Inhibition Of MAPK Pathway Research Articles

Total Flavonoids from Camellia oleifera Alleviated Mycoplasma pneumoniae-Induced Lung Injury via Inhibition of the TLR2-Mediated NF-κB and MAPK Pathways.

Mycoplasma pneumoniae (M. pneumoniae) is an atypical bacterial pathogen responsible for community-acquired pneumonia primarily among school-aged children and young adults. Camellia oleifera (C. oleifera) has been used as a medicinal and edible plant in China for centuries, the constituents from which possessed various bioactivities. Notably, flavonoids existing in residues of C. oleifera defatted seeds exhibited significant anti-inflammatory activities. In the present study, we investigated the impact of total flavonoids from C. oleifera (TFCO) seed extract on M. pneumoniae pneumonia. TFCO was obtained using multiple column chromatography methods and identified as kaempferol glycosides via UPLC-HRESIMS. In a M. pneumoniae pneumonia mouse model, TFCO significantly reduced the lung damage, suppressed IL-1β, IL-6, and TNF-α production, and curbed TLR2 activation triggered by M. pneumoniae. Similarly, in RAW264.7 macrophage cells stimulated by lipid-associated membrane proteins (LAMPs), TFCO suppressed the generation of proinflammatory cytokines and TLR2 expression. Moreover, TFCO diminished the phosphorylation of IκBα, JNK, ERK, p38, and p65 nuclear translocation in vitro. In conclusion, TFCO alleviated M. pneumoniae-induced lung damage via inhibition of TLR2-mediated NF-κB and MAPK pathways, suggesting its potential therapeutic application in M. pneumoniae-triggered lung inflammation.

Inhibiting BRAF/EGFR/MEK suppresses cancer stemness and drug resistance of primary colorectal cancer cells.

Drug resistance is a major barrier against successful treatments of cancer patients. Gain of stemness under drug pressure is a major mechanism that renders treatments ineffective. Identifying approaches to target cancer stem cells (CSCs) is expected to improve treatment outcomes for patients. To elucidate the role of cancer stemness in resistance of colorectal cancer cells to targeted therapies, we developed spheroid cultures of patient-derived BRAFmut and KRASmut tumor cells and studied resistance mechanisms to inhibition of MAPK pathway through phenotypic and gene and protein expression analysis. We found that treatments enriched the expression of CSC markers CD166, ALDH1A3, CD133, and LGR5 and activated PI3K/Akt pathway in cancer cells. We examined various combination treatments to block these activities and found that a triple combination against BRAF, EGFR, and MEK significantly reduced stemness and activities of oncogenic signaling pathways. This study demonstrates the feasibility of blocking stemness-mediated drug resistance and tumorigenic activities in colorectal cancer.

Mosaic BRAF Fusions Are a Recurrent Cause of Congenital Melanocytic Nevi Targetable by MAPK Pathway Inhibition

Among children with multiple congenital melanocytic naevi (CMN), 25% have no established genetic cause, of which many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of CMN. Here, we study 169 CMN patients, 38 of whom were double wild-type for NRAS/BRAF mutations. Nineteen of these 38 patients had sufficient tissue to undergo RNAseq, which revealed mosaic BRAF fusions in 11/19 patients and mosaic RAF1 fusions in 1/19. Recurrently, fusions involved the loss of the 5' regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate naevi in 5/12 patients, confirming clonality. The absence of the fusion in blood in 8/12 patients indicated mosaicism. Primary culture of BRAF-fusion naevus cells from 3/12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement, and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes.

Abietic acid ameliorates pancreatic injury in acute pancreatitis by modulating MAPK pathway

Purpose: To examine the effect of abietic acid (AA) in the treatment of acute pancreatitis (AP) in mice.Methods: C57BL/6J mice were randomly assigned to 4 groups: sham, AP, AP + 20 mg/kg AA, and AP + 40 mg/kg AA groups. To induce AP mouse model, the mice received intraperitoneal (IP) injections of cerulein. The extent of pancreatic tissue damage was evaluated by histological examination. Serum ALT, lipase, and amylase levels were determined by commercial kits while TUNEL assay was used to assess the apoptosis of pancreatic cells. The contents of Bax, Caspase-3, Bcl-2, p-ERK/ERK, p-JNK/JNK, and p-P38/P38 in pancreatic tissues were evaluated by western blot while the contents of IL-6, TNF-α, IL-1β, nitrite, MDA, and GSH in the tissues were evaluated by enzyme-linked immunosorbent assay (ELISA) kits.Results: AA relieved pancreatic damage and reduced ALT, lipase, and amylase levels in ceruleintreated AP mouse (p < 0.001). AA repressed apoptosis of pancreatic cells in cerulein-induced AP mouse, and inhibited oxidative stress and inflammatory response in the mice by reducing IL-6, TNF-α, IL-1β, nitrite, and MDA contents; it also enhanced the levels of GSH in the tissues (p < 0.001). In addition, AA inhibited MAPK pathway activity in the mice (p < 0.001).Conclusion: AA ameliorates pancreatic damage, pancreatic cell apoptosis, oxidative stress, and inflammation in cerulein-induced AP mouse by inhibiting MAPK pathway. This study offers a new potential drug for the management of AP and expands the relevant molecular regulatory mechanisms.

A novel hepatocellular carcinoma-specific mTORC1-related signature for anticipating prognosis and immunotherapy.

Tumor oncogenesis, cancer metastasis, and immune evasion were substantially impacted by the mammalian target of the rapamycin complex 1 (mTORC1) pathway. However, in hepatocellular carcinoma (HCC), no mTORC1 signaling-based gene signature has ever been published. mTORC1 scores were computed employing a single sample gene set enrichment analysis based on databases including the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). The PAG1, LHFPL2, and FABP5 expression levels were obtained to construct a mTORC1 pathway-related model. In two databases, the overall survival (OS) rate was shorter for high-mTORC1 score patients compared to those with low scores. The activation of TFs in the group with high risk was enhanced, such as the HIF-1 pathway. Additionally, it was discovered that a high mTORC1 score was linked to an immune exclusion phenotype and enhanced immunosuppressive cell infiltration. Notably, it was discovered that high-mTORC1 scores patients had poorer immunotherapeutic results and might not gain benefit from immunotherapy. When compared to the low HCC metastatic cell lines, the high HCC metastatic cell lines have overexpressed levels of PAG1, LHFPL2, and FABP5 expression. The expression of PAG1, LHFPL2, and FABP5 was inhibited by the MAPK and mTORC1 pathway inhibitors. Our study identified mTORC1 score signature can aid in the development of individualized immunotherapy protocols and predict the HCC patients' prognoses.

Puerarin improves busulfan-induced disruption of spermatogenesis by inhibiting MAPK pathways

Male infertility is a global concern, with a noticeable increase in the decline of spermatogenesis and sperm quality. However, there are limited clinically effective treatments available. This study aimed to investigate the potential effectiveness of puerarin in treating male infertility, which leads to gonadal changes. The results obtained from various analyses such as CASA, immunofluorescence, DIFF-Quick, hematoxylin and eosin (H&E), and periodic acid-Schiff (PAS) staining demonstrated that puerarin supplementation significantly alleviated the busulfan-induced reduction in spermatogenesis and sperm quality in both young and adult mice. Furthermore, puerarin exhibited a marked improvement in the damage caused by busulfan to the architecture of seminiferous tubules, causal epididymis, blood-testicular barrier (BTB), as well as spermatogonia and Sertoli cells. Similarly, puerarin significantly reduced the levels of total antioxidant capacity (T-AOC), malondialdehyde (MDA), and caspase-3 in the testes of busulfan-induced mice, as determined by microplate reader analysis. Additionally, RNA-seq data, RT-qPCR, and western blotting revealed that puerarin restored the abnormal gene expressions induced by busulfan to nearly healthy levels. Notably, puerarin significantly reversed the impact of busulfan on the expression of marker genes involved in spermatogenesis and oxidative stress. Moreover, puerarin suppressed the phosphorylation of p38, ERK1/2, and JNK in the testes, as observed through testicular analysis. Consequently, this study concludes that puerarin may serve as a potential alternative for treating busulfan-induced damage to male fertility by inactivating the testicular MAPK pathways. These findings may pave the way for the use of puerarin in addressing chemotherapy- or other factors-induced male infertility in humans.

Gene signature reveals decreased SOX10-dependent transcripts in malignant cells from immune checkpoint inhibitor-resistant cutaneous melanomas

Evidence is mounting for cross-resistance between immune checkpoint and targeted kinase inhibitor therapies in cutaneous melanoma patients. Since the loss of the transcription factor, SOX10, causes tolerance to MAPK pathway inhibitors, we used bioinformatic techniques to determine if reduced SOX10 expression/activity is associated with immune checkpoint inhibitor resistance. We integrated SOX10 ChIP-seq, knockout RNA-seq, and knockdown ATAC-seq data from melanoma cell models to develop a robust SOX10 gene signature. We used computational methods to validate this signature as a measure of SOX10-dependent activity in independent single-cell and bulk RNA-seq SOX10 knockdown, cell line panel, and MAPK inhibitor drug-resistant datasets. Evaluation of patient single-cell RNA-seq data revealed lower levels of SOX10-dependent transcripts in immune checkpoint inhibitor-resistant tumors. Our results suggest that SOX10-deficient melanoma cells are associated with cross-resistance between targeted and immune checkpoint inhibitors and highlight the need to identify therapeutic strategies that target this subpopulation.

A phase I/IB trial of binimetinib in combination with erlotinib in NSCLC harboring activating KRAS or EGFR mutations

BackgroundActivating mutations in EGFR or KRAS are highly prevalent in NSCLC, share activation of the MAPK pathway and may be amenable to combination therapy to prevent negative feedback activation. MethodsIn this phase 1/1B trial, we tested the combination of binimetinib and erlotinib in patients with advanced NSCLC with at least 1 prior line of treatment (unless with activating EGFR mutation which could be treatment-naïve). A subsequent phase 1B expansion accrued patients with either EGFR- or KRAS-mutation using the recommended phase 2 dose (RP2D) from Phase 1. The primary objective was to evaluate the safety of binimetinib plus erlotinib and establish the RP2D. Results43 patients enrolled (dose-escalation = 23; expansion = 20). 17 harbored EGFR mutation and 22 had KRAS mutation. The RP2D was erlotinib 100 mg daily and binimetinib 15 mg BID × 5 days/week. Common AEs across all doses included diarrhea (69.8%), rash (44.2%), fatigue (32.6%), and nausea (32.6%), and were primarily grade 1/2. Among KRAS mutant patients, 1 (5%) had confirmed partial response and 8 (36%) achieved stable disease as best overall response. Among EGFR mutant patients, 9 were TKI-naïve with 8 (89%) having partial response, and 8 were TKI-pretreated with no partial responses and 1 (13%) stable disease as best overall response. ConclusionsBinimetinib plus erlotinib demonstrated a manageable safety profile and modest efficacy including one confirmed objective response in a KRAS mutant patient. While clinical utility of this specific combination was limited, these results support development of combinations using novel small molecule inhibitors of RAS, selective EGFR- and other MAPK pathway inhibitors, many of which have improved therapeutic indices. Clinical Trial RegistrationNCT01859026

MAPK-negative feedback regulation confers dependence to JAK2V617F signaling.

Despite significant advances in developing selective JAK2 inhibitors, JAK2 kinase inhibitor (TKI) therapy is ineffective in suppressing the disease. Reactivation of compensatory MEK-ERK and PI3K survival pathways sustained by inflammatory cytokine signaling causes treatment failure. Concomitant inhibition of MAPK pathway and JAK2 signaling showed improved in vivo efficacy compared to JAK2 inhibition alone but lacked clonal selectivity. We hypothesized that cytokine signaling in JAK2V617F induced MPNs increases theapoptotic threshold that causes TKI persistence or refractoriness. Here, we show that JAK2V617F and cytokine signaling converge to induce MAPK negative regulator, DUSP1. Enhanced DUSP1 expression blocks p38 mediated p53 stabilization. Deletion of Dusp1 increases p53 levels in the context of JAK2V617F signaling that causes synthetic lethality to Jak2V617F expressing cells. However, inhibition of Dusp1 by a small molecule inhibitor (BCI) failed to impart Jak2V617F clonal selectivity due to pErk1/2 rebound caused by off-target inhibition of Dusp6. Ectopic expression of Dusp6 and BCI treatment restored clonal selectively and eradicated the Jak2V617F cells. Our study shows that inflammatory cytokines and JAK2V617F signaling converge to induceDUSP1, which downregulates p53 and establishes a higher apoptotic threshold. These data suggest that selectively targeting DUSP1 may provide a curative response in JAK2V617F-driven MPN.

IMPemBra: a phase 2 study comparing pembrolizumab with intermittent/short-term dual MAPK pathway inhibition plus pembrolizumab in patients with melanoma harboring the BRAFV600 mutation

BackgroundContinuous combination of MAPK pathway inhibition (MAPKi) and anti-programmed death-(ligand) 1 (PD-(L)1) showed high response rates, but only limited improvement in progression-free survival (PFS) at the cost of a high...

Antigen presentation by clonally diverse CXCR5+ B cells to CD4 and CD8 T cells is associated with durable response to immune checkpoint inhibitors.

Increased T cell infiltration and interferon gamma (IFNγ) pathway activation are seen in tumors of melanoma patients who respond to ICI (immune checkpoint inhibitor) or MAPK pathway inhibitor (MAPKi) therapies. Yet, the rate of durable tumor control after ICI is almost twice that of MAPKi, suggesting that additional mechanisms may be present in patients responding to ICI therapy that are beneficial for anti-tumor immunity. We used transcriptional analysis and clinical outcomes from patients treated with ICI or MAPKi therapies to delineate immune mechanisms driving tumor response. We discovered response to ICI is associated with CXCL13-driven recruitment of CXCR5+ B cells with significantly higher clonal diversity than MAPKi. Our in vitro data indicate that CXCL13 production was increased in human peripheral blood mononuclear cells by anti-PD1, but not MAPKi, treatment. Higher B cell infiltration and B cell receptor (BCR) diversity allows presentation of diverse tumor antigens by B cells, resulting in activation of follicular helper CD4 T cells (Tfh) and tumor reactive CD8 T cells after ICI therapy. Higher BCR diversity and IFNγ pathway score post-ICI are associated with significantly longer patient survival compared to those with either one or none. Response to ICI, but not to MAPKi, depends on the recruitment of CXCR5+ B cells into the tumor microenvironment and their productive tumor antigen presentation to follicular helper and cytotoxic, tumor reactive T cells. Our study highlights the potential of CXCL13 and B cell based strategies to enhance the rate of durable response in melanoma patients treated with ICI.

Nimbolide Targets Multiple Signalling Pathways to Reduce Neuroinflammation in BV-2 Microglia

Nimbolide, a limonoid compound found in the neem plant, was investigated for effects on neuroinflammation in BV-2 microglia activated with lipopolysaccharide (LPS). Cultured BV-2 cells were treated with nimbolide (125, 250 and 500 nM) followed by stimulation with LPS (100 ng/ml). Results showed that nimbolide caused a significant reduction in the levels of TNFα, IL-6, IFNγ, NO/iNOS and PGE2/COX-2 in LPS-activated BV-2 cells. Further experiments revealed that LPS-induced increased expression of phospho-p65 and phospho-IκBα proteins were reduced in the presence of nimbolide. Also, LPS-induced NF-κB acetylation, increased binding to consensus sites and transactivation, as well as phosphorylation of p38 and JNK MAPKs were reduced by nimbolide. Reduction of cellular ROS generation by nimbolide was accompanied by a reduction in gp91phox protein levels, while antioxidant effects were also observed through elevation in protein levels of HO-1 and NQO-1. It was observed that treatment of BV-2 microglia with nimbolide resulted in reduced levels of cytoplasmic Nrf2, which was accompanied by increased levels in the nucleus. Furthermore, treatment with this compound resulted in increased binding of Nrf2 to antioxidant responsive element (ARE) consensus sites accompanied by enhanced ARE luciferase activity. Knockdown experiments revealed a loss of anti-inflammatory activity by nimbolide in cells transfected with Nrf2 siRNA. Treatment with nimbolide resulted in nuclear accumulation of SIRT-1, while siRNA knockdown of SIRT-1 resulted in the reversal of anti-inflammatory activity of nimbolide. It is proposed that nimbolide reduces neuroinflammation in BV-2 microglia through mechanisms resulting in dual inhibition of NF-κB and MAPK pathways. It is also proposed that activation of Nrf2 antioxidant mechanisms may be contributing to its anti-inflammatory activity.Graphical

LGG-03. EXPLORING THE SIGNALING NETWORK INVOLVED IN MAPK PATHWAY INHIBITION BY THE MEK INHIBITOR TRAMETINIB IN BRAF-FUSION-DRIVEN PEDIATRIC PILOCYTIC ASTROCYTOMA

Abstract INTRODUCTION Pilocytic astrocytomas (PA) are the most common pediatric brain tumors. They are characterized by MAPK pathway alterations, leading to its constitutive activation and modulating the balance between proliferation and oncogene-induced senescence (OIS) sustained by senescence-associated secretory phenotype (SASP). Little is known about the molecular implications of MAPK pathway inhibition in the proliferating and senescent tumor compartments. METHODS DKFZ-BT66 cells derived from a primary KIAA:BRAF-fusion positive PA cell line and BT40 cells derived from pleomorphic xanthoastrocytoma with a BRAFV600E mutation and CDKN2A/B deletion, were used as model systems. RNA-sequencing and phospho-/proteomic datasets were generated in both the proliferative and senescent cells, and treated with the MEKi trametinib for different time-spans. A multi-omics factor analysis tool (MEFISTO) was used to identify key OIS/proliferation effectors. RESULTS Differential gene expression analysis revealed that MEK inhibition leads to the inhibition of the OIS/SASP gene program in senescent DKFZ-BT66. In addition, the protein level of several SASP factors was decreased. This translated in reduced sensitivity towards senolytics drugs, indicating inhibition of senescence features upon MEKi. MEFISTO analysis allowed to identify key transcription factors, genes and proteins involved in MAPK-induced OIS in the senescent PA cells, that were mapped using a prior knowledge network approach. Finally, single sample geneset enrichment analysis showed that most MAPK-related signatures were downregulated upon MEKi treatment, while pathways related to upstream MAPK activators (including several RTK pathways) were predicted to be upregulated, in both proliferating and senescent cells. CONCLUSION This data suggests that MAPKi reverses OIS in senescent PA cells, while inducing the activation of MAPK upstream regulators, identifying putative co-targets for the treatment of PA. Further validation of the targetability of these pathways is pending. Furthermore, the identification of the MAPK-related OIS/SASP genes provide insight about the regulation of OIS/SASP by the MAPK pathway.

RPS15 interacted with IGF2BP1 to promote esophageal squamous cell carcinoma development via recognizing m6A modification

Increased rates of ribosome biogenesis have been recognized as hallmarks of many cancers and are associated with poor prognosis. Using a CRISPR synergistic activation mediator (SAM) system library targeting 89 ribosomal proteins (RPs) to screen for the most oncogenic functional RPs in human esophageal squamous cell carcinoma (ESCC), we found that high expression of RPS15 correlates with malignant phenotype and poor prognosis of ESCC. Gain and loss of function models revealed that RPS15 promotes ESCC cell metastasis and proliferation, both in vitro and in vivo. Mechanistic investigations demonstrated that RPS15 interacts with the K homology domain of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), which recognizes and directly binds the 3′-UTR of MKK6 and MAPK14 mRNA in an m6A-dependent manner, and promotes translation of core p38 MAPK pathway proteins. By combining targeted drug virtual screening and functional assays, we found that folic acid showed a therapeutic effect on ESCC by targeting RPS15, which was augmented by the combination with cisplatin. Inhibition of RPS15 by folic acid, IGF2BP1 ablation, or SB203580 treatment were able to suppress ESCC metastasis and proliferation via the p38 MAPK signaling pathway. Thus, RPS15 promotes ESCC progression via the p38 MAPK pathway and RPS15 inhibitors may serve as potential anti-ESCC drugs.

Neutrophil extracellular traps induced by the hypoxic microenvironment in gastric cancer augment tumour growth

BackgroundInflammation-related predisposition to cancer plays an essential role in cancer progression and is associated with poor prognosis. A hypoxic microenvironment and neutrophil infiltration are commonly present in solid tumours, including gastric cancer (GC). Neutrophil extracellular traps (NETs) have also been demonstrated in the tumour immune microenvironment (TIME), but how NETs affect GC progression remains unknown. Here, we investigated the role of NET formation in the TIME and further explored the underlying mechanism of NETs in GC tumour growth.MethodsHypoxia-induced factor-1α (HIF-1α), citrulline histone 3 (citH3) and CD66b expression in tumour and adjacent nontumor tissue samples was evaluated by western blotting, immunofluorescence and immunohistochemical staining. The expression of neutrophil-attracting chemokines in GC cells and their hypoxic-CM was measured by qRT‒PCR and ELISA. Neutrophil migration under hypoxic conditions was evaluated by a Transwell assay. Pathway activation in neutrophils in a hypoxic microenvironment were analysed by western blotting. NET formation was measured in vitro by immunofluorescence staining. The protumour effect of NETs on GC cells was identified by Transwell, wound healing and cell proliferation assays. In vivo, an lipopolysaccharide (LPS)-induced NET model and subcutaneous tumour model were established in BALB/c nude mice to explore the mechanism of NETs in tumour growth.ResultsGC generates a hypoxic microenvironment that recruits neutrophils and induces NET formation. High mobility group box 1 (HMGB1) was translocated to the cytoplasm from the nucleus of GC cells in the hypoxic microenvironment and mediated the formation of NETs via the toll-like receptor 4 (TLR4)/p38 MAPK signalling pathway in neutrophils. HMGB1/TLR4/p38 MAPK pathway inhibition abrogated hypoxia-induced neutrophil activation and NET formation. NETs directly induced GC cell invasion and migration but not proliferation and accelerated the augmentation of GC growth by increasing angiogenesis. This rapid tumour growth was abolished by treatment with the NET inhibitor deoxyribonuclease I (DNase I) or a p38 MAPK signalling pathway inhibitor.ConclusionsHypoxia triggers an inflammatory response and NET formation in the GC TIME to augment tumour growth. Targeting NETs with DNase I or HMGB1/TLR4/p38 MAPK pathway inhibitors is a potential therapeutic strategy to inhibit GC progression.Ca2HLG-e4J4ceVPYPV7JaoVideo

Abstract IA26: Exploiting altered metabolism as a therapeutic strategy for RAS-driven cancers

Abstract We recently determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo (Bryant et al., 2019, Nat Med 25:628). Our findings, together with similar observations by McMahon, Kinsey and colleagues (Kinsey et al., 2019, Nat Med 25:620), provided the rationale for our initiation of Phase I and Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that combined inhibition of the ERK MAPK pathway and autophagy is effective in additional cancers driven by KRAS/NRAS, BRAF, or EGFR. Additionally, we have also shown that the combined inhibition of KRASG12C or KRASG12D and autophagy was effective in KRAS-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting metabolic resistance mechanisms to KRAS or ERK MAPK targerted therapies. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers as well as mediators of resistance to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Second, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition and hypothesize that further dissection of this regulation will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Claire E. Gates, Noah L. Pieper, Runying Yang, Clint A. Stalnecker, Kirsten L. Bryant. Exploiting altered metabolism as a therapeutic strategy for RAS-driven cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA26.

Abstract B003: Combined inhibition of the MAPK and Hippo pathways drives efficacious tumor suppression in a faithful model of mutant Kras (KPC) PDAC

Abstract Oncogenic mutations in the KRAS oncogene hyperactivate the MAPK pathway, a key driver of oncogenesis in the majority of human malignancies. KRAS-driven tumors are aggressive and highly refractory to standard-of-care treatments, thus intense efforts have been devoted to developing inhibitors of its activity. However, pioneering studies in engineered conditional mKras driven mouse models of pancreatic ductal adenocarcinoma (KPC) have demonstrated that while mKras is necessary for tumor maintenance, the anti-neoplastic effects of mKras extinction can be counteracted by activation of the Hippo pathway. These data strongly suggest that the full realization of MAPK pathway (including KRAS) inhibitors’ clinical potential requires the concomitant suppression of the function of YAP/TEAD, the effectors of Hippo pathway. Here, we directly test this hypothesis utilizing Sporos BioDiscovery’s next generation TEAD inhibitor (SPR1) - optimized to maximize anti-neoplastic activity and minimize toxicity through fine-tuning of TEAD paralog specificity. Using mKras murine (KrasG12D mutant, p53 null, PDAC) and human cell lines - we demonstrated strong interaction between SPR1 TEAD inhibitors and the clinical MEK inhibitors (since no G12D inhibitor is yet approved) Trametinib/Binimetinib in vitro. Functional TEAD inhibition was also shown by the reduced expression of the YAP/TEAD transcriptional targets CTGF and CYR61. Further, using a well-established murine orthotopic model, KPC (KrasG12D;p53-/-;p48Cre), that faithfully mimics human PDAC, we demonstrated robust combined efficacy of Trametinib and SPR1 TEAD inhibitors in vivo in suppressing tumor growth and strikingly doubling the survival time of the tumor-bearing mice. To our knowledge, this is the first preclinical demonstration of the therapeutic potential of a MAPK + TEAD inhibitor combination in a mKras G12D-driven orthotopic PDAC; given the high barrier for efficacy of this KPC model –we believe these results favorably presage generalization to MAPK/TEAD inhibitor combinations in other cancer setting and translatability to the clinic. Citation Format: Deepavali Chakravarti, Florian Muller, Jeno Gyuris, Erkun Baloglu, Selvi Kunnimalayiaan, Jill Olson, Sharon Shacham. Combined inhibition of the MAPK and Hippo pathways drives efficacious tumor suppression in a faithful model of mutant Kras (KPC) PDAC [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B003.

Abstract B025: The RAF/MEK clamp avutometinib (VS-6766) enhances antitumor efficacy of KRAS G12C and G12D inhibitors through vertical inhibition of RAS, RAF and MEK. </title>

Abstract KRAS is the most frequently mutated RAS family member. Within non-small cell lung cancer (NSCLC), G12C is the most prevalent KRAS mutation (~13%) and the KRAS G12C inhibitors (G12Ci) sotorasib and adagrasib are now FDA approved. However, it has been shown that simultaneous targeting of multiple nodes in the MAPK pathway may be optimal for deeper and more durable responses. Furthermore, acquired mutations in the MAPK pathway occur clinically upon progression on sotorasib or adagrasib, altogether supporting the need for clinical combinations with G12Ci. Based on the clinical success of G12Ci, several KRAS G12D inhibitors (G12Di) are now being developed as G12D is the most prevalent KRAS mutation across human cancers including pancreatic (~28%) and colorectal (~11%) cancers. Avutometinib (VS-6766; avuto) is a RAF/MEK clamp that potently inhibits MEK kinase activity and induces a dominant negative RAF-MEK complex preventing phosphorylation of MEK by ARAF, BRAF and CRAF. This unique mechanism allows avuto to block MEK signaling without the compensatory re-activation of MEK that appears to limit the efficacy of MEK-only inhibitors. Here, we tested whether addition of avuto to G12Ci or G12Di could improve MAPK pathway blockade and anti-tumor efficacy. Avuto was synergistic with sotorasib and adagrasib in reducing viability of a panel of KRAS G12C cancer cell lines in 3D culture, and the combination of avuto + G12Ci showed improved depth and duration of MAPK pathway inhibition, stronger inhibition of cell cycle/proliferation markers and stronger activation of pro-apoptotic markers relative to G12Ci alone. In mouse models, avuto enhanced efficacy of G12Ci in KRAS G12C NSCLC xenograft models. Furthermore, avuto potentiated G12Ci efficacy in a KRAS(+/G12C);p53null NSCLC GEMM model with avuto + G12Ci combination inducing complete response in 25% of the mice. To evaluate efficacy of avuto against KRAS mutations that have been observed clinically upon acquired resistance to G12Ci, proliferation of Ba/F3 cells expressing these mutations was assessed. While avuto potently inhibited (IC50 < 15 nM) proliferation of cells bearing KRAS G12C/R68S, G12C/H95D or G12C/Y96C mutations, adagrasib showed diminished potency (IC50 ≥ 85 nM) against all 3 of these mutations and sotorasib showed diminished potency (IC50 ≥ 150 nM) against G12C/R68S and G12C/Y96C. Accordingly, avuto strongly inhibited tumor growth of an in vivo model expressing KRAS G12C/Y96D, while sotorasib was ineffective. Similarly, avuto was found to be synergistic with the G12Di MRTX1133 in reducing viability of a panel of KRAS G12D cancer cell lines, and avuto enhanced anti-tumor efficacy of MRTX1133 in KRAS G12D colorectal (CR3300) and pancreatic (PA1252) cancer PDX models. These results support ongoing clinical studies of avutometinib in combination with sotorasib (NCT05074810) and adagrasib (NCT05375994) for patients with KRAS G12C NSCLC. Additionally, these preclinical data support combination of avutometinib with a G12D inhibitor for patients with KRAS G12D colorectal or pancreatic cancers. Citation Format: Silvia Coma, Monica Musteanu, Cristina Caffarra, Alessia Mira, Enrico Patrucco, Julien Dilly, Andrew J. Aguirre, Mariano Barbacid, Chiara Ambrogio, Jonathan A. Pachter. The RAF/MEK clamp avutometinib (VS-6766) enhances antitumor efficacy of KRAS G12C and G12D inhibitors through vertical inhibition of RAS, RAF and MEK. </title> [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B025.

GMI, a fungal immunomodulatory protein, ameliorates SARS-CoV-2 envelope protein-induced inflammation in macrophages via inhibition of MAPK pathway

Clinically, COVID-19 is often accompanied by a severe immune response (cytokine storm) which produces a large number of cytokines, such as TNF-α, IL-6 and IL-12, and consequently causes acute respiratory distress syndrome (ARDS). GMI is a type of fungal immunomodulatory protein that is cloned from Ganoderma microsporum and acts as modulating immunocyte for various inflammatory diseases. This study identifies GMI as a potential anti-inflammatory agent and determines the effects of GMI on the inhibition of SARS-CoV-2-induced cytokine secretion. Functional studies showed that SARS-CoV-2 envelope (E) protein induces inflammatory process in murine macrophages RAW264.7 and MH-S cells and in phorbol 12-myristate 13-acetate (PMA)-stimulated human THP-1 cells. GMI exhibits a strong inhibitory effect for SARS-CoV-2-E-induced pro-inflammatory mediators, including NO, TNF-α, IL-6, and IL-12 in macrophages. GMI reduces SARS-CoV-2-E-induced intracellular inflammatory molecules, such as iNOS and COX-2, and inhibits SARS-CoV-2-E-stimulated phosphorylation of ERK1/2 and P38. GMI also downregulates pro-inflammatory cytokine levels in lung tissue and serum after the mice inhale SARS-CoV-2-E protein. In conclusion, this study shows that GMI acts as an agent to alleviate SARS-CoV-2-E-induced inflammation.

Whole exome sequencing analysis of canine urothelial carcinomas without BRAF V595E mutation: Short in-frame deletions in BRAF and MAP2K1 suggest alternative mechanisms for MAPK pathway disruption.

Molecular profiling studies have shown that 85% of canine urothelial carcinomas (UC) harbor an activating BRAF V595E mutation, which is orthologous to the V600E variant found in several human cancer subtypes. In dogs, this mutation provides both a powerful diagnostic marker and a potential therapeutic target; however, due to their relative infrequency, the remaining 15% of cases remain understudied at the molecular level. We performed whole exome sequencing analysis of 28 canine urine sediments exhibiting the characteristic DNA copy number signatures of canine UC, in which the BRAF V595E mutation was undetected (UDV595E specimens). Among these we identified 13 specimens (46%) harboring short in-frame deletions within either BRAF exon 12 (7/28 cases) or MAP2K1 exons 2 or 3 (6/28 cases). Orthologous variants occur in several human cancer subtypes and confer structural changes to the protein product that are predictive of response to different classes of small molecule MAPK pathway inhibitors. DNA damage response and repair genes, and chromatin modifiers were also recurrently mutated in UDV595E specimens, as were genes that are positive predictors of immunotherapy response in human cancers. Our findings suggest that short in-frame deletions within BRAF exon 12 and MAP2K1 exons 2 and 3 in UDV595E cases are alternative MAPK-pathway activating events that may have significant therapeutic implications for selecting first-line treatment for canine UC. We developed a simple, cost-effective capillary electrophoresis genotyping assay for detection of these deletions in parallel with the BRAF V595E mutation. The identification of these deletion events in dogs offers a compelling cross-species platform in which to study the relationship between somatic alteration, protein conformation, and therapeutic sensitivity.