Inhibition Of MAPK Pathway Research Articles

BRAFV600E induces key features of LCH in iPSCs with cell type-specific phenotypes and drug responses.

Langerhans cell histiocytosis (LCH) is a clonal hematopoietic disorder defined by tumorous lesions containing CD1a+/CD207+ cells. Two severe complications of LCH are systemic hyperinflammation and progressive neurodegeneration. The scarcity of primary samples and lack of appropriate models limit our mechanistic understanding of LCH pathogenesis and affect patient care. We generated a human in vitro model for LCH using induced pluripotent stem cells (iPSCs) harboring the BRAFV600E mutation, the most common genetic driver of LCH. We show that BRAFV600E/WT iPSCs display myelo-monocytic skewing during hematopoiesis and spontaneously differentiate into CD1a+/CD207+ cells that are similar to lesional LCH cells and are derived from a CD14+ progenitor. We show that BRAFV600E modulates the expression of key transcription factors regulating monocytic differentiation and leads to an upregulation of pro-inflammatory molecules and LCH marker genes early during myeloid differentiation. In vitro drug testing revealed that BRAFV600E-induced transcriptomic changes are reverted upon treatment with MAPK-pathway inhibitors (MAPKi). Importantly, MAPKi do not affect myeloid progenitors but reduces only the mature CD14+ cell population. Furthermore, iPSC-derived neurons (iNeurons) co-cultured with BRAFV600E/WT iPSC-derived microglia-like cells (iMGL), differentiated from iPSC-derived CD34+ progenitors, exhibit signs of neurodegeneration with neuronal damage and release of neurofilament light chain. In summary, the iPSC-based model described here provides a platform to investigate the effects of BRAFV600E in different hematopoietic cell types and provides a tool to compare and identify novel approaches for the treatment of BRAFV600E-driven diseases.

Baliosperoid A attenuates lipopolysaccharide-induced acute lung injury by targeting SHP2 to inhibit inflammation and oxidative stress

Acute lung injury (ALI) remains a devastating clinical condition with limited therapeutic options. While Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2) has emerged as a critical mediator in ALI pathogenesis, effective SHP2-targeting therapeutics remain largely elusive. Baliospermum solanifolium (Burm.) is a traditional medicine used to treat various diseases such as asthma, edema, bronchitis, jaundice, and constipation. Baliosperoid A (BA), a diterpenoid compound derived from Baliospermum solanifolium’s roots, exhibits potent NO inhibitory activity in RAW264.7 cells. However, its anti-inflammatory activity and potential targets have never been reported. Here, we report BA acts as a selective SHP2 inhibitor with remarkable therapeutic potential against ALI. Through comprehensive molecular and functional analyses, we demonstrate that BA directly binds to and inhibits SHP2 phosphatase activity with high specificity (IC50 = 1.638 ± 0.324 μM). Mechanistically, BA orchestrates a dual-action therapeutic effect by simultaneously suppressing inflammatory cascades through SHP2-mediated MAPK and NF-κB pathway inhibition while activating the Nrf2-dependent antioxidant response. In preclinical models of ALI and sepsis, BA treatment significantly improved survival rates, preserved lung architecture, and prevented multi-organ dysfunction. Notably, BA demonstrated superior efficacy to the existing SHP2 inhibitor SHP099, particularly in sepsis survival outcomes (90 % vs 50 % survival at 24 h). Our findings not only identify BA as a promising therapeutic candidate for ALI but also establish a novel paradigm for targeting SHP2 in inflammatory diseases.

High Levels of Uric Acid Upregulate Endothelin Receptors: The Role of MAPK Pathways in an in vitro Study

IntroductionUric acid (UA) is the end product of purine compounds' metabolism. There is overwhelming evidence linking hyperuricemia (high levels of UA) and cerebrovascular diseases, but the effect of high levels of UA on cerebral vessels is not fully understood. The aim of this research is to clarify how UA affect the endothelin (ET) receptor in rat cerebral arteries and the related mechanism.Material and methodsIn an in vitro setting, segments of rat cerebral arteries (n=12) were exposed to high levels of UA, either alone or in conjunction with MAPK pathway inhibitors. ET agonists were used to induce contractions that were then measured with a myograph. ET receptor expression was measured using RT-PCR (n=6), Western Blot (n=3), or immunohistochemistry (n=3) to quantify mRNA and protein levels.ResultsThe study revealed that high levels of UA notably increase ETA and ETB receptor-induced contractions and boost the expression of ET receptors in cerebral arteries when compared to that are fresh or cultured alone, suggesting that UA enhances ETA and ETB receptors. Additionally, the up-regulation of ETB receptors induced by UA was inhibited by the p38 inhibitor SB203580, the JNK inhibitor SP600125, and the ERK1/2 inhibitor U0126. SB203580 significantly blocked the increase in ETA receptor-mediated contractions induced by UA and the upregulation of ETA receptor. Neither SP600125 nor U0126 had such effect.ConclusionsHigh levels of UA stimulate the up-regulation of ET receptors in rat cerebral arteries in vitro through MAPK pathways. This study may offer novel perspectives on hyperuricemia-associated cerebrovascular diseases.

Hosta plantaginea (Lam.) Aschers flower modulates inflammation and amino acid metabolism by inhibiting NF-κB/MAPK/JAK-STAT/PI3K-Akt and AMPK pathways to alleviate benign prostatic hyperplasia in rats

Ethnopharmacological relevanceBenign prostatic hyperplasia (BPH) is the most common urogenital disease in men with no definitive treatment. Inflammation, androgen imbalance, and oxidative stress play crucial roles in the pathogenesis of BPH. The flower of Hosta plantaginea (Lam.) Ascher is a pivotal medicinal plant in China, used to treat BPH. However, its effect and mechanism against BPH have not been clear. Aim of the studyOur aim was to decipher the pharmacodynamics and mechanisms of H. plantaginea flower against BPH. Materials and methodsThe extract yields and HPLC-based chemoprofile of ethanolic extract (HP) and total flavonoid (TF) of H. plantaginea flowers were used as reference standard to ensure their quality. The testosterone propionate-induced BPH rat model was used to assess the effects of HP and TF. Protein expression, metabolomics, and network pharmacology analyses were performed. ResultsTwenty constituents were identified in both HP and TF, with four quantitatively analyzed using the HPLC method. HP and TF demonstrated significant therapeutic effects on BPH, including reduced prostate size and prostatic index, improved pathological injury of prostate, as well as increased levels of testosterone, superoxide dismutase, glutathione, and glutathione peroxidase, along with decreased levels of dihydrotestosterone, 5 alpha-reductase, epidermal growth factor, TNF-α, IL-1β, IL-6, and malondialdehyde. Western blotting assay indicated that HP and TF prominently inhibited the protein expression of phosphorylated p65, IκBα, JNK, p38, Erk1/2, JAK1, STAT3, PI3K, Akt, and AMPKα1 in a dose-dependent manner. Integrating metabolomics and network pharmacology analyses revealed that HP and TF observably regulated 30 differential metabolites and 11 hub genes across the aforementioned pathways, which are closely associated with amino acid metabolism. ConclusionThe proposed comprehensive strategy of in vivo experiments, metabolomics, and network pharmacology studies has demonstrated that HP and TF could alleviate BPH injury in rats by suppressing inflammation, androgen imbalance, oxidative stress, and amino acid metabolism through the inhibition of NF-κB, MAPK, JAK-STAT, PI3K-Akt, and AMPK pathways, which provides novel insights into the potential of H. plantaginea flower as a treatment for BPH.

Dectin-1 participates in the immune-inflammatory response to mouse Aspergillus fumigatus keratitis by modulating macrophage polarization.

The aim of this study was to investigate whether Dectin-1 influences the immune-inflammatory response in A. fumigatus keratitis by modulating macrophage polarization. 1. The models of 1-day, 3-day, and 5-day of fungal keratitis were established in SPF C57BL/6 mice after stimulation by A. fumigatus. Dectin-1 agonist (curdlan) and antagonist (laminaran) were injected separately in the mouse subconjunctivae for 1 day in the established mouse model of A. fumigatus keratitis; PBS was used as the control. Inflammation of the mouse cornea was observed under a slit lamp to obtain a clinical score. 2. The expression of M1 (TNF-α, INOS, IL-6, IL-12) and M2 (Arg-1, IL-10, Fizz-1, Ym-1) cytokine-encoding mRNAs was quantified by RT-PCR. 3. Changes in the number of macrophages and expression of M1 and M2 macrophages in mouse corneas detected by immunofluorescence and flow cytometry. 4. Pre-treatment of RAW264.7 cells with MAPK cell signaling pathway inhibitors SB203580 (p38 inhibitor, 10µM), U0126 (ERK inhibitor, 20µM), SP600125 (JNK inhibitor, 10µM) and DMSO separately for 2h, and stimulated by A. fumigatus for 12h. Changes in the mRNA expression of M1 and M2 cytokines in the macrophages were quantified by RT-PCR. 1. With curdlan pre-treatment, mouse corneal inflammation worsened, and the clinical score increased after infection. In contrast, in the laminaran pre-treated group, corneal inflammation was alleviated and the clinical score decreased significantly compared to the PBS group after infection. 2. Compared with the control group, the expression levels of macrophage phenotype-related M1 and M2 cytokine mRNAs increased significantly 1, 3, and 5 days after A. fumigatus infected the corneas of mice. 3. With curdlan pre-treatment, the expression of mRNAs encoding M1 cytokines increased, while those encoding M2 cytokines decreased in the cornea compared to the PBS group. In contrast, after infection, mRNA levels for M1 cytokines decreased significantly and those for M2 cytokines increased in the cornea of the laminaran pre-treated group compared to the PBS group. 4. The number of macrophages in the corneal stroma of mice in the curdlan pretreatment group increased significantly compared with the PBS group, while in the laminaran pretreatment group this number decreased significantly. 5. The results of flow cytometry showed that after 3 days of mouse corneal A. fumigatus infection, the number of macrophages in the mouse A. fumigatus model in the curdlan pretreatment group was increased (10.4%) and the number of macrophages in the mouse A. fumigatus model in the laminaran pretreatment group (6.31%), when compared with the AF+FBS group (7.91%). The proportion of M1-type macrophages was increased in the curdlan pretreated group (55.6%) compared to the AF+FBS group (51.2%), the proportion of laminaran pretreatment group had a decreased proportion of M1-type macrophages (46.8%); while M2-type macrophages were the opposite of M1-type: the proportion of M2-type macrophages was 49.2% in the AF+FBS group, the proportion of M2-type macrophages was decreased in the curdlan pretreatment group (44.0%), and the proportion of M2-type macrophages was increased in the laminaran pretreatment group (53.5%). 6. Expression of M1 and M2 cytokine-encoding mRNAs decreased and increased, respectively, after infection, in the RAW264.7 cells pre-treated with MAPK pathway inhibitors, compared to the control. In a mouse model of A. fumigatus keratitis, Dectin-1 can affect macrophage recruitment and polarization, may regulate macrophage phenotype-associated factor changes through the MAPK signaling pathway.

Targetable treatment resistance in thyroid cancer with clonal hematopoiesis.

Anaplastic thyroid cancer (ATC) is a clinically aggressive malignancy with a dismal prognosis. Combined BRAF/MEK inhibition offers significant therapeutic benefit in patients with BRAF V600E -mutant ATCs. However, relapses are common and overall survival remains poor. Compared with differentiated thyroid cancer, a hallmark of ATCs is significant infiltration with myeloid cells, particularly macrophages. ATCs are most common in the aging population, which also has an increased incidence of TET2 -mutant clonal hematopoiesis (CH). CH-mutant macrophages have been shown to accelerate CH-associated pathophysiology including atherosclerosis. However, the clinical and mechanistic contribution of CH-mutant clones to solid tumour biology, prognosis and therapeutic response has not been elucidated. Here we show that TET2 -mutant CH is enriched in the tumour microenvironment of patients with solid tumours and associated with adverse prognosis in ATC patients. We find that Tet2 -mutant macrophages selectively infiltrate mouse Braf V600E -mutant ATC and that their overexpression of Tgfβ-family ligands mediates resistance to BRAF/MEK inhibition. Importantly, inhibition of Tgfβ signaling restores sensitivity to MAPK pathway inhibition, opening a path for synergistic strategies to improve outcomes of patients with ATCs and concurrent CH.

Abstract C036: Vertical inhibition of the autophagy pathway enhances sensitization to RAS MAPK pathway inhibition in pancreatic ductal adenocarcinoma

Abstract PDAC growth is characterized by dependencies on both mutant KRAS signaling and autophagy. Our group and others previously demonstrated that inhibition of the RAF/MEK/ERK pathway in PDAC induces an increased dependence on autophagy, a metabolic nutrient scavenging process by which cellular components are recycled in times of nutrient stress. Combined inhibition of the ERK MAPK pathway and autophagy inhibited the growth of multiple preclinical models of PDAC. Based on these findings, combined ERK/MEK inhibition and hydroxychloroquine (HCQ) is currently under clinical evaluation (NCT03825289, NCT04386057). However, resistance to this combination has already been described. To identify sensitizers to CQ treatment, our lab performed a CRISPR-Cas9 mediated loss-of- function screen using an sgRNA library targeting genes associated with cancer signaling pathways. Surprisingly, we identified multiple autophagy related genes, indicating that concurrent inhibition of two nodes of the autophagy pathway may be a more effective method of inhibiting autophagy in PDAC. For example, we identified, PIK3C3, the gene that encodes for VPS34, a protein essential for the nucleation of autophagosomes, as a potential sensitizer to CQ treatment. Inhibition of VPS34 with the compound SAR405, resulted in both decreased autophagic flux and impaired proliferation. Additionally, VPS34 inhibition sensitized cells to CQ treatment, leading to further reduced proliferation. These results prompted us to hypothesize that inhibition of ULK1, a serine/threonine kinase critical for the initiation of autophagy, could also sensitize PDAC cells to CQ treatment. We found that ULK1 inhibition further reduced CQ-mediated anti-proliferative effects. Additionally, both ULK1 and VPS34 inhibitor treatment sensitized cells to PIKfyve inhibition with apilimod, a chemically and mechanistically distinct inhibitor of the termination phase of the autophagic pathway. Previous studies have indicated that autophagy inhibition alone is not a viable therapeutic avenue for the treatment of PDAC. Thus, we next determined whether vertical inhibition of the autophagy pathway improves the efficacy of combined inhibition of the RAS and autophagy pathway. We found that both VPS34 inhibition and ULK1 inhibition can decrease RAS inhibitor induced autophagic flux. Finally, vertical inhibition of the autophagic pathway via VPS34 inhibition and CQ sensitized PDAC cells to RAS inhibition to further reduce PDAC proliferation. Ongoing and future studies are aimed at elucidating the mechanism leading to decreased proliferation of combined anti-autophagy and RAS inhibitor therapies, as well as mechanistically understanding the effect of autophagy inhibition at multiple nodes on autophagic flux. Citation Format: Mallory K Roach, Jonathan M DeLiberty, Elyse G Schechter, Runying Yang, Noah L Pieper, Clint A Stalnecker, Kirsten L Bryant. Vertical inhibition of the autophagy pathway enhances sensitization to RAS MAPK pathway inhibition in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C036.

Abstract C023: PDAC tumors from patients treated with combination MEK and autophagy inhibition reveal a treatment sensitive epithelial subtype that is enriched in murine models

Abstract Combining MEK and autophagy inhibitors in preclinical PDAC mouse models resulted in tumor regressions and improved rodent survival. These findings provided the rationale for clinical investigation of this combination. The phase 1 MEKiAUTO trial evaluated combination cobimetinib, HCQ, with or without atezolizumab in 14 patients (pts) with KRAS-mutated pancreatic adenocarcinoma (PDAC). The study was closed due to a lack of preliminary efficacy and poor tolerability; however, three patients experienced progression free survival (PFS) beyond 12 weeks (two with KRAS G12R , one with KRAS G12D ). Correlative analyses on twenty-one biopsies (8-paired FFPE and 6-paired fresh frozen specimens) with multiplex immunofluorescence (mIF) demonstrated decreased phosphorylation of ERK and accumulation of p62 indicating a pharmacodynamic effect. Reverse phase protein array (RPPA) also confirmed MAPK pathway inhibition, with a decrease in pERK, and autophagy inhibition, with decreased autophagy signature score, within the tumor compartment of on-treatment biopsies compared to baseline. Single nucleus RNAseq with protein expression activity extrapolated using ARACNe (Algorithm for the Reconstruction of Accurate Cellular Networks) and VIPER (Virtual Inference of Protein-activity by Enriched Regulon) pipelines identified five distinct tumor subpopulations. Comparing prevalence of tumor clusters before and after treatment identified differential responses to treatment. Gene set enrichment analysis revealed that the most sensitive tumor cluster, tumor cluster 2, was enriched in autophagy and MAPK signaling. Pre- treatment frequency of tumor cluster 2 was higher in patients who experienced a clinical benefit and independently correlated with PFS. Among the non-tumor cell clusters identified, patients with clinical benefit also had increased immune cell infiltration. To explain the discordance in efficacy with this combination in preclinical models and humans, we interrogated the preclinical tumor model systems for tumor heterogeneity. Single cell RNA-Seq analysis of tumors from Kras LSL.G12D/+ ; p53 LSL.R172H/+ ; Pdx1-Cre tg/+ (KPC) mice treated with the combination confirmed presence of similar treatment-sensitive and treatment-resistant clusters, with the former being enriched for MAPK and autophagy signatures. Parallel analysis of cell lines previously shown to be sensitive to combination MEK and autophagy inhibition, including MiaPACA2 and PANC1, showed they were mostly enriched in the treatment-sensitive human cluster 2 phenotype but not treatment-resistant clusters. Our results suggest that some preclinical models may overrepresent subtypes which are sensitive to MEK and autophagy inhibitors and thus do not recapitulate the extent of intertumoral heterogeneity in human PDAC. Observed preclinical responses likely represented elimination of treatment-sensitive cluster(s), with the remaining clusters responsible for maintained resistance. Citation Format: Brian W Labadie, Aleksandar Z Obradovic, Sarah Sta Ana, Michael May, Naomi Sender, Isabelle V Ross, Monica Rodriguez-Mendez, Urszula Wasko, Lorenzo Tomassoni, Li Li, Manan Vij, Sinan Abuzaid, Julia Wulfkuhle, Winston Wong, Ilenia Pellicciotta, Benjamin Izar, Emanuel F Petricoin, Aik Choon Tan, Kenneth P Olive, Alex G Raufi, Gulam A Manji. PDAC tumors from patients treated with combination MEK and autophagy inhibition reveal a treatment sensitive epithelial subtype that is enriched in murine models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C023.

The Novel Application of EUK-134 in Retinal Degeneration: Preventing Mitochondrial Oxidative Stress-Triggered Retinal Pigment Epithelial Cell Apoptosis by Suppressing MAPK/p53 Signaling Pathway.

Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by mitochondrial dysfunction of retinal pigment epithelium (RPE) cells. EUK-134 is a mimetic of SOD2 and catalase, widely used for its antioxidant properties in models of light-induced damage or oxidative stress. However, its effects on the retina are not yet clear. Here, we investigated the capability of EUK-134 in averting AMD using sodium iodate (NaIO3)-induced Balb/c mouse and ARPE-19 cells (adult RPE cell line). In vivo, EUK-134 effectively antagonized NaIO3-induced retinal deformation and prevented outer and inner nuclear layer thinning. In addition, it was found that the EUK-134-treated group significantly down-regulated the expression of cleaved caspase-3 compared with the group treated with NaIO3 alone. Our results found that EUK-134 notably improved cell viability by preventing mitochondrial ROS accumulation-induced membrane potential depolarization-mediated apoptosis in NaIO3-inducted ARPE-19 cells. Furthermore, we found that EUK-134 could inhibit p-ERK, p-p38, p-JNK, p-p53, Bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP by increasing Bcl-2 protein expression. Additionally, we employed MAPK pathway inhibitors by SB203580 (a p38 inhibitor), U0126 (an ERK inhibitor), and SP600125 (a JNK inhibitor) to corroborate the aforementioned observation. The results support that EUK-134 may effectively prevent mitochondrial oxidative stress-mediated retinal apoptosis in NaIO3-induced retinopathy.

1931P Phase II study of PDR001 in combination with MAPK pathway inhibitors in patients with radioactive-refractory differentiated thyroid cancer (DTC)

1931P Phase II study of PDR001 in combination with MAPK pathway inhibitors in patients with radioactive-refractory differentiated thyroid cancer (DTC)

Non-saponin from Panax ginseng maintains blood-brain barrier integrity by inhibiting NF-κB and p38 MAP kinase signaling pathways to prevent the progression of experimental autoimmune encephalomyelitis

BackgroundThe non-saponin (NS) fraction is an important active component of Panax ginseng, with multifunctional pharmacological activities including neuroprotective, immune regulatory, anti-inflammatory, and antioxidant effects. However, the effects of NSs on multiple sclerosis (MS), a chronic and autoimmune demyelinating disorder, have not yet been demonstrated. Purposeand Methods: The goal of the present study was to demonstrate the pharmacological actions of NSs on movement dysfunctions and the related mechanisms of action using an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. ResultsNSs (p.o.) alleviated movement dysfunctions in EAE mice related to reduced demyelination in the lumbar spinal cord (LSC). NSs attenuated the recruitment of microglia (CD11b+/CD45low) and macrophages (CD11b+/CD45high) in LSCs from EAE model mice, consistent with the decreased mRNA expression levels of the main proinflammatory mediators (IL-1β, COX-2, MCP-1, MIP-1α, and RANTES). NSs blocked the migration of Th17 cells (CD4+/IL17A+) and mRNA expression levels of IL-17A (product of Th17 cells) in LSCs from EAE mice. NSs suppressed alterations in blood-brain barrier (BBB) components, such as astrocytes and cell adhesion molecules, associated with inhibiting NF-κB and p38 MAPK pathways in LSCs of EAE mice and lipopolysaccharide-induced bEND.3 cells. ConclusionsNSs could attenuate movement dysfunctions and related pathological/inflammatory changes by reducing BBB permeability through NF-κB and p38 MAPK pathway inhibition in LSCs of EAE model mice. These are the first results suggesting that NSs can be potential therapeutic agents for MS by reducing BBB permeability.

Evidence for Radiation Therapy in Stage III Locoregionally Advanced Cutaneous Melanoma in the Post-Immunotherapy Era: A Literature Review.

In the landscape of Stage III locoregionally advanced cutaneous melanoma treatment, the post-immunotherapy era has sparked a number of questions on the management of the nodal basin. However, much of the available literature is not focused on radiation therapy as an adjuvant therapy. This literature review aims to illuminate the evidence surrounding radiation therapy's potential to mitigate regional recurrences in the adjuvant setting for melanoma. Additionally, it seeks to identify adjunct systemic therapy options and explore the synergy between systemic therapy and radiation. Despite strides in surgical techniques and systemic therapies, controlling regional Stage III melanoma remains a formidable clinical hurdle. While historical data strongly suggest the efficacy of adjuvant radiation therapy in reducing regional recurrence risk, its evaluation predates the advent of MAPK pathway inhibitors and robust immunotherapy options. Notably, clinical trials have yet to definitively demonstrate a survival advantage with adjuvant radiation therapy. Additional research should focus on refining the definition of high risk for regional recurrence through gene expression profiling or tumor immune profiling scores and elucidate the optimal role of adjuvant radiation therapy in patients treated with neoadjuvant systemic therapy.

Recurrent adamantinomatous craniopharyngiomas show MAPK pathway activation, clonal evolution and rare TP53-loss-mediated malignant progression

The two types of craniopharyngioma, adamantinomatous (ACP) and papillary (PCP), are clinically relevant tumours in children and adults. Although the biology of primary craniopharyngioma is starting to be unravelled, little is known about the biology of recurrence. To fill this gap in knowledge, we have analysed through methylation array, RNA sequencing and pERK1/2 immunohistochemistry a cohort of paired primary and recurrent samples (32 samples from 14 cases of ACP and 4 cases of PCP). We show the presence of copy number alterations and clonal evolution across recurrence in 6 cases of ACP, and analysis of additional whole genome sequencing data from the Children’s Brain Tumour Network confirms chromosomal arm copy number changes in at least 7/67 ACP cases. The activation of the MAPK/ERK pathway, a feature previously shown in primary ACP, is observed in all but one recurrent cases of ACP. The only ACP without MAPK activation is an aggressive case of recurrent malignant human craniopharyngioma harbouring a CTNNB1 mutation and loss of TP53. Providing support for a functional role of this TP53 mutation, we show that Trp53 loss in a murine model of ACP results in aggressive tumours and reduced mouse survival. Finally, we characterise the tumour immune infiltrate showing differences in the cellular composition and spatial distribution between ACP and PCP. Together, these analyses have revealed novel insights into recurrent craniopharyngioma and provided preclinical evidence supporting the evaluation of MAPK pathway inhibitors and immunomodulatory approaches in clinical trials in against recurrent ACP.

A kinome drug screen identifies multi-TKI synergies and ERBB2 signaling as a therapeutic vulnerability in MYC/TYR subgroup atypical teratoid rhabdoid tumors.

Atypical teratoid rhabdoid tumor (ATRT) is a rare, devastating, and largely incurable pediatric brain tumor. Although recent studies have uncovered 3 molecular subgroups of ATRTs with distinct disease patterns, and signaling features, the therapeutic profiles of ATRT subgroups remain incompletely elucidated. We examined the effect of 465 kinase inhibitors on a panel of ATRT subgroup-specific cell lines. We then applied multiomics analyses to investigate the underlying molecular mechanism of kinase inhibitor efficacy in ATRT subgroups. We observed that ATRT cell lines are broadly sensitive to inhibitors of the PI3K and MAPK signaling pathways, as well as CDKs, AURKA/B kinases, and polo-like kinase 1. We identified 2 classes of multikinase inhibitors predominantly targeting receptor tyrosine kinases including PDGFR and EGFR/ERBB2 in MYC/TYR ATRT cells. The PDGFRB inhibitor, Dasatinib, synergistically affected MYC/TYR ATRT cell growth when combined with broad-acting PI3K and MAPK pathway inhibitors, including Rapamycin and Trametinib. We observed that MYC/TYR ATRT cells were also distinctly sensitive to various inhibitors of ERBB2 signaling. Transcriptional, H3K27Ac ChIPSeq, ATACSeq, and HiChIP analyses of primary MYC/TYR ATRTs revealed ERBB2 expression, which correlated with differential methylation and activation of a distinct enhancer element by DNA looping. Significantly, we show the brain penetrant EGFR/ERBB2 inhibitor, Afatinib, specifically inhibited in vitro and in vivo growth of MYC/TYR ATRT cells. Taken together, our studies suggest combined treatments with PDGFR and ERBB2-directed TKIs with inhibitors of the PI3K and MAPK pathways as an important new therapeutic strategy for the MYC/TYR subgroup of ATRTs.

A novel approach to alleviate acetaminophen-induced hepatotoxicity with hybrid balloon flower root-derived exosome-like nanoparticles (BDEs) with silymarin via inhibition of hepatocyte MAPK pathway and apoptosis

IntroductionBalloon flower root-derived exosome-like nanoparticles (BDEs) have recently been proposed as physiologically active molecules with no cytotoxicity. However, the therapeutic effects of drug-induced hepatotoxicity of BDEs have not been elucidated. BDEs contain a large amount of platycodin D, which is widely known to be effective in regulating inflammation and ameliorating systemic toxicity. Thus, the main therapeutic activity of BDEs is attributed to inhibiting the inflammatory response and alleviating toxicity. In this study, we fabricated the hybrid BDEs fused with liposomes containing silymarin (SM) to enhance the synergistic effect on inhibition of acetaminophen-induced hepatotoxicity (APAP).ObjectiveConsidering the potential therapeutic effects of BDEs, and the potential to achieve synergistic effects to improve therapeutic outcomes, we constructed hybrid BDEs with a soy lecithin-based liposome loaded with SM. Since liposomes can provide higher thermal stability and have greater structural integrity, these might be more resistant to clearance and enzymatic degradation of drug molecules.MethodsHybrid BDEs with liposome-loaded SM (BDEs@lipo-SM) were fabricated by thin-film hydration and extrusion. BDEs@lipo-SM were characterized using dynamic light scattering and high-performance liquid chromatography. After confirmation of the physical properties of BDEs@lipo-SM, various therapeutic properties were evaluated.ResultsBDEs@lipo-SM were internalized by hepatocytes and immune cells and significantly decreased mRNA expression of apoptosis and inflammation-relevant cytokines by inhibiting the hepatocyte MAPK pathway. BDEs@lipo-SM significantly induced an increase in glutathione levels and inhibited APAP-induced hepatotoxicity.ConclusionFrom this study, we know that BDEs are reliable and safe nanovesicles containing natural metabolites derived from balloon flower, and they can facilitate intercellular communication. BDEs are also easily modified to enhance drug loading capacity, targeting effects, and long-term accumulation in vivo. BDEs@lipo-SM have therapeutic benefits for acute liver injury and can alleviate cell death and toxicity. They can be efficiently delivered to the liver and effectively inhibit APAP-induced hepatotoxicity by inhibiting the MAPK signaling pathway and apoptosis, which accelerates liver recovery in the APAP-induced acute liver injury model. These findings highlight that BDEs represent an attractive delivery vehicle for drug delivery.Graphical abstract

CP-05. MAPK PATHWAY ACTIVATION, CLONAL EVOLUTION, INCLUDING RARE TP53 LOSS, AND THE IMMUNE ENVIRONMENT OF RECURRENT CRANIOPHARYNGIOMA

Abstract BACKGROUND Craniopharyngiomas are rare challenging tumours, with around 25% of cases recurring despite surgery and/or radiotherapy. Relatively little is known about the biology of recurrence and there is an urgent need to develop new therapies. Our previous studies have suggested preclinical efficacy of MEKinhition with trametinib in human and murine adamantinomatous craniopharyngoioma (ACP) tissue. At ISPNO2022 we reported methylation and expression profiling results from a cohort of relapsed craniopharyngioma, identifying acquisition of chromosomal abnormalities across recurrence, the persistent activation of MAPK pathway at recurrence, the presence of myeloid cells and a rare case of malignant transformation associated with TP53 loss. METHODS Here we present an update from this study through exploring additional datasets, preclinical drug testing, and genetic manipulation of genetic engineered mouse models of ACP RESULTS Exploration of whole genome sequencing data from 67 cases of ACP from Children’s Brain Tumour Network has confirmed the presence of chromosomal arm changes in 7 (10%) of cases, including at diagnosis, confirming that the genomic landscape of ACP is more complex than previously thought. To further explore the potential of MAPK pathway inhibiton we have demonstrated efficacy of a further two clinically available MEK inbhiibtors, Selumetinib and Binimetinib, in reducing proliferation and inducing cell death in ex vivo explants of murine ACP. To explore the role of TP53 loss in aggressive craniopharyngioma, we show that Trp53 loss in a murine ACP model results in very aggressive tumours and reduced mouse survival. Finally, we have further characterised the tumour immune infiltrate showing differences in the cellular composition between ACP and Papillary CP, and revealing a diverse phenotype of macrophages in ACP. CONCLUSIONS Together, this research provides further preclinical support for the ongoing evaluation of MAPK pathway inhibitors and immunomodulatory approaches in clinical trials in patients with craniopharyngioma.

LGG-29. BEYOND MAPK SIGNALING - GENOME-SCALE CRISPR/CAS9 SCREENS REVEAL NEW TARGETS FOR TREATMENT OF KIAA1549::BRAF-DRIVEN PEDIATRIC LOW-GRADE GLIOMA

Abstract Since the KIAA1549::BRAF fusion was discovered as the most common driver of pediatric low-grade glioma (pLGG), it has been hypothesized that the fusion induces oncogenicity through BRAF activation as a result of KIAA1549 replacing the BRAF negative regulatory N-terminus. This led to the rapid translation of MAPK pathway inhibitors into the clinical setting. Despite most tumors exhibiting promising initial responses, some tumors are not sensitive and about half of responsive tumors grow back after treatment cessation. Therefore, strategies that result in sustained tumor responses are desperately needed. We have recently performed genome-scale CRISPR/Cas9 screens across isogenic neural stem cell models transduced to express pLGG-associated oncogenes (KIAA1549::BRAF, BRAFV600E, and multiple FGFR1 and MYB family alterations) to generate a dependency map of genetic vulnerabilities associated with expression of these oncogenes. We also included normal neural stem cells to allow identification of genetic dependencies specifically induced by the expression of each oncogene. Surprisingly, through these efforts, we have discovered KIAA1549::BRAF expressing cells to harbor striking and specific dependency on multiple members of an enzymatic complex that exerts its activity outside of the MAPK signaling axis*. Interestingly, this enzymatic complex has been described to modify only a few substrates, including KIAA1549, suggesting specificity. We have now validated this dependency across other isogenic models of KIAA1549::BRAF-expressing cells. Finally, our KIAA1549::BRAF-expressing models exhibit preferential sensitivity to a tool compound that targets our novel enzyme, suggesting novel therapeutic potential for KIAA1549::BRAF. These findings highlight a MAPK pathway-independent avenue for therapeutically targeting the most frequent genomic alterations in pediatric brain tumors. Consequently, we also propose that the fusion partner KIAA1549 is instrumental for the aberrant BRAF signaling driving pLGGs. (* we are currently in the process of working with our IP offices to ensure that we can disclose the names of the genes and proteins at the ISPNO meeting)

LGG-14. INTEGRATED MULTI-OMICS EXPLORATION OF MAPK PATHWAY INHIBITION IN PEDIATRIC PILOCYTIC ASTROCYTOMA

Abstract Pilocytic astrocytomas (PA), the most common pediatric brain tumors, exhibit MAPK pathway alterations leading to its constitutive activation. They are also associated with low proliferation index due to the oncogene-induced senescence (OIS), sustained by senescence-associated secretory phenotype (SASP) factors. Little is known about the downstream consequences of MAPK activation leading to OIS-SASP, and of the molecular implications of MAPK pathway inhibition in senescent PA cells. Senescent DKFZ-BT66 cells derived from a primary KIAA::BRAF-fusion positive PA were used to generate RNA-sequencing and phospho-/proteomic datasets, before and after treatment with the MEK inhibitor (MEKi) trametinib for different time-spans. A multi-omics factor analysis tool (MEFISTO) and single sample gene set enrichment analysis (ssGSEA) were used to identify key OIS effectors and differentially regulated pathways upon MAPK inhibition. Trametinib treatment inhibited OIS and SASP signatures across all omics levels in the senescent DKFZ-BT66 cells, associated with reduced sensitivity towards senolytics drugs, indicating inhibition of senescence features upon MEKi. MEFISTO analysis identified key effectors involved in MAPK-induced OIS in the senescent PA cells. We built a pathway network using a prior knowledge network approach allowing to map the direct downstream effectors of MAPK leading to OIS-SASP. We identified several parallel pathways (JAK/STAT, p38, GSK3B) potentially involved in OIS and under the control of the MAPK pathway. In addition, ssGSEA showed that pathways related to upstream MAPK activators (SOS1, CK2, several RTKs) were predicted to be upregulated upon treatment, highlighting putative targets to be further investigated. Our data suggest that MAPKi reverses OIS signaling 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 provides insight into the regulation of OIS-SASP by the MAPK pathway.

Recent Advances in Molecular and Genetic Research on Uveal Melanoma.

Uveal melanoma (UM), a distinct subtype of melanoma, presents unique challenges in its clinical management due to its complex molecular landscape and tendency for liver metastasis. This review highlights recent advancements in understanding the molecular pathogenesis, genetic alterations, and immune microenvironment of UM, with a focus on pivotal genes, such as GNAQ/11, BAP1, and CYSLTR2, and delves into the distinctive genetic and chromosomal classifications of UM, emphasizing the role of mutations and chromosomal rearrangements in disease progression and metastatic risk. Novel diagnostic biomarkers, including circulating tumor cells, DNA and extracellular vesicles, are discussed, offering potential non-invasive approaches for early detection and monitoring. It also explores emerging prognostic markers and their implications for patient stratification and personalized treatment strategies. Therapeutic approaches, including histone deacetylase inhibitors, MAPK pathway inhibitors, and emerging trends and concepts like CAR T-cell therapy, are evaluated for their efficacy in UM treatment. This review identifies challenges in UM research, such as the limited treatment options for metastatic UM and the need for improved prognostic tools, and suggests future directions, including the discovery of novel therapeutic targets, immunotherapeutic strategies, and advanced drug delivery systems. The review concludes by emphasizing the importance of continued research and innovation in addressing the unique challenges of UM to improve patient outcomes and develop more effective treatment strategies.

Efficacy and toxicity of KIT targeted therapy in elderly patients with melanoma.

e23269 Background: Most cases of malignant melanoma are cutaneous, with ≈50% having a BRAF driver mutation that predicts response to MAPK pathway inhibitors. A small minority of melanoma cases harbor an activating KIT mutation, most notably in mucosal and acral melanomas (15-20%) and occasionally in those arising from chronic sun-damaged skin (2-3%). Only about a third of KIT mutated melanomas respond to targeted therapy with Imatinib, yet data is scarce and limited, especially in older patients. We sought to characterize the efficacy and toxicity of Imatinib in KIT-mutated melanoma patients over 65. Methods: We identified electronic medical records of patients over 65 that were treated with Imatinib for KIT mutated melanoma at our institution over the years 2017-2023. Data was retrospectively collected including demographics, disease characteristics, response patterns and toxicity. Results: 10 patients were identified, 50% were males. The median age at treatment initiation was 75 years (71-100). 3 patients had cutaneous primary, 3 had Vulvar/vaginal primary, 3 had acral primary and 1 had anal canal primary. All patients had normal Lactate dehydrogenase at treatment initiation and ECOG performance status was 0-2 (median 0). 9 patients received Imatinib for stage IV disease with an overall response rate (ORR) of 55% (44% partial response, 11% complete response). Median duration of response was 4 months. Responses were seen in acral melanoma (67%), vulvar/vaginal melanoma (67%) and cutaneous melanoma (33%). Median Progression free survival was 4.3 months (1.8-44), median overall survival from treatment initiation was 22.6 months (4-64). 6 patients were treated as 2nd line with an ORR of 67% and 3 were treated as 3rd line with an ORR of 33%. There was one patient with M1d disease who did not respond to treatment either systematically or intracranially. 6 patients had Exon 11 mutations (V560D/G/E, N564S and L576P) of which 4 responded (67%). 2 had Exon 17 mutations (N822C/K) of which 1 responded (50%) and the 2 others had an Exon 14 (F681I) and Exon 13 (K642E) mutation that did not respond to treatment at all. Toxicity was moderate with 90% of patients experiencing any grade AE, yet only 1 experienced toxicity requiring dose interruption with two Grade 3 toxicities - Pneumonitis and decreased neutrophil count, leading to temporary treatment interruption, dose reduction and a course of corticosteroids. The rest of the patients experienced only Grade 1-2 toxicity. 4 patients needed dose reductions, there were no treatment discontinuations due to toxicity. Conclusions: Treatment with Imatinib for elderly KIT mutated melanoma patients is safe and well tolerated with no treatment discontinuations recorded. The response rate is encouraging yet seems to vary according to the KIT mutation present as previously reported. The duration of response is usually short-lived and alternative/combination treatments are still needed for this patient population.