Mitogen-activated Protein Kinase Inhibitor Research Articles

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.

In asthma, sputum group 2 innate lymphoid cells (ILC2) are activated within 7h after allergen challenge. Neuroimmune interactions mediate rapid host responses at mucosal interfaces. In murine models of asthma, lung ILC2 co-localize to sensory neuronal termini expressing the neuropeptide, neuromedin U (NMU) and NMU stimulates type 2 cytokines secretion by ILC2 with additive effects to alarmins, in vitro. Investigate effect of NMU/NMUR1 axis on early activation of ILC2 in asthma. M ild asthmatics (n=8) were enrolled in a diluent-controlled, allergen-inhalation challenge study. Sputum ILC2 expression of NMU receptor 1 (NMUR1) and T2 cytokines were enumerated by flow cytometry and airway NMU levels were assessed by ELISA. This was compared to samples from moderate-severe asthmatics (n=9). Flow sort-purified and ex-vivo expanded ILC2 were used for functional assays and transcriptomic analyses. Significant increases in sputum ILC2 expressing NMUR1 were detected 7h post- allergen versus diluent challenge where the majority of NMUR1+ILC2 expressed IL-5/IL-13. Sputum NMUR1+ILC2 were significantly greater in mild versus moderate-severe asthmatics and NMUR1+ILC2 correlated inversely with the dose of inhaled corticosteroid in the latter group. Co-culturing with alarmins upregulated NMUR1 in ILC2, which was attenuated by dexamethasone. NMU stimulated T2 cytokine expression by ILC2, maximal at 6h was abrogated by dexamethasone or specific signaling inhibitors for mitogen-activated protein kinase ½, phospho-inositol 3 kinase but not IL-33 signaling moiety MyD88, in vitro. The NMU/NMUR1 axis stimulates rapid effects on ILC2, and maybe 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.

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.

Abstract 5063: Exploring the mutational landscape of KRAS and NRAS in tumors with non-V600 BRAF mutations

Abstract Background: BRAF, a key modulator of the Mitogen-Activated Protein Kinase (MAPK) pathway, is observed in 7% of all cancers. Therapeutic response to MAPK inhibition (MAPKi) often relies on molecular distinctions between members of varying classes: Class 1 (V600), Class 2 and 3 (non-V600) BRAF mutants. Preliminary data indicates that co-occurring RAS mutations in non-V600 BRAF mutant cancers are less responsive to MAPKi treatment. This emphasizes the need to investigate the characteristics of RAS co-mutations in non-V600 BRAF mutant tumors. Methods: Genomic data was obtained from a cohort of 183,292 patients provided by the AACR GENIE database (v14.1). Patient samples were clustered according to their BRAF mutation status and co-occurring K/NRAS mutations: WT BRAF (n=60,845) vs. non-V600 BRAF (n=1009). Samples were grouped based on cancer type: melanoma (n=3841), colorectal (n=15,434), and non-small cell lung cancer (n=14640) and were further categorized according to key biochemical features of RAS GTPase function and overall GTPase activity. Results: This dataset revealed a diverse array of allelic variants of K/NRAS between cancer types and their underlying BRAF mutation status (Table 1). Non-V600 BRAF mutant cancers showed an enrichment for KRAS mutations linked to amplified nucleotide exchange (37.9% vs. 13.4%; p<0.0001) and hydrolysis-impairing NRAS mutations (41.9% vs. 23%; p<0.0001), compared to WT BRAF cancers. Rare allelic variants including KRAS L19F, KRAS A146T, and NRAS G60E were seen in Class 2/3 BRAF mutants. Conclusion: This data suggests that non-V600 BRAF mutant tumors are characterized by a unique distribution of RAS mutations. More research into the difference in downstream effectors of RAS mutants overrepresented in non-V600 BRAF mutant tumors could provide important insights into how these tumors develop and resist targeted therapies. Table 1. Classification of KRAS and NRAS mutations in WT BRAF vs. non-V600 BRAF mutant tumors GTPase Function Cancer Type All cancers NSCLC Colorectal Melanoma BRAF mutation WT non-V600 p-value WT non-V600 p-value WT non-V600 p-value WT non-V600 p-value KRASmutation class Impaired hydrolysis n=20731 (79.8%) n=167 (51.9%) <0.0001 n=6068 (86.1%) n=60 (56.6%) <0.0001 n=4799 (68.2%) n=16 (29.1%) <0.0001 n=38 (48.1%) n=13 (50.0%) 0.4047 Nucleotide Exchange n=3475 (13.4%) n=122 (37.9%) n=530 (7.5%) n=36 (34.0%) n=1842 (26.2%) n=36 (65.5%) n=30 (38.0%) n=12 (46.2%) Hybrid n=1765 (6.8%) n=33 (10.2%) n=446 (6.3%) n=10 (9.4%) n=395 (5.6%) n=3 (5.5%) n=11 (13.9%) n=1 (3.8%) NRAS mutation class Impaired hydrolysis n = 974 (23.0%) n=78 (41.9%) <0.0001 n=29 (18.7%) n=9 (39.1%) 0.0196 n=188 (32.6%) n=17 (68.0%) <0.0001 n=86 (5.5%) n=22 (27.8%) <0.0001 Nucleotide Exchange n=459 (10.8%) n=22 (11.8%) n=5 (3.2%) n=2 (8.7%) n=48 (8.3%) n=4 (16.0%) n=93 (5.9%) n=10 (12.7%) Hybrid n=2811 (66.2%) n=86 (46.2%) n=121 (78.1%) n=12 (52.2%) n=341 (59.1%) n=4 (16.0%) n=1385 (88.6%) n=47 (59.5%) GTPase Activity BRAF mutation WT non-V600 p-value WT non-V600 p-value WT non-V600 p-value WT non-V600 p-value KRASmutation class Intermediate activity n=14408 (55.3%) n=142 (44.4%) 0.0001 n=5082 (72.3%) n=54 (50.5%) <0.0001 n=3540 (49.9%) n=17 (31.5%) 0.0089 n=46 (56.8%) n=13 (50.0%) 0.6516 High activity n=11630 (44.7%) n=178 (55.6%) n=1947 (27.7%) n=53 (49.5%) n=3552 (50.1%) n=37 (68.5%) n=35 (43.2%) n=13 (50.0%) NRAS mutation class Intermediate activity n=1364 (32.2%) n=101 (55.8%) <0.0001 n=36 (23.4%) n=12 (54.5%) 0.0041 n=239 (41.8%) n=20 (87.0%) <0.0001 n=190 (12.3%) n=34 (43.0%) <0.0001 High activity n=2868 (67.8%) n=80 (44.2%) n=118 (76.6%) n=10 (45.5%) n=333 (58.2%) n=3 (13.0%) n=1357 (87.7%) n=45 (57.0%) Citation Format: Chantel L. Mukonoweshuro, Emmanuelle Rousselle, April A. Rose. Exploring the mutational landscape of KRAS and NRAS in tumors with non-V600 BRAF mutations [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 5063.

Abstract 3081: Targeting lipid kinases to enhance autophagy inhibition for the treatment of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized as the most KRAS-addicted cancer type, with KRAS mutations found in >95% of cases. Mutant KRAS relies on dysregulated metabolism to support growth. One dysregulated pathway upregulated in PDAC is autophagy, a lysosome-mediated process that degrades proteins and organelles to supply macromolecules for growth. Our lab and others have shown that inhibition of KRAS or the mitogen activated protein kinase (MAPK) components, MEK or ERK, further increases PDAC dependency on autophagy as a resistance mechanism. Cotreatment with ERK MAPK inhibitors and the autophagy inhibitor chloroquine (CQ) decreased autophagic flux, inhibited proliferation, and induced apoptosis in PDAC cells. CQ is the only FDA-approved autophagy inhibitor, and it demonstrates low potency in vivo, motivating our studies to identify strategies to improve autophagy inhibition in PDAC. We utilized a CRISPR-Cas9 loss-of-function screen to identify sensitizers to CQ in PDAC and identified 25 autophagy-related targets. We hypothesize that targeting the autophagy pathway at multiple nodes will increase the efficacy of CQ treatment in PDAC. To validate hits identified in the CRISPR-Cas9 screen, I designed an siRNA array that contained three distinct oligos for each target. I quantified changes in proliferation in a panel of four PDAC cell lines after siRNA-mediated suppression of the target genes following 3- or 6-day time points. I hypothesized that targets identified to be non-essential on their own would allow for potential synthetic lethality with CQ. To test this, I performed a 3-day knockdown followed by 3-day exposure to a dose response of CQ. I found that suppression of lipid kinases PIP4K2A, PIP4K2C, or PIK3C3 shift the dose response to CQ in a panel of PDAC lines. This indicates that loss of those targets sensitizes PDAC cells to CQ treatment. Furthermore, we found that pharmacological inhibition of VPS34, the lipid kinase encoded by PIK3C3 by the ATP-competitive inhibitor SAR-405 synergizes with CQ. My future studies will be focused on validating other targets identified in the loss-of-function CRISPR-Cas9 screen. I will measure changes in autophagic flux and apoptosis due to the loss of validated targets. The long-term goal of this project is to enhance the translatability of autophagy inhibition for patient care through identifying combinational approaches to improve CQ response in PDAC. Citation Format: Elyse G. Schechter, Jonathan M. DeLiberty, Clint A. Stalnecker, Kirsten L. Bryant. Targeting lipid kinases to enhance autophagy inhibition for the treatment of pancreatic 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 3081.

Combined KRAS-MAPK pathway inhibitors and HER2-directed drug conjugate is efficacious in pancreatic cancer

Targeting the mitogen-activated protein kinase (MAPK) cascade in pancreatic ductal adenocarcinoma (PDAC) remains clinically unsuccessful. We aim to develop a MAPK inhibitor-based therapeutic combination with strong preclinical efficacy. Utilizing a reverse-phase protein array, we observe rapid phospho-activation of human epidermal growth factor receptor 2 (HER2) in PDAC cells upon pharmacological MAPK inhibition. Mechanistically, MAPK inhibitors lead to swift proteasomal degradation of dual-specificity phosphatase 6 (DUSP6). The carboxy terminus of HER2, containing a TEY motif also present in extracellular signal-regulated kinase 1/2 (ERK1/2), facilitates binding with DUSP6, enhancing its phosphatase activity to dephosphorylate HER2. In the presence of MAPK inhibitors, DUSP6 dissociates from the protective effect of the RING E3 ligase tripartite motif containing 21, resulting in its degradation. In PDAC patient-derived xenograft (PDX) models, combining ERK and HER inhibitors slows tumour growth and requires cytotoxic chemotherapy to achieve tumour regression. Alternatively, MAPK inhibitors with trastuzumab deruxtecan, an anti-HER2 antibody conjugated with cytotoxic chemotherapy, lead to sustained tumour regression in most tested PDXs without causing noticeable toxicity. Additionally, KRAS inhibitors also activate HER2, supporting testing the combination of KRAS inhibitors and trastuzumab deruxtecan in PDAC. This study identifies a rational and promising therapeutic combination for clinical testing in PDAC patients.

CHF6297: a novel potent and selective p38 MAPK inhibitor with robust anti-inflammatory activity and suitable for inhaled pulmonary administration as dry powder.

Inhibition of p38 mitogen-activated protein kinase (MAPKs) is a potential therapeutic approach for the treatment of acute and chronic pulmonary inflammatory conditions. Here, we report the in vitro and in vivo characterization of the anti-inflammatory effects of CHF6297, a novel potent and selective p38α inhibitor designed for inhalation delivery as a dry powder formulation. CHF6297 has been proven to inhibit p38α enzymatic activity with sub-nanomolar potency (IC50 = 0.14 ± 0.06nM), with >1,000-fold selectivity against p38γ and p38δ. In human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharides (LPS), as well as in human bronchial epithelial cells (BEAS2B) stimulated with TNF-α or cigarette smoke extract (CSE), CHF6297 inhibited interleukin (IL)-8 release with low nanomolar potency. CHF6297 administered to rats by using a nose-only inhalation device as a micronized dry powder formulation blended with lactose dose-dependently inhibited the LPS-induced neutrophil influx in the bronchoalveolar lavage fluid (BALF). CHF6297 administered intratracheally to rats dose-dependently counteracted the IL-1β (0.3mg/kg)-induced neutrophil influx (ED50 = 0.22mg/kg) and increase in IL-6 levels (ED50 = 0.82mg/kg) in the BALF. In mice exposed to tobacco smoke (TS), CHF6297, administered intranasally (i.n.) for 4 days at 0.03 or 0.3mg/kg, dose-dependently inhibited the corticosteroid-resistant TS-induced neutrophil influx in the BALF. In a murine house dust mite (HDM) model of asthma exacerbated by influenza virus A (IAV) (H3N3), CHF6297 (0.1mg/kg, i.n.) significantly decreased airway neutrophilia compared to vehicle-treated IAV/HDM-challenged mice. When CHF6297, at a dose ineffective per se (0.03mg/kg), was added to budesonide, it augmented the anti-inflammatory effects of the steroid. Overall, CHF6297 effectively counteracted lung inflammation in experimental models where corticosteroids exhibit limited anti-inflammatory activity, suggesting a potential for the treatment of acute exacerbations associated with chronic obstructive pulmonary disease (COPD) and asthma, acute lung injury (ALI), and viral-induced hyperinflammation.

Cutaneous toxicities of mitogen-activated protein kinase inhibitors in children and young adults with neurofibromatosis-1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder which commonly causes neoplasms leading to disfigurement or dysfunction. Mitogen-activated protein kinase inhibitors (MEKi) are generally well-tolerated treatments which target neural tumor progression in patients with NF1. However, cutaneous adverse events (CAEs) are common and may hinder patients' abilities to remain on treatment, particularly in children. We aim to characterize CAEs secondary to MEKi treatment in pediatric and young adult patients with NF1. We reviewed institutional medical records of patients under 30 years with a diagnosis of "NF1," "NF2," or "other neurofibromatoses" on MEKi therapy between January 1, 2019 and June 1, 2022. We recorded the time-to-onset, type, and distribution of CAEs, non-cutaneous adverse events (AEs), AE management, and tumor response. Our cohort consisted of 40 patients with NF1 (median age, 14 years). Tumor types included low-grade gliomas (51%) and plexiform neurofibromas (38%). MEKi used included selumetinib (69%), trametinib (25%), and mirdametinib (6%). A total of 74 CAEs occurred, with 28 cases of acneiform rash (38%). Other common CAEs were paronychia, seborrheic dermatitis, eczema, xerosis, and oral mucositis. The most common treatments included oral antibiotics and topical corticosteroids. Most patients had clinical (stable or improved) tumor response (71%) while 29% had tumor progression while on a MEKi. There was no significant association between CAE presence and tumor response (p = 0.39). Improvement in characterization of MEKi toxicities and their management is important to develop treatment guidelines for pediatric and young adult patients with NF1 on MEKi therapy.