Inhibition Of ERK Signaling Research Articles

Identification of signaling pathways mediating human chondrocyte degeneration induced by the chemokine CCL-2

Purpose: Increased expression of chemokines in cartilage, synovial membrane and subchondral bone are believed to be linked to osteoarthritis (OA). The chemokine receptor CCR2 and its major human circulating ligand CCL2 (a.k.a. MCP-1), have been recognized as important potential targets in OA and have been shown also to mediate OA pain. In previous clinical studies, we determined that CCL2 serum levels at baseline were significantly associated with radiographic knee OA progression and joint space narrowing at 5-year follow up. Our findings in a murine model of OA based on the destabilization of the medial meniscus (DMM), showed that the articular cartilage of OA knees had increased protein levels of the chemokine CCL12, a murine CCL2 homologue that binds the same CCR2 receptor, while it was undetectable in controls; CCL12 protein levels increased progressively with OA severity. Interestingly, systemic blockage of the CCR2 at early OA stages, led to decreased levels of matrix metalloproteinases (MMPs) in articular cartilage, accompanied by reduced joint damage and pain. The aim of the present study was to analyze downstream mediators of CCL2/CCR2 signaling in human articular chondrocytes that lead to the activation of genes involved in cartilage degradation. Methods: We used human articular chondrocytes from normal cartilage obtained from tissue donors to determine the signaling pathways activated by CCL2 (through CCR2) that lead to production of MMPs. Human chondrocyte primary cultures were treated with or without recombinant CCL2 (20ng/ml) in serum free medium for 5, 10 and 15 minutes. Cell lysates were collected and subjected to Immunoblotting (IB) using antibodies against phospho-ERK, phospho-p38 and phospho-JNK. To determine whether CCL2 treatments led to changes in the expression of genes involved in cartilage degeneration, human articular chondrocytes with or without recombinant CCL2 were incubated in serum free medium for mRNA collection; two different incubations were performed, a short treatment (6h) and a long treatment (18-24h). Quantitative RT-PCR analyses were performed to quantify the gene expression of mmp-1, mmp-3, mmp-13, comp and timp-1 using TaqMan method. Gene expression for the ccr2 receptor was also assessed. In some experiments, human articular chondrocytes were pre-incubated with the ERK inhibitor U0126 (10 μM) for 1 hour prior to CCL2 treatment, and then IB (for phospho-Erk) or qRT-PCR analyses (for mmp-13 and mmp-3) were performed. Results: CCL2 treatments of human chondrocytes led to activation of phospho-ERK and phospho-p38, but not phospho-JNK. When gene expression was assessed by RT-PCR, both short (6h) and long (18-24h) CCL2 treatments led to an upregulation of ccr2 expression compared to untreated controls, indicating a CCL2 positive feedback on its receptor. Both short and long CCL2 treatments also led to an upregulation in the expression of mmp’s genes (mmp-1, mmp-3, and mmp-13), as well as an increased expression of the tissue inhibitor of metalloproteinases-1 (timp-1) and comp, which has been demonstrated to be a biomarker for cartilage breakdown. Inhibition of ERK signaling by U0126 abolished the CCL-2-induced upregulation of mmp-13, at both the short and long incubation times (Figure 1). Interestingly, inhibition of ERK signaling did not significantly affect mmp-3 upregulation by CCL-2. Conclusions: Our data suggest that CCL2 stimulation of articular cartilage chondrocytes acts as positive feedback on its CCR2 receptor leading to cartilage degradation. The triggering of the CCL2/CCR2 axis leads to the activation of specific MAPK signaling pathways, such as ERK and p38 signaling. In particular, our results suggest that ERK signaling mediates the CCL-2-induced mmp-13 activation but may not be involved, or only partially involved, in the mmp-3 upregulation. Additional studies are currently in progress in our laboratory to determine whether inhibition of ERK and p38 is correlated to the expression of different genes involved in cartilage metabolism and degradation (such as mmp-1, timp-1, comp), following short or long CCL-2 treatments.

Coordination between TGF-\u03b2 cellular signaling and epigenetic regulation during epithelial to mesenchymal transition

BackgroundEpithelial to mesenchymal transition (EMT) plays a crucial role in cancer propagation. It can be orchestrated by the activation of multiple signaling pathways, which have been found to be highly coordinated with many epigenetic regulators. Although the mechanism of EMT has been studied over decades, cross talk between signaling and epigenetic regulation is not fully understood.ResultsHere, we present a time-resolved multi-omics strategy, which featured the identification of the correlation between protein changes (proteome), signaling pathways (phosphoproteome) and chromatin modulation (histone modifications) dynamics during TGF-β-induced EMT. Our data revealed that Erk signaling was activated in 5-min stimulation and structural proteins involved in cytoskeleton rearrangement were regulated after 1-day treatment, constituting a detailed map of systematic changes. The comprehensive profiling of histone post-translational modifications identified H3K27me3 as the most significantly up-regulated mark. We thus speculated and confirmed that a combined inhibition of Erk signaling and Ezh2 (H3K27me3 methyltransferase) was more effective in blocking EMT progress than individual inhibitions.ConclusionsIn summary, our data provided a more detailed map of cross talk between signaling pathway and chromatin regulation comparing to previous EMT studies. Our findings point to a promising therapeutic strategy for EMT-related diseases by combining Erk inhibitor (singling pathway) and Ezh2 inhibitor (epigenetic regulation).

SHP2 Drives Adaptive Resistance to ERK Signaling Inhibition in Molecularly Defined Subsets of ERK-Dependent Tumors

SUMMARYPharmacologic targeting of components of ERK signaling in ERK-dependent tumors is often limited by adaptive resistance, frequently mediated by feedback-activation of RTK signaling and rebound of ERK activity. Here, we show that combinatorial pharmacologic targeting of ERK signaling and the SHP2 phosphatase prevents adaptive resistance in defined subsets of ERK-dependent tumors. In each tumor that was sensitive to combined treatment, p(Y542) SHP2 induction was observed in response to ERK signaling inhibition. The strategy was broadly effective in TNBC models and tumors with RAS mutations at G12, whereas tumors with RAS(G13D) or RAS(Q61X) mutations were resistant. In addition, we identified a subset of BRAF(V600E) tumors that were resistant to the combined treatment, in which FGFR was found to drive feedback-induced RAS activation, independently of SHP2. Thus, we identify molecular determinants of response to combined ERK signaling and SHP2 inhibition in ERK-dependent tumors.

RAF inhibitor PLX8394 selectively disrupts BRAF dimers and RAS-independent BRAF-mutant-driven signaling.

Activating BRAF mutants and fusions signal as RAS-independent constitutively active dimers with the exception of BRAF V600 mutant alleles which can function as active monomers1. Current RAF inhibitors are monomer selective, they potently inhibit BRAF V600 monomers but their inhibition of RAF dimers is limited by induction of negative cooperativity when bound to one site in the dimer1–3. Moreover, acquired resistance to these drugs is usually due to molecular lesions that cause V600 mutants to dimerize4–8. We show here that PLX8394, a new RAF inhibitor9, inhibits ERK signaling by specifically disrupting BRAF-containing dimers, including BRAF homodimers and BRAF-CRAF heterodimers, but not CRAF homodimers or ARAF-containing dimers. Differences in the amino acid residues in the N-terminal portion of the kinase domain of RAF isoforms are responsible for this differential vulnerability. As a BRAF-specific dimer breaker, PLX8394 selectively inhibits ERK signaling in tumors driven by dimeric BRAF mutants, including BRAF fusions and splice variants and as well BRAF V600 monomers, but spares RAF function in normal cells in which CRAF homodimers can drive signaling. Our work suggests that drugs with these properties will be safe and useful for treating tumors driven by activating BRAF mutants or fusions.

Epigenetic silencing of TMEM176A activates ERK signaling in human hepatocellular carcinoma

BackgroundThe role of TMEM176A in human hepatocellular carcinoma (HCC) is unknown. This study explored the epigenetic regulation and function of TMEM176A in human HCC.Materials and methodsTwelve HCC cell lines and 126 cases of primary cancer were analyzed. Methylation-specific PCR, immunohistochemistry, flow cytometry, and xenograft mouse models were employed.ResultsTMEM176A was highly expressed in SNU387, SNU182, Huh1, and SNU475 cells; reduced expression was observed in HepG2 and PLC/PRF/5 cells; and no expression was found in SNU449, HBXF344, SMMC7721, Huh7, and LM3 cells. Unmethylation of the TMEM176A promoter was detected in SNU387, SNU182, Huh1, and SNU475 cells; partial methylation was observed in HepG2 and PLC/PRF/5 cells; and complete methylation was found in SNU449, HBXF344, SMMC7721, Huh7, and LM3 cells. Upon treatment with 5-Aza-2-deoxycytidine, re-expression of TMEM176A was detected in SNU449, HBXF344, SMMC7721, Huh7, and LM3 cells; increased expression of TMEM176A was observed in HepG2 and PLC/PRF/5 cells; and no expression changes were found in SNU387, SNU182, Huh1, and SNU475 cells. The TMEM176A promoter region was methylated in 75.4% (95/126) of primary human HCC. Reduced expression of TMEM176A was associated with promoter region methylation (P < 0.05). No association was found between TMEM176A promoter methylation and age, gender, HBV infection, liver cirrhosis, tumor size, lymph node metastasis, vessel cancerous embolus, number of lesions, and TNM stage (all P > 0.05). These results demonstrated that the expression of TMEM176A is regulated by promoter region methylation. Methylation of the TMEM176A promoter was significantly associated with tumor cell differentiation (P < 0.05) and was an independent prognostic factor for poor 3-year overall survival (OS, P < 0.05). TMEM176A expression induced cell apoptosis; inhibited cell proliferation, migration, and invasion; suppressed human HCC cell xenograft growth in mice; and inhibited ERK signaling in HCC cells.ConclusionThe promoter region of TMEM176A is frequently methylated in human HCC, and the expression of TMEM176A is regulated by promoter region methylation. Methylation of the TMEM176A promoter may serve as a diagnostic and prognostic marker in HCC. TMEM176A suppresses HCC growth by inhibiting the ERK signaling pathway.

Balancing RAF, MEK, and EGFR inhibitor doses to achieve clinical responses and modulate toxicity in BRAF V600E colorectal cancer.

Recent years have seen dramatic clinical advances in targeting the ERK pathway with the FDA approval of several selective inhibitors of RAF and MEK1–5. Clinical trials of these agents indicate that near complete inhibition of pathway signaling is necessary to effectively inhibit tumor growth6. Targeted approaches have been most successful in BRAF V600E tumors because of the relatively wide therapeutic index of RAF inhibitors (vemurafenib and dabrafenib), which inhibit signaling in cells with BRAF V600 mutants but cause “paradoxical activation” of ERK signaling in normal cells that are wild-type for RAF kinases. In contrast, MEK inhibitors (trametinib and cobimetinib) suppress ERK signaling in all cells, and their clinical activity has been limited by a narrow therapeutic index. Combining these agents – with RAF and MEK inhibitors for BRAF V600E melanoma or lung cancer and with RAF, MEK, and EGFR inhibitors for BRAF V600E colorectal cancer – to profoundly inhibit ERK signaling has led to improved antitumor activity7. Combination therapy has also been shown to offset the toxicities caused by RAF inhibitors, such as the development of keratoacanthomas and squamous cell carcinoma, resulting from paradoxical ERK activation with these agents. The role of RAF inhibitors to offset MEK inhibitor toxicity and the need for dose intensity to modulate opposing toxicities is less clear. Recent observations in clinical trials have suggested that RAF inhibitors offset dermatologic toxicity from MEK or EGFR inhibitors. In the phase III trial of trametinib in melanoma, grade 3 or 4 acneiform dermatitis occurred in 8% of trametinib-treated patients, whereas in the phase III trial of the combination of dabrafenib and trametinib, no patient had grade 3 or 4 acneiform dermatitis8,9. Combinations of RAF and EGFR inhibitors have also had a lower incidence of acneiform rash than seen with EGFR inhibitors alone7,10,11. This is also likely due to the opposite effects of RAF and EGFR inhibitors on MEK activation in normal cells. However, the doses of RAF inhibitors needed for these clinically opposing effects and how these doses compare to clinically efficacious doses have not been studied. We now report the course of a patient with BRAF V600E CRC treated with dabrafenib, trametinib, and panitumumab in a phase II clinical trial, and within this patient characterize the effect on toxicities of different dose levels of these agents. Further, we find that, within the clinical dose range, there is a RAF inhibitor dose that is an inflection point for the toxicity and efficacy of this regimen.

BRAF Oncogenic Activation Induces Radioresistance Through Non-homologous End-Joining Repair Which is Abrogated by Targeted Inhibition of BRAF Mutational Activity

BRAF activating mutations, most commonly BRAFV600E/K, are driver mutations commonly found in solid tumors for which ionizing radiation (IR) is used, including thyroid, melanoma, colorectal, and lung cancer. Whether BRAFV600E mutation fosters radioresistance through nuclear DNA repair mechanisms is unclear. We hypothesize that BRAFV600E promotes radioresistance through heightened double-strand break repair mechanisms, which can be attenuated by targeted inhibition of the BRAF oncogene. We tested the effects of a BRAFV600E mutation using papillary and anaplastic thyroid cancer cell lines that possessed either wild-type or mutant BRAFV600E. We evaluated the role of BRAF and downstream targets in radiation clonogenic, neutral comet, nuclear foci, immunofluorescence, immunoblotting, RT-PCR, functional DNA repair reporter, and in vivo tumor growth assays. Radiation clonogenic assay showed BRAFV600E thyroid cancer cell lines were resistant to IR, and introduction of BRAFV600E into wild-type BRAF thyroid cancer TPC-1 cells led to significant IR resistance (dose enhancement factor, DEF 0.70). Selectively targeting BRAFV600E with a potent mutant BRAF inhibitor (vemurafenib, Vem) significantly sensitized BRAFV600E to IR, but not wild-type BRAF thyroid cancer cells (DEFs 1.37-1.78). Immunoblotting demonstrates that vemurafenib potently inhibited ERK signaling in BRAFV600E mutants but not BRAF wild-type cells. Vem delayed DNA repair in BRAFV600E cells after IR as evidenced by comet tail assays at 15 min, 1, 6, and 24 hrs (p<0.005) and γ-H2AX nuclear foci assays at 6 and 24 hrs (p<0.01). Moreover, Vem altered the kinetics of 53BP and RAD51 nuclear foci in BRAFV600E cells at 6 and 24 hrs after IR (p<0.05). Assessment of double-strand DNA repair using functional reporter assays revealed that BRAFV600E mutant was associated with enhanced non-homologous end-joining (NHEJ) repair activity, while Vem significantly reduced NHEJ repair activity by ∼40% (p<0.01). BRAFV600E mutation led to upregulation of XLF, a nuclear protein involved in NHEJ-mediated DNA repair, in thyroid cancer cell lines and in the publicly-available TCGA dataset (p<2.4e-14). Additionally, Vem treatment resulted in decreased XLF expression and genetic down-regulation of XLF sensitized BRAFV600E cells to IR (p<0.05). Importantly, Vem in combination with IR resulted in sustained tumor regression of BRAFV600E thyroid tumor xenografts, with minimal evidence of tumor regrowth after 60 days. BRAFV600E mutation promotes NHEJ activity leading to radioresistance, and Vem selectively radiosensitizes BRAFV600E thyroid cancer cells by inhibiting NHEJ repair. Our findings suggest that combining BRAF inhibition and IR may improve the therapeutic outcome for patients with BRAFV600E mutant thyroid cancer.

ERK Signaling Controls Innate-like CD8+ T Cell Differentiation via the ELK4 (SAP-1) and ELK1 Transcription Factors

In mouse thymocyte development, signaling by the TCR through the ERK pathway is required for positive selection of conventional naive T cells. The Ets transcription factor ELK4 (SAP-1), an ERK-regulated cofactor of the SRF transcription factor, plays an important role in positive selection by activating immediate-early genes such as the Egr transcription factor family. The role of ELK4–SRF signaling in development of other T cell types dependent on ERK signaling has been unclear. In this article, we show that ELK4, and its close relative ELK1, act cell autonomously in the thymus to control the generation of innate-like αβ CD8+ T cells with memory-like characteristics. Mice lacking ELK4 and ELK1 develop increased numbers of innate-like αβ CD8+ T cells, which populate the periphery. These cells develop cell autonomously rather than through expansion of PLZF+ thymocytes and concomitantly increased IL-4 signaling. Their development is associated with reduced TCR-mediated activation of ELK4–SRF target genes and can be partially suppressed by overexpression of the ELK4–SRF target gene EGR2. Consistent with this, partial inhibition of ERK signaling in peripheral CD8+T cells promotes the generation of cells with innate-like characteristics. These data establish that low-level ERK signaling through ELK4 (and ELK1) promotes innate-like αβ CD8+ T cell differentiation, tuning conventional versus innate-like development.

Aloin induces apoptosis via regulating the activation of MAPKs signaling pathway in human gastric cancer cells in vitro

To investigate the effect of aloin on apoptosis of human gastric cancer cells and explore the molecular mechanism. Gastric cancer MKN-28 and HGC-27 cells were cultured routinely in 1640 medium supplemented with 10% fetal bovine serum and 10% non-essential amino acids (for HGC-27 cells) and treated with different concentrations of aloin for different durations. The cell viability, cell nuclear morphology, and apoptotic rate of the cells were detected using CCK-8 assay, DAPI staining and AnnexinV-FITC/PI, respectively; Western blotting was used to detect the expression levels of PARP, procaspase 3 and the phosphorylation of p38, ERK and JNK. The cells were treated with specific inhibitors of p38, ERK and JNK, and the inhibitory effects on these pathways were detected with Western blotting; DAPI staining was used to detect the effects of inhibitors on apoptosis of gastric cancer cells. Aloin dose-dependently inhibited the viability and induced apoptosis of HGC-27 and MKN-28 cells. Alion treatment obvious enhanced the phosphorylation of p38 and JNK but decreased ERK phosphorylation in the cells. Blocking ERK activation with the ERK inhibitor obviously enhanced aloin-induced cell apoptosis, where inhibiting p38 and JNK activation partly reversed alion-induced apoptosis in the cells. Aloin induces apoptosis of human gastric cancer cells in vitro by activating p38 and JNK signaling pathways and inhibiting ERK signaling pathway.

Lin28a overexpression reveals the role of Erk signaling in articular cartilage development.

Adult articular cartilage shows limited tissue turnover, and therefore development of the proper structure of articular cartilage is crucial for life-long joint function. However, the mechanism by which the articular cartilage structure is developmentally regulated is poorly understood. In this study, we show evidence that activation of extracellular signal-regulated kinases (Erk1/2) in articular chondrocyte progenitors during developmental stages control articular cartilage thickness. We found that overexpression of Lin28a, an RNA-binding protein that regulates organismal growth and metabolism, in articular chondrocyte progenitor cells upregulated Erk signaling and increased articular cartilage thickness. Overexpression of a constitutively active Kras mimicked Lin28a overexpression, and inhibition of Erk signaling during embryonic stages normalized the cartilage phenotype of both Kras- and Lin28a-overexpressing mice. These results suggest that articular cartilage thickness is mainly determined during the process of embryonic synovial joint development, which is positively regulated by Erk signaling.

3078 - Jam3b Regulates Hemato-Vascular Development Through the Repression of the Erk Signaling Pathway

Hematopoietic and endothelial cells develop in close proximity to each other within the mesoderm. It has been proposed that these two cell types share a common precursor, termed the hemangioblast. The zebrafish cloche mutant, in which both hematopoietic and endothelial development are defective, has been identified as a unique model for the study of hemangioblast development. Recently, it was determined that the cloche gene encodes a PAS-domain-containing bHLH transcription factor, Npas4l, and plays a central role in the regulation of hemangioblast specification. Here, we show in zebrafish that Junctional adhesion molecule 3b (Jam3b) plays dual roles in hemato-vascular development during early embryogenesis: (1) promotes hemato-vascular differentiation via the up-regulation of npas4l, and (2) confers the hematopoietic identity in endothelial precursors through the regulation of Leucine rich-repeat containing 15 (Lrrc15). Mutation of jam3b (jam3bsa37) led to the down-regulation of npas4l in the posterior lateral plate mesoderm (PLPM) and the cloche-like phenotype, a defect of both hematopoietic and endothelial development. We found that jam3bsa37 embryos remained the high level of Erk activation in the PLPM. Inhibition of Erk signaling by chemical treatment in jam3bsa37 embryos rescued the expression of npas4l in the PLPM and led to the recovery of the vascular formation, but not hematopoietic stem cell (HSC) formation. Transcriptome analysis and loss-of-function studies revealed that a membrane protein gene, lrrc15, is essential for HSC specification and is largely reduced in endothelial precursors in jam3bsa37 embryos. Enforced expression of lrrc15 in combination with Erk inhibition in jam3bsa37 embryos rescued HSCs in the dorsal aorta. Collectively, our data reveal a novel molecular mechanism for HSC specification, in which Jam3b mediates the repression of the Erk signaling pathway to promote npas4l expression followed by the up-regulation of lrrc15 in endothelial precursors.

Dissecting RAF Inhibitor Resistance by Structure-based Modeling Reveals Ways to Overcome Oncogenic RAS Signaling

Clinically used RAF inhibitors are ineffective in RAS mutant tumors because they enhance homo- and heterodimerization of RAF kinases, leading to paradoxical activation of ERK signaling. Overcoming enhanced RAF dimerization and the resulting resistance is a challenge for drug design. Combining multiple inhibitors could be more effective, but it is unclear how the best combinations can be chosen. We built a next-generation mechanistic dynamic model to analyze combinations of structurally different RAF inhibitors, which can efficiently suppress MEK/ERK signaling. This rule-based model of the RAS/ERK pathway integrates thermodynamics and kinetics of drug-protein interactions, structural elements, posttranslational modifications, and cell mutational status as model rules to predict RAF inhibitor combinations for inhibiting ERK activity in oncogenic RAS and/or BRAFV600E backgrounds. Predicted synergistic inhibition of ERK signaling was corroborated by experiments in mutant NRAS, HRAS, and BRAFV600E cells, and inhibition of oncogenic RAS signaling was associated with reduced cell proliferation and colony formation.

Role of ERK in IRF3-Mediated Immune Responses

Insulin resistance precedes and contributes to the development of type 2 diabetes mellitus, and it is now believed that chronic inflammation is a major contributor to insulin resistance. Both cellular and secreted factors participate in the pathological and physiological changes that occur to promote inflammation. However, the molecular signaling pathways that drive these processes remain elusive. The mitogen activated protein kinase, extracellular signal-regulated kinase 1 and 2 module (ERK1/2), and the transcription factor, interferon regulatory factor-3 (IRF3), are both activated downstream of Toll-like receptor 4 and associated with the development of insulin resistance. We examined whether inhibition of ERK activity blocked IRF3-mediated immune responses. ERK1 and ERK2 isoforms can have different cellular functions; thus, to determine which ERK isoform is involved in the regulation of IRF3 activity, we performed siRNA to knockdown either ERK1, ERK2, or both in RAW 264.7 macrophages and saw 70% knockdown of each ERK1 and ERK2. Knockdown of ERK1 or ERK2 or both blocked IRF3 translocation to the nucleus with LPS treatment (100 ng/ml, 1 hour), as determined by immunofluorescence using an antibody specific for nuclear IRF3. Because phosphorylated and dimerized IRF3 translocates to the nucleus to regulate the transcription of interferon (IFN)-beta expression, we investigated whether ERK1 or ERK2 inhibited IFN-beta release. Macrophages were treated with LPS (100 ng/ml, 6 hour). LPS induced an IFN-beta production of 791±18.0 pg/ml, but knockdown of either ERK1 or ERK2 decreased the release of IFN-beta to 199±12.3 and 282±32.7 pg/ml, respectively. However, double knockdown of ERK1 and ERK 2 had the greatest inhibition of IFN-beta release (121±15.2 pg/ml). In summary, both ERK1 and ERK2 regulate IRF3 nuclear translocation and signaling in macrophages. We propose that ERK positively regulates IRF3-mediated immune responses and inhibition of ERK signaling will protect against insulin resistance. Disclosure P.F. Chang: None. D. Acevedo: None. S.M. Reyna: None.

Pharmacological Inhibition of ERK Signaling Rescues Pathophysiology and Behavioral Phenotype Associated with 16p11.2 Chromosomal Deletion in Mice.

The human 16p11.2 microdeletion is one of the most common gene copy number variations linked to autism, but the pathophysiology associated with this chromosomal abnormality is largely unknown. The 593 kb deletion contains the ERK1 gene and other genes that converge onto the ERK/MAP kinase pathway. Perturbations in ERK signaling are linked to a group of related neurodevelopmental disorders hallmarked by intellectual disability, including autism. We report that mice harboring the 16p11.2 deletion exhibit a paradoxical elevation of ERK activity, cortical cytoarchitecture abnormalities and behavioral deficits. Importantly, we show that treatment with a novel ERK pathway inhibitor during a critical period of brain development rescues the molecular, anatomical and behavioral deficits in the 16p11.2 deletion mice. The ERK inhibitor treatment administered to adult mice ameliorates a subset of these behavioral deficits. Our findings provide evidence for potential targeted therapeutic intervention in 16p11.2 deletion carriers.SIGNIFICANCE STATEMENT The ERK/MAPK pathway is genetically linked to autism spectrum disorders and other syndromes typified by intellectual disability. We provide direct evidence connecting the ERK/MAP kinases to the developmental abnormalities in neurogenesis and cortical cytoarchitecture associated with the 16p11.2 chromosomal deletion. Most importantly, we demonstrate that treatment with a novel ERK-specific inhibitor during development rescues aberrant cortical cytoarchitecture and restores normal levels of cell-cycle regulators during cortical neurogenesis. These treatments partially reverse the behavioral deficits observed in the 16p11.2del mouse model, including hyperactivity, memory as well as olfaction, and maternal behavior. We also report a rescue of a subset of these deficits upon treatment of adult 16p11.2del mice. These data provide a strong rationale for therapeutic approaches to this disorder.

Overexpression of DNMT1 leads to hypermethylation of H19 promoter and inhibition of Erk signaling pathway in disuse osteoporosis.

Disuse osteoporosis (DOP) is a common complication of the lack of mechanical loading. The precise mechanism underlying DOP remains unknown, although epigenetic modifications may be a major cause. Recently, cumulative research has revealed that DNA methyltransferase (DNMT) proteins can catalyze the conversion of cytosine to 5-methylcytosine (5mC), altering the epigenetic state of DNA. Here, we report that DNMT1 expression and lncRNA-H19 methylation are upregulated in the femoral tissues of DOP rats, accompanied with inhibited Erk signaling pathway. Overexpression of DNMT1 in UMR-106 cells mimics 5mC enrichment in the H19 promoter, inhibition of Erk signaling and impairment of osteogenesis, which can be rescued by 5'-aza-deoxycytidine (5'-Aza) treatment. Moreover, local intramedullary injection of Dnmt1 siRNA (siDNMT1) in Sprague-Dawley (SD) rats abrogated disuse lncRNA-H19 (H19) downregulation, Erk signaling inhibition, histopathological changes, and bone microstructure declines in the distal femur in vivo. Therefore, our data identify for the first time a new signaling cascade in DOP: mechanical unloading causes upregulation of DNMT1 and hypermethylation of H19 promoter, which subsequently leads to downregulation of lncRNA-H19 and inhibition of the ERK signaling, suggesting a new potential therapeutic target.

17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model.

Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy alone. Our studies support a role for 17-AAG and HSP90 inhibition in enhancing cellular immunotherapy for melanoma.

Cholic Acid Attenuates ER Stress-Induced Cell Death in Coxsackievirus-B3 Infection.

Coxsackievirus Type B3 (CVB3) is an enterovirus that belongs to the Picornaviridae and causes various diseases such as myocarditis and hand-foot-mouth disease. However, an effective antiviral drug is still not developed. In this study, we looked for potential inhibitors of CVB3 replication by examining the survival of CVB3-infected HeLa cells. We detected an antiviral effect by cholic acid and identified it as a candidate inhibitor of CVB3 replication. Cholic acid circulates in the liver and intestines, and it helps the digestion and absorption of lipids in the small intestine. HeLa cells were cultured in 12-well plates and treated with cholic acid (1 and 10 μg/ml) and 10⁶ PFU/ml of CVB3. After 16 h post-infection, the cells were lysed and subjected to western blot analysis and RT-PCR. The production of the viral capsid protein VP1 was dramatically decreased, and translation initiation factor eIF4G1 cleavage was significantly inhibited by treatment with 10 μg/ml cholic acid. Moreover, cholic acid inhibited ERK signaling in CVB3-infected HeLa cells. RT-PCR showed that the amounts of the CVB3 RNA genome and mRNA for the ER stress-related transcription factor ATF4 were significantly reduced. These results showed that cholic acid strongly reduced ER stress and CVB3 proliferation. This compound can be developed as a safe natural therapeutic agent for enterovirus infections.

The MEK1/2 Inhibitor AZD6244 Sensitizes BRAF-Mutant Thyroid Cancer to Vemurafenib.

BackgroundBRAFV600E mutation occurs in approximately 45% of papillary thyroid cancer (PTC) cases, and 25% of anaplastic thyroid cancer (ATC) cases. Vemurafenib/PLX4032, a selective BRAF inhibitor, suppresses extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) signaling and shows beneficial effects in patients with metastatic melanoma harboring the BRAFV600E mutation. However, the response to vemurafenib is limited in BRAF-mutant thyroid cancer. The present study evaluated the effect of vemurafenib in combination with the selective MEK1/2 inhibitor AZD6244 on cell survival and explored the mechanism underlying the combined effect of vemurafenib and AZD6244 on thyroid cancer cells harboring BRAFV600E.Material/MethodsThyroid cancer 8505C and BCPAP cells harboring the BRAFV600E mutation were exposed to vemurafenib (0.01, 0.1, and 1 μM) and AZD6244 (0.01, 0.1, and 1 μM) alone or in the indicated combinations for the indicated times. Cell viability was detected by the MTT assay. Cell cycle distribution and induction of apoptosis were detected by flow cytometry. The expression of cyclin D1, P27, (P)-ERK1/2 was evaluated by Western blotting. The effect of vemurafenib or AZD6244 or their combination on the growth of 8505C cells was examined in orthotopic xenograft mouse models in vivo.ResultsVemurafenib alone did not increase cell apoptosis, whereas it decreased cell viability by promoting cell cycle arrest in BCPAP and 8505C cells. AZD6244 alone increased cell apoptosis by inducing cell cycle arrest in BCPAP and 8505C cells. Combination treatment with AZD6244 and vemurafenib significantly decreased cell viability and increased apoptosis in both BCPAP and 8505C cells compared with the effects of each drug alone. AZD6244 alone abolished phospho-ERK1/2 (pERK1/2) expression at 48 h, whereas vemurafenib alone downregulated pERK1/2 at 4–6 h, with rapid recovery of expression, reaching the highest level at 24–48 h. Combined treatment for 48 h completely inhibited pERK1/2 expression. Combination treatment with vemurafenib and AZD6244 inhibited cell growth and induced apoptosis by causing cell-cycle arrest, with the corresponding changes in the expression of the cell cycle regulators p27Kip1 and cyclin D1. Co-administration of vemurafenib and AZD6244 in vivo had a significant synergistic antitumor effect in a nude mouse model.ConclusionsVemurafenib activated pERK1/2 and induced vemurafenib resistance in thyroid cancer cells. Combination treatment with vemurafenib and AZD6244 inhibited ERK signaling and caused cell cycle arrest, resulting in cell growth inhibition. Combination treatment in patients with thyroid cancer harboring the BRAFV600E mutation may overcome vemurafenib resistance and enhance the therapeutic effect.

Taurocholate Induces Connective Tissue Growth Factor Expression in Hepatocytes Through ERK-YAP Signaling

Background/Aims: Cholestasis is characterized by intrahepatic accumulation of cytotoxic bile acids (BAs), ultimately leading to fibrosis and cirrhosis, but the precise role of BAs in cholestasis-induced liver fibrosis remains largely elusive. In this study, we investigated the role and the potential mechanisms of BAs during cholestasis in vivo and in vitro. Methods: The effect of BAs during cholestasis was studied in bile duct ligation (BDL) rat models in vivo. We performed immunohistochemistry, Western blotting, and quantitative RT-PCR to investigate the expression of connective tissue growth factor (CTGF/CCN2) in rat liver during cholestasis. The hepatic cell lines AML12 and BRL were stimulated with taurocholate (TC) and the level of CTGF/CCN2, and activation of ERK, Akt, p38 MAPK, JNK, YAP, and TGF-β/Smad signaling were examined using Western blotting. Next, to elucidate the mechanism underlying bile acid-induced CTGF/CCN2, we treated the cells with MEK1/2 inhibitor (U0126), YAP function inhibitor (verteporfin), p38 kinase inhibitor (SB203580), Akt inhibitor (MK2206), and small interfering RNA (siRNA) targeting mek1, erk, and yap in cooperation with TC. Besides, we confirmed the activation of these signaling pathways in BDL and sham rat livers by immunohistochemistry, Western blotting, and quantitative RT-PCR. Results: In this study, we confirmed that the expression of CTGF/CCN2 was increased in BDL-induced rodent cholestatic liver fibrosis. In addition, we showed that TC, the main component of BAs, enhanced the synthesis of CTGF/ CCN2 in AML12 and BRL hepatic cell lines. Moreover, we demonstrated that TC activated ERK, Akt, and YAP signaling in hepatocytes, but the precise roles of these signaling cascades in the expression of CTGF/CCN2 were different: TC-induced expression of CTGF/CCN2 was mediated by ERK-YAP signaling, whereas Akt signaling inhibited ERK signaling and YAP and subsequently the expression of CTGF/CCN2 in hepatocytes. Furthermore, YAP functioned as a downstream regulator of ERK signaling in TC-induced CTGF/CCN2 expression in hepatocytes. Conclusion: Our report provides evidence for the role of conjugated BAs in liver fibrosis and suggests that the production of CTGF/CCN2, induced by conjugated BAs via ERK-YAP axis activation, may be a therapeutic target in cholestasis-induced liver fibrosis.

Abstract LB-A35: Discovery of a new kinase inhibitor targeting BRAF monomers and dimers

Abstract Increased activation of the RAS/RAF/MEK/ERK signaling pathway, through mutations in upstream activators or in components of the pathway, can lead to uncontrolled cellular growth and proliferation leading to tumor development. BRAF mutations are present in more than 50% of melanoma patients as well as in colorectal (5-10%), thyroid carcinomas (25-45%), hairy-cell leukemia (~100%) and less commonly in ovarian and lung malignancies. Specifically, activating BRAF mutation V600E in the kinase domain of BRAF accounts for almost 50% of melanoma. Although, FDA-approved RAF inhibitors vemurafenib and dabrafenib have elicited remarkable responses and improved survival of melanoma patients with BRAFV600E/K mutations, acquired resistance limits their effectiveness as patients relapse. Several studies have demonstrated that resistance in melanoma and other tumors involve increased formation of RAF dimers that are not effectively inhibited by clinical inhibitors. Hence, underscoring the need for improved RAF inhibitors. Toward this goal, we developed an in-cell-western assay to screen for small molecule kinase inhibitors of the ERK signaling pathway, using melanoma cells that express BRAFV600E monomers and dimers. Because several inhibitors have been identified to inhibit ERK signaling that led us to investigate the precise mechanism of inhibition. We performed structural characterization and biochemical analysis of the RAS/RAF/MEK/ERK signaling pathway in cells with different RAS/BRAF mutational background, expressing BRAF monomers and dimers. Our study enabled us to identify a kinase inhibitor that potently inhibits BRAF monomers and dimers, with distinct structural properties compared to current RAF inhibitors. Further development of this new class of RAF kinase inhibitor may lead to more effective ERK signaling inhibition and improved treatments for BRAF-dependent tumors. Citation Format: Xiomaris M. Cotto Rios, Paul Agianian, Yang Wu, Evripidis Gavathiotis. Discovery of a new kinase inhibitor targeting BRAF monomers and dimers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A35.