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Introduction Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. Methods Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC–MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. Results We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. Conclusion Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.

G.B., D.B., K.W., A.P., R.F., T.R.H., S.K., and J.A.A. received support from Fundacja na rzecz Nauki Polskiej (FNP) (grant ID: MAB/3/2017). D.R.G. received support from Genome Canada & Genome BC (grant ID: 264PRO). D.J.H. received support from NuCana plc (grant ID: SMD0-ZIUN05). H.A. received support from Swedish Cancer Foundation (grant ID: 211709). H.G. received support from United Kingdom Research and Innovation (UKRI) (grant ID: EP/S02431X/1). C.P. received support from Fundação para a Ciência e a Tecnologia (FCT) through LASIGE Research Unit (grant ID: UIDB/00408/2020 and UIDP/00408/2020). A.L. F.M.Z., C.P., A.R., A.P., and J.A.A. received support from European Union’s Horizon 2020 research and innovation programme (grant ID: 101017453). C.B. received support from Agence Nationale de la Recherche (ANR) through GRAL LabEX (grant ID: ANR-10-LABX-49-01) and CBH-EUR-GS 32 (grant ID: ANR-17-EURE0003). S.N.S. received support from Cancer Research UK (CRUK) and the Chief Scientist's Office of Scotland (CSO): Experimental Cancer Medicine Centre (ECMC) (grant ID: ECMCQQR-2022/100017). A.L. received support from Chief Scientist's Office of Scotland (CSO) NRS Career Researcher Fellowship. R.O.N. received support from CRUK Cambridge Centre Thoracic Cancer Programme (grant ID: CTRQQR-2021\100012). Tumor antigens can emerge through multiple mechanisms, including translation of non-coding genomic regions. This non-canonical category of antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry to enable the discovery of non-canonical MHC-associated peptides (ncMAPs) from non-coding regions. Considering that the emergence of tumor antigens can also involve post-translational modifications, we included an open search component in our pipeline. Leveraging the wealth of mass spectrometry-based immunopeptidomics, we analyzed 26 MHC class I immunopeptidomic studies of 9 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant post-translational modifications, using spectral matching and controlled their false discovery rate (FDR) to 1%. Interestingly, the non-canonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 54.85%. Here, we reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targeting agents for T-cell therapies or vaccine development. Publisher PDF

Purpose: Nucleoside analogues form the backbone of many therapeutic regimens in oncology and require the presence of intracellular enzymes for their activation. A ProTide is comprised of a nucleoside fused to a protective phosphoramidate cap. ProTides are easily incorporated into cells whereupon the cap is cleaved and a preactivated nucleoside released. 3′-Deoxyadenosine (3′-dA) is a naturally occurring adenosine analogue with established anticancer activity in vitro but limited bioavailability due to its rapid in vivo deamination by the circulating enzyme adenosine deaminase, poor uptake into cells, and reliance on adenosine kinase for its activation. In order to overcome these limitations, 3′-dA was chemically modified to create the novel ProTide NUC-7738. Experimental Design: We describe the synthesis of NUC-7738. We determine the IC50 of NUC-7738 using pharmacokinetics (PK) and conduct genome-wide analyses to identify its mechanism of action using different cancer model systems. We validate these findings in patients with cancer. Results: We show that NUC-7738 overcomes the cancer resistance mechanisms that limit the activity of 3′-dA and that its activation is dependent on ProTide cleavage by the enzyme histidine triad nucleotide-binding protein 1. PK and tumor samples obtained from the ongoing first-in-human phase I clinical trial of NUC-7738 further validate our in vitro findings and show NUC-7738 is an effective proapoptotic agent in cancer cells with effects on the NF-κB pathway. Conclusions: Our study provides proof that NUC-7738 overcomes cellular resistance mechanisms and supports its further clinical evaluation as a novel cancer treatment within the growing pantheon of anticancer ProTides.

Although Immune Checkpoint Inhibitors (ICIs) have significantly improved Clear Cell Renal Cell Carcinoma (ccRCC) prognosis, about one third of patients experience recurrence. Current prognostic algorithms like the Leibovich Score (LS) rely on morphological features manually assessed by pathologists, and are therefore subject to bias. Moreover, these tools do not consider the heterogeneous molecular milieu present in the Tumour Microenvironment (TME), which may have prognostic value. We systematically developed a semi-automated method to investigate 62 markers and their combinations in 150 primary ccRCCs using multiplex Immunofluorescence (mIF), NanoString GeoMx®Digital Spatial Profiling (DSP) and Artificial Intelligence (AI)-assisted image analysis in order to find novel prognostic signatures and investigate their spatial relationship. We found that coexpression of Cancer Stem Cell (CSC) and Epithelial-to-Mesenchymal Transition (EMT) markers such as OCT4 and ZEB1 are indicative of poor outcome. OCT4 and the immune markers CD8, CD34 and CD163 significantly stratified patients at intermediate LS. Furthermore, augmenting the LS with OCT4 and CD34 improved patient stratification by outcome. Our results support the hypothesis that combining molecular markers has prognostic value and can be integrated with morphological features to improve risk stratification and personalised therapy. To conclude, GeoMx®DSP and AI image analysis are complementary tools providing high multiplexing capability required to investigate the TME of ccRCC, while reducing observer bias.Simple SummaryCancer is a complex ensemble of morphological and molecular features whose role is still unclear. Moreover, their role may change in different areas of the same tumour. Artificial intelligence (AI) allows pathologists to go beyond human perception and bias, and may help better understand how these features influence disease progression. Furthermore, by capturing variation intrinsic to the tumour, AI may improve the accuracy of current prognostic tools, such as Leibovich Score (LS), in predicting patient outcome and response to therapy. For these reasons, we studied in clear cell renal cell carcinoma (ccRCC) tissue, in which molecular features and their coexpression in the same cell were quantified and mapped using AI-based image analysis software. We demonstrated a novel approach for investigating ccRCC and revealed new potential biomarkers of prognosis which may also be able to direct patients towards the most appropriate personalised therapy.

Background: Metabolic dysregulation, a hallmark of cancer, allows tumor cells to sustain high rates of growth and proliferation in unfavorable conditions, including hypoxia. Many cancers, including clear cell renal cell carcinoma (ccRCC), demonstrate the Warburg effect with high rates of glycolysis and some are dependent on glutamine. These cancers express high levels of glutaminase (GLS), the enzyme responsible for conversion of glutamine to glutamate. GLS exists as two distinct isoforms, GAC and KGA, which are generated by alternative polyadenylation. The presence of GAC favors more metabolically active cell growth, and the GAC:KGA ratio negatively correlates with survival in ccRCC. Selectively inhibiting the GAC isoform may be a target for therapy. NUC-7738, a ProTide transformation of 3'-deoxyadenosine (3’-dA), has shown encouraging efficacy signals in several solid tumor types in a Phase ½ clinical study (NuTide:701 NCT03829254). It generates 3’-dATP which profoundly affects transcription by altering polyadenylation and has been shown to alter expression of mitochondrial electron transport chain genes. The aim of this study was to investigate the impact of NUC-7738 on GLS alternative polyadenylation and the ratio of GAC:KGA isoforms. Material and Methods: ccRCC (Caki-1 [VHL mutant];786-O [wildtype]) and pancreatic (PANC1; MIAPACA) cancer cell lines were treated with NUC-7738 at IC50 doses in hypoxic and normoxic conditions. NUC-7738 and 3’-dATP were measured by LC-MS. mRNA levels of glutaminase isoforms were determined by RT-qPCR using primers targeting exon 14-15 junction for GAC and exon 16-18 junction for KGA. GAC and KGA protein expression determined by JESS Western analysis. Results: NUC-7738 was converted into 3’-dATP within 6 hours of treatment with an average concentration of 8 pmol/106 cells, which was maintained for ~24 hours and decreased by 50% by 48 hours. The levels are comparable to those measured in PBMCs from patients treated with NUC-7738. In ccRCC cells (VHL wildtype and mutant), NUC-7738 reduced transcript expression of GAC isoform, with no change in KGA, indicating a change in polyadenylation site usage. This was reflected in altered protein levels, with decreased GAC in ccRCC and pancreatic cell lines in hypoxic and normoxic conditions after NUC-7738 treatment. Conclusion: NUC-7738 generates sustained levels of 3’-dATP in cells, which is associated with alternative polyadenylation site usage changes. In a variety of cell types, mRNA and protein levels of the more metabolically active GAC isoform were reduced, with an increase in the relative amount of KGA isoform. Increased glutamate production and GAC:KGA ratio are key for survival of some tumors. As alternative polyadenylation is implicated in the control of expression of many genes in cancer, the ability to influence it may lead to new strategies for developing anti-cancer treatments interfering with cellular metabolism. Citation Format: Mustafa Elshani, Ying Zhang, Tia Hawkins, Mary Kudsy, In Hwa Um, Greice Zickuhr, Alison L. Dickson, David J. Harrison. NUC-7738 promotes alternative polyadenylation site usage and reduces glutaminase GAC isoform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 277.

Background: Immune checkpoint inhibitors (ICIs), including PD-(L)1 pathway inhibitors, are now standard of care for a variety of cancers. Characterization of PD-L1 protein has revealed multiple secreted forms; soluble (sPD-L1) and exosomal (ExoPD-L1) variants. In the tumor microenvironment these interfere with T-cell activation, facilitating tumor immune evasion. High circulating levels of sPD-L1 are correlated with advanced disease, a worse prognosis and/or poor response to ICIs. High levels of ExoPD-L1 are reported to contribute to T-cell dysfunction. NUC-7738, a ProTide transformation of 3'-deoxyadenosine (3’-dA), currently in Phase 1/2 in patients with solid tumors (NuTide:701 NCT03829254), has shown encouraging efficacy signals in several tumor types, including melanoma. This study investigates the dynamic between NUC-7738 and secreted forms of PD-L1 in a melanoma cell line and patients. Material and Methods: A375 malignant melanoma cells were treated with NUC-7738 (10 μM) or DMSO for 6 - 72 hours. Cell supernatant was collected for sPD-L1 and ExoPD-L1 analysis and cells were measured for mRNA and metabolites. sPD-L1 mRNA and protein expression were measured in supernatant using RT-qPCR and sandwich ELISA, respectively. NUC-7738 and 3’-dATP were measured by LC-MS. ExoPD-L1 protein levels were assessed by Jess Western analysis (normalized to CD81) and PD-L1 cell surface expression by flow cytometry. Findings were validated in patient plasma samples taken before and 24 hours after C1D1 treatment with NUC-7738 and prior to C2D1of a 14-day cycle. Results: NUC-7738 was converted into anti-cancer metabolite 3’-dATP within 6 hours of treatment with an average concentration of 80 pmoles/106 cells, which was maintained for at least 24 hours and decreased by approximately 50% by 72 hours. The levels are comparable to those measured in PBMCs from patients treated with NUC-7738. NUC-7738 treatment reduced sPD-L1 mRNA expression by up to 40% and caused a time dependent decrease in sPD-L1 protein in the media supernatant by up to 3-fold. NUC-7738 also reduced ExoPD-L1 protein by 50%. NUC-7738 did not alter cell surface PD-L1 expression. Preliminary studies in serum from 4 study patients treated with NUC-7738 showed reductions in Exo-PD-L1 of ≤50% compared to pre-treatment levels. Conclusion: NUC-7738 reduces secreted forms of PD-L1 whilst having no effect on cell surface protein levels. These in vitro results (melanoma cell line) were validated in the clinical setting, whereby ExoPD-L1 was reduced in plasma samples from patients treated with NUC-7738. These findings indicate that, by reducing sPD-L1 levels, NUC-7738 may have the potential to act as an immune sensitizer. We propose that NUC-7738 given in combination with PD-(L)1 pathway inhibitors may offer a promising treatment option, and are currently exploring this in patients who have experienced therapeutic resistance to ICI treatment. Citation Format: Mustafa Elshani, Ying Zhang, Boback Kaghazchi, Alison L. Dickson, Sarah P. Blagden, Stefan Symeonides, Ruth Plummer, David J. Harrison. NUC-7738 causes reduction of soluble and exosome-associated PD-L1 in melanoma cell lines and patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5962.

Simple Summary Desmoplastic reaction (DR) has previously been shown to be a promising prognostic factor in colorectal cancer (CRC). However, its manual reporting can be subjective and consequently consistency of reporting might be affected. The aim of our study was to develop a deep learning algorithm that would facilitate the objective and standardised DR assessment. By applying this algorithm on a CRC cohort of 528 patients, we demonstrate how deep learning methodologies can be used for the accurate and reproducible reporting of DR. Furthermore, this study showed that the prognostic significance of DR was superior when assessed through the use of the deep learning classifier than when assessed manually. In this study, we demonstrate how the application of machine learning approaches can help by not only identifying complex patterns present within histopathological images in a standardised and reproducible manner, but also report a more accurate patient stratification. Abstract The categorisation of desmoplastic reaction (DR) present at the colorectal cancer (CRC) invasive front into mature, intermediate or immature type has been previously shown to have high prognostic significance. However, the lack of an objective and reproducible assessment methodology for the assessment of DR has been a major hurdle to its clinical translation. In this study, a deep learning algorithm was trained to automatically classify immature DR on haematoxylin and eosin digitised slides of stage II and III CRC cases (n = 41). When assessing the classifier’s performance on a test set of patient samples (n = 40), a Dice score of 0.87 for the segmentation of myxoid stroma was reported. The classifier was then applied to the full cohort of 528 stage II and III CRC cases, which was then divided into a training (n = 396) and a test set (n = 132). Automatically classed DR was shown to have superior prognostic significance over the manually classed DR in both the training and test cohorts. The findings demonstrated that deep learning algorithms could be applied to assist pathologists in the detection and classification of DR in CRC in an objective, standardised and reproducible manner.

Background: Over the past 60 years, numerous strategies have been employed to enhance the generation of the anticancer metabolite FUDR-MP (FdUMP) by 5-FU and to reduce the generation of toxic metabolites. FUDR-MP, the primary anticancer metabolite of 5-FU, exerts its activity via TS inhibition which results in increased dUMP, depletion of dTMP and DNA damage. 5-FU can also generate FdUTP, which is incorporated into DNA, resulting in DNA damage. 5-FU can also be metabolized to FUTP, which results in the RNA mediated dose-limiting toxicities of myelosuppression and GI tract inflammation. NUC-3373 is a phosphoramidate transformation of FUDR designed specifically to inhibit TS. It generates higher intracellular levels of FUDR-MP, lower levels of toxic metabolites and through its favorable PK profile can be administered via a short infusion. NUC-3373 is a more potent inhibitor of TS than 5-FU and we tested the hypothesis that it more effectively targets DNA than 5-FU. Methods: CRC cells (HCT116 & SW480) were exposed to sub-IC50 doses of NUC-3373 or 5-FU for 6 or 24h. Cells were harvested and analyzed as follows: metabolite levels and incorporation of FdUTP into DNA and FUTP into RNA (using FdUr and FUr as surrogates) by LC-MS and LC-MS/MS. Gene expression of dUTPase was knocked down by siRNA and protein assessed by western blot. Cytotoxicity of NUC-3373 and 5-FU was determined by IC50, measured with sulforhodamine B. Results: NUC-3373 generated significantly higher levels of FUDR-MP compared to 5-FU (AUC >45x greater) resulting in a pronounced increase in dUMP levels (AUC >162x compared to 5-FU). NUC-3373 treated cells incorporated FdUTP into DNA, while incorporation by 5-FU treated cells was below the LLOQ (0.1 nM). NUC-3373 was effective in the presence of dUTPase, but its potency increased after dUTPase knockdown demonstrating that FdUTP incorporation contributes to NUC-3373’s cytotoxicity. At equimolar doses, 42x more FUTP was incorporated into RNA in 5-FU treated cells compared to NUC-3373. Conclusion: NUC-3373 generates higher intracellular levels of the anticancer metabolite FUDR-MP and is a more potent inhibitor of TS than 5-FU resulting in markedly greater accumulation of intracellular dUMP. Additionally, in contrast to 5-FU, NUC-3373 treatment results in incorporation of FdUTP into DNA which indicates that NUC-3373 is a more efficient DNA damaging agent. Through bypassing the FUTP-generation pathway, NUC-3373 avoids the RNA damage that is known to be associated with dose-limiting toxicities such as myelosuppression and GI tract inflammation. This is consistent with the very low rates of FUTP-related toxicities observed in patients treated with NUC-3373 (NCT03428958). By exploiting a more DNA-directed approach, NUC-3373 provides a potentially more effective, safer and convenient therapeutic option than 5-FU for patients with cancer. Citation Format: Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Clarissa M. Czekster, David J. Harrison. NUC-3373 targets the DNA-directed pathway more effectively than 5-FU [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1835.

Background: NUC-3373 is a phosphoramidate modification of fluorodeoxyuridine-monophosphate (FUDR-MP), the active anti-cancer metabolite of fluorouracil (5-FU), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373 is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, reduce generation of toxic metabolites, and deliver higher levels of active metabolite FUDR-MP to tumors. Our previous work demonstrated that NUC-3373 causes colorectal cancer (CRC) cell lines to release damage associated molecular patterns (DAMPs)- molecular signals that activate circulating immune cells leading to immunogenic cell death (ICD). NUC-3373 promotes activation of co-cultured NK-92 MI cells, a natural killer cell line, by upregulating degranulation and IFN-γ production and downregulating the immune checkpoint molecule TIGIT. This study aims to test the hypothesis that NUC-3373 induced DAMPs promote ICD in co-cultures of CRC cells with peripheral blood mononuclear cells (PBMCs). We also explore the dynamics of PD-L1 and cytokine expression and combination with a PD-1 checkpoint inhibitor. Methods: HCT116 cells were exposed to increasing concentrations of NUC-3373 or vehicle control for 24 hours prior to co-culture with healthy patient-derived PBMCs. At the time of co-culture, cells were treated with 10 υg/ml nivolumab to assess the effect of an anti-PD-1 antibody being combined with NUC-3373. CRC and PBMC co-cultures were continued for up to 72 hours. Gene expression of PD-L1 and cytokines (TNF-α, IFN-γ, and IL-2) was assessed by RT-PCR. PD-L1 surface expression was determined by flow cytometry. Confluence was determined by automated cytometry analysis (Celigo) and cell viability of CRC cells was determined by sulforhodamine-B assay (SRB). Results: NUC-3373 increased the gene expression of cytokines and PD-L1 over 48 hours of co-culture. Surface expression of PD-L1 was increased on both PBMCs and CRC cells in co-culture, as well as on CRC cells cultured without PBMCs, indicating that global increase in PD-L1 is NUC-3373 mediated. CRC cells pre-treated with NUC-3373 and co-cultured with PBMCs displayed a reduction in cell viability compared to monocultured CRC cells. Furthermore, CRC and PBMC co-cultures treated with anti-PD-1 antibody showed increased cytotoxicity when CRC cells were pre-treated with NUC-3373. Conclusions: NUC-3373 induces DAMPs and PD-L1 expression in CRC cells and promotes pro-immune cytokine production from PBMCs resulting in ICD in vitro. Furthermore, the addition of anti-PD-1 antibody to co-cultures of NUC-3373-treated CRC cells and PMBCs enhanced this ICD. With an improved clinical safety profile (NCT03428958), including less haematologic toxicity, and a more convenient dosing regimen than 5-FU, NUC-3373 is an attractive combination partner for immune checkpoint inhibitors. Citation Format: Oliver James Read, Jennifer Bré, David J. Harrison. NUC-3373 potentiates immune-mediated cytotoxicity of CRC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1113.

Purpose: NUC-1031 is a first-in-class ProTide modification of gemcitabine. In PRO-002, NUC-1031 was combined with carboplatin in recurrent ovarian cancer. Patients and Methods: NUC-1031 was administered on days 1 and 8 with carboplatin on day 1 every 3 weeks for up to six cycles. Four dose cohorts of NUC-1031 (500, 625, and 750 mg/m2) with carboplatin (AUC4 or 5) were investigated. Primary endpoint was recommended phase II combination dose (RP2CD). Secondary endpoints included safety, investigator-assessed objective response rate (ORR), clinical benefit rate (CBR), progression-free survival (PFS), and pharmacokinetics. Results: A total of 25 women with recurrent ovarian cancer, a mean of 3.8 prior lines of chemotherapy, and a median platinum-free interval of 5 months (range: 7–451 days) were enrolled; 15 of 25 (60%) were platinum resistant, 9 (36%) were partially platinum sensitive, and 1 (4%) was platinum sensitive. Of the 23 who were response evaluable, there was 1 confirmed complete response (4%), 5 partial responses (17%), and 8 (35%) stable disease. The ORR was 26% and CBR was 74% across all doses and 100% in the RP2CD cohort. Median PFS was 27.1 weeks. NUC-1031 was stable in the plasma and rapidly generated high intracellular dFdCTP levels that were unaffected by carboplatin. Conclusions: NUC-1031 combined with carboplatin is well tolerated in recurrent ovarian cancer. Highest efficacy was observed at the RP2CD of 500 mg/m2 NUC-1031 on days 1 and 8 with AUC5 carboplatin day 1, every 3 weeks for six cycles. The ability to deliver carboplatin at AUC5 and the efficacy of this schedule even in patients with platinum-resistant disease makes this an attractive therapeutic combination.

Background NUC-3373 is a novel anti-cancer agent designed to replace 5-FU, one of the most widely used therapies across a broad range of tumors, including colorectal cancer (CRC). 5-FU exerts its main anti-cancer activity via the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), preventing the conversion of dUMP into dTMP and disrupting DNA synthesis and repair. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, and reduce generation of toxic metabolites including FUTP, which is incorporated into RNA causing myelosuppression and gastrointestinal toxicity, and FBAL which causes hand-foot syndrome. Thymidine supplementation rescues cells from NUC-3373 induced cytotoxicity, supporting a DNA targeted mode of action. The aim of this study was to further evaluate the differences between NUC-3373 and 5-FU. Methods CRC cell lines HCT116 and SW480 were exposed to sub-IC50 doses of NUC-3373 or 5-FU (0.1-25 μM) for 6h. At specific time-points, cells were harvested and analyzed as follows: TS inhibition by western blot, metabolite levels by mass spectrometry (LC-MS & LC-MS/MS) and cell cycle by flow cytometry. In rescue experiments, NUC-3373 and 5-FU were supplemented with thymidine (8 μg/mL) for 24h and cell survival assessed at 96h post-treatment. Results In both cell lines, NUC-3373 was a more potent inhibitor of TS than 5-FU with a higher proportion of TS protein bound to FUDR-MP at low concentrations. At 24h, 10 µM 5-FU was required to achieve the same level of TS binding as 0.1 µM NUC-3373 in HTC116 cells and as 0.5 µM NUC-3373 in SW480 cells. TS inhibition by NUC-3373 was almost maximal by 6 hours and was sustained for at least 48 hours. NUC-3373 generated significantly higher levels (50x) of free FUDR-MP compared to 5-FU, resulting in a more pronounced increase in dUMP levels (5x compared to 5-FU). At 48h, NUC-3373 treated cells remained arrested in S phase, while 5-FU treated cells had reverted to a normal cell cycle. FUTP was present in cells exposed to low doses of 5-FU (0.5 μM) but was not detectable in NUC-3373 treated cells. Finally, thymidine supplementation did not alter cell sensitivity to 5-FU but rescued cells treated with NUC-3373. Conclusion NUC-3373 generates higher intracellular levels of FUDR-MP and is a more potent inhibitor of TS than 5-FU, leading to more pronounced effects on cell cycle arrest and perturbation of the nucleotide pool that can result in misincorporation of uracil into DNA. Furthermore, NUC-3373 did not generate FUTP, consistent with the observation that patients treated with NUC-3373 in clinical studies have experienced much lower rates of FUTP-related toxicities. NUC-3373 is a potent TS inhibitor with a favorable safety profile. Citation Format: Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Peter Mullen, Clarissa M. Czekster, David J. Harrison. NUC-3373 is a more potent inhibitor of thymidylate synthase than 5-FU and reduces generation of toxic metabolites [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P025.

Background NUC-7738, a phosphoramidate transformation of 3’deoxyadenosine (3’dA), is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine monophosphate (3’-dAMP) directly in cells, bypassing key cancer resistance mechanisms of transport, activation and breakdown. NUC-7738 is currently in a Phase I clinical study to assess safety and determine the recommended dose in patients with advanced solid tumors and lymphoma. Acute myeloid leukemia (AML) cells exhibit impaired differentiation, uncontrolled proliferation and are highly dependent on mitochondrial metabolic processes for survival and chemoresistance. The aim of this study was to determine the effect of NUC-7738 on oxidative phosphorylation (OXPHOS) proteins and resulting changes in mitochondria morphology and activation of the intrinsic apoptotic pathway. Methods AML cell lines OCI-AML3 and HL-60 were treated with NUC-7738 for 48 hours. Cell differentiation was determined by morphologic modification with Giemsa stain and assessing the expression of surface markers CD11b and CD163 by flow cytometry. Immunoblotting was performed for Mitochondrial Electron Transport Chain Complex I subunit NDUFB8, Complex II subunit SDHB, Complex III subunit UQCRC2, Complex IV subunit COX II and ATP synthase subunit ATP5A. Mitochondria morphology was assessed by confocal microscopy with MitoTracker-Deep Red CMXRos dye. Annexin-V, DAPI, Bcl-2, cytochrome c, and cleaved-caspase 7 were assessed by flow cytometry to determine intrinsic apoptosis. Results NUC-7738 increased the proportion of CD11b+ and CD163+ cells in a dose-dependent manner from 6.1% to 28.7% and 9.6% to 26.9%, respectively. CD11b positivity was also increased in HL-60 cells from 14.5% to 31.1%. NUC-7738 induced morphological differentiation as indicated by decreased nucleo-cytoplasmic ratio, appearance of multi-lobed nuclei and vacuolization of the cytoplasm in OCI-AML3 cells. Decreased nucleo-cytoplasmic ratio was also evident in HL-60 cells. NUC-7738 decreased the protein expression of all 5 complexes of oxidative phosphorylation in OCI-AML3 cells at 48 hours and this metabolic disruption was also evident from distinct changes in mitochondria morphology. NUC-7738 activated the intrinsic apoptotic pathway, causing sequestration of BCL-2 and release of cytochrome c, resulting in a decrease in the percentage of live OCI-AML3 cells from 76% to 37% at 48 hours. Conclusion Through the ability of NUC-7738 to inhibit OXPHOS complexes with consequent terminal differentiation and activation of mitochondrial-mediated apoptosis, it exploits the mitochondrial characteristics of certain cancer cells, including their greater dependence on OXPHOS for survival than normal cells. NUC-7738 offers a potential treatment option for patients with a variety of malignancies. Citation Format: Akbar M. Shahid, In Hwa Um, Oliver J. Read, David J. Harrison. NUC-7738 alters oxidative phosphorylation and causes terminal differentiation in acute myeloid leukemia cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P026.

This work has been supported by the Instituto de Salud Carlos III and co-funded by the European Regional Development Fund (ERDF) [CP13/00160, PI14/00783, PI17/01304 to JC]; the Agència de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya [2017 SGR 1035]; PERIS Generalitat de Catalunya [SLT002/16/00398 to JC]; Fundación Científica de la Asociación Española Contra el Cáncer [GCB13131592CAST]; the intramural program of the National Institutes of Health. CIBEREHD is funded by the Instituto de Salud Carlos III. This article is based upon work from COST Action [CA17118], supported by COST (European Cooperation in Science and Technology). RB is supported by a REACH HIGH Scholars Programme-PostDoctoral Grants. The grant is part-financed by the EU, Operational Programme II-Cohesion Policy 2014–2020 investing in human capital to create more opportunities and promote the wellbeing of society-European Social Fund. Aneuploidy, the unbalanced state of the chromosome content, represents a hallmark of most solid tumors, including colorectal cancer. Such aneuploidies result in tumor specific genomic imbalances, which emerge in premalignant precursor lesions. Moreover, increasing levels of chromosomal instability have been observed in adenocarcinomas and are maintained in distant metastases. A number of studies have systematically integrated copy number alterations with gene expression changes in primary carcinomas, cell lines, and experimental models of aneuploidy. In fact, chromosomal aneuploidies target a number of genes conferring a selective advantage for the metabolism of the cancer cell. Copy number alterations not only have a positive correlation with expression changes of the majority of genes on the altered genomic segment, but also have effects on the transcriptional levels of genes genome-wide. Finally, copy number alterations have been associated with disease outcome; nevertheless, the translational applicability in clinical practice requires further studies. Here, we (i) review the spectrum of genetic alterations that lead to colorectal cancer, (ii) describe the most frequent copy number alterations at different stages of colorectal carcinogenesis, (iii) exemplify their positive correlation with gene expression levels, and (iv) discuss copy number alterations that are potentially involved in disease outcome of individual patients. Publisher PDF

We are grateful to Wyeth/Pfizer (ONC-EU-150) and to the Scottish Funding Council (SRDG HR07005) for support of this study. This study investigated the antitumour effects of two dual mTOR/PI3K inhibitors, gedatolisib (WYE-129587/PKI-587/PF-05212384) and PF-04691502 against a panel of six human patient derived ovarian cancer xenograft models. Both dual mTOR/PI3K inhibitors demonstrated antitumour activity against all xenografts tested. The compounds produced tumour stasis during the treatment period and upon cessation of treatment, tumours re-grew. In several models, there was an initial rapid reduction of tumour volume over the first week of treatment before tumour stasis. No toxicity was observed during treatment. Biomarker studies were conducted in two xenograft models; phospho-S6 (Ser235/236) expression (as a readout of mTOR activity) was reduced over the treatment period in the responding xenograft but expression increased to control (no treatment) levels on cessation of treatment. Phospho-AKT (Ser473) expression (as a readout of PI3K) was inhibited by both drugs but less markedly so than phospho-S6 expression. Initial tumour volume reduction on treatment and regrowth rate after treatment cessation was associated with phospho-S6/total S6 expression ratio. Both drugs produced apoptosis but minimally influenced markers of proliferation (Ki67, phospho-histone H3). These results indicate that mTOR/PI3K inhibition can produce broad spectrum tumour growth stasis in ovarian cancer xenograft models during continuous chronic treatment and this is associated with apoptosis. Publisher PDF

A.S. is the recipient of a Medical Research Scotland PhD Studentship awarded to P.A.R. Edinburgh Genomics is partly supported through core grants from Natural Environment Research Council (R8/H10/56), Medical Research Council (MR/K001744/1) and Biotechnological and Biological Research Council (BB/J004243/1). Publication of this article was funded in part by the University of St Andrews Open Access Publishing Fund. Gemcitabine is a fluoropyrimidine analogue that is used as a mainstay of chemotherapy treatment for pancreatic and ovarian cancers, amongst others. Despite its widespread use, gemcitabine achieves responses in less than 10% of patients with metastatic pancreatic cancer and has a very limited impact on overall survival due to intrinsic and acquired resistance. NUC-1031 (Acelarin), a phosphoramidate transformation of gemcitabine, was the first anti-cancer ProTide to enter the clinic. We find it displays important in vitro cytotoxicity differences to gemcitabine, and a genome-wide CRISPR/Cas9 genetic screening approach identified only the pyrimidine metabolism pathway as modifying cancer cell sensitivity to NUC-1031. Low deoxycytidine kinase expression in tumour biopsies from patients treated with gemcitabine, assessed by immunostaining and image analysis, correlates with a poor prognosis, but there is no such correlation in tumour biopsies from a Phase I cohort treated with NUC-1031. Publisher PDF

Both immune profiling and tumor budding significantly correlate with colorectal cancer patient outcome but are traditionally reported independently. This study evaluated the association and interaction between lymphocytic infiltration and tumor budding, coregistered on a single slide, in order to determine a more precise prognostic algorithm for patients with stage II colorectal cancer. Multiplexed immunofluorescence and automated image analysis were used for the quantification of CD3+CD8+ T cells, and tumor buds (TBs), across whole slide images of three independent cohorts (training cohort: n = 114, validation cohort 1: n = 56, validation cohort 2: n = 62). Machine learning algorithms were used for feature selection and prognostic risk model development. High numbers of TBs [HR = 5.899; 95% confidence interval (CI) 1.875–18.55], low CD3+ T-cell density (HR = 9.964; 95% CI, 3.156–31.46), and low mean number of CD3+CD8+ T cells within 50 μm of TBs (HR = 8.907; 95% CI, 2.834–28.0) were associated with reduced disease-specific survival. A prognostic signature, derived from integrating TBs, lymphocyte infiltration, and their spatial relationship, reported a more significant cohort stratification (HR = 18.75; 95% CI, 6.46–54.43), than TBs, a lymphocytic infiltration score, or pT stage. This was confirmed in two independent validation cohorts (HR = 12.27; 95% CI, 3.524–42.73; HR = 15.61; 95% CI, 4.692–51.91). The investigation of the spatial relationship between lymphocytes and TBs within the tumor microenvironment improves accuracy of prognosis of patients with stage II colorectal cancer through an automated image analysis and machine learning workflow.

Background: NUC-3373 is a novel anti-cancer agent developed to replace 5-FU, one of the most widely used anti-cancer drugs globally. Used across a broad range of tumors, including colorectal cancer (CRC), 5-FU (and oral formulation, capecitabine) exerts its main anti-cancer activity via conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373, a targeted inhibitor of TS, is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown and avoid the generation of toxic metabolites. NUC-3373 has a longer plasma t1/2 (~10 hours) than 5-FU (8-14 minutes), generating substantially higher intracellular levels of FUDR-MP and lower levels of toxic metabolites FUTP and FBAL. Methods: NuTide:302 is a 3-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of fluoropyrimidine-containing therapies. Part 1: patients receive NUC-3373 with or without leucovorin (LV). Part 2: NUC-3373 + LV is administered in dose-escalation cohorts with either oxaliplatin (NUFOX) or irinotecan (NUFIRI). Part 3: NUFOX and NUFIRI regimens from Part 2 will be combined with biologics targeting VEGF and EGFR pathways. Results: As of 26 November 2020, 37 patients have been treated in Part 1: 21 received 1500 mg/m2 NUC-3373 ± LV q2w; 11 received 1500 mg/m2 NUC-3373 + LV q1w; and 5 received 2500 mg/m2 NUC-3373 + LV q1w. Patients had a median of 4 prior lines of therapy (range: 2-13). NUC-3373 was well tolerated, with 10 patients experiencing Grade 3 treatment-related AEs (2 ALT elevations, 1 AST elevation, 1 ALP elevation, 1 hyponatremia, 1 anemia, 1 fatigue, 1 nausea, 1 fever, 1 bilirubin elevation) and 1 patient experiencing a Grade 4 treatment-related AE (bilirubin elevation). NUC-3373's favorable plasma PK profile was unaffected by LV. Clinical activity observed to date includes a 4th-line patient who achieved a partial response (40% reduction in tumor volume) and a fluoropyrimidine-refractory patient who achieved a 28% reduction in tumor volume and over 5 months disease stabilization. Among the efficacy evaluable population, a disease control rate of 62% was achieved. The RP2D and schedule has not yet been defined. Conclusions: NUC-3373 ± LV (Part 1) has shown encouraging clinical activity in heavily pre-treated CRC patients, including a partial response in a 4th-line patient and a 28% tumor shrinkage and 5-month disease stabilization in a fluoropyrimidine-refractory patient. The safety profile of NUC-3373 ± LV is very encouraging with no NUC-3373 induced neutropenia or hand-foot syndrome of any grade. NUFOX and NUFIRI combinations are currently being investigated in Part 2.Clinical trial information: NCT03428958 Citation Format: Farasat Kazmi, Kristen Ciombor, Janet Graham, Andrew Coveler, Michelle Myers, Jordan Berlin, David Harrison, Sarah Blagden, TR Jeffry Evans. NUC-3373, a targeted inhibitor of thymidylate synthase, in patients with advanced 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 CT140.

Background: 3'-deoxyadenosine (3'-dA; also known as cordycepin) is a nucleoside analog that has shown potent anti-cancer activity in non-clinical studies but has not been clinically developed because of its vulnerability to rapid degradation by the circulating enzyme adenosine deaminase (ADA) and its poor uptake into cancer cells. The ProTide NUC-7738 is a pre-activated and protected nucleotide analog (3'-dA 5'monophosphate; 3'-dAMP) specifically designed to overcome the limitations of 3'-dA. NUC-7738's phosphoramidate moiety renders it resistant to ADA degradation. Here we compared NUC-7738 to 3'-dA in several model systems prior to conducting a first-in-class dose-escalation/expansion study of NUC-7738 in patients with advanced cancers. Materials and Methods: To determine the potency of NUC-7738, IC50 values were measured in multiple cancer cell lines and compared to the parent compound, 3'-dA. Chemical inhibitors of ADA and other 3'-dA processing enzymes were applied to assess the relative ability of NUC-7738 to bypass these pathways. Using genome-wide gene-trap screens and RNA sequencing we compared mechanisms of action (MOA) for NUC-7738 and 3'-dA. Results: NUC-7738 demonstrated up to 185x greater anti-cancer potency than 3'-dA across a variety of cancer cells lines. Gene trap experiments showed that the intracellular activating enzyme adenosine kinase (ADK) and the hENT1 transporter were amongst the highest enriched genes for 3'-dA, whilst no enrichments for these genes were observed in NUC-7738 treated cells. In support of this, in vitro inhibition assays showed that unlike 3'-dA, NUC-7738 is resistant to ADA breakdown, is not reliant on hENT1 transport for its cellular uptake, and is independent of ADK for its activity. As expected, RNA sequencing analysis demonstrated overlap between the MOA of NUC-7738 and 3'-dA; both cause cancer cell death via the intrinsic apoptosis pathway and suppression of pro-survival signaling. Further investigation of gene candidates was employed in ex-vivo cancer kidney cancer samples. Conclusion: Phosphoramidate chemistry was used to transform the nucleoside analog 3'-dA into NUC-7738, rendering it resistant to degradation by ADA and enabling it to enter cancer cells independent of nucleoside transporters, both of which contribute to NUC-7738's substantially greater in vitro potency compared to 3'-dA. The gene trap approach allowed a sophisticated comparison of the MOA of NUC-7738 with 3'-dA. By overcoming the resistance mechanisms associated with 3'-dA, NUC-7738 generates higher levels of the active anti-cancer metabolite in cancer cells. These data supported the initiation of NuTide:701, a first-in-human Phase I study assessing the safety, tolerability, pharmacokinetics and pharmacodynamics of NUC-7738 in patients with advanced solid tumors that are resistant to conventional treatment. Citation Format: Hagen Schwenzer, Michaela Serpi, Valentina Ferrari, James Chettle, Josephine Morris, Ruud van Stiphout, Erica de Zan, Sebastian Nijman, Mustafa Elshani, Mary Kudsy, David Harrison, Gareth Bond, Sarah P. Blagden. From bench to bedside: Using ProTide chemistry to transform 3'-deoxyadenosine into the novel anti-cancer agent Nuc-7738 [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 931.

Background: NUC-1031 is a ProTide transformation of the nucleoside analog gemcitabine, designed to overcome key cancer resistance mechanisms and generate significantly higher levels of the active metabolite, dFdCTP. Its incorporation into DNA interrupts cell replication and induces DNA damage, leading to cell death. NUC-1031 has shown broad clinical activity across a range of solid tumors as both a single agent and in combination with platinum agents. Increasing evidence in the clinical setting in both ovarian and biliary tract cancer populations suggests potential synergy between NUC-1031 and platinum agents, which may lead to increased anti-tumor activity. The aim of this study was to investigate the dose and time relationships between the incorporation of dFdCTP into DNA, the effect on the cell cycle and DNA damage response in biliary tract cancer cells treated with NUC-1031. Methods: Human intrahepatic cholangiocarcinoma HuCCT1 cells were treated with 500 nM (half-IC50 dose) or 1 µM (IC50 dose) NUC-1031 for 24h and samples were collected every 24h up to 96h. The intracellular conversion of NUC-1031 to its active metabolite dFdCTP was assessed by liquid chromatography mass spectrometry (LC-Q-TOF). This technique was also used to determine the incorporation of dFdCTP into DNA, using the dFdC signature as a surrogate and deoxyguanosine (2dG) for normalization. Flow cytometry was used to measure cell cycle populations and to analyse histone variant H2AX phosphorylation (γH2AX) as a marker for response to DNA damage, where increased signal is related to increase in response. Results: NUC-1031 generated dFdCTP in HuCCT1 cells. The incorporation of this fluorinated deoxycytidine into DNA was dose-dependent over time. At 48h post-treatment, the ratio of dFdC:2dG was 0.70 at half-IC50 dose, with comparable levels at IC50 dose. After 48h, the ratio continued to rise for the 1 µM dose but decreased with 500 nM. The incorporation of the active metabolite was accompanied by an increase in cells in S phase, up to 46% for 500 nM and 64% for 1 µM. This dose-dependent increase coincides with H2AX phosphorylation over time up to 48h. Active metabolite incorporation, S phase population and yH2AX signals reduced towards the 96h time point. Conclusion: NUC-1031 is a potent cytotoxic agent that causes DNA damage through the incorporation of its active metabolite into DNA, in a cell cycle dependant manner. This incorporation induces S phase arrest and evokes the DNA damage response via generation of double-strand breaks. The cytotoxic effect of NUC-1031 is prolonged post-treatment. Ongoing studies are investigating NUC-1031 in combination with cisplatin, where relative levels of dFdCTP incorporated in tumor DNA may act as a pharmacodynamic biomarker to determine synergy in patients who receive the combination treatment. Citation Format: Dillum Patel, Alison L. Dickson, Oliver J. Read, Clarissa M. Czekster, Essam A. Ghazaly, David J. Harrison, Jennifer Bré. NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells [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 1003.

Background: NUC-3373 is a novel anti-cancer agent designed to replace 5-FU, one of the most widely used anti-cancer drugs globally. Used across a broad range of tumors, including colorectal cancer (CRC), 5-FU (and oral formulation, capecitabine) exerts its main anti-cancer activity via conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds and inhibits thymidylate synthase (TS), disrupting DNA synthesis and repair. NUC-3373, a phosphoramidate transformation of FUDR-MP, is a targeted inhibitor of TS is designed to bypass 5-FU resistance mechanisms associated with transport, activation and breakdown, and reduce the generation of toxic metabolites. Drug-induced cellular stress leads to the release of damage associated molecular patterns (DAMPs), including calreticulin, HMGB1 and ATP, from cancer cells. These DAMPs can be detected by immune cells in the surrounding tumor microenvironment and promote immunogenic cell death (ICD) of the cancer cells. Previous data showed that NUC-3373 induces the release of DAMPs in the human CRC lines HCT116 and SW480. This study aims to demonstrate the association between DAMPs release caused by NUC-3373 and the activation of natural killer (NK) cells. Methods: CRC cells HCT116, HT-29 and SW480 were exposed to several sub-IC50 doses of NUC-3373. The negative control was DMSO in medium, and the positive controls were oxaliplatin, a known inducer of ICD, and PMA/ionomycin, which causes NK cell degranulation. After 24 h, the culture medium was changed to wash out NUC-3373 and subsequently, cells were co-cultured with NK-92 MI cells for 4 h. NK cells were harvested from the supernatant and labelled with anti-CD56 for analysis by flow cytometry. NK cell activation was assessed by measuring expression of LAMP1 on the cell surface as an indication of degranulation, and intracellular accumulation of interferon gamma (IFNγ) in cells treated with GolgiStop. Results: Following treatment of HCT116 cells with sub-IC50 doses of NUC-3373, co-cultured NK-92 MI cells displayed a 125% increase in cell surface LAMP1 and a 96% increase in intracellular IFNγ expression compared to the DMSO-treated control. PMA/ionomycin-treated samples displayed a 463% and 271% upregulation in LAMP1 and IFNγ respectively. Degranulation was also observed in NK cells that had been co-cultured with HT-29 and SW480 cells treated at NUC-3373 doses closer to the respective IC50. Conclusions: NUC-3373 induces endoplasmic reticulum stress in CRC cells, followed by release of DAMPs. This promotes NK activation as shown by increased degranulation and upregulation of IFNγ. In an in vivo system, activation of NK cells can recruit and activate other immune cell subtypes to create a more immunogenic environment. This study suggests that NUC-3373 can modulate the tumor microenvironment to promote ICD, making it an attractive combination partner for immunotherapies such as PD-1/PD-L1 blockade therapy. Citation Format: Oliver James Read, David James Harrison, Jennifer Bré. NUC-3373 induced DAMPs release in CRC cells promotes natural killer cell activation [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 1655.

The clinical staging and prognosis of muscle-invasive bladder cancer (MIBC) routinely includes the assessment of patient tissue samples by a pathologist. Recent studies corroborate the importance of image analysis in identifying and quantifying immunological markers from tissue samples that can provide further insight into patient prognosis. In this paper, we apply multiplex immunofluorescence to MIBC tissue sections to capture whole-slide images and quantify potential prognostic markers related to lymphocytes, macrophages, tumour buds, and PD-L1. We propose a machine-learning-based approach for the prediction of 5 year prognosis with different combinations of image, clinical, and spatial features. An ensemble model comprising several functionally different models successfully stratifies MIBC patients into two risk groups with high statistical significance (p value &lt, 1×10−5). Critical to improving MIBC survival rates, our method correctly classifies 71.4% of the patients who succumb to MIBC, which is significantly more than the 28.6% of the current clinical gold standard, the TNM staging system.

Background and Objectives: The tumor microenvironment (TME) plays an important role on tumor progression and patient survival outcome. The TME varies significantly amongst patients as well as within individual tumors. Although a number of studies have reported the prognostic significance of the various TME components, only a very small number of those address the issue of intra-tumor heterogeneity. In this study, we evaluate the densities and interactions of tumor infiltrating lymphocytes, macrophages and tumor buds (TBs) in order to create a more personalized prognosis for patients with stage II colorectal cancer (CRC). This was achieved through the use of multiplexed immunofluorescence, automated image analysis and machine learning approaches. In addition, we developed an objective methodology for studying the intra-tumor heterogeneity and assess its impact on patient survival outcome. Methods: Multiplexed immunofluorescence and automated image analysis using HALO® software were applied for the quantification of CD3+, CD8+ T cells, CD68+, CD163+ macrophages and TBs, across 2 sequential whole slide images (WSI). This was performed on 230 stage II CRC patient samples from Scotland and Japan. Density and spatial relationships between the cellular subpopulations were averaged across the WSI to form input for a prognostic model. To evaluate the intra-patient heterogeneity a further analysis method was developed which divided the WSI into grids with a fixed tile area of 0.785mm2. Tiles with significantly small or large numbers of the feature of interest were considered hot or coldspots respectively. The number of each objects' hot or coldspots within each patient were then calculated. Two machine learning algorithms were employed for the analysis of the data from each analysis method, which lead to the development of two new prognostic risk models. Results: The first combinatorial prognostic model, utilizing the averaged data, consisted of lymphocyte infiltration, the number of lymphocytes within 50µm of TBs and CD68+ /CD163+ macrophage cell ratio. This model was shown to identify a subpopulation of patients who exhibit 100% survival over a 5-year follow-up period. This finding was confirmed in an independent and international validation cohort. The second prognostic model using the results from the spatial heatmap analysis, included the number of TB hotspots as well as the number of hotspots for the proximity of lymphocytes to TBs. This model was shown to be of high prognostic significance. Conclusion: This work demonstrates how by applying digital pathology and machine learning approaches it is possible to identify stage II CRC patients for whom surgical resection alone may be curative. Furthermore, we report a new methodology to evaluate the intra-tumor heterogeneity which was found to improve stage II CRC patient stratification when compared to the current clinical gold standards. Citation Format: Ines P. Nearchou, Daniel A. Soutar, Kate Lillard, Ueno Hideki, Ognjen Arandjelović, David J. Harrison, Peter D. Caie. Applications of automated image analysis, machine learning and spatial statistics for the improvement of stage II colorectal cancer prognosis [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 LB-368.

Background: The nucleoside analog gemcitabine is used for the treatment of patients with lung cancer. Its cytotoxic effect is largely attributed to generation of the active metabolite dFdCTP, which causes DNA damage. NUC-1031, a ProTide transformation of gemcitabine, is specifically designed to overcome the key cancer resistance mechanisms associated with poor survival following gemcitabine treatment. NUC-1031 has shown high response rates in clinical studies, and efficacy in patients who were refractory to or had relapsed on gemcitabine. Certain cytotoxic agents are known to promote the release of damage-associated molecular patterns (DAMPs) from cancer cells, including cell surface exposure of calreticulin (CRT) and re-localization of nuclear high mobility group box protein 1 (HMGB1). DAMPs enhance cancer cell recognition by the immune system, promoting immunogenic cell death (ICD). Conversely, studies have shown that cancer cells can upregulate programmed death-ligand 1 (PD-L1) as an adaptive, protective response to cell stress resulting from drug exposure, potentially leading to evasion from immune cell-mediated cytotoxicity. This study investigates the effect of NUC-1031 on the release of DAMPs and modulation of PD-L1 in lung cancer cells. Methods: Non-small cell lung cancer (NSCLC) cell lines were treated with NUC-1031 for 24h and cytotoxicity was assessed at 96h post-treatment to determine half-maximal inhibitory concentrations (IC50). Cells were treated with increasing concentrations of NUC-1031 and cell surface expression of CRT and PD-L1 were analyzed by flow cytometry and complemented with RT-qPCR. IFN-γ was used as a positive control for PD-L1 induction. Localization of HMGB1 was assessed by immunofluorescence microscopy. Results: NUC-1031 induced the release of HMGB1 from the nucleus and increased cell surface expression of CRT in a dose-dependent manner. We confirmed that IFN-γ strongly and uniformly induced cell surface PD-L1 (270% increase). By comparison, NUC-1031-induced cell surface expression of PD-L1 was modest (67% increase). This effect was dose-dependent, with no further increase in PD-L1 expression above 1 μM. Conclusions: NUC-1031 is a potent cytotoxic agent that directly causes DNA damage through generation of dFdCTP. In addition, these data demonstrate the NUC-1031 promotes adaptive changes that alter the recognition of cancer cells by the immune system. The release of DAMPs by NUC-1031 and increase in PD-L1 is predicted to engender an immune response, which might be further potentiated by immune checkpoint inhibition. Citation Format: Jennifer Bré, Oliver J. Read, David J. Harrison. NUC-1031 causes release of DAMPs and upregulates PD-L1 expression in lung cancer cells [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 1840.

Introduction Renal Cell Carcinoma (RCC) is the deadliest urological malignancy. Profiling its complex microenvironment (TME) in situ is crucial to understand the mechanisms of progression and immune evasion that lead to metastasis and death. NanoString® GeoMx™ Digitial Spatial Profiling (DSP) platform facilitates these studies by enabling highly multiplexed, spatially resolved characterisation of proteins and RNA from FFPE tissue. DSP visualises and quantifies targets from areas of interest (AOI) using oligonucleotide-conjugated antibodies. Here, DSP is combined with automated image analysis (IA). When coupled with multiplexed immunofluorescence (IF), IA is able to automatically segment tumor from stroma and profile marker co-expression at single cell level. We present the advantages of using a combinatorial strategy, applied to clear cell RCC (ccRCC) tissue sections, in order to predict patient outcome. Methods 165 patients, grouped into 11 tumour microarray (TMA) slides were labelled with multiplex IF and scanned with a Zeiss Axioscan.z1. Scans were imported into Definiens Tissue Studio® IA software. Multiple TMA cores were sampled from matched non-cancerous kidney, primary, and venous thrombus (VTT) ccRCC. Tumor regions (labelled with Pan-cadherin and CA9) and stroma were segmented prior to automated immune quantification, where CD3, CD163, PD-1 and PD-L1 antibodies were used to profile the immune contexture. DSP was performed on the corresponding serial sections, where a 60-plex antibody panel was applied to each TMA core. Statistical analysis was performed on R Studio, where cox-proportional hazard ratios and Kaplan-Meier curves were used to correlate marker densities to risk of metastasis and cancer-related death. Results Both IA and DSP associated M2 macrophages (CD163) and T cells (CD3) to increased risk of metastasis and poor survival. IA demonstrated that tumor/stroma segmentation and single cell marker co-registration complements DSP analysis by allowing a more detailed profiling of the TME. In particular, a high density of PD-L1 positive tumor cells and PD-1 positive T-cells were correlated to poor survival in VTT and non-cancerous cores, respectively. DSP's high-plex ability is useful to investigate the relationship among the proteins of interest. It confirmed the T-cell exhaustion marker TIM-3 as a poor prognostic factor, thus demonstrating that quantifying only CD3 positive T cells may be insufficient to predict a precise prognosis. Conclusions This data demonstrates that both co-registration of cellular protein expression and highly plexed analysis can add value to the prediction of patient outcome and the risk of metastasis. We further report the prognostic significance of analysing the molecular signature of the immune contexture in both ccRCC tumorous and its adjacent non-cancerous tissue. Citation Format: Raffaele De Filippis, Sarah Warren, Youngmi Kim, Andrew White, Jason Reeves, Grant D. Stewart, David J. Harrison, Joe M. Beechem, Peter D. Caie. Combining automated image analysis and digital spatial profiling to investigate prognostic immune signatures in clear cell renal cell carcinoma [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 2670.

Background: NUC-3373 is a ProTide transformation of fluorodeoxyuridine monophosphate (FUDR-MP), the active anti-cancer metabolite of 5-fluorouracil (5-FU). NUC-3373 can overcome key cancer resistance mechanisms and the rapid degradation known to impact 5-FU's clinical utility. Clinical studies are currently investigating NUC-3373 as monotherapy (solid tumors) and in combination with agents commonly used to treat patients with colorectal cancer (CRC). Previous studies revealed ultrastructural adaptations in CRC cell lines exposed to NUC-3373 that were indicative of endoplasmic reticulum (ER) stress. NUC-3373 was also shown to induce the formation of long-lasting ternary thymidylate synthase (TS) complexes. The induction of ER stress may promote the release of damage-associated molecular patterns (DAMPs) which have the potential to evoke immunogenic cell death (ICD). This study examines the ability of NUC-3373 to induce both ER stress and the release of DAMPs in CRC. Methods: Immunoglobulin-binding protein (BiP) was used as a marker of ER stress induction. BiP and TS (free and ternary complex) expression were measured by Western blot and quantified using Licor Odyssey. TS was knocked down using TYMS-targeting siRNA and lipofectamine-based transfection. DAMPs, active release of nuclear high mobility group box protein 1 (HMGB1) and cell surface exposure of calreticulin (CRT), were assessed by fluorescence microscopy and flow cytometry respectively. Results: NUC-3373 resulted in the rapid formation of TS ternary complexes, which was associated with ER stress as assessed by BiP protein expression. siRNA knockdown studies confirmed that TS ternary complex formation is necessary for the induction of ER stress. A strong correlation between BiP and TS ternary complex formation (R2=0.97) was observed. Furthermore, NUC-3373 induced the release of DAMPs, with increased expression of CRT at the cell surface and concomitant loss of nuclear HMGB1. Conclusions: NUC-3373, a potent inhibitor of TS activity, also induces ER stress, upregulates cell surface CRT, and decreases nuclear HMGB1 in CRC cells. The aforementioned DAMPs have been shown to cause ICD. In addition to being an effective cytotoxic agent, these findings suggest that NUC-3373 has the potential to evoke a host immune response and enhance the clinical utility of immunotherapy. Citation Format: Fiona G. McKissock, Oliver J. Read, David J. Harrison. NUC-3373 induces ER stress and the release of damage associated molecular patterns in colorectal cancer cells [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 1848.

This study was supported by Cancer Research UK, AstraZeneca and Pfizer UK. Introduction: Phase I of the Cancer Research UK Stratified Medicine Programme (SMP1) was designed to roll out molecular pathology testing nationwide at the point of cancer diagnosis, as well as facilitate an infrastructure where surplus cancer tissue could be used for research. It offered a non-trial setting to examine common UK cancer genetics in a real-world context. Methods: A total of 26 sites in England, Wales and Scotland, recruited samples from 7814 patients for genetic examination between 2011 and 2013. Tumour types involved were breast, colorectal, lung, prostate, ovarian cancer and malignant melanoma. Centralised molecular testing of surplus material from resections or biopsies of primary/metastatic tissue was performed, with samples examined for 3-5 genetic alterations deemed to be of key interest in site-specific cancers by the National Cancer Research Institute Clinical Study groups. Results: 10 754 patients (98% of those approached) consented to participate, from which 7814 tumour samples were genetically analysed. In total, 53% had at least one genetic aberration detected. From 1885 patients with lung cancer, KRAS mutation was noted to be highly prevalent in adenocarcinoma (37%). In breast cancer (1873 patients), there was a striking contrast in TP53 mutation incidence between patients with ductal cancer (27.3%) and lobular cancer (3.4%). Vast inter-tumour heterogeneity of colorectal cancer (1550 patients) was observed, including myriad double and triple combinations of genetic aberrations. Significant losses of important clinical information included smoking status in lung cancer and loss of distinction between low-grade and high-grade serous ovarian cancers. Conclusion: Nationwide molecular pathology testing in a non-trial setting is feasible. The experience with SMP1 has been used to inform ongoing CRUK flagship programmes such as the CRUK National Lung MATRIX trial and TRACERx. Publisher PDF

Background The approval of novel targeted treatments for epidermal growth factor receptor (EGFR)-positive and anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer has led to the increased requirement for mutation testing. Results We report our experience of ALK testing with immunohistochemistry (IHC) and fluorescence in-situ hybridisation (FISH) and present the prevalence of EGFR, Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) and ALK mutations. From January 2011 to May 2014, we found mutation rates of EGFR, KRAS and ALK to be 10.4% (67/643), 35.8% (86/240) and 2.3% (7/304), respectively. ALK-rearrangements were found to be associated with never smokers (p < 0.001) and younger patients (≤ 50 years old) (p < 0.001). ALK IHC protein expression in tumour cells is 100% sensitive (7 IHC+/7 FISH+) and 96.6% specific (113 IHC-/117 FISH-) for ALK-rearrangements by FISH. ALK-rearranged tumours were wild-type for EGFR and KRAS. Conclusion Our findings support the use of ALK protein expression and KRAS mutation testing as part of the molecular diagnostic algorithm for lung adenocarcinomas.

This work was supported by the Chief Scientist Office, Scotland (ETM37; GDS, DJH), Cancer Research UK (Experimental Cancer Medicine Centre; TP, London, DJH, Edinburgh), Medical Research Council (AL, DJH), Royal College of Surgeons of Edinburgh Robertson Trust (AL), Melville Trust AL), Renal Cancer Research Fund (GDS), Kidney Cancer Scotland (GDS) and an educational grant from Pfizer (TP). CVN and TDM were funded by Ghent University Multidisciplinary Research Partnership 'Bioinformatics: from nucleotides to networks' Background: Sample collection for clinical epigenetics research often occurs at the time of surgical excision of a diseased organ. However, this approach may compromise the epigenetic profile under study. The use of different sampling procedures during a study can often not be avoided, but may in theory lead to biased results. The effect of tissue sampling approach on DNA methylation is studied here using clear cell renal cell cancer (ccRCC) as a model. A comparison of the DNA methylation profiles between vascularised tumour biopsy samples and subsequent devascularised nephrectomy samples obtained from the same two individuals (total of 6 samples per individual) was undertaken. Validation of the results was performed using biopsy and nephrectomy samples obtained from 14 patients included in a ccRCC clinical trial (SuMR; ClinicalTrials.gov identifier: NCT01024205). Findings: Using MBD2 sequencing, the methylome was analysed for all samples and differential methylome regions were retrieved. The results, from the test set, show six differentially methylated genes, of which four were clearly linked to ischaemia or hypoxia (REXO1L1, TLR4, hsa-mir-1299, and ANKRD2). To validate these findings, it was evaluated whether these loci were also featured by differential methylation in the clinical trial cohort with a similar experimental design to the test set. Three of the six genes are again significantly differentially methylated, showing an overall clear impact of renal artery clamping on DNA methylation. Conclusions: Renal artery ligation modulates the ccRCC methylome, impacting methylation of ischaemia and hypoxia associated genes. Results from devascularised surgical resection specimens do not accurately reflect findings from tumour biopsies. Postprint

Flavonoids are a large group of ubiquitous polyphenolic secondary metabolites in plants with a wide range of properties, including a widely reported anti-cancer effect. The present review focuses on the different known mechanisms partaking in said anti-tumour effects, with particular emphasis on breast cancer. Their structure and reactivity allows flavonoids to work as antioxidant agents and phyto-oestrogens, modulating oestrogen signalling and metabolism to induce an overall anti-proliferative response. Other effects include the ability of flavonoids to modulate the CYP1 (cytochrome P450 1) and ABC (ATP-binding cassette) protein families, involved in carcinogenesis and drug delivery respectively. They can also induce apoptosis and cell cycle arrest and regulate other signalling pathways involved in the development and progression of cancer. In conclusion, there is accumulating evidence on the versatility of flavonoids and the numerous activities contributing to their anti-tumour effect. The complex, yet effective, mechanism of action of flavonoids, together with their interesting pharmacological properties, is the basis for their potential application in breast and other cancers. This rationale has led to the current interest in the application of flavonoids, including clinical trials currently underway and the development of novel flavonoids with improved properties, which hold great promise for tackling breast cancer.

Background: Although 5-FU-based regimens such as FOLFOX and FOLFIRI remain the standard of care for treatment of patients with colorectal cancer (CRC), their clinical utility is limited by resistance mechanisms and the production of toxic by-products. Anti-cancer activity of 5-FU requires its conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds to and inhibits thymidylate synthase (TS), depleting dTMP, resulting in DNA damage. Due to breakdown by dihydropyrimidine dehydrogenase (DPD), a reliance on enzymatic activation and a short plasma half-life; 5-FU is a sub-optimal inhibitor of TS. NUC-3373, a phosphoramidate transformation of FUDR-MP, was designed to bypass the key resistance mechanisms associated with the activation and breakdown of 5-FU. Herein, we characterize the impact of DPD on NUC-3373 and the role of NUC-3373 on TS biology in vitro. The NuTide:302 clinical study (NCT03428958) is investigating NUC-3373 pharmacokinetics and dTMP levels. Methods: Sensitivity of nine CRC cell lines was assessed by sulforhodamine B assay and IC50values established, with two (one sensitive and one less sensitive) selected for further evaluation. Pharmacological inhibition by gimeracil was used to investigate DPD-induced catabolism in cells treated with 5-FU and NUC-3373. Thymidine supplementation was used to indirectly assess the effect of NUC-3373 on the de novopathway of dTMP synthesis. Immunocytochemistry and Western blot analysis were used to assess the localization and expression of TS. NuTide:302 is a three-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of 5-FU-containing therapies. Pharmacokinetic analyses via LC-MS from patient PBMCs are intended to determine whether leucovorin (LV) augments NUC-3373 inhibition of TS and its depletion of the dTMP pool. Results: Unlike 5-FU-treated cells, inhibition of DPD by gimeracil had no significant effect on survival following NUC-3373 treatment. Supplementation with exogenous thymidine rescued cells from NUC-3373-induced death. No correlation was found between pre-treatment TS protein expression and the IC50for NUC-3373. In selected cell lines treated with NUC-3373, TS ternary complexes were detected for at least 72 hours. Treatment with NUC-3373 was also associated with increased cytoplasmic TS expression. In the clinical setting, 22 patients have received NUC-3373 in NuTide:302. The clinical impact of LV on TS inhibition and dTMP pool depletion in patient samples will be presented. Conclusions: Unlike 5-FU, NUC-3373 was not catabolised by DPD, highlighting the ability of NUC-3373 to bypass a key rate-limiting factor associated with 5-FU. NUC-3373 targets the de novo pathway of dTMP synthesis and its cytotoxicity is not dependent on basal TS expression. Formation of long-lasting ternary complexes and increased cytoplasmic expression suggest that NUC-3373 is a potent inhibitor of TS activity. Data describing whether LV potentiates the impact of NUC-3373 on TS in patients will be presented. Citation Format: Sarah P Blagden, Fiona G McKissock, Janet S Graham, Kristen K Ciombor, Francesca Aroldi, Lisa J Rodgers, Michelle Myers, Jordan Berlin, T.R. Jeffry Evans, David J Harrison. Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C059. doi:10.1158/1535-7163.TARG-19-C059

Background: Cordycepin or 3′-deoxyadenosine inhibits the growth of cancer cells by several possible mechanisms. The ProTide NUC-7738, a phosphoramidate transformation of cordycepin, is designed to overcome cancer resistance mechanisms through direct release of 3’-deoxyadenosine monophosphate in cells. We hypothesized that it might activate AMP-activated protein kinase (AMPK), the key cellular energy sensor, and thus disrupt metabolic homeostasis in cancer cells. Clear cell renal cell carcinoma (ccRCC) is characterized by lipid accumulation due to dysregulation of the genes fundamental in metabolic pathways and so this disease might be a promising target for metabolic treatment. Phosphorylation of AMPK (pAMPK) is associated with downregulation of mTOR signalling, highlighting the potential of NUC-7738 to regulate this important cancer pathway. Methods: The expression of pAMPK and AMPK in ccRCC from 293 patients was analyzed by immunofluorescence, images were captured digitally and were analyzed using QuPath software. The effect of NUC-7738 on the growth and confluence of nine renal cancer cell lines was assessed using SRB assay and Celigo scanner, under both 0.5% oxygen and normoxic conditions. Western blotting was used to assess changes in the ratio of pAMPK:AMPK caused by NUC-7738 and results read using LiCor Odyssey. Effect of NUC-7738 on AMPK activation in ex vivo ccRCC tissue slices was analyzed using immunofluorescence and QuPath software. Results: Whereas AMPK was widely expressed in ccRCC, pAMPK was focal and very heterogeneous. Cell lines by contrast strongly expressed pAMPK, but this was reduced when culture conditions were altered to be more physiologically appropriate through reduction of oxygen tension and lowering glucose levels. NUC-7738 inhibited the growth of renal cancer cell lines under both hypoxic and normoxic conditions, and increased pAMPK levels were noted after 1 hour, 6 hours, 24 hours, and 48 hours treatment, with inhibition of mTOR activity predominantly observed after 48 hours. NUC-7738 also increased pAMPK levels in ex vivo ccRCC tissue slices. Conclusion: Activation of AMPK was generally low in both primary ccRCC tissue and cell lines grown under physiologically appropriate conditions. NUC-7738 caused activation of AMPK in ex vivo ccRCC tissue slices and in cell lines, and demonstrated efficacy against cell lines in both normoxic and hypoxic conditions. Renal cancer tissue typically has low expression of pAMPK, raising the prospect that AMPK modulation may offer a therapeutic option for ccRCC. These results suggest that inhibition of the mTOR pathway may be one of the anti-cancer mechanisms through which NUC-7738 exerts its activity. Citation Format: Mary Kudsy, Mustafa Elshani, Sarah Puthur, In Hwa Um, Grant D. Stewart, Maeve Rahilly, Alex Chapman, Michelle Myers, David J. Harrison. NUC-7738, a novel ProTide modification of 3’-deoxyadenosine, activates AMPK and kills renal cancer cells in vitro [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C122. doi:10.1158/1535-7163.TARG-19-C122

BACKGROUND: Although 5-fluorouracil-based (5-FU) chemotherapies remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. This includes the intracellular conversion of 5-FU into its active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), before core anti-cancer activity can be exerted via inhibition of the enzyme thymidylate synthase (TS). Although widely studied, there are mixed reports as to whether high or low basal tumor TS levels are prognostic of response to 5-FU therapy. NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass the key resistance mechanisms associated with 5-FU, resulting in a potentially more effective and safer treatment option. This study aimed to determine the prognostic value of cancer cell TS protein expression in predicting therapeutic response to NUC-3373 and to further explore the effects of NUC-3373 on TS biology. METHODS: IC50 values for NUC-3373 were established in nine CRC cell lines by sulforhodamine B assay, after which one sensitive (HCT116) and one resistant (SW480) cell line were selected for further characterization. In these two cell lines, endogenous, induced, and ternary complex TS expression was measured by Western blot analysis and the cellular localization of TS was confirmed by immunocytochemistry and assessed by stereological methods. RESULTS: TS protein expression was measured in nine CRC cell lines treated with NUC-3373 and no correlation was found between TS protein levels and the IC50 for NUC-3373. This indicates that the sensitivity of these cell lines to NUC-3373 is not dependent on basal TS protein expression levels. When the two cell lines selected for their sensitivity/resistance to NUC-3373 were treated with NUC-3373, TS expression was induced after 12 hours and remained elevated at 48 hours. In addition, treatment with NUC-3373 was associated with a translocation of TS from the nucleus to the cell cytoplasm, which was more pronounced in the NUC-3373 sensitive cells. Translocated TS did not localize to vesicles or the Golgi apparatus; the cytoplasmic localization remains to be determined. CONCLUSIONS: These results suggest that basal TS levels are not prognostic of response to NUC-3373 in CRC cells, instead NUC-3373 induces TS expression and causes nuclear to cytoplasmic translocation. This novel finding is indicative of an alternative mechanism of action for FUDR-MP in promoting anti-cancer activity that is independent of the DNA damage pathway. Citation Format: Fiona G. McKissock, In Hwa Um, Peter Mullen, David J. Harrison. NUC 3373 induces a cytoplasmic translocation of thymidylate synthase in colorectal carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2082.

Background Although 5-fluorouracil-based (5-FU) chemotherapies remain the cornerstone of combination therapies for colorectal cancer (CRC), their clinical utility is limited by key cancer resistance mechanisms associated with breakdown, transport, and activation. This includes the intracellular conversion of 5-FU into its active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), before core anti-cancer activity can be exerted via inhibition of the enzyme thymidylate synthase (TS). NUC-3373, a phosphoramidate transformation of FUDR-MP, is designed to bypass the key resistance mechanisms associated with 5-FU, resulting in a potentially more effective and safer treatment option. In a Phase I dose-finding study, plasma obtained from patients with advanced cancers revealed that NUC-3373 has a longer half-life than 5-FU (9.7 hours vs 8-14 minutes). Herein, we aimed to investigate the potential role of endoplasmic reticulum (ER) stress pathways in the cancer cell response to NUC-3373. Methods IC50 values for NUC-3373 were established in nine CRC cell lines by sulforhodamine B assay, after which one sensitive (HCT116) and one resistant (SW480) cell line were selected for further characterization. Control experiments were conducted with thapsigargin and tunicamycin. Cells were examined by transmission electron microscopy and, using stereological principles, changes in cell ultrastructure were quantified. Expression of the ER chaperone protein binding immunoglobulin protein (BiP) was detected by Western blot analysis. Results Ultrastructural analysis revealed that NUC-3373 had differential effects on ER stress that were dependent on cell line sensitivity to the drug. Following treatment, NUC-3373-sensitive HCT116 cells responded by dilation of their ER, while the SW480 resistant cells responded by lengthening of ER but without dilation. These changes were reflected with the two classical ER stress inducers, thapsigargin and tunicamycin. NUC-3373 activated the unfolded protein response (UPR), as evidenced by upregulation of BiP. Both the magnitude and the duration of the BiP response were higher in SW480 cells. Conclusions This study supports a novel mechanism of action for NUC-3373-induced anti-cancer activity through modulation of cell stress. NUC-3373 induces UPR and ER stress in colorectal cancer cells. Results of the ultrastructural analysis suggest that the sensitivity of cells to NUC-3373 may be determined by the differential adaptation of ER to stress conditions. Citation Format: Fiona G. McKissock, Asmithaa Prabhakaran, Peter Mullen, David J. Harrison. NUC 3373 induces endoplasmic reticulum stress in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2081.

Introduction: E3 ligases are responsible for tagging ubiquitin onto substrates to either activate, relocate or target the substrate for degradation via the proteasome. Previous studies have shown that genetic knockdown of the E3 ubiquitin ligase Itch caused an increase in sensitivity to gemcitabine in pancreatic cancer models both in-vitro (cell lines) and in-vivo (mouse xenografts). Here we test the hypothesis that Itch knockdown sensitises pancreatic cell lines to existing cancer therapeutics, using both transient and stable knockdown methods. Experimental Procedures: Knockdown of Itch in MiaPaCa2 (p53 mutant) and Capan 2 (p53 WT) cells was performed using both siRNA and CRISPR-Cas9 techniques; validated by western blot and RT-PCR. Alongside parental and scrambled controls, cells were treated with either doxorubicin, gemcitabine or γ-radiation. For doxorubicin and gemcitabine treatment, cells were exposed to the drug for 24 hours before it was removed. In the case of irradiation; cells were subject to 0-4Gy of radiation before being passaged onto plates. Cell viability was assessed in 96-well plates using a combination of a Sulforhodamine B assay (SRB) to quantify live cells 4 days post-treatment, and automated cytometry analysis (Celigo) to assess well confluence over each of the 4 days. Clonogenicity was also assessed by aliquoting cells at low initial density and treating the cells with distinct doses dependent on mode of treatment and cell line. After 7 days number of colonies using a colony formation algorithm on Celigo. Data Summary: Transient knockdown of Itch in p53-/- cells was sufficient to decrease cell viability. This decrease in cell viability was further potentiated by the application of gemcitabine, doxorubicin or radiation. Stable knockdown using CRISPR-Cas9 increased MiaPaCa2 cell sensitivity to low-dose doxorubicin and radiation (5-10nM and 0.5-1Gy respectively). Whilst statistically significant, the change in response was not as large as that observed with the transient knockdown. In p53 WT cells there was no significant difference in cell sensitivity upon Itch knockdown. Conclusions: The data from this investigation support the hypothesis of Itch being a possible target for sensitising pancreatic cell lines to cancer therapeutics. However the discrepancies in response to treatment between the transient and stable Itch knockdown scenarios raises the possibility that, in the case of the stable knockdown, the cells have adapted to the absence of Itch. This presents a potential weakness in using CRISPR-Cas9 in long-term experiments where the downstream consequences of gene silencing are not immediate. Citation Format: Oliver J. Read, David J. Harrison, Paul A. Reynolds. Genetic knockdown of the E3 ubiquitin ligase Itch increases pancreatic cell line sensitivity to cancer therapeutics: A comparison between different gene editing techniques [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4440.

// James Meehan 1 , Carol Ward 1 , Arran Turnbull 1 , Jimi Bukowski-Wills 2 , Andrew J. Finch 2 , Edward J. Jarman 1 , Chrysi Xintaropoulou 1 , Carlos Martinez-Perez 1 , Mark Gray 1 , Matthew Pearson 3 , Peter Mullen 4 , Claudiu T. Supuran 5 , Fabrizio Carta 5 , David J. Harrison 4 , Ian H. Kunkler 1 and Simon P. Langdon 1 1 Cancer Research UK Edinburgh Centre and Division of Pathology Laboratory, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom 2 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom 3 IGMM Advanced Imaging Resource, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, United Kingdom 4 School of Medicine, University of St Andrews, Fife KY16 9TF, United Kingdom 5 Department of Neurofarba, Sez. Chimica Farmaceutica e Nutraceutica, University of Florence, Sesto Fiorentino 50019, Italy Correspondence to: James Meehan, email: J.Meehan@sms.ed.ac.uk Keywords: carbonic anhydrase IX, NHE1, V-ATPase, breast cancer, hypoxia Received: April 14, 2016 Accepted: March 15, 2017 Published: April 17, 2017 ABSTRACT Hypoxic cancer cells exhibit resistance to many therapies. This study compared the therapeutic effect of targeting the pH regulatory proteins (CAIX, NHE1 and V-ATPase) that permit cancer cells to adapt to hypoxic conditions, using both 2D and 3D culture models. Drugs targeting CAIX, NHE1 and V-ATPase exhibited anti-proliferative effects in MCF-7, MDA-MB-231 and HBL-100 breast cancer cell lines in 2D. Protein and gene expression analysis in 2D showed that CAIX was the most hypoxia-inducible protein of the 3 targets. However, the expression of CAIX differed between the 3 cell lines. This difference in CAIX expression in hypoxia was consistent with a varying activity of FIH-1 between the cell lines. 3D expression analysis demonstrated that both CAIX and NHE1 were up-regulated in the hypoxic areas of multicellular tumor spheroids. However, the induction of CAIX expression in hypoxia was again cell line dependent. 3D invasion assays conducted with spheroids showed that CAIX inhibition significantly reduced the invasion of cells. Finally, the capability of both NHE1 and CAIX inhibitors to combine effectively with irradiation was exhibited in clonogenic assays. Proteomic-mass-spectrometric analysis indicated that CAIX inhibition might be combining with irradiation through stimulating apoptotic cell death. Of the three proteins, CAIX represents the target with the most promise for the treatment of breast cancer.

Radiation and drug resistance remain major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Metabolic reprogramming is a recently recognised hallmark of cancer with the hypoxic acidic extracellular environment as a major driver of invasion and metastases. Nearly 40% of all breast cancers and 50% of locally advanced breast cancers are hypoxic and their altered metabolism is strongly linked to resistance to radiotherapy and systemic therapy. The dependence of metabolically adapted breast cancer cells on alterations in cell function presents a wide range of new therapeutic targets such as carbonic anhydrase IX (CAIX). This review highlights preclinical approaches to evaluating an array of targets against tumour metabolism in breast cancer and early phase clinical studies on efficacy. © 2012 Elsevier Ltd.

We thank SULSA (Scottish Universities Life Science Alliance) for supporting this project through a SULSA BioSkape Industry PhD Studentship and Antoxis Limited for providing additional funding. We also thank the 7th Framework Programme of the European Union (METOXIA project; HEALTH-F2-2009-222741) for support. Background: The natural polyphenol myricetin induces cell cycle arrest and apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with enhanced redox properties, improved cell uptake and mitochondrial targeting might have increased potential as antitumour agents. Methods: We studied the effect of a second-generation flavonoid analogue, Oncamex, in a panel of 7 breast cancer cell lines, applying western blotting, gene expression analysis, fluorescence microscopy, and immunohistochemistry to xenograft tissue to investigate its mechanism of action. Results: Proliferation assays showed that Oncamex: treatment for 8 h reduced cell viability and induced cytotoxicity and apoptosis, concomitant with increased caspase activation. Microarray analysis showed that Oncamex was associated with changes in expression of genes controlling cell cycle and apoptosis . Fluorescence microscopy showed the compound’s mitochondrial targeting and ROS-modulating properties, inducing superoxide production at concentrations associated with anti-proliferative effects. A preliminary in vivo study in mice implanted with the MDA-MB-231 breast cancer xenograft showed that Oncamex inhibited tumour growth, reduced tissue viability and Ki-67 proliferation, with no overall systemic toxicity. Conclusion: Oncamex is a novel flavonoid capable of specific mitochondrial delivery and redox modulation. It has shown antitumour activity in preclinical models of breast cancer, supporting the potential of this prototypic candidate for its continued development as an anticancer agent. Publisher PDF

High expression of Rac small GTPases in invasive breast ductal carcinoma is associated with poor prognosis, but its therapeutic value in human cancers is not clear. The aim of the current study was to determine the response of human primary breast cancers to Rac-based drug treatments ex vivo. Three-dimensional organotypic cultures were used to assess candidate therapeutic avenues in invasive breast cancers. Uniquely, in these primary cultures, the tumour is not disaggregated, with both epithelial and mesenchymal components maintained within a 3-dimensional matrix of type I collagen. EHT 1864, a small molecule inhibitor of Rac GTPases, prevents spread of breast cancers in this setting, and also reduces proliferation at the invading edge. Rac1+ epithelial cells in breast tumours also contain high levels of the phosphorylated form of the transcription factor STAT3. The small molecule Stattic inhibits activation of STAT3 and induces effects similar to those seen with EHT 1864. Pan-Rac inhibition of proliferation precedes down-regulation of STAT3 activity, defining it as the last step in Rac activation during human breast cancer invasion. Our data highlights the potential use of Rac and STAT3 inhibition in treatment of invasive human breast cancer and the benefit of studying novel cancer treatments using 3-dimensional primary tumour tissue explant cultures. Publisher PDF

Advances in predicting responses to therapies in ovarian cancer have not matched progress seen in other solid-organ tumours: ovarian cancer remains a poor-prognosis disease. There has been a paradigm shift in molecular therapeutics away from targeting individual molecules to whole biological pathways. The aim of this study was to quantitatively measure the activation state of druggable oncogenic pathways by generating a phosphoprotein profile in cancer tissues, in order to establish associations with clinicopathological parameters and to identify treatment groups for targeted therapy. In total we analysed the expression of ten phosphoproteins within eight signalling pathways (PI3K, MAPK, β-catenin, STAT, NFκB, ER, cell cycle and DNA damage response), proliferation (phospho-histone H3 and Ki67) and apoptosis (activated caspase 3), in two independent cohorts of ovarian cancers using quantitative immunofluorescence image analysis. Data were analysed by unsupervised and K-means clustering to determine new biologically relevant groups. Expression of markers of the five main pathways deregulated by mutation or copy number changes was different between histological subtypes. Four main clusters with distinct phosphoprotein profiles were identified, which were significantly associated with survival in univariate analysis, and which had distinct patterns of pathway expression reproducible between clinical cohorts. These pathway profiles suggest novel therapeutic regimens for the treatment of ovarian cancer, such as MAPK-inhibition in serous or clear cell carcinomas, or combined inhibition of STAT, NFκB and WNT signalling.

Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG. Publisher PDF

Tumour cells employ a variety of mechanisms to invade their environment and to form metastases. An important property is the ability of tumour cells to transition between individual cell invasive mode and collective mode. The switch from collective to individual cell invasion in the breast was shown recently to determine site of subsequent metastasis. Previous studies have suggested a range of invasion modes from single cells to large clusters. Here, we use a novel image analysis method to quantify and categorise invasion. We have developed a process using automated imaging for data collection, unsupervised morphological examination of breast cancer invasion using cognition network technology (CNT) to determine how many patterns of invasion can be reliably discriminated. We used Bayesian network analysis to probabilistically connect morphological variables and therefore determine that two categories of invasion are clearly distinct from one another. The Bayesian network separated individual and collective invading cell groups based on the morphological measurements, with the level of cell-cell contact the most discriminating morphological feature. Smaller invading groups were typified by smoother cellular surfaces than those invading collectively in larger groups. Interestingly, elongation was evident in all invading cell groups and was not a specific feature of single cell invasion as a surrogate of epithelial-mesenchymal transition. In conclusion, the combination of cognition network technology and Bayesian network analysis provides an insight into morphological variables associated with transition of cancer cells between invasion modes. We show that only two morphologically distinct modes of invasion exist.

NUC-1031 (NuCana plc), is a phosphoramidate transformation of gemcitabine, which bypasses cancer cell resistance mechanisms to gemcitabine. This compound showed promising results in clinical studies (phase I/II), demonstrating tumor reduction and durable disease control in patients who were refractory to or had relapsed on gemcitabine. Here, we adopted an unbiased approach to uncover genes and pathways involved in gemcitabine and NUC-1031 resistance. We performed a genome-wide knockdown screen using CRISPR/Cas9 technology in the pancreatic cancer cell line MiaPaCa2. Potential candidates from the screen were validated by gene inactivation using individual or combined single guide (sg)RNA targeting sequences. The effect of gene knockdown on NUC-1031 and gemcitabine resistance was assessed by monitoring cell survival after drug treatment using Celigo cytometry to assess cell number. EC50 values were determined using the resulting survival curves. This screen generated a list of 4 candidate genes potentially involved in NUC-1031 resistance. Two candidates were investigated further: DCK and DCTPP1, both involved in pyrimidine metabolism, especially in the maintenance of the dCMP/dCTP pool. DCK knockdown induced significant resistance to gemcitabine (EC50 350 times higher than in control cells) and a degree of resistance to NUC-1031 (EC50 7 times higher than in control cells). A less robust effect was observed when DCTPP1 was inactivated. DCTPP1 knockdown induced a small decrease in sensitivity to NUC-1031 (EC50 1.5 times higher than in control cells). These results highlight the potential role of dCMP/dCTP pool regulation by DCK and DCTPP1 in sensitivity to pyrimidine nucleoside analogs and may provide a useful biomarker for patient selection. Citation Format: Awa SARR, Jennifer Bré, Peter Mullen, Sarah Blagden, In Hwa Um, David J. Harrison, Paul A. Reynolds. Genome-scale CRISPR/Cas9 screen identifies factors required for sensitivity to pyrimidine nucleoside analogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 572.

NUC-1031 (NuCana plc) is a phosphoramidate transformation of gemcitabine and the first anti-cancer compound based on ProTide technology (Slusarczyk et al., 2014). It overcomes the main resistance mechanisms associated with gemcitabine and is currently in Phase I, II and III trials for biliary, ovarian, and pancreatic cancers respectively. High expression of dCK is required for activation of gemcitabine, which competes directly with deoxycytidine (dC) as a substrate for dCK. We aimed to compare the mode of action of NUC-1031 with gemcitabine. A panel of pancreatic and ovarian cancer cell lines were treated for 2h to 24h with NUC-1031 or gemcitabine at the EC50 dose and were observed for 96h. Cell morphology and death were observed using time-lapse microscopy. Cell cycle analysis was performed using BrdU labeling and flow cytometry. Exogenous dC was added to the cells either when they were plated or at the time of the treatment, to observe the effect on the sensitivity of cells to NUC-1031 and gemcitabine. Variation of expression of dCK was determined using RT-qPCR and western blotting. Cytospin, followed by immunocytochemistry, was performed to determine the intracellular localization of dCK. Expression of dCK was assessed in tissue samples from patients in the clinical studies using a modified Allred scoring system. After 48h, most cells treated with NUC-1031 show a 24h period when they neither divide nor die, after which they then undergo apoptosis. By contrast, cells treated with gemcitabine start to die sooner, showing features of necrosis and apoptosis. A higher proportion of cells treated with NUC-1031 exhibited a G2/M arrest compared to those treated with gemcitabine or control samples. Protein expression of dCK was increased in MiaPaCa2 cells soon after treatment with gemcitabine compared with NUC-1031. The pool of endogenous nucleotides was altered by addition of dC, inducing significant resistance to gemcitabine, both when dC was added at the time of the treatment and when cells were plated (EC50 respectively 2.5 and 9 times higher than in control cells). NUC-1031 was associated with a moderate decrease in sensitivity when dC was added at the time of the treatment and when cells were plated (EC50 3 times higher than in control cells). Immunohistochemistry of 50 primary tumours from patients enrolled in a Phase 1 study for NUC-1031 showed low-level dCK expression in almost all tumours studied. Our findings indicate that NUC-1031 has a more targeted mode of action to induce cell death than gemcitabine. Also, although a small amount of dCK may be required for maximal sensitivity to NUC-1031, dCK levels are not a limiting step for NUC-1031 efficacy. Citation Format: Jennifer Bré, Awa Sarr, Peter Mullen, In Hwa Um, Sarah P. Blagden, Paul A. Reynolds, David J. Harrison. NUC-1031 overcomes resistance associated with gemcitabine in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1855.

BACKGROUND: C35 is a 12 kDa membrane-anchored protein endogenously over-expressed in many invasive breast cancers. C35 (C17orf37) is located on the HER2 amplicon, between HER2 and GRB7. The function of over-expressed C35 in invasive breast cancer is unknown. METHODS: Tissue microarrays containing 122 primary human breast cancer specimens were used to examine the association of C35 with HER2 expression. Cell lines over-expressing C35 were generated and tested for evidence of cell transformation in vitro. RESULTS: In primary breast cancers high levels of C35 mRNA expression were associated with HER2 gene amplification. High levels of C35 protein expression were associated with hallmarks of transformation, such as, colony growth in soft agar, invasion into collagen matrix and formation of large acinar structures in three-dimensional (3D) cell cultures. The transformed phenotype was also associated with characteristics of epithelial to mesenchymal transition, such as adoption of spindle cell morphology and down-regulation of epithelial markers, such as E-cadherin and keratin-8. Furthermore, C35-induced transformation in 3D cell cultures was dependent on Syk kinase, a downstream mediator of signalling from the immunoreceptor tyrosine-based activation motif, which is present in C35. CONCLUSION: C35 functions as an oncogene in breast cancer cell lines. Drug targeting of C35 or Syk kinase might be helpful in treating a subset of patients with HER2-amplified breast cancers. British Journal of Cancer (2010) 103, 401-410. doi:10.1038/sj.bjc.6605763 www.bjcancer.com Published online 13 July 2010 (C) 2010 Cancer Research UK Publisher PDF

To examine the roles of endogenous K-ras 4A and K-ras 4B splice variants in tumorigenesis, murine lung carcinogenesis was induced by N-methyl-N-nitrosourea (MNU), which causes a K-ras mutation (G12D) that jointly affects both isoforms. Compared with age-matched K-ras(tmDelta4A/-) mice (where tumours can express mutationally activated K-ras 4B only), tumour number and size were significantly higher in K-ras(+/-) mice (where tumours can also express mutationally activated K-ras 4A), and significantly lower in K-ras(tmDelta4A/tmDelta4A) mice (where tumours can express both wild-type and activated K-ras 4B). MNU induced significantly more, and larger, tumours in wild-type than K-ras(tmDelta4A/tmDelta4A) mice which differ in that only tumours in wild-type mice can express wild-type and activated K-ras 4A. Lung tumours in all genotypes were predominantly papillary adenomas, and tumours from K-ras(+/-) and K-ras(tmDelta4A/-) mice exhibited phospho-Erk1/2 and phospho-Akt staining. Hence (1) mutationally activated K-ras 4B is sufficient to activate the Raf/MEK/ERK(MAPK) and PI3-K/Akt pathways, and initiate lung tumorigenesis, (2) when expressed with activated K-ras 4B, mutationally activated K-ras 4A further promotes lung tumour formation and growth (both in the presence and absence of its wild-type isoform) but does not affect either tumour pathology or progression, and (3) wild-type K-ras 4B, either directly or indirectly, reduces tumour number and size.

Background/Aims The chromosome locus 3p21.3 is a "hot-spot" for chromosomal aberrations and loss of heterozygosity in cancers. The 35 genes mapped to the AP20 subregion of this locus were screened for their expression to identify candidate tumor suppressor genes. DLEC1 was selected for further characterization in primary hepatocellular carcinomas and cell lines. Methods RT-PCR and methylation-specific PCR were performed to examine the expression and methylation. Stable clones with DLEC1 overexpression were established to analyze cell proliferation and cell cycle. Results DLEC1 was silenced and hypermethylated in 9 of 11 cell lines examined. Treatment with 5-aza-2′-deoxycytidine reversed the methylation and restored DLEC1 expression. The correlation between hypermethylation and expression was also demonstrated in 10 pairs of hepatocellular carcinoma and adjacent normal tissues ( t -test, p DLEC1 was detected in 70.6% of tumors, compared to 10.3% in normal tissues ( n =68, p χ 2 ). Of interest, DLEC1 methylation was associated with the AJCC staging of the tumors ( p =0.036, χ 2 ). DLEC1 over-expression in cell lines decreased colony formation, cell growth and cell size, and induced a G1 arrest in cell cycle. Conclusions Our data indicate that DLEC1 is a candidate tumor suppressor gene that plays an important role in the development and progression of hepatocellular carcinoma.

MBD4 binds to methylated DNA and acts as a thymine DNA glycosylase in base excision repair. Deficiency of MBD4 in mice enhances mutation at CpG sites and alters apoptosis in response to DNA damage, but does not increase tumorigenesis in mismatch repair-deficient mice. However, in humans, frameshift mutation of MBD4, rather than deletion, is what occurs in up to 43% of microsatellite unstable colon cancers. There is no murine equivalent of this mutation. We now show that recombinant truncated MBD4 (MBD4(tru)) inhibits glycosylase activities of normal MBD4 or Uracil DNA glycosylase in cell-free assays as a dominant negative effect. Furthermore, overexpression of MBD4(tru) in Big Blue (lacI)-transfected, MSI human colorectal carcinoma cells doubled mutation frequency, indicating that the modest dominant negative effect on DNA repair can occur in living cells in short-term experiments. Intriguingly, the whole mutation spectrum was increased, not only at CpG sites, suggesting that truncated MBD4 has a more widespread effect on genomic stability. This demonstration of a dominant negative effect may be of significance in tumour progression and acquisition of drug resistance. Publisher PDF

This study was supported by financial support from the University of Edinburgh/China Scholarship Council joint Scholarship Scheme. We are also grateful to support from the Scottish Funding Council. Chromatin undergoes structural changes in response to extracellular and environmental signals. We observed changes in nuclear morphology in cancer tissue biopsied after chemotherapy and hypothesised that these DNA damage-induced changes are mediated by histone deacetylases (HDACs). Nuclear morphological changes in cell lines and a xenograft model (OV1002) weremeasured in response to platinum chemotherapy by image analysis of nuclear texture. HDAC2 expression increased in PEO1 cells treated with cisplatin at 24h, which was accompanied by increased expression of heterochromatin protein 1 (HP1). HDAC2 and HP1 expression were also increased after carboplatin treatment in the OV1002 carboplatin-sensitive xenograft model but notin the insensitive HOX424 model. Expression of DNA damage response pathways (pBRCA1, γH2AX, pATM, pATR) showed time-dependent changes after cisplatin treatment. HDAC2 knockdown by siRNA reduced HP1 expression, induced DNA double strand breaks (DSB) measured by γH2AX, and interfered with the activation of DNA damage responses induced by cisplatin. Furthermore, HDAC2depletion affected γH2AX foci formation, cell cycle distribution, and apoptosis triggered by cisplatin and was additive to the inhibitory effect of cisplatin in cell lines. By inhibiting HDAC2 expression, reversible alterations in chromatin patterns during cisplatin treatment were observed. These results demonstrate quantifiable alterations in nuclear morphology after chemotherapy and implicateHDAC2 in higher-order chromatin changes and cellular DNA damage responses in ovarian cancer cells in vitro and in vivo. Publisher PDF

Liver inflammation, for example caused by hepatitis viruses, is a major cause of morbidity and mortality worldwide. It is characterized by hepatocyte injury, death and regeneration, Kupfer cell activation, an influx of leucocytes and increased cellular metabolic demands. A complex cytokine network generates and sustains the cellular response to the initial stimulus (Simpson et al. 1997). Reorganization of extracellular matrix components occurs rapidly following hepatic injury (Kim et al. 1997). Growth factors are bound to the hepatic extracellular matrix and are released or activated via proteolytic degradation of the substratum (Pediaditakis et al. 2001). Resident and migrant cells produce further growth factors. These factors act as survival signals for immune cells and mediate hepatocyte regeneration (Stolz et al. 1999). Epidermal growth factor (EGF), hepatocyte growth factor (HGF) (Stolz et al. 1999), platelet-derived growth factor (PDGF) and the insulin-like growth factors I and II (IGF-I and -II) (Alexia et al. 2004) have been implicated in the hepatic response to injury. Repeated cycles of cellular injury, regeneration and repair lead to hepatic fibrosis and eventually cirrhosis, which predisposes to the development of hepatocellular carcinoma (HCC) (Nissen & Martin 2002). Interferon gamma (IFNγ) is a critical mediator of inflammatory liver damage in clinical studies (Morshed et al. 1993, Fukuda et al. 1995) and in animal models (Toyonaga et al. 1994). In vitro, IFNγ induces apoptosis of primary murine hepatocytes (Kano et al. 1997), an effect attenuated by HGF (Morita et al. 1995). In many cell types, survival signalling is effected via activation of the phosphatidylinositol 3 kinase (PI(3)K) and protein kinase B/Akt signalling pathways (Coffer et al. 1998, Datta et al. 1999) Recent elucidation of genetic regulators of the IFNγ-mediated apoptosis has contributed to understanding of the molecular basis of inflammatory liver disease. Binding of IFNγ to its cell surface receptor activates the STAT-1 transcription factor via 701 tyrosine phosphorylation as a result of sequential phosphorylation of the JAK kinases (Bach etal. 1997, Pellegrini & Dusanter-Fourt 1997). Maximal transcriptional activation of STAT-1 requires additional MAPK-directed serine 727-phosphorylation (Chung et al. 1997). STAT1 is critical proximal mediator of IFNγ signalling and acts in part by inducing expression of interferon regulatory factors (IRF), of which the tumour suppressor IRF-1 has been identified as a positive regulator of apoptotic genes (Mamane et al. 1999). p53 is not required for IFNγ-mediated apoptosis in hepatocytes while loss of IRF-1 renders cells resistant (Kano et al. 1999). We established a primary cell culture system as an in vitro model of inflammatory hepatocyte injury. We assess the importance of p53, IRF-1 and p21 in IFNγ-mediated hepatocyte apoptosis. We hypothesize that extra-cellular survival factors attenuate the apoptotic response to IFNγ in non-transformed hepatocytes and that cytoprotective growth factors signalling is mediated by the PI(3)K pathway.