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.     

Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition

Background: Inhibition of acetylcholinesterase (AChE), a biomarker of organophosphorous and carbamate exposure in environmental and occupational human health, has been commonly used to identify potential safety liabilities. So far, many environmental chemicals, including drug candidates, food additives, and industrial chemicals, have not been thoroughly evaluated for their inhibitory effects on AChE activity. AChE inhibitors can have therapeutic applications (e.g., tacrine and donepezil) or neurotoxic consequences (e.g., insecticides and nerve agents).Objectives: The objective of the current study was to identify environmental chemicals that inhibit AChE activity using in vitro and in silico models.Methods: To identify AChE inhibitors rapidly and efficiently, we have screened the Toxicology in the 21st Century (Tox21) 10K compound library in a quantitative high-throughput screening (qHTS) platform by using the homogenous cell-based AChE inhibition assay and enzyme-based AChE inhibition assays (with or without microsomes). AChE inhibitors identified from the primary screening were further tested in monolayer or spheroid formed by SH-SY5Y and neural stem cell models. The inhibition and binding modes of these identified compounds were studied with time-dependent enzyme-based AChE inhibition assay and molecular docking, respectively.Results: A group of known AChE inhibitors, such as donepezil, ambenonium dichloride, and tacrine hydrochloride, as well as many previously unreported AChE inhibitors, such as chelerythrine chloride and cilostazol, were identified in this study. Many of these compounds, such as pyrazophos, phosalone, and triazophos, needed metabolic activation. This study identified both reversible (e.g., donepezil and tacrine) and irreversible inhibitors (e.g., chlorpyrifos and bromophos-ethyl). Molecular docking analyses were performed to explain the relative inhibitory potency of selected compounds.Conclusions: Our tiered qHTS approach allowed us to generate a robust and reliable data set to evaluate large sets of environmental compounds for their AChE inhibitory activity. https://doi.org/10.1289/EHP6993     

Small group health insurance: ranking the states on the depth of reform, 1999

States are ranked based on the potential of their small group health insurance reforms to enhance health insurance uptake. Reforms were quantified based on their market impact potential. Five dimensions of reforms were identified, Access Improvement, Premium Reduction, Premium Differential Reduction, Continuity of Coverage, and Enhancing Valued Plan Features. The reform indices representing these dimensions were developed based on document review of state statutes, combined with actuarial judgment to identify and assign scores to market-relevant regulations in line with their impact potential. The distribution of the states' reform scores and rankings show wide variation in the depth and focus of their reforms. Only seven of the top ten states on the Total Reform index had consistently higher scores on two or more reform dimensions. The conceptual linkages between specific regulations and the documented small group market problems lead to normative expectations of an association between the depth of state reforms and the prevalence of uninsurance.     

Evaluation of micro-parallel liquid chromatography as a method for HTS-coupled actives verification

The identification of biologically active compounds from high-throughput screening (HTS) can involve considerable postscreening analysis to verify the nature of the sample activity. In this study we evaluated the performance of micro-parallel liquid chromatography (microPLC) as a separation-based enzyme assay platform for follow-up of compound activities found in quantitative HTS of two different targets, a hydrolase and an oxidoreductase. In an effort to couple secondary analysis to primary screening we explored the application of microPLC immediately after a primary screen. In microPLC, up to 24 samples can be loaded and analyzed simultaneously via high-performance liquid chromatography within a specially designed cartridge. In a proof-of-concept experiment for screen-coupled actives verification, we identified, selected, and consolidated the contents of "active" wells from a 1,536-well format HTS experiment into a 384-well plate and subsequently analyzed these samples by a 24-channel microPLC system. The method utilized 0.6% of the original 6-microl 1,536-well assay for the analysis. The analysis revealed several non-biological-based "positive" samples. The main examples included "false" enzyme activators resulting from an increase in well fluorescence due to fluorescent compound or impurity. The microPLC analysis also provided a verification of the activity of two activators of glucocerebrosidase. We discuss the benefits of microPLC and its limitations from the standpoint of ease of use and integration into a seamless postscreen workflow.     

Edmonton, Canada: a regional model of palliative care development

Palliative care developed unevenly in Edmonton in the 1980s and early 1990s. Health care budget cuts created an opportunity for innovative redesign of palliative care service delivery. This report describes the components that were developed to build an integrated comprehensive palliative care program, the use of common clinical assessments and outcome evaluation that has been key to establishing credibility and ongoing support. Our program has continued to develop and grow with an ongoing focus on the core areas of clinical care, education, and research.     

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.     

Accreditation across cultures: a case study

Accreditation of academic programs is one of the most significant developments in the evolution of professional education in the United States. Efforts in several fields to extend programmatic accreditation to institutions outside the United States have had mixed results. This report describes such an accreditation experience in health services administration, its pitfalls, and the lessons that the site visit team (the authors) learned. The authors hope that others undertaking such tasks can benefit from this experience.     

Diagnosis of pneumonia in the ED has poor accuracy despite diagnostic uncertainty

Background

In 2007, the Centers for Medicare and Medicaid Services created a measure known as “diagnostic uncertainty” in emergency department (ED) pneumonia admissions. This documentation excludes the antibiotic timing measure, as pressure to quickly diagnose pneumonia may serve to reduce overall accuracy.

Study objectives

The objective of the study was to determine the correlation between ED and final discharge diagnosis of pneumonia and measure the effect of invoking the diagnostic uncertainty documentation on accuracy.

Methods

We retrospectively reviewed all ED pneumonia admissions among adults from July to October 2008. We analyzed the effect of invoking the diagnostic uncertainty documentation in the ED by comparing against final outcomes. We then performed a multivariate analysis to adjust for the potential effects of sex, age, Emergency Severity Index (ESI) score, weekend arrival, and level of ED-attending physician staffing.

Results

Of 401 patients who were admitted with pneumonia, 297 (74%) had a discharge diagnosis of pneumonia, with 72 (18%) of those diagnoses being the primary outcome. Diagnostic uncertainty documentation was used in 11% (45/401). This documentation did not significantly alter the odds of a primary pneumonia discharge diagnosis (odds ratio, 0.68; 95% confidence interval, 0.28-1.7) but did reduce the odds of pneumonia being diagnosed (odds ratio, 0.43; 95% confidence interval, 0.23-0.81). Sex, age, day of week, and (ESI) score remained nonsignificant predictors.

Conclusions

Correlation between ED and discharge diagnosis of pneumonia was limited. Use of diagnostic uncertainty documentation decreased the likelihood of a hospital discharge diagnosis of pneumonia. Further analysis of the effects of artificially imposed time constraints on ED diagnoses appears warranted.