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[Image: see text] Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit 6a. We identified compound 6j as the most potent and selective hHDAC6 inhibitor of the series. Biological investigation of compounds 6b, 6h, and 6j demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. 6j induced HDAC6-dependent pSTAT3 inhibition.

Background Only 10–30% of oesophageal and rectal adenocarcinoma patients treated with neoadjuvant chemoradiotherapy have a complete pathological response. Inflammatory and angiogenic mediators in the tumour microenvironment (TME) may enable evasion of anti-tumour immune responses. Methods The TME influence on infiltrating dendritic cells (DCs) was modelled by treating immature monocyte-derived DCs with Tumour Conditioned Media (TCM) from distinct gastrointestinal sites, prior to LPS-induced maturation. Results Cell line conditioned media from gastrointestinal cell lines inhibited LPS-induced DC markers and TNF-α secretion. TCM generated from human tumour biopsies from oesophageal, rectal and colonic adenocarcinoma induced different effects on LPS-induced DC markers - CD54, CD80, HLA-DR, CD86 and CD83 were enhanced by oesophageal cancer; CD80, CD86 and CD83 were enhanced by rectal cancer, whereas CD54, HLA-DR, CD86, CD83 and PD-L1 were inhibited by colonic cancer. Notably, TCM from all GI cancer types inhibited TNF-α secretion. Additionally, TCM from irradiated biopsies inhibited DC markers. Profiling the TCM showed that IL-2 levels positively correlated with maturation marker CD54, while Ang-2 and bFGF levels negatively correlated with CD54. Conclusion This study identifies that there are differences in DC maturational capacity induced by the TME of distinct gastrointestinal cancers. This could potentially have implications for anti-tumour immunity and response to radiotherapy.

Introduction: Five-year survival rates for oesophageal adenocarcinoma (OAC) remain poor at just 15%. The development of drug-resistance limits the effectiveness of current chemotherapies, so the discovery of underlying resistance mechanisms and novel agents to target these pathways is crucial. Materials and Methods: 273 pre-treatment OAC biopsies were transcriptionally profiled using the Almac Diagnostics Xcel array™. The Broad Institute's Gene Set Enrichment Analysis (GSEA) software was used to identify pathways enriched in non-responders to chemotherapy compared to responders. A focused siRNA screen of 80 target genes identified from the GSEA results was then devised and completed, highlighting the role of Src in chemotherapy non-responders. An Enzo compound screen of FDA-approved drugs was carried out in OE33 and FLO-1 cell lines in combination with cisplatin, and cell viability was measured by CellTiter Glo. Src inhibition using dasatinib and saracatinib was assessed with MTT viability assays, western blotting and flow cytometry to investigate the mechanisms of action. CI values were generated to assess synergy. Results: GSEA identified pathways associated with resistance to chemotherapy in OAC. Candidate genes were selected according to predefined criteria and 80 genes were taken forward in a siRNA screen to study the effects on cell viability of gene silencing alone or in combination with cisplatin or 5-FU. Twelve genes were found to have an additive effect with chemotherapy to improve chemo-efficacy across an OAC cell line panel, including Src. A high-throughput drug screen highlighted a number of novel compounds that displayed additive effects when combined with cisplatin in OAC cell lines including dasatinib, a Src inhibitor. Src was then validated in an in-vitro OAC setting, investigating the effects of Src inhibition using siRNA, dasatinib and saracatinib. In-vitro combination experiments showed that combining Src inhibitors with traditional chemotherapies provides synergistic effects. Conclusions: Using a functional genomic approach Src was identified as a novel target associated with reduced cell viability following siRNA-mediated knockdown. By the use of small molecule inhibitors, the results of the siRNA screen were reinforced, verifying the clinical potential and synergistic effects of targeting Src alongside traditional chemotherapy treatments. Citation Format: Niamh Helen McCabe, Leanne Stevenson, Enya Scanlon, Oliver Keminer, Richard D. Kennedy, Bjoern Windshuegel, Jaine K. Blayney, Richard C. Turkington. Identification of Src's role in governing chemotherapy resistance in oesophageal adenocarcinoma through functional genomic and high-throughput drug screening approaches [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 2999.

e15535 Background: The incidence of Esophageal Adenocarcinoma (EAC) has risen in the western world but response rates to chemotherapy are low and survival is poor. Increased molecular understanding is needed to develop novel treatments. Methods: Transcriptional profiling of 274 treatment naïve EAC biopsies was performed using the Almac Diagnostics Xcel array™. All patients received platinum-based neo-adjuvant chemotherapy prior to surgical resection at four United Kingdom centers between 2004-2012. Semi-supervised clustering was performed followed by functional enrichment using DAVID. Cluster membership was assessed for independence of prognostic factors using Cox proportional hazards. Candidate targets were identified by siRNA screening in OE33 cells. Treatment with the survivin inhibitor, YM155 in EAC cell lines was also assessed. Results: Semi-supervised hierarchical clustering identified two groups with significant differences in RFS [HR = 0.54 (0.29-0.99), p = 0.05] and OS [HR = 0.52 (0.28-0.96), p = 0.04]. There were significant associations between the clusters and both nodal and TNM downstaging but not with pathological response. The PI3K-AKT, p53, tight junction and HIF-1 signaling pathways were upregulated in the poor prognostic group. Eighty-four genes were selected and taken forward in a functional genomic siRNA screen. Twenty-seven genes showed a significant reduction in viability following siRNA-mediated knockdown and further verification resulted in twelve candidate genes. Finally, target knockdown in seven EAC cell lines resulted in four interrelated hits- BIRC5, JAK1, OSMR and SLC2A1. Knock down of BIRC5 (Survivin) induced apoptosis, as evidenced by PARP cleavage, in both the parental OE33 and cisplatin-resistant OE33CDDPR cell lines. YM155, a survivin inhibitor, is shown to induce apoptosis at nanomolar concentrations across a panel of parental and cisplatin-resistant EAC cell lines and further mechanistic work is ongoing. Conclusions: We have performed clustering of a large transcriptomic dataset and defined a poor prognostic group of EAC patients. We identified Survivin (BIRC5) as a mediator of cisplatin resistance in EAC and a potential novel drug target. Further pre-clinical and clinical work to assess the benefit of survivin inhibition in EAC is warranted.