Mitogen-activated Protein Kinase Pathway Genes Research Articles

Potential molecular mechanism of photosynthesis regulation by PeMPK7 in poplar under para-hydroxybenzoic acid stress

Due to continuous plantation of poplar, its growth and biomass accumulation may be negatively affected by the accumulation of allelochemicals such as para-hydroxybenzoic acid (pHBA) in soil. As photosynthesis is the most fundamental process in plants, it can be negatively impacted by pHBA stress. Therefore, it is crucial to improve photosynthetic capacity under pHBA stress to facilitate poplar plant growth. The mitogen-activated protein kinase (MAPK) cascade pathway is widely involved in environmental stress responses in plants. However, the regulation mechanisms of photosynthesis-related pathways by MAPK pathway genes under pHBA stress are still unclear. In this study, through transcriptome analysis and weighted gene co-expression network analysis, we observed that PeMPK7 overexpression in poplar can regulate the expression of photosynthesis-related genes and transcription factor genes, namely, WRKY1, WRKY33, and ERF3, during the early stage of pHBA stress. In addition, PeMPK7 can improve photosynthesis in poplar under long-term pHBA stress. Moreover, yeast two-hybrid and pull-down assays confirmed the interaction between PeMPK7 and PeMKK7/10. Based on these results, a schematic diagram of the pathways involved in the regulation of photosynthesis by PeMPK7 was constructed. This study provided novel insights into the molecular mechanisms of regulation of pHBA stress via MAPK cascade pathway.

FsCGBP, a Cutinase G-Box Binding Protein, Regulates the Growth, Development, and Virulence of Fusarium sacchari, the Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari is a causal agent of sugarcane Pokkah boeng, an important fungal disease that causes a considerable reduction in yield and sugar content in susceptible varieties of sugarcane worldwide. Despite its importance, the fungal factors that regulate the virulence of this pathogen remain largely unknown. In our previous study, mapping of an insertional mutant defect in virulence resulted in the identification of a cutinase G-box binding protein gene, designated FsCGBP, that encodes a C2H2-type transcription factor (TF). FsCGBP was shown to localize in the nuclei, and the transcript level of FsCGBP was significantly upregulated during the infection process or in response to abiotic stresses. Deletion or silencing of FsCGBP resulted in a reduction in mycelial growth, conidial production, and virulence and a delay in conidial germination in the F. sacchari. Cutinase genes FsCUT2, FsCUT3, and FsCUT4 and the mitogen-activated protein kinase (MAPK) genes FsHOG1, FsMGV1, and FsGPMK1, which were significantly downregulated in ΔFsCGBP. Except for FsHOG1, all of these genes were found to be transcriptionally activated by FsCGBP using the yeast one-hybrid system in vitro. The deletion of individual cutinase genes did not result in any of the phenotypes exhibited in the ΔFsCGBP mutant, except for cutinase activity. However, disruption of the MAPK pathway upon deletion of FsMGV1 or FsGPMK1 resulted in phenotypes similar to those of the ΔFsCGBP mutant. The above results suggest that FsCGBP functions by regulating the MAPK pathway and cutinase genes, providing new insights into the mechanism of virulence regulation in F. sacchari.

MAPK signaling pathway orchestrates and fine-tunes the pathogenicity of Colletotrichum falcatum

Colletotrichum falcatum is the causal organism of red rot, the most devastating disease of sugarcane. Mitogen-activated protein kinase (MAPK) signaling pathway plays pivotal role in coordinating the process of pathogenesis. We identified eighteen proteins implicated in MAPK signaling pathway in C. falcatum, through nanoLCMS/MS based proteomics approach. Twelve of these proteins were the part of core MAPK signaling pathway, whereas remaining proteins were indirectly implicated in MAPK signaling. Majority of these proteins had enhanced abundance in C. falcatum samples cultured with host sugarcane stalks. To validate the findings, core MAPK pathway genes (MAPKKK-NSY1, MAPK 17-MAPK17, MAPKKK 5-MAPKKK5, MAPK-HOG1B, MAPKKK-MCK1/STE11, MAPK-MST50/STE50, MAPKK-SEK1, MAPKK-MEK1/MST7/STE7, MAPKK-MKK2/STE7, MAPKKK-MST11/STE11, MAPK 5-MPK5, and MAPK-MPK-C) were analyzed by qPCR to confirm the real-time expression in C. falcatum samples cultured with host sugarcane stalks. The results of qPCR-based expression of genes were largely in agreement with the findings of proteomics. String association networks of MAPKK- MEK1/MST7/STE7, and MAPK- MPK-C revealed strong association with plenty of assorted proteins implicated in the process of pathogenesis/virulence. This is the novel and first large scale study of MAPK proteins in C. falcatum, responsible for red rot epidemics of sugarcane various countries. Key messageOur findings demonstrate the pivotal role of MAPK proteins in orchestrating the pathogenicity of Colletotrichum falcatum, responsible devastating red rot disease of sugarcane. SignificanceOur findings are novel and the first large scale study demonstrating the pivotal role of MAPK proteins in C. falcatum, responsible devastating red rot disease of sugarcane. The study will be useful for future researchers in terms of manipulating the fungal pathogenicity through genome editing.

The highs and lows of serous ovarian cancer.

Low-grade serous ovarian cancer was initially described as a distinct type of rare epithelial ovarian cancer 20 years ago; however, only recently have physicians begun to leverage the understanding of the clinical behavior and molecular profile of this disease for treatment. The use of routine next-generation sequencing has allowed a deeper understanding of the molecular drivers of this disease and shown how molecular alterations in mitogen-activated protein kinase pathway genes such as KRAS and BRAF can affect overall prognosis and disease behavior. The use of targeted therapies, including MEK inhibitors, BRAF kinase inhibitors, and other investigational targeted therapies are changing the way this disease is viewed and treated. In addition, endocrine therapy can provide prolonged disease stability with generally mild toxicity, as well as promising response rates in recent studies examining combination therapy with CDK 4/6 inhibitors in the upfront and recurrent setting. Once seen merely as a chemo-resistant form of ovarian cancer, recent studies have worked to harness the unique features of low-grade serous ovarian cancer to provide individualized treatment options for patients with this disease.

Activation of MAPK signaling pathway genes leads to promoted thermotolerance in a high-temperature adapted predatory mite Neoseiulus barkeri (Hughes)

Predatory mites are important biological control agents for phytophagous mites and several small insect pests on vegetables, while the control efficiency is often limited by the changeable or fluctuating environment, especially under the threat of global warming. To mitigate the undesired impacts of high temperature environment, a high-temperature adapted strain (HTAS) of the predatory mite Neoseiulus barkeri (Hughes) was screened from a conventional strain (CS) by long-term heat acclimation (35°C) and regular heat hardenings (45°C). In this study, to investigate the molecular mechanisms of enhanced thermotolerance in HTAS N. barkeri, four mitogen-activated protein kinase (MAPK) pathway genes were identified from the predatory mite N. barkeri. The expression levels at different developmental stages showed four MAPK pathway genes were highest at adults and lowest at eggs or nymphs in both strains. Under 42℃ high temperature stress, the expression levels of four identified MAPK pathway genes increased rapidly at a short time in both N. barkeri strains and the magnitude of expression increase in HTAS N. barkeri was even more evident. In addition, the MAPK pathway genes expression in CS N. barkeri sharply decreased following longer exposure periods at 4 h, whereas the expression in HTAS N. barkeri remained elevated after 4 h. These results suggested that MAPK signaling pathway participated in the formation of thermotolerance in predatory phytoseiid mites, which provided new insights in promoting the biological control efficiency of predatory mites under changeable environment.

Histiocytic Disorders of Childhood.

Histiocytic disorders of childhood represent a wide spectrum of conditions that share the common histologic feature of activated or transformed "histiocytes." Langerhans cell histiocytosis (LCH) is the most common, with an incidence of approximately 5 per million children. LCH may be difficult to distinguish from more ubiquitous causes of skin rashes, bone pain, or fever. Current chemotherapy fails to cure more than 50% of children with multifocal disease, and treatment failure is associated with increased risks of long-term sequelae. Somatic activating mitogen-activated protein kinase (MAPK) pathway-activating mutations (most often BRAFV600E) have been identified in hematopoietic precursors in patients with LCH. Opportunities to improve outcomes with targeted therapies are under investigation. Juvenile xanthogranuloma (JXG) and Rosai-Dorfman disease (RDD) are less common than LCH and are distinguished by specific histologic and clinical features. Recurrent MAPK pathway gene mutations are also identified in JXG and RDD. In many cases, these conditions spontaneously resolve, but disseminated disease can be fatal. Although there has been historic debate regarding the nature of these conditions as inflammatory versus neoplastic, LCH, JXG, and RDD are now considered myeloid neoplastic disorders. In contrast, hemophagocytic lymphohistiocytosis (HLH) is clearly a disorder of immune dysregulation. HLH is characterized by extreme immune activation driven by hyperactivated T cells. HLH arises in approximately 1 child per million and is nearly universally fatal without prompt recognition and immune suppression. Outcomes of treated children are poor, with approximately 60% survival. Emapalumab, which targets interferon-γ signaling, was recently approved for patients with recurrent or refractory HLH, and additional cytokine-directed therapies are under investigation.

Time and age dependent regulation of neuroinflammation in a rat model of mesial temporal lobe epilepsy: Correlation with human data.

Acute brain insults trigger diverse cellular and signaling responses and often precipitate epilepsy. The cellular, molecular and signaling events relevant to the emergence of the epileptic brain, however, remain poorly understood. These multiplex structural and functional alterations tend also to be opposing - some homeostatic and reparative while others disruptive; some associated with growth and proliferation while others, with cell death. To differentiate pathological from protective consequences, we compared seizure-induced changes in gene expression hours and days following kainic acid (KA)-induced status epilepticus (SE) in postnatal day (P) 30 and P15 rats by capitalizing on age-dependent differential physiologic responses to KA-SE; only mature rats, not immature rats, have been shown to develop spontaneous recurrent seizures after KA-SE. To correlate gene expression profiles in epileptic rats with epilepsy patients and demonstrate the clinical relevance of our findings, we performed gene analysis on four patient samples obtained from temporal lobectomy and compared to four control brains from NICHD Brain Bank. Pro-inflammatory gene expressions were at higher magnitudes and more sustained in P30. The inflammatory response was driven by the cytokines IL-1β, IL-6, and IL-18 in the acute period up to 72 h and by IL-18 in the subacute period through the 10-day time point. In addition, a panoply of other immune system genes was upregulated, including chemokines, glia markers and adhesion molecules. Genes associated with the mitogen activated protein kinase (MAPK) pathways comprised the largest functional group identified. Through the integration of multiple ontological databases, we analyzed genes belonging to 13 separate pathways linked to Classical MAPK ERK, as well as stress activated protein kinases (SAPKs) p38 and JNK. Interestingly, genes belonging to the Classical MAPK pathways were mostly transiently activated within the first 24 h, while genes in the SAPK pathways had divergent time courses of expression, showing sustained activation only in P30. Genes in P30 also had different regulatory functions than in P15: P30 animals showed marked increases in positive regulators of transcription, of signaling pathways as well as of MAPKKK cascades. Many of the same inflammation-related genes as in epileptic rats were significantly upregulated in human hippocampus, higher than in lateral temporal neocortex. They included glia-associated genes, cytokines, chemokines and adhesion molecules and MAPK pathway genes. Uniquely expressed in human hippocampus were adaptive immune system genes including immune receptors CDs and MHC II HLAs. In the brain, many immune molecules have additional roles in synaptic plasticity and the promotion of neurite outgrowth. We propose that persistent changes in inflammatory gene expression after SE leads not only to structural damage but also to aberrant synaptogenesis that may lead to epileptogenesis. Furthermore, the sustained pattern of inflammatory genes upregulated in the epileptic mature brain was distinct from that of the immature brain that show transient changes and are resistant to cell death and neuropathologic changes. Our data suggest that the epileptogenic process may be a result of failed cellular signaling mechanisms, where insults overwhelm the system beyond a homeostatic threshold.

Abstract 3365: Transcriptomics-based stratification of response to MAPK inhibition in vitro better predicts sensitivity to single agent and combination treatment than MAPK genomic alteration status alone

Abstract Overactivation of mitogen-activated protein kinase (MAPK) signaling is a frequently observed driver of many cancers. Targeted MAPK pathway inhibitors have been approved as therapies in patient populations with genomic alterations to MAPK pathway genes (e.g. dabrafenib in BRAF-mutant melanoma), with multiple other MAPK-targeted therapies in development. We have previously demonstrated that cell lines with MAPK pathway genomic alterations show increased sensitivity to ASTX029, our potent and selective dual-mechanism ERK inhibitor which is currently undergoing clinical development. However, MAPK oncogene status alone is not a universal indicator of sensitivity, nor is it clear that genomic markers of sensitivity to single agent inhibition represent the optimal approach to predicting response to MAPK targeted agents in the combination setting (e.g. sotorasib + trametinib). We therefore analysed MAPK pathway related transcriptional signatures to assess predictability for response to ASTX029 in combination with a SHP2 inhibitor. We previously presented data from large-scale viability screens assessing the response to ASTX029 as a single agent treatment (437 cell lines), as well as in combination with an inhibitor of SHP2 (491 cell lines), an upstream adaptor protein in the MAPK pathway. Using these data, several published transcriptional signatures related to the MAPK pathway were assessed for their ability to stratify sensitive vs resistant cell lines when benchmarked against MAPK oncogene genomic status alone. All assessed transcriptomics-based scores had a significant positive correlation with growth inhibition for ERK inhibitor single agent treatment as well as in combination with a SHP2 inhibitor. Transcriptomics-based scores were shown to be more sensitive than the use of mutant MAPK oncogene status alone based on assessment of binomial regression models. The significant increase in sensitivity enables the identification of additional cell lines responsive to MAPK pathway inhibition. The sensitivity was further increased when considering response to the combination of an ERK and SHP2 inhibitor compared to the ERK inhibitor as a single agent. This analysis demonstrates the potential use of MAPK pathway-related transcriptomics-based scores for the stratification of MAPK pathway inhibitor response. ASTX029 is currently undergoing clinical development in advanced solid tumours (NCT03520075) and transcriptomics-based methods may provide utility in refining patient selection in the clinic for single agent or combination approaches. Citation Format: Jessica Brothwood, Andrea Biondo, Kim-Hien Dao, Keisha Hearn, Chris Hindley, Navrohit Kandola, Harold Keer, John Lyons, Yoko Nakatsuru, Marc O'Reilly, Nicola Wallis, Harpreet Saini. Transcriptomics-based stratification of response to MAPK inhibition in vitro better predicts sensitivity to single agent and combination treatment than MAPK genomic alteration status alone [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 3365.

Recurrent Alterations in the MAPK pathway in Sporadic Pyogenic Granuloma of Childhood.

Pyogenic granuloma is one of the most common vascular tumours. The cause of pyogenic granuloma was previously thought to be an inflammatory reaction with consecutive stimulation of endothelial cell proliferation. However, recent studies suggest that pyogenic granuloma may be driven by constitutive activation of the mitogen-activated protein kinase pathway. The aim of this study was to investigate the molecular profile of sporadic pyogenic granuloma of childhood, using a systematic approach scrutinizing potential aberrations within different oncogenic pathways. Within a retrospective setting pyogenic granuloma of 15 patients was analysed by targeted next generation sequencing using the Oncomine Focus Assay, which includes genes of key tumorigenic signalling pathways. Activating mutations were found in 4 out of 15 cases (27%). Two HRAS hotspot mutations (p.Gly13Arg, p.Ala59Thr), 1 BRAF (p.Val600Glu) mutation and a novel, previously not reported, MAP2K1 hotspot mutation (p.Glu203Lys) were identified. It is notable that all of these genes are involved in constitutive mitogen-activated protein kinase signalling. This study increases the range of underlying genetic alterations in pyogenic granuloma by identifying novel oncogenic mutations in crucial mitogen-activated protein kinase pathway genes. The results provide supporting evidence that activated mitogen-activated protein kinase signalling is a key driver in the pathogenesis of pyogenic granuloma, which might be exploited by targeted treatment approaches for selected cases.

Cyclin D1 Expression and Molecular Genetic Findings in Periocular Histiocytoses and Neoplasms of Macrophage-Dendritic Cell Lineage

Frequent activating mutations in the mitogen-activated protein kinase (MAPK) pathway genes have been identified in histiocytoses. MAPK signaling consistently upregulates cyclin D1. The goal of this study was to determine whether cyclin D1 expression by immunohistochemistry is a useful diagnostic marker for periocular histiocytoses and to further characterize their genetic basis. Retrospective observational case series. Pathology records were searched for all patients with histiocytoses diagnosed between 1995 and 2020. Eleven histiocyte-rich inflammatory lesions and 10 xanthelasma served as controls. Cyclin D1 immunohistochemistry was performed on all tissues. A subset of histiocytoses was evaluated by next-generation sequencing (NGS) and droplet digital PCR (ddPCR). There were 36 patients, 15 males (42%) and 21 females (58%), with histiocytoses: 9 juvenile xanthogranuloma (25%), 8 adult-onset asthma and periocular xanthogranuloma (22%), 7 Langerhans cell histiocytosis (19%), 5 Rosai-Dorfman disease (14%), 5 xanthogranuloma-not otherwise specified (14%), 1 Erdheim-Chester disease (3%), and 1 histiocytic sarcoma (3%). Moderate to strong nuclear cyclin D1 expression was present in ≥50% of lesional cells in histiocytoses (23/36, 64%), significantly more when compared to histiocyte-rich inflammatory lesions (0/11, 0%, P<.001) and xanthelasma (0/10, 0%, P<.001). Cyclin D1 was expressed in <10% of lesional cells in all 11 histiocyte-rich inflammatory lesions (P<.001) and all 10 xanthelasma lesions (P<.001). MAPK pathway gene mutations were detected in 12 of 14 (86%) histiocytoses successfully assayed by NGS and/or ddPCR. Our study confirms that the cyclin D1 immunohistochemical stain is a useful diagnostic marker for periocular histiocytoses, correlating with underlying mutations in MAPK pathway genes.

Astroblastomas exhibit radial glia stem cell lineages and differential expression of imprinted and X-inactivation escape genes

Astroblastomas (ABs) are rare brain tumors of unknown origin. We performed an integrative genetic and epigenetic analysis of AB-like tumors. Here, we show that tumors traceable to neural stem/progenitor cells (radial glia) that emerge during early to later brain development occur in children and young adults, respectively. Tumors with MN1-BEND2 fusion appear to present exclusively in females and exhibit overexpression of genes expressed prior to 25 post-conception weeks (pcw), including genes enriched in early ventricular zone radial glia and ependymal tumors. Other, histologically classic ABs overexpress or harbor mutations of mitogen-activated protein kinase pathway genes, outer and truncated radial glia genes, and genes expressed after 25 pcw, including neuronal and astrocyte markers. Findings support that AB-like tumors arise in the context of epigenetic and genetic changes in neural progenitors. Selective gene fusion, variable imprinting and/or chromosome X-inactivation escape resulting in biallelic overexpression may contribute to female predominance of AB molecular subtypes.

Dual-Specificity Phosphatase 14 Regulates Zebrafish Hair Cell Formation Through Activation of p38 Signaling Pathway.

Most cases of acquired hearing loss are due to degeneration and subsequent loss of cochlear hair cells. Whereas mammalian hair cells are not replaced when lost, in zebrafish, they constantly renew and regenerate after injury. However, the molecular mechanism among this difference remains unknown. Dual-specificity phosphatase 14 (DUSP14) is an important negative modulator of mitogen-activated protein kinase (MAPK) signaling pathways. Our study was to investigate the effects of DUSP14 on supporting cell development and hair cell regeneration and explore the potential mechanism. Our results showed that dusp14 gene is highly expressed in zebrafish developing neuromasts and otic vesicles. Behavior analysis showed that dusp14 deficiency resulted in hearing defects in zebrafish larvae, which were reversed by dusp14 mRNA treatment. Moreover, knockdown of dusp14 gene caused a significant decrease in the number of neuromasts and hair cells in both neuromast and otic vesicle, mainly due to the inhibition of the proliferation of supporting cells, which results in a decrease in the number of supporting cells and ultimately in the regeneration of hair cells. We further found significant changes in a series of MAPK pathway genes through transcriptome sequencing analysis of dusp14-deficient zebrafish, especially mapk12b gene in p38 signaling. Additionally, inhibiting p38 signaling effectively rescued all phenotypes caused by dusp14 deficiency, including hair cell and supporting cell reduction. These results suggest that DUSP14 might be a key gene to regulate supporting cell development and hair cell regeneration and is a potential target for the treatment of hearing loss.

Somatic mosaicism in the MAPK pathway in sporadic brain arteriovenous malformation and association with phenotype.

Sporadic brain arteriovenous malformation (BAVM) is a tangled vascular lesion characterized by direct artery-to-vein connections that can cause life-threatening intracerebral hemorrhage (ICH). Recently, somatic mutations in KRAS have been reported in sporadic BAVM, and mutations in other mitogen-activated protein kinase (MAPK) signaling pathway genes have been identified in other vascular malformations. The objectives of this study were to systematically evaluate somatic mutations in MAPK pathway genes in patients with sporadic BAVM lesions and to evaluate the association of somatic mutations with phenotypes of sporadic BAVM severity. The authors performed whole-exome sequencing on paired lesion and blood DNA samples from 14 patients with sporadic BAVM, and 295 genes in the MAPK signaling pathway were evaluated to identify genes with somatic mutations in multiple patients with BAVM. Digital droplet polymerase chain reaction was used to validate KRAS G12V and G12D mutations and to assay an additional 56 BAVM samples. The authors identified a total of 24 candidate BAVM-associated somatic variants in 11 MAPK pathway genes. The previously identified KRAS G12V and G12D mutations were the only recurrent mutations. Overall, somatic KRAS G12V was present in 14.5% of BAVM lesions and G12D was present in 31.9%. The authors did not detect a significant association between the presence or allelic burden of KRAS mutation and three BAVM phenotypes: lesion size (maximum diameter), age at diagnosis, and age at ICH. The authors confirmed the high prevalence of somatic KRAS mutations in sporadic BAVM lesions and identified several candidate somatic variants in other MAPK pathway genes. These somatic variants may contribute to understanding of the etiology of sporadic BAVM and the clinical characteristics of patients with this condition.

New therapeutic opportunities for women with low-grade serous ovarian cancer.

Low-grade serous ovarian cancer (LGSC) is a morphologically and molecularly distinct subtype of ovarian cancer, accounting for ~10% of serous carcinomas. Women typically present at a younger age and have a protracted clinical course compared with the more common, high-grade serous ovarian cancer. Currently, the primary treatment of LGSC is the same as other epithelial ovarian cancer subtypes, with treatment for most patients comprised of debulking surgery and platinum/taxane chemotherapy. Primary surgical cytoreduction to no visible residual disease remains a key prognostic factor; however, the use of platinum-based chemotherapy in both upfront and relapsed setting is being questioned due to low response rates in LGSC. Most LGSC expresses steroid hormone receptors, and selected patients may benefit from endocrine maintenance therapy following chemotherapy, in particular, those with evidence of residual disease at completion of surgery. In the recurrent setting, while hormonal therapies may offer disease stabilisation with relatively low toxicity, objective response rates remain low. Strategies to increase response rates, including combining with CDK4/6 inhibitors, are being investigated. LGSC has a high prevalence of activating somatic mutations in mitogen-activated protein kinase pathway genes, most commonly in KRAS, BRAF and NRAS. Trametinib, a MEK inhibitor, has shown efficacy over chemotherapy and endocrine therapy. The use of combination targeted therapies, immunotherapy and anti-angiogenic agents, remain active areas of investigation for the treatment of LGSC.

Simultaneous Detection of Single Nucleotide Variants and Expression of RNA and Protein in Multiple Myeloma Bone Marrow Aspirates

Introduction: Multiple myeloma (MM) is generally an incurable plasma cell neoplasm that evolves from a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS). Primary genomic events leading to the development of MGUS include hyperdiploidy, translocations involving the immunoglobulin heavy chain genes, and copy number variations. Single-nucleotide variants (SNVs) as well as insertions and deletions are secondary events that trigger progression to active MM. Identifying nucleotide variants that lead to changes in RNA and protein expression is critically important to understanding the biology of the cancer and ultimately identifying therapeutic targets. Analysis of DNA, RNA, and protein has traditionally involved integration of three separate assays and is frequently limited by the availability of sufficient material from MM patient specimens. We used a more efficient approach that simultaneously detects DNA variants, RNA and protein expression in a single sample from as little as 5 ng DNA, 25 ng RNA, and 250 ng protein. We evaluated the NanoString nCounter® Vantage 3D™ DNA:RNA:Protein Heme Assay performed on 1 x 106cells from bone marrow biopsies obtained from patients with MM.Methods: Bone marrow aspirates from 15 patients diagnosed with MM were obtained through an Institutional Review Board approved protocol. Seven patients had newly diagnosed MM and 8 had relapsed disease. DNA, RNA, and protein were extracted from unsorted, cryopreserved, ficoll-hypaque separated bone marrow mononuclear cells and hybridized with optical barcodes designed to detect 124 DNA somatic variants common to hematologic malignancies as well as 180 mRNA transcripts and 38 total and phospho-proteins. We also used the nCounter PanCancer Pathways Panel Assay to measure gene expression of 770 additional genes from 13 cancer-associated canonical pathways. The hybridized probes in each sample were counted using the nCounter Digital Analyzer and statistical analysis was performed using nSolver™ v4.0 alpha software.Results: The percentage of CD138+ bone marrow plasma cells measured by immunohistochemistry at the time of biopsy varied from 44 to 100% in each sample. One or more DNA variants were detected in 3 pre-treatment and 4 relapse samples. Mutations in KRAS, NRAS, and BRAF were most common and 2 patients were found to have variants in both KRAS and NRAS or KRAS and BRAF . Additionally, variants in DNMT3A and IDH2 were detected in 1 patient each. Since KRAS, NRAS, and BRAF are all members of the mitogen-activated protein kinase (MAPK) signaling pathway, a differential gene expression analysis of both RNA and protein was performed comparing samples with and without mutations in these genes. Findings confirmed that a higher level of expression was detectable in MAPK pathway genes, although this result did not reach statistical significance following correction for replicate testing, potentially the consequence of a small sample size.Conclusions: The Vantage 3D DNA:RNA:Protein Heme Assay is a rapid, sensitive and resource sparing platform for the simultaneous detection of DNA variants, RNA transcripts, and protein expression. We show that this method can feasibly be performed on as few as 1 x 106 cryopreserved bone marrow cells in patients with MM and our findings confirm detection of 3 of the most common MM variants. This technology should have broad application in other hematological malignancies and further evaluation of blood and lymph node biopsies from fresh, frozen and formalin-fixed paraffin-embedded (FFPE) samples is warranted. DisclosuresDemirkan:NanoString Technologies: Employment. Meredith:NanoString Technologies: Employment. Meredith:NanoString Technologies: Employment.

Genetic variations of rs6928 and rs5999521 of ERK2 were found to have correlation with the risk of brain metastasis in patients with lung adenocarcinoma.

BackgroundPatients with lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutations have a high risk of brain metastasis (BM). Mitogen-activated protein kinase (MAPK) is an important mediator of EGFR/c-MET crosstalk, which is involved in development of BM in non-small cell lung cancer. Here, we investigated the association of MAPK genetic variations with the risk of BM in patients with lung adenocarcinoma.MethodsPatients with pathologically confirmed lung adenocarcinoma from two Hospitals (n=120, discovery cohort; n=213, validation cohort) were enrolled. Magnetic resonance imaging (MRI) was employed for BM follow-up after the completion of planned therapy. Single nucleotide polymorphisms (SNPs) of MAPK pathway genes were tested with blood samples.ResultsAfter adjustment for sex, age, staging, smoking status, surgery, and thoracic radiotherapy, extracellular signal-regulated kinase 2 (ERK2) rs6928 and rs5999521 SNPs were found to be associated with increased risk of BM. The rs6928 GG and CG genotypes were associated with 2.033-fold (P=0.033) and 1.910-fold (P=0.012) increases in the risk of developing BM compared with the CC genotype. For rs5999521, the risk of developing BM was increased by 1.993-fold (P=0.037) in patients with the GG genotype and 1.834-fold (P=0.019) in patients with the AG genotype compared with patients with the AA genotype. Furthermore, patients with genotypes of higher risk of BM showed higher EGFR mutation rates and tended to have >1 BM lesions.ConclusionsIn patients with lung adenocarcinoma, ERK2 rs6928 and rs5999521 SNPs contributed to BM risk, particularly in patients with specific genotypes.

Swertiamarin from Enicostemma littorale, counteracts PD associated neurotoxicity via enhancement α-synuclein suppressive genes and SKN-1/NRF-2 activation through MAPK pathway

The elusive targets and the multifactorial etiology of Parkinson’s disease (PD) have hampered the discovery of a potent drug for PD. Furthermore, the presently available medications provide only symptomatic relief and have failed to mitigate the pathogenesis associated with PD. Therefore, the current study was aimed to evaluate the prospective of swertiamarin (SW), a secoiridoid glycoside isolated from a traditional medicinal plant, Enicostemma littorale Blume to ameliorate the characteristic features of PD in Caenorhabditis elegans. SW (25 μM) administration decreased the α-synuclein (α-syn) deposition, inhibited apoptosis and increased dopamine level mediated through upregulating the expression of genes linked to ceramide synthesis, mitochondrial morphology and function regulation, fatty acid desaturase genes along with stress responsive MAPK (mitogen-activated protein kinase) pathway genes. The neuroprotective effect of SW was evident from the robust reduction of 6-hydroxydopamine (6-OHDA) induced dopaminergic neurodegeneration independent of dopamine transporter (dat-1). SW mediated translational regulation of MAPK pathway genes was observed through increase expression of SKN-1 and GST-4. Further, in-silico molecular docking analysis of SW with C. elegans MEK-1 showed a promising binding affinity affirming the in-vivo results. Overall, these novel finding supports that SW is a possible lead for drug development against the multi- factorial PD pathologies.

Integrated molecular characterisation of the MAPK pathways in human cancers reveals pharmacologically vulnerable mutations and gene dependencies

The mitogen-activated protein kinase (MAPK) pathways are crucial regulators of the cellular processes that fuel the malignant transformation of normal cells. The molecular aberrations which lead to cancer involve mutations in, and transcription variations of, various MAPK pathway genes. Here, we examine the genome sequences of 40,848 patient-derived tumours representing 101 distinct human cancers to identify cancer-associated mutations in MAPK signalling pathway genes. We show that patients with tumours that have mutations within genes of the ERK-1/2 pathway, the p38 pathways, or multiple MAPK pathway modules, tend to have worse disease outcomes than patients with tumours that have no mutations within the MAPK pathways genes. Furthermore, by integrating information extracted from various large-scale molecular datasets, we expose the relationship between the fitness of cancer cells after CRISPR mediated gene knockout of MAPK pathway genes, and their dose-responses to MAPK pathway inhibitors. Besides providing new insights into MAPK pathways, we unearth vulnerabilities in specific pathway genes that are reflected in the re sponses of cancer cells to MAPK targeting drugs: a revelation with great potential for guiding the development of innovative therapies.

CIC Is a Mediator of the ERK1/2-DUSP6 Negative Feedback Loop.

SummaryDUSP6 functions as an important negative feedback component of the MAPK/ERK signaling pathway. Although DUSP6 expression is tightly regulated by ERK1/2 signaling, the molecular mechanism of this regulation remains partially understood. In this work, we show that the transcriptional repressor CIC functions downstream of the ERK1/2 signaling to negatively regulate DUSP6 expression. CIC directly represses DUSP6 transcription by binding to three cis-regulatory elements (CREs) in DUSP6 promoter. p90RSK, a downstream target of ERK1/2, phosphorylates CIC at S173 and S301 sites, which creates a 14-3-3 recognition motif, resulting in 14-3-3-mediated nuclear export of CIC and derepression of DUSP6. Finally, we demonstrate that the oncogenic CIC-DUX4 fusion protein acts as a transcriptional activator of DUSP6 and its nuclear/cytoplasmic distribution remains regulated by ERK1/2 signaling. These results complete an ERK1/2/p90RSK/CIC/DUSP6 negative feedback circuit and elucidate the molecular mechanism of how RTK/MAPK signaling harnesses the transcriptional repressor activity of CIC in mammalian cells.

Frequency of MicroRNA Response Elements Identifies Pathologically Relevant Signaling Pathways in Triple-Negative Breast Cancer.

SummaryComplex interactions between mRNAs and microRNAs influence cellular functions. The mRNA-microRNA interactions also determine the post-transcriptional availability of mRNAs and unbound microRNAs. MicroRNAs binds to one or more microRNA response elements (MREs) located on the 3′UTR of mRNAs. In this study, we leveraged MREs and their frequencies in cancer and matched normal tissues to obtain insights into disease-specific interactions between mRNAs and microRNAs. We developed a bioinformatics method “ReMIx” that utilizes RNA sequencing (RNA-Seq) data to quantify MRE frequencies across the transcriptome. We applied ReMIx to triple-negative (TN) breast cancer tumor-normal adjacent pairs and identified MREs specific to TN tumors. ReMIx identified candidate mRNAs and microRNAs in the MAPK signaling cascade. Further analysis of MAPK gene regulatory networks revealed microRNA partners that influence and modulate MAPK signaling. In conclusion, we demonstrate a novel method of using MREs in the identification of functionally relevant mRNA-microRNA interactions in TN breast cancer.