Putative Inv Is Essential for Basolateral Invasion of Caco-2 Cells and Acts Synergistically with OmpA To Affect In Vitro and In Vivo Virulence of Cronobacter sakazakii ATCC 29544

Cronobacter sakazakii is an opportunistic pathogen that causes neonatal meningitis and necrotizing enterocolitis. Its interaction with intestinal epithelium is important in the pathogenesis of enteric infections. In this study, we investigated the involvement of the inv gene in the virulence of C. sakazakii ATCC 29544 in vitro and in vivo. Sequence analysis of C. sakazakii ATCC 29544 inv revealed that it is different from other C. sakazakii isolates. In various cell culture models, an Δinv deletion mutant showed significantly lowered invasion efficiency, which was restored upon genetic complementation. Studying invasion potentials using tight-junction-disrupted Caco-2 cells suggested that the inv gene product mediates basolateral invasion of C. sakazakii ATCC 29544. In addition, comparison of invasion potentials of double mutant (ΔompA Δinv) and single mutants (ΔompA and Δinv) provided evidence for an additive effect of the two putative outer membrane proteins. Finally, the importance of inv and the additive effect of putative Inv and OmpA were also proven in an in vivo rat pup model. This report is the first to demonstrate two proteins working synergistically in vitro, as well as in vivo in C. sakazakii pathogenesis.     

Co-targeting bromodomain and extra-terminal proteins and MCL1 induces synergistic cell death in melanoma

The treatment of melanoma has been markedly improved by the introduction of targeted therapies and checkpoint blockade immunotherapy. Unfortunately, resistance to these therapies remains a limitation. Novel anticancer therapeutics targeting the MCL1 anti-apoptotic protein have shown impressive responses in haematological cancers but are yet to be evaluated in melanoma. To assess the sensitivity of melanoma to new MCL1 inhibitors, we measured the response of 51 melanoma cell lines to the novel MCL1 inhibitor, S63845. Additionally, we assessed combination of this drug with inhibitors of the bromodomain and extra-terminal (BET) protein family of epigenetic readers, which we postulated would assist MCL1 inhibition by downregulating anti-apoptotic targets regulated by NF-kB such as BCLXL, BCL2A1 and XIAP, and by upregulating pro-apoptotic proteins including BIM and NOXA. Only 14% of melanoma cell lines showed sensitivity to S63845, however, combination of S63845 and I-BET151 induced highly synergistic apoptotic cell death in all melanoma lines tested and in an in vivo xenograft model. Cell death was dependent on caspases and BAX/BAK. Although the combination of drugs increased the BH3-only protein, BIM, and downregulated anti-apoptotic proteins such as BCL2A1, the importance of these proteins in inducing cell death varied between cell lines. ABT-199 or ABT-263 inhibitors against BCL2 or BCL2 and BCLXL, respectively, induced further cell death when combined with S63845 and I-BET151. The combination of MCL1 and BET inhibition appears to be a promising therapeutic approach for metastatic melanoma, and presents opportunities to add further BCL2 family inhibitors to overcome treatment resistance.     

Combining BET and HDAC inhibitors synergistically induces apoptosis of melanoma and suppresses AKT and YAP signaling

Histone acetylation marks have an important role in controlling gene expression and are removed by histone deacetylases (HDACs). These marks are read by bromodomain and extra-terminal (BET) proteins and novel inhibitiors of these proteins are currently in clinical development. Inhibitors of HDAC and BET proteins have individually been shown to cause apoptosis and reduce growth of melanoma cells. Here we show that combining the HDAC inhibitor LBH589 and BET inhibitor I-BET151 synergistically induce apoptosis of melanoma cells but not of melanocytes. Induction of apoptosis proceeded through the mitochondrial pathway, was caspase dependent and involved upregulation of the BH3 pro-apoptotic protein BIM. Analysis of signal pathways in melanoma cell lines resistant to BRAF inhibitors revealed that treatment with the combination strongly downregulated anti-apoptotic proteins and proteins in the AKT and Hippo/YAP signaling pathways. Xenograft studies showed that the combination of inhibitors was more effective than single drug treatment and confirmed upregulation of BIM and downregulation of XIAP as seen in vitro. These results support the combination of these two classes of epigenetic regulators in treatment of melanoma including those resistant to BRAF inhibitors.     

HDAC inhibitors restore BRAF‐inhibitor sensitivity by altering PI3K and survival signalling in a subset of melanoma

Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non‐genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient‐ and in vivo‐derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase‐dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro‐apoptotic BIM or NOXA together with reduced expression of anti‐apoptotic proteins. Independent of these changes, panobinostat reduced c‐Myc and pre‐treatment of cells with siRNA against c‐Myc reduced BRAFi/HDACi drug‐induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK‐independent pathways.     

Heavy cannabis users at elevated risk of stroke: evidence from a general population survey

Objective: Case reports and hospital‐based case–control studies suggest that cannabis use may increase the risk of stroke. We examined the risk of non‐fatal stroke or transient ischemic attack (TIA) among cannabis users in the general community. Method: A general population survey of Australians aged 20–24 years (n=2,383), 40–44 years (n=2,525) and 60–64 years (n=2,547) was used to determine the odds of lifetime stroke or TIA among participants who had smoked cannabis in the past year while adjusting for other stroke risk factors. Results: There were 153 stroke/TIA cases (2.1%). After adjusting for age cohort, past year cannabis users (n=1,043) had 3.3 times the rate of stroke/TIA (95% CI 1.8–6.3, p<0.001). The incidence rate ratio (IRR) reduced to 2.3 after adjustment for covariates related to stroke, including tobacco smoking (95% CI 1.1–4.5). Elevated stroke/TIA was specific to participants who used cannabis weekly or more often (IRR 4.7, 95% CI 2.1–10.7) with no elevation among participants who used cannabis less often. Conclusions: Heavy cannabis users in the general community have a higher rate of non‐fatal stroke or transient ischemic attack than non‐cannabis users.