Hearing loss is one of the most common types of disability; however, there is only one FDA-approved drug to prevent any type of hearing loss. Treatment with the highly effective chemotherapy agent, cisplatin, and exposure to high-decibel noises are two of the most common causes of hearing loss. The mitogen-activated protein kinase (MAPK) pathway, a phosphorylation cascade consisting of RAF, MEK1/2, and ERK1/2, has been implicated in both types of hearing loss. Pharmacologically inhibiting BRAF or ERK1/2 is protective against noise- and cisplatin-induced hearing loss in multiple mouse models. Trametinib, a MEK1/2 inhibitor, protects from cisplatin-induced outer hair cell death in mouse cochlear explants; however, to the best of our knowledge, inhibiting MEK1/2 has not yet been shown to be protective against hearing loss in vivo. In this study, we demonstrate that trametinib protects against cisplatin-induced hearing loss in a translationally relevant mouse model and does not interfere with cisplatin\'s tumor-killing efficacy in cancer cell lines. Higher doses of trametinib were toxic to mice when combined with cisplatin, but lower doses of the drug were protective against hearing loss without any known toxicity. Trametinib also protected mice from noise-induced hearing loss and synaptic damage. This study shows that MEK1/2 inhibition protects against both insults of hearing loss, as well as that targeting all three kinases in the MAPK pathway protects mice from cisplatin- and noise-induced hearing loss.

Hearing loss is one of the most common types of disability; however, there is only one FDA-approved drug to prevent any type of hearing loss. Treatment with the highly effective chemotherapy agent, cisplatin, and exposure to high decibel noises are two of the most common causes of hearing loss. The mitogen activated protein kinase (MAPK) pathway, a phosphorylation cascade consisting of RAF, MEK1/2, and ERK1/2, has been implicated in both types of hearing loss. Pharmacologically inhibiting BRAF or ERK1/2 is protective from noise and cisplatin-induced hearing loss in multiple mouse models. Trametinib, a MEK1/2 inhibitor, protects from cisplatin induced outer hair cell death in mouse cochlear explants; however, to the best of our knowledge, inhibiting MEK1/2 has not yet been shown to be protective from hearing loss in vivo. In this study, we demonstrate that trametinib protects from cisplatin-induced hearing loss in a translationally relevant mouse model and does not interfere with cisplatin\'s tumor killing efficacy in cancer cell lines. Higher doses of trametinib were toxic to mice when combined with cisplatin but lower doses of the drug were protective from hearing loss without any known toxicity. Trametinib also protected mice from noise-induced hearing loss and synaptic damage. This study shows that MEK1/2 inhibition protects from both insults of hearing loss and that targeting all three kinases in the MAPK pathway protect from cisplatin and noise-induced hearing loss in mice.

Preclinical studies rarely test the efficacy of therapies in both sexes. The field of oncology is no exception in this regard. In a model of syngeneic, orthotopic, metastasized pancreatic ductal adenocarcinoma we evaluated the impact of sex on pathological features of this disease as well as on the efficacy and possible adverse side effects of a novel, small molecule-based therapy inhibiting KRAS:SOS1, MEK1/2 and PI3K signaling in male and female C57BL/6J mice. Male mice had less tumor infiltration of CD8-positive cells, developed bigger tumors, had more lung metastasis and a lower probability of survival compared to female mice. These more severe pathological features in male animals were accompanied by higher distress at the end of the experiment. The evaluated inhibitors BI-3406, trametinib and BKM120 showed synergistic effects in vitro. This combinatorial therapy reduced tumor weight more efficiently in male animals, although the drug concentrations were similar in the tumors of both sexes. These results underline the importance of sex-specific preclinical research and at the same time provide a solid basis for future studies with the tested compounds.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the leading causes of digestive system tumor related death in the world. Unfortunately, effective chemopreventive agent is lack for patients with ESCC in clinical practice, which leads to the extremely high mortality rate.
METHODS: A library of prescribed drugs was screened for finding critical anti-tumor properties in ESCC cells. The phosphoproteomics, kinase array, pulldown assay and drug affinity responsive target stabilization assay (DARTS) were applied to explore mechanisms and searched for synergistic targets. Established models of PDX in mice were used to determine the therapeutic effect of domperidone.
RESULTS: After screening a library of prescribed drugs, we discovered that domperidone has anti-tumor properties. Domperidone, acting as a gastroprokinetic agent, has been widely used in clinic for gastrointestinal motility disorders. Despite limited research, there are indications that domperidone may have anti-tumor properties. In this study, we determined that domperidone significantly inhibited ESCC proliferation in vitro and in vivo. We employed phosphoproteomics to reveal p-ERK, and p-SMAD3 down-regulation upon domperidone treatment. Then, the results of kinase assay and pulldown assay further validated that domperidone directly combined with MEK1/2 and CDK4, leading to the inhibition of their kinase activity. Furthermore, our results revealed that MEK/ERK and CDK4/SMAD3 signal pathway were major pathways in domperidone against ESCC.
CONCLUSIONS: Collectively, these findings suggest that domperidone serves as an effective \"multi-target\" inhibitor of MEK1/2 and CDK4, offering potential benefits for the chemoprevention of ESCC.

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging \'hits\' from drug discovery screens.

Disrupted germline differentiation or compromised testis development can lead to subfertility or infertility and are strongly associated with testis cancer in humans. In mice, SRY and SOX9 induce expression of Fgf9, which promotes Sertoli cell differentiation and testis development. FGF9 is also thought to promote male germline differentiation but the mechanism is unknown. FGFs typically signal through mitogen-activated protein kinases (MAPKs) to phosphorylate ERK1/2 (pERK1/2). We explored whether FGF9 regulates male germline development through MAPK by inhibiting either FGF or MEK1/2 signalling in the foetal testis immediately after gonadal sex determination and testis cord formation, but prior to male germline commitment.
pERK1/2 was detected in Sertoli cells and inhibition of MEK1/2 reduced Sertoli cell proliferation and organisation and resulted in some germ cells localised outside of the testis cords. While pERK1/2 was not detected in germ cells, inhibition of MEK1/2 after somatic sex determination profoundly disrupted germ cell mitotic arrest, dysregulated a broad range of male germline development genes and prevented the upregulation of key male germline markers, DPPA4 and DNMT3L. In contrast, while FGF inhibition reduced Sertoli cell proliferation, expression of male germline markers was unaffected and germ cells entered mitotic arrest normally. While male germline differentiation was not disrupted by FGF inhibition, a range of stem cell and cancer-associated genes were commonly altered after 24 h of FGF or MEK1/2 inhibition, including genes involved in the maintenance of germline stem cells, Nodal signalling, proliferation, and germline cancer.
Together, these data demonstrate a novel role for MEK1/2 signalling during testis development that is essential for male germline differentiation, but indicate a more limited role for FGF signalling. Our data indicate that additional ligands are likely to act through MEK1/2 to promote male germline differentiation and highlight a need for further mechanistic understanding of male germline development.

Macrophages undergo different cellular states upon activation that can be hyporesponsive (tolerated) or hyperresponsive (primed or trained) to subsequent stimuli. Epigenetic modifications are known to play key roles in determining these cellular states. However, little is known about the role of signaling pathways that lead to these epigenetic modifications. Here, we examined the effects of various inhibitors targeting key signaling pathways induced by lipopolysaccharide (LPS) on tolerance and priming in murine macrophages. We found that a prolonged inhibition (>18 h) of the mitogen-activated protein kinase (MEK)1/2-extracellular signal-regulated kinase (ERK)1/2 signaling axis reversed tolerance and primed cells in expressing interleukin (IL)-1β and other inflammatory cytokines such as IL-6, tumor necrosis factor (TNF)α, and CXCL10. The ectopic expression of catalytically active and inactive MEK1 mutants suppressed and enhanced IL-1β expression, respectively. A transcriptomic analysis showed that cells primed by the MEK1/2 inhibitor U0126 expressed higher levels of gene sets associated with immune responses and cytokine/chemokine production, but expressed lower levels of genes with cell cycle progression, chromosome organization, and heterochromatin formation than non-primed cells. Of interest, the mRNA expressions of the histone 3 lysine 9 (H3K9) methyltransferase Suv39h1 and the H3K9 methylation reader Cbx5 were substantially suppressed, whereas the H3K9 demethylase Kdm7a was enhanced, suggesting a role of the MEK1/2-ERK signaling axis in H3K9 demethylation. The H3K9 trimethylation levels in the genomic regions of IL-1β, TNFα, and CXCL10 were decreased by U0126. Also, the H3K9 methyltransferase inhibitor BIX01294 mimicked the U0126 training effects and the overexpression of chromobox homolog (CBX)5 prevented the U0126 training effects in both RAW264.7 cells and bone-marrow-derived macrophages. Collectively, these data suggest that the prolonged inhibition of the MEK1/2-ERK signaling axis reverses tolerance and primed macrophages likely through decreasing the H3K9 methylation levels.

Glioblastoma (GBM) remains an incurable disease with an extremely high five-year recurrence rate. We studied apoptosis in glioma stem cells (GSCs) in response to HDAC inhibition (HDACi) combined with MEK1/2 inhibition (MEKi) or BCL-2 family inhibitors. MEKi effectively combined with HDACi to suppress growth, induce cell cycle defects, and apoptosis, as well as to rescue the expression of the pro-apoptotic BH3-only proteins BIM and BMF. A RNAseq analysis of GSCs revealed that HDACi repressed the pro-survival BCL-2 family genes MCL1 and BCL-XL. We therefore replaced MEKi with BCL-2 family inhibitors and observed enhanced apoptosis. Conversely, a ligand for the cancer stem cell receptor CD44 led to reductions in BMF, BIM, and apoptosis. Our data strongly support further testing of HDACi in combination with MEKi or BCL-2 family inhibitors in glioma.

Stress triggers relapses in cocaine use that engage the activity of memory-related nuclei, such as the basolateral amygdala (BLA) and dentate gyrus (DG). Preclinical research suggests that D3 receptor (D3R) antagonists may be a promising means to attenuate cocaine reward and relapse. As D3R regulates the activity of the Akt/mTOR and MEK/ERK1/2 pathways, we assessed the effects of SB-277011-A, a D3R antagonist, on the activity of these kinases during the reinstatement of cocaine-induced conditioned place preference (CPP) induced by psychological (restraint) and physiological (tail pinch) stress. Both stimuli reactivated an extinguished cocaine-CPP, but only restrained animals decreased their locomotor activity during reinstatement. Cocaine-seeking behavior reactivation was correlated with decreased p-Akt, p-mTOR, and p-ERK1/2 activation in both nuclei of restrained animals. While a D3R blockade prevented stress-induced CPP reinstatement and plasma corticosterone enhancement, SB-277011-A distinctly modulated Akt, mTOR, and ERK1/2 activation depending on the stressor and the dose used. Our data support the involvement of corticosterone in the SB-277011-A effects in restrained animals. Additionally, the ratios p-mTOR/mTOR and/or p-ERK1/2 /ERK1/2 in the BLA during stress-induced relapse seem to be related to the locomotor activity of animals receiving 48 mg/kg of the antagonist. Hence, our study indicates the D3R antagonist\'s efficacy to prevent stress-induced relapses in drug use through distinct modulation of Akt/mTOR and MEK/ERK1/2 pathways in memory-processing nuclei.

Homo sapiens chromosome 2 clone RP11-339H12 (AC010883.5) is a dysregulated long non-coding RNA (lncRNA) that has never been investigated in cervical cancer (CC). Thus, the potential function and molecular mechanism remain unclear. Our study explored the biological function of AC010883.5 to determine the underlying mechanisms in CC and provide potential therapeutic targets for improving the clinical treatment strategy. We used quantitative real-time polymerase chain reaction to measure mitochondrial RNA levels and western blot to measure the protein levels of target genes. Further, we used Cell Counting Kit-8 and 5-Bromo-2\'-deoxyuridine incorporation assays to evaluate cell proliferation in vitro. Cell apoptosis was analyzed by flow cytometry. Cell invasion was analyzed by wound healing and Transwell migration assays was ued to analyze cell migration. Finally, the biological function and mechanism of AC010883.5 in CC growth were evaluated by in vivo xenograft assay. AC010883.5 was enhanced in CC tissues and cell lines, and enhanced AC010883.5 expression accelerated CC cell proliferation, migration, and invasion and induced epithelial-mesenchymal transition in vitro and in vivo. AC010883.5 also activated the mitogen-activated protein kinase (MAPK) signaling pathway by promoting phosphorylation of extracellular signal-regulated kinase 1/2 (i.e., ERK1/2) and MAPK kinase 1/2 (i.e., MEK1/2). Blocking the MAPK signaling pathway could counteract the pro-proliferative, pro-migrative, and pro-invasive effects of AC010883.5 over-expression. We found that the lncRNA, AC010883.5, is an oncogenic molecule involved in CC tumor progression via dysregulation of the MAPK signaling pathway, implying that AC010883.5 could be a tumor progression and therapeutic response biomarker.