Targeting MAPK Pathway Research Articles

Expression of Concern: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways.

Expression of Concern: Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways.

Nigella sativa Averts 5-Fluorouracil Induced Kidney Injury via Targeting Redox Imbalance and MAPK Pathway

Nigella sativa Averts 5-Fluorouracil Induced Kidney Injury via Targeting Redox Imbalance and MAPK Pathway

Secretory products of DPSC mitigate inflammatory effects in microglial cells by targeting MAPK pathway

Activated microglial cells in the central nervous system (CNS) are the main contributors to neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Inhibiting their activation will help in reducing inflammation and oxidative stress during pathogenesis, potentially limiting the progression of the diseases. The immunomodulation properties of dental pulp-derived stem cells (DPSC) make it a promising therapy for neurodegenerative disorders. This study aims to determine whether secretory factors of DPSC (DPSC℗) inhibit inflammation and proliferation of microglial cells and define the molecular mechanisms. Our quantitative RT-PCR analysis showed that the DPSC℗ reduced the markers of the inflammation and induced anti-inflammatory molecules in microglial cells. DPSC ℗ reduced the intracellular and mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential in microglial cells. In addition, DPSC ℗ decreased the cellular bioenergetics parameters related to oxygen consumption rate (OCAR) and extracellular acidification rate (ECAR). We found that DPSC℗ inhibited microglial cell proliferation by activating a checkpoint molecule, Chk1 leading an arrest at the G1 phase of the cell cycle. To define the mechanism, we performed the western blot analysis and observed that the MAPK P38 pathway was inhibited by DPSC℗. Furthermore, a System biology analysis revealed that the BDNF and GDNF, secretory factors of DPSC, blocked at the phosphorylation site (Tyr 182) of the P38 molecule resulting in the inhibition of downstream signaling of inflammation. These data suggest that the DPSC℗ may be a potential therapeutic agent for neurodegenerative diseases.

Curative Effect of AD-MSCs against Cisplatin-Induced Hepatotoxicity in Rats is Potentiated by Azilsartan: Targeting Oxidative Stress, MAPK, and Apoptosis Signaling Pathways.

Despite its clinical value, cisplatin (CISP) is complicated by marked hepatotoxicity via inducing oxidative stress, inflammatory, and apoptotic pathways. This study aims to explore the protective impact of azilsartan (AZIL), an antihypertensive drug, in addition to adipose tissue-derived mesenchymal stem cells (AD-MSCs) on CISP-induced hepatotoxicity. After characterization and labeling of AD-MSCs by PKH26 dye, 54 Wistar male albino rats were randomly divided into nine groups: I (CONT), II (AZIL.H), III (CISP), IV (CISP + AZIL.L), V (CISP + AZIL.H), VI (CISP + AD-MSCs), VII (CISP + AZIL.L + AD-MSCs), VIII (CISP + AZIL.H + AD-MSCs), and IX (CISP + VITA C). Serum alanine aminotransferase (ALT), alanine aminotransferase (AST), and albumin levels were determined. Assessment of reactive oxygen species, malondialdehyde, and glutathione contents, and superoxide dismutase activity and histopathological evaluations were done on hepatic tissue. Quantitative real-time PCR was utilized to estimate the expression of TNF-α and IL-6 genes. Cell homing of labeled AD-MSCs to the liver tissues was investigated. Hepatic expression of JNK1/2, ERK1/2, p38, Bax, Bcl-2, and cleaved caspase-3 proteins was investigated by western blot analysis. CISP elevated serum ALT and AST activities, reduced albumin level, and remarkably changed the hepatic architecture. It increased the expression TNF-α and IL-6 genes, raised the expression of JNK1/2, ERK1/2, p38, Bax, and cleaved caspase-3 proteins, and diminished the Bcl-2 protein. By contrast, treatment of animals with either AZIL or AD-MSCs dramatically reduced the effects of CISP injection. Moreover, treatment with combination therapy (AZIL.L or H + AD-MSCs) considerably mitigated all previously mentioned alterations superior to AZIL or AD-MSCs alone, which might be attributed to the AZIL-enhanced homing ability of AD-MSCs into the injured liver tissue. In conclusion, the present findings demonstrated that AZIL improves the hepatoprotective potential of AD-MSCs against CISP-induced hepatotoxicity by modulating oxidative stress, mitogen-activated protein kinase, and apoptotic pathways.

Combination Treatment Targeting mTOR and MAPK Pathways Has Synergistic Activity in Multiple Myeloma

Multiple myeloma (MM) is an incurable, malignant B cell disorder characterized by frequent relapses and a poor prognosis. Thus, new therapeutic approaches are warranted. The phosphatidylinositol-3-kinase (PI3K) pathway plays a key role in many critical cellular processes, including cell proliferation and survival. Activated PI3K/AKT (protein kinases B)/mTOR (mammalian target of rapamycin) signaling has been identified in MM primary patient samples and cell lines. In this study, the efficacy of PI3K and mTOR inhibitors in various MM cell lines representing three different prognostic subtypes was tested. Whereas MM cell lines were rather resistant to PI3K inhibition, treatment with the mTOR inhibitor temsirolimus decreases the phosphorylation of key molecules in the PI3K pathway in MM cell lines, leading to G0/G1 cell cycle arrest and thus reduced proliferation. Strikingly, the efficacy of temsirolimus was amplified by combining the treatment with the Mitogen-activated protein kinase kinase (MEK) inhibitor trametinib. Our findings provide a scientific rationale for the simultaneous inhibition of mTOR and MEK as a novel strategy for the treatment of MM.

1066P Tumor microenvironment (TME) of HRAS mutated non-small cell lung cancer (NSCLC)

RAS pathway alterations in NSCLC have been linked to worse prognosis and remain a challenging therapeutic target. HRAS, a RAS family members, is under-investigated and its activation depends on farnesylation, making it an appealing target for farnesyltransferase inhibitors. Although KRAS has been associated with immune modulation in NSCLC, the role of HRAS remains unclear. We aimed to investigate the relationship of HRAS gene mutations and the TME. Molecular profiling of 29,767 NSCLC tumor samples was performed at Caris Life Sciences to obtain whole exom sequencing, whole transcriptome sequencing and immunohistochemistry. MAPK signaling activity (MPAS) and immune cell fraction were assessed by mRNA analysis. Wilcoxon, Fisher’s exact were used for statistical significance. Overall survival was calculated using the Kaplan-Meier method. Comparisons were conducted between HRAS mutated (mt) tumors and the entire NSCLC general cohort (GC). HRAS mt were detected in 0.4% of NSCLC tumors (n=128), with a majority among smokers. HRAS mt displayed less B cell, macrophage M2, NK cell, CD4+ and CD8+ T cells infiltrates compared to GC (q<0.05 ). In addition, HRAS Q61 was found to be correlated with worse prognosis in pts treated with pembro (HR = 2.779, 95% CI [1.04-7.423], p =0.03) but not G12 or G13, and displayed the highest MPAS score (p = 0.05), which had previously been demonstrated to indicate immune evasion. The median difference for survival curves was -246 days between HRAS mt squamous cell carcinomas (SCC, all smokers) and GC SCC in pts treated with pembro (HR 1.72, 95% CI 0.85-3.64, p =0.126). HRAS mt SCC showed less infiltration with M1, B cells, myeloid dendritic cells (mDC) and also displayed a significantly higher MPAS score and LAG3 expression as compared to GC (0.53 vs -0.31, p = 0.03; 2.10 vs 1.03 TPM, p = 0.04). No similar trends were observed in adenocarcinoma histology. HRAS mt NSCLC displayed a relatively immune-cold pattern with less CD4+ and CD8+ T cells infiltrates compared to GC. HRAS mt SCC also showed an immune-cold TME associated with high MAPK pathway activation and high LAG3 expression. This warrants further investigation, in particular in HRAS Q61 or HRAS mt SCC, with combination therapies targeting MAPK pathway or LAG3 protein.

Icariin protects bone marrow mesenchymal stem cells in aplastic anemia by targeting MAPK pathway.

Icariin, the main pharmacological active flavonoid extracted from Epimedi herba, can regulate cellular processes in diverse diseases. The aim of this study was to explore the effects and mechanisms of icariin on proliferation and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) in aplastic anemia (AA). Bone marrow mesenchymal stem cells were isolated from posterior tibias and femurs of AA rats that were induced by benzene and cyclophosphamide and gavaged with icariin. The isolated BMSCs were characterized morphologically and immunologically by positive markers (CD29 and CD90) and negative markers (CD34 and CD45). CCK-8 assay was performed to examine the BMSCs proliferation. Cell apoptosis and cell cycle were detected by flow cytometry. Oil red O staining was carried out to evaluate the adipogenesis of BMSCs. The mRNA expression of PPARγ, C/EBP-α, and FABP4 was measured by qRT-PCR. The protein levels of p-p38/p38, p-JNK/JNK, p-ERK/ERK, PPARγ, C/EBP-α, and FABP4 were detected using Western blotting. Icariin promoted the proliferation of BMSCs from AA rats in a dose-dependent manner. The protein levels of p-p38/p38, p-JNK/JNK, and p-ERK/ERK were downregulated in BMSCs from AA rats after icariin treatment. Icariin inhibited the apoptosis and arrested cell cycle at G/S phase of BMSCs from AA rats. The adipogenesis of BMSCs from AA rats was also suppressed after icariin treatment. However, the effects of icariin on BMSCs were weakened by p38 agonist addition. Icariin promoted the proliferation and inhibited the apoptosis and adipogenesis of BMSCs in AA by suppressing MAPK pathway.

Targeting mTOR signaling overcomes acquired resistance to combined BRAF and MEK inhibition in BRAF-mutant melanoma

Targeting MAPK pathway using a combination of BRAF and MEK inhibitors is an efficient strategy to treat melanoma harboring BRAF-mutation. The development of acquired resistance is inevitable due to the signaling pathway rewiring. Combining western blotting, immunohistochemistry, and reverse phase protein array (RPPA), we aim to understanding the role of the mTORC1 signaling pathway, a center node of intracellular signaling network, in mediating drug resistance of BRAF-mutant melanoma to the combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) therapy. The mTORC1 signaling pathway is initially suppressed by BRAFi and MEKi combination in melanoma but rebounds overtime after tumors acquire resistance to the combination therapy (CR) as assayed in cultured cells and PDX models. In vitro experiments showed that a subset of CR melanoma cells was sensitive to mTORC1 inhibition. The mTOR inhibitors, rapamycin and NVP-BEZ235, induced cell cycle arrest and apoptosis in CR cell lines. As a proof-of-principle, we demonstrated that rapamycin and NVP-BEZ235 treatment reduced tumor growth in CR xenograft models. Mechanistically, AKT or ERK contributes to the activation of mTORC1 in CR cells, depending on PTEN status of these cells. Our study reveals that mTOR activation is essential for drug resistance of melanoma to MAPK inhibitors, and provides insight into the rewiring of the signaling networks in CR melanoma.

The potential regulatory role of hsa_circ_0004104 in the persistency of atrial fibrillation by promoting cardiac fibrosis via TGF-\u03b2 pathway

IntroductionThe progression of paroxysmal AF (PAF) to persistent AF (PsAF) worsens the prognosis of AF, but its underlying mechanisms remain elusive. Recently, circular RNAs (circRNAs) were reported to be associated with cardiac fibrosis. In case of the vital role of cardiac fibrosis in AF persistency, we hypothesis that circRNAs may be potential regulators in the process of AF progression.Materials and methods6 persistent and 6 paroxysmal AF patients were enrolled as derivation cohort. Plasma circRNAs expressions were determined by microarray and validated by RT-PCR. Fibrosis level, manifested by serum TGF-β, was determined by ELISA. Pathways and related non-coding RNAs involving in the progression of AF regulated were predicted by in silico analysis.ResultsPsAF patients showed a distinct circRNAs expression profile with 92 circRNAs significantly dysregulated (fold change ≥ 2, p < 0.05), compared with PAF patients. The validity of the expression patterns was subsequently validated by RT-PCR in another 60 AF patients (30 PsAF and PAF, respectively). In addition, all the 5 up and down regulated circRNAs were clustered in MAPK and TGF-beta signaling pathway by KEGG pathway analysis. Among the 5 circRNAs, hsa_circ_0004104 was consistently downregulated in PsAF group (0.6 ± 0.33 vs 1.46 ± 0.41, p < 0.001) and predicted to target several AF and/or cardiac fibrosis related miRNAs reported by previous studies. In addition, TGF-β1 level was significantly higher in the PsAF group (5560.23 ± 1833.64 vs 2236.66 ± 914.89, p < 0.001), and hsa_circ_0004104 showed a significant negative correlation with TGF-β1 level (r = − 0.797, p < 0.001).ConclusionCircRNAs dysregulation plays vital roles in AF persistency. hsa_circ_0004104 could be a potential regulator and biomarker in AF persistency by promoting cardiac fibrosis via targeting MAPK and TGF-beta pathways.

The Evolutionary Landscape of Treatment for BRAFV600E Mutant Metastatic Colorectal Cancer.

Simple SummaryThe BRAFV600E mutation accounts for 8–10% of metastatic colorectal cancer (mCRC) patients and it is an established prognostic factor. Median overall survival of this subset of patients is indeed so poor that it is similar to first line PFS of patients without this molecular alteration. An exception is represented by patients displaying concomitant MSI-H status who can benefit from immunotherapy with checkpoint inhibitors (CPIs). Recently, a targeted therapy with the combination of encorafenib and cetuximab provided for the first time a survival gain and thus translation in the clinic, even though acquired resistance limits the possibility of more than an incremental benefit. Many studies exploiting other different strategies are ongoing. In this review we present current therapies specifically headed to BRAFV600E mutant mCRC and systematically review ongoing clinical trials identifying different approaches under investigations: targeting MAPK pathway (monotherapy or combinations), targeting MAPK pathway combined with cytotoxic agents, intensive cytotoxic regimen combinations, targeted agents combined with CPIs, oxidative stress induction, and cytotoxic agents combined with antiangiogenic drugs and CPIs.The BRAFV600E mutation is found in 8–10% of metastatic colorectal cancer (mCRC) patients and it is recognized as a poor prognostic factor with a median overall survival inferior to 20 months. At present, besides immune checkpoint inhibitors (CPIs) for those tumors with concomitant MSI-H status, recommended treatment options include cytotoxic chemotherapy + anti-VEGF in the first line setting, and a combination of EGFR and a BRAF inhibitor (cetuximab plus encorafenib) in second line. However, even with the latter targeted approach, acquired resistance limits the possibility of more than an incremental benefit and survival is still dismal. In this review, we discuss current treatment options for this subset of patients and perform a systematic review of ongoing clinical trials. Overall, we identified six emerging strategies: targeting MAPK pathway (monotherapy or combinations), targeting MAPK pathway combined with cytotoxic agents, intensive cytotoxic regimen combinations, targeted agents combined with CPIs, oxidative stress induction, and cytotoxic agents combined with antiangiogenic drugs and CPIs. In the future, the integration of new therapeutic strategies targeting key players in the BRAFV600E oncogenic pathways with current treatment approach based on cytotoxic chemotherapy and surgery is likely to redefine the treatment landscape of these CRC patients.

Luteolin and Apigenin Attenuate LPS-Induced Astrocyte Activation and Cytokine Production by Targeting MAPK, STAT3, and NF-κB Signaling Pathways.

Astrocytes release biologically active substances that cause inflammation in neurodegenerative diseases. The present study investigated the effects of two flavonoids (apigenin and luteolin) on the production of IL-31 and IL-33 in lipopolysaccharide (LPS)-activated astrocytes. Cell viability was investigated using EZ-Cytox assay, mRNA expressions of IL-31 and IL-33 were analyzed by RT-PCR, protein expressions were analyzed by western blot, and cytokine secretion was analyzed by ELISA. Apigenin and luteolin prevented astrocyte activation and inhibited mRNA and protein expression and secretion of IL-31 and IL-33 in the LPS-treated astrocytes. Apigenin's suppression of ERK, NF-κB, and STAT3 activations was responsible for the inhibition of IL-31 and IL-33, while luteolin's suppression of JNK, p38, ERK, NF-κB, and STAT3 activations was responsible for the inhibition of IL-31 in the astrocytes. Also, luteolin's suppression of ERK, NF-κB, and STAT3 activations inhibited IL-33 production in the activated astrocytes. In addition, apigenin and luteolin also prevented the translocation of activated STAT3 and NF-κB to the nucleus of the activated astrocytes and subsequently affected their DNA binding activities. The results suggest that apigenin and luteolin may have potentials as neuroprotective agents for the treatment of diseases involving astrocyte activation and detrimental production of IL-31 and IL-33.

Molecular correlates of PD-L1 expression in patients (pts) with gastroesophageal (GE) cancers.

4558 Background: The increased PD-L1 expression evaluated by combined positive score (CPS) is associated with improved efficacy of immunotherapy in GE cancers. The impact of tumor molecular alterations on PD-L1 expression is still not well-studied. We aimed to characterize specific molecular features of tumors with different CPS levels in GE cancers. Methods: 2,707 GE tumors [1,662 gastric/GE junction adenocarcinoma (GA), 856 esophageal adenocarcinoma (EA), 75 esophageal squamous (ES) and 114 GE unspecified] collected between 2000.8 and 2019.7 were analyzed using NextGen DNA sequencing (NGS), immunohistochemistry (IHC) and fragment analysis (FA) (Caris Life Sciences, Phoenix, AZ). Tumor mutation burden (TMB) was calculated based on somatic nonsynonymous missense mutations. dMMR/MSI status was evaluated by a combination of IHC, FA and NGS. PD-L1 expression measured by IHC (22c3) was evaluated by CPS scores. Molecular alterations were compared in three groups (CPS ≥ 10, H; CPS = 1~9, M; CPS = 0, L) using Fisher-Exact or Chi-square and adjusted for multiple comparison by Benjamini-Hochberg. Significance was determined by adjusted (adj) p &lt; .05. Results: Overall, CPS-H, M, and L were seen in 18% (n = 494), 28% (n = 765) and 53% (n = 1,448) of GE tumors respectively. CPS-H was the most prevalent in ES (43%) followed by GA (19%) and lowest in EA (14%). Overall, TMB was similar between CPS-L and M, but was significantly increased in H (average TMB = 8.4 vs. 8.6 vs. 11 mt/MB, adj p &lt; .0001); the effect was seen in EA and GA, but not in ES. An overall significant association between MSI/dMMR status and PD-L1 expression levels was seen (2%, 3.2% and 12% in CPS-L, M and H, adj p &lt; .05) in GE tumors; the significance was seen in GA, but not in EA or ES. Amplifications of PD-L1 (H: 1.5%, M: 0.1% and L: 0) and PD-L2 (H: 1.1%, M: 0.1%, L: 0) were the highest in CPS-H, while ASPSCR1 (H: 0, M: 0, L: 1%) and TNFRSF14 (H: 0, M: 0.4, L: 2%) were the lowest (adj p &lt; .01). Genes involved in epigenetic modification (top 5: ARID1A, ASXL1, BCL9, BCOR, CREBBP), MAPK ( KRAS, MAP2K1) and mismatch repair ( MLH1, MSH6) had the highest mutation rates in CPS-H, compared to M and L ( p &lt; .0001). In contrast, CDH1 had higher mutation rates in CPS-L (12%), compared to M and H (5% and 5%) ( p &lt; .0001). Conclusions: This is the largest study to investigate the distinct molecular landscape of pts with different PD-L1 expression levels in GE cancers. Our data may provide novel insights for pt selection (e.g. pts with gene mutations involved in epigenetic modification) and the development of rational combination immunotherapy (e.g. drugs targeting MAPK pathway).

Predictive Gene Signature for Pyrazolopyrimidine Derivative c-Src Inhibitor 10a Sensitivity in Melanoma Cells.

Melanoma is a highly aggressive cancer with poor prognosis. Although more than 80% of melanomas harbor an activating mutation in genes within the MAPK pathway, which are mutually exclusive, usefulness of therapies targeting MAPK pathway are impeded by innate and/or acquired resistance in most patients. In this study, using melanoma cells, we report the efficacy of a recently developed pyrazolo[3,4-d]pyrimidine derived c-Src inhibitor 10a and identify a molecular signature which is predictive of 10a chemosensitivity. We show that the expression of TMED7, PLOD2, XRCC5, and NSUN5 are candidate biomarkers for 10a sensitivity. Although an undifferentiated/mesenchymal/invasive status of melanoma cells is associated with resistance to 10a, we show here for the first time that melanoma cells can be sensitized to 10a via treatment with valproic acid, a histone deacetylase inhibitor.

Co-occurring Alterations in the RAS-MAPK Pathway Limit Response to MET Inhibitor Treatment in MET Exon 14 Skipping Mutation-Positive Lung Cancer.

Although patients with advanced-stage non-small cell lung cancers (NSCLC) harboring MET exon 14 skipping mutations (METex14) often benefit from MET tyrosine kinase inhibitor (TKI) treatment, clinical benefit is limited by primary and acquired drug resistance. The molecular basis for this resistance remains incompletely understood. Targeted sequencing analysis was performed on cell-free circulating tumor DNA obtained from 289 patients with advanced-stage METex14-mutated NSCLC. Prominent co-occurring RAS-MAPK pathway gene alterations (e.g., in KRAS, NF1) were detected in NSCLCs with METex14 skipping alterations as compared with EGFR-mutated NSCLCs. There was an association between decreased MET TKI treatment response and RAS-MAPK pathway co-occurring alterations. In a preclinical model expressing a canonical METex14 mutation, KRAS overexpression or NF1 downregulation hyperactivated MAPK signaling to promote MET TKI resistance. This resistance was overcome by cotreatment with crizotinib and the MEK inhibitor trametinib. Our study provides a genomic landscape of co-occurring alterations in advanced-stage METex14-mutated NSCLC and suggests a potential combination therapy strategy targeting MAPK pathway signaling to enhance clinical outcomes.

Abstract 1283: Targeting c-MYC and MAPK pathway to overcome pancreatic cancer drug resistance

Abstract Background: Acquired resistance to systemic chemotherapy is the main complication in pancreatic ductal adenocarcinoma (PDAC) treatment. Although there are studies focused on gemcitabine resistance mechanisms, our understanding of the mechanisms of nab-paclitaxel (n-PTX) treatment failure remains extremely limited. To enhance the use of properly powered patient-derived platforms, we adopted the conditionally reprogrammed (CR) cell culture technique in order to develop both parental and nab-PTX-resistant cells. The CR approach allowed us to identify the critical role of c-MYC and ERK in the PDAC drug response. Small molecule activators of PP2A (SMAPS) have showed activity in inhibiting lung KRAS-mutant tumor growth. We used SMAPS as new therapeutic agents in PDAC, for its ability to alter c-MYC activity through PP2A dysregulation and enhance PDAC sensitivity to n-PTX. Methods: Long-term cultures of PDAC CRs were established from treatment-naive PDAC patients’ biopsies, and used to generate drug-resistant cells. Zebrafish and mouse model were used to test the cells’ ability to form tumors and to verify the drug resistance in vivo. Molecular analyses were used to characterize the drug-resistant cells and to identify key pathways involved in the drug resistance evolution. Genomic and chemical alterations of the key proteins were used to confirm the involvement in the drug resistance mechanism. We regulated the expression of c-MYC and ERK using SMAPS as a new targeting agent and trametenib to verify the direct correlation between c-MYC and ERK and the drug resistance mechanism. Results: Using the credentialed KRAS-mutant CR cultures, we generated n-PTX-resistant cell lines. The parental and nab-PTX resistant cells were subjected to subcutaneous injections in nude mice, and formed tumors in 2-3 weeks. Histological evaluation showed that the CRs self-assembled into ductal structures, surrounded by a desmoplastic stromal microenvironment that faithfully recapitulates human PDAC. Resistant profiles were verified both in mouse and Zebrafish model. RNA microarrays identified a sustained induction of a pro-inflammatory pathway leading to c-MYC overexpression. c-MYC silencing and overexpression confirmed the role of c-MYCin the evolution of nab-PTX resistance. Treatment of the resistant CRs with either trametenib or with SMAPS resulted in enhanced sensitivity to nab-PTX. We furtherverified that the enhanced sensitivity was commensurate with a reduction in p-Erk and c-Myc. Conclusion: The CR methodology addresses the need for a reliable method for generating primary cell lines on a single patient basis. The ability to rapidly model in vitro, and verify in vivo, that the overexpression of c-MYC contributes to the development of n-PTX resistance is a significant advancement in the field. Our data showed that SMAPs or trametanib overcome a significant component of the n-PTX resistance providing new hope for refractory PDAC. Citation Format: Erika Maria Parasido, George S. Avetian, Jonathan Brody, Jordan Winter, Eric Londin, Michael Pishvaian, Eric Glasgow, Stephen Byers, Goutham Narla, Christopher Albanese. Targeting c-MYC and MAPK pathway to overcome pancreatic cancer drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1283.

Abstract 4475: Targeting c-MET and MAPK pathway in metastatic castration-resistant prostate cancer

Abstract Identification of “agnostic” genetic drivers in cancer is foreseen as a major step forward in precision medicine. Thus, treatment selection could be based on the molecular background of the disease rather than on tumor histology in the near future. However clinical validation of most alterations is lacking and only preclinical models are available. We describe a prostate cancer patient refractory to standard therapies that was found to harbor both a c-Met activating mutation and a BRAF fusion that greatly responded to the MEK inhibitor (MEKi) trametinib. EXPERIMENTAL PROCEDURES The Next Generation Sequencing (NGS) platforms (Oncomine Assay and Archer Variant Plex) were used to characterize molecularly FFPE tissue from the primary tumor of a 73 years old man with metastatic castration-resistant prostate cancer (mCRPC) refractory to Docetaxel, Enzalutamide and Radium 223. Immunostaining was performed to evaluate the status of protumoral pathways activated by the identified genomic alterations. RESULTS Oncomine assay detected a pathogenic c-MET mutation that affected the splicing of exon 14 and the patient was included in a clinical trial with a c-MET inhibidor. He developed Grade 3 anemia, G3 bone pain which required major opioids, a rise of PSA over 900 ng/ml and progressive disease as best response. An extended screening using Archer platform detected a SND1/BRAF fusion. Immunohistochemistry revealed homogeneous tumor positive staining for c-MET, and a punctured staining on pERK in FFPE regions with perineural tumor invasion suggesting a critical role of the BRAF fusion in the spread of the disease. After a comprehensive review of the literature a similar model was identified, where the SND1/BAF function raised in a cell culture of gastric cancer as mechanism of resistance to MET inihibitors (Lee NV et al PLoS One 2012). In preclinical experiments the MEKi trametinib induced significant cell mortality. After given written consent, the patient was put on trametinib (2mg/day) under compassionate use. We observed a large decrease of PSA levels to 90 ng/ml after two weeks of treatment, an increase of hemoglobin levels and significant clinical benefit, with no need of analgesics. After two months on treatment the patient continues on tumor response by the time of communication of this abstract. CONCLUSIONS According to our knowledge, this is the first published study able to demonstrate that cancer patients harboring c-MET exon 14 splicing mutation and SND1-BRAF fusion genes could benefit from therapies with MEK inhibitors. Further experimental procedures are needed to validate this hypothesis. 1. Lee NV, Lira ME, Pavlicek A, Ye J, Buckman D, et al. (2012) A Novel SND1-BRAF Fusion Confers Resistance to c-Met Inhibitor PF-04217903 in GTL16 Cells though MAPK Activation. PLoS ONE 7(6): e39653. doi:10.1371/journal.pone.0039653. Citation Format: Sergio Ruiz, María Dolores Fenor de la Maza, Juan Francisco Rodríguez Moreno, Elena Sevillano, Paloma Navarro, Sandra Amarilla, Teresa García Donas, Eduardo Caleiras, Juan Carlos Torrego, Jesus García Donas. Targeting c-MET and MAPK pathway in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4475.

Targeting MAPK Pathways by Naringenin Modulates Microglia M1/M2 Polarization in Lipopolysaccharide-Stimulated Cultures.

Neuroinflammation is considered to be an important and inevitable pathological process associated with all types of damages to, and disorders of, the central nervous system. The hallmark of neuroinflammation is the microglia activation. In response to different micro-environmental disturbances, microglia could polarize into either an M1 pro-inflammatory phenotype, exacerbating neurotoxicity, or an M2 anti-inflammatory phenotype, exerting neuroprotection. Therefore, shifting the polarization of microglia toward the M2 phenotype could possess a more viable strategy for the neuroinflammatory disorders treatment. Naringenin (NAR) is naturally a grapefruit flavonoid and possesses various kinds of pharmacological activities, such as anti-inflammatory and neuroprotective activities. In the present study, we aimed to investigate the potential effects of NAR on microglial M1/M2 polarization and further reveal the underlying mechanisms of actions. First, NAR inhibited lipopolysaccharide (LPS)-induced microglial activation. Then, NAR shifted the M1 pro-inflammatory microglia phenotype to the M2 anti-inflammatory M2 microglia state as demonstrated by the decreased expression of M1 markers (i.e., inducible TNF-α and IL-1β) and the elevated expression of M2 markers (i.e., arginase 1, IL-4, and IL-10). In addition, the effects of NAR on microglial polarization were dependent on MAPK signaling, particularly JNK inactivation, as evidenced by the fact that the selective activator of JNK abolished NAR-promoted M2 polarization and further NAR-inhibited microglial activation. Together, this study demonstrated that NAR promoted microglia M1/M2 polarization, thus conferring anti-neuroinflammatory effects via the inhibition of MAPK signaling activation. These findings might provide new alternative avenues for neuroinflammation-related disorders treatment.

Acquired resistance to BRAFi reverses senescence-like phenotype in mutant BRAF melanoma.

Targeting MAPK pathway in mutant BRAF melanoma with the specific BRAF inhibitor vemurafenib showed robust initial responses in the majority of patients followed by relapses due to acquired resistance to the drug. In V600EBRAF melanoma cell lines, senescence-associated β-galactosidase activity is often encountered in a constitutive manner or induced after MAPK inhibition. However, the link between the senescence-like phenotype and the resistance to BRAF inhibition is not fully understood yet. Our data validate a senescence-like phenotype (low cell proliferation, high cell volume, and high β-Gal activity) in mutant BRAF cells. Vemurafenib increased β-Gal activity in 4 out of 5 sensitive lines and in 2 out of 5 lines with intrinsic resistance to the drug. Interestingly, the 3 lines with acquired resistance to vemurafenib became depending on the drug for proliferation. In absence of drug, these lines showed a lower cell proliferation rate together with a substantial increase of β-Gal activity both in vitro and in vivo. In all settings, the senescence-like phenotype was significantly associated with an inhibition of pRB and cyclin D1, explaining the inhibition of cell proliferation. In conclusion, β-Gal activity is increased by V600EBRAF inhibition in the majority of sensitive and intrinsically resistant melanoma cells. Acquired resistance to vemurafenib is associated with a dependence to the drug for cell proliferation and tumor growth, and, in this case, drug removal stimulate β-Gal activity suggesting that the senescence-like phenotype could contribute to the acquired resistance to BRAF inhibition.

The pyrazolyl-urea GeGe3 inhibits tumor angiogenesis and reveals dystrophia myotonica protein kinase (DMPK)1 as a novel angiogenesis target.

The limitation of targeting VEGF/VEGFR2 signalling to stop angiogenesis in cancer therapy has been blamed on re-activation of alternative receptor tyrosine kinases by compensatory angiogenic factors. Targeting MAPK and PI3K signaling pathways in endothelial cells may be an alternative or complementary approach. Herein we aimed to evaluate the antitumor and antiangiogenic potential of a novel pyrazolyl-urea kinase inhibitor, GeGe3, and to identify its kinase targets.We found GeGe3 to inhibit the proliferation of HUVEC and endothelial tube formation. GeGe3 impaired inter-segmental angiogenesis during development of zebrafish embryos. In mice, GeGe3 blocked angiogenesis and tumor growth in transplanted subcutaneous Lewis Lung Carcinomas. Screening for GeGe3-targeted kinases revealed Aurora B, Aurora C, NEK10, polo-like kinase (PLK)2, PLK3, DMPK1 and CAMK1 as candidate targets. Biochemical analysis of these kinases showed DMPK1 regulation upon VEGF challenge. Investigation of the role of DMPK1 in endothelial cells revealed DMPK1 as a novel mediator of angiogenesis that controls the activation of MAPK signaling, proliferation and migration. GeGe3 alters angiogenesis by targeting DMPK in tumor endothelial cells and pericytes.The pyrazolyl-urea GeGe3, a novel blocker of MAPK and PI3K pathways, strongly inhibits physiological and tumor angiogenesis. We also report GeGe3-targeted kinase DMPK as a novel mediator of angiogenesis.

Gap junction protein connexin43 deregulation contributes to bladder carcinogenesis via targeting MAPK pathway

High expression of connexins was found in a variety of cancers, but their role is still controversial. We investigated whether connexin43 (Cx43) contributed to bladder carcinogenesis through MAPK activation. In this study, we found that Cx43 expression was significantly increased in bladder cancer tissues and cell line. Overexpression of Cx43 in bladder cancer 5637 cells increased cell proliferation, promoted cell cycle progression, and inhibited apoptosis. Western blot showed that JNK and ERK pathways were dramatically activated in Cx43-overexpressed cells. Conversely, knockdown of Cx43 inhibited cell proliferation by increasing apoptosis and causing cell cycle arrest, concomitant with inhibition of JNK and ERK signaling. In addition, JNK and ERK pathways were also activated in bladder cancer tissues. In conclusion, abnormal high expression and cytoplasmic localization of Cx43 contributed to bladder cancer. Inhibition of Cx43 activity could be a potential therapeutic strategy for preventing the progression of bladder cancer.