Induction of macrophage efferocytosis in pancreatic cancer via PI3Kγ inhibition and radiotherapy promotes tumour control.

Background: The immune suppression mechanisms in pancreatic ductal adenocarcinoma (PDAC) remain unknown, but preclinical studies have implicated macrophage-mediated immune tolerance. Hence, pathways that regulate macrophage phenotype are of strategic interest, with reprogramming strategies focusing on inhibitors of phosphoinositide 3-kinase-gamma (PI3Kγ) due to restricted immune cell expression. Inhibition of PI3Kγ alone is ineffective in PDAC, despite increased infiltration of CD8+ T cells.

Development of human pancreatic cancer avatars as a model for dynamic immune landscape profiling and personalized therapy.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer, a disease with dismal overall survival. Advances in treatment are hindered by a lack of preclinical models. Here, we show how a personalized organotypic "avatar" created from resected tissue allows spatial and temporal reporting on a complete in situ tumor microenvironment and mirrors clinical responses.

TET2 Drives 5hmc Marking of GATA6 and Epigenetically Defines Pancreatic Ductal Adenocarcinoma Transcriptional Subtypes.

Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is characterized by advanced disease stage at presentation, aggressive disease biology, and resistance to therapy, resulting in an extremely poor 5-year survival rate of <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression, but exactly why is unclear and is hindered by the technical limitations of analyzing clinical samples.

Circulating biomarkers and outcomes from a randomised phase 2 trial of gemcitabine versus capecitabine-based chemoradiotherapy for pancreatic cancer.

Background: The Phase 2 SCALOP trial compared gemcitabine with capecitabine-based consolidation chemoradiotherapy (CRT) in locally advanced pancreatic cancer (LAPC).

Methods: Thirty-five systematically identified circulating biomarkers were analysed in plasma samples from 60 patients enroled in SCALOP. Each was measured in triplicate at baseline (prior to three cycles of gemcitabine-capecitabine induction chemotherapy) and, for a subset, prior to CRT.

Dual-specificity protein phosphatase DUSP4 regulates response to MEK inhibition in BRAF wild-type melanoma.

Background: Aiming to improve treatment options for BRAF wild-type melanoma, we previously conducted the DOC-MEK study of docetaxel with MEK inhibitor (MEKi) selumetinib or placebo, revealing trends to prolongation of progression-free survival (hazard ratio 0.75, P = 0.130), and improved response rates (32% vs 14%, P = 0.

RAPTOR up-regulation contributes to resistance of renal cancer cells to PI3K-mTOR inhibition.

The outlook for patients with advanced renal cell cancer (RCC) has been improved by targeted agents including inhibitors of the PI3 kinase (PI3K)-AKT-mTOR axis, although treatment resistance is a major problem. Here, we aimed to understand how RCC cells acquire resistance to PI3K-mTOR inhibition. We used the RCC4 cell line to generate a model of in vitro resistance by continuous culture in PI3K-mTOR kinase inhibitor NVP-BEZ235 (BEZ235, Dactolisib).

Perfused Three-dimensional Organotypic Culture of Human Cancer Cells for Therapeutic Evaluation.

Pharmaceutical research requires pre-clinical testing of new therapeutics using both in-vitro and in-vivo models. However, the species specificity of non-human in-vivo models and the inadequate recapitulation of physiological conditions in-vitro are intrinsic weaknesses. Here we show that perfusion is a vital factor for engineered human tissues to recapitulate key aspects of the tumour microenvironment.

Synthesis and bioactivity of fused- and spiro-β-lactone-lactam systems.

An investigation of the formation of fused- and spiro-β-lactone annulate to γ-lactams has shown that the fused systems are formed preferentially, under standard conditions, but that spiro systems are accessible only when the formation of the fused system is blocked and require careful optimisation of reaction conditions. These systems display both weak antibacterial activity and proteasome inhibition.

Novel automated tracking analysis of particles subjected to shear flow: kindlin-3 role in B cells.

Shear flow assays are used to mimic the influence of physiological shear force in diverse situations such as leukocyte rolling and arrest on the vasculature, capture of nanoparticles, and bacterial adhesion. Analysis of such assays usually involves manual counting, is labor-intensive, and is subject to bias. We have developed the Leukotrack program that incorporates a novel (to our knowledge) segmentation routine capable of reliable detection of cells in phase contrast images.

The Gαq/11 proteins contribute to T lymphocyte migration by promoting turnover of integrin LFA-1 through recycling.

The role of Gαi proteins coupled to chemokine receptors in directed migration of immune cells is well understood. In this study we show that the separate class of Gαq/11 proteins is required for the underlying ability of T cells to migrate both randomly and in a directed chemokine-dependent manner. Interfering with Gαq or Gα11 using dominant negative cDNA constructs or siRNA for Gαq causes accumulation of LFA-1 adhesions and stalled migration.

The insider's guide to leukocyte integrin signalling and function.

The activation of leukocyte integrins through diverse receptors results in transformation of the integrin from a bent, resting form to an extended conformation, which has at least two states of ligand-binding activity. This highly regulated activation process is essential for T cell migration and the formation of an immunological synapse. The signalling events that drive integrin activation are complex.

Kinetic analysis of the inhibition of matrix metalloproteinases: lessons from the study of tissue inhibitors of metalloproteinases.

Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and "tight-binding" nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges for the determination of both equilibrium and dissociation rate constants of these inhibitory events. Methodologies that enable some of these challenges to be overcome are described in this chapter and represent valuable lessons for the in vitro assessment of MMP or ADAM inhibitors within a drug discovery context.

The specificity of TIMP-2 for matrix metalloproteinases can be modified by single amino acid mutations.

Residues 1-127 of human TIMP-2 (N-TIMP-2), comprising three of the disulfide-bonded loops of the TIMP-2 molecule, is a discrete protein domain that folds independently of the C-terminal domain. This domain has been shown to be necessary and sufficient for metalloproteinase inhibition and contains the major sites of interaction with the catalytic N-terminal domain of active matrix metalloproteinases (MMPs). Residues identified as being involved in the interaction with MMPs by NMR chemical shift perturbation studies and TIMP/MMP crystal structures have been altered by site-directed mutagenesis.

Human tissue inhibitor of metalloproteinases 3 interacts with both the N- and C-terminal domains of gelatinases A and B. Regulation by polyanions.

We compared the association constants of tissue inhibitor of metalloproteinases (TIMP)-3 with various matrix metalloproteinases with those for TIMP-1 and TIMP-2 using a continuous assay. TIMP-3 behaved more like TIMP-2 than TIMP-1, showing rapid association with gelatinases A and B. Experiments with the N-terminal domain of gelatinase A, the isolated C-terminal domain, or an inactive progelatinase A mutant showed that the hemopexin domain of gelatinase A makes an important contribution to the interaction with TIMP-3.

Kinetic analysis of the mechanism of interaction of full-length TIMP-2 and gelatinase A: evidence for the existence of a low-affinity intermediate.

We have undertaken a detailed analysis of the mechanism of inhibition of matrix metalloproteinase-2 (gelatinase A) by tissue inhibitor of metalloproteinase-2 (TIMP-2). Quenched fluorescent substrates have been used to analyze the rate of inhibition of gelatinase A by TIMP-2 over a wide range of TIMP-2 concentrations. When the values of the observed rate constant for the inhibition are plotted against TIMP-2 concentration, saturation is observed at high concentrations, providing evidence for formation of an intermediate in the pathway.

Evidence for the importance of weakly bound water for matrix metalloproteinase activity.

The effects of organic cosolvents on the kinetic characteristics of two matrix metalloproteinases, gelatinase A and stromelysin 1, were investigated. In each case, addition of the cosolvent resulted in a decrease in the apparent kcat/Km for the catalyzed hydrolysis of fluorogenic peptide substrates. Two factors were identified as being responsible for this decrease in catalytic activity: hydrophobic partitioning of the substrate in favor of the bulk solvent and decrease in the water content of the enzyme.

The second zinc atom in the matrix metalloproteinase catalytic domain is absent in the full-length enzymes: a possible role for the C-terminal domain.

Domain deletion mutants of the matrix metalloproteinases consisting of the catalytic domain only contain two zinc atoms per molecule. One is essential for catalysis, while the other may fulfil a structural role. We have analysed the zinc contents of both the full-length and the truncated mutants of prostromelysin-1 and progelatinase A and report that the second zinc atom is not present in the full-length form of the proenzymes.

Structure-function relationships in the tissue inhibitors of metalloproteinases.

The tissue inhibitors of metalloproteinases (TIMPs) are proteins that specifically inhibit the matrix metalloproteinases. They consist of two distinct structural and functional domains. In order to elucidate the role of these domains, we have prepared mutants of TIMP-1 and TIMP-2 that lack a C-terminal domain.