Vemurafenib and dabrafenib downregulates RIPK4 level

Vemurafenib and dabrafenib are BRAF kinase inhibitors (BRAFi) used for the treatment of patients with melanoma carrying the V600E BRAF mutation. However, melanoma cells develop resistance to both drugs when used as monotherapy. Therefore, mechanisms of drug resistance are investigated, and new molecular targets are sought that could completely inhibit melanoma progression. Since receptor-interacting protein kinase (RIPK4) probably functions as an oncogene in melanoma and its structure is similar to the BRAF protein, we analyzed the impact of vemurafenib and dabrafenib on RIPK4 in melanomas. The in silico study confirmed the high similarity of BRAF kinase domains to the RIPK4 protein at both the sequence and structural levels and suggests that BRAFi could directly bind to RIPK4 even more strongly than to ATP. Furthermore, BRAFi inhibited ERK1/2 activity and lowered RIPK4 protein levels in BRAF-mutated melanoma cells (A375 and WM266.4), while in wild-type BRAF cells (BLM and LoVo), both inhibitors decreased the level of RIPK4 and enhanced ERK1/2 activity. The phosphorylation of phosphatidylethanolamine binding protein 1 (PEBP1) - a suppressor of the BRAF/MEK/ERK pathway - via RIPK4 observed in pancreatic cancer did not occur in melanoma. Neither downregulation nor upregulation of RIPK4 in BRAF- mutated cells affected PEBP1 levels or the BRAF/MEK/ERK pathway. The downregulation of RIPK4 inhibited cell proliferation and the FAK/AKT pathway, and increased BRAFi efficiency in WM266.4 cells. However, the silencing of RIPK4 did not induce apoptosis or necroptosis. Our study suggests that RIPK4 may be an off-target for BRAF inhibitors

Effect of BRAF kinase inhibitors on the level of RIPK4 protein in melanoma cells

Niskocząsteczkowe inhibitory zmutowanej kinazy BRAF (wemurafenib i dabrafenib) wykorzystuje się w terapii czerniaka złośliwego z mutacją V600EBRAF. Są to jednak leki na które komórki czerniaka się uodparniają, prowadząc do wznowy nowotworu. Stąd też bada się mechanizmy lekooporności i poszukuje się nowych celów molekularnych, które całkowicie zahamują rozwój czerniaka. Z uwagi na sugerowaną onkogenną funkcję kinazy RIPK4, której rola w czerniaku nie jest poznana oraz podobieństwo strukturalne białka BRAF i RIPK4, w niniejszej pracy przeanalizowano ekspresję genów białek BRAF, MAP2K1, RIPK4 oraz PEBP1 w komórkach czerniaka wykorzystując ogólnodostępne bazy mikromacierzowe GEO, porównano struktury domen kinazowych dzikiej i zmutowanej kinazy BRAF oraz białka RIPK4 za pomocą programu BLAST i PyMOL. Dodatkowo sprawdzono wpływ dabrafenibu i wemurafenibu na przeżywalność komórek czerniaka testem MTT oraz aktywność ERK1/2 i poziom kinazy RIPK4 za pomocą Western blot. W pracy potwierdzono wysoki stopień podobieństwa struktur BRAF i RIPK4 oraz wykazano, wyższy poziomu ekspresji białek szlaku MAPK i RIPK4 w komórkach melanomy względem prawidłowych melanocytów. Ponadto wykazano doświadczalnie, że zarówno wemurafenib jak i dabrafenib hamuje aktywność ERK1/2 i obniża poziom kinazy RIPK4, co koreluje ze spadkiem przeżywalności komórek czerniaka.Small-molecular weight inhibitors of mutant BRAF kinase (vemurafenib and dabrafenib) are used for the treatment of malignant melanoma patients carrying V600EBRAF mutation. However, melanoma cells develops resistance for both drugs when used as monotherapy. Therefore mechanisms of drug resistance are being investigated and new molecular targets are studied being sought that will could completely inhibit the development of melanoma. Because of suggested oncogenic function of RIPK4 kinase, which role in melanoma remain unknown and its structural similarity with BRAF protein, in the present work the domains of wild and mutant BRAF kinase, and RIPK4 protein were compared using the BLAST and PyMOL programs. Furthermore, we analyzed relative expression of know genes such as MAP2K1, and PEBP1 in melanoma cells and compare with RIPK4 genes. The gene expression microarray data of melanoma were obtained from (NCBI) Gene Expression Omnibus and were accessible through Gene Expression Omnibus series accession number GDS1965, GDS429S and GSE31909 microarray bases. In addition, the effect of dabrafenib and vemurafenib on melanoma cell survival was tested by MTT. ERK1/2 activity and RIPK4 kinase levels were analyzed by Western blot technique. The results confirms the high degree of similarity of RIPK4 with BRAF structures and shows a higher level of expression of the MAPK pathway and RIPK4 proteins in melanoma cells compared to normal melanocytes. We demonstrated that both vemurafenib and dabrafenib inhibits ERK1/2 activity and reduce RIPK4 kinase level in melanoma cells in the time and dose-dependent way what correlates with a decrease of melanoma cell survival

Identification and management of alcohol withdrawal syndrome.

Symptoms of alcohol withdrawal syndrome (AWS) may develop within 6-24 h after the abrupt discontinuation or decrease of alcohol consumption. Symptoms can vary from autonomic hyperactivity and agitation to delirium tremens. The gold-standard treatment for AWS is with benzodiazepines (BZDs). Among the BZDs, different agents (i.e., long-acting or short-acting) and different regimens (front-loading, fixed-dose or symptom-triggered) may be chosen on the basis of patient characteristics. Severe withdrawal could require ICU admission and the use of barbiturates or propofol. Other drugs, such as \u3b12-agonists (clonidine and dexmetedomidine) and \u3b2-blockers can be used as adjunctive treatments to control neuroautonomic hyperactivity. Furthermore, neuroleptic agents can help control hallucinations. Finally, other medications for the treatment for AWS have been investigated with promising results. These include carbamazepine, valproate, sodium oxybate, baclofen, gabapentin and topiramate. The usefulness of these agents are discussed

Ticagrelor in patients with diabetes and stable coronary artery disease with a history of previous percutaneous coronary intervention (THEMIS-PCI) : a phase 3, placebo-controlled, randomised trial

Background: Patients with stable coronary artery disease and diabetes with previous percutaneous coronary intervention (PCI), particularly those with previous stenting, are at high risk of ischaemic events. These patients are generally treated with aspirin. In this trial, we aimed to investigate if these patients would benefit from treatment with aspirin plus ticagrelor. Methods: The Effect of Ticagrelor on Health Outcomes in diabEtes Mellitus patients Intervention Study (THEMIS) was a phase 3 randomised, double-blinded, placebo-controlled trial, done in 1315 sites in 42 countries. Patients were eligible if 50 years or older, with type 2 diabetes, receiving anti-hyperglycaemic drugs for at least 6 months, with stable coronary artery disease, and one of three other mutually non-exclusive criteria: a history of previous PCI or of coronary artery bypass grafting, or documentation of angiographic stenosis of 50% or more in at least one coronary artery. Eligible patients were randomly assigned (1:1) to either ticagrelor or placebo, by use of an interactive voice-response or web-response system. The THEMIS-PCI trial comprised a prespecified subgroup of patients with previous PCI. The primary efficacy outcome was a composite of cardiovascular death, myocardial infarction, or stroke (measured in the intention-to-treat population). Findings: Between Feb 17, 2014, and May 24, 2016, 11 154 patients (58% of the overall THEMIS trial) with a history of previous PCI were enrolled in the THEMIS-PCI trial. Median follow-up was 3·3 years (IQR 2·8–3·8). In the previous PCI group, fewer patients receiving ticagrelor had a primary efficacy outcome event than in the placebo group (404 [7·3%] of 5558 vs 480 [8·6%] of 5596; HR 0·85 [95% CI 0·74–0·97], p=0·013). The same effect was not observed in patients without PCI (p=0·76, p interaction=0·16). The proportion of patients with cardiovascular death was similar in both treatment groups (174 [3·1%] with ticagrelor vs 183 (3·3%) with placebo; HR 0·96 [95% CI 0·78–1·18], p=0·68), as well as all-cause death (282 [5·1%] vs 323 [5·8%]; 0·88 [0·75–1·03], p=0·11). TIMI major bleeding occurred in 111 (2·0%) of 5536 patients receiving ticagrelor and 62 (1·1%) of 5564 patients receiving placebo (HR 2·03 [95% CI 1·48–2·76], p<0·0001), and fatal bleeding in 6 (0·1%) of 5536 patients with ticagrelor and 6 (0·1%) of 5564 with placebo (1·13 [0·36–3·50], p=0·83). Intracranial haemorrhage occurred in 33 (0·6%) and 31 (0·6%) patients (1·21 [0·74–1·97], p=0·45). Ticagrelor improved net clinical benefit: 519/5558 (9·3%) versus 617/5596 (11·0%), HR=0·85, 95% CI 0·75–0·95, p=0·005, in contrast to patients without PCI where it did not, p interaction=0·012. Benefit was present irrespective of time from most recent PCI. Interpretation: In patients with diabetes, stable coronary artery disease, and previous PCI, ticagrelor added to aspirin reduced cardiovascular death, myocardial infarction, and stroke, although with increased major bleeding. In that large, easily identified population, ticagrelor provided a favourable net clinical benefit (more than in patients without history of PCI). This effect shows that long-term therapy with ticagrelor in addition to aspirin should be considered in patients with diabetes and a history of PCI who have tolerated antiplatelet therapy, have high ischaemic risk, and low bleeding risk