Inhibition Of Mitogen-activated Protein Kinase Signaling Research Articles

Tectorigenin inhibits inflammatory responses in murineinflammatory bowel disease and LPS-stimulated macrophages via inactivating MAPK signaling pathway.

Considering the antihepatitis effects of Tectorigenin (TEC), and the same adenosine mitogen-activated protein kinase (MAPK) pathway in both hepatitis and inflammatory bowel disease (IBD) models, exploring the role of TEC in IBD is contributive to develop a new treatment strategy against IBD. The IBD mouse model was constructed by feeding with dextran sodium sulfate (DSS) and injection of TEC. Afterward, the mouse body weight, colon length, and disease activity index (DAI) were tested to assess the enteritis level. Mouse intestine lesions were detected by hematoxylin and eosin staining. Murine macrophages underwent lipopolysaccharide (LPS) induction to establish an inflammation model. Cell viability was determined by cell counting kit-8 assay. Enzyme-linked immunosorbent assay was performed to measure interleukin 6(IL-6) and tumor necrosis factor-α (TNF-α) levels. Cyclooxygenase-2(COX-2) and inducible nitric oxide synthase(iNOS) expressions were quantified via quantitative reverse transcription polymerase chain reaction. Levels of MAPK pathway-related proteins (p-P38, P38, p-Jun N-terminal kinase(JNK), JNK, signal-regulated kinase(ERK), p-ERK), COX-2 and iNOS were quantitated by Western blot. TEC improved the inflammatory response through ameliorating weight loss, shortening colon, and increasing DAI score in IBD mouse. Expressions of intestinal inflammatory factors (IL-6, TNF-α, iNOS and COX-2) and MAPK pathway-related proteins (p-P38, p-JNK, and p-ERK) were increased both in DSS-induced mouse intestinal tissue, but TEC inhibited expressions of inflammatory factors. The same increased trend was identified in LPS-induced macrophages, but TEC improved macrophage inflammation, as evidenced by downregulation of inflammatory factors. TEC mitigates IBD and LPS-induced macrophage inflammation in mice via inhibiting MAPK signaling pathway.

Abstract 6506: SOS1 inhibition in combination with HER2 inhibition in models of HER2-driven cancer

Abstract Cancers driven by amplifications or mutations of the gene ERBB2 (coding for the receptor tyrosine kinase (RTK) HER2) have been the focus of HER2-targeting antibodies as well as small molecule inhibitors. Despite initial efficacy, resistance to these HER2-targeted therapies inevitably develops. One strategy to tackle this resistance is to identify rational combinations to address common pathways/factors that contribute to resistance. One such common mechanism of resistance is reactivation of the Mitogen-activated protein kinase (MAPK) pathway. SOS1 is a guanine nucleotide exchange factor (GEF) which regulates the MAPK pathway by catalyzing the exchange of RAS-GDP (inactive) to RAS-GTP (active). SOS1 is activated by multiple different RTKs - upon RTK-ligand binding, autophosphorylation of the RTK recruits the GRB2-SOS1 protein complex to the plasma membrane, enabling the complex to bind and activate RAS-GDP. Given SOS1’s role downstream of RTKs and upstream of the MAPK pathway, we sought to investigate the potential of a SOS1 inhibitor (SOS1i) in combination with a HER2 inhibitor (HER2i) in preclinical models of cancer driven by HER2 mutation or gene amplification. Using in vitro and in vivo models we found evidence of added benefit of SOS1i in combination with HER2i, as well as enhanced inhibition of MAPK signaling, as seen by decrease in ERK activation and expression of MAPK effector genes such as DUSP6. Taken together, we propose that SOS1 inhibition combined with HER2 inhibition may be a rational strategy for deeper and more prolonged responses to HER2 inhibitor treatment by preventing resistance via the MAPK pathway. Citation Format: Robyn L. Schenk, Kaja Kostyrko, Johannes Popow, Christina Gewinner, Anke Baum, Christoph Albrecht, Melanie Hinkel, Robin Jacob, Tom Madensky, Regina Ruzicka, Eva Strauss, Hengyu Lyu, Anastasia M. Lopez, Ningping Feng, Christopher P. Vellano, Joseph R. Marszalek, Timothy P. Heffernan, Mark Pearson, Marco H. Hofmann. SOS1 inhibition in combination with HER2 inhibition in models of HER2-driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6506.

Tolperisone hydrochloride improves motor functions in Parkinson’s disease via MMP-9 inhibition and by downregulating p38 MAPK and ERK1/2 signaling cascade

The mitogen-activated protein kinase (MAPK) signaling pathway, particularly the p38 MAPK and ERK1/2, has been implicated in the pathogenesis of Parkinson's disease (PD). Recent studies have shown that MAPK signaling pathway can influence the expression of matrix metalloproteinase 9 (MMP-9), known for its involvement in various physiological and pathological processes, including neurodegenerative diseases. This study explores the modulation of MMP-9 expression via the MAPK/ERK signaling cascade and its potential therapeutic implications in the context of PD-associated motor dysfunction. Here, tolperisone hydrochloride (TL), a muscle relaxant that blocks voltage-gated sodium and calcium channels, was used as a treatment to observe its effect on MAPK signaling and MMP-9 expression. Rotenone (RT) exposure in mice resulted in a significant reduction in substantia nigra and primary motor cortex neurons, which were further evidenced by impairments in motor function. When TL was administered, neuron count was restored (89.0 ± 4.78 vs 117.0 ± 4.46/mm2), and most of the motor dysfunction was alleviated. Mechanistically, TL reduced the protein expression of phospho-p38MAPK (1.06 fold vs 1.00 fold) and phospho-ERK1/2 (1.16 fold vs 1.02 fold), leading to the inhibition of MAPK signaling, as well as reduced MMP-9 concentrations (2.76 ± 0.10 vs 1.94 ± 0.10 ng/mL) in the process of rescuing RT-induced neuronal cell death and motor dysfunction. Computational analysis further revealed TL’s potential inhibitory properties against MMP-9 along with N and L-type calcium channels. These findings shed light on TL's neuroprotective effects via MMP-9 inhibition and MAPK signaling downregulation, offering potential therapeutic avenues for PD-associated motor dysfunction.

Two Main Cancer Biomarkers as Molecular Targets of Binase Antitumor Activity.

Cancer is frequently coupled with the disturbance of key signaling pathways. Aberrant activation of the mitogen-activated protein kinase (MAPK) signaling cascade, occurring in over 85% of cancers, is mainly caused by the genetic alterations of its main components-oncogenes EGFR and RAS, and plays a crucial role in cell fate. The importance of EGFR and RAS proteins in a variety of tumors suggests that they would be good therapeutic targets, but at present, no effective targeted therapy against these two oncogenes has been proven. Here, we show that ribonuclease from Bacillus pumilus (binase) inhibits MAPK signaling through direct interaction with EGFR and RAS proteins. This effect contributes to the antitumor potential of binase along with its enzymatic activity. Multitargeticity of binase prevents the development of drug resistance, which is considered a major obstacle to effective anticancer treatment.

Effect of miR-21 Inhibitor Coated with Ferric Oxide on Ovarian Cancer Cells Through Mitogen-Activated Protein Kinase Signaling Pathway

Ovarian cancer (OC) incidence has in recent years been on the rise among common gynecological cancers. Iron oxide nanoparticles (IONPs), as emerging nanomedicines, have been widely used in research on cancer therapy. However, with in-depth research on microRNA (miRNA), the effect of combining miRNA with nanoparticles on ovarian cancer is not yet clear. We deeply explored the mechanism of IONPs loading miRNA to regulate ovarian cancer cells. In this study, novelmiR-21 inhibitor-IONPs nanoparticles were prepared by loading miR-21 inhibitor into IONPs. Electron microscopy was used to observe nano-encapsulation and miR-21 expression was measured along with analysis of cell proliferation, apoptosis by flow cytometry, and phosphorylation of apoptotic proteins and mitogen-activated protein kinase (MAPK) signaling pathways by Western blot. Our results showed that miR-21 inhibitor-IONPs reduced miR-21 expression, thereby inhibiting ovarian cancer cells activities and promoting apoptosis. miR-21 inhibitor-IONPs also inhibited p-p38MAPK and p-ERK levels, which were increased after addition of MAPK agonist (U-46619). Moreover, ovarian cancer cell proliferation increased and apoptosis decreased. miR-21 inhibitor-IONPs can thus inhibit MAPK signaling, thereby reducing the activities of ovarian cancer cells. This study provides theoretical support for application of miR-21 inhibitor-IONPs as novel nanoparticles for the treatment of ovarian cancer.

The SMAC mimetic birinapant alleviates lipopolysaccharide-induced acute lung injury by inhibiting MAPK signaling

The second mitochondria-derived activator of caspases (SMAC) mimetic birinapant attenuated liver injury by inhibited the degradation of tumor necrosis factor receptor-associated factor 3 (TRAF3) and activation of mitogen-activated protein kinase (MAPK) signaling pathway in liver macrophage, but its role in LPS induced acute lung injury (ALI) is not understood. The present study was to investigate the effects of birinapant on ALI and its possible mechanism. A dose of birinapant (30 mg/kg) or a vehicle was administered intravenously 24 hours before LPS (100 μg) stimulation in mice. The levels of TNF-α, IL-6 and IL-1β in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The infiltrated macrophages and expression of monocyte chemoattractant protein-1 (MCP-1) was determined by immunohistochemistry staining in the lung tissues. The JNK and p38 MAPK activation, protein expression and K48-linked polyubiquitination of TRAF3 were determined in alveolar macrophage cell line (MH-S cells) after 1μg/ml LPS stimulation. The results showed that the birinapant down-regulated the levels of TNF-α, IL-6 and IL-1β in the BALF. In addition, birinapant markedly inhibited macrophages infiltration and MCP-1 protein expression in lung tissues. At last, birinapant suppressed the MAPKsignaling pathway and K48-linked ubiquitinated degradation of TRAF3 in MH-S cells after LPS administration. In conclusion, the results proved that birinapant protected against LPS-induced ALI through inhibiting MAPK activation and K48-linked ubiquitination of TRAF3 in alveolar macrophages.

An NFATc1/SMAD3/cJUN Complex Restricted to SMAD4-Deficient Pancreatic Cancer Guides Rational Therapies

Background and AimsThe highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology but contradicts the current concept of one size fits all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and the therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the Nuclear Factor of Activated T-cells (SMAD4-/-/NFATc1High). MethodsTranscriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of the SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. ResultsOur findings determine the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition (MEKi) normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. ConclusionOur results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a MEKi/gemcitabine combination therapy.

Development of a MEK inhibitor, NFX-179, as a chemoprevention agent for squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. Although cSCC contributes to substantial morbidity and mortality in high-risk individuals, deployment of otherwise effective chemoprevention of cSCC is limited by toxicities. Our systematic computational drug repurposing screen predicted that selumetinib, a MAPK (mitogen-activated protein kinase) kinase inhibitor (MEKi), would reverse transcriptional signatures associated with cSCC development, consistent with our genomic analysis implicating MEK as a chemoprevention target. Although systemic MEKi suppresses the formation of cSCC in mice, systemic MEKi can cause severe adverse effects. Here, we report the development of a metabolically labile MEKi, NFX-179, designed to potently and selectively suppress the MAPK pathway in the skin before rapid metabolism in the systemic circulation. NFX-179 was identified on the basis of its biochemical and cellular potency, selectivity, and rapid metabolism upon systemic absorption. In our ultraviolet-induced cSCC mouse model, topical application of NFX-179 gel reduced the formation of new cSCCs by an average of 60% at doses of 0.1% and greater at 28 days. We further confirmed the localized nature of these effects in an additional split-mouse randomized controlled study where suppression of cSCC was observed only in drug-treated areas. No toxicities were observed. NFX-179 inhibits the growth of human SCC cell lines in a dose-dependent manner, and topical NFX-179 application penetrates human skin and inhibits MAPK signaling in human cSCC explants. Together, our data provide a compelling rationale for using topical MEK inhibition through the application of NFX-179 gel as an effective strategy for cSCC chemoprevention.

PKC-δ Promotes IL-1β-Induced Apoptosis of Rat Chondrocytes and Via Activating JNK and P38 MAPK Pathways.

Protein kinase C-delta (PKC-δ) is involved in apoptosis. This study aimed to establish whether PKC-δ can further promote IL-1β-induced chondrocyte apoptosis by mediating the phosphorylation of the JNK and p38 mitogen-activated protein kinase (MAPK) signaling pathways In osteoarthritis (OA). We employed chondrocyte staining to determine the extent of cartilage degeneration. PKC-δ and p38 signal expressions were used in the immunohistochemical (IHC) test and apoptosis was assayed at the TUNEL test in human osteoarthritic and controls. We stimulated rat cartilage cells using IL-1β (10 ng/ml)/rottlerin (10 μM) or lentivirus. To determine the apoptosis rate, we employed flow cytometry. The mRNA of both BCL2-related X (BAX) and cysteine aspartate protease 3 (caspase-3) could be measured via qRT-PCR. Western blot measured the protein levels of BAX, caspase-3, PKC-δ, p-JNK/JNK and p-p38/p38. The positive rate of PKC-δ and the apoptotic rate of chondrocytes in OA were higher than controls. The manifestation of PKC-δ was positively related to the degree of cartilage degeneration, p38 protein expression, and apoptosis rate. IL-1β exposure upregulated PKC-δ expression in chondrocytes in a dose-dependent manner. Decreasing PKC-δ expression and its phosphorylation in OA can inhibit MAPK signaling pathway activation (phosphorylation) by downregulating JNK and p38 protein phosphorylation and expression. This inhibition decreases caspase-3 and BAX levels, consequently lowering the apoptosis rate in chondrocytes. PKC-δ activation by IL-1β in OA promotes chondrocyte apoptosis via activation of the JNK and p38 MAPK signal pathways, thereby promoting the OA progression.

TRIM22 promotes the proliferation of glioblastoma cells by activating MAPK signaling and accelerating the degradation of Raf-1

The tripartite motif (TRIM) 22 and mitogen-activated protein kinase (MAPK) signaling pathways play critical roles in the growth of glioblastoma (GBM). However, the molecular mechanism underlying the relationship between TRIM22 and MAPK signaling remains unclear. Here, we found that TRIM22 binds to exon 2 of the sphingosine kinase 2 (SPHK2) gene. An ERK1/2-driven luciferase reporter construct identified TRIM22 as a potential activator of MAPK signaling. Knockout and overexpression of TRIM22 regulate the inhibition and activation of MAPK signaling through the RING-finger domain. TRIM22 binds to Raf-1, a negative regulator of MAPK signaling, and accelerates its degradation by inducing K48-linked ubiquitination, which is related to the CC and SPRY domains of TRIM22 and the C1D domain of Raf-1. In vitro and in vivo, an SPHK2 inhibitor (K145), an ERK1/2 inhibitor (selumetinib), and the nonphosphorylated mutant Raf-1S338A inhibited GBM growth. In addition, deletion of the RING domain and the nuclear localization sequence of TRIM22 significantly inhibited TRIM22-induced proliferation of GBM cells in vivo and in vitro. In conclusion, our study showed that TRIM22 regulates SPHK2 transcription and activates MAPK signaling through posttranslational modification of two critical regulators of MAPK signaling in GBM cells.

Neferine alleviates ovalbumin-induced asthma via MAPK signaling pathways in mice.

To investigate the role of neferine in ovalbumin (OVA)-induced asthma, and to reveal the possible mechanism. In OVA-induced asthmatic mice, enzyme-linked-immunosorbent serologic assay was performed to evaluate the level of interleukin (IL)-4, IL-5, IL-13, immunoglobulin E (IgE) in serum and tumor necrosis factor-α (TNF-α), IL-6, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) in bronchoalveolar lavage fluid (BALF). Eosinophil, neutrophil, and lymphocyte counts in BALF were calculated to assess inflammation. The pulmonary function was measured by airway resistance, peak expiratory flow (PEF) and forced expiratory volume/forced vital capacity (FEV0.4/FVC) ratio, and respiratory rate. Hematoxylin and eosin staining and Masson staining were used to evaluate lung injury. Further, Western blot analysis was conducted to detect phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 of mitogen-activated protein kinase (MAPK) signaling pathways. Neferine, 20 mg/kg or 40 mg/kg, could significantly decrease the levels of IL-4, IL-5, IL-13, and IgE in OVA-induced serum, and that of TNF-α, IL-6, IL-1β, and MCP-1 in OVA-induced BALF. Moreover, neferine could significantly decline eosinophil, neutrophil, and lymphocyte counts in BALF. Neferine contributed to improve OVA-induced airway resistance, promoted the value of PEF and FEV0.4/FVC ratio, and recovered the respiratory rate. It also reduced mucus secretion, distribution of inflammatory and goblet cells around bronchi, and attenuated collagen deposition in lung tissues. Furthermore, neferine reduced the phosphorylation of p38, JNK, and ERK to inhibit MAPK signaling pathways. Neferine relieves asthma-induced inflammatory reaction, airway resistance, and lung injury by inhibiting MAPK signaling pathways. This could serve neferine as a novel therapeutic candidate for treating asthma.

Roles of p38 MAPK signalling in intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is a common degenerative disease mediated by multiple factors. Because of its complex aetiology and pathology, no specific molecular mechanisms have yet been identified and no definitive treatments are currently available for IVDD. p38 mitogen-activated protein kinase (MAPK) signalling, part of the serine and threonine (Ser/Thr) protein kinases family, is associated with the progression of IVDD, by mediating the inflammatory response, increasing extracellular matrix (ECM) degradation, promoting cell apoptosis and senescence and suppressing cell proliferation and autophagy. Meanwhile, the inhibition of p38 MAPK signalling has a significant effect on IVDD treatment. In this review, we first summarize the regulation of p38 MAPK signalling and then highlight the changes in the expression of p38 MAPK signalling and their impact on pathological process of IVDD. Moreover, we discuss the current applications and future prospects of p38 MAPK as a therapeutic target for IVDD treatment.

Osteoprotegerin deficiency aggravates methionine–choline-deficient diet-induced nonalcoholic steatohepatitis in mice

Clinical studies have shown that osteoprotegerin (OPG) is reduced in patients with nonalcoholic steatohepatitis (NASH), but the underlying mechanisms are unclear. The current study focuses on the role of OPG in the NASH pathogenesis. OPG knockout mice and wild-type control mice fed a methionine choline-deficient diet (MCD) for 4 weeks resulted in an animal model of NASH. Measurement of triglycerides (TG) in serum and liver to assess steatosis. Hematoxylin eosin (HE), Sirius Red and Masson staining were used to assess the liver damage. Transcriptome sequencing analysis, qPCR and western blot were to analyze changes in lipid metabolism and inflammation-related indicators in the liver. In vivo knockout of OPG resulted in a reduction of TG levels in the liver and a significant increase in serum ALT and AST. The expression of inflammatory factors and fibrosis genes was significantly upregulated in the livers of OPG knockout mice. Transcriptome sequencing analysis showed that OPG knockout significantly enhanced MCD diet-induced activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Mechanistically, OPG may inhibit MAPK signaling pathway activity by upregulating the expression of dual specificity phosphatase 14 (DUSP14), thereby reducing inflammatory injury. OPG could regulate the activity of the MAPK signaling pathway via DUSP14, thus regulating the expression of some inflammatory factors in NASH, it may be a promising target for the treatment of NASH.

Aspergillus cristatus attenuates DSS-induced intestinal barrier damage through reducing the oxidative stress, regulating short-chain fatty acid and inhibiting MAPK signaling pathways.

Probiotics are regarded as a promising strategy for relieving colitis caused by dextran sulfate sodium (DSS). One of the dominant probiotic fungi in Fuzhuan brick tea is identified as Aspergillus cristatus, but whether it can effectively improve colitis remains poorly understood. Here, the improving effect of A. cristatus on colitis was investigated. Our results showed that A. cristatus intervention prominently alleviated gut damage as evidenced by the inhibition of shortened colon length, goblet cell depletion, and histological injury. Mechanistically, after administration with low concentrations of A. cristatus H-1 and A. cristatus S-6, the expression of interleukin-6, tumor necrosis factor-α, interleukin-1β, nitric oxide, and malondialdehyde were significantly downregulated, and the content of glutathione, catalase, interleukin-10, immunoglobulin G, claudin-1, occludin, and zonula occludens-1 were effectively upregulated. More importantly, live A. cristatus supplementation lightened DSS-induced gut barrier damage by suppressing activation of the mitogen-activated protein kinase (MAPK) signaling pathway, increasing the synthesis of short-chain fatty acids (SCFAs) and stimulating the increase in peroxisomeproliferator-activated receptor γ expression. Together, A. cristatus can attenuate DSS-induced intestinal barrier damage through reducing the oxidative stress, regulating SCFA and inhibiting MAPK signaling pathways (P38/JNK/ERK). Our findings indicate that A. cristatus replenishment has potential as a new probiotic fungi to reduce DSS-induced colitis. © 2022 Society of Chemical Industry.

Exercise training combined with alprostadil improves myocardial infarction and coronary microcirculation disorder in aged rats by inhibiting mitogen-activated protein kinase (MAPK) signaling pathway activation.

We aimed to explore the effects and mechanisms of exercise training combined with alprostadil (ALPR) treatment on myocardial infarction (MI) in aged rats. Male Wistar rats were randomly divided into five groups. One day after MI induction, an automatic biochemical analyzer was used to measure cardiac troponin I (cTnI), cardiac troponin T (cTnT), and creatine kinase MB isoenzyme (CK-MB) serum levels. One week after MI induction, echocardiography was performed to examine the left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF), and left ventricular fraction shortening (LVFS) rates of the rats. Parameters such as body weight (BW), heart mass index, and the heart weight (HW)/tibia length (TL) ratio of the rats were also calculated. Western blot was performed to assess angiogenesis and mitogen-activated protein kinase (MAPK) signal-related protein expression. Compared with the MI group, the LVEDD and LVESD in the Trained + ALPR group were significantly decreased, while LVEF, LVFS, HW/BW, and HW/TL were significantly increased. Additionally, the Trained + ALPR group exhibited decreased levels of cTnI, cTnT, and CK-MB and significantly reduced MI size and myocardial injury. Moreover, compared with the Trained or ALPR group, the Trained + ALPR group showed upregulated energy metabolism, increased microvessel density, and better efficacy. Finally, the Trained + ALPR group showed a significant increase in angiogenesis-related proteins and a significant reduction in MAPK signaling pathway-related protein activity. Exercise training combined with ALPR improved MI in elderly rats by inhibiting MAPK signaling, promoting angiogenesis, and increasing metabolism.

Abstract A021: Development of a novel MEK inhibitor, NFX-179, as a chemoprevention agent for squamous cell carcinoma

Abstract Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer comprising at least 20% of all non-melanoma skin cancers. While cSCC contributes to significant morbidity and mortality in high-risk individuals, deployment of otherwise effective chemoprevention of cSCC is limited by toxicities. To help address this, we conducted a systematic computational drug repositioning screen which predicted that selumetinib, an FDA-approved MEK inhibitor (MEKi), would reverse transcriptional signatures associated with cSCC development. This is consistent with our genomic analysis implicating ETS2, a transcription factor in the canonical RAS/RAF/MEK/ERK Mitogen-Activated Protein Kinase (MAPK) pathway, as an upstream regulator of cSCC development. Therefore, as a key regulator of the MAPK pathway, we reasoned MEK would be a viable chemopreventive target. Although systemic MEK inhibition suppresses the formation of cSCC in mice, systemic MEKi administration causes significant adverse effects, including diarrhea, peripheral edema, cardiomyopathy and retinal toxicity. Here, we report the development of a topically formulated, metabolically labile, novel MEKi, NFX-179, designed to potently and selectively suppress the MAPK pathway in the skin prior to rapid metabolism in the systemic circulation. NFX-179 was identified from a targeted drug discovery effort based on its biochemical and cellular potency, selectivity, and rapid metabolism upon systemic absorption. In our UV-induced cSCC mouse model, topical application of NFX- 179 gel reduced the formation of new cSCCs by an average of 60% at doses of 0.1% and greater at 28 days. No systemic or skin toxicities were observed in this model. Furthermore, we conducted a second split-mouse randomized controlled study in which NFX-179 0.5% gel was applied to one half of the back and vehicle was applied to the opposite half of each of the UV-irradiated mice. Near complete suppression of cSCC was observed only in the drug-treated area, demonstrating the targeted and dermal effect of the intervention. NFX-179 inhibits the growth of human SCC cell lines in a dose-dependent manner and topical NFX-179 application penetrates human skin and inhibits MAPK signaling in human cSCC explants. Together our data provide compelling rationale for using topical MEK inhibition through application of NFX-179 gel as an effective strategy for cSCC chemoprevention. Citation Format: Kavita Y. Sarin, John Kincaid, Brittney Sell, Jahanbanoo Shahryari, Matthew A. J. Duncton, Elaine Morefield, Wenchao Sun, Omar Chavez-Chiang, Scott R. Plotkin, Gerd G. Kochendoerfer, Peter Fenn, Christopher Powala, Kenneth Y. Tsai. Development of a novel MEK inhibitor, NFX-179, as a chemoprevention agent for squamous cell carcinoma [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A021.

DDDR-05. ERK INHIBITION INCREASES SENSITIVITY TO ANTI-PD-1 THERAPY IN A MOUSE MODEL OF BRAF MUTANT MELANOMA BRAIN METASTASIS

Abstract BACKGROUND Immune checkpoint inhibitors (ICI) have revolutionized the landscape of cancer treatment, including brain metastases. However, only a subset of patients with brain metastases respond to ICI. Activating mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, a key regulator of cell processes, are frequently found in brain metastases. LY3214996 (LY321) is a selective ERK 1/2 inhibitor that inhibits MAPK signaling and leads to regression of extracranial disease in preclinical models. Our objective was to evaluate whether with ERK inhibition (ERKi) can improve the efficacy of ICI in preclinical models of brain metastases. METHODS We tested sensitivity to LY321 in a BRAFV600E mutant, ICI resistant murine melanoma cell line (D4M) both in vitro and in vivo. To model brain metastases in vivo, we implanted D4M cells subcutaneously with subsequent intracranial implantation three days later. The treatment arms included LY321 and anti-PD-1 combination therapy, single agent therapies, and respective controls. We tested the efficacies of 2-week versus 4-week LY321 treatments, as well as 3-week treatment in an independent experiment. RESULTS D4M demonstrates in vitro sensitivity to LY321 (IC50 value of 4.75 µM). Our model demonstrated that LY321 in combination with anti-PD-1 improves both intracranial and extracranial disease control. Increased survival with combination therapy was observed with 3-week and 4-week LY321 administration. While individually, LY321 and anti-PD-1 produced better survival outcomes than control groups, combination therapy significantly outperformed both monotherapies. CONCLUSION Previous studies demonstrated that LY321 has minimal intracranial activity. However, we found that ERKi reduces intracranial tumor burden, with improved efficacy in combination with ICI in immunocompetent mice. Together, these data suggest that ERKi increases sensitivity to ICI therapy in pre-clinical brain metastasis models. Further investigation is needed to elucidate the mechanisms of this therapeutic approach, and to ultimately determine whether this combination will improve patient outcomes.

Mefunidone ameliorates lipopolysaccharide-induced acute lung injury through inhibiting MAPK signaling pathway and enhancing Nrf2 pathway

Background and ObjectiveAcute lung injury (ALI) is a life-threatening disease which has high mortality and lacks effective pharmacological treatments. Excessive inflammation and oxidative stress are the key pathogenesis of ALI. Mefunidone (MFD), a novel small molecule compound, displayed anti-inflammation and anti-oxidative stress effects on streptozocin (STZ) and db/db mice in our previous studies. In this study, we aimed to investigate the effects of MFD on lipopolysaccharide (LPS)-induced ALI and explore the potential molecular mechanisms. MethodsWe investigated the effects of MFD on LPS-induced ALI mouse model and LPS-stimulated immortalized mouse bone marrow-derived macrophages (iBMDMs). ResultsMFD could alleviate pulmonary structure disorder and attenuate pulmonary neutrophils infiltration induced by LPS. MFD could also decreased proinflammatory cytokines release and reduce reactive oxygen species (ROS) generation stimulated by LPS. Further, MFD could significantly reduce LPS-induced phosphorylation levels of mitogen-activated protein kinase (MAPK), increase expression of nuclear factor-erythroid 2 related factor 2 (Nrf2) and restore the expressions of antioxidant enzymes. ConclusionOur results firstly supported that MFD effectively protected LPS-induced ALI against inflammation and oxidative stress through inhibiting MAPK signaling pathway and activating Nrf2 pathway.

Blockades of effector T cell senescence and exhaustion synergistically enhance antitumor immunity and immunotherapy

BackgroundCurrent immunotherapies still have limited successful rates among cancers. It is now recognized that T cell functional state in the tumor microenvironment (TME) is a key determinant for effective antitumor...

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.