Genetic analyses in mouse fibroblast and melanoma cells demonstrate novel roles for PDGF-AB ligand and PDGF receptor alpha

Platelet Derived Growth Factor Receptor (PDGFR) signaling is a central mitogenic pathway in development, as well as tissue repair and homeostasis. The rules governing the binding of PDGF ligand to the receptor to produce activation and downstream signaling have been well defined over the last several decades. In cultured cells after a period of serum deprivation, treatment with PDGF leads to the rapid formation of dramatic, actin-rich Circular Dorsal Ruffles (CDRs). Using CDRs as a robust visual readout of early PDGFR signaling, we have identified several contradictory elements in the widely accepted model of PDGF activity. Employing CRISPR/Cas9 gene editing to disrupt the Pdgfra gene in two different murine cell lines, we show that in addition to the widely accepted function for PDGFR-beta in CDR formation, PDGFR-alpha is also clearly capable of eliciting CDRs. Moreover, we demonstrate activity for heterodimeric PDGF-AB ligand in the vigorous activation of PDGFR-beta homodimers to produce CDRs. These findings are key to a more complete understanding of PDGF ligand-receptor interactions and their downstream signaling consequences. This knowledge will allow for more rigorous experimental design in future studies of PDGFR signaling and its contributions to development and disease.

Whole transcriptome in-silico screening implicates cardiovascular and infectious disease in the mechanism of action underlying atypical antipsychotic side-effects

BACKGROUND Stroke/thromboembolic events, infections and death are all significantly increased by antipsychotics in dementia but little is known about why they can be harmful. Using a novel application of a drug repurposing paradigm, we aimed to identify potential mechanisms underlying adverse events. METHODS Whole transcriptome signatures were generated for SH-SY5Y cells treated with amisulpride, risperidone and volinanserin using RNA-sequencing. Bioinformatic analysis was performed which scored the association between antipsychotic signatures and expression data from 415,252 samples in the NCBI GEO repository. RESULTS Atherosclerosis, venous thromboembolism and influenza NCBI GEO-derived samples scored positively against antipsychotic signatures. Pathways enriched in antipsychotic signatures were linked to the cardiovascular and immune systems (e.g. BDNF, PDGFR-beta, TNF, TGF-beta, selenoamino acid metabolism and influenza infection). CONCLUSIONS These findings for the first time mechanistically link antipsychotics to specific cardiovascular and infectious diseases which are known side effects of their use in dementia, providing new information to explain related adverse events.

Whole transcriptome in-silico screening implicates cardiovascular and infectious disease in the mechanism of action underlying atypical antipsychotic side-effects

INTRODUCTIONStroke/thromboembolic events, infections and death are all significantly increased by antipsychotics in dementia but little is known about why they can be harmful. Using a novel application of a drug repurposing paradigm, we aimed to identify potential mechanisms underlying adverse events.METHODWhole transcriptome signatures were generated for SH-SY5Y cells treated with amisulpride, risperidone and volinanserin using RNA-sequencing. Bioinformatic analysis was performed which scored the association between antipsychotic signatures and expression data from 415,252 samples in the NCBI GEO repository.RESULTSAtherosclerosis, venous thromboembolism and influenza NCBI GEO-derived samples scored positively against antipsychotic signatures. Pathways enriched in antipsychotic signatures were linked to the cardiovascular and immune systems (e.g. BDNF, PDGFR-beta, TNF, TGF-beta, selenoamino acid metabolism and influenza infection).CONCLUSIONThese findings for the first time mechanistically link antipsychotics to specific cardiovascular and infectious diseases which are known side effects of their use in dementia, providing new information to explain related adverse events.COMPETING INTERESTSCB has received grants and personal fees from ACADIA Pharmaceuticals and Lundbeck, and personal fees from Heptares, Roche, Lilly, Otsuka, Orion, GlaxoSmithKline and Pfizer. DAC is an employee of Eli Lilly and Company Ltd.

ErbB2 and EphA3 as a novel prognostic and therapeutic target of Recurrent Non-functioning Pituitary Adenomas: A pilot study

Non-functioning pituitary adenomas (NFPA) are 30% of all pituitary adenomas. Although benign in nature but they may be invasive and recurrent. Markers of recurrence are needed to guide patient management. Recteptor Tyrosine Kinases (RTK) may sereve as therapeutic marker for recurrence as they can targeted by already available tyrosine kinase inhibitors. To examine differential RTK phosphorylation pattern of recurrent NFPAs, we recruited 20 patients and grouped them as non-recurrent (n=10), and recurrent (n=10). Recurrence was determined by follow-up of 15 years. We then performed a membrane-based antibody array (ARY001B) for the determination of the relative phosphorylation of 49 tyrosine kinases in recurrent NFPAs. As the experiment was replicated on two set of membranes, each tyrosine kinase was represented in quadruples. Student’s t-test was performed to compare the means between two groups. We found ErbB2, PDGFR beta, SCFR, Trk, VEGFR1, VEGFR2, EphA3, and Alk significantly hyperphosphorylated in recurrent NFPAs. Out of these eight hyperphosphorylated tyrosine kinases ErbB2 and EphA3 were 1.6 (p=0.01) and 1.9 (p=0.002) times hyperphosphorylated in recurrent NFPAs. This result indicates that EphA3 may be an effective therapeutic target in recurrent NFPAs.

Effects of Sunitinib and Other Kinase Inhibitors on Cells Harboring a PDGFRB Mutation Associated with Infantile Myofibromatosis

Infantile myofibromatosis represents one of the most common proliferative fibrous tumors of infancy and childhood. More effective treatment is needed for drug-resistant patients, and targeted therapy using specific protein kinase inhibitors could be a promising strategy. To date, several studies have confirmed a connection between the p.R561C mutation in gene encoding platelet-derived growth factor receptor beta (PDGFR-beta) and the development of infantile myofibromatosis. This study aimed to analyze the phosphorylation of important kinases in the NSTS-47 cell line derived from a tumor of a boy with infantile myofibromatosis who harbored the p.R561C mutation in PDGFR-beta. The second aim of this study was to investigate the effects of selected protein kinase inhibitors on cell signaling and the proliferative activity of NSTS-47 cells. We confirmed that this tumor cell line showed very high phosphorylation levels of PDGFR-beta, extracellular signal-regulated kinases (ERK) 1/2 and several other protein kinases. We also observed that PDGFR-beta phosphorylation in tumor cells is reduced by the receptor tyrosine kinase inhibitor sunitinib. In contrast, MAPK/ERK kinases (MEK) 1/2 and ERK1/2 kinases remained constitutively phosphorylated after treatment with sunitinib and other relevant protein kinase inhibitors. Our study showed that sunitinib is a very promising agent that affects the proliferation of tumor cells with a p.R561C mutation in PDGFR-beta.