Activation Of MAPK Pathway Research Articles

Abstract 302: MEKi-FGFRi combination nanoparticles for use against KRASmt/FGFR-compensatory lung tumors

Abstract Targeting oncogenic signaling in the MAPK pathway can be achieved with small molecules such as trametinib, a MEK inhibitor (MEKi). Trametinib-induced MEK inhibition in KRAS-mutant (KRASmt) lung cancer, however, results in compensatory MAPK pathway re-activation through fibroblast growth factor receptor 1 (FGFR1). Combining MEK inhibition with FGFR1 inhibition should thereby combat this compensation; nonetheless, systemic administration of combination therapy faces its own set of challenges. Such challenges include delivering appropriate kinase inhibitor concentrations to target tumor tissue while avoiding dose-limiting additive toxicities. Although previous work has identified that combination therapy involving trametinib and FGFR inhibitor (FGFRi) ponatinib synergistically inhibits growth and proliferation of KRASmt cells, ponatinib interacts with a variety of targets and may mediate its synergistic effects via mechanisms not specific to FGFR1. To improve the efficacy at which we target KRASmt/FGFR-compensatory lung tumors, we encapsulated trametinib with a variety of FGFR1-specific inhibitors to form P-selectin-targeted nanoparticles. By formulating these nanoparticles, compensatory MAPK pathway activation through FGFR1 can be more efficaciously inhibited. Concurrently, greater concentrations of kinase inhibitor combinations can be locally delivered to lung tumors while mitigating toxic side effects that occur from systemic free drug. Here we show that these MEKi-FGFRi combination nanoparticles can be formulated to efficaciously inhibit growth and proliferation in KRASmt/FGFR-compensatory cancer cells. Citation Format: Arianna R. Izawa-Ishiguro, Ramya Sridharan, Christopher Wun, Tabassum J. Sami, Daniel A. Heller. MEKi-FGFRi combination nanoparticles for use against KRASmt/FGFR-compensatory lung tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 302.

Abstract 2668: Targeting of ERK and HDAC in the treatment of uveal melanoma

Abstract Uveal melanoma is one of the most common intraocular malignancies of the adult eye. GNAQ/GNA11 genetic alterations are present in 85% of cases resulting in activation of the MAPK pathway. Single agent approaches targeting the MAPK pathway and histone deacetylases (HDACs) have shown very modest activity in uveal melanoma. We hypothesize that a combination strategy may be an effective, novel therapeutic approach in uveal melanoma. We examined the activity of ASTX029, a novel dual-mechanism ERK inhibitor, which inhibits both the catalytic activity of ERK and its phosphorylation by MEK, and belinostat, a pan-HDAC inhibitor, in one GNAQQ209L- (92.1) and GNAQQ209P- (OMM1.3) and two GNA11Q209L-mutant (MP41, OMM1) uveal melanoma cell lines. The uveal melanoma cell lines were sensitive to ASTX029 with IC50 values between 50 to 100nM and to belinostat with IC50 values between 100 to 250nM. Combination index analysis of ASTX029 and belinostat by the Chou-Talalay method indicated synergism, with CI values ranging from 0.34 to 0.97. We explored other drug candidates for combinatorial approaches including the BCL-2 inhibitor navitoclax, but no combination effect was observed. After 48h of combination treatment with ASTX029 and belinostat, we examined the biochemical effects by western blot which demonstrated a sustained decrease in levels of p-ERK with the combination treatment. Western blot analysis confirmed reduction of downstream markers of MAPK pathway activity including cell cycle markers p-RB (780), cyclin D1 and c-myc with combination treatment. Apoptosis was evident from an increase in the subG1 population by flow cytometry analysis, which correlated with BIM induction and PARP cleavage on western blot. We also observed increased surface expression of immunological markers HLA-A, B, C (MHC Class 1), PD-L1, gp100 and MART-1. These results support that a combination strategy targeting ERK and HDAC in uveal melanoma should be investigated further. Citation Format: Elgilda Musi, Kim-Hien Dao, Chris Hindley, Gary K. Schwartz. Targeting of ERK and HDAC in the treatment of uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2668.

Abstract 5625: Molecular and immunologic characterization of HRAS mutations in a cohort of 6,329 patients with cutaneous melanoma

Abstract Background: Activation in RAS pathway has been associated with cancer development. Three RAS family members, including NRAS, KRAS and HRAS are frequently mutated across various cancer types, where NRAS mutations are present in 15-20% of melanomas. NRAS-mutant melanomas (NRASm) have been extensively characterized. However, molecular and clinical implications of HRAS mutations (HRASm) in melanoma are less well understood. Methods: A total of 6329 melanoma samples were subjected to comprehensive molecular profiling at Caris Life Sciences. Analyses included next generation sequencing of DNA (592 Gene Panel, NextSeq; whole exome sequencing, NovaSEQ), RNA (NovaSeq, whole transcriptome sequencing, WTS) and IHC. MPAS scores to evaluate MAPK pathway activation, IFN scores, QuantiSeq, neoantigen load (high, intermediate, low binding affinity: HBA, IBA and LBA) and GSEA were calculated from mRNA expression data. Wilcoxon, Fisher’s exact were used to determined statistical significance (p value without and q value with multi comparison correction; FDR for GSEA). The reference cohort was the entire melanoma cohort (MC). Results: HRASm were identified in 69 (1.09%) of melanoma samples (hotspots mutations: G13, 40%; Q61, 34%; G12, 18% and others, 9%). HRASm and NRASm had different genomic landscapes: HRASm were significantly associated with a higher mutation rate of NF1 (43.2% vs 27.7%, p<.05), ARID1A (17.2% vs 6.3%, p<.05), B2M (14.3% vs 2.4%, p<.05), RAF1 (12.2% vs 1.4, p<.0001), CTNNB1 (9.1% vs 3.3%, p<.05) and higher amplifications of EMSY (11.8% vs 1.8%, p<.01), MRE11 (4.3% vs 0.5%, p<.05), whereas NRASm harbored less NF1 (14.8% vs 27.7%, q<.0001 ), BRAF (6.9% vs 39.9%, p <.0001), PTEN (3.9% vs 6.9%, q <.05), KIT (0.8% vs 4.4%, q <.0001) mutations and less amplification PDGFR (0.4% vs 1.3%, p<.05), BRAF (0.2% vs 1.4%, p<.05), KIT (0.2% vs 2.1%, q<.05) when comparing to MC. Both HRASm and NRASm had higher MPAS scores than MC (HRASm, 0.24; NRASm, 0.11; MC, -0.41, q<.001). In addition, HRASm showed higher TMB (HRASm, 68.1%; NRASm, 56.9%; MC, 50.0%, q<.05), relatively higher IFN scores (HRASm, 0.16; NRASm, -0.23; MC, -0.24, q = .16) and higher neoantigen load (HBA: HRASm, 10.5; NRASm, 4; MC, 4, p < .05; IBA: HRASm, 17.5; NRASm, 8 MC, 7, p < .05; LBA: HRASm, 37.5; NRASm, 21; MC, 19, p =.1) when compared to NRASm and MC. Lastly, suppression of angiogenesis pathway was observed in both HRASm (NES = 1.7, FDR<.05) and NRASm with respect to MC (NES = 1.4, FDR<.25). Conclusions: The genomic landscape of HRASm are significantly different from that of NRASm, implying their distinct roles in tumorigenesis. HRASm also demonstrated higher MAPK activation, suggesting that they could potentially benefit from agents targeting on this pathway. In addition, HRASm displayed more immunogenic features, associated with down-regulation of angiogenesis pathway, revealing a potential higher susceptibility of HRASm to immunotherapy. Citation Format: Leonel F. Hernandez-Aya, Estelamari Rodriguez, Aparna Nallagangula, Jun Yin, Phillip Walker, Joanne Xiu, Justin Moser, Gino K. In, David Spetzler, Geoffery T. Gibney, Matthew Oberley, Thuy Phung, Michael Atkins, Dave S. Hoon, Wolfgang Michael Korn, Jose Lutzky, Gilberto Lopes. Molecular and immunologic characterization of HRAS mutations in a cohort of 6,329 patients with cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5625.

Abstract 90: STAT3-driven MAPK activation represents a therapeutic vulnerability in ASCL1 high SCLC

Abstract Background Small cell lung cancer (SCLC) is an aggressive high grade neuroendocrine tumor (Hann et al. 2019; Bernhardt et al. 2016). Whilst MAPK mutations can be found in roughly 30% of human cancers (Schubbert et al. 2007) including non-small cell lung cancer (NSCLC), genomic and proteomic analyses have indicated suppression of MAPK pathway activity in SCLC (Cerami et al. 2012; Gao et al. 2013). Previous attempts to determine whether this might be therapeutically important (Ravi et al, 1998; Cristea et al. 2016/2020) have had conflicting conclusions. SCLC has recently been defined by the relative expression of four major transcriptional regulators (ASCL1, NEUROD1, POU2F3, YAP1) (Rudin et al., 2019). In this study we aimed to elucidate the effect of MAPK activation in these different SCLC subtypes and explore its therapeutic vulnerability. Results ASCL1-, NEUROD1- and POU2F3- driven SCLC cell lines were transduced with a doxycycline-inducible vector for expression of MEKDDS217D/S221D (MEK1). Activation through MEK1 in ASCL1-driven SCLC cell lines resulted in a significant decrease in cell growth over 9 days. This was associated with G2 cell-cycle arrest and senescence. Expression of MEK1 in the cells of other SCLC subtypes and NSCLC failed to show any appreciable changes in cell growth. Remarkably, athymic mice injected with a MEK1 expressing ASCL1-driven cell line showed significantly slower tumor formation and longer survival than when MEK1 was not expressed. Notably, we observed the opposite when MEK1 expressing NEUROD1-driven cells were injected into athymic mice. We observed strong upregulation of negative feedback regulators DUSP6 and SPRY2 upon MAPK activation which has previously been described in solid tumors and pre-B ALL (Courtois-Cox et al 2006; Shojaee et al. 2015). Interestingly, phosphokinase array demonstrated that, almost exclusively, STAT3 through phosphorylation at S727 was strongly upregulated in the ASCL1-driven subtype after MEK1 expression. This prompted us to look at other regulators of the JAK-STAT pathway and found an increase in phosphorylation of the inhibitory phosphatase PTPN6 (SHP1) in the ASCL1-driven subtype and no STAT5 phosphorylation in any of the subtypes. We next examined whether these cells were sensitive to STAT3 inhibition. Upon treatment with a STAT3 inhibitor, stattic, ASCL1-driven SCLC cells reached their IC50 after 3-5 days in comparison to 9 days for other SCLC subtypes. MEK inhibition through PD0325901 rescued growth inhibition upon MAPK activation and was further associated with a decrease in DUSP6, SPRY2 and pS727 STAT3. JAK1/2 inhibition through ruxolitinib had no effect on cell viability. Summary These findings suggest that ASCL1-driven SCLC in vitro and in vivo is sensitive to activation of MAPK signaling. Our data provides additional understanding of SCLC biology and its complex signaling networks and potential subtype-specific drug susceptibilities. Citation Format: Rebecca Caeser, Christopher Hulton, Emily Costa, Vidushi Durani, Megan Little, Xiaoping Chen, Sam E. Tischfield, Marina Asher, Faruk Erdem Kombak, Shweta S. Chavan, Nisargbhai S. Shah, Metamia Ciampricotti, Elisa de Stanchina, John T. Poirier, Charles M. Rudin, Triparna Sen. STAT3-driven MAPK activation represents a therapeutic vulnerability in ASCL1 high SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 90.

Abstract 6130: Comprehensive genomic and transcriptomic profiling of acral lentiginous melanoma

Abstract Background: Acral lentiginous melanoma (ALM) is a rare melanoma subtype found on the palms, soles and nailbeds. Outcomes are poor for patients with advanced ALM, and novel treatment approaches are needed. Here, we seek to explore the global genomic and transcriptomic landscape of ALM. Methods: A total of 699 primary CM (non-ALM cutaneous melanoma) and 18 primary ALM samples underwent next generation sequencing of DNA (592 Gene Panel, NextSeq, or WES, NovaSeq), and whole transcriptome sequencing (NovaSeq, WTS). Wilcoxon, Fisher’s exact test were used to determine statistical significance (displayed as p value without and q value with multi comparison correction). xCell, HLA subtyping, neoantigen load (HBA: high binding affinity; IBA: intermediate binding affinity; LBA: low binding affinity), Interferon gamma score (IFNγ), MAPK pathway activity score (MPAS), and Innate anti-PD-1 Resistance score (IPRES) were calculated by mRNA expression. Global differentially regulated genes were assessed via limma R package (C: log fold change). Results: The most common alterations in ALM included NRAS (22.2%), NF1 (20.0%), BRAF (11.1%) and CDKN2A (11.1%) mutations, and EMSY (22.2%), ELL (11.1%), MAML2 (11.1%), MRE11(11.1%) and PIK3R2 (11.1%) amplifications. ALM had lower TMB (1.5 v 9 Mut/Mb, q<.0001), lower rates of TERT (0 v 66.7%, q<.01) and a trend towards lower BRAF (11.1 v 39.8%, p<.05) mutations, compared to CM. Neoantigen load was lower in ALM compared to CM, regardless of MHC binding affinity (HBA: 1 vs 4, q <.01; IBA: 2 vs 7, q <.001; LBA: 7 vs 18, q <.001). HLA-G RNA expression was upregulated in ALM with respect to CM (C = 1.14, q <.001). ALM showed less CD4+ T cell Th1 (C = -0.8, p <.05), B cell plasma (C = -1.8, p<.05), and γδ T cells (C = -5.9, p<.05), but more CD4+ T cell central memory cell (C = 9.7, p<.05), stroma score (C = 1.7, p<.05), and endothelial cells (C = 1.7, p<.05), versus CM. There was a trend towards lower IFNγ in ALM (-0.4 vs -0.3, p = .1), but no difference in IPRES, compared to CM (-0.09 vs 0.1, p=.9). MPAS scores were lower for ALM compared to CM (-1.6 vs -0.4, q<.001), even when stratifying by BRAF (q<.05) or NF1 (q<.05) status, but not NRAS (p = .22). Pathways related to keratinization (p <.0001) and amyloid fiber formation (p<.0001) were enriched in ALM, due to overexpression of KRT16 (C = 3.5, q <.01), KRT6B (C = 3.4, q <.01), and KRT17 (C = 3.2, q <.05), among others. Conclusion: ALM has distinct immunologic features, including upregulation of HLA-G, as well as lower MAPK activation in ALM, compared to CM, highlighting the need for novel therapeutic approaches in the treatment of this rare subtype. Citation Format: Gino K. In, Jun Yin, Phillip Walker, Justin Moser, Joanne Xiu, Kelsey Poorman, Geoffery T. Gibney, Matthew Oberley, Thuy Phung, Leonel F. Hernandez-Aya, Jose Lutzky, Wolfgang Michael Korn, Michael B. Atkins. Comprehensive genomic and transcriptomic profiling of acral lentiginous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6130.

Sophoridine Suppresses Herpes Simplex Virus Type 1 Infection by Blocking the Activation of Cellular PI3K/Akt and p38 MAPK Pathways.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous and important human pathogen capable of causing significant clinical diseases ranging from skin damage to encephalitis, particularly in immunocompromised and neonatal hosts. Currently, widely used nucleoside analogs, including acyclovir and penciclovir, have some limitations in their use due to side effects and drug resistance. Herein, we report sophoridine's (SRI) dramatic inhibition of HSV-1 replication in vitro. SRI exhibited a remarkable inhibitory influence on HSV-1 virus-induced cytopathic effect and plaque formation, as well as on progeny viruses in Vero and HeLa cells, with selection indexes (SI) of 38.96 and 22.62, respectively. Moreover, SRI also considerably suppressed HSV-1 replication by hindering the expression of viral immediate-early (ICP0 and ICP22), early (ICP8 and TK), and late (gB and gD) genes and the expression of viral proteins ICP0, gB, and gD. We suggest that SRI can directly inactivate viral particles and block some stages in the life cycle of HSV-1 after adsorption. Further experiments showed that SRI downregulated the cellular PI3K/Akt signaling pathway and obstructed HSV-1 replication even more. Most importantly, SRI markedly repressed HSV-1-induced p38 MAPK pathway activation. Collectively, this natural bioactive alkaloid could be a promising therapeutic candidate against HSV-1 via the modulation of cellular PI3K/Akt and p38 MAPK pathways.

The Role of Microglia/Macrophages Activation and TLR4/NF-κB/MAPK Pathway in Distraction Spinal Cord Injury-Induced Inflammation.

Distraction spinal cord injuries (DSCIs) often occur as the neurological complication of distraction forces following the implantation of internal fixation devices during scoliosis correction surgery. However, the underlying mechanism behind these injuries remains unclear. The present study aimed to explore the activation of microglia and macrophages, as well as changes in TLR4-mediated NF-κB and MAPK pathway activity after DSCIs in Bama miniature pigs. Prior to surgical intervention, the pigs were randomly divided into three groups: the sham group, the complete distraction spinal cord injury (CDSCI) group, and the incomplete distraction spinal cord injury (IDSCI) group. After surgery, the Tarlov scale and individual limb motor scale (ILMS) were used to evaluate changes in the pigs’ behavior. All pigs were euthanized 7 days after surgery, and histopathological examinations of the spinal cord tissues were performed. Immunohistochemistry was used to detect Caspase-3 expression in the anterior horn of spinal gray matter tissues. Immunofluorescence staining was utilized to assess the M1/M2 phenotype changes in microglia/macrophages and NF-κB P65 expression in central DSCI lesions, while western blotting was performed to determine the expression of TLR4/NF-κB/MAPK pathway-related proteins. The results of the present study showed that the Tarlov and ILMS scores decreased significantly in the two DSCI groups compared with the sham group. Hematoxylin and eosin (HE) and Nissl staining revealed that the tissue structure and nerve fiber tracts in the distracted spinal cord tissues were destroyed. Both DSCI groups showed the number of survived neurons decreased and the Caspase-3 expression increased. The results of the immunofluorescence staining indicated that the CD16 and CD206 expression in the microglia/macrophages increased. Between the two DSCI groups, the CDSCI group showed increased CD16 and decreased CD206 expression levels. The intensity of the fluorescence of NF-κB P65 was found to be significantly enhanced in pigs with DSCIs. Moreover, western blot results revealed that the expression of TLR4, p-IκBα, NF-κB P65, p-JNK, p-ERK, and p-P38 proteins increased in spinal cord tissues following DSCI. The present study was based on a porcine DSCI model that closely mimicked clinical DSCIs while clarifying DSCI-associated neuroinflammation mechanisms, in turn providing evidence for identifying potential anti-inflammatory targets.

Pulegone ameliorates inflammation and oxidative stress in L-arginine-induced acute pancreatitis in mice by regulating the activation of p38 MAPK pathway

Purpose: To evaluate the effects of pulegone (PLG) on inflammation and oxidative stress in L-arginineinduced acute pancreatitis (AP), and determine its molecular mechanism.Methods: Hematoxylin-Eosin (H & E) staining assay were performed, and histopathological score, blood glucose concentration, and serum amylase level were evaluated in order to assess the effects of PLG on pancreatic injury in L-arginine-induced AP mice. Enzyme linked immunosorbent assay (ELISA) was conducted to assess the levels of myeloperoxidase (MPO), malonaldehyde (MDA) and inflammatory factors (IL-6, TNF-α and IL-1β) in L-arginine-induced AP mice. Serum lactate dehydrogenase (LDH) and relative levels of ROS generation in L-arginine-induced AP mice were determined using an LDH kit and immunofluorescence assay, respectively. The effect of pulegone (PLG) on the activation of p38 MAPK pathway in L-arginine-induced AP mice was evaluated by Western blot.Results: Significant pancreatic tissue injury occurred in L-arginine-induced AP mice was revealed. PLG alleviated the pathological injury of pancreatitis, and decreased the blood glucose concentration and serum amylase level in L-arginine-induced AP mice. In addition, PLG inhibited oxidative stress and inflammatory responses, and was enabled to inhibit the activation of p38 MAPK pathway in L-arginineinduced AP mice. Furthermore, PLG exhibited protective effect against the development of pancreatic injury in L-arginine-induced AP mice.Conclusion: PLG ameliorates L-arginine-induced inflammation and oxidative stress in AP mice in vivo by inhibiting p38 MAPK pathway, and therefore, is a potential therapeutic agent for the management of acute pancreatitis.

DIPG-40. Combined pharmacological and genetic screening to identify dependencies and combinations in ACVR1-mutant diffuse midline glioma

Somatic mutations in ACVR1, which encodes the serine/threonine kinase ALK2, are found in 20-25% of DMG-H3K27 patients. Treatment of ACVR1-mutant patient-derived models with multiple chemotypes of ALK2 inhibitors (ALK2i) results in reduced cell viability in vitro and extended survival in orthotopic xenografts in vivo but, as single agents, these inhibitors were unable to achieve a complete anti-tumour response. Recently we reported that combinatorial treatment of ACVR1-mutant DIPG cells with vandetanib (RTK inhibitor) and everolimus (mTOR/ABC transporter inhibitor) was synergistic both in vitro and in vivo and was shown to be a feasible combination to trial clinically in this setting. To identify specific dependencies in ACVR1-mutant cells which may be translatable with novel synergistic drug combinations alongside ALK2i, we have implemented both candidate and unbiased drug and genetic screening approaches. Using a panel of patient-derived ACVR1-mutant and wild-type models, we identified synergy between multiple chemotypes of ALK2i (M4K2009/LDN-214117) and PI3K/mTOR (AZD8055/everolimus) and MEK inhibitors (trametinib), reflecting the common co-segregation of PIK3CA/PIK3R1 alterations in these tumours. Whole-genome CRISPR/Cas9 screening of ACVR1-mutant SU-DIPG-IV cells in combination with two ALK2i (M4K2009/LDN-193189), confirmed a specific MTOR genetic dependency, as well as for the protein phosphatase regulatory subunit PPP2R1A, known to play a role in MAPK pathway activation. Additional hits include the serine/threonine kinase PKMYT1, a negative regulator of the G2/M checkpoint via a functionally redundant phosphorylation of CDK1/CCNB1 alongside WEE1; confirmatory drug assays with the WEE1 inhibitor AZD1775 resulted in a synergistic interaction with ALK2i in ACVR1-mutant cells. Hits were integrated with DepMap using ‘gene-effect’ scores (Chronos) enabling filtering of common essential genes. Preliminary pathway enrichment analysis (MAGeCKFlute) identified ALK2i-specific vulnerabilities involving TGFB1/SMAD signalling and histone deacetylation. These data highlight functionally rational and novel combinatorial possibilities for children with ACVR1-mutant DMG, with systematic preclinical assessment required for prioritisation for the clinic.

LGG-17. Preventing recurrence: targeting molecular mechanisms driving tumor growth rebound after MAPKi withdrawal in pediatric low-grade glioma

Pediatric low-grade gliomas, a diverse group of WHO grade 1 and 2 glial or glioneural tumors, comprise the most common category of primary brain tumors in children. The majority of these tumors are driven by alterations in the MAPK pathway, making them in principle susceptible to MAPKi therapy. While patients often benefit from MAPKi during treatment, tumor rebound may occur once treatment is stopped, constituting a significant clinical challenge. BT-40, patient-derived cells with molecular features of pleomorphic xanthoastrocytoma (BRAFV600E, CDKN2Adel), were used to model the rebound growth in vitro, based on viable cell counts in response to treatment and withdrawal of the clinically relevant BRAFV600E specific inhibitor dabrafenib. Standard-of-care chemotherapy (vincristine and carboplatin) was used as a reference. MAPK pathway reactivation upon withdrawal was assessed by WB and qPCR analysis. Based on the observed cell-regrowth and MAPK-reactivation pattern, key-timepoints during withdrawal were identified, which are currently being further analyzed through RNAseq and phospho-/proteomics. BT-40 cells started to proliferate again two days after dabrafenib withdrawal, and earliest five days after chemotherapy withdrawal. MAPK pathway activity, based on Mek and Erk phosphorylation, reached baseline levels three hours after dabrafenib withdrawal, this was associated with 2.5-fold increased c-Fos gene expression two hours after withdrawal. The earlier cell regrowth after dabrafenib withdrawal compared to chemotherapy withdrawal matches clinical observations, making the model suitable to study the rebound. The observed MAPK overactivation suggests the growth rebound might not only be caused by a fast reactivation of the pathway but also by other mechanisms, e.g. accumulation of upstream activators due to loss of negative feedback or parallel pathways. To investigate this, key-timepoints during treatment withdrawal will be analyzed using a multi-omics approach. Based on these findings, possible rebound-driving mechanisms will be identified and further validated using BT-40 and additional PXA models in vitro and in vivo.

LGG-22. SJ901: Phase I/II evaluation of single agent mirdametinib (PD-0325901), a brain-penetrant MEK1/2 inhibitor, for the treatment of children, adolescents, and young adults with low-grade glioma (LGG)

BACKGROUND: MEK inhibitor trials in pediatric low-grade glioma (pLGG) have yielded promising results, but there remains room for improvement since objective responses are rarely complete and disease recurrence after completion of therapy is common. Mirdametinib (PD-0325901) is a highly selective MEK1/MEK2 inhibitor that, in preclinical studies, has been reported to have superior blood-brain-barrier penetration compared to other MEK inhibitors. As such, we recently launched the SJ901 clinical trial (NCT04923126) to determine the safety, recommended phase 2 dose, pharmacokinetics, and preliminary efficacy of mirdametinib in patients with pLGG when administered continuously. Here, we present preliminary phase 1 data. METHODS: SJ901 is a multi-arm phase I/II trial of mirdametinib in patients >2 and <25 years with LGG. Phase I requires participants to have no prior exposure to MEK inhibitors and recurrent/progressive disease with biopsy-proven evidence of MAPK pathway activation. Three escalating dose levels (2 mg/m2/dose BID, 2.5mg/m2/dose BID and 3mg/m2/dose BID) are planned using a rolling 6 design. RESULTS: Accrual began in June 2021. As of Jan 13, 2022, eleven patients enrolled: 5 on dose level 1 (DL1) and 6 on dose level 2 (DL2). Median age is 10 (3-21) years. Ten patients have somatic gene rearrangements (7 BRAF, 1 MYB, 1 RAF1, 1 FGFR1) and one has an NF1 germline mutation. Four have metastatic disease. No dose-limiting toxicities occurred for DL1 (whereas data are pending for DL2) and only grade 1/2 treatment-related adverse events have been observed. No MEK-related retinopathy or cardiopathy has been observed. Four of the six patients with at least one follow-up disease evaluation have a minor response (>25%-<50% decrease). Median time on therapy is 6.6 (2.2-7) months. No disease progressions have occurred. CONCLUSION: Thus far, mirdametinib is well-tolerated and clinically promising when dosed continuously in patients with recurrent/progressive pLGG. More information will be forthcoming.

CCL25 Inhibition Alleviates Sepsis-Induced Acute Lung Injury and Inflammation.

PurposeAcute lung injury (ALI) is a common clinical syndrome with high mortality. The chemokine ligand 25 (CCL25) is involved in inflammation, leukocyte trafficking and immunoregulation. However, the role and mechanism of CCL25 in ALI are not fully understood yet. The aim of this study was to explore the relationship between acute lung injury and CCL25.Patients and MethodsIn this study, we first examined chemokine expression in sepsis patients and found that serum CCL25 expression levels were relatively high in sepsis patients compared to healthy individuals. Based on this, we designed in vitro and in vivo experiments to verify the validity of the theory. In vitro, we used lipopolysaccharide-stimulated human pulmonary microvascular endothelial cells (HPMECs). In vivo, we established male C57BL/6 mice cecal ligation puncture (CLP) model of sepsis.ResultsIn vitro, we used lipopolysaccharide-stimulated human pulmonary microvascular endothelial cells (HPMECs) and found significantly higher expression of CCL25 by enzyme-linked immunosorbent assay. Inhibition of CCL25 resulted in a significant decrease in the expression of inflammatory cytokines in HPMECs. In addition, we found that CCL25 promoted increased endothelial permeability by reducing the expression of tight junction proteins and was associated with activation of the P38 MAPK pathway by measuring the transepithelial electrical resistance and fluorescence intensity of fluorescein isothiocyanate. Results from luciferase assays and chromatin immunoprecipitation assays showed that inhibition of NF-κB activity in HPMECs decreased CCL25 expression, but addition of recombinant CCL25 increased cell permeability and inflammatory cytokine expression. In vivo, we established male C57BL/6 mice cecal ligation puncture (CLP) model of sepsis. We found that inhibition of CCL25 significantly reduced inflammatory cytokine expression in a CLP-induced sepsis model, thereby alleviating lung tissue damage in mice.ConclusionOur study suggests that CCL25 contributed to the development of ALI by modulating the functions of microvascular endothelial cells.

Progress in the Treatment of Adult Langerhans Cell Histiocytosis --Review

Langerhans cell histiocytosis (LCH) is a rare proliferative disease dominated by the proliferation of Langerhans cells, which is inflammatory myeloid neoplasms. Its clinical manifestations are variable, occurring at any age and at any site, and it is rarer in adults than in children. The gold standard for diagnosis is histopathological biopsy. Due to the rarity of adult LCH and the heterogeneity of this disease, treatment of adult LCH should be developed according to the extent of the disease and risk stratification. With the discovery of MAPK, PI3K and c-KIT signaling pathway activation, especially BRAF V600E and MAP2K1 mutations, targeted therapy has become a hot spot for therapeutic research. Meanwhile, the discovery of high expression of M2-polarized macrophages and Foxp3+ regulatory T cells (Treg) in LCH has provided an important basis for the immunotherapy. In this article, we will focus on reviewing the latest research progress in the treatment of adult LCH in recent years, and provide a reference for clinical research on the treatment of adult LCH patients.

Development of combination strategies for focal adhesion kinase inhibition in diffuse gastric cancer.

e16057 Background: Diffuse gastric cancer (DGC) is an aggressive and frequently lethal pathological subtype of gastric cancer (GC). Since DGC often lacks genomic aberrations that indicate clear candidate therapeutic targets, it has been challenging to develop target therapies for this GC subtype. Our previous study highlighted the contribution of focal adhesion kinase (FAK) in the tumorigenesis of DGC, and the potential for small molecule FAK inhibitors to have efficacy in DGC. Currently, FAK inhibitors are under active clinical evaluation in several tumor types. To date, FAK inhibitor monotherapy has been tolerated in the clinic, but has shown only modest clinical efficacy in cancer treatment, which inspired us to explore targets for the combination with FAK inhibitors in DGC. Methods: We constructed the engineered murine DGC model of Cdh1-/- RHOAY42C/+ organoids, with Cdh1 (E-Cadherin) loss and RHOA Y42C mutation, in our previous study. Using this DGC organoids model, we performed genome-scale libraries of open reading frames (ORF) screen to identify candidate mechanisms of resistance to FAK inhibitors (defactinib or PF-573228). For the top hits found in ORF screen, further validations were performed in Cdh1-/- RHOAY42C/+ organoids and human DGC cell lines of NUGC4 and SNU668 to check whether they could function as candidates for the combination with FAK inhibitors. Besides, we used Digiwest, a high-throughput protein analysis assay, to investigate pathway alterations of Cdh1-/- RHOAY42C/+ organoids treated with FAK inhibitors (defactinib or PF-573228). We then explored whether the activated pathways were the candidates for the drug combination. Zero Interaction Potency (ZIP) model was used to measure the synergistic effect between the drugs. The combination strategies were also tested in vivo. Results: We identified that cyclin-dependent kinase 6 (CDK6) promotes FAK inhibitors (defactinib and PF-573228) resistance according to ORF screen, and CDK4/6 inhibitor, Palbociclib, enhances the efficacy of defactinib for DGC models. Furthermore, we demonstrated that FAK inhibitors (defactinib and PF-573228) treatment in DGC models leads to compensatory activation of MAPK pathway in DGC, and the RAF/MEK dual inhibitor VS-6766 effectively enhances the antitumor efficacy of FAK inhibitors in vitro and in vivo. Conclusions: These data suggest that combinations of FAK inhibitors with MAPK inhibitors or CDK4/6 inhibitors are promising treatment strategies for DGC that warrant further testing in clinical trials.

Molecular characterization of NF1-mutated NSCLC and clinical outcomes.

9086 Background: NF1 is a tumor suppressor gene that regulates the RAS-MAPK and mTOR pathways. Co-mutations previously observed with NF1-mutations (mt) include TP53, KRAS, EGFR and rarely HER2, STK11, and PIK3CA mutations. We report a comprehensive molecular characterization with clinical outcomes analyses for NF1-mt non-small cell lung cancer (NSCLC). Methods: Next-generation sequencing (NGS) of DNA (592-gene or whole exome) and RNA (whole transcriptome) was performed for NSCLC patient (pt) samples (n = 10,310) submitted to a CLIA-certified laboratory (Caris Life Sciences, Phoenix, AZ).. RAS-MAPK and PI3K-AKT-MTOR signaling were assessed by transcriptional signatures of pathway activation (MAPK pathway Activation Score [MPAS], Wagle, 2018; and GSEA Hallmarks collection, respectively)., PD-L1 by immunohistochemistry (IHC, positive: TPS ≥1%), high tumor mutational burden (TMB) defined as ≥10 mut/Mb, and deficient mismatch repair/high microsatellite instability (dMMR/MSI-High) was assessed by IHC/NGS. Overall survival (OS) was obtained from insurance claims. Statistical significance was determined using Chi-square &amp; Wilcoxon rank sum tests. P-value adjust for multiple hypothesis testing (Benjamini-Hochberg). Results: NF1-mt were identified in 1,045 NSCLC samples (10.1%). Concurrent KRAS, EGFR, ERBB2, BRAF or MET alterations are noted in Table 1, with no ROS1, RET or ALK fusions identified. Compared to NF1-wt, NF1-mt NSCLC was associated with increased RAS-MAPK expression (3.0-fold, P &lt; 0.0001), while PI3K-AKT-MTOR-signaling was not significantly increased (2.1-fold, P = 0.12). Rates of TMB-High (51.7% vs 32.5%, P &lt; 0.0001), PD-L1+ (69.1% vs 58.8%, P = 0.06), and dMMR/MSI-High (1.7 vs 0.7%, P &lt; 0.05) were higher in NF1-mt samples. OS and duration on treatment from the start of Pembrolizumab (HR: 1.0 and 1.0, respectively) or other IOs (HR: 0.9 and 1.0, respectively) were not significantly different between NF1-mt and NF1-wt patients, However, among NF1-mt samples, high TMB and TP53-wt were associated with better OS (HR 0.6, P &lt; 0.05 each). Conclusions: NF1-mt patients rarely harbored actionable NSCLC driver co-alterations. NF1-mt cases showed increased activation of RAS-MAPK axis, which may represent a potential pathway to target with MEK inhibitors. NF1- mt are responsive to immunotherapy and better outcomes are seen with high TMB and absence of TP53 mutations. Further work is warranted to determine the influence of actionable drivers on targeted therapy outcomes in NF1-mt NSCLC. [Table: see text]

HMGB1-NLRP3-P2X7R pathway participates in PM2.5-induced hippocampal neuron impairment by regulating microglia activation

Neuroinflammation is a key mechanism underlying the cognitive impairment induced by PM2.5, and activated microglia plays an important role in this process. However, the mechanisms by which activated microglia induced by PM2.5 impair hippocampal neurons have not been fully elucidated. In this study, we focused on the role of HMGB1-NLRP3-P2X7R pathway which mediated the microglia activation in hippocampal neurons impairment induced by PM2.5 using a co-culture model of microglia and hippocampal neurons. We found that PM2.5 resulted in activated microglia and HMGB1-NLRP3 inflammatory pathway, and elevated proinflammatory cytokines of IL-18 and IL-1β in a dose-dependent manner. Notably, we next utilized previously reported pharmacological inhibitors or siRNA for HMGB1 and found that they significantly inhibited the activation of downstream NLRP3 and MAPK pathways derived from PM2.5 exposure, and down-regulated IL-18 and IL-1β in microglia. Furthermore, we employed co-cultured hippocampal neurons and microglia and found that reducing HMGB1 significantly decreased neuron impairment, apoptosis related protein of cl-caspase3, synaptic damage, and neurotransmitter receptor of 5-HT2A, along with notably elevated presynaptic and postsynaptic proteins of SYP and PSD-95, as well as learning and memory related proteins of p-CREB and BDNF. The neuronal impairment induced by PM2.5 could not be prevented in the case of simultaneous employment of HMGB1 siRNA and NLRP3 agonist. After silencing NLRP3 alone in microglia, hippocampal neurons demonstrated decreased excessive autophagy and up-regulated synaptic protein of GAP43 as well as learning and memory related protein of NCAM1. Therefore, we further studied how hippocampal neurons affected microglia under PM2.5 exposure, Further investigation indicated that silencing HMGB1 could affect the activation of P2X7R and reduce the release of ATP from hippocampal neurons, thus protecting the interaction between microglia and hippocampal neurons. The present work suggests that regulation of HMGB1-NLRP3-P2X7R pathway can inhibit the microglia activation induced by PM2.5 to alleviate hippocampal neuron impairment and stabilize the microenvironment between microglia and neurons. This contributes to maintaining the normal function of hippocampal neurons and alleviating the cognitive impairment derived from PM2.5 exposure.

Spiropachysine A suppresses hepatocellular carcinoma proliferation by inducing methuosis in vitro and in vivo

BackgroundSpiropachysine A is the extracted compound of traditional Chinese ethnic medicine Pachysandra axillaries Franch. var. styiosa (Dunn) M. Cheng. Spiropachysine A is the primary active steroidal alkaloids (SAs) widely used to facilitate blood circulation and relieve pain and inflammation. Few previous studies have investigated the anti-cancer activity of Spiropachysine A to treat hepatocellular carcinoma (HCC), and its molecular mechanism remains unknown. PurposeThis study aims to investigate the anti-cancer activity of Spiropachysine A and the underlying mechanisms by inducing methuosis in vitro and in vivo. MethodsHere, the activity of Spiropachysine A against cancer was evaluated by the experiments with MHCC-97H cells and the xenografted mice model. The cell proliferation was examined using MTT assay, and cell morphological characteristics were observed by microscope cellular imaging. The effects of autophagy, paraptosis, and oncosis on cytoplasmic vacuolisation were detected using immunofluorescence staining, transmission electron microscopy (TEM) and western blotting (WB). The cell cycle distribution and apoptosis were analysed by flow cytometry. Hematoxylin eosin (H & E) staining was used to observe the pathological changes of the tissues. ResultsThe in vitro and in vivo results indicated that Spiropachysine A could inhibit HCC cells proliferation (IC50 = 2.39 ± 0.21 μM against MHCC-97H cells) and tumor growth (TGI = 32.81 ± 0.23% at 25 mg/kg and 50.32 ± 0.26% at 50 mg/kg). The morphological changes of the treated cells showed that cell proliferation inhibition caused by Spiropachysine A was associated with numerous cytoplasmic vacuolization. Mechanistically, Spiropachysine A-induced methuosis rather than autophagy or arapaptic because the autophagy flux was blocked, leading to the increased LC3-II/I value and an accumulation of selective autophagy substrate p62. And, there was no activation of the regulatory parapaptic MAPK pathway. Additionally, the TEM and Lucifer yellow (LY) accumulation data confirmed that Spiropachysine A significantly triggered methuosis instead of oncosis. Further, the study indicated that the anti-proliferative activity of Spiropachysine A was independent of PCD since no alterations in apoptosis and cell cycle arrest-related proteins were observed after Spiropachysine A treatment. Impressively, the increased expression of Rac1 was observed in Spiropachysine A-treated MHCC-97H cells and its xenograft tumours, confirming that Spiropachysine A inhibited cell proliferation and induced methuosis through Ras/Rac1 signal pathways. ConclusionsSpiropachysine A was collectively identified as a novel methuosis inducer that suppresses HCC in vitro and in vivo. The underlying mechanisms might be involved in the Ras/Rac1 pathway. Such data predict that Spiropachysine A is a promising candidate for developing novel chemotherapeutic agents as a methuosis inducer for cancer therapy.

Synergistic anti-inflammatory effects of peimine, peiminine, and forsythoside a combination on LPS-induced acute lung injury by inhibition of the IL-17-NF-κB/MAPK pathway activation

Ethnopharmacological relevanceForsythia suspensa (Thunb.) Vahl and Fritillaria thunbergii Miq are traditional Chinese medicines that exhibit the ability to clear heat and toxic material effects. In China, the combination of these two medicines is widely used to treat mucopurulent sputum and bloody phlegm, arising due to phlegm-heat obstruction in respiratory diseases. However, very limited information is available regarding the combined anti-inflammatory effect of important effective components of Forsythia suspensa (Thunb.) Vahl and Fritillaria thunbergii Miq, namely peimine, peiminine, and forsythoside A.Aim of this study: To investigate synergistic anti-inflammatory effects of combined administration of peimine, peiminine, and forsythoside A on LPS-induced acute lung injury compared to combined administration of two compounds or individual administration, and unravel the underlying mechanism. Material and methodsIn the present study, male BALB/c mice received an oral dosage of sodium carboxymethylcellulose (CMC-Na) (0.5%, 1 mL/100 g), peimine, peiminine, forsythoside A, peimine + forsythoside A, peiminine + forsythoside A, and peimine + peiminine + forsythoside A (suspended in CMC-Na; 0.5%), once daily for 7 days. Subsequently, intratracheal instillation of LPS was applied to establish acute lung injury model. After 6 h of administration, the mice were sacrificed, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. These samples were further used to determine lung W/D (wet/dry) weight ratio, total protein (TP) levels, inflammatory cytokines (IL-6, TNF-α, IL-1β, and IL-17), and expression of proteins involved in TLR4/MAPK/NF-κB pathway and IL-17 pathway. Further, tissue sections were subjected to H&E staining to assess the pathological alterations induced by LPS. The expression of IL-6 and TNF-α proteins in lung tissues was also analyzed using immunohistochemical staining. ResultsA synergistic anti-inflammatory effect of peimine, peiminine, and forsythoside A was observed when administered in combination to LPS-induced acute lung injury. The combined administration of peimine, peiminine, and forsythoside A had a strongly inhibitory effects on the W/D weight ratio, total protein (TP) level and the inflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-17) level in acute lung injury mice, compared to combined administration of two compounds or individual administration. The infiltration of inflammatory cells and thickened bronchoalveolar walls induced by LPS were also ameliorated through the combined administration of peimine, peiminine, and forsythoside A. More importantly, the upregulation of protein related to TLR4/MAPK/NF-κB signaling pathway and the activation of IL-17 were significantly suppressed by pretreatment with each of the three compounds alone, while the effects of individual compounds were synergistically augmented by the combined pretreatment of these three compounds. ConclusionThe combined administration of peimine, peiminine, and forsythoside A ameliorated inflammatory response in acute lung injury mice induced by LPS in a synergistic manner, the mechanism may be related to the dampening of the TLR4/MAPK/NF-κB signaling pathway and IL-17 activation.

CgHMGB1 functions as a broad-spectrum recognition molecule to induce the expressions of CgIL17-5 and Cgdefh2 via MAPK or NF-κB signaling pathway in Crassostrea gigas

High-mobility group box 1 (HMGB1), a highly conserved nucleoprotein, functions in immune recognition, inflammation and antibacterial immunization in vertebrates. In the present study, the mediation mechanism of CgHMGB1 in activating MAPK and NF-κB/Rel signaling pathways to induce the expressions of immune effectors was investigated. CgHMGB1 mRNA was detected in all tested developmental stages from fertilized egg to D-larvae, with the higher expressions in 4 cells and 8 cells stages. CgHMGB1 proteins were mainly distributed in haemocyte granulocytes. The expressions of CgHMGB1 mRNA in haemocytes increased significantly after Vibrio splendidus stimulation, and CgHMGB1 protein translocated into the haemocyte cytoplasm and release into cell-free haemolymph. The phosphorylation of CgERK and CgP38 were induced, the nuclear translocation of CgRel were promoted, and the mRNA expressions of CgIL17-5 and Cgdefh2 increased significantly after rCgHMGB1 treatment. Obvious branchial swelling and cilium shedding were observed after rCgHMGB1 treatment. rCgHMGB1 exhibited binding activity to different polysaccharides, bacteria, and fungi. rCgHMGB1 also displayed obvious antibacterial activity to V. splendidus and E. coli. These results indicated that CgHMGB1 functioned as an immune recognition molecule to recognize various PAMPs and bacteria to induce the mRNA expressions of CgIL17-5 and Cgdefh2 via the activation of MAPK and NF-κB signaling pathways in oysters.

RUVBL1 promotes enzalutamide resistance of prostate tumors through the PLXNA1-CRAF-MAPK pathway.

Although enzalutamide improves the overall survival of patients with metastatic prostate cancers, enzalutamide resistance (ENZR) will be inevitably developed. Emerging evidence support that alternative oncogenic pathways may bypass the androgen receptor (AR) signaling to promote ENZR progression, however, the underpinning mechanisms remain poorly defined. Here, we report that the expression of RuvB like AAA ATPase 1 (RUVBL1) is upregulated in ENZR cells and xenograft models and prostate tumors in patients. Enzalutamide increases RUVBL1 accumulation in the cytoplasm, which in turn enhances the recruitment of CRAF proto-oncogene serine/threonine kinase protein to plexin A1 (PLXNA1) and the subsequent activation of the downstream MAPK pathway. Co-overexpression of RUVBL1 and PLXNA1 defines a subgroup of prostate cancer (PCa) patients with a poor prognosis. Furthermore, pharmacological inhibition of RUVBL1 by CB-6644 suppresses ENZR cell proliferation and xenograft growth and allows re-sensitization of ENZR cells and xenografts to enzalutamide, indicating that RUVBL1 may act to substitute the AR signaling to promote cancer cell survival and ENZR development. Together, these findings may lead to the identification of RUVBL1 as a potential therapeutic target for ENZR tumors.