown. Previous studies suggest a functional interaction between FANCD2 and the protein kinase ATM (ataxia telangiectasia mutated). Recently we have shown that the C-terminus of FANCD2 is required for its function (Montes de Oca et al., Blood2005:105, 1003). A C-terminal truncation of FANCD2 (FANCD2-C) fails to correct the MMC sensitivity FANCD2−/− cells despite its monoubiquitination and assembly into DNA damage foci. In the present study we demonstrate that FANCD2 forms a protein complex with the ATM kinase and that this interaction requires the FANCD2 C-terminus. The association of ATM with FANCD2 occurs in the absence of DNA damage, and does not require FANCD2 to be monoubiquitinated. In immunoprecipitation experiments involving the FANCD2-C mutant, ATM fails to co-precipitate, suggesting the C-terminus of FANCD2 is required for recruiting ATM to sites of DNA damage. In addition, we have identified a new ATM phosphorylation site on FANCD2, Thr691. Using a phospho-specific antibody to FANCD2 Thr691 we have determined that siRNA knockdown of ATM results in loss of phosphorylation of Thr691, and ionizing radiation (IR) activates the phosphorylation of Thr691. Mutation of Thr691 to Alanine destroys this ATM phosphorylation site. Interestingly, this FANCD2 mutant also fails to correct the MMC sensitivity FANCD2−/− cells even though it can be monoubiquitinated and targeted to DNA foci following DNA damage, a phenotype observed for the FANCD2-C mutant. Next we examined the phosphorylation of Thr691 in FANCD2-C. In FANCD2 constructs expressing a mutated or truncated C-terminus, phosphorylation of Thr691 is delayed following exposure to MMC and the cells are sensitive to DNA damage. Following exposure to ionizing radiation the ATM kinase phosphorylates itself on Ser1981. Using a phospho-specific antibody to ATM Ser1981 we evaluated the phosphorylation of ATM in response to DNA damage caused by IR and MMC. Western analysis shows that phosphorylation of ATM on Ser1981 following IR is the same in FANCD2 and FANCD2-C cells as well as uncorrected FAND2−/− cells. ATM foci formation following IR also appears identical in all three FANCD2 cell lines based upon immunoflourescence microscopy. However in cells damaged with MMC, there is a delay in Ser1981 phosphorylation and fewer ATM foci in the uncorrected and FANCD2-C cells compared to cells corrected with intact FANCD2. We propose a model for FANCD2 function where the targeting of FANCD2 to DNA damage inducible foci is required for the proper coordination of some ATM-dependent DNA repair and checkpoint responses. For instance, FANCD2 may be required for targeting ATM to a subset of double strand breaks - namely, those breaks associated with an adjacent interstrand crosslink. Thus targeting of ATM to MMC-induced DNA damage requires FANCD2 but recruitment to IR-induced double strand breaks does not, depending instead on NBS1 and the M/R/N complex.

ObjectiveCurrent strategies to guide selection of neoadjuvant therapy in oesophageal adenocarcinoma (OAC) are inadequate. We assessed the ability of a DNA damage immune response (DDIR) assay to predict response following neoadjuvant chemotherapy in OAC.DesignTranscriptional profiling of 273 formalin-fixed paraffin-embedded prechemotherapy endoscopic OAC biopsies was performed. All patients were treated with platinum-based neoadjuvant chemotherapy and resection between 2003 and 2014 at four centres in the Oesophageal Cancer Clinical and Molecular Stratification consortium. CD8 and programmed death ligand 1 (PD-L1) immunohistochemical staining was assessed in matched resection specimens from 126 cases. Kaplan-Meier and Cox proportional hazards regression analysis were applied according to DDIR status for recurrence-free survival (RFS) and overall survival (OS).ResultsA total of 66 OAC samples (24%) were DDIR positive with the remaining 207 samples (76%) being DDIR negative. DDIR assay positivity was associated with improved RFS (HR: 0.61; 95% CI 0.38 to 0.98; p=0.042) and OS (HR: 0.52; 95% CI 0.31 to 0.88; p=0.015) following multivariate analysis. DDIR-positive patients had a higher pathological response rate (p=0.033), lower nodal burden (p=0.026) and reduced circumferential margin involvement (p=0.007). No difference in OS was observed according to DDIR status in an independent surgery-alone dataset.DDIR-positive OAC tumours were also associated with the presence of CD8+ lymphocytes (intratumoural: pConclusionThe DDIR assay is strongly predictive of benefit from DNA-damaging neoadjuvant chemotherapy followed by surgical resection and is associated with a proinflammatory microenvironment in OAC.