An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer.

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

Prognostic Value of BAP1 Protein Expression in Uveal Melanoma.

The prognostic value of the traditional pathologic parameters that form part of the American Joint Committee on Cancer staging system and genetic classifications using monosomy chromosome 3 and structural alterations in chromosome 8 are well established and are part of the diagnostic workup of uveal melanoma (UM). However, it has not been fully clarified whether nuclear protein expression of the tumor suppressor gene BAP1 (nBAP1) by immunohistochemistry alone is as powerful a predictor of overall survival (OS) and/or disease-specific survival (DSS) as chromosome analysis. The protein expression of nBAP1 was evaluated in a retrospective cohort study of 308 consecutive patients treated by primary enucleation between January 1974 and December 2022. We correlated clinical, pathologic, and cytogenetic characteristics to identify the best prognostic indicators for OS and DSS. Loss of nBAP1 was detected in 144/308 (47%) of patients. Loss of nBAP1 expression was significantly associated with poor survival. In patients with disomy chromosome 3, nBAP1 negative is significantly associated with poorer OS but not DSS. We observed that older age (>63 years), presence of metastasis, and nBAP1 negative remained independent prognostic factors in multivariate analysis. nBAP1 protein expression proved to be a more reliable prognostic indicator for OS than the American Joint Committee on Cancer staging, M3 status, or The Cancer Genome Atlas classification in this cohort. This study provides support for accurate prognostication of UM patients in routine histology laboratories by immunohistochemistry for nBAP1 alone.

Gastrointestinal parasites in captive olive baboons in a UK safari park.

From the safety inside vehicles, Knowsley Safari offers visitors a close-up encounter with captive olive baboons. As exiting vehicles may be contaminated with baboon stool, a comprehensive coprological inspection was conducted to address public health concerns. Baboon stools were obtained from vehicles, and sleeping areas, inclusive of video analysis of baboon­vehicle interactions. A purposely selected 4-day sampling period enabled comparative inspections of 2662 vehicles, with a total of 669 baboon stools examined (371 from vehicles and 298 from sleeping areas). As informed by our pilot study, front-line diagnostic methods were: QUIK-CHEK rapid diagnostic test (RDT) (Giardia and Cryptosporidium), Kato­Katz coproscopy (Trichuris) and charcoal culture (Strongyloides). Some 13.9% of vehicles were contaminated with baboon stool. Prevalence of giardiasis was 37.4% while cryptosporidiosis was <0.01%, however, an absence of faecal cysts by quality control coproscopy, alongside lower than the expected levels of Giardia-specific DNA, judged RDT results as misleading, grossly overestimating prevalence. Prevalence of trichuriasis was 48.0% and strongyloidiasis was 13.7%, a first report of Strongyloides fuelleborni in UK. We advise regular blanket administration(s) of anthelminthics to the colony, exploring pour-on formulations, thereafter, smaller-scale indicator surveys would be adequate.

Phototoxicity of Tridentate Ru(II) Polypyridyl Complex with Expanded Bite Angles toward Mammalian Cells and Multicellular Tumor Spheroids.

Tridentate ligand-coordinated ruthenium (II) polypyridyl complexes with large N-Ru-N bite angles have been shown to promote ligand field splitting and reduce singlet-triplet state mixing leading to dramatically extended emission quantum yields and lifetimes under ambient conditions. These effects are anticipated to enhance their photoinduced singlet oxygen production, promoting prospects for such complexes as type II phototherapeutics. In this contribution, we examined this putative effect for [Ru(bqp)(bqpCOOEt)]2+, Ru-bqp-ester, a heteroleptic complex containing bqp = [2,6-bi(quinolin-8-yl)pyridine], a well-established large bite angle tridentate ligand, as well as its peptide conjugates [Ru(bqp)(bqpCONH-ahx-FrFKFrFK(Ac)-CONH2)]5+ (Ru-bqp-MPP) and [Ru(bqp) (bqp)(CONH-ahx-RRRRRRRR-CONH2)]10+ (Ru-bqp-R8) that were prepared in an effort to promote live cell/tissue permeability and targeting of the parent. Membrane permeability of both parent and peptide conjugates were compared across 2D cell monolayers; A549, Chinese hamster ovary, human pancreatic cancer (HPAC), and 3D HPAC multicellular tumor spheroids (MCTS) using confocal microscopy. Both the parent complex and peptide conjugates showed exceptional permeability with rapid uptake in both 2D and 3D cell models but with little distinction in permeability or distribution in cells between the parent or peptide conjugates. Unexpectedly, the uptake was temperature independent and so attributed to passive permeation. Both dark and photo-toxicity of the Ru(II) complexes were assessed across cell types, and the parent showed notably low dark toxicity. In contrast, the parent and conjugates were found to be highly phototoxic, with impressive phototoxic indices (PIs) toward HPAC cell monolayers in particular, with PI values ranging from ∼580 to 760. Overall, our data indicate that the Ru(II) parent complex and its peptide conjugates show promise at both cell monolayers and 3D MCTS as photosensitizers for photodynamic therapy.

ON LIVER FLUKE (FASCIOLA HEPATICA) IN CAPTIVE VICUÑAS (VICUGNA VICUGNA) AT KNOWSLEY SAFARI, PRESCOT, UNITED KINGDOM.

Knowsley Safari (KS), Prescot, United Kingdom houses a variety of captive exotic ungulates. As part of their animal welfare plan, a prospective coprological survey was undertaken for liver fluke. In June 2021, 330 fecal samples, representative of 18 exotic ungulate species, were processed by sedimentation and filtration, with examination by coproscopy. Finding fascioliasis in all five vicuña alone, with fecal egg counts ranging from one to eight eggs per gram, anthelminthic treatment was attempted twice, with three coprological reviews. While the first anthelminthic treatment (oxyclozanide) was equivocal, the second anthelminthic treatment (triclabendazole) was proven effective upon two later follow-ups. An initial malacological survey of 16 freshwater sites in KS, first found Galba truncatula at two sites in June 2021, then upon more extensive searching subsequently within the vicuña's enclosure. It appears that F. hepatica was locally acquired, being the first report of fascioliasis within captive vicuñas in the United Kingdom. To develop a better fluke-management plan, regular coprological and malacological surveillance is justified, perhaps with molecular xenomonitoring of snails, alongside prompt administration of appropriate flukicide as required.

The Impact of COVID-19 Zoo Closures on Behavioural and Physiological Parameters of Welfare in Primates.

Primates are some of the most cognitively advanced species held in zoos, and their interactions with visitors are complex. The COVID-19 pandemic provided a unique opportunity to understand the impact of zoo visitors on animals, in comparison to "empty zoos". This study sought to understand the impact of facility closures and subsequent reopenings on behavioural and physiological parameters of welfare in four primate species housed in the UK: bonobos (Pan paniscus) (n = 8), chimpanzees (Pan troglodytes) (n = 11), and western lowland gorillas (Gorilla gorilla gorilla) (n = 6) held at Twycross Zoo (TZ); and olive baboons (Papio anubis) (n = 192) held at Knowsley Safari (KS). Behavioural data were collected from April-September 2020 (KS) and November 2020-January 2021 (TZ). Faecal samples were collected during morning checks from October-November (TZ) and July-November 2020 (KS). Faecal glucocorticoid metabolites (FGMs) were measured using ELISA kits. Statistical analysis for behavioural observations was undertaken using general linear models. Enclosure usage was assessed using t-tests and Mann-Whitney U-tests as appropriate. Bonobos and gorillas spent less time alone when facilities were open to the public (p = 0.004, p = 0.02 respectively). Gorillas spent less time resting when the facility was open to the public (p = 0.04), and chimpanzees engaged in more feeding (p = 0.02) and engagement with enrichment (p = 0.03) when the zoo was open to the public than when it was closed. Olive baboons performed less sexual and dominance behaviour and approached visitor cars more frequently when the safari park was opened to the public than they did the ranger's vehicle during closure periods. There were no significant changes in physiological parameters for any of the study species. The results suggest variable impacts of the zoo closures on zoo-housed primates. We recommend future work that seeks to understand the impact of individual-level differences on "visitor effects" and that differences between animal experiences in zoos and safari parks are further explored in a range of species.

Clinical Impact of Immune Checkpoint Inhibitor (ICI) Response, DNA Damage Repair (DDR) Gene Mutations and Immune-Cell Infiltration in Metastatic Melanoma Subtypes.

Molecular and histopathological analysis of melanoma subtypes has revealed distinct epidemiological, genetic, and clinical features. However, immunotherapy for advanced metastatic melanoma patients does not differ based on subtype. Response to immune checkpoint inhibitors (ICI) has been shown to vary, therefore, predictive biomarkers are needed in the design of precision treatments. Targeted sequencing and histopathological analysis (CD8 and CD20 immunohistochemistry) were performed on subtypes of metastatic melanoma (cutaneous melanoma (CM, n = 10); head and neck melanoma (HNM, n = 7); uveal melanoma (UM, n = 4); acral lentiginous melanoma (AM, n = 1) and mucosal melanoma (MM, n = 1) treated with ICI). Progression-free survival (PFS) was significantly associated with high CD8 expression (p = 0.025) and mutations in DNA damage repair (DDR) pathway genes (p = 0.012) in all subtypes but not with CD20 expression. Our study identified that immune cell infiltration and DDR gene mutations may have an impact in response to ICI treatment in metastatic melanoma but differs among subtypes. Therefore, a comprehensive understanding of the immune infiltration cells' role and DDR gene mutations in metastatic melanoma may identify prognostic biomarkers.

DNA Damage Repair Deficiency in Pancreatic Ductal Adenocarcinoma: Preclinical Models and Clinical Perspectives.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide, and survival rates have barely improved in decades. In the era of precision medicine, treatment strategies tailored to disease mutations have revolutionized cancer therapy. Next generation sequencing has found that up to a third of all PDAC tumors contain deleterious mutations in DNA damage repair (DDR) genes, highlighting the importance of these genes in PDAC. The mechanisms by which DDR gene mutations promote tumorigenesis, therapeutic response, and subsequent resistance are still not fully understood. Therefore, an opportunity exists to elucidate these processes and to uncover relevant therapeutic drug combinations and strategies to target DDR deficiency in PDAC. However, a constraint to preclinical research is due to limitations in appropriate laboratory experimental models. Models that effectively recapitulate their original cancer tend to provide high levels of predictivity and effective translation of preclinical findings to the clinic. In this review, we outline the occurrence and role of DDR deficiency in PDAC and provide an overview of clinical trials that target these pathways and the preclinical models such as 2D cell lines, 3D organoids and mouse models [genetically engineered mouse model (GEMM), and patient-derived xenograft (PDX)] used in PDAC DDR deficiency research.

Hindsight: Review of Preclinical Disease Models for the Development of New Treatments for Uveal Melanoma.

The molecular, histopathological, genomic and transcriptomic characteristics of uveal melanoma (UM) have identified four molecular subgroups with different clinical outcomes. Despite the improvements in UM classification and biological pathology, current treatments do not reduce the occurrence of metastasis. The development of effective adjuvant and metastatic therapies for UM has been slow and extremely limited. Preclinical models that closely resemble the molecular and genetic UM subgroups are essential for translating molecular findings into improved clinical treatment. In this review, we provide a retrospective view of the existing preclinical models used to study UM, and give an overview of their strengths and limitations. We review targeted therapy clinical trial data to evaluate the gap in the translation of preclinical findings to human studies. Reflecting on the current high attrition rates of clinical trials for UM, preclinical models that effectively recapitulate the human in vivo situation and/or accurately reflect the subtype classifications would enhance the translational impact of experimental data and have crucial implications for the advancement of personalised medicine.

Os(II)-Bridged Polyarginine Conjugates: The Additive Effects of Peptides in Promoting or Preventing Permeation in Cells and Multicellular Tumor Spheroids.

The preparation of two polyarginine conjugates of the complex Os(II) [bis-(4'-(4-carboxyphenyl)-2,2':6',2″-terpyridine)] [Os-(Rn)2]x+ (n = 4 and 8; x = 10 and 18) is reported, to explore whether the R8 peptide sequence that promotes cell uptake requires a contiguous amino acid sequence for membrane permeation or if this can be accomplished in a linearly bridged structure with the additive effect of shorter peptide sequences. The conjugates exhibit NIR emission centered at 754 nm and essentially oxygen-insensitive emission with a lifetime of 89 ns in phosphate-buffered saline. The uptake, distribution, and cytotoxicity of the parent complex and peptide derivatives were compared in 2D cell monolayers and a three-dimensional (3D) multicellular tumor spheroid (MCTS) model. Whereas, the bis-octaarginine sequences were impermeable to cells and spheroids, and the bis-tetraarginine conjugate showed excellent cellular uptake and accumulation in two 2D monolayer cell lines and remarkable in-depth penetration of 3D MCTSs of pancreatic cancer cells. Overall, the data indicates that cell permeability can be promoted via non-contiguous sequences of arginine residues bridged across the metal centre.

Whole-exome sequencing of long-term, never relapse exceptional responders of trastuzumab-treated HER2+ metastatic breast cancer.

Trastuzumab has significantly improved the overall survival of patients with HER2+ metastatic breast cancer (MBC). However, outcomes can vary, with patients progressing within 1 year of treatment or exceptional cases of complete response to trastuzumab for ≥10 years. Identification of the underlying genomic aberrations of "exceptional responders (ExRs)" compared to "rapid non-responders (NRs)" increases our understanding of the mechanisms involved in MBC progression and identification of biomarkers of trastuzumab response and resistance. Whole-exome sequencing was performed on six ExRs compared to five NR. The overall fraction of genome copy number alteration (CNA) burden was higher in NR patients (P = 0.07), while more significantly pronounced in copy number gains (P = 0.03) in NR compared to ExRs. Delineation of the distribution of CNA burden across the genome identified a greater degree of CNA burden in NR within Chr8 (P = 0.02) and in Chr17 (P = 0.06) and conferred a statistically significant benefit in overall survival. Clinical trial number: NCT01722890 [ICORG 12/09].

Genome-Wide Association Study Data Reveal Genetic Susceptibility to Chronic Inflammatory Intestinal Diseases and Pancreatic Ductal Adenocarcinoma Risk.

Registry-based epidemiologic studies suggest associations between chronic inflammatory intestinal diseases and pancreatic ductal adenocarcinoma (PDAC). As genetic susceptibility contributes to a large proportion of chronic inflammatory intestinal diseases, we hypothesize that the genomic regions surrounding established genome-wide associated variants for these chronic inflammatory diseases are associated with PDAC. We examined the association between PDAC and genomic regions (±500 kb) surrounding established common susceptibility variants for ulcerative colitis, Crohn's disease, inflammatory bowel disease, celiac disease, chronic pancreatitis, and primary sclerosing cholangitis. We analyzed summary statistics from genome-wide association studies data for 8,384 cases and 11,955 controls of European descent from two large consortium studies using the summary data-based adaptive rank truncated product method to examine the overall association of combined genomic regions for each inflammatory disease group. Combined genomic susceptibility regions for ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis were associated with PDAC at P values < 0.05 (0.0040, 0.0057, 0.011, and 3.4 × 10-6, respectively). After excluding the 20 PDAC susceptibility regions (±500 kb) previously identified by GWAS, the genomic regions for ulcerative colitis, Crohn disease, and inflammatory bowel disease remained associated with PDAC (P = 0.0029, 0.0057, and 0.0098, respectively). Genomic regions for celiac disease (P = 0.22) and primary sclerosing cholangitis (P = 0.078) were not associated with PDAC. Our results support the hypothesis that genomic regions surrounding variants associated with inflammatory intestinal diseases, particularly, ulcerative colitis, Crohn disease, inflammatory bowel disease, and chronic pancreatitis are associated with PDAC. SIGNIFICANCE: The joint effects of common variants in genomic regions containing susceptibility loci for inflammatory bowel disease and chronic pancreatitis are associated with PDAC and may provide insights to understanding pancreatic cancer etiology.

Genetic Alterations Featuring Biological Models to Tailor Clinical Management of Pancreatic Cancer Patients.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death worldwide. This high mortality rate is due to the disease's lack of symptoms, resulting in a late diagnosis. Biomarkers and treatment options for pancreatic cancer are also limited. In order to overcome this, new research models and novel approaches to discovering PDAC biomarkers are required. In this review, we outline the hereditary and somatic causes of PDAC and provide an overview of the recent genome wide association studies (GWAS) and pathway analysis studies. We also provide a summary of some of the systems used to study PDAC, including established and primary cell lines, patient-derived xenografts (PDX), and newer models such as organoids and organ-on-chip. These ex vitro laboratory systems allow for critical research into the development and progression of PDAC.

Targeting c-Met in triple negative breast cancer: preclinical studies using the c-Met inhibitor, Cpd A.

Introduction Triple negative breast cancer (TNBC) represents a heterogeneous subtype of breast cancer that carries a poorer prognosis. There remains a need to identify novel drivers of TNBC, which may represent targets to treat the disease. c-Met overexpression is linked with decreased survival and is associated with the basal subtype of breast cancer. Cpd A, a kinase inhibitor selective/specific for Met kinase has demonstrated preclinical anti-cancer efficacy in TNBC. We aimed to assess the anti-cancer efficacy of Cpd A when combined with Src kinase, ErbB-family or hepatocyte growth factor (HGF) inhibitors in TNBC cell lines. Methods We determined the anti-proliferative effects of Cpd A, rilotumumab, neratinib and saracatinib tested alone and in combination in a panel of TNBC cells by acid phosphatase assays. We performed reverse phase protein array analysis of c-Met and IGF1Rß expression and phosphorylation of c-Met (Y1234/1235) in TNBC cells and correlated their expression/phosphorylation with Cpd A sensitivity. We examined the impact of Cpd A, neratinib and saracatinib tested alone and in combination on invasive potential and colony formation.Results TNBC cells are not inherently sensitive to Cpd A, and neither c-Met expression nor phosphorylation are biomarkers of sensitivity to Cpd A. Cpd A enhanced the anti-proliferative effects of neratinib in vitro; however, this effect was limited to cell lines with innate sensitivity to Cpd A. Cpd A had limited anti-invasive effects but it reduced colony formation in the TNBC cell line panel.Conclusions Despite Cpd A having a potential role in reducing cancer cell metastasis, identification of strong predictive biomarkers of c-Met sensitivity would be essential to the development of a c-Met targeted treatment for an appropriately selected cohort of TNBC patients.

Modelling of pancreatic cancer biology: transcriptomic signature for 3D PDX-derived organoids and primary cell line organoid development.

With a five-year survival rate of 9%, pancreatic ductal adenocarcinoma (PDAC) is the deadliest of all cancers. The rapid mortality makes PDAC difficult to research, and inspires a resolve to create reliable, tractable cellular models for preclinical cancer research. Organoids are increasingly used to model PDAC as they maintain the differentiation status, molecular and genomic signatures of the original tumour. In this paper, we present novel methodologies and experimental approaches to develop PDAC organoids from PDX tumours, and the simultaneous development of matched primary cell lines. Moreover, we also present a method of recapitulating primary cell line cultures to organoids (CLOs). We highlight the usefulness of CLOs as PDAC organoid models, as they maintain similar transcriptomic signatures as their matched patient-derived organoids and patient derived xenografts (PDX)s. These models provide a manageable, expandable in vitro resource for downstream applications such as high throughput screening, functional genomics, and tumour microenvironment studies.

Genomic Profiling and Functional Analysis of let-7c miRNA-mRNA Interactions Identify SOX13 to Be Involved in Invasion and Progression of Pancreatic Cancer.

BACKGROUND: Pancreatic cancer is a devastating disease; its lethality is related to rapid growth and tendency to invade adjacent organs and metastasize at an early stage. OBJECTIVE: The aim of this study was to identify miRNAs and their gene targets involved in the invasive phenotype in pancreatic cancer to better understand the biological behaviour and the rapid progression of this disease. METHODS: miRNA profiling was performed in isogenic matched high invasive and low-invasive subclones derived from the MiaPaCa-2 cell line and validated in a panel of pancreatic cancer cell lines, tumour, and normal pancreas. Online miRNA target prediction algorithms and gene expression arrays were used to predict the target genes of the differentially expressed miRNAs. miRNAs and potential target genes were subjected to overexpression and knockdown approaches and downstream functional assays to determine their pathological role in pancreatic cancer. RESULTS: Differential expression analysis revealed 10 significantly dysregulated miRNAs associated with invasive capacity (Student's t-tests; P value <0.05; fold change = ±2). The expression of top upregulated miR-135b and downregulated let-7c miRNAs correlated with the invasive abilities of eight pancreatic cancer cell lines and displayed differential expression in pancreatic cancer and adjacent normal tissue specimens. Ectopic overexpression of let-7c decreased proliferation, invasion, and colony formation. Integrated analysis of miRNA-mRNA using in silico algorithms and experimental validation databases identified four putative gene targets of let-7c. One of these targets, SOX13, was found to be upregulated in PDAC tumour compared with normal tissue in TCGA and an independent data set by qPCR and immunohistochemistry. RNAi knockdown of SOX13 reduced the invasion and colony formation ability of pancreatic cancer cells. CONCLUSION: The identification of key miRNA-mRNA gene interactions and networks provide potential diagnostic and therapeutic strategies for better treatment options for pancreatic cancer patients.

Infectious particle identity determines dissemination and disease outcome for the inhaled human fungal pathogen Cryptococcus.

The majority of invasive human fungal pathogens gain access to their human hosts via the inhalation of spores from the environment into the lung, but relatively little is known about this infectious process. Among human fungal pathogens the most frequent cause of inhaled fatal fungal disease is Cryptococcus, which can disseminate from the lungs to other tissues, including the brain, where it causes meningoencephalitis. To determine the mechanisms by which distinct infectious particles of Cryptococcus cause disseminated disease, we evaluated two developmental cell types (spores and yeast) in mouse models of infection. We discovered that while both yeast and spores from several strains cause fatal disease, there was a consistently higher fungal burden in the brains of spore-infected mice. To determine the basis for this difference, we compared the pathogenesis of avirulent yeast strains with their spore progeny derived from sexual crosses. Strikingly, we discovered that spores produced by avirulent yeast caused uniformly fatal disease in the murine inhalation model of infection. We determined that this difference in outcome is associated with the preferential dissemination of spores to the lymph system. Specifically, mice infected with spores harbored Cryptococcus in their lung draining lymph nodes as early as one day after infection, whereas mice infected with yeast did not. Furthermore, phagocyte depletion experiments revealed this dissemination to the lymph nodes to be dependent on CD11c+ phagocytes, indicating a critical role for host immune cells in preferential spore trafficking. Taken together, these data support a model in which spores capitalize on phagocytosis by immune cells to escape the lung and gain access to other tissues, such as the central nervous system, to cause fatal disease. These previously unrealized insights into early interactions between pathogenic fungal spores and lung phagocytes provide new opportunities for understanding cryptococcosis and other spore-mediated fungal diseases.

Dasatinib Treatment Increases Sensitivity to c-Met Inhibition in Triple-Negative Breast Cancer Cells.

In pre-clinical studies, triple-negative breast cancer (TNBC) cells have demonstrated sensitivity to the multi-targeted kinase inhibitor dasatinib; however, clinical trials with single-agent dasatinib showed limited efficacy in unselected populations of breast cancer, including TNBC. To study potential mechanisms of resistance to dasatinib in TNBC, we established a cell line model of acquired dasatinib resistance (231-DasB). Following an approximately three-month exposure to incrementally increasing concentrations of dasatinib (200 nM to 500 nM) dasatinib, 231-DasB cells were resistant to the agent with a dasatinib IC50 value greater than 5 µM compared to 0.04 ± 0.001 µM in the parental MDA-MB-231 cells. 231-DasB cells also showed resistance (2.2-fold) to the Src kinase inhibitor PD180970. Treatment of 231-DasB cells with dasatinib did not inhibit phosphorylation of Src kinase. The 231-DasB cells also had significantly increased levels of p-Met compared to the parental MDA-MB-231 cells, as measured by luminex, and resistant cells demonstrated a significant increase in sensitivity to the c-Met inhibitor, CpdA, with an IC50 value of 1.4 ± 0.5 µM compared to an IC50 of 6.8 ± 0.2 µM in the parental MDA-MB-231 cells. Treatment with CpdA decreased p-Met and p-Src in both 231-DasB and MDA-MB-231 cells. Combined treatment with dasatinib and CpdA significantly inhibited the growth of MDA-MB-231 parental cells and prevented the emergence of dasatinib resistance. If these in vitro findings can be extrapolated to human cancer treatment, combined treatment with dasatinib and a c-Met inhibitor may block the development of acquired resistance and improve response rates to dasatinib treatment in TNBC.

Agnostic Pathway/Gene Set Analysis of Genome-Wide Association Data Identifies Associations for Pancreatic Cancer.

BACKGROUND: Genome-wide association studies (GWAS) identify associations of individual single-nucleotide polymorphisms (SNPs) with cancer risk but usually only explain a fraction of the inherited variability. Pathway analysis of genetic variants is a powerful tool to identify networks of susceptibility genes. METHODS: We conducted a large agnostic pathway-based meta-analysis of GWAS data using the summary-based adaptive rank truncated product method to identify gene sets and pathways associated with pancreatic ductal adenocarcinoma (PDAC) in 9040 cases and 12 496 controls. We performed expression quantitative trait loci (eQTL) analysis and functional annotation of the top SNPs in genes contributing to the top associated pathways and gene sets. All statistical tests were two-sided. RESULTS: We identified 14 pathways and gene sets associated with PDAC at a false discovery rate of less than 0.05. After Bonferroni correction (P ≤ 1.3 × 10-5), the strongest associations were detected in five pathways and gene sets, including maturity-onset diabetes of the young, regulation of beta-cell development, role of epidermal growth factor (EGF) receptor transactivation by G protein-coupled receptors in cardiac hypertrophy pathways, and the Nikolsky breast cancer chr17q11-q21 amplicon and Pujana ATM Pearson correlation coefficient (PCC) network gene sets. We identified and validated rs876493 and three correlating SNPs (PGAP3) and rs3124737 (CASP7) from the Pujana ATM PCC gene set as eQTLs in two normal derived pancreas tissue datasets. CONCLUSION: Our agnostic pathway and gene set analysis integrated with functional annotation and eQTL analysis provides insight into genes and pathways that may be biologically relevant for risk of PDAC, including those not previously identified.

Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL.

BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.