Supplementary Table S2. DAVID gene ontology analysis.

Supplementary Table S4. Synthetic lethal siRNAs that are associated with the drug classes identified as being CDH1 synthetic lethal in the known drug screen.

Supplementary Figure S3. Alternative real time assays.

Supplementary Figure S1. Cell viability normalized to mock for a range of siRNAs tested as potential synthetic lethal controls.

Supplementary Figure S4. Additional HDAC inhibitor assays.

Supplementary Figure S2. HDAC inhibitor and Taxol combinations.

Interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by persistent micro-injuries to alveolar epithelial tissues accompanied by aberrant repair processes. IPF is currently treated with pirfenidone and nintedanib, compounds which slow the rate of disease progression but fail to target underlying pathophysiological mechanisms. The DNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1) has significant roles in the modulation of inflammation and metabolic syndromes. Currently, no pharmaceutical solutions targeting OGG1 have been utilized in the treatment of IPF. In this study we show Ogg1 -targeting siRNA mitigates bleomycin-induced pulmonary fibrosis in male mice, highlighting OGG1 as a tractable target in lung fibrosis. The small molecule OGG1 inhibitor, TH5487, decreases myofibroblast transition and associated pro-fibrotic gene expressions in fibroblast cells. In addition, TH5487 decreases levels of pro-inflammatory mediators, inflammatory cell infiltration, and lung remodeling in a murine model of bleomycin-induced pulmonary fibrosis conducted in male C57BL6/J mice. OGG1 and SMAD7 interact to induce fibroblast proliferation and differentiation and display roles in fibrotic murine and IPF patient lung tissue. Taken together, these data suggest that TH5487 is a potentially clinically relevant treatment for IPF but further study in human trials is required.

Supplementary Table S1. List SL1. Candidates meeting synthetic lethal criteria of <50% death in MCF10A and {greater than or equal to}15% increased death in CDH1-/- cells compared to MCF10A cells.

Supplementary Table S3. List SL2. Candidates meeting the more stringent criteria of {less than or equal to}85% death in MCF10A and {greater than or equal to} 15% increased death in CDH1-/- cells compared to MCF10A cells.

The development of most solid cancers, including pancreatic, breast, lung, liver, and ovarian cancer, involves a desmoplastic reaction: a process of major remodeling of the extracellular matrix (ECM) affecting the ECM composition, mechanics, and microarchitecture. These properties of the ECM influence key cancer cell functions, including treatment resistance. Furthermore, emerging data show that various chemotherapeutic treatments lead to alterations in ECM features and ECM-cell communication. Here, we summarize the current knowledge around the effects of chemotherapy on both the ECM remodeling and ECM-cell signaling and discuss the implications of these alterations on distinct mechanisms of chemoresistance. Additionally, we provide an overview of current therapeutic strategies and ongoing clinical trials utilizing anti-cancer drugs to target the ECM-cell communication and explore the future challenges of these strategies.

Background and aim: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing irreparable scarring of lung tissue, with most patients succumbing rapidly after diagnosis. The mevalonate pathway, which is involved in the regulation of cell proliferation, survival, and motility, is targeted by the bisphosphonate zoledronic acid (ZA). The aim of this study was to assess the antifibrotic effects of ZA and to elucidate the mechanisms by which potential IPF treatment occurs. Methods: A series of in vitro and in vivo models were employed to identify the therapeutic potential of ZA in treating IPF. In vitro transwell assays were used to assess the ability of ZA to reduce fibrotic-related immune cell recruitment. Farnesyl diphosphate synthase (FDPS) was screened as a potential antifibrotic target using a bleomycin mouse model. FDPS-targeting siRNA and ZA were administered to mice following the onset of experimentally-induced lung fibrosis. Downstream analyses were conducted on murine lung tissues and lung fluids including 23-plex cytokine array, flow cytometry, histology, Western blotting, immunofluorescent staining, and PCR analysis. Results: In vitro administration of ZA reduced myofibroblast transition and blocked NF-κB signaling in macrophages leading to impaired immune cell recruitment in a transwell assay. FDPS-targeting siRNA administration significantly attenuated profibrotic cytokine production and lung damage in a murine lung fibrosis model. Furthermore, ZA treatment of mice with bleomycin-induced lung damage displayed decreased cytokine levels in the BALF, plasma, and lung tissue, resulting in less histologically visible fibrotic scarring. Bleomycin-induced upregulation of the ZA target, FDPS, was reduced in lung tissue and fibroblasts upon ZA treatment. Confirmatory increases in FDPS immunoreactivity was seen in human IPF resected lung samples compared to control tissue indicating potential translational value of the approach. Additionally, ZA polarized macrophages towards a less profibrotic phenotype contributing to decreased IPF pathogenesis. Conclusion: This study highlights ZA as an expedient and efficacious treatment option against IPF in a clinical setting.

BACKGROUND:Cystic fibrosis (CF), involves excessive airway accumulation of neutrophils, often in parallel with severe infection caused by Pseudomonas aeruginosa. Free histones are known to possess bactericidal properties, but the degree of antibacterial activity exerted on specific lung-based pathogens is largely unknown. Neutrophils have a high content of peptidyl deiminase 4 (PADI4), which citrullinate cationic peptidyl-arginines. In histone H3.1, several positions in the NH2-terminal tail are subject to citrullination.METHODS:Full-length and segmented histone subunit H3.1 was investigated for bactericidal activity towards P. aeruginosa (strain PAO1). PADI4-induced citrullination of histone H3.1 was assessed for antibacterial activity towards P. aeruginosa. Next, the effect of neutrophil elastase (NE)-mediated proteolysis of histone H3.1 was investigated. Finally, PADI4, H3.1, and citrullinated H3.1 were examined in healthy control and CF patient lung tissues.RESULTS:Full-length histone H3.1 and sections of the histone H3.1 tail, displayed bactericidal activity towards P. aeruginosa. These antibacterial effects were reduced following citrullination by PADI4 or proteolysis by NE. Interestingly, citrullination of histone H3.1 exacerbated NE-mediated degradation. In CF lung tissue, citrullinated histone H3.1 and PADI4 immunoreactivity was abundant. Degraded histone H3.1 was detected in the sputum of CF patients but was absent in the sputum of healthy controls.CONCLUSIONS:Citrullination impairs the antibacterial activity of histone H3.1 and exacerbates its proteolytic degradation by NE. Citrullination is likely to play an important role during resolution of acute inflammation. However, in chronic inflammation akin to CF, citrullination may dampen host defense and promote pathogen survival, as exemplified by P. aeruginosa.

The CDH1 gene, encoding the cell adhesion protein E-cadherin, is one of the most frequently mutated genes in gastric cancer and inactivating germline CDH1 mutations are responsible for hereditary diffuse gastric cancer syndrome (HDGC). Using cell viability assays, we identified that breast (MCF10A) and gastric (NCI-N87) cells lacking CDH1 expression are more sensitive to allosteric AKT inhibitors than their CDH1-expressing isogenic counterparts. Apoptosis priming and total apoptosis assays in the isogenic MCF10A cells confirmed the enhanced sensitivity of E-cadherin-null cells to the AKT inhibitors. In addition, two of these inhibitors, ARQ-092 and MK2206, preferentially targeted mouse-derived gastric Cdh1−/− organoids for growth arrest. AKT protein expression and activation (as measured by phosphorylation of serine 473) were differentially regulated in E-cadherin-null MCF10A and NCI-N87 cells, with downregulation in the normal breast cells, but upregulation in the gastric cancer cells. Bioinformatic analysis of the TCGA STAD dataset revealed that AKT3, but not AKT1 or AKT2, is upregulated in the majority of E-cadherin-deficient gastric cancers. In conclusion, allosteric AKT inhibitors represent a promising class of drugs for chemoprevention and chemotherapy of cancers with E-cadherin loss.

Chronic obstructive pulmonary disease (COPD) affects the lives of an ever-growing number of people worldwide. The lack of understanding surrounding the pathophysiology of the disease and its progression has led to COPD becoming the third leading cause of death worldwide. COPD is incurable, with current treatments only addressing associated symptoms and sometimes slowing its progression, thus highlighting the need to develop novel treatments. However, this has been limited by the lack of experimental standardization within the respiratory disease research area. A lack of coherent animal models that accurately represent all aspects of COPD clinical presentation makes the translation of promising in vitrodata to human clinical trials exceptionally challenging. Here, we review current knowledge within the COPD research field, with a focus on current COPD animal models. Moreover, we include a set of advantages and disadvantages for the selection of pre-clinical models for the identification of novel COPD treatments.

The cell-cell adhesion protein E-cadherin ( CDH1) is a tumor suppressor that is required to maintain cell adhesion, cell polarity and cell survival signalling. Somatic mutations in CDH1 are common in diffuse gastric cancer (DGC) and lobular breast cancer (LBC). In addition, germline mutations in CDH1 predispose to the autosomal dominant cancer syndrome Hereditary Diffuse Gastric Cancer (HDGC). One approach to target cells with mutations in specific tumor suppressor genes is synthetic lethality. To identify novel synthetic lethal compounds for the treatment of cancers associated with E-cadherin loss, we have undertaken a high-throughput screening campaign of ~114,000 lead-like compounds on an isogenic pair of human mammary epithelial cell lines – with and without CDH1 expression. This unbiased approach identified 12 novel compounds that preferentially harmed E-cadherin-deficient cells. Validation of these compounds using both real-time and end-point viability assays identified two novel compounds with significant synthetic lethal activity, thereby demonstrating that E-cadherin loss creates druggable vulnerabilities within tumor cells. In summary, we have identified novel synthetic lethal compounds that may provide a new strategy for the prevention and treatment of both sporadic and hereditary LBC and DGC.

BACKGROUND:The E-cadherin gene (CDH1) is frequently mutated in diffuse gastric cancer and lobular breast cancer, and germline mutations predispose to the cancer syndrome Hereditary Diffuse Gastric Cancer. We are taking a synthetic lethal approach to identify druggable vulnerabilities in CDH1-mutant cancers.METHODS:Density distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1-/- cells were used to identify protein classes affected by CDH1 mutation. The synthetic lethal relationship between selected protein classes and E-cadherin was characterised by drug sensitivity assays in both the isogenic breast MCF10A cells and CDH1-isogenic gastric NCI-N87. Endocytosis efficiency was quantified using cholera toxin B uptake. Pathway metagene expression of 415 TCGA gastric tumours was statistically correlated with CDH1 expression.RESULTS:MCF10A-CDH1-/- cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10A-CDH1-/- and NCI-N87-CDH1-/- cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. The MCF10A-CDH1-/- cell lines showed reduced capacity to endocytose cholera toxin B. Pathway metagene analysis identified 20 Reactome pathways that were potentially synthetic lethal in tumours. Genes involved in GPCR signalling, vesicle transport and the metabolism of PI3K and membrane lipids were strongly represented amongst the candidate synthetic lethal genes.CONCLUSIONS:E-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities.

Abstract Germline mutation of the E-cadherin gene (CDH1) genetically defines the inherited cancer syndrome hereditary diffuse gastric cancer (HDGC). HDGC is characterized by highly penetrant diffuse gastric cancer (DGC) and an elevated rate of lobular breast cancer (LBC). Somatic CDH1 mutations also occur frequently in the sporadic forms of these cancers. We propose that the loss of E-cadherin, a tumor suppressor gene, creates vulnerabilities in the cancer cell that can be exploited with drugs (“synthetic lethal” interactions). We are particularly interested in identifying drugs that can be used for the chemoprevention of advanced disease in HDGC family members by targeting the multifocal precursor lesions that occur in stomach and breast tissue. In the stomach, these early lesions are mucosally-confined stage T1a signet ring cell carcinomas. Up to 400 independent foci have been identified in the stomachs of CDH1 mutation carriers; these foci are genetically homogenous and are likely to only require the 2nd CDH1 hit for their initiation. To identify the vulnerabilities created by E-cadherin loss, we have conducted a genome-wide siRNA knockdown screen, a 4000 compound known drug screen and a 114,000 compound library screen in isogenic MCF10a breast cell lines with and without E-cadherin expression. The functional screen has shown that GPCR signaling proteins are highly enriched amongst the candidate synthetic lethal proteins, as well as many protein classes associated cell signaling and cytoskeletal function. Drugs that show increased activity against the E-cadherin-deficient cells include the JAK inhibitor LY2784544, the c-SRC inhibitor saracatanib, the beta-2 adrenoreceptor (GPCR) agonist formoterol, several HDAC inhibitors and statins. Detail on synergistic combinations involving many of these drugs is provided in the abstract entitled “Statins show synthetic lethality in E-cadherin-deficient cells and are synergistic with SRC and HDAC inhibitors”. To identify mechanisms associated with E-cadherin synthetic lethality, we used the cell viability data from the siRNA screen to examine the distributions of cell viabilities for the genes that make up the major KEGG signaling pathways. Using the Kolomogorov-Smirnof method to test for significance, we identified the PI3K-AKT survival pathway as being central to E-cadherin's synthetic lethal associations. These findings pave the way for the development of rationally designed drug combinations for the chemoprevention and treatment of E-cadherin-negative cancers. Citation Format: Parry J. Guilford, Augustine Chen, Bryony Telford, Andrew Single, Henry Beetham, Tanis Godwin. Synthetic lethal targeting of E-cadherin-deficient cancers. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B04.

Abstract E-cadherin (CDH1) is a cell-cell adhesion protein implicated in the epithelial-mesenchymal transition and frequently dysregulated in diffuse gastric cancer (DGC) and invasive lobular breast cancer (ILBC). Germline CDH1 mutations define hereditary diffuse gastric cancer (HDGC), a rare cancer syndrome characterised by highly penetrant DGC and an elevated rate of ILBC in females. There is a strong need to develop specific chemopreventative strategies for CDH1 germline mutation carriers which are capable of reducing the viability of early stage CDH1-deficient cancers whilst minimizing side effects. We have previously applied a synthetic lethal approach to an isogenic pair of MCF10A breast cell lines, one with and one without functional CDH1 expression (MCF10A CDH1-/-), to screen for drugs which preferentially reduce the viability of CDH1-deficient cells. This led to our finding that histone deacetylase inhibitors (HDACi; entinostat and vorinostat) and SRC inhibitors (SRCi; saracatinib) are involved in synthetic lethal interactions with MCF10A CDH1-/- cells. Single agent therapy, however, only produces a modest synthetic lethal effect, leading to our interest in developing synergistic drug combinations. We have now shown that statins, inhibitors of the HMG-CoA reductase enzyme, also show a synthetic lethal phenotype in the MCF10A isogenic cell line pair. This result suggests that survival of E-cadherin-deficient cells may be enhanced by the mevalonate pathway, a metabolic pathway responsible for synthesizing prenyl groups and subsequently activating the small GTPase proteins Rho, Rac, and Cdc42. SRC is also a known activator of GTPase proteins and combined statin and saracatinib treatment caused an enhanced reduction in cell viability in the isogenic MCF10A cells whilst maintaining the synthetic lethal phenotype. This drug combination was shown to be synergistic in both cell lines using the Chou-Talalay median effect analysis and is the first known example of synergy between a statin and SRC inhibitor. Combined statin and HDACi treatment also enhanced cell viability inhibition and was synthetic lethal in the MCF10A pair. This effect was observed using both class-I specific (entinostat) and pan- (vorinostat) HDACi and was synergistic in both cell lines using median effect analysis. These are the first known examples of synergistic drug combinations being used in the field of synthetic lethality and serve as a foundation for novel DGC and ILBC treatment options. Citation Format: Andrew Single, Augustine Chen, Bryony Telford, Henry Beetham, Parry Guilford. Statins show synthetic lethality in E-cadherin-deficient cells and are synergistic with SRC and HDAC inhibitors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B41.

Abstract E-cadherin is a cellular adhesion protein that is frequently mutated in lobular breast cancer and diffuse gastric cancer. The E-cadherin protein which is encoded by the CDH1 gene has key roles in establishing and maintaining cell polarity and differentiation, the organization of cell migration and architecture and the mediation of signaling through various proliferation and survival pathways. E-cadherin also has a tumor suppressor role and its loss in cancer cells would preclude drug targeting by conventional therapy. To circumvent this, we have taken a synthetic lethal approach to exploit any vulnerability created by the loss of E-cadherin. In the therapeutic setting, synthetic lethality refers to a combination of a mutated gene and a drug targeted at a second gene or protein causing cell death (specifically in cancer cells). To identify the vulnerabilities created by E-cadherin loss, we performed a genome-wide siRNA knockdown screen in isogenic MCF10A cell lines with and without E-cadherin expression. From the functional screen, we identified broad classes of G-protein-coupled receptor (GPCR) signaling proteins and families of cytoskeletal proteins which were highly enriched among the synthetic lethal candidates. Indeed, we identified drug classes with linkages to several of the GPCR and cytoskeletal targets that showed evidence of E-cadherin synthetic lethality when we performed a 4,057 known drug screen. These included certain PI3K inhibitors (PI-103), anti-glucocorticoid (mifepristone), tyrosine kinase inhibitor (saracatinib) and multiple histone deacetylase inhibitors (vorinostat and entinostat). Interestingly, the combination of saracatinib and mifepristone gave a synergistic effect (combination index < 1.0) in targeting E-cadherin-deficient MCF10A cells. These results demonstrate that E-cadherin loss creates druggable vulnerabilities that have the potential to improve the management of both of sporadic and familial lobular breast cancer and diffuse gastric cancer. Citation Format: Augustine Chen, Bryony J. Telford, Andrew Single, Henry Beetham, Kaylene J. Simpson, Parry Guilford. Synthetic lethal approaches targeting E-cadherin-deficient cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3814.