ERK Inhibitor Research Articles

ERK is involved in the regulation of CpG ODN 2395 on the expression levels of anti-lipopolysaccharide factors in Chinese mitten crab, Eriocheir sinensis

CpG oligodeoxynucleotides (ODN), as an effective adjuvant or immunopotentiator, activate the immune system and induce various immune responses. Recently, it has also been reported that high dose of CpG ODN can lead to immunosuppression. However, the underlying mechanism of CpG ODN-mediated immune response remains largely unknown in invertebrates. In the present study, the role of ERK in regulating expression levels of anti-lipopolysaccharide factors (ALFs) induced by different doses of CpG ODN 2395 was analyzed in Chinese mitten crab, Eriocheir sinensis. The mRNA expression levels of EsALFs (EsALF1, EsALF2 and EsALF3) and EsERK in haemocytes were observed to increase from 6 h to 48 h post low doses of CpG ODN 2395 (0.5 μg and 2.5 μg) stimulation, while they were suppressed after high dose of CpG ODN 2395 (12.5 μg) injection. Meanwhile, the phosphorylation levels of ERK in haemocytes were significantly promoted after low doses of CpG ODN 2395 injection, and a reduce level of ERK phosphorylation was observed after high dose of CpG ODN 2395 injection. Further investigation showed that the expression levels of EsALFs induced by CpG ODN 2395 were markedly down-regulated after knocking down the expression of EsERK. Similarly, the EsALFs mRNA expression were also inhibited post different doses of CpG ODN 2395 stimulation in PD98059 (ERK inhibitor) injection crabs. These results collectively suggest that ERK is involved in regulating the expression level of EsALFs induced by different dose of CpG ODN 2395 in Chinese mitten crab, which contribute to the understanding of the regulation of CpG ODN involving in immune response in crustaceans.

RAF-Independent MEK Mutations Drive Histiocytic Neoplasms In Vivo and Are Sensitive to Single-Agent ERK Inhibition in Patients

Histiocytic neoplasms are clonal disorders of the monocyte/macrophage lineage that include Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD). Histiocytoses are defined by recurrent, mutually exclusive mutations activating MAP kinase signaling, including BRAFV600E mutations in ~50% of LCH and ECD patients, which confer robust lasting clinical responses to BRAF inhibition. Mutations in MEK1/2, ARAF, and a diverse array of other kinases are present in BRAF wild-type histiocytoses patients and are broadly responsive to the allosteric MEK1/2 inhibitor cobimetinib. Following genomic analysis of ~300 pediatric and adult patients with diverse histiocytoses, the single most common genomic alteration following BRAFV600E mutations are MEK1 E102_I103 in-frame deletions. In contrast to BRAFV600E, the biological impact of activating mutations in MEK1/2 kinases are unknown, and no cancer associated MEK1/2 mutation has ever been modeled in vivo. MEK1/2 mutations are recurrent across a wide variety of human cancers and RASopathies and are now often found at resistance to emerging KRAS-mutant selective inhibitors. These findings motivated us to generate Map2k1E102_I103del conditional knock-in mice within the endogenous locus of Map2k1 (encoding MEK1 kinase), which represent the first genetically engineered mouse model of MEK1/2 mutations. We first crossed Map2k1E102_I103del mice to inducible Mx1-cre driver mice. Interestingly, Mx1-cre Map2k1E102_I103del knock-in mice developed a 100% penetrant lethal myelomonocytic disorder by 5-6 weeks of age while their cre-negative Map2k1E102_I103del littermates did not develop disease (Figure A). Notably, recombination of the Map2k1E102_I103del alleleoccurred spontaneously without pIpC administration and resulted in strong activation of phosphorylated ERK1/2 in bone marrow and spleen mononuclear cells. Mx1-cre Map2k1E102_I103del mice developed a myelomonocytic neoplasm with suppression of B-lymphopoiesis and expansion of classical and non-classical monocyte subsets, macrophages, and Cd11b+ conventional dendritic cells. Monocyte, macrophage, and dendritic cells infiltrated hematopoietic organs (bone marrow, spleen, and peripheral blood), as well as liver and skin, each of which are common sites of LCH and ECD disease involvement. The Mx1-cre Map2k1E102_I103del disease was hematopoietic cell autonomous and could be propagated by serial hematopoietic reconstitution in irradiated recipient mice, as well as using Vav1-cre Map2k1E102_I103del knock-in mice (where Cre is expressed only in hematopoietic cells from earliest hematopoietic development). Finally, restricting MEK1 mutant expression to monocyte and dendritic cell precursors using CD11c-cre resulted in similar lethal expansions of monocyte, macrophage, and dendritic cells as seen in the Mx1-cre and Vav1-cre models. Prior in vitro work has demonstrated that MEK1 p.E102_I103del activates MEK and ERK without requiring activated GTP-bound RAS and is associated with resistance to allosteric MEK inhibitors. Consistent with this, we identified nine histiocytosis patients with this specific MEK1 mutation, three of which had de novo resistance to cobimetinib. These three patients and one treatment-naïve patient were treated with an oral ERK1/2 inhibitor ulixertinib under the FDA single-patient compassionate use program. Three of 4 patients had a clinical or radiological partial or complete response to ulixertinib (Figure B). Overall, these data identify the impact of oncogenic MEK kinase mutations in vivo as found in children and adults with histiocytoses and RASopathies. Expression of a RAF-independent MEK1 mutation in vivo gave rise to disease reminiscent of human systemic histiocytic neoplasms. Importantly, this molecular entity is resistant to MEK inhibition in some patients, a phenomenon previously undocumented, but responsive to ERK inhibition, which may be a promising therapeutic approach to histiocytic neoplasms unresponsive to allosteric MEK inhibition. The clinical observation of efficacy of ERK inhibition in MEK inhibitor resistant histiocytoses patients has resulted in a soon-to-be initiated focused phase 2 clinical trial of ulixertinib in adults with histiocytoses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract B002: ARNTL2 is a Kras-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma

Abstract We employed regulatory network analysis to identify master transcriptional regulators whose activity drives key pancreatic cancer phenotypes. Regulatory network analysis is a systems biology approach that computationally infers the activity of transcriptional regulators based on the integrated expression of their positive and negative target genes. To focus on epithelial cell biology, we performed laser capture microdissection and RNA sequencing on 200 human PDAC samples and 45 low-grade precursors (PanIN and IPMN), generating a rich dataset with clinical, epidemiological, and histopathological annotation. Using a transcriptional regulatory network derived from this dataset, we performed master regulator analysis to identify master regulators of four phenotypes: precursors vs. PDAC (initiation), low-grade vs. high grade histopathology (progression), survival post resection, and association with KRAS activity. Changes in the activity of multiple established PDAC drivers and suppressors were associated with tumor initiation and progression, providing a strong validation from existing biology. Integrating across these phenotypes, the top master regulator of PDAC malignancy was found to be ARNTL2, a member of the PAS family of bHLH transcription factors. The canonical function of ARNTL2 is as the heterodimeric binding partner of the circadian rhythm protein CLOCK. However, functional analysis ARNTL2 inferred transcriptional targets highlighted a potential role in hypoxia response and prior studies have demonstrated the ability of ARNTL2 to heterodimerize with HIF1A. Indeed, ARNTL2 was inferred to directly or indirectly regulate every single hypoxia-responsive protein in a previously published hypoxia signature. We therefore studied the role of ARNTL2 in both normoxic and hypoxic conditions and found that its knockdown in PDAC cells led to a selective decrease in viability and invasion under hypoxic conditions. ARNTL2 activity was induced in response to hypoxia and inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its computationally-ingerred association with RAS activity. Strikingly, we found that ARNTL2 was required in PDAC cells for the stabilization of HIF1A in response to hypoxia. Conversely, HIF2A was further stabilized in response to hypoxia upon ARNTL2 deletion, suggesting that ARNTL2 serves to control the balance between HIF1A- and HIF2A dependent metabolic programs in PDAC. Consistent with this, loss of ARNTL2 had sizeable impacts on profiles in PDAC cells and led to a significant reduction of lactate dehydrogenase A under hypoxic conditions. Together these data demonstrate that ARNTL2 is a previously underappreciated master regulator of hypoxia response in pancreatic ductal adenocarcinoma. Citation Format: H. Carlo Maurer, Alvaro Curiel-Garcia, Pasquale Laise, Carmine F. Palermo, Stephen A. Sastra, Gulam A. Manji, Alina Iuga, Kristen Johnson, Jeanine Genkinger, Costas A. Lyssiotis, Andrea Califano, Kenneth P. Olive. ARNTL2 is a Kras-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B002.

Abstract A053: Inhibition of the MEK5-ERK5 signaling pathway blocks pancreatic tumorigenesis

Abstract Mutant KRAS represents one of the most common driving events in pancreatic ductal adenocarcinomas (PDAC), yet therapies targeting this pathway have proven largely ineffective. We identified ERK5 activation as critical component of KRAS-driven pancreatic tumorigenesis. ERK5 belongs to the MAP kinase family and is phosphorylated and activated by the MAPK kinase MEK5. Therefore, the aim of this study is to elucidate the function of the MEK5-ERK5 signaling axis in PDAC pathogenesis. Several novel genetically engineered mouse models with pancreas-specific deletion or activation of the MEK5-ERK5 signaling were generated to study pancreatic cancer initiation and progression. Caerulein-induced pancreatitis was performed in p48Cre/+ KrasG12D/+ (Kras) and p48Cre/+ KrasG12D/+ Erk5fl/fl (Kras;Erk5) mice to study early steps in pancreatic tumorigenesis. To investigate if ERK5 ablation impacts tumor progression a two-stage tumor model utilizing dual recombinase (Cre/Flp) mouse model was utilized. Next, the impact of ERK5 depletion or pharmacological inhibition on survival of pancreatic cancer mice was analyzed. Furthermore, human patient-derived PDAC xenograft models in immunodeficient mice were utilized to evaluate efficacy of a small molecule ERK5 inhibitor which does not exhibit off-target activity on bromodomains. Deletion of ERK5 or inhibition of its kinase activity resulted in blocked tumorigenesis in Kras mutant mice using caerulein-induced pancreatitis. Upon pancreatitis induction pancreas in Kras;Erk5 mice exhibit normal tissue architecture with significantly fewer proliferating cells compared to control mice. Intriguingly, pancreas-specific expression of constitutively active MEK5 (MEK5DD) even without oncogenic Kras mutations was sufficient in promoting development of precancerous lesions after pancreatitis induction. Next, we observed that genetic depletion or pharmacological inhibition of ERK5 significantly prolonged survival of the KPC PDAC mouse model, whereas expression of MEK5DD in KPC mice resulted in significantly accelerated tumorigenesis and reduced survival compared to control mice. Furthermore, depletion of ERK5 in already established PDAC resulted in significantly prolonged survival and reduced tumor burden indicating an important role of ERK5 in tumor maintenance. Finally, ERK5 inhibitor treatment of patient-derived PDAC xenografts in immunocompromised mice resulted in tumor growth arrest. Our studies demonstrate that the MEK5-ERK5 signaling pathway plays an important role in pancreatic cancer initiation and progression. Deletion of ERK5 or pharmacological inhibition of ERK5 kinase activity results in significantly reduced tumor burden and prolonged survival in autochthonous PDAC models and patient-derived xenografts. Conversely, activation of MEK5-ERK5 signaling is sufficient to drive pancreatic tumorigenesis indicating an essential role of the ERK5 pathway as downstream effector of oncogenic KRAS. Further studies will be performed to identify the mechanisms of ERK5 action including downstream kinase targets. Citation Format: Simone Hausmann, Asli Ece Kasapoglu, Ana Benitez Morales, Shane Lofgren, Pawel K. Mazur. Inhibition of the MEK5-ERK5 signaling pathway blocks pancreatic tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A053.

Pharmacologic Inhibition of DYRK1A Results in Hyperactivation and Hyperphosphorylation of MYC and ERK Rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

Background: B-cell acute lymphoblastic leukemia (ALL) harboring KMT2A-rearrangements (KMT2A-R) is a high-risk ALL subtype with significant relapse risk and poor overall survival across the age spectrum. While new antibody-based and cellular immunotherapies have revolutionized the therapeutic paradigm for many patients with relapsed ALL, application of these therapies has proven particularly challenging to date in infants given their immune system immaturity and in older adults prone to high-grade immunotherapy toxicities. Unlike other ALL subtypes, KMT2A-R ALL has an extremely low frequency of co-mutations, which makes it particularly difficult to find new treatment options. To identify targetable kinases in KMT2A-R ALL, we performed a domain-specific kinome-wide CRISPR screen and identified the serine/threonine kinase DYRK1A as required for the survival of KMT2A-R ALL. Importantly, DYRK1A appears to be non-essential for normal tissues per the Cancer Dependency MAP, suggesting potential for a therapeutic window via DYRK1A targeting. Results:In vitro treatment studies using three different menin inhibitors (MI-2-2 / MI-503 / VTP50469) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A at the RNA and the protein level. We further analyzed ChIP-Seq data and identified that oncogenic KMT2A-R directly binds to the DYRK1A promoter, indicating that DYRK1A is directly regulated by the oncogenic KMT2A fusion. Interestingly, the DYRK1A RNA and protein levels were elevated in KMT2A-R ALL cell lines and patient-derived xenograft (PDX) samples compared to non-KMT2A-R ALL cases. To test the consequence of direct pharmacologic DYRK1A inhibition on cell proliferation and apoptosis, we treated KMT2A-R ALL cells with the DYRK1A inhibitors EHT1610 and harmine. Interestingly, we observed that pharmacologic inhibition of DYRK1A with both drugs did not induce apoptosis but induced potent leukemic cell growth inhibition in vitro and in vivo. In preliminary studies, we further observed that pharmacologic inhibition of DYRK1A or genetic deletion resulted in MYC upregulation and hyperphosphorylation of ERK. It has been demonstrated that premature signaling activation in developing B cells induces cell cycle arrest as a protective mechanism to prevent the development of autoimmune diseases. We hypothesized that DYRK1A inhibitor-induced ERK hyperactivation and consequent cell cycle arrest remains a conserved mechanism in KMT2A-R ALL. Supporting our hypothesis, combining DYRK1A inhibitors with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibition does not induce apoptosis and cells restart to proliferate after DYRK1A inhibitor withdrawal, we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. Interestingly, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. BIM is a pro-apoptotic molecule that is inactive when bound by the anti-apoptotic molecule BCL2. Given that DYRK1A inhibition results in MYC upregulation, we performed a new Western blot analysis and validated that EHT1610 and harmine both induce BIM overexpression in KMT2A-R ALL cell lines. To test if combined DYRK1A and BCL2 inhibition will lead to enhanced KMT2A-R ALL apoptosis, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, combining both drugs synergistically killed KMT2A-R ALL cells in vitro. More importantly, we validated the anti-leukemic effect of harmine in combination with venetoclax in mice injected with a KMT2A-R PDX case. Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract B007: Enhancing autophagy inhibition as a therapeutic strategy for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth. We determined the role of mutationally activated KRAS, found in ~95% of PDAC, in supporting autophagy. Surprisingly, acute KRAS suppression was associated with increased autophagic flux. Pharmacologic inhibition of ERK MAPK phenocopied the genetic silencing of KRAS and increased autophagic flux. We speculated that the loss of ERK-driven metabolic processes may induce compensatory mechanisms to increase autophagy. We then addressed whether ERK inhibition increased PDAC dependence on autophagy. Supporting this possibility, we found that cotreatment with the autophagy inhibitor chloroquine (CQ) synergistically enhanced ERK inhibitor-mediated anti-proliferative activity. Our findings, together with similar observations by others, provided the rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. Our ongoing studies are centered on developing additional combinations for targeting autophagy. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers as well as mediators of resistance to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Second, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition and hypothesize that further dissection of this regulation will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Claire E. Gates, Noah L. Pieper, Runying Yang, Clint A. Stalnecker, Kirsten L. Bryant. Enhancing autophagy inhibition as a therapeutic strategy for pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B007.

DDDR-05. ERK INHIBITION INCREASES SENSITIVITY TO ANTI-PD-1 THERAPY IN A MOUSE MODEL OF BRAF MUTANT MELANOMA BRAIN METASTASIS

Abstract BACKGROUND Immune checkpoint inhibitors (ICI) have revolutionized the landscape of cancer treatment, including brain metastases. However, only a subset of patients with brain metastases respond to ICI. Activating mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, a key regulator of cell processes, are frequently found in brain metastases. LY3214996 (LY321) is a selective ERK 1/2 inhibitor that inhibits MAPK signaling and leads to regression of extracranial disease in preclinical models. Our objective was to evaluate whether with ERK inhibition (ERKi) can improve the efficacy of ICI in preclinical models of brain metastases. METHODS We tested sensitivity to LY321 in a BRAFV600E mutant, ICI resistant murine melanoma cell line (D4M) both in vitro and in vivo. To model brain metastases in vivo, we implanted D4M cells subcutaneously with subsequent intracranial implantation three days later. The treatment arms included LY321 and anti-PD-1 combination therapy, single agent therapies, and respective controls. We tested the efficacies of 2-week versus 4-week LY321 treatments, as well as 3-week treatment in an independent experiment. RESULTS D4M demonstrates in vitro sensitivity to LY321 (IC50 value of 4.75 µM). Our model demonstrated that LY321 in combination with anti-PD-1 improves both intracranial and extracranial disease control. Increased survival with combination therapy was observed with 3-week and 4-week LY321 administration. While individually, LY321 and anti-PD-1 produced better survival outcomes than control groups, combination therapy significantly outperformed both monotherapies. CONCLUSION Previous studies demonstrated that LY321 has minimal intracranial activity. However, we found that ERKi reduces intracranial tumor burden, with improved efficacy in combination with ICI in immunocompetent mice. Together, these data suggest that ERKi increases sensitivity to ICI therapy in pre-clinical brain metastasis models. Further investigation is needed to elucidate the mechanisms of this therapeutic approach, and to ultimately determine whether this combination will improve patient outcomes.

HPV16 E6E7 up-regulates KIF2A expression by activating JNK/c-Jun signal, is beneficial to migration and invasion of cervical cancer cells.

Cervical cancer is the fourth most common cancer and the fourth leading cause of cancer death in women. Human papillomavirus (HPV16) E6/E7 heterogenous expression in C33A cells increased the mRNA and protein levels of KIF2A, while siRNA deletion of endogenous E6/E7 reduced the mRNA and protein levels of KIF2A in SiHa cells. KIF2A promoted cell migration and invasion, and regulated the expression of epithelial-mesenchymal transition-related proteins in C33A and SiHa cells. The exogenous expression of E6/E7 in C33A cells increased the phosphorylation of Akt, ERK, and JNK. However, Akt (API-2) and ERK (PD98059) inhibitors had no effect on the increase in KIF2A expression induced by E6/E7, while JNK inhibitors (JNK-IN-8 and SP600125) blocked the increase in KIF2A expression induced by E6/E7. The exogenous expression of E6/E7 increased the levels of transcription factor c-Jun, which is the classic substrate of JNK. Knockdown of c-Jun reduced the increase in KIF2A expression induced by E6/E7. In summary, KIF2A plays a key role in the motility and metastasis of cervical cancer. HPV16 E6/E7 can increase the levels of transcription factor c-Jun by activating the JNK signal, thereby up-regulating the transcriptional expression of KIF2A.

Treponema pallidum FlaA2 inducing the release of pro-inflammatory cytokines is mediated via TLR2 in keratinocytes

BackgroundSyphilis, caused by Treponema pallidum (T. pallidum), is a multi-organ, multiple systems, multi-stage sexually transmitted diseases with various clinical manifestations, among of which pathological lesions of skin and mucosa are the typical clinical manifestations of syphilis. However, the immunopathogenesis of this process is poorly understood. T. pallidum flagellin FlaA2, as a part of the important organelle responsible for the causative agent's motility, may contributes to the host skin inflammatory response. ObjectivesTo determine the mechanisms of T. pallidum FlaA2 stimulating the expression of pro-inflammatory cytokines in human keratinocytes. MethodsRecombinant FlaA2 protein was performed to stimulate human keratinocytes. The mRNA transcription levels and protein expression levels of IL-6 and IL-8 were detected by qRT-PCR and ELISA, respectively. Western blot was used to detect the total protein and phosphorylation levels of ERK, p38, JNK and NF-κB, respectively. The intracellular location of NF-κB p65 was detected by immunofluorescence staining. ResultsRecombinant FlaA2 could considerably induced the expression of pro-inflammation cytokines IL-6 and IL-8 in HaCaT cells, and FlaA2-induced IL-6 and IL-8 secretion could be decreased by inhibiting TLR2 using pZERO-hTLR2. Further investigation showed that FlaA2 could activate the phosphorylation of ERK, p38 and IκBα and FlaA2-stimulated secretion of IL-6, IL-8 were attenuated by ERK, p38 and NF-κB inhibitors in HaCaT cells. Moreover, FlaA2 activates the ERK, p38 and NF-κB pathways through TLR2 signaling pathway in HaCaT cells. ConclusionsFrom the findings above, these results confirm that T. pallidum FlaA2 activates ERK, p38 and NF-κB signaling pathway through TLR2 pathway to induce the production of IL-6 and IL-8, which could contribute to enhance the understanding of the skin inflammatory response induced by the pathogen in syphilis patients.

Thrombin induces IL-8/CXCL8 expression by DCLK1-dependent RhoA and YAP activation in human lung epithelial cells

BackgroundDoublecortin-like kinase 1 (DCLK1) has been recognized as a marker of cancer stem cell in several malignancies. Thrombin is crucial in asthma severity as it can promote IL-8/CXCL8 production in lung epithelial cells, which is a potent chemoattractant for neutrophils. However, the pathologic role of DCLK1 in asthma and its involvement in thrombin-stimulated IL-8/CXCL8 expression remain unknown.MethodsIL-8/CXCL8, thrombin, and DCLK1 expression were observed in the lung tissues of severe asthma patients and ovalbumin (OVA)-induced asthmatic mice model. A549 and BEAS-2B cells were either pretreated with inhibitors or small interfering RNAs (siRNAs) before being treated with thrombin. IL-8/CXCL8 expression and the molecules involved in signaling pathway were performed using ELISA, luciferase activity assay, Western blot, or ChIP assay.ResultsIL-8/CXCL8, thrombin, and DCLK1 were overexpressed in the lung tissues of severe asthma patients and ovalbumin (OVA)-induced asthmatic mice model. Our in vitro study found that DCLK siRNA or LRKK2-IN-1 (DCLK1 inhibitor) attenuated IL-8/CXCL8 release after thrombin induction in A549 and BEAS-2B cells. Thrombin activated DCLK1, RhoA, and YAP in a time-dependent manner, in which DCLK1 siRNA inhibited RhoA and YAP activation. YAP was dephosphorylated on the Ser127 site after thrombin stimulation, resulting in YAP translocation to the nucleus from the cytosol. DCLK1, RhoA and YAP activation following thrombin stimulation were inhibited by U0126 (ERK inhibitor). Moreover, DCLK1 and YAP siRNA inhibited κB-luciferase activity. Thrombin stimulated the recruitment of YAP and p65 to the NF-κB site of the IL-8/CXCL8 promoter and was inhibited by DCLK1 siRNA.ConclusionsThrombin activates the DCLK1/RhoA signaling pathway, which promotes YAP activation and translocation to the nucleus from the cytosol, resulting in YAP/p65 formation, and binding to the NF-κB site, which enhances IL-8/CXCL8 expression. DCLK1 might be essential in thrombin-stimulated IL-8/CXCL8 expression in asthmatic lungs and indicates a potential therapeutic strategy for severe asthma treatment.

Abstract 11032: Peptide Lv Promotes the Trafficking and Membrane Insertion of Endothelial K Ca 31 Through the MEK1/ERK Pathway

Background: Peptide Lv is an endogenous secretory peptide that is upregulated in the eyes of patients with diabetic retinopathy, an ocular disease with pathological angiogenesis. Peptide Lv promotes vascular endothelial cell proliferation, migration, and sprouting, and blocking peptide Lv with a specific antibody, anti-Lv, dampens neovascularization, so peptide Lv is an angiogenic factor. However, the molecular mechanism of how peptide Lv promotes angiogenesis is not clear. We found that peptide Lv causes endothelial cell membrane hyperpolarization, a step that is essential in endothelium-dependent angiogenesis. Peptide Lv augments the protein expression and current densities of K Ca 3.1, an ion channel underlying the endothelial hyperpolarization. As ion channels require trafficking and insertion to the plasma membrane to be functional after they are expressed, we aimed to determine the downstream signaling of peptide Lv that is involved in the trafficking of K Ca 3.1 in endothelial cells. Hypothesis: Peptide Lv activates the MEK1/ERK signaling pathway to promote the trafficking and membrane insertion of K Ca 3.1 in endothelial cells. Blocking MEK1/ERK will prevent augmentation of K Ca 3.1 by peptide Lv. Methods: Cultured human umbilical vein endothelial cells (HUVECs) were treated with peptide Lv (500 ng/ml) or PBS (vehicle control) in the presence/absence of FR180402 (10 μM; an ERK inhibitor) or PD98059 (10 μM, a MEK1 inhibitor) followed by the patch-clamp electrophysiological recordings and biotinylation assays. Results: Blocking ERK activation attenuated the peptide Lv-meditated membrane hyperpolarization and increase of K Ca 3.1 currents in endothelial cells. Blocking ERK activation did not affect peptide Lv-elicited increases of K Ca 3.1 protein expression, but it reduced the level of membrane-bound K Ca 3.1 Conclusions: The MEK1/ERK signaling pathway mediates peptide Lv-elicited trafficking and membrane insertion of K Ca 3.1 but does not affect the increase of K Ca 3.1 expression by peptide Lv.

Insulin-binding protein-5 down-regulates the balance of Th17/Treg

The inflammatory response plays critical important role in tissue hemostasis. Our previous study showed insulin-binding protein-5 (IGFBP5) could enhance the regeneration of tissue defect under inflammation condition, but the function of IGFBP5 in controlling inflammation and regulating immune responses remains unclear. In present study, we studied the regulatory effect of IGFBP5 on T cell immune response in vitro, and the maintenance of Th17/Treg balance in vivo by using dextran sulfate sodium salt (DSS)-induced colitis in mice. The results showed that IGFBP5 inhibited the differentiation of CD4+ T cells into Th17 subset while promoted its differentiation into Treg subsets. Further results of animal experiments demonstrated that recombinant IGFBP5 reversed the imbalance of Th17/Treg and alleviated the severity of DSS-induced colitis. The percentage of Th17 cells decreased and the percentage of Treg cells increased in the inflamed colon tissue and mesenteric lymph nodes of mice with colitis after IGFBP5 treatment. Besides, pro-inflammatory cytokines such as TNF-α, IL-1β and IFN-γ in serum were suppressed after the treatment of IGFBP5. Moreover, the function of IGFBP5 in regulating Th17/Treg balance could be inhibited by the inhibitors of ERK or JNK pathway. In conclusion, all these data showed that IGFBP5 could regulate Th17/Treg balance via ERK or JNK pathways. The findings of our study provide a theoretical basis for the application of IGFBP5 in inflammatory diseases.

Extensive preclinical validation of combined RMC-4550 and LY3214996 supports clinical investigation for KRAS mutant pancreatic cancer

Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity invitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. Invivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.

Effect of sulfasalazine on endothelium-dependent vascular response by the activation of Nrf2 signalling pathway.

Background: Diabetes mellitus leads to endothelial dysfunction and accumulation of oxygen radicals. Sulfasalazine-induced Nrf2 activation reduces oxidative stress in vessels. Thus, in the present study, we investigated the effects of sulfasalazine on endothelial dysfunction induced by high glucose. We also ascribed the underlying mechanism involved in glucose-induced endothelial dysfunction. Methods: For this experiment we used 80 Wistar Albino rats thoracic aorta to calculate the dose response curve of noradrenaline and acetylcholine. Vessels were incubated in normal and high glucose for 2h. To investigate glucose and sulfasalazine effects the vessels of the high glucose group were pre-treated with sulfasalazine (300mM), JNK inhibitor (SP600125), and ERK inhibitor (U0126) for 30min. The dose response curve was calculated through organ bath. The eNOS, TAS, TOS, and HO-1 levels were estimated by commercially available ELISA kits. Results: In the high glucose group, the Emax for contraction was significantly higher (p < 0.001), and Emax for relaxation was lower than that of control. These functional changes were parallel with the low levels of eNOS (p < 0.05). High glucose vessel treated with sulfasalazine showed low Emax value for contraction (p < 0.001) however, the Emax for relaxation was significantly high (p < 0.001) when compared to high glucose group. In the JNK group, Emax for contraction and relaxation was inhibited (p < 0.001) compared to sulfasalazine treated vessels. HO-1 enzyme levels were significantly low (p < 0.01) with sulfasalazine but higher with ERK inhibitor (p < 0.05). Conclusion: High glucose induced endothelial dysfunction and sulfasalazine reduced damage in high glucose vessels by activating eNOS, antioxidant effect through HO-1 enzymes and particularly inducing Nrf2 via the ERK and JNK pathways.

Neuroprotective Action of Coumarin Derivatives through Activation of TRKB-CREB-BDNF Pathway and Reduction of Caspase Activity in Neuronal Cells Expressing Pro-Aggregated Tau Protein.

Hyperphosphorylation and aggregation of the microtubule binding protein tau is a neuropathological hallmark of Alzheimer's disease/tauopathies. Tau neurotoxicity provokes alterations in brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB)/cAMP-response-element binding protein (CREB) signaling to contribute to neurodegeneration. Compounds activating TRKB may therefore provide beneficial effects in tauopathies. LM-031, a coumarin derivative, has demonstrated the potential to improve BDNF signaling in neuronal cells expressing pro-aggregated ΔK280 tau mutant. In this study, we investigated if LM-031 analogous compounds provide neuroprotection effects through interaction with TRKB in SH-SY5Y cells expressing ΔK280 tauRD-DsRed folding reporter. All four LMDS compounds reduced tau aggregation and reactive oxygen species. Among them, LMDS-1 and -2 reduced caspase-1, caspase-6 and caspase-3 activities and promoted neurite outgrowth, and the effect was significantly reversed by knockdown of TRKB. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in these cells, implying that the neuroprotective effects of LMDS-1/2 are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. Furthermore, LMDS-1/2 demonstrated their ability to quench the intrinsic fluorescence of tryptophan residues within the extracellular domain of TRKB, thereby consolidating their interaction with TRKB. Our results suggest that LMDS-1/2 exert neuroprotection through activating TRKB signaling, and shed light on their potential application in therapeutics of Alzheimer's disease/tauopathies.

Tanshinone increases Hemopexin expression in lung cells and macrophages to protect against cigarette smoke-induced COPD and enhance antiviral responses

ABSTRACT Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease, while respiratory infections can elicit exacerbations in COPD patients to mediate increased mortality. Administration of Tanshinones (TS) derivatives has been demonstrated to protect against cigarette smoking (CS) and lipopolysaccharide (LPS)-induced COPD progression. However, the underlying molecular mechanisms and the roles of TS in mitigating the severity of viral-mediated exacerbations of COPD have not been elucidated. Here, we found that TS treatments significantly attenuated lung function decline, inflammatory responses and oxidative stress in CS and LPS-induced COPD mice. Subsequent RNA-seq analysis revealed significantly upregulated Hemopexin expression and enriched interferons (IFNs) signaling pathways in lung tissues of COPD mice upon TS treatments. Moreover, TS administration demonstrated Hemopexin-dependent beneficial roles in BEAS-2B lung cells and RAW264.7 macrophages, which was associated with the suppression of oxidative stress and ERK, NF-κB, and NLRP3 inflammasome signaling pathways-mediated inflammation. Furthermore, TS promoted IFN signaling and rescued impaired antiviral responses in CS and LPS-exposed lung cells that were infected by influenza virus. Notably, hemopexin over-expression in lung cells and macrophages recapitulated the pharmacological activities of TS. Taken together, these results indicate that TS administration is a promising and potential therapeutic strategy for treating COPD and preventing COPD exacerbations.

Chitosan Oligosaccharide Prevents Afatinib-Induced Barrier Disruption and Chloride Secretion through Modulation of AMPK, PI3K/AKT, and ERK Signaling in T84 Cells

Diarrhea is an important adverse effect of epidermal growth factor receptor-tyrosine kinase inhibitors, especially afatinib. Novel antidiarrheal agents are needed to reduce epidermal growth factor receptor-tyrosine kinase inhibitor-associated diarrhea to improve the quality of life and treatment outcome in cancer patients. This study aimed to investigate the anti-diarrheal activity of chitosan oligosaccharide against afatinib-induced barrier disruption and chloride secretion in human intestinal epithelial cells (T84 cells). Chitosan oligosaccharide (100 μg/mL) prevented afatinib-induced barrier disruption determined by changes in transepithelial electrical resistance and FITC-dextran flux in the T84 cell monolayers. In addition, chitosan oligosaccharide prevented afatinib-induced potentiation of cAMP-induced chloride secretion measured by short-circuit current analyses in the T84 cell monolayers. Chitosan oligosaccharide induced the activation of AMPK, a positive regulator of epithelial tight junction and a negative regulator of cAMP-induced chloride secretion. Moreover, chitosan oligosaccharide partially reversed afatinib-induced AKT inhibition without affecting afatinib-induced ERK inhibition via AMPK-independent mechanisms. Collectively, this study reveals that chitosan oligosaccharide prevents the afatinib-induced diarrheal activities in T84 cells via both AMPK-dependent and AMPK-independent mechanisms. Chitosan oligosaccharide represents a promising natural polymer-derived compound for further development of treatment for afatinib-associated diarrheas.

224 (PB104) - Preliminary results from HERKULES-1: a phase 1b/2, open-label, multicenter study of ERAS-007, an oral ERK1/2 inhibitor, in patients with advanced or metastatic solid tumors

Background: The RAS/MAPK pathway is dysregulated in a broad range of cancers, resulting in downstream activation of ERK1/2. ERAS-007 is a novel, potent, and orally bioavailable inhibitor of ERK with a prolonged target residence time (550 min). The first-in-human Phase 1 study of ERAS-007 identified the 250 mg once weekly (QW) schedule for further evaluation based on a manageable toxicity profile and encouraging signs of clinical activity (NCT03415126). Pharmacokinetic (PK) modeling suggests that an alternative intermittent regimen (twice a day, once a week; BID-QW) is expected to have lower peak concentrations (Cmax) and similar overall PK exposures (area under the curve, AUC) compared to QW, which may improve Cmax-driven adverse events (AE) while maintaining comparable efficacy.

TNFAIP3 promotes ALDH-positive breast cancer stem cells through FGFR1/MEK/ERK pathway.

Breast cancer stem cells (BCSCs) are a tiny population of self-renewing cells that may contribute to cancer initiation, progression, and resistance to therapy in patients. In our prior study, we found that tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is necessary for fibroblast growth factors receptor 1 (FGFR1) signaling-promoted tumor growth and progression in breast cancer (BC). This study aims to investigate the involvement of TNFAIP3 in regulating BCSCs. In this work, we showed that ALDH-positive BCSCs were increased by activating the FGFR1-MEK-ERK pathway, meanwhile utilizing FGFR1 inhibitor, MEK inhibitor, or ERK inhibitor reversed the phenomenon in BC cells. Moreover, ALDH-positive BCSCs were decreased in TNFAIP3-knockout or TNFAIP3-depressing cells. In vivo analysis displayed that TNFAIP3-silenced MDA-MB-231 xenografts developed smaller tumors and ALDH immunostaining levels were significantly lower in TNFAIP3-depressing or TNFAIP3-knockout tumor tissues. Besides, our results also revealed that TNFAIP3 influences the transcription stemness factors gene expression. Taken together, TNFAIP3 could be a potential regulator in FGFR1-MEK-ERK-promoted ALDH-positive BCSCs.

Extracellular vesicles of Clonorchis sinensis promote IL-6 and TNF-α secretion via the Toll-like receptor 9-mediated ERK pathway in biliary epithelial cells

Clonorchis sinensis is closely associated with cholangitis, cholecystitis, biliary fibrosis and cholangiocarcinoma. The present study elucidated the role of extracellular vesicles of C. sinensis (CsEVs) in activating Toll-like receptor 9 (TLR9) and regulating inflammatory responses. The results showed that TLR9 expression was increased in the livers of C. sinensis-infected mice. CsEVs were cup-shaped or saucer-shaped and 80–120 nm in diameter. CsEVs activated TLR9 and promoted IL-6 and TNF-α expression in mouse biliary epithelial cells (BECs), and TLR9 siRNA interference reduced the secretion of the two cytokines. CsEV stimulation promoted the phosphorylation of ERK, p38, AKT, and p65, and TLR9 siRNA interference regulated the phosphorylated ERK, AKT and p65 levels. The ERK inhibitor decreased the CsEVs-induced IL-6 and TNF-α secretion. The present study elucidated for the first time that CsEVs induced IL-6 and TNF-α production in BECs via the TLR9-mediated ERK pathway.