Biochemical features important for D6 function

Chemokines are the principle regulators of leukocyte migration in vivo and function during both normal (homeostatic) and inflammatory conditions to direct leukocytes to appropriate tissue locales. Chemokines mediate their affects by binding to their cognate G-protein coupled receptors (GPCRs) which are expressed on the surface of cells, and generate a signal upon ligand binding resulting in the initiation of a response such as chemotaxis. As well as the classical chemokine receptors which generate a conventional GPCR signal upon ligand binding, there exists a small family of atypical chemokine receptors that are characterised by an inability to mount classical receptor signalling. One of the most prominent members of this family is the atypical chemokine receptor, D6, which can bind at least 14 inflammatory CC chemokines with high affinity, but instead of the generation of a classical G-protein signalling response, D6 internalises ligands and targets them for lysosomal-mediated degradation. This functional attribute makes D6 a highly efficient binder, internaliser and scavenger of inflammatory CC chemokines that has been shown to be important for the resolution of inflammatory responses in vivo. Despite its well-studied biological role, very little is known about the structure/function relationships within and around D6 which regulate ligand binding and scavenging. Glycosaminoglycans have been demonstrated to be important for chemokine sequestration and presentation to many of the conventional chemokine receptors. Consequently, the role of glycosaminoglycans (GAGs) in chemokine presentation to D6 was studied using a cell line which is deficient in the synthesis of proteoglycans (CHO 745). Transfection of these cells with D6 and comparison to transfected WT CHO cells revealed that D6-mediated uptake and internalisation of chemokine is significantly reduced in the absence of GAGs. The N-terminus of D6 is thought to be the principle site for ligand binding, and the ability of D6 to bind all inflammatory CC chemokines makes this region an attractive target for therapeutic manipulation. Therefore a sulphated peptide representing the first 35 amino acids of D6 (D6-N (s)) was synthesised and investigated for its ability to bind D6 ligands. D6-N (s) was shown to neutralise the activity of the inflammatory CC chemokine CCL2 and prevent its interaction with its cognate receptor CCR2 in vitro. Importantly D6-N (s) was active, only in a specifically sulphated form, highlighting the importance of sulphated tyrosines for ligand binding. Considering the functional significance of the synthetic D6 peptide, attempts were made to identify a naturally ‘shed’ D6 N-terminal peptide which had been reported previously. Further study demonstrated the ability of the bacterial protease staphopain A, released from Staphylococcus aureus, to cleave the N-terminus of D6 and suppress its ligand internalisation activity. Finally, the conserved tyrosine motif present on the N-terminus of D6 was investigated more closely. Site-directed mutagenesis and sulphation inhibition of this region revealed the importance of post-translational tyrosine sulphation for ligand binding, internalisation and scavenging of inflammatory chemokines and alluded to the existence of an optimal sulphation pattern for ligand binding. Overall the results presented in this thesis shed new light on the nature of the molecules around, and the structural features within D6 that contribute to ligand binding and function of this extraordinary receptor. Furthermore, it was shown that a sulphated peptide derived from the N-terminus of D6 has the potential to be used therapeutically as a broad-based chemokine scavenger, which may be useful for dampening the effects of excessive chemokine production in chronic inflammatory conditions

Repurposing screen identifies mebendazole as a clinical candidate to synergise with docetaxel for prostate cancer treatment

BACKGROUND: Docetaxel chemotherapy in prostate cancer has a modest impact on survival. To date, efforts to develop combination therapies have not translated into new treatments. We sought to develop a novel therapeutic strategy to tackle chemoresistant prostate cancer by enhancing the efficacy of docetaxel. METHODS: We performed a drug-repurposing screen by using murine-derived prostate cancer cell lines driven by clinically relevant genotypes. Cells were treated with docetaxel alone, or in combination with drugs (n = 857) from repurposing libraries, with cytotoxicity quantified using High Content Imaging Analysis. RESULTS: Mebendazole (an anthelmintic drug that inhibits microtubule assembly) was selected as the lead drug and shown to potently synergise docetaxel-mediated cell killing in vitro and in vivo. Dual targeting of the microtubule structure was associated with increased G2/M mitotic block and enhanced cell death. Strikingly, following combined docetaxel and mebendazole treatment, no cells divided correctly, forming multipolar spindles that resulted in aneuploid daughter cells. Liposomes entrapping docetaxel and mebendazole suppressed in vivo prostate tumour growth and extended progression-free survival. CONCLUSIONS: Docetaxel and mebendazole target distinct aspects of the microtubule dynamics, leading to increased apoptosis and reduced tumour growth. Our data support a new concept of combined mebendazole/docetaxel treatment that warrants further clinical evaluation

Atypical chemokine receptor ACKR2 controls branching morphogenesis in the developing mammary gland

Macrophages are important regulators of branching morphogenesis during development and postnatally in the mammary gland. Regulation of macrophage dynamics during these processes can therefore have a profound impact on development. We demonstrate here that the developing mammary gland expresses high levels of inflammatory CC-chemokines, which are essential in vivo regulators of macrophage migration. We further demonstrate that the atypical chemokine receptor ACKR2, which scavenges inflammatory CC-chemokines, is differentially expressed during mammary gland development. We have previously shown that ACKR2 regulates macrophage dynamics during lymphatic vessel development. Here, we extend these observations to reveal a novel role for ACKR2 in regulating the postnatal development of the mammary gland. Specifically, we show that Ackr2−/− mice display precocious mammary gland development. This is associated with increased macrophage recruitment to the developing gland and increased density of the ductal epithelial network. These data demonstrate that ACKR2 is an important regulator of branching morphogenesis in diverse biological contexts and provide the first evidence of a role for chemokines and their receptors in postnatal development processes

A functional genomics screen reveals a strong synergistic effect between docetaxel and the mitotic gene DLGAP5 that is mediated by the androgen receptor

Based on a molecular classification of prostate cancer using gene expression pathway signatures, we derived a set of 48 genes in critical pathways that significantly predicts clinical outcome in all tested patient cohorts. We tested these genes in a functional genomics screen in a panel of three prostate cancer cell lines (LNCaP, PC3, DU145), using RNA interference. The screen revealed several genes whose knockdown caused strong growth inhibition in all cell lines. Additionally, we tested the gene set in the presence of docetaxel to see whether any gene exhibited additive or synergistic effects with the drug. We observed a strong synergistic effect between DLGAP5 knockdown and docetaxel in the androgen-sensitive line LNCaP, but not in the two other androgen-independent lines. We then tested whether this effect was connected to androgen pathways and found that knockdown of the androgen receptor by si-RNA attenuated the synergy significantly. Similarly, androgen desensitized LNCaP-AI cells had a higher IC50 to docetaxel and did not exhibit the synergistic interaction. Short-term exposure to enzalutamide did not significantly alter the behaviour of parental LNCaP cells. An immunofluorescence analysis in LNCaP cells suggests that under the double insult of DLGAP5 knockdown and docetaxel, cells predominantly arrest in metaphase. In contrast, the knockdown of the androgen receptor by siRNA appears to assist cells to progress through metaphase in to anaphase, even in the presence of docetaxel. Our data suggest that DLGAP5 has a unique function in stabilizing spindle formation and surviving microtubule assault from docetaxel, in an androgen-regulated cell cycle system

Pancreas-derived mesenchymal stromal cells share immune response-modulating and angiogenic potential with bone marrow mesenchymal stromal cells and can be grown to therapeutic scale under GMP conditions

Background aims: Mesenchymal stromal cells (MSCs) isolated from various tissues are under investigation as cellular therapeutics in a wide range of diseases. It is appreciated that the basic biological functions of MSCs vary depending on tissue source. However, in-depth comparative analyses between MSCs isolated from different tissue sources under Good Manufacturing Practice (GMP) conditions are lacking. Human clinical-grade low-purity islet (LPI) fractions are generated as a byproduct of islet isolation for transplantation. MSC isolates were derived from LPI fractions with the aim of performing a systematic, standardized comparative analysis of these cells with clinically relevant bone marrow-derived MSCs (BM MSCs). Methods: MSC isolates were derived from LPI fractions and expanded in platelet lysate-supplemented medium or in commercially available xenogeneic-free medium. Doubling rate, phenotype, differentiation potential, gene expression, protein production and immunomodulatory capacity of LPIs were compared with those of BM MSCs. Results: MSCs can be readily derived in vitro from non-transplanted fractions resulting from islet cell processing (i.e., LPI MSCs). LPI MSCs grow stably in serum-free or platelet lysate-supplemented media and demonstrate in vitro self-renewal, as measured by colony-forming unit assay. LPI MSCs express patterns of chemokines and pro-regenerative factors similar to those of BM MSCs and, importantly, are equally able to attract immune cells in vitro and in vivo and suppress T-cell proliferation in vitro. Additionally, LPI MSCs can be expanded to therapeutically relevant doses at low passage under GMP conditions. Conclusions: LPI MSCs represent an alternative source of GMP MSCs with functions comparable to BM MSCs

Identification of a Clinically Relevant Signature for Early Progression in KRAS-Driven Lung Adenocarcinoma

Inducible genetically defined mouse models of cancer uniquely facilitate the investigation of early events in cancer progression, however, there are valid concerns about the ability of such models to faithfully recapitulate human disease. We developed an inducible mouse model of progressive lung adenocarcinoma (LuAd) that combines sporadic activation of oncogenic KRasG12D with modest overexpression of c-MYC (KM model). Histological examination revealed a highly reproducible spontaneous transition from low-grade adenocarcinoma to locally invasive adenocarcinoma within 6 weeks of oncogene activation. Laser-capture microdissection coupled with RNA-SEQ (ribonucleic acid sequencing) was employed to determine transcriptional changes associated with tumour progression. Upregulated genes were triaged for relevance to human LuAd using datasets from Oncomine and cBioportal. Selected genes were validated by RNAi screening in human lung cancer cell lines and examined for association with lung cancer patient overall survival using KMplot.com. Depletion of progression-associated genes resulted in pronounced viability and/or cell migration defects in human lung cancer cells. Progression-associated genes moreover exhibited strong associations with overall survival, specifically in human lung adenocarcinoma, but not in squamous cell carcinoma. The KM mouse model faithfully recapitulates key molecular events in human adenocarcinoma of the lung and is a useful tool for mechanistic interrogation of KRAS-driven LuAd progression

Chemokines as novel and versatile reagents for flow cytometry and cell sorting

Cell therapy regimens are frequently compromised by low-efficiency cell homing to therapeutic niches. Improvements in this regard would enhance effectiveness of clinically applicable cell therapy. The major regulators of tissue-specific cellular migration are chemokines, and therefore selection of therapeutic cellular populations for appropriate chemokine receptor expression would enhance tissue-homing competence. A number of practical considerations preclude the use of Abs in this context, and alternative approaches are required. In this study, we demonstrate that appropriately labeled chemokines are at least as effective in detecting their cognate receptors as commercially available Abs. We also demonstrate the utility of biotinylated chemokines as cell-sorting reagents. Specifically, we demonstrate, in the context of CCR7 (essential for lymph node homing of leukocytes), the ability of biotinylated CCL19 with magnetic bead sorting to enrich for CCR7-expressing cells. The sorted cells demonstrate improved CCR7 responsiveness and lymph node–homing capability, and the sorting is effective for both T cells and dendritic cells. Importantly, the ability of chemokines to detect CCR7, and sort for CCR7 positivity, crosses species being effective on murine and human cells. This novel approach to cell sorting is therefore inexpensive, versatile, and applicable to numerous cell therapy contexts. We propose that this represents a significant technological advance with important therapeutic implications

Targeting BCR-ABL-Independent TKI Resistance in Chronic Myeloid Leukemia by mTOR and Autophagy Inhibition

Background Imatinib and second-generation tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib have statistically significantly improved the life expectancy of chronic myeloid leukemia (CML) patients; however, resistance to TKIs remains a major clinical challenge. Although ponatinib, a third-generation TKI, improves outcomes for patients with BCR-ABL-dependent mechanisms of resistance, including the T315I mutation, a proportion of patients may have or develop BCR-ABL-independent resistance and fail ponatinib treatment. By modeling ponatinib resistance and testing samples from these CML patients, it is hoped that an alternative drug target can be identified and inhibited with a novel compound. Methods Two CML cell lines with acquired BCR-ABL-independent resistance were generated following culture in ponatinib. RNA sequencing and gene ontology (GO) enrichment were used to detect aberrant transcriptional response in ponatinib-resistant cells. A validated oncogene drug library was used to identify US Food and Drug Administration–approved drugs with activity against TKI-resistant cells. Validation was performed using bone marrow (BM)–derived cells from TKI-resistant patients (n = 4) and a human xenograft mouse model (n = 4–6 mice per group). All statistical tests were two-sided. Results We show that ponatinib-resistant CML cells can acquire BCR-ABL-independent resistance mediated through alternative activation of mTOR. Following transcriptomic analysis and drug screening, we highlight mTOR inhibition as an alternative therapeutic approach in TKI-resistant CML cells. Additionally, we show that catalytic mTOR inhibitors induce autophagy and demonstrate that genetic or pharmacological inhibition of autophagy sensitizes ponatinib-resistant CML cells to death induced by mTOR inhibition in vitro (% number of colonies of control[SD], NVP-BEZ235 vs NVP-BEZ235+HCQ: 45.0[17.9]% vs 24.0[8.4]%, P = .002) and in vivo (median survival of NVP-BEZ235- vs NVP-BEZ235+HCQ-treated mice: 38.5 days vs 47.0 days, P = .04). Conclusion Combined mTOR and autophagy inhibition may provide an attractive approach to target BCR-ABL-independent mechanism of resistance