PDGFRα depletion attenuates glioblastoma stem cells features by modulation of STAT3, RB1 and multiple oncogenic signals.

Platelet derived growth factor receptors (PDGFRs) play an important role in tumor pathogenesis, and they are frequently overexpressed in glioblastoma (GBM). Earlier we have shown a higher protein expression of PDGFR isoforms (α and β) in peritumoral-tissue derived cancer stem cells (p-CSC) than in tumor core (c-CSC) of several GBM affected patients. In the current study, in order to assess the activity of PDGFRα/PDGF-AA signaling axis, we performed time course experiments to monitor the effects of exogenous PDGF-AA on the expression of downstream target genes in c-CSC vs p-CSC. Interestingly, in p-CSC we detected the upregulation of Y705-phosphorylated Stat3, concurrent with a decrement of Rb1 protein in its active state, within minutes of PDGF-AA addition. This finding prompted us to elucidate the role of PDGFRα in self-renewal, invasion and differentiation in p-CSC by using short hairpin RNA depletion of PDGFRα expression. Notably, in PDGFRα-depleted cells, protein analysis revealed attenuation of stemness-related and glial markers expression, alongside early activation of the neuronal marker MAP2a/b that correlated with the induction of tumor suppressor Rb1. The in vitro reduction of the invasive capacity of PDGFRα-depleted CSC as compared to parental cells correlated with the downmodulation of markers of epithelial-mesenchymal transition phenotype and angiogenesis. Surprisingly, we observed the induction of anti-apoptotic proteins and compensatory oncogenic signals such as EDN1, EDNRB, PRKCB1, PDGF-C and PDGF-D. To conclude, we hypothesize that the newly discovered PDGFRα/Stat3/Rb1 regulatory axis might represent a potential therapeutic target for GBM treatment.BRC, Q

Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids.

Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.Cancer Research UK; Royal Marsden/Institute of Cancer Research National Institute for Health Research Biomedical Research Centre (M.L.)

Delayed mucosal anti-viral responses despite robust peripheral inflammation in fatal COVID-19

Background While inflammatory and immune responses to SARS-CoV-2 infection in peripheral blood are extensively described, responses at the upper respiratory mucosal site of initial infection are relatively poorly defined. We sought to identify mucosal cytokine/chemokine signatures that distinguished COVID-19 severity categories, and relate these to disease progression and peripheral inflammation. Methods We measured 35 cytokines and chemokines in nasal samples from 274 patients hospitalised with COVID-19. Analysis considered the timing of sampling during disease, as either the early (0-5 days post-symptom onset) or late (6-20 days post-symptom onset). Results Patients that survived severe COVID-19 showed IFN-dominated mucosal immune responses (IFN-γ, CXCL10 and CXCL13) early in infection. These early mucosal responses were absent in patients that would progress to fatal disease despite equivalent SARS-CoV-2 viral load. Mucosal inflammation in later disease was dominated by IL-2, IL-10, IFN-γ, and IL-12p70, which scaled with severity but did not differentiate patients who would survive or succumb to disease. Cytokines and chemokines in the mucosa showed distinctions from responses evident in the peripheral blood, particularly during fatal disease. Conclusions Defective early mucosal anti-viral responses anticipate fatal COVID-19 but are not associated with viral load. Early mucosal immune responses may define the trajectory of severe COVID-19

Pseudokinase drug intervention: a potentially poisoned chalice

Pseudokinases, the catalytically impaired component of the kinome, have recently been found to share more properties with active kinases than previously thought. In many pseudokinases, ATP binding and even some activity is preserved, highlighting these proteins as potential drug targets. In both active kinases and pseudokinases, binding of ATP or drugs in the nucleotide-binding pocket can stabilize specific conformations required for activity and protein-protein interactions. We discuss the implications of locking particular conformations in a selection of (pseudo)kinases and the dual potential impact on the druggability of these proteins.</p

Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion

In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality

Interleukin-6 Receptor Antagonists in Critically Ill Patients with Covid-19 – Preliminary report

Abstract Background The efficacy of interleukin-6 receptor antagonists in critically ill patients with coronavirus disease 2019 (Covid-19) is unclear. Methods We evaluated tocilizumab and sarilumab in an ongoing international, multifactorial, adaptive platform trial. Adult patients with Covid-19, within 24 hours of commencing organ support in an intensive care unit, were randomized to receive either tocilizumab (8mg/kg) or sarilumab (400mg) or standard care (control). The primary outcome was an ordinal scale combining in-hospital mortality (assigned −1) and days free of organ support to day 21. The trial uses a Bayesian statistical model with pre-defined triggers to declare superiority, efficacy, equivalence or futility. Results Tocilizumab and sarilumab both met the pre-defined triggers for efficacy. At the time of full analysis 353 patients had been assigned to tocilizumab, 48 to sarilumab and 402 to control. Median organ support-free days were 10 (interquartile range [IQR] −1, 16), 11 (IQR 0, 16) and 0 (IQR −1, 15) for tocilizumab, sarilumab and control, respectively. Relative to control, median adjusted odds ratios were 1.64 (95% credible intervals [CrI] 1.25, 2.14) for tocilizumab and 1.76 (95%CrI 1.17, 2.91) for sarilumab, yielding >99.9% and 99.5% posterior probabilities of superiority compared with control. Hospital mortality was 28.0% (98/350) for tocilizumab, 22.2% (10/45) for sarilumab and 35.8% (142/397) for control. All secondary outcomes and analyses supported efficacy of these IL-6 receptor antagonists. Conclusions In critically ill patients with Covid-19 receiving organ support in intensive care, treatment with the IL-6 receptor antagonists, tocilizumab and sarilumab, improved outcome, including survival. ( ClinicalTrials.gov number: NCT02735707