Inhibition Of Mitogen-activated Protein Kinase Signaling Research Articles

Ovatodiolide and antrocin synergistically inhibit the stemness and metastatic potential of hepatocellular carcinoma via impairing ribosome biogenesis and modulating ERK/Akt-mTOR signaling axis

Activation of mitogen-activated protein kinase (MAPK) and PI3K signaling confers resistance against sorafenib, a mainstay treatment for advanced hepatocellular carcinoma (HCC). Antrocin and ovatodiolide constitute as the most potent secondary metabolites isolated from Antrodia camphorata and Anisomeles indica, respectively. Both natural compounds have recently gained a lot of attention due to their putative inhibition of MAPK and PI3K signaling in various solid cancers. However, whether their combination is effective in HCC remains unknown. Here, we investigated their effect, alone or in various combinations, on MAPK and PI3K signaling pathways in HCC cells. An array of in vitro study were used to investigate anticancer and stemness effects to treat HCC, such as cytotoxicity, drug combination index, migration, invasion, colony formation, and tumor sphere formation. Drug effect in vivo was evaluated using mouse xenograft models. In this study, antrocin and ovatodiolide synergistically inhibited the SNU387, Hep3B, Mahlavu, and Huh7 cell lines. Sequential combination treatment of Huh7 and Mahlavu with ovatodiolide followed by antrocin resulted stronger cytotoxic effect than did treatment with antrocin followed by ovatodiolide, their simultaneous administration, antrocin alone, or ovatodiolide alone. In the Huh7 and Mahlavu cell lines, ovatodiolide→antrocin significantly suppressed colony formation and proliferation as well as markedly downregulated ERK1/2, Akt, and mTOR expression. Inhibition of ERK1/2 and Akt/mTOR signaling by ovatodiolide→antrocin suppressed ribosomal biogenesis, autophagy, and cancer stem cell-like phenotypes and promoted apoptosis in Huh7 and Mahlavu cells. The sorafenib-resistant clone of Huh7 was effectively inhibited by synergistic combination of both compound in vitro. Eventually, the ovatodiolide→antrocin combination synergistically suppressed the growth of HCC xenografts. Taken together, our findings suggested that ovatodiolide→antrocin combination may represent potential therapeutic approach for patients with advanced HCC.

Eupalinolide B attenuates lipopolysaccharide-induced acute lung injury through inhibition of NF-κB and MAPKs signaling by targeting TAK1 protein

Acute lung injury (ALI) is a life-threatening disease characterized by severe inflammatory response, which has no pharmacological therapy in clinic. In this study, we found that eupalinolide B (EB), a sesquiterpene lactone isolated from Eupatorium lindleyanum, significantly ameliorated lipopolysaccharide (LPS)-induced ALI in mice, which manifests as reduction in lung injury score, activity of myeloperoxidase, and release of cytokines interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein-1 (MCP-1). In RAW264.7 murine macrophages, EB effectively inhibited LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) by down-regulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2), respectively. Mechanistically, EB not only blocked LPS-induced phosphorylation of inhibitor of nuclear factor kappa B kinase-α/β (IKKα/β), phosphorylation and degradation of inhibitor of nuclear factor‐kappa B alpha (IκBα), and phosphorylation and nuclear translocation of nuclear factor‐kappa B (NF-κB) P65, but also suppressed LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) in vitro or in vivo. Through cellular thermal shift assay and western blotting, EB was demonstrated to target and inactivate transforming growth factor β activated kinase-1 (TAK1), which is an important upstream kinase for the activation of NF-κB and MAPKs pathways. Additionally, EB-mediated actions were markedly abolished by dithiothreitol in LPS-exposed RAW264.7 cells, suggesting a crucial role of the α,γ-unsaturated lactone for the anti-inflammatory activity of EB. In conclusion, our findings showed that EB could effectively alleviate ALI in mice, and attenuate inflammatory response by inhibiting the activation of TAK1, and TAK1-mediated activation of NF-κB and MAPKs cascades.

Mangiferin Alleviates Postpartum Depression-Like Behaviors by Inhibiting MAPK Signaling in Microglia.

Postpartum depression (PPD), a severe mental health disorder, is closely associated with decreased gonadal hormone levels during the postpartum period. Mangiferin (MGF) possesses a wide range of pharmacological activities, including anti-inflammation. Growing evidence has suggested that neuroinflammation is involved in the development of depression. However, the role of MGF in the development of PPD is largely unknown. In the present study, by establishing a hormone-simulated pregnancy PPD mouse model, we found that the administration of MGF significantly alleviated PPD-like behaviors. Mechanistically, MGF treatment inhibited microglial activation and neuroinflammation. Moreover, we found that MGF treatment inhibited mitogen-activated protein kinase (MAPK) signaling in vivo and in vitro. Together, these results highlight an important role of MGF in microglial activation and thus give insights into the potential therapeutic strategy for PPD treatment.

α-Bisabolol Mitigates Colon Inflammation by Stimulating Colon PPAR-γ Transcription Factor: In Vivo and In Vitro Study.

The incidence and prevalence of inflammatory bowel disease (IBD, Crohn's disease, and ulcerative colitis) are increasing worldwide. The etiology of IBD is multifactorial, including genetic predisposition, dysregulated immune response, microbial dysbiosis, and environmental factors. However, many of the existing therapies are associated with marked side effects. Therefore, the development of new drugs for IBD treatment is an important area of investigation. Here, we investigated the anti-inflammatory effects of α-bisabolol, a naturally occurring monocyclic sesquiterpene alcohol present in many aromatic plants, in colonic inflammation. To address this, we used molecular docking and dynamic studies to understand how α-bisabolol interacts with PPAR-γ, which is highly expressed in the colonic epithelium: in vivo (mice) and in vitro (RAW264.7 macrophages and HT-29 colonic adenocarcinoma cells) models. The molecular docking and dynamic analysis revealed that α-bisabolol interacts with PPAR-γ, a nuclear receptor protein that is highly expressed in the colon epithelium. Treatment with α-bisabolol in DSS-administered mice significantly reduced Disease Activity Index (DAI), myeloperoxidase (MPO) activity, and colonic length and protected the microarchitecture of the colon. α-Bisabolol treatment also reduced the expression of proinflammatory cytokines (IL-6, IL1β, TNF-α, and IL-17A) at the protein and mRNA levels. The expression of COX-2 and iNOS inflammatory mediators were reduced along with tissue nitrite levels. Furthermore, α-bisabolol decreased the phosphorylation of activated mitogen-activated protein kinase (MAPK) signaling and nuclear factor kappa B (NFκB) proteins and enhanced colon epithelial PPAR-γ transcription factor expression. However, the PPAR-α and β/δ expression was not altered, indicating α-bisabolol is a specific stimulator of PPAR-γ. α-Bisabolol also increased the PPAR-γ transcription factor expression but not PPAR-α and β/δ in pretreated in LPS-stimulated RAW264.7 macrophages. α-Bisabolol significantly decreased the expression of proinflammatory chemokines (CXCL-1 and IL-8) mRNA in HT-29 cells treated with TNF-α and HT-29 PPAR-γ promoter activity. These results demonstrate that α-bisabolol mitigates colonic inflammation by inhibiting MAPK signaling and stimulating PPAR-γ expression.

Dexmedetomidine represses TGF-β1-induced extracellular matrix production and proliferation of airway smooth muscle cells by inhibiting MAPK signaling pathway.

Airway remodeling is implicated in the pathogenesis of asthma, and abnormal proliferation of airway smooth muscle cells (ASMCs) contribute to airway remodeling. Inflammatory mediator, transforming growth factor-β1 (TGF-β1), stimulates the proliferation of ASMCs, and is associated with airway remodeling in asthma. Dexmedetomidine (DEX) has been widely used in the adjuvant therapy of acute asthma. The potential effects of DEX on extracellular matrix (ECM) production and proliferation of ASMCs were investigated in this study. Human ASMCs were incubated with TGF-β1 for 48 hours, and then treated with different concentrations of DEX for another 24 hours. Cell proliferation was detected by MTT and BrdU (5'-bromo-2'-deoxyuridine) staining. Flow cytometry was used to assess cell apoptosis, and western blot was applied to identify the underlying mechanism. TGF-β1 induced increase in cell viability and bromodeoxyuridine (BrdU) positive cells in ASMCs while repressed cell apoptosis. Second, TGF-β1-induced ASMCs were then treated with different concentrations of DEX. Cell viability of TGF-β1-induced ASMCs was decreased by incubation of DEX. The number of BrdU positive cells in TGF-β1-induced ASMCs was reduced by incubation of DEX. Moreover, incubation of DEX promoted cell apoptosis of TGF-β1-induced ASMCs. Third, incubation of DEX attenuated TGF-β1-induced increase in fibronectin, collagen I, MMP9, and versican in ASMCs. Lastly, the up-regulation of phosphorylated extracellular receptor kinase (p-ERK), phosphorylated Jun N-terminal Kinase (p-JNK), and p-p38 in TGF-β1-induced ASMCs was reversed by incubation of DEX. DEX suppressed TGF-β1-induced ECM production and proliferation of ASMCs through inactivation of p38 mitogen-activated protein kinase (MAPK) pathway, providing a potential strategy for prevention of asthma.

Sulforaphane inhibits cytokine-stimulated chemokine and adhesion molecule expressions in human corneal fibroblasts: Involvement of the MAPK, STAT, and NF-κB signaling pathways

Chemokines and adhesion molecules are major inflammatory mediators of chronic and recurrent vernal keratoconjunctivitis (VKC). Sulforaphane (SFN) is a natural plant extract that is known to have anti-inflammatory and antioxidant properties. SFN is demonstrated to be effective against a variety of human diseases. The current investigation examines the effects and the molecular mechanisms of SFN on cytokine-induced human corneal fibroblasts (HCFs) expression of adhesion molecules and chemokines. HCFs were exposed to both interleukin (IL)-4 and tumor necrosis factor (TNF)-α in the absence or presence of SFN treatment. The levels of thymus- and activation-regulated chemokine (TARC) and eotaxin-1 in culture supernatants were evaluated using enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction analysis (RT-PCR) enabled quantification of mRNA levels of vascular cell adhesion molecule (VCAM)-1, eotaxin-1, and TARC along with cytokine receptors. An immunoblotting assay was used to evaluate the activities of VCAM-1, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), signal transducer and activator of transcription factor (STAT)6 pathways, along with the expression of the cytokine receptors including IL-4 receptor (R)α, IL-13Rα1, TNFRI, as well as TNFRII. SFN inhibited TARC and eotaxin-1 release in HCFs stimulated by TNF-α and IL-4 in a manner dependent on dose and time. SFN suppressed transcriptions of TARC, eotaxin-1, and VCAM-1. Furthermore, the mRNA and protein expression levels of IL-4Rα, TNFRI, and TNFRII were also attenuated by SFN exposure, however, those of IL-13Rα1 remained unaffected. In addition, SFN downregulated the expression of VCAM-1 and the phosphorylation of MAPKs, IκBα, and STAT6. These results suggest that SFN inhibited cytokine-stimulated TARC, eotaxin-1 secretion as well as VCAM-1 expression in HCFs, with these effects likely occurring as a result of cytokine receptor inhibition and attenuation of MAPK, NF-κB, and STAT6 signaling. SFN may therefore have therapeutic potential in VKC treatment.

RETRACTED ARTICLE: The myocardial infarction-associated transcript 2 inhibits lipid accumulation and promotes cholesterol efflux in oxidized low-density lipoprotein-induced THP-1-derived macrophages via inhibiting mitogen-activated protein kinase signaling and activating the nuclear factor erythroid-related factor 2 signaling pathway

Statement of Retraction Fangxiang Mu, Yuqing Wang, Hong Wu, Qingxia You, and Daimin Zhng- 2022, The myocardial infarction-associated transcript 2 inhibits lipid accumulation and promotes cholesterol efflux in oxidized low-density lipoprotein-induced THP-1-derived macrophages via inhibiting mitogen-activated protein kinase signaling and activating the nuclear factor erythroid-related factor 2 signaling pathway, Bioengineered 13(1), doi: 10.1080/21655979.2021.2005932 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. The corresponding author listed in this publication has been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

BTB and CNC homology 1 (Bach1) induces lung cancer stem cell phenotypes by stimulating CD44 expression

BackgroundGrowing evidence suggests that cancer stem cells (CSCs) are responsible for cancer initiation in tumors. Bach1 has been identified to contribute to several tumor progression, including lung cancer. The role of Bach1 in CSCs remains poorly known. Therefore, the function of Bach1 on lung CSCs was focused currently.MethodsThe expression of Bach1, CD133, CD44, Sox2, Nanog and Oct4 mRNA was assessed using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). Protein expression of Bach1, CD133, CD44, Sox2, Nanog, Oct4, p53, BCL2, BAX, p-p38, p-AKT1, c-Fos and c-Jun protein was analyzed by western blotting. 5-ethynyl-29-deoxyuridine (EdU), colony formation, Flow cytometry analysis and transwell invasion assay were carried out to analyze lung cancer cell proliferation, apoptosis and invasion respectively. Tumor sphere formation assay was utilized to evaluate spheroid capacity. Flow cytometry analysis was carried out to isolate CD133 or CD44 positive lung cancer cells. The relationship between Bach1 and CD44 was verified using ChIP-qPCR and dual-luciferase reporter assay. Xenograft tumor tissues were collected for hematoxylin and eosin (HE) staining and IHC analysis to evaluate histology and Ki-67.ResultsThe ratio of CD44 + CSCs from A549 and SPC-A1 cells were significantly enriched. Tumor growth of CD44 + CSCs was obviously suppressed in vivo compared to CD44− CSCs. Bach1 expression was obviously increased in CD44 + CSCs. Then, via using the in vitro experiment, it was observed that CSCs proliferation and invasion were greatly reduced by the down-regulation of Bach1 while cell apoptosis was triggered by knockdown of Bach1. Loss of Bach1 was able to repress tumor-sphere formation and tumor-initiating CSC markers. A repression of CSCs growth and metastasis of shRNA-Bach1 was confirmed using xenograft models and caudal vein injection. The direct interaction between Bach1 and CD44 was confirmed by ChIP-qPCR and dual-luciferase reporter assay. Furthermore, mitogen-activated protein kinases (MAPK) signaling pathway was selected and we proved the effects of Bach1 on lung CSCs were associated with the activation of the MAPK pathway. As manifested, loss of Bach1 was able to repress p-p38, p-AKT1, c-Fos, c-Jun protein levels in lung CSCs. Inhibition of MAPK signaling remarkably restrained lung CSCs growth and CSCs properties induced by Bach1 overexpression.ConclusionIn summary, we imply that Bach1 demonstrates great potential for the treatment of lung cancer metastasis and recurrence via activating CD44 and MPAK signaling.

MiR-143-3p Inhibits the Differentiation of Osteoclast Induced by Synovial Fibroblast and Monocyte Coculture in Adjuvant-Induced Arthritic Rats.

Osteoclast, which mediates overactive bone resorption, is one of the key factors for bone destruction in rheumatoid arthritis (RA). Existing studies have shown that abnormal miR-143-3p expression was observed in both RA patients and arthritis animals, which can participate in osteoclast differentiation, and mitogen-activated protein kinase (MAPK) signaling pathway was closely related to osteoclast differentiation. The primary objective of the current study was to determine the role of miR-143-3p in the progression of osteoclast differentiation and its relationship with MAPK signaling pathways. The results showed that miR-143-3p inhibited osteoclast differentiation and decreased the levels of M-CSF and RANKL during osteoclast differentiation. miR-143-3p inhibited the expression of MAPK signaling proteins, which is ERK1/2 in the early stage and JNK in the later stage of osteoclast differentiation. It was also observed that MAPK inhibitors upregulated miR-143-3p expression in osteoclast differentiation. Taken together, our results suggested that miR-143-3p could inhibit the differentiation of osteoclast, which was related to inhibiting MAPK signaling pathways. This may provide a novel strategy for curing RA.

A method for the expression of fibroblast growth factor 14 and assessment of its neuroprotective effect in an Alzheimer's disease model.

BackgroundFibroblast growth factor (FGF) 14 is a member of the FGF family that is mainly expressed in the central nervous system. FGF14 has a close association with the occurrence of neurodegenerative conditions; however, its significance in Alzheimer’s disease (AD) has yet to be evaluated. Therefore, we sought to obtain a large amount of exogenous FGF14 protein and explore its effect in a cellular model of AD.MethodsFGF14 protein was expressed in an Escherichia coli system using gene recombination technology. Purified protein was obtained through washing and renaturation of inclusion bodies combined with nickel column affinity chromatography. The AD model was established via Aβ25-35-induced injury in PC12 cells. Changes in the levels of lactate dehydrogenase and malondialdehyde were detected, and the neuroprotective effect of recombinant human FGF14 (rhFGF14) was evaluated through double-fluorescence staining and flow cytometry apoptosis detection. For further exploration of rhFGF14-mediated regulation of mitogen-activated protein kinase (MAPK) signaling, western blot was employed.ResultsWe successfully induced large amounts of insoluble rhFGF14. Following solubilization and refolding of the rhFGF14 from inclusion bodies, high purity rhFGF14 was purified by Nickel affinity column chromatography. The results showed that rhFGF14 alleviated Aβ25-3-induced PC12 cell injury by inhibiting the phosphorylation of p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase, thus suppressing the MAPK signaling pathway.ConclusionsFGF14 performed a neuroprotective role in our in vitro AD model via its inhibition of MAPK signaling, highlighting its potential as a therapeutic drug for neurodegenerative conditions.

Combined VEGFR and MAPK pathway inhibition in angiosarcoma

Angiosarcoma is an aggressive malignancy of endothelial cells that carries a high mortality rate. Cytotoxic chemotherapy can elicit clinical responses, but the duration of response is limited. Sequencing reveals multiple mutations in angiogenesis pathways in angiosarcomas, particularly in vascular endothelial growth factor (VEGFR) and mitogen-activated protein kinase (MAPK) signaling. We aimed to determine the biological relevance of these pathways in angiosarcoma. Tissue microarray consisting of clinical formalin-fixed paraffin embedded tissue archival samples were stained for phospho- extracellular signal-regulated kinase (p-ERK) with immunohistochemistry. Angiosarcoma cell lines were treated with the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, pan-VEGFR inhibitor cediranib, or combined trametinib and cediranib and viability was assessed. Reverse phase protein array (RPPA) was performed to assess multiple oncogenic protein pathways. SVR angiosarcoma cells were grown in vivo and gene expression effects of treatment were assessed with whole exome RNA sequencing. MAPK signaling was found active in over half of clinical angiosarcoma samples. Inhibition of MAPK signaling with the MEK inhibitor trametinib decreased the viability of angiosarcoma cells. Combined inhibition of the VEGF and MAPK pathways with cediranib and trametinib had an additive effect in in vitro models, and a combinatorial effect in an in vivo model. Combined treatment led to smaller tumors than treatment with either agent alone. RNA-seq demonstrated distinct expression signatures between the trametinib treated tumors and those treated with both trametinib and cediranib. These results indicate a clinical study of combined VEGFR and MEK inhibition in angiosarcoma is warranted.

Therapeutic and prognostic implications of NOTCH and MAPK signaling in bladder cancer.

Signaling pathways that drive bladder cancer (BC) progression may be promising and specific targets for systemic therapy. Here, we investigated the clinical significance and targetability of NOTCH and mitogen‐activated protein kinase (MAPK) signaling for this aggressive malignancy. We assessed NOTCH1 and MAPK activity in 222 stage III and IV BC specimens of patients that had undergone radical cystectomy, and tested for clinical associations including cancer‐specific and overall survival. We examined therapeutic effects of NOTCH and MAPK repression in a murine xenograft model of human bladder cancer cells and evaluated tumor growth and tumor cell plasticity. In BC, NOTCH1 and MAPK signaling marked two distinct tumor cell subpopulations. The combination of high NOTCH1 and high MAPK activity indicated poor cancer‐specific and overall survival in univariate and multivariate analyses. Inhibition of NOTCH and MAPK in BC xenografts in vivo depleted targeted tumor cell subpopulations and revealed strong plasticity in signaling pathway activity. Combinatorial inhibition of NOTCH and MAPK signaling most strongly suppressed tumor growth. Our findings indicate that tumor cell subpopulations with high NOTCH and MAPK activity both contribute to tumor progression. Furthermore, we propose a new concept for BC therapy, which advocates specific and simultaneous targeting of these different tumor cell subpopulations through combined NOTCH and MAPK inhibition.

Betulinic Acid Alleviates Spleen Oxidative Damage Induced by Acute Intraperitoneal Exposure to T-2 Toxin by Activating Nrf2 and Inhibiting MAPK Signaling Pathways.

T-2 toxin, which is mainly produced by specific strains of Fusarium in nature, can induce immunotoxicity and oxidative stress, resulting in immune organ dysfunction and apoptosis. Betulinic acid (BA), a pentacyclic triterpenoids from nature plants, has been demonstrated to possess immunomodulating and antioxidative bioactivities. The purpose of the study was to explore the effect of BA on T-2 toxin-challenged spleen oxidative damage and further elucidate the underlying mechanism. We found that BA not only ameliorated the contents of serum total cholesterol (TC) and triglyceride (TG) but also restored the number of lymphocytes in T-2 toxin-induced mice. BA dose-dependently reduced the accumulation of reactive oxygen species (ROS), enhanced superoxide dismutase (SOD) activity, and decreased malondialdehyde (MDA) content, as well as increased the total antioxidant capacity (T-AOC) in the spleen of T-2-toxin-exposed mice. Moreover, BA reduced inflammatory cell infiltration in the spleen, improved the morphology of mitochondria and enriched the number of organelles in splenocytes, and dramatically attenuated T-2 toxin-triggered splenocyte apoptosis. Furthermore, administration of BA alleviated the protein phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases (ERK); decreased the protein expression of kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein1 (Keap1); and increased the protein expression of nuclear factor erythroid 2 [NF-E2]-related factor (Nrf2) and heme oxygenase-1 (HO-1) in the spleen. These findings demonstrate that BA defends against spleen oxidative damage associated with T-2 toxin injection by decreasing ROS accumulation and activating the Nrf2 signaling pathway, as well as inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway.

Anti-Tumor Activity of ASTX029, a Dual Mechanism Inhibitor of ERK1/2, in Preclinical AML Models

Oncogenic mutations in genes such as the RAS family (KRAS, NRAS or HRAS) or receptor tyrosine kinases (RTKs) drive tumor growth through aberrant activation of the mitogen activated protein kinase (MAPK) signaling pathway. Acute myeloid leukemia (AML) patients frequently exhibit activating mutations in MAPK pathway members, such as NRAS and KRAS, suggesting that these malignancies may be driven by aberrant activation of the MAPK pathway. Targeting of the MAPK pathway has been clinically validated in solid tumors, with agents targeting BRAF and MEK approved for the treatment of BRAF-mutant melanoma. However, there is currently no approved therapy directly targeting activated RAS family members and resistance to MAPK pathway inhibitors is frequently associated with reactivation of MAPK signaling. ERK1/2 (ERK) is a downstream node in the MAPK pathway and therefore represents an attractive therapeutic target for inhibition of MAPK signaling in these settings. We have recently described in vivo anti-tumor activity in MAPK-activated solid tumor models following treatment with ASTX029, a highly potent ERK inhibitor developed using fragment-based drug design. ASTX029 has a distinctive ERK binding mode which confers dual mechanism inhibition of ERK, inhibiting both the catalytic activity of ERK and its phosphorylation by MEK. Here, we demonstrate that ASTX029 is also active in AML models and potently inhibits in vitro and in vivo MAPK signaling and growth in these models. Using a panel of 15 AML cell lines, we investigated sensitivity to ASTX029 in vitro. We observed that 8 cell lines bearing mutations leading to increased MAPK pathway signaling were sensitive to treatment with ASTX029 with an average IC50 value of 47 nM, in contrast to an average IC50 value of 1800 nM for cell lines without activating mutations. The phosphorylation of RSK, a direct substrate of ERK, was suppressed for up to 24 h following treatment with ASTX029 in vitro. We have previously demonstrated good oral bioavailability for ASTX029 and once daily dosing resulted in significant tumor growth inhibition in AML cell line xenograft models. To confirm target engagement in vivo, we examined MAPK signaling in xenograft tissue and observed inhibition of the phosphorylation of RSK and of ERK itself, consistent with the dual mechanism of action proposed for ASTX029. In summary, the ERK inhibitor, ASTX029, has potent activity against MAPK-activated tumor models, including AML models, and is now being tested in a Phase 1/2 clinical trial in advanced solid tumors (NCT03520075). These data highlight its therapeutic potential for the treatment of AML in patients with mutations leading to MAPK pathway activation and support further investigation in these patient populations. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Fazal:Astex Pharmaceuticals: Current Employment. Munck:Astex Pharmaceuticals: Current Employment. Martins:Astex Pharmaceuticals: Current Employment. Shah:Astex Pharmaceuticals: Current Employment. Wilsher:Astex Pharmaceuticals: Current Employment. Wallis:Astex Pharmaceuticals: Current Employment. Keer:Astex Pharmaceuticals, Inc.: Current Employment. Lyons:Astex Pharmaceuticals: Current Employment.

Long non-coding RNA00844 inhibits MAPK signaling to suppress the progression of hepatocellular carcinoma by targeting AZGP1.

BackgroundPrevious data have confirmed that disordered long non-coding ribonucleic acid (lncRNA) expression is evident in many cancers and is correlated with tumor progression. The present study aimed to investigate the function of long non-coding RNA00844 (LINC00844) in hepatocellular carcinoma (HCC).MethodsThe expression levels of target genes were detected with real-time polymerase chain reaction (PCR) and western blotting. The biologic function of HCC cells was determined with cell viability assay, colony formation assay, cell cycle analysis, apoptosis detection, and Transwell migration assay in vitro. Tumorigenesis was performed with cell injection in vivo. The relationship between LINC00844 and survival outcomes was determined with the Cox proportional hazards model. A RNA precipitation assay was conducted to reveal the types of LINC00844 that potentially bind with proteins.ResultsLINC00844 was found to be significantly decreased in HCC tissue and was correlated with poor tumor characteristics, such as portal vein invasion, high α-fetoprotein (AFP), and a high rate of tumor recurrence. Exotic LINC00844 expression in HCC cell lines significantly suppressed proliferation and migration, as well as invasiveness, whereas LINC00844 deletion had the opposite effect. LINC00844 overexpression significantly inhibited HCC tumorigenesis in vivo. Mechanistic analyses indicated that the mitogen-activated protein kinase (MAPK) signaling pathway was remarkably inactivated by LINC00844. Furthermore, the immunoprecipitation assay verified that LINC00844 can bind to zinc-alpha-2-glycoprotein (AZGP1) and interfere with its translocation. LINC00844 can also promote AZGP1 expression, leading to the suppression of the transforming growth factor-β1 (TGF-β1)-extracellular signal-regulated kinase (ERK) pathway.ConclusionsLINC00844 is a novel anti-oncogene in the development of HCC and a potentially promising therapeutic target in HCC.

Contributions of HO-1-Dependent MAPK to Regulating Intestinal Barrier Disruption.

The mitogen-activated protein kinase (MAPK) pathway controls intestinal epithelial barrier permeability by regulating tight junctions (TJs) and epithelial cells damage. Heme oxygenase-1 (HO-1) and carbon monoxide (CO) protect the intestinal epithelial barrier function, but the molecular mechanism is not yet clarified. MAPK activation and barrier permeability were studied using monolayers of Caco-2 cells treated with tissue necrosis factor α (TNF-α) transfected with FUGW-HO-1 or pLKO.1-sh-HO-1 plasmid. Intestinal mucosal barrier permeability and MAPK activation were also investigated using carbon tetrachloride (CCl4) administration with CoPP (a HO-1 inducer), ZnPP (a HO-1 inhibitor), CO releasing molecule 2 (CORM-2), or inactived-CORM-2-treated wild-type mice and mice with HO-1 deficiency in intestinal epithelial cells. TNF-α increased epithelial TJ disruption and cleaved caspase-3 expression, induced ERK, p38, and JNK phosphorylation. In addition, HO-1 blocked TNF-α-induced increase in epithelial TJs disruption, cleaved caspase-3 expression, as well as ERK, p38, and JNK phosphorylation in an HO-1-dependent manner. CoPP and CORM-2 directly ameliorated intestinal mucosal injury, attenuated TJ disruption and cleaved caspase-3 expression, and inhibited epithelial ERK, p38, and JNK phosphorylation after chronic CCl4 injection. Conversely, ZnPP completely reversed these effects. Furthermore, mice with intestinal epithelial HO-1 deficient exhibited a robust increase in mucosal TJs disruption, cleaved caspase-3 expression, and MAPKs activation as compared to the control group mice. These data demonstrated that HO-1-dependent MAPK signaling inhibition preserves the intestinal mucosal barrier integrity by abrogating TJ dysregulation and epithelial cell damage. The differential targeting of gut HO-1-MAPK axis leads to improved intestinal disease therapy.

DUSP4 appears to be a highly localized endogenous inhibitor of epileptic signaling in human neocortex

BackgroundWe previously identified the Mitogen Activated Protein Kinase (MAPK) pathway as focally upregulated in brain regions with high epileptic activity and showed that inhibition of MAPK signaling reduces epileptic spiking in an animal model. Here we examined how activators and inhibitors of the MAPK pathway are expressed in human epileptic cortex and how these could contribute to the localization of epileptic signaling. MethodsWe localized gene and protein expression in human epileptic neocortical tissues based on epileptic activities from 20 patients based on long-term intracranial recordings. Follow-up mechanistic studies by depolarization of human Sh-SY5Y cell line were used to model epileptic activity in the human brain. ResultsA clustering algorithm of differentially expressed genes identified a unique gene expression cluster distinct from other MAPK genes. Within this cluster was dual specificity phosphatase 4 (DUSP4), a potent MAPK inhibitor. In situ hybridization studies revealed focal patches of DUSP4 mRNA in layer 2/3 brain regions associated with a dramatic reduction in MAPK signaling genes. In vitro depolarization led to the rapid and transient induction of DUSP4 protein, which, in turn, reduced MAPK activity. Activity-dependent induction of DUSP4 protein was transient and required MAPK signaling. Human epileptic brain regions with lower epileptic activity had lower MAPK activity and higher DUSP4 protein levels. DiscussionDUSP4 is a highly localized, endogenous feedback inhibitor of pro-epileptogenic MAPK signaling in the human epileptic brain. Increasing DUSP4 expression could therefore be a novel therapeutic approach to prevent the development and spread of epileptic circuits. Significance statementEpilepsy is a chronic debilitating disease. Once it develops, epileptic circuits often persist throughout life. Fortunately, in focal forms of epilepsy, these circuits can remain highly localized and are amenable to surgical resections, suggesting that endogenous mechanisms restrict their spread to other brain regions. Using a high-throughput genomic analysis of human epileptic brain regions, we identified DUSP4 as an activity-dependent inhibitor of MAPK signaling expressed in focal patches surrounding human neocortical epileptic brain regions. Our results suggest that DUSP4, through local inhibition of MAPK signaling, acts as an endogenous, spatially segregated safety mechanism to prevent the spread of epileptic activity. Augmenting DUSP4 expression could be a novel disease-modifying approach to prevent or treat human epilepsy.

Response to trametinib treatment in progressive pediatric low-grade glioma patients

IntroductionA hallmark of pediatric low-grade glioma (pLGG) is aberrant signaling of the mitogen activated protein kinase (MAPK) pathway. Hence, inhibition of MAPK signaling using small molecule inhibitors such as MEK inhibitors (MEKi) may be a promising strategy.MethodsIn this multi-center retrospective centrally reviewed study, we analyzed 18 patients treated with the MEKi trametinib for progressive pLGG as an individual treatment decision between 2015 and 2019. We have investigated radiological response as per central radiology review, molecular classification and investigator observed toxicity.ResultsWe observed 6 partial responses (PR), 2 minor responses (MR), and 10 stable diseases (SD) as best overall responses. Disease control rate (DCR) was 100% under therapy. Responses were observed in KIAA1549:BRAF- as well as neurofibromatosis type 1 (NF1)-driven tumors. Median treatment time was 12.5 months (range: 2 to 27 months). Progressive disease was observed in three patients after cessation of trametinib treatment within a median time of 3 (2–4) months. Therapy related adverse events occurred in 16/18 patients (89%). Eight of 18 patients (44%) experienced severe adverse events (CTCAE III and/or IV; most commonly skin rash and paronychia) requiring dose reduction in 6/18 patients (33%), and discontinuation of treatment in 2/18 patients (11%).ConclusionsTrametinib was an active and feasible treatment for progressive pLGG leading to disease control in all patients. However, treatment related toxicity interfered with treatment in individual patients, and disease control after MEKi withdrawal was not sustained in a fraction of patients. Our data support in-class efficacy of MEKi in pLGGs and necessity for upfront randomized testing of trametinib against current standard chemotherapy regimens.

Abstract 6415: RAF dimer inhibitor lifirafenib enhances the antitumor activity of MEK inhibitor mirdametinib in RAS mutant tumors

Abstract K-RAS mutation represents one of the most frequent genetic alterations in cancers. Monotherapy approaches targeting RAS downstream effectors such as MEK and ERK have had limited clinical success in patients with K-RAS-mutated cancers. The reduced sensitivity of K-RAS-mutated cells to certain MEK inhibitors is associated with feedback phosphorylation of MEK and reactivation of mitogen-activated protein kinase (MAPK) signaling. Herein, we report that RAF dimer inhibitor lifirafenib (BGB-283) shows strong synergistic effect with MEK inhibitor mirdametinib (PD-0325901) in suppressing proliferation of K-RAS-mutated cancer cell lines. This synergistic effect was not observed using vemurafenib, a first-generation B-RAFV600E selective inhibitor. Mechanistic analysis revealed that RAF dimer inhibition could suppress RAF-dependent MEK reactivation and led to sustained inhibition of MAPK signaling in K-RAS-mutated cells. Furthermore, combination synergy was observed in K-RAS mutant xenograft models. Pharmacodynamic analysis supported the role of synergistic phospho-ERK blockade in enhancing the antitumor activity in the K-RAS mutant models. These findings support the rationale to combine a RAF dimer inhibitor and a MEK inhibitor to treat K-RAS-mutated cancers and led to the ongoing a Phase Ib/II clinical trial of lifirafenib and mirdametinib in patients with K-RAS mutations or other MAPK pathway aberrations (Clinical Trial ID: NCT03905148). Citation Format: Xi Yuan, Xinwen Zhang, Rong Du, Shing-Hu Cheung, Min Wei, Changyou Zhou, Lai Wang, Todd Shearer, Badreddin Edris, Mary Smith, Zhan Yao, Claire N. Thant, Neal Rosen, Lusong Luo. RAF dimer inhibitor lifirafenib enhances the antitumor activity of MEK inhibitor mirdametinib in RAS mutant tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6415.

RAF dimer inhibition enhances the antitumor activity of MEK inhibitors in K-RAS mutant tumors.

The mutation of K‐RAS represents one of the most frequent genetic alterations in cancer. Targeting of downstream effectors of RAS, including of MEK and ERK, has limited clinical success in cancer patients with K‐RAS mutations. The reduced sensitivity of K‐RAS‐mutated cells to certain MEK inhibitors (MEKi) is associated with the feedback phosphorylation of MEK by C‐RAF and with the reactivation of mitogen‐activated protein kinase (MAPK) signaling. Here, we report that the RAF dimer inhibitors lifirafenib (BGB‐283) and compound C show a strong synergistic effect with MEKi, including mirdametinib (PD‐0325901) and selumetinib, in suppressing the proliferation of K‐RAS‐mutated non‐small‐cell lung cancer and colorectal cancer (CRC) cell lines. This synergistic effect was not observed with the B‐RAFV600E selective inhibitor vemurafenib. Our mechanistic analysis revealed that RAF dimer inhibition suppresses RAF‐dependent MEK reactivation and leads to the sustained inhibition of MAPK signaling in K‐RAS‐mutated cells. This synergistic effect was also observed in several K‐RAS mutant mouse xenograft models. A pharmacodynamic analysis supported a role for the synergistic phospho‐ERK blockade in enhancing the antitumor activity observed in the K‐RAS mutant models. These findings support a vertical inhibition strategy in which RAF dimer and MEKi are combined to target K‐RAS‐mutated cancers, and have led to a Phase 1b/2 combination therapy study of lifirafenib and mirdametinib in solid tumor patients with K‐RAS mutations and other MAPK pathway aberrations.