[Abstract unavailable

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.

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Epidermal barrier acquisition during late murine gestation is accompanied by an increase in Akt kinase activity and cJun dephosphorlyation. The latter is directed by the Ppp2r2a regulatory subunit of the Pp2a phosphatase. This was accompanied by a change of Claudin-1 localisation to the cell surface and interaction between Occludin and Claudin-1 which are thought to be required for tight junction formation.The aim of this study was to determine the nature of the barrier defect caused by the loss of AKT/Ppp2r2a function. There was a paracellular barrier defect in rat epidermal keratinocytes expressing a Ppp2r2a siRNA. In Ppp2r2a knockdown cells, Claudin-1 was located to the cytoplasm and its expression was increased. Inhibiting cJun phosphorylation restored barrier function and plasma membrane localisation of Claudin-1. Expression of the Rab3 GTPase activating protein, Rab3Gap1, was restored in Ppp2r2a siRNA cells when cJun phosphorylation was inhibited. During normal mouse epidermal development, Claudin-1 plasma membrane localisation and Rab3Gap1 cell surface expression were co-incident with Akt activation in mouse epidermis, strongly suggesting a role of Rab3Gap1 in epidermal barrier acquisition. Supporting this hypothesis, siRNA knockdown of Rab3Gap1 prevented plasma membrane Claudin-1 expression and the formation of a barrier competent epithelium. Replacing Rab3Gap1 in Ppp2r2a knockdown cells was sufficient to rescue Claudin-1 transport to the cell surface. Therefore these data suggest Rab3Gap1 mediated exocytosis of Claudin-1 is an important component of epidermal barrier acquisition during epidermal development.

Epidermal barrier acquisition during late mammalian development is a prerequisite for terrestrial existence. Over a 24-h period, the epidermis goes from being a barrier-deficient, dye permeable epithelium to a barrier-competent epithelium. We have previously shown that Akt signalling is necessary for barrier acquisition in the mouse and that the protein phosphatase 2A regulatory subunit Ppp2r2a causes barrier acquisition by dephosphorylation of cJun. Here, we demonstrate that there is transient interaction between the gap junction protein Connexin 43 (Cx43) and Zonula occludins-1 (Zo-1) during epidermal barrier acquisition. Ppp2r2a knockdown prevented plasma membrane co-localisation and interaction between the two proteins. Ppp2r2a knockdown also increased phosphorylation at Serine 368 of Connexin 43. Cx43 phosphorlyation at Serine368 occurred just prior to the interaction between Connexin 43 and Zo-1. We therefore propose a model in which Ppp2r2a is required both for the initial interaction between Zo-1 and Cx43 and the consequent dephosphorylation of Connexin 43, preventing interaction of Zo-1 and allowing Zo-1 to initiate tight junction formation and barrier acquisition.

BACKGROUND: Neoantigens are ideal for developing personalized cancer vaccines as they are tumor specific and not subject to central tolerance. Targeting neoantigens to generate potent tumor-specific T-cell responses has shown both promising preclinical efficacy as well as clinical responses. VB10.NEO is a DNA plasmid vaccine with intrinsic adjuvant effect designed for efficient delivery of personalized neoepitopes and can hold up unique neoepitopes specific for each patient’s tumor. Here we describe VB N-01 (DIRECT-01), an open-label phase I/IIa study, designed to evaluate the safety, immunogenicity and efficacy of administrating personalized VB10.NEO in combination with checkpoint blockade in up to 40 patients with locally advanced or metastatic solid tumors including melanoma, NSCLC, renal cell carcinoma, urothelial cancer or head and neck cancer. The study is open at 3 clinical study sites in Germany (NCT03548467). METHODS: The primary objectives are to assess the safety/tolerability of VB10.NEO immunotherapy and determine the feasibility of the VB10.NEO vaccine manufacturing. Secondary objectives are to make a preliminary assessment of the efficacy, to assess the immunogenicity of VB10.NEO immunotherapy, and the correlation between immunological response and clinical efficacy. Eligible patients who have not achieved a confirmed complete response by week 12 on checkpoint blockade as respective standard of care (SOC) will be enrolled in the study. A tumor biopsy and blood sample will be collected and used to identify the patient-specific tumor mutations. A personalized VB10.NEO holding up to 20 unique neoantigens will be designed based on Vaccibody’s proprietary neoepitope selection algorithm NeoSelect™. Patients will receive multiple vaccinations of VB10.NEO at pre-specified time points; an induction phase consisting of 3 injections during the first 6 weeks followed by a maintenance phase with vaccinations every 4th week up to one year from first immunization. VB10.NEO will be administered IM using the PharmaJet® Stratis Needle-free Injection System. A 24 month follow up period will follow the treatment phase. Patients will be assessed for safety, including AEs, vital signs, safety laboratory assessments and ECOG status. Tumor response will be assessed according to the iRECIST criteria. The study aims to characterize the cellular immune response against the individual neoepitopes by IFNγ ELISpot or other immune assays. A descriptive analysis of serial biopsies will also be assessed, including analysis of the mutagenic landscape by exome and RNA sequencing, and IHC to study tumor microenvironment. The trial is currently recruiting patients and initial accrual goal was meet. Citation Format: Jurgen Krauss, Angela Krackhardt, Elke Jager, Anja Williams, Hedda Wold, Lisa Gerner, Monika Sekelja, Karoline Schjetne, Agnete B. Fredriksen, Mads Axelsen. An open-label, Phase I/IIa study of VB10.NEO (DIRECT-01) in combination with checkpoint blockade in patients with locally advanced or metastatic solid tumors including melanoma, NSCLC, renal cell carcinoma, urothelial cancer or SSCHN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT217.

(2016). Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2. Biochemical and Biophysical Research Communications, 476(2) This manuscript is distributed under a Creative Commons Attribution-NonCommercial-NoDerivs License (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits distribution and reproduction for non-commercial purposes, provided the author and source are cited. General rightsCopyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In conclusion, the intrinsic fluorescent properties of STO-609 provide a great opportunity to utilise this drug to label the ATP-binding pocket and probe the impact of mutations and other regulatory modifications and interactions on the pocket. It is however clear that the number of phosphorylation sites on CaMKK2 will pose a challenge in studying the impact of phosphorylation on the pocket unless the field can develop approaches to control the spectrum of modifications that occur during recombinant protein expression in E. coli.

Calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) has been implicated in a range of conditions and pathologies from prostate to hepatic cancer. Here, we describe the expression in Escherichia coli and the purification protocol for the following constructs: full-length CaMKK2 in complex with CaM, CaMKK2 ‘apo’, CaMKK2 (165-501) in complex with CaM, and the CaMKK2 F267G mutant. The protocols described have been optimized for maximum yield and purity with minimal purification steps required and the proteins subsequently used to develop a fluorescence-based assay for drug binding to the kinase, “Using the fluorescent properties of STO-609 as a tool to assist structure-function analyses of recombinant CaMKK2” [1].