Mitogen-activated Protein Kinase Inhibitor Research Articles

P38 MAPK as a gatekeeper of reprogramming in mouse migratory primordial germ cells.

Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing ex vivo analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our ex vivo model in mimicking in vivo teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.

Alantolactone enhances the sensitivity of melanoma to MAPK pathway inhibitors by targeting inhibition of STAT3 activation and down-regulating stem cell markers

Mitogen-activated protein kinase inhibitors (MAPKi) were the first line drugs for advanced melanoma patients with BRAF mutation. Targeted therapies have significant therapeutic effects; however, drug resistance hinders their long-term efficacy. Therefore, the development of new therapeutic strategies against MAPKi resistance is critical. Our previous results showed that MAPKi promote feedback activation of STAT3 signaling in BRAF-mutated cancer cells. Studies have shown that alantolactone inhibited the activation of STAT3 in a variety of tumor cells. Our results confirmed that alantolactone suppressed cell proliferation and promoted apoptosis by inhibiting STAT3 feedback activation induced by MAPKi and downregulating the expression of downstream Oct4 and Sox2. The inhibitory effect of alantolactone combined with a MAPKi on melanoma cells was significantly stronger than that on normal cells. In vivo and in vitro experiments showed that combination treatment was effective against drug-resistant melanomas. Our research indicates a potential novel combination therapy (alantolactone and MAPKi) for patients with BRAF-mutated melanoma.

P38-MAPK is prerequisite for the synthesis of SARS-CoV-2 protein.

The inhibition of p38 mitogen-activated protein kinase (p38-MAPK) by small molecule chemical inhibitors was previously shown to impair severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, however, mechanisms underlying antiviral activity remains unexplored. In this study, reduced growth of SARS-CoV-2 in p38-α knockout Vero cells, together with enhanced viral yield in cells transfected with construct expressing p38α, suggested that p38-MAPK is essential for the propagation of SARS-CoV-2. The SARS-CoV-2 was also shown to induce phosphorylation (activation) of p38, at time when transcription/translational activities are considered to be at the peak levels. Further, we demonstrated that p38 supports viral RNA/protein synthesis without affecting viral attachment, entry, and budding in the target cells. In conclusion, we provide mechanistic insights on the regulation of SARS-CoV-2 replication by p38 MAPK.

Protective effects of orientin against spinal cord injury in rats.

We aimed to study the reparative effects of orientin against spinal cord injury (SCI) in rats and explore its potential mechanisms. Sprague-Dawley rats were divided into Sham, SCI, Orientin, and SB203580 [an inhibitor of p38 mitogen-activated protein kinase (p38MAPK)] groups. In the SCI group, rats underwent Allen's beat. SCI animals in Orientin and SB203580 groups were respectively treated with 40 mg kg-1 orientin and 3 mg kg-1 SB203580 once daily. Functional recovery was evaluated based on Basso, Beattie, and Bresnahan scoring. Histopathological analysis was performed using hematoxylin-eosin and Nissl staining. Cell apoptosis was examined by TUNEL staining. The relative quantity of apoptosis-related proteins, glial fibrillary acidic protein (GFAP), neurofilament 200 (NF200), and brain derived neurotrophic factor (BDNF) was detected via western blotting. The indices related to inflammation and oxidation were measured using agent kits. The p38MAPK/inducible nitric oxide synthase (iNOS) signaling activity was detected using real-time quantitative PCR, western blotting, and immunohistochemical staining. Orientin was revealed to effectively mitigate cell apoptosis, neuroinflammation, and oxidative stress in impaired tissues. Meanwhile, orientin exerted great neuroprotective effects by abating GFAP expression, and up-regulating the expression of NF200 and BDNF, and significantly suppressed the p38MAPK/iNOS signaling. Orientin application could promote the repair of secondary SCI through attenuating oxidative stress and inflammatory response, reducing cell apoptosis and suppressing p38MAPK/iNOS signaling.

Green tea extract suppresses airway inflammation via oxidative stress-driven MAPKs/MMP-9 signaling in asthmatic mice and human airway epithelial cells.

The anti-inflammatory effect of green tea extract (GTE) has been confirmed in asthmatic mice, however, the pharmacological mechanism is not fully elucidated. To investigate the therapeutic efficacy of GTE in asthma and identify specific pathways, murine model of allergic asthma was established by ovalbumin (OVA) sensitization and the challenge for 4 weeks, with oral treatment using GTE and dexamethasone (DEX). Inflammatory cell counts, cytokines, OVA-specific IgE, airway hyperreactivity, and antioxidant markers in the lung were evaluated. Also, pulmonary histopathological analysis and western blotting were performed. In vitro, we established the model by stimulating the human airway epithelial cell line NCI-H292 using lipopolysaccharide, and treating with GTE and mitogen-activated protein kinases (MAPKs) inhibitors. The GTE100 and GTE400 groups showed a decrease in airway hyperresponsiveness and the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) compared to the OVA group. GTE treatment also reduced interleukin (IL)-13, IL-5, and IL-4 levels in the BALF, and OVA-specific immunoglobulin E levels in the serum compared to those in the OVA group. GTE treatment decreased OVA-induced mucus secretion and airway inflammation. In addition, GTE suppressed the oxidative stress, and phosphorylation of MAPKs, which generally occurs after exposure to OVA. GTE administration also reduced matrix metalloproteinase-9 activity and protein levels. GTE effectively inhibited asthmatic respiratory inflammation and mucus hyperproduction induced by OVA inhalation. These results suggest that GTE has the potential to be used for the treatment of asthma.

The hyperplasia of the mammary gland effect of the Mongolian Remedy RuXian-I in rats reduced by estrogen and progesterone via inhibition of MAPK and NF-κB pathways

Background: The aim of the present study was to evaluate whether the HMG (Hyperplasia of the Mammary Gland) effect of RuXian-I in estrogen- and progestogen- induced HMG rats is mediated through the activation of MAPK (Mitogen activated protein kinase) and NF-κB signaling pathways. Materials and methods: Fifty virgin female Wistar rats were randomly divided into in control and HMG, RuXian-I (0.75 g·kg−1, 1.5 g·kg−1, 3 g·kg−1, respectively) groups, 10 in each. Injections of estrogen and progestogen were given to establish rat models of HMG and RuXian-I at the same time. Changes in nipple heights were measured; pathologic changes of HMG in rats were also observed under a light microscope; the phosphorylation levels of MAPK and NF κB and the expression levels of TNF-α and IL-1β were measured. Results: Compared with the control group, the nipple diameters and height were increased significantly, the numbers of MG lobules were increased, there were changes in breast histopathology, the levels of TNF-α and IL-1β increased significantly, and MAPK and NF-κB were activated in HMG rats. Compared with the HMG model group, the increased nipple height was decreased, the numbers of MG lobules were reduced, the degree of HMG in rats was alleviated obviously, and the phosphorylation level of MAPK and NF-κB and cytokines TNF- α and IL-1β levels in serum were decreased by RuXian-I treatment. Conclusion: Those results suggest RuXian-I has protective and therapeutic effects on HMG rats induced by estrogen and progestogen and is likely to activate MAPK and NF-κB signaling pathways.

Evaluation of antioxidant activity and fermentation properties of potential probiotic strain Lactiplantibacillus plantarum HY7720 in plant-based materials

People on vegan diets are at risk of being deficient in varied nutrients such as vitamin B12 and certain amino acids. In this study, we investigated vitamin B12-producing lactic acid bacteria (LAB) as well as the probiotic and antioxidant properties. Lactiplantibacillus plantarum HY7720 was screened from 22 strains of LAB that were isolated from different plant foods, and its growth ability and extracellular vitamin B12-producing capacity in vitamin B12-deficient medium were investigated. To determine whether HY7720 functions as a probiotic, survival rate in the simulated gastrointestinal tract and adhesion property to human intestinal epithelial cells of HY7720 were compared with positive control, Lacticaseibacillus rhamnosus GG (LGG). Moreover, the results showed that HY7720 recovered the gene expression levels of tight junction-associated proteins (TJPs), including TJP1, TJP2, occludin (OCLN), and claudin-1 (CLDN1) and inhibited the secretion levels of pro-inflammatory cytokines, including interleukin (IL)-6 and IL-8, in tumor necrosis factor (TNF)-α-stimulated Caco-2 cells. Furthermore, we verified that HY7720 exhibit the antioxidant potential, by showing its intracellular reactive oxygen species (ROS) scavenging ability in hydrogen peroxide (H2O2)-stimulated Caco-2 cells. The ability of HY7720 to ameliorate H2O2-induced cytotoxicity in Caco-2 cells was inhibited by mitogen-activated protein kinase (MAPK) inhibitors, indicating that its antioxidant responses are related to extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK). This study also investigated the nutritional qualities of three plant-based materials (brown rice, white rice, and soy milk) fermented using HY7720. Collectively, HY7720 could be used as a promising probiotic strain for the prevention of nutritional deficiencies among people on vegetarian diets.

Arenobufagin induces cell apoptosis by modulating the cell cycle regulator claspin and the JNK pathway in nasopharyngeal carcinoma cells

ABSTRACT Background The high recurrence rate and incidence of distant metastasis of nasopharyngeal carcinoma (NPC) result in poor prognosis. It is necessary to identify natural compounds that can complement combination radiation therapy. Arenobufagin is commonly used for heart diseases and liver cancer, but its effectiveness in NPC is unclear. Study Design and Methods The effect of arenobufagin-induced apoptosis was measured by a cell viability assay, tumorigenic assay, fluorescence assay, and Western blot assay through NPC-039 and NPC-BM cell lines. The protease array, Western blot assay, and transient transfection were used to investigate the underlying mechanism of arenobufagin-induced apoptosis. An NPC xenograft model was established to explore the antitumor activity of arenobufagin in vivo. Results Our findings indicated that arenobufagin exerted cytotoxic effects on NPC cells, inhibiting proliferation through apoptosis activation. Downregulation of claspin was confirmed in arenobufagin-induced apoptosis. Combined treatment with arenobufagin and mitogen-activated protein kinase inhibitors demonstrated that arenobufagin induced NPC apoptosis through the c-Jun N-terminal kinases (JNK) pathway inhibition. Furthermore, arenobufagin suppressed NPC tumor proliferation in vivo. Conclusion Our results revealed the antitumor effect of arenobufagin in vitro and in vivo. Arenobufagin may have clinical utility in treating NPC due to its suppression of claspin and inhibition of the JNK pathway.

Sustained activation of the FGF1-MEK-ERK pathway inhibits proliferation, invasion and migration and enhances radiosensitivity in mouse angiosarcoma cells.

Angiosarcoma is a rare refractory soft-tissue tumor with a poor prognosis and is treated by radiotherapy. The fibroblast growth factor 1 (FGF1) mutant, with enhanced thermostability due to several substituted amino acids, inhibits angiosarcoma cell metastasis, yet the mechanism of action is unclear. This study aims to clarify the FGF1 mutant mechanism of action using ISOS-1 mouse angiosarcoma cells. The wild-type FGF1 or FGF1 mutant was added to ISOS-1 cells and cultured, evaluating cell numbers over time. The invasive and migratory capacity of ISOS-1 cells was assessed by transwell analysis. ISOS-1 cell radiosensitivity was assessed by colony formation assay after X-ray irradiation. To examine whether mitogen-activated protein kinase (MEK) inhibitor counteracts the FGF1 mutant effects, a combination of MEK inhibitor and FGF1 mutant was added to ISOS-1 cells and cultured. The FGF1 mutant was observed to inhibit ISOS-1 cell proliferation, invasion and migration by sustained FGF1 signaling activation. A MEK inhibitor suppressed the FGF1 mutant-induced inhibition of proliferation, invasion and migration of ISOS-1 cells. Furthermore, the FGF1 mutant enhanced radiosensitivity of ISOS-1 cells, but MEK inhibition suppressed the increased radiosensitivity. In addition, we found that the FGF1 mutant strongly inhibits actin polymerization, suggesting that actin cytoskeletal dynamics are closely related to ISOS-1 cell radiosensitivity. Overall, this study demonstrated that in ISOS-1 cells, the FGF1 mutant inhibits proliferation, invasion and migration while enhancing radiosensitivity through sustained activation of the MEK-mediated signaling pathway.

Sub-acute oral exposure to lowest observed adverse effect level of nivalenol exacerbates atopic dermatitis in mice via direct activation of mitogen-activated protein kinase signal in antigen-presenting cells.

This study investigated the immunotoxic effects of the mycotoxin nivalenol (NIV) using antigen-presenting cells and a mouse model of atopic dermatitis (AD). In vitro experiments were conducted using a mouse macrophage cell line (RAW 264.7) and mouse dendritic cell line (DC 2.4). After cells were exposed to NIV (0.19-5µmol) for 24h, the production of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα) was quantified. To further investigate the inflammatory cytokine production pathway, the possible involvement of mitogen-activated protein kinase (MAPK) pathways, such as ERK1/2, p-38, and JNK, in NIV exposure was analyzed using MAPK inhibitors and phosphorylation analyses. In addition, the pro-inflammatory effects of oral exposure to NIV at low concentrations (1 or 5ppm) were evaluated in an NC/Nga mouse model of hapten-induced AD. In vitro experiments demonstrated that exposure to NIV significantly enhanced the production of TNFα. In addition, it also directly induced the phosphorylation of MAPK, indicated by the inhibition of TNFα production following pretreatment with MAPK inhibitors. Oral exposure to NIV significantly exacerbated the symptoms of AD, including a significant increase in helper T cells and IgE-produced B cells in auricular lymph nodes and secretion of pro-inflammatory cytokines, such as IL-4, IL-5, and IL-13, compared with the vehicle control group. Our findings indicate that exposure to NIV directly enhanced the phosphorylation of ERK1/2, p-38, and JNK, resulting in a significant increase in TNFα production in antigen-presenting cells, which is closely related to the development of atopic dermatitis.

Galangin Prevents Against Ethanol-Induced Intestinal Barrier Dysfunction and NLRP3 Inflammasome Activation via NF-κB/MAPK Signaling Pathways in Mice and Caco-2 Cells.

The potential of natural phytochemicals in addressing ethanol-related public safety concerns has been garnering attention. Galangin, a potent flavonoid renowned for its antioxidative and anti-inflammatory characteristics, is derived from the galanga plant, and propolis is derived from bees. Here, we documented the effects of galangin on ethanol-stimulated intestinal tight junction damage and investigated its potential protective mechanism in both in vivo and in vitro models, which has not been extensively investigated. Our results revealed that galangin efficaciously mitigated ethanol-induced intestine injury and dysfunction of the intestinal barrier. Concurrently, galangin significantly counteracted the ethanol-induced upregulation of NLRP3 inflammasome-associated proteins and activated the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways in both the mouse colon and Caco-2 cells. Interestingly, similar to galangin, inhibitors of MAPKs and the NF-κB p65 reduced ethanol-induced NLRP3 inflammasome activation and intestinal tight junction damage. To sum up, our results showed that galangin blocks the ethanol-induced perturbation of the intestinal barrier and activation of the NLRP3 inflammasome via the NF-κB/MAPK signaling pathways.

Neuromedin-U Mediates Rapid Activation of Airway Group 2 Innate Lymphoid Cells in Mild Asthma.

Rationale: In asthma, sputum group 2 innate lymphoid cells (ILC2s) are activated within 7 hours after allergen challenge. Neuroimmune interactions mediate rapid host responses at mucosal interfaces. In murine models of asthma, lung ILC2s colocalize to sensory neuronal termini expressing the neuropeptide neuromedin U (NMU), which stimulates type 2 (T2) cytokine secretion by ILC2s, with additive effects to alarmins invitro. Objectives: To investigate the effect of the NMU/NMUR1 (NMU receptor 1) axis on early activation of ILC2s in asthma. Methods: Subjects with mild asthma (n = 8) were enrolled in a diluent-controlled allergen inhalation challenge study. Sputum ILC2 expression of NMUR1 and T2 cytokines was enumerated by flow cytometry, and airway NMU levels were assessed by ELISA. This was compared with samples from subjects with moderate to severe asthma (n = 9). Flow sort-purified and ex vivo-expanded ILC2s were used for functional assays and transcriptomic analyses. Measurements and Main Results: Significant increases in sputum ILC2s expressing NMUR1 were detected 7 hours after allergen versus diluent challenge whereby the majority of NMUR1+ ILC2s expressed IL-5/IL-13. Sputum NMUR1+ ILC2 counts were significantly greater in mild versus moderate to severe asthma, and NMUR1+ ILC2s correlated inversely with the dose of inhaled corticosteroid in the latter group. Coculturing with alarmins upregulated NMUR1 in ILC2s, which was attenuated by dexamethasone. NMU-stimulated T2 cytokine expression by ILC2s, maximal at 6 hours, was abrogated by dexamethasone or specific signaling inhibitors for mitogen-activated protein kinase 1/2 and phosphoinositol 3-kinase but not the IL-33 signaling moiety MyD88 invitro. Conclusions: The NMU/NMUR1 axis stimulates rapid effects on ILC2s and may be an important early activator of these cells in eosinophilic inflammatory responses in asthma.

Abstract LB250: Dynamics of transcript alternations and differential splicing in the evolution of acquired MAPKi resistance in melanoma

Abstract The primary therapy for melanoma with a BRAF mutation involves Mitogen-Activated Protein Kinase inhibitors (MAPKi), targeting the dysregulated MAPK signaling pathway. Despite the initial response, many patients often develop acquired resistance. Resistance to MAPKi could be linked to various altered signaling pathways, including reactivation of the MAPK pathway, activation of alternative survival pathways like PI3K/PTEN/AKT, engagement of receptor tyrosine kinases (RTKs) such as PDGFRβ and EGFR, and developmental pathways. While numerous omics studies have aimed to understand the mechanism of acquired resistance, how these pathways are altered remains elusive. To gain further insights into the acquired MAPKi resistance in melanoma, we explored the role of RNA splicing events in this context by analyzing publicly available datasets of pre- and post-MAPKi treated melanoma from patients and in vitro studies using cell lines. We investigated the differential transcript usage (DTU) to detect the specific splice variants that are altered during the development of resistance. We find that the genes involving DTU are enriched in various pathways related to MAPKi resistance, such as the MAPKi signaling pathway, PI3K/AKT pathway, signaling by RTKs, etc. Furthermore, the DTU-centered analysis provides better insights into MAPKi resistance mechanisms compared to standard differential gene expression analysis. Next, we analyzed the developmental splicing signatures during MAPKi resistance development, revealing developmental reprogramming as a key factor in MAPKi resistance. Further, we identified the top 10 transcription factors as potential upstream regulators of the perturbed transcripts, among which five have been previously reported to be involved in various stages of embryonic development. This finding suggests a link between the reactivation of developmental pathways and MAPKi resistance development. Further, by analyzing MAPKi-treated melanoma cell-line models at different time points, such as no-treatment, on-treatment, and at the resistant stage, we observed significant alterations in transcript isoforms during the development of resistance. These changes involve lipid metabolic reprogramming and PI3K/Akt/mTOR signaling, providing further insights into the mechanisms of MAPKi resistance. Our study also uncovers a global shift in transcript profiles towards shorter 3’ and 5’ UTRs in response to MAPKi treatment, suggesting the existence of a master regulatory mechanism influencing global splicing changes during therapy resistance. In summary, our study deciphered the post-transcriptional mechanism coordinating the signaling changes, underscoring the complexity of MAPKi resistance in melanoma, as revealed by the joint analysis of both transcript alterations and splicing events. Citation Format: Sumit Mukherjee, Arashdeep Singh, Hyunjeong Joo, Sumeet Patiyal, Hyungsoo Kim, Lipika R. Pal, Kun Wang, Chi-Ping Day, Ze’ev A. Ronai, Eytan Ruppin, Sridhar Hannenhalli. Dynamics of transcript alternations and differential splicing in the evolution of acquired MAPKi resistance in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB250.

5-Hydroxytryptamine Enhances the Pacemaker Activity of Interstitial Cells of Cajal in Mouse Colon.

We examined the localization of the 5-hydroxytryptamine (5-HT) receptor and its effects on mouse colonic interstitial cells of Cajal (ICCs) using electrophysiological techniques. Treatment with 5-HT increased the pacemaker activity in colonic ICCs with depolarization of membrane potentials in a dose-dependent manner. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers blocked pacemaker activity and 5-HT-induced effects. Moreover, an adenylate cyclase inhibitor inhibited 5-HT-induced effects, and cell-permeable 8-bromo-cAMP increased the pacemaker activity. Various agonists of the 5-HT receptor subtype were working in colonic ICCs, including the 5-HT4 receptor. In small intestinal ICCs, 5-HT depolarized the membrane potentials transiently. Adenylate cyclase inhibitors or HCN blockers did not show any influence on 5-HT-induced effects. Anoctamin-1 (ANO1) or T-type Ca2+ channel blockers inhibited the pacemaker activity of colonic ICCs and blocked 5-HT-induced effects. A tyrosine protein kinase inhibitor inhibited pacemaker activity in colonic ICCs under controlled conditions but did not show any influence on 5-HT-induced effects. Among mitogen-activated protein kinase (MAPK) inhibitors, a p38 MAPK inhibitor inhibited 5-HT-induced effects on colonic ICCs. Thus, 5-HT's effect on pacemaker activity in small intestinal and colonic ICCs has excitatory but variable patterns. ANO1, T-type Ca2+, and HCN channels are involved in 5-HT-induced effects, and MAPKs are involved in 5-HT effects in colonic ICCs.

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Pirfenidone ameliorates alcohol-induced promotion of breast cancer in mice.

Alcohol consumption increases the risk of breast cancer and promotes cancer progression. Alcohol exposure could affect both processes of the mammary carcinogenesis, namely, the cell transformation and onset of tumorigenesis as well as cancer aggressiveness including metastasis and drug resistance/recurrence. However, the cellular and molecular mechanisms underlying alcohol tumor promotion remain unclear. There are four members of the mammalian p38 mitogen-activated protein kinase (MAPK) family, namely, p38α, p38β, p38γ and p38δ. We have previously demonstrated alcohol exposure selectively activated p38γ MAPK in breast cancer cells in vitro and in vivo. Pirfenidone (PFD), an antifibrotic compound approved for the treatment of idiopathic pulmonary fibrosis, is also a pharmacological inhibitor of p38γ MAPK. This study aimed to determine whether PFD is useful to inhibit alcohol-induced promotion of breast cancer. Female adolescent (5 weeks) MMTV-Wnt1 mice were exposed to alcohol with a liquid diet containing 6.7% ethanol. Some mice received intraperitoneal (IP) injection of PFD (100 mg/kg) every other day. After that, the effects of alcohol and PFD on mammary tumorigenesis and metastasis were examined. Alcohol promoted the progression of mammary tumors in adolescent MMTV-Wnt1 mice. Treatment of PFD blocked tumor growth and alcohol-promoted metastasis. It also significantly inhibited alcohol-induced tumorsphere formation and cancer stem cell (CSC) population. PFD inhibited mammary tumor growth and alcohol-promoted metastasis. Since PFD is an FDA-approved drug, the current findings may be helpful to re-purpose its application in treating aggressive breast cancer and alcohol-promoted mammary tumor progression.

Insights into the mechanism of action of pterostilbene against influenza A virus-induced acute lung injury

BackgroundSevere respiratory system illness caused by influenza A virus infection is associated with excessive inflammation and abnormal apoptosis in alveolar epithelial cells (AEC). However, there are limited therapeutic options for influenza-associated lung inflammation and apoptosis. Pterostilbene (PTE, trans-3,5-dimethoxy-4-hydroxystilbene) is a dimethylated analog of resveratrol that has been reported to limit influenza A virus infection by promoting antiviral innate immunity, but has not been studied for its protective effects on virus-associated inflammation and injury in AEC. PurposeOur study aimed to investigate the protective effects and underlying mechanisms of PTE in modulating inflammation and apoptosis in AEC, as well as its effects on macrophage polarization during influenza virus infection. Study design and methodsA murine model of influenza A virus-mediated acute lung injury was established by intranasal inoculation with 5LD50 of mouse-adapted H1N1 viruses. Hematoxylin and eosin staining, immunofluorescence, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, western blotting, Luminex and flow cytometry were performed. ResultsPTE effectively mitigated lung histopathological changes and injury induced by H1N1 viruses in vivo. These beneficial effects of PTE were attributed to the suppression of inflammation and apoptosis in AEC, as well as the modulation of M1 macrophage polarization. Mechanistic investigations revealed that PTE activated the phosphorylated AMP-activated protein kinase alpha (P-AMPKα)/sirtui1 (Sirt1)/PPARγ coactivator 1-alpha (PGC1α) signal axis, leading to the inhibition of nuclear factor kappa-B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signaling induced by H1N1 viruses, thereby attenuating inflammation and apoptosis in AEC. PTE also forced activation of the P-AMPKα/Sirt1/PGC1α signal axis in RAW264.7 cells, counteracting the activation of phosphorylated signal transducer and activator of transcription 1 (P-STAT1) induced by H1N1 viruses and the augment of P-STAT1 activation in RAW264.7 cells with interferon-gamma (IFN-γ) pretreatment before viral infection, thereby reducing H1N1 virus-mediated M1 macrophage polarization as well as the enhancement of macrophages into M1 phenotypes elicited by IFN-γ pretreatment. Additionally, the promotion of the transition of macrophages towards the M2 phenotype by PTE was also related to activation of the P-AMPKα/Sirt1/PGC1α signal axis. Moreover, co-culturing non-infected AEC with H1N1 virus-infected RAW264.7 cells in the presence of PTE inhibited apoptosis and tight junction disruption, which was attributed to the suppression of pro-inflammatory mediators and pro-apoptotic factors in an AMPKα-dependent manner. ConclusionIn conclusion, our findings suggest that PTE may serve as a promising novel therapeutic option for treating influenza-associated lung injury. Its ability to suppress inflammation and apoptosis in AEC, modulate macrophage polarization, and preserve alveolar epithelial cell integrity highlights its potential as a therapeutic agent in influenza diseases.

Abstract 620: Screening of small-molecule MAPK pathway inhibitors on a panel of 130 human cancer cell lines identifies determinants of response in RAS-mutant subgroups

Abstract Oncogenic mutations in members of the RAS family, HRAS, NRAS, and KRAS, are frequently identified in various cancers and often lead to increased signaling of the mitogen-activated protein kinase (MAPK) pathway. KRAS is the most frequently mutated RAS isoform, with a mutation incidence of >10% across all cancers, and up to 70% and 20% in pancreatic and non-small cell lung cancer (NSCLC), respectively. RAS has long been considered to be undruggable, and direct inhibition of RAS with small molecule inhibitors has only recently been achieved, with the approval of the KRAS G12C-mutant selective inhibitors sotorasib and adagrasib for treatment of NSCLC. Indirect targeting of RAS-mutated cancers by inhibitors of downstream components such as MEK and ERK has shown variable responses. Novel insights into MAPK signaling have renewed interest into targeting the MAPK pathway in RAS-mutant cancer, with pan-RAF dimer inhibitors and dual RAF/MEK inhibitors reaching clinical trials. To improve the chances of success, it is important to determine which patient populations are most likely to respond to these inhibitors. To gain more insight into the cellular targeting of MAPK pathway inhibitors, eighteen inhibitors of the MAPK components KRAS, RAF, MEK, and ERK were profiled on a panel of 130 cell lines in cell viability assays (the Oncolines® panel). The cell lines represented a diverse range of tumor tissues and had different genomic backgrounds. Many of the cell lines had mutations in KRAS, NRAS, or BRAF. Drug responses were determined by exposing the cell lines to nine-point duplicate dose ranges of the inhibitors. Using bioinformatics, cell line responses to the inhibitors were related to genomic and transcriptomic features, to identify determinants of drug response in both tissue-agnostic and tissue-specific contexts. The results of our analyses revealed variable responses of KRAS-mutant cell lines to the inhibitors, reflecting clinical responses. Tissue-focused analyses showed that the different responses could be traced back to the different KRAS amino acid substitutions. For instance, in pancreatic cancer cell lines, the KRAS Q61H mutation predicted increased sensitivity to MAPK pathway inhibitors compared to other KRAS amino acid substitutions. Additionally, in tissue groups with a more heterogenous mutation profile, co-mutations explained the decreased effectivity of MAPK pathway inhibitors in KRAS-mutant cell lines, such as PIK3CA mutations in colorectal cancer cell lines. Lastly, a gene expression-based signature of MAPK activity predicted response to MAPK pathway inhibitors, independent of mutation status. Our findings have uncovered determinants of response to MAPK pathway inhibitors in RAS-mutant cell lines. These determinants can serve as markers to select patients who are most likely to benefit from MAPK inhibitors. Citation Format: Jeffrey J. Kooijman, Awan Al Koerdi, Daphne J. Kluitmans, Esmee van den Bossche, Jelle Dylus, Jeroen A. de Roos, Janneke J. Melis, Guido J. Zaman. Screening of small-molecule MAPK pathway inhibitors on a panel of 130 human cancer cell lines identifies determinants of response in RAS-mutant subgroups [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 620.

Abstract 142: HDAC3 is required for pathognomonic features of Langerhans cell histiocytes

Abstract Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm that develops due to dysregulated dendritic cell (DC) development and most commonly affects the children with an incidence of 2.6 to 8.9 cases per million individuals per year. BRAFV600E, the most common disease-driving mutation, activates the mitogen-activated protein kinase (MAPK) pathway causing pathognomonic features that lead to the accumulation of LCH cells and formation of granulomatous lesions. These features include enhanced myelopoiesis, reduced tissue-egress and acquisition of an oncogene induced senescence-associated secretory phenotype (SASP) (marked by increased expression of anti-apoptotic proteins and inflammatory cytokines). Accumulation of LCH cells can occur in any organ causing a wide range of clinical sequelae, which is directly related to the originating cell bearing the oncogenic mutation. Frontline therapy for LCH involves combination chemotherapy and steroidal anti-inflammatories, or MAPK inhibitors, which have significant toxicity and fail to eliminate disease causing precursors. We have found that HDAC3 is required for epidermal LC homeostasis and hypothesized that LCH cells rely on similar epigenetic factors to LCs. CD11cCre LSL-BRAFV600E (BRAFV600ECD11c) mice develop severe multifocal LCH with pronounced lesion development in their livers and lungs, hepatosplenomegaly, and a reduced lifespan due to the accumulation of LCH cells. To test our hypothesis, we generated BRAFV600ECD11c HDAC3fl/fl (BRAFV600EHD3KO) mice, which produce LCH cells that harbor a conditional deletion in the deacetylase domain of HDAC3. Compared to BRAFV600ECD11c mice, BRAFV600EHD3KO mice exhibited significantly less hepatosplenomegaly, reduced lesional burden, and improved survival indicating reduced LCH disease burden. Compared to BRAFV600ECD11c, flow cytometry showed BRAFV600EHD3KO had reduced numbers of LCH cells in lungs and livers, which were more apoptotic. We also found decreased frequencies of circulating LCH cells and LCH precursors, indicating that a lack of HDAC3 activity prevents the development of LCH cells. Bone-marrow derived LCH-like cells provide a valuable drug screening tool. We treated Bone-marrow derived LCH-like cells with RGFP966, an HDAC3-specific inhibitor, increased apoptosis indicated by annexin-V and DAPI staining, reduced expression of Bcl-2, increased CCR7 expression, and decreased S6 phosphorylation (an indication of a loss of the SASP), thus inhibition of HDAC3 may prove therapeutically efficacious by abrogating pathognomonic features of LCH cells. Together, our findings identify HDAC3 as a critical epigenetic regulator LCH cell pathophysiology and HDAC3 blockade would address a great need in treatment of patients with LCH. Furthermore, our results indicate that LCH cells rely on similar epigenetic regulators as epidermal LCs despite coming from a different myeloid lineage. Citation Format: Peter Dimitrion, Jugmohit Toor, James Ge, Qiyan Wang, Carl Allen, Li Zhou, Qing-Sheng Mi. HDAC3 is required for pathognomonic features of Langerhans cell histiocytes [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 142.

Abstract 3157: The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis

Abstract Brain metastasis (BM) affects approximately 10% of cancer patients with metastatic disease, representing a major cause of morbidity and mortality. BM is a complex multi-step process involving various immune checkpoint proteins. Mitogen-activated protein kinase (MAPK), particularly extracellular signal-regulated kinases 1/2 (ERK1/2), and signal transducer and activator of transcription 3 (STAT3) are implicated in tumorigenesis and are critical upstream regulators of Programmed Death Ligand 1 (PD-L1) expression, a common immunotherapy target. Tumor suppressor p53 is shown to regulate STAT3 and ERK1/2 signaling, which are dysregulated in various malignancies. We hypothesize that p53 along with STAT3 and ERK1/2 play a critical role in the initiation and maintenance of BM. Twenty-six BM tumors with various primary sites of origin were used following IRB guidelines. pSTAT3Tyr705, pERK1/2Thr202/Tyr204, mutant p53 (p53mt), and PD-L1 expression was determined by immunohistochemistry. Gene expression analysis of BM was performed on a cDNA microarray. Human brain metastatic breast cancer cells (MDA-MB-231) were treated with STAT3 inhibitor (NSC74859) or MAPK inhibitor (U0126) in regular or astrocytic media to determine cell proliferation and migration by MTT assay and scratch-wound, respectively. ERK1/2 signaling pathway status was determined by Western blotting. Results demonstrated: 1) pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 were expressed profusely at tumor margins, while p53mt was uniformly expressed throughout. 81% of BM expressed p53mt while 86% and 63% expressed pERK1/2Thr202/Tyr204 and pSTAT3Tyr705, respectively. Venn analysis displayed significant overlap between expression, with 62% overlap between p53mt and pERK1/2Thr202/Tyr204 and a 50% overlap between both pSTAT3Tyr705 and pERK1/2Thr202/Tyr204 with p53mt. Expression of PD-L1 was seen in a majority of BM tumors. 2) Gene expression analysis demonstrated alterations in 32 STAT3- and MAPK-associated genes; these included upregulation of stathmin-1 and stathmin-like 3, which control microtubule dynamics in STAT3-related cell migration, and changes in cytokine-related genes, suggesting altered immune responses. 3) Cell proliferation showed augmented growth in astrocytic media (p≤0.05) and was significantly decreased following treatment with NSC74850 or U0126 (p≤0.01). While migration showed a trend towards significance in reduction following NSC74859 (p=0.08), migration stagnated following U0126 treatment in astrocytic media (p≤0.01). 4) pERK1/2Thr202/Tyr204 expression was reduced following U0126 or NSC74859 treatment. These findings suggest activation of STAT3 and ERK1/2 promote BM and suppression of these pathways may lead to a reduction in cell viability and motility. Therefore, this provides compelling evidence for the use of STAT3 and ERK1/2 along with p53 as potential targets for immunotherapy in brain metastatic disease. Citation Format: Sabrina L. Zeller, Eris Spirollari, John V. Wainwright, Simon J. Hanft, Chirag D. Gandhi, Meena Jhanwar-Uniyal. The crosstalk between STAT3 and p53/ERK signaling regulates brain metastasis [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 3157.