KRAS Allelic Imbalance Enhances Fitness and Modulates MAP Kinase Dependence in Cancer.

Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.

Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma.

PD-1 immune checkpoint blockade provides significant clinical benefits for melanoma patients. We analyzed the somatic mutanomes and transcriptomes of pretreatment melanoma biopsies to identify factors that may influence innate sensitivity or resistance to anti-PD-1 therapy. We find that overall high mutational loads associate with improved survival, and tumors from responding patients are enriched for mutations in the DNA repair gene BRCA2. Innately resistant tumors display a transcriptional signature (referred to as the IPRES, or innate anti-PD-1 resistance), indicating concurrent up-expression of genes involved in the regulation of mesenchymal transition, cell adhesion, extracellular matrix remodeling, angiogenesis, and wound healing. Notably, mitogen-activated protein kinase (MAPK)-targeted therapy (MAPK inhibitor) induces similar signatures in melanoma, suggesting that a non-genomic form of MAPK inhibitor resistance mediates cross-resistance to anti-PD-1 therapy. Validation of the IPRES in other independent tumor cohorts defines a transcriptomic subset across distinct types of advanced cancer. These findings suggest that attenuating the biological processes that underlie IPRES may improve anti-PD-1 response in melanoma and other cancer types.

Non-genomic and Immune Evolution of Melanoma Acquiring MAPKi Resistance.

Clinically acquired resistance to MAPK inhibitor (MAPKi) therapies for melanoma cannot be fully explained by genomic mechanisms and may be accompanied by co-evolution of intra-tumoral immunity. We sought to discover non-genomic mechanisms of acquired resistance and dynamic immune compositions by a comparative, transcriptomic-methylomic analysis of patient-matched melanoma tumors biopsied before therapy and during disease progression. Transcriptomic alterations across resistant tumors were highly recurrent, in contrast to mutations, and were frequently correlated with differential methylation of tumor cell-intrinsic CpG sites. We identified in the tumor cell compartment supra-physiologic c-MET up-expression, infra-physiologic LEF1 down-expression and YAP1 signature enrichment as drivers of acquired resistance. Importantly, high intra-tumoral cytolytic T cell inflammation prior to MAPKi therapy preceded CD8 T cell deficiency/exhaustion and loss of antigen presentation in half of disease-progressive melanomas, suggesting cross-resistance to salvage anti-PD-1/PD-L1 immunotherapy. Thus, melanoma acquires MAPKi resistance with highly dynamic and recurrent non-genomic alterations and co-evolving intra-tumoral immunity.

The Ras-Erk-ETS-Signaling Pathway Is a Drug Target for Longevity.

Identifying the molecular mechanisms that underlie aging and their pharmacological manipulation are key aims for improving lifelong human health. Here, we identify a critical role for Ras-Erk-ETS signaling in aging in Drosophila. We show that inhibition of Ras is sufficient for lifespan extension downstream of reduced insulin/IGF-1 (IIS) signaling. Moreover, direct reduction of Ras or Erk activity leads to increased lifespan. We identify the E-twenty six (ETS) transcriptional repressor, Anterior open (Aop), as central to lifespan extension caused by reduced IIS or Ras attenuation. Importantly, we demonstrate that adult-onset administration of the drug trametinib, a highly specific inhibitor of Ras-Erk-ETS signaling, can extend lifespan. This discovery of the Ras-Erk-ETS pathway as a pharmacological target for animal aging, together with the high degree of evolutionary conservation of the pathway, suggests that inhibition of Ras-Erk-ETS signaling may provide an effective target for anti-aging interventions in mammals.

Targeting Interleukin-6 Signaling in Clinic.

Interleukin-6 (IL-6) is a pleiotropic cytokine with roles in immunity, tissue regeneration, and metabolism. Rapid production of IL-6 contributes to host defense during infection and tissue injury, but excessive synthesis of IL-6 and dysregulation of IL-6 receptor signaling is involved in disease pathology. Therapeutic agents targeting the IL-6 axis are effective in rheumatoid arthritis, and applications are being extended to other settings of acute and chronic inflammation. Recent studies reveal that selective blockade of different modes of IL-6 receptor signaling has different outcomes on disease pathology, suggesting novel strategies for therapeutic intervention. However, some inflammatory diseases do not seem to respond to IL-6 blockade. Here, we review the current state of IL-6-targeting approaches in the clinic and discuss how to apply the growing understanding of the immunobiology of IL-6 to clinical decisions.

ERK inhibition rescues defects in fate specification of Nf1-deficient neural progenitors and brain abnormalities.

Germline mutations in the RAS/ERK signaling pathway underlie several related developmental disorders collectively termed neuro-cardio-facial-cutaneous (NCFC) syndromes. NCFC patients manifest varying degrees of cognitive impairment, but the developmental basis of their brain abnormalities remains largely unknown. Neurofibromatosis type 1 (NF1), an NCFC syndrome, is caused by loss-of-function heterozygous mutations in the NF1 gene, which encodes neurofibromin, a RAS GTPase-activating protein. Here, we show that biallelic Nf1 inactivation promotes Erk-dependent, ectopic Olig2 expression specifically in transit-amplifying progenitors, leading to increased gliogenesis at the expense of neurogenesis in neonatal and adult subventricular zone (SVZ). Nf1-deficient brains exhibit enlarged corpus callosum, a structural defect linked to severe learning deficits in NF1 patients. Strikingly, these NF1-associated developmental defects are rescued by transient treatment with an MEK/ERK inhibitor during neonatal stages. This study reveals a critical role for Nf1 in maintaining postnatal SVZ-derived neurogenesis and identifies a potential therapeutic window for treating NF1-associated brain abnormalities.

Diverse alterations associated with resistance to KRAS(G12C) inhibition.

Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.

IKKα Kinase Regulates the DNA Damage Response and Drives Chemo-resistance in Cancer.

Phosphorylated IKKα(p45) is a nuclear active form of the IKKα kinase that is induced by the MAP kinases BRAF and TAK1 and promotes tumor growth independent of canonical NF-κB signaling. Insights into the sources of IKKα(p45) activation and its downstream substrates in the nucleus remain to be defined. Here, we discover that IKKα(p45) is rapidly activated by DNA damage independent of ATM-ATR, but dependent on BRAF-TAK1-p38-MAPK, and is required for robust ATM activation and efficient DNA repair. Abolishing BRAF or IKKα activity attenuates ATM, Chk1, MDC1, Kap1, and 53BP1 phosphorylation, compromises 53BP1 and RIF1 co-recruitment to sites of DNA lesions, and inhibits 53BP1-dependent fusion of dysfunctional telomeres. Furthermore, IKKα or BRAF inhibition synergistically enhances the therapeutic potential of 5-FU and irinotecan to eradicate chemotherapy-resistant metastatic human tumors in vivo. Our results implicate BRAF and IKKα kinases in the DDR and reveal a combination strategy for cancer treatment.

Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms.

Janus kinases (JAKs) mediate responses to cytokines, hormones and growth factors in haematopoietic cells1,2. The JAK gene JAK2 is frequently mutated in the ageing haematopoietic system3,4 and in haematopoietic cancers5. JAK2 mutations constitutively activate downstream signalling and are drivers of myeloproliferative neoplasm (MPN). In clinical use, JAK inhibitors have mixed effects on the overall disease burden of JAK2-mutated clones6,7, prompting us to investigate the mechanism underlying disease persistence. Here, by in-depth phosphoproteome profiling, we identify proteins involved in mRNA processing as targets of mutant JAK2. We found that inactivation of YBX1, a post-translationally modified target of JAK2, sensitizes cells that persist despite treatment with JAK inhibitors to apoptosis and results in RNA mis-splicing, enrichment for retained introns and disruption of the transcriptional control of extracellular signal-regulated kinase (ERK) signalling. In combination with pharmacological JAK inhibition, YBX1 inactivation induces apoptosis in JAK2-dependent mouse and primary human cells, causing regression of the malignant clones in vivo, and inducing molecular remission. This identifies and validates a cell-intrinsic mechanism whereby differential protein phosphorylation causes splicing-dependent alterations of JAK2-ERK signalling and the maintenance of JAK2V617F malignant clones. Therapeutic targeting of YBX1-dependent ERK signalling in combination with JAK2 inhibition could thus eradicate cells harbouring mutations in JAK2.

KSR1 Knockout Mouse Model Demonstrates MAPK Pathway's Key Role in Cisplatin- and Noise-induced Hearing Loss.

Hearing loss is a major disability in everyday life and therapeutic interventions to protect hearing would benefit a large portion of the world population. Here we found that mice devoid of the protein kinase suppressor of RAS 1 (KSR1) in their tissues (germline KO mice) exhibit resistance to both cisplatin- and noise-induced permanent hearing loss compared with their wild-type KSR1 littermates. KSR1 is a scaffold protein that brings in proximity the mitogen-activated protein kinase (MAPK) proteins BRAF, MEK1/2 and ERK1/2 and assists in their activation through a phosphorylation cascade induced by both cisplatin and noise insults in the cochlear cells. KSR1, BRAF, MEK1/2, and ERK1/2 are all ubiquitously expressed in the cochlea. Deleting the KSR1 protein tempered down the MAPK phosphorylation cascade in the cochlear cells following both cisplatin and noise insults and conferred hearing protection of up to 30 dB SPL in three tested frequencies in male and female mice. Treatment with dabrafenib, an FDA-approved oral BRAF inhibitor, protected male and female KSR1 wild-type mice from both cisplatin- and noise-induced hearing loss. Dabrafenib treatment did not enhance the protection of KO KSR1 mice, providing evidence dabrafenib works primarily through the MAPK pathway. Thus, either elimination of the KSR1 gene expression or drug inhibition of the MAPK cellular pathway in mice resulted in profound protection from both cisplatin- and noise-induced hearing loss. Inhibition of the MAPK pathway, a cellular pathway that responds to damage in the cochlear cells, can prove a valuable strategy to protect and treat hearing loss.

Scutellarin alleviates renal ischemia-reperfusion injury by inhibiting the MAPK pathway and pro-inflammatory macrophage polarization.

Renal ischemia-reperfusion injury (IRI) is an integral process in renal transplantation, which results in compromised graft survival. Macrophages play an important role in both the early inflammatory period and late fibrotic period in response to IRI. In this study, we investigated whether scutellarin (SCU) could protect against renal IRI by regulating macrophage polarization. Mice were given SCU (5-50 mg/kg) by gavage 1 h earlier, followed by a unilateral renal IRI. Renal function and pathological injury were assessed 24 h after reperfusion. The results showed that administration of 50 mg/kg SCU significantly improved renal function and renal pathology in IRI mice. In addition, SCU alleviated IRI-induced apoptosis. Meanwhile, it reduced macrophage infiltration and inhibited pro-inflammatory macrophage polarization. Moreover, in RAW 264.7 cells and primary bone marrow-derived macrophages (BMDMs) exposed to SCU, we found that 150 µM SCU inhibited these cells to polarize to an inflammatory phenotype induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). However, SCU has no influence on anti-inflammatory macrophage polarization in vivo and in vitro induced by in interleukin-4 (IL-4). Finally, we explored the effect of SCU on the activation of the mitogen-activated protein kinase (MAPK) pathway both in vivo and in vitro. We found that SCU suppressed the activation of the MAPK pathway, including the extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38. Our results demonstrated that SCU protects the kidney against IRI by inhibiting macrophage infiltration and polarization toward pro-inflammatory phenotype via the MAPK pathway, suggesting that SCU may be therapeutically important in treatment of IRI.

Efficacy of MEK inhibition in patients with histiocytic neoplasms.

Histiocytic neoplasms are a heterogeneous group of clonal haematopoietic disorders that are marked by diverse mutations in the mitogen-activated protein kinase (MAPK) pathway1,2. For the 50% of patients with histiocytosis who have BRAFV600 mutations3-5, RAF inhibition is highly efficacious and has markedly altered the natural history of the disease6,7. However, no standard therapy exists for the remaining 50% of patients who lack BRAFV600 mutations. Although ERK dependence has been hypothesized to be a consistent feature across histiocytic neoplasms, this remains clinically unproven and many of the kinase mutations that are found in patients who lack BRAFV600 mutations have not previously been biologically characterized. Here we show ERK dependency in histiocytoses through a proof-of-concept clinical trial of cobimetinib, an oral inhibitor of MEK1 and MEK2, in patients with histiocytoses. Patients were enrolled regardless of their tumour genotype. In parallel, MAPK alterations that were identified in treated patients were characterized for their ability to activate ERK. In the 18 patients that we treated, the overall response rate was 89% (90% confidence interval of 73-100). Responses were durable, with no acquired resistance to date. At one year, 100% of responses were ongoing and 94% of patients remained progression-free. Cobimetinib treatment was efficacious regardless of genotype, and responses were observed in patients with ARAF, BRAF, RAF1, NRAS, KRAS, MEK1 (also known as MAP2K1) and MEK2 (also known as MAP2K2) mutations. Consistent with the observed responses, the characterization of the mutations that we identified in these patients confirmed that the MAPK-pathway mutations were activating. Collectively, these data demonstrate that histiocytic neoplasms are characterized by a notable dependence on MAPK signalling-and that they are consequently responsive to MEK inhibition. These results extend the benefits of molecularly targeted therapy to the entire spectrum of patients with histiocytosis.

TRIM29 facilitates gemcitabine resistance via MEK/ERK pathway and is modulated by circRPS29/miR-770-5p axis in PDAC.

AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Chemotherapy based on gemcitabine (GEM) remains the first-line drug for patients with advanced PDAC. However, GEM resistance impairs its therapeutic effectiveness. Therefore, identifying effective therapeutic targets are urgently needed to overcome GEM resistance. METHODS: The clinical significance of Tripartite Motif Containing 29 (TRIM29) was identified by exploring GEO datasets and TCGA database and its potential biological functions were predicted by GSEA analysis. The regulatory axis was established by bioinformatics analysis and validated by mechanical experiments. Then, in vitro and in vivo assays were performed to validate the roles of TRIM29 in PDAC GEM resistance. RESULTS: High TRIM29 expression was associated with poor prognosis of PDAC and functional experiments demonstrated that TRIM29 promoted GEM resistance in PDAC GEM-resistant (GR) cells. Furthermore, we revealed that circRPS29 promoted TRIM29 expression via competitive interaction with miR-770-5p and then activated MEK/ERK signaling pathway. Additionally, both in vitro and in vivo functional experiments demonstrated that circRPS29/miR-770-5p/TRIM29 axis promoted PDAC GEM resistance via activating MEK/ERK signaling pathway. CONCLUSION: Our results identify the significance of the signaling axis, circRPS29/miR-770-5p/TRIM29-MEK/ERK, in PDAC GEM resistance, which will provide novel therapeutic targets for PDAC treatment.

MAPK-mediated transcription factor GATAd contributes to Cry1Ac resistance in diamondback moth by reducing PxmALP expression.

The benefits of biopesticides and transgenic crops based on the insecticidal Cry-toxins from Bacillus thuringiensis (Bt) are considerably threatened by insect resistance evolution, thus, deciphering the molecular mechanisms underlying insect resistance to Bt products is of great significance to their sustainable utilization. Previously, we have demonstrated that the down-regulation of PxmALP in a strain of Plutella xylostella (L.) highly resistant to the Bt Cry1Ac toxin was due to a hormone-activated MAPK signaling pathway and contributed to the resistance phenotype. However, the underlying transcriptional regulatory mechanism remains enigmatic. Here, we report that the PxGATAd transcription factor (TF) is responsible for the differential expression of PxmALP observed between the Cry1Ac susceptible and resistant strains. We identified that PxGATAd directly activates PxmALP expression via interacting with a non-canonical but specific GATA-like cis-response element (CRE) located in the PxmALP promoter region. A six-nucleotide insertion mutation in this cis-acting element of the PxmALP promoter from the resistant strain resulted in repression of transcriptional activity, affecting the regulatory performance of PxGATAd. Furthermore, silencing of PxGATAd in susceptible larvae reduced the expression of PxmALP and susceptibility to Cry1Ac toxin. Suppressing PxMAP4K4 expression in the resistant larvae transiently recovered both the expression of PxGATAd and PxmALP, indicating that the PxGATAd is a positive responsive factor involved in the activation of PxmALP promoter and negatively regulated by the MAPK signaling pathway. Overall, this study deciphers an intricate regulatory mechanism of PxmALP gene expression and highlights the concurrent involvement of both trans-regulatory factors and cis-acting elements in Cry1Ac resistance development in lepidopteran insects.

Inactivation of Capicua in adult mice causes T-cell lymphoblastic lymphoma.

CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4 Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancy.

Mechanistic Understanding of Dexamethasone-Mediated Protection against Remdesivir-Induced Hepatotoxicity.

Remdesivir (RDV), a broad-spectrum antiviral agent, is often used together with dexamethasone (DEX) for hospitalized COVID-19 patients requiring respiratory support. Potential hepatic adverse drug reaction is a safety concern associated with the use of RDV. We previously reported that DEX cotreatment effectively mitigates RDV-induced hepatotoxicity and reduces elevated serum alanine aminotransferase and aspartate aminotransferase levels in cultured human primary hepatocytes (HPH) and hospitalized COVID-19 patients, respectively. Yet, the precise mechanism behind this protective drug-drug interaction remains largely unknown. Here, we show that through the activation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling, RDV induces apoptosis (cleavage of caspases 8, 9, and 3), autophagy (increased autophagosome and LC3-II), and mitochondrial damages (decreased membrane potential, respiration, ATP levels, and increased expression of Bax and the released cytosolic cytochrome C) in HPH. Importantly, cotreatment with DEX partially reversed RDV-induced apoptosis, autophagy, and cell death. Mechanistically, DEX deactivates/dephosphorylates p38, JNK, and ERK1/2 signaling by enhancing the expression of dual specificity protein phosphatase 1 (DUSP1), a mitogen-activated protein kinase (MAPK) phosphatase, in a glucocorticoid receptor (GR)-dependent manner. Knockdown of GR in HPH attenuates DEX-mediated DUSP1 induction, MAPK dephosphorylation, as well as protection against RDV-induced hepatotoxicity. Collectively, our findings suggest a molecular mechanism by which DEX modulates the GR-DUSP1-MAPK regulatory axis to alleviate the adverse actions of RDV in the liver. SIGNIFICANCE STATEMENT: The research uncovers the molecular mechanisms by which dexamethasone safeguards against remdesivir-associated liver damage in the context of COVID-19 treatment.

Overexpression of olfactory receptor 78 ameliorates brain injury in cerebral ischaemia-reperfusion rats by activating Prkaca-mediated cAMP/PKA-MAPK pathway.

Ischemic stroke is one of the main causes of disability and death. However, recanalization of occluded cerebral arteries is effective only within a very narrow time window. Therefore, it is particularly important to find neuroprotective biological targets for cerebral artery recanalization. Here, gene expression profiles of datasets GSE160500 and GSE97537 were downloaded from the GEO database, which were related to ischemic stroke in rats. Olfactory receptor 78 (Olfr78) was screened, and which highly associated with Calcium signalling pathway and MAPK pathway. Interacting protein of Olfr78, Prkaca, was predicted by STRING, and their interaction was validated by Co-IP analysis. Then, a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) and a neuronal cell model stimulated by oxygen-glucose deprivation/reoxygenation (OGD/R) were constructed, and the results showed that expression of Olfr78 and Prkaca was downregulated in MCAO rats and OGD/R-stimulated neurons. Overexpression of Olfr78 or Prkaca inhibited the secretion of inflammatory factors, Ca2+ overload, and OGD/R-induced neuronal apoptosis. Moreover, Overexpression of Prkaca increased protein levels of cAMP, PKA and phosphorylated p38 in OGD/R-stimulated neurons, while SB203580, a p38 inhibitor, treatment inhibited activation of the cAMP/PKA-MAPK pathway and counteracted the effect of Olfr78 overexpression on improvement of neuronal functions. Meanwhile, overexpression of Olfr78 or Prkaca markedly inhibited neuronal apoptosis and improved brain injury in MCAO/R rats. In conclusion, overexpression of Olfr78 inhibited Ca2+ overload and reduced neuronal apoptosis in MCAO/R rats by promoting Prkaca-mediated activation of the cAMP/PKA-MAPK pathway, thereby improving brain injury in cerebral ischaemia-reperfusion.

Unveiling the role of RAC3 in the growth and invasion of cisplatin-resistant bladder cancer cells.

Bladder cancer is one of the most prevalent cancers worldwide, and its morbidity and mortality rates have been increasing over the years. However, how RAC family small GTPase 3 (RAC3) affects the proliferation, migration and invasion of cisplatin-resistant bladder cancer cells remains unclear. Bioinformatics techniques were used to investigate the expression of RAC3 in bladder cancer tissues. Influences of RAC3 in the grade, stage, distant metastasis, and survival rate of bladder cancer were also examined. Analysis of the relationship between RAC3 expression and the immune microenvironment (TIME), genomic mutations, and stemness index. In normal bladder cancer cells (T24, 5637, and BIU-87) and cisplatin-resistant bladder cancer cells (BIU-87-DDP), the expression of RAC3 was detected separately with Western blotting. Plasmid transfection was used to overexpress or silence the expression of RAC3 in bladder cancer cells resistant to cisplatin (BIU-87-DDP). By adding activators and inhibitors, the activities of the JNK/MAPK signalling pathway were altered. Cell viability, invasion, and its level of apoptosis were measured in vitro using CCK-8, transwell, and flow cytometry. The bioinformatics analyses found RAC3 levels were elevated in bladder cancer tissues and were associated with a poor prognosis in bladder cancer. RAC3 in BIU-87-DDP cells expressed a higher level than normal bladder cancer cells. RAC3 overexpression promoted BIU-87-DDP proliferation. The growth of BIU-87-DDP cells slowed after the knockdown of RAC3, and RAC3 may have had an impact on the activation of the JNK/MAPK pathway.

CRKL but not CRKII contributes to hemin-induced erythroid differentiation of CML.

Destruction of erythropoiesis process leads to various diseases, including thrombocytopenia, anaemia, and leukaemia. miR-429-CT10 regulation of kinase-like (CRKL) axis involved in development, progression and metastasis of cancers. However, the exact role of miR-429-CRKL axis in leukaemic cell differentiation are still unknown. The current work aimed to uncover the effect of miR-429-CRKL axis on erythropoiesis. In the present study, CRKL upregulation was negatively correlated with miR-429 downregulation in both chronic myeloid leukaemia (CML) patient and CR patient samples. Moreover, CRKL expression level was significantly decreased while miR-429 expression level was increased during the erythroid differentiation of K562 cells following hemin treatment. Functional investigations revealed that overexpression and knockdown of CRKL was remarkably effective in suppressing and promoting hemin-induced erythroid differentiation of K562 cells, whereas, miR-429 exhibited opposite effects to CRKL. Mechanistically, miR-429 regulates erythroid differentiation of K562 cells by downregulating CRKL via selectively targeting CRKL-3'-untranslated region (UTR) through Raf/MEK/ERK pathway. Conversely, CRKII had no effect on erythroid differentiation of K562 cells. Taken together, our data demonstrated that CRKL (but not CRKII) and miR-429 contribute to development, progression and erythropoiesis of CML, miR-429-CRKL axis regulates erythropoiesis of K562 cells via Raf/MEK/ERK pathway, providing novel insights into effective diagnosis and therapy for CML patients.

Protein interaction network analysis of mTOR signaling reveals modular organization.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (phosphoinositide 3-kinases), protein kinases (AKT), and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex coimmunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum-deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after 1 h, despite phosphorylation changes observed after only 5 min. Using small molecule inhibitors of phosphoinositide 3-kinase, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed "modules", that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to the activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.