Target Protein Levels Research Articles

Expression of antibody-drug conjugates (ADC) biomarkers in colorectal cancer.

17 Background: Multiple ADCs are in clinical trials for CRC and the optimal strategy for selecting patients who may benefit from the treatment is evolving. Due to the unique mechanism of ADCs, patient selection should involve screening not only for the presence of the antibody target, but also for markers of resistance or response to the payload. We have built a multiplexed ADC biomarker panel in FFPE tumor tissue that simultaneously quantifies the protein levels of the antibody targets and also the payload markers. Methods: FFPE tumor tissues from 363 CRC patients were microdissected and solubilized for mass spectrometry-based targeted proteomic analysis in our CLIA certified laboratory. We quantified protein levels of EGFR, HER2, HER3, Axl, Mesothelin, FRalpha, Trop2 (antibody targets), tubulin-beta3 and TOPO1 (payload resistance and response markers, respectively) simultaneously. The multiplexed assay also quantified additional 22 clinically relevant proteins. Results: Expression of EGFR(83%), HER2(52%), HER3(21.5%), Axl(3.7%), Mesothelin(26.5%), FRalpha(3.7%), and Trop2(59.8%) may indicate likely response to ADCs. Expression of TUBB3(+) and TOPO1 (>1350amol/µg) in antibody target-positive subset may suggest resistance or response to payloads, such as taxanes and irinotecan, respectively (Table). Previously we identified that HER2 expression >750amol/µg correlated with HER2 positivity. Accordingly, 1.4% (5/355) of CRC patients were HER2 positive, of which 40% (2/5) had TOPO1 expression >1350amol/µg (75th percentile) suggesting that these 2 patients may receive benefit from a HER2/TOPO1 ADC. (+) indicates expression ≥LOQ; (-) indicates expression <LOQ. Conclusions: In patients with CRC, quantitative proteomics identified both antibody targets and markers of resistance or response to the payloads for multiple approved and investigational ADC therapies. [Table: see text]

First small-molecule PROTACs for G protein-coupled receptors: inducing α1A-adrenergic receptor degradation

Proteolysis targeting chimeras (PROTACs) are dual-functional hybrid molecules that can selectively recruit an E3 ubiquitin ligase to a target protein to direct the protein into the ubiquitin-proteasome system (UPS), thereby selectively reducing the target protein level by the ubiquitin-proteasome pathway. Nowadays, small-molecule PROTACs are gaining popularity as tools to degrade pathogenic protein. Herein, we present the first small-molecule PROTACs that can induce the α1A-adrenergic receptor (α1A-AR) degradation, which is also the first small-molecule PROTACs for G protein-coupled receptors (GPCRs) to our knowledge. These degradation inducers were developed through conjugation of known α1-adrenergic receptors (α1-ARs) inhibitor prazosin and cereblon (CRBN) ligand pomalidomide through the different linkers. The representative compound 9c is proved to inhibit the proliferation of PC-3 cells and result in tumor growth regression, which highlighted the potential of our study as a new therapeutic strategy for prostate cancer.

Temozolomide induces activation of Wnt/β-catenin signaling in glioma cells via PI3K/Akt pathway: implications in glioma therapy.

Glioblastoma (GBM) is the most aggressive type of glioma. Temozolomide (TMZ) is currently the drug of choice used for post-operative chemotherapy of GBM. However, the presence of intrinsic and acquired resistance hinders the success of chemotherapy. To understand the TMZ resistant mechanisms in glioma, we investigated the alterations in cellular signaling pathways by performing transcriptome analysis of TMZ treated glioma cells. Gene Set Enrichment Analysis (GSEA) indicated a significant enrichment of Wnt/β-catenin signaling besides many other pathways in TMZ treated cells. Further, we demonstrate that TMZ treatment increased the activity from TOPflash reporter, (a Wnt responsive reporter), enhanced the levels of pGSK-3β (S9) and reduced the levels of p-β-catenin (S33/37/T41) with a concomitant increase in transcript and protein levels of Wnt targets in a concentration and time-dependent manner. While TMZ treated cells did not show alteration in any of the Wnt ligands, PI3K inhibitor (LY294002) treatment repressed Akt activation and abolished the TMZ-mediated induction of Wnt/β-catenin pathway. In addition, we show that Wnt/β-catenin signaling activation by TMZ is independent of ATM/Chk2 pathway. Further, we also demonstrate the activation of mTOR pathway after TMZ treatment. Thus, our results demonstrate that activation of Wnt/β-catenin pathway involves an ATM/Chk2- independent PI3K/Akt/GSK-3 cascade in TMZ treated cells and further provides mechanistic basis for the chemoresistance of glioma to TMZ.

APOE-knockout in rabbits causes loss of cells in nucleus pulposus and enhances the levels of inflammatory catabolic cytokines damaging the intervertebral disc matrix.

Rabbits with naturally high levels of cholesterol ester transfer protein (CETP), unlike rodents, have become an interesting animal model for the study of lipid metabolism and atherosclerosis, as they have similarities to humans in lipid metabolism, cardiovascular physiology and susceptibility to develop atherosclerosis. Rodents, such as mice, are not prone to atherosclerosis as they lack the mass and activity of CETP, as a key player in lipoprotein metabolism. Recently, APOE-knockout in rabbits has been shown to promote atherosclerosis and associated premature IVD degeneration that mimic the symptoms of atherosclerosis and structural changes of IVDs in humans. Here we examined whether APOE-knockout promoted IVD degeneration in rabbits is associated with imbalanced inflammatory catabolic activities, as the underlying problem of biological deterioration that mimic the symptoms of advanced IVD degeneration in humans. We analysed in lumbar nucleus pulposus (NP) of APOE-knockout rabbits the cell viabilities and the intracellular levels of inflammatory, catabolic, anti-catabolic and anabolic proteins derogating IVD matrix. Grades of IVD degeneration were evaluated by magnetic resonance imaging. NP cells were isolated from homozygous APOE-knockout and wild-type New Zealand White rabbits of similar age. Three-dimensional cell culture with low-glucose was completed in alginate hydrogel. Cell proliferation and intracellular levels of target proteins were examined by MTT and ELISA assays. Alike human NP cells of different disc degeneration grades, NP cells of APOE-knockout and wild-type rabbits showed significantly different in vivo cell population densities (p<0.0001) and similar in vitro proliferation rates. Furthermore, they showed differences in overexpression of selective inflammatory and catabolic proteins (p<0.0001) similar to those found in human NP cells of different disc degeneration grades, such as IL-1β, TNF-α, ADAMTS-4, ADAMTS-5 and MMP-3. This study showed that premature IVD degeneration in APOE-knockout rabbits was promoted by the accumulation of selective inflammatory catabolic factors that enhanced imbalances between catabolic and anabolic factors mimicking the symptoms of advanced IVD degeneration in humans. Thus, APOE-knockout rabbits could be used as a promising model for therapeutic approaches of degenerative disc disorders.

Combining CDK2/9 Inhibitor CYC065 with Venetoclax, a BCL2 Inhibitor, to Treat Patients with Relapsed or Refractory AML or MDS

Cyclin dependent kinases (CDKs) are critical for cell cycle regulation and transcriptional elongation. Dysregulated CDKs have been linked to the cancer hallmarks of uncontrolled proliferation and increased survival. CYC065 is a potent and orally‐available inhibitor of CDK2 and CDK9. CDK9 regulates transcription of genes through phosphorylation of RNA polymerase II (RNAP II) C-terminal domain (CTD). Through inhibition of CDK9, CYC065 suppresses CDK9-dependent gene expression and reduces the level of MCL1, a key anti-apoptotic protein. In the first-in-human study, CYC065 was administered by 4-hour infusion every 3 weeks in patients with advanced cancers. Biomarkers related to CYC065 target inhibition, e.g. phosphorylation of RNAP II CTD Ser2, a direct substrate of CDK9, and protein levels of downstream targets, such as MCL1, were determined in patient's peripheral blood mononuclear cells (PBMCs). Durable MCL1 suppression was observed after a single dose in 11 out of 13 patients treated at the recommended phase 2 dose (RP2D) of 192 mg/m2. Five of these 13 patients achieved stable disease lasting ≥ 6 cycles (Do, KT et al, AACR Annual Meeting 2018 Abs CT037). Acute myeloid leukemia (AML) frequently relapses after initial treatment by intensive or low-intensive therapy. Drug resistance has been attributed to dysregulation in apoptotic pathways. AML cells often upregulate pro-survival members of the BCL2 family, such as BCL2 and MCL1, to avoid apoptosis (Grundy M et al, Oncotarget, 2018). Suppression of MCL1 triggered rapid apoptosis in AML, and cured AML-afflicted mice (Glaser SP et al, Genes Dev. 2012). Preclinically, CYC065 has demonstrated potent anti-tumor effect in various AML cell lines, including those with MLL rearrangements, and xenograft models (Frame S et al, AACR, 2010 Abs 3886; Frame S et al, SOHO, 2014 Abs 209). Venetoclax has modest single agent activity in AML. MCL1 dependence appeared to correlate with resistance to venetoclax (Konopleva M et al, Cancer Discovery, 2016). Preclinical study confirmed synergy of CYC065 and venetoclax, suggesting that the suppression of both BCL2 and MCL1 may be more beneficial than inhibiting either one alone (MacKay C et al. AACR-NCI-EORTC 2015 Abs B182). Based on the above rationale, a clinical study (NCT04017546) has been initiated to evaluate a combination of CYC065 with venetoclax in relapsed/refractory AML and MDS. CYC065 will be administered intravenously via 4-hour infusion on Day 1 and Day 15 in combination with daily venetoclax every 4 weeks. Initial dose escalation is 33% and then 25% upon occurrence of the first dose limiting toxicity (DLT). RP2D is the highest dose level at which less than one-third of at least 6 patients experience a DLT during the first treatment cycle. Eligible patients are ≥18 years with previously treated AML or MDS and ≥10% blasts in bone marrow or peripheral blood; adequate bone marrow, renal and liver functions are required. All patients will be asked to participate in the pharmacokinetic and pharmacodynamic studies. Plasma levels of CYC065 and its metabolites as well as venetoclax will be determined. PBMCs will be collected to assess MCL1 levels and the phosphorylation and protein levels of other downstream targets of CDK9 inhibition. Treatment will continue until progression of disease, unacceptable toxicity or changes in patient condition that renders patients ineligible for further treatment. Laboratory tests and bone marrow aspirate/biopsy will be performed to assess response according to standard criteria. Disclosures Borthakur: AbbVie: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; GSK: Research Funding; Polaris: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Incyte: Research Funding; Cyclacel: Research Funding; Janssen: Research Funding; Bayer Healthcare AG: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Oncoceutics, Inc.: Research Funding; Strategia Therapeutics: Research Funding; PTC Therapeutics: Consultancy; BMS: Research Funding; Oncoceutics: Research Funding; Eisai: Research Funding; NKarta: Consultancy; Xbiotech USA: Research Funding; Cantargia AB: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Eli Lilly and Co.: Research Funding; Agensys: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Arvinas: Research Funding. Kadia:BMS: Research Funding; Bioline RX: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees. Al Azzawi:Cyclacel LTD: Research Funding. Zheleva:Cyclacel Ltd: Employment, Equity Ownership, Patents &amp; Royalties. Blake:Cyclacel Ltd: Employment, Equity Ownership, Patents &amp; Royalties. Chiao:Cyclacel Ltd: Employment, Equity Ownership, Patents &amp; Royalties.

A Phase I Study Combining CDK2/9 Inhibitor CYC065 with Venetoclax, a BCL2 Inhibitor, to Treat Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL)

Cyclin dependent kinases (CDKs) are critical for cell cycle regulation and transcriptional elongation. Dysregulated CDKs have been linked to the cancer hallmarks of uncontrolled proliferation and increased survival. CYC065 is a potent and orally‐available inhibitor of CDK2 and CDK9. CDK9 regulates transcription of genes through phosphorylation of RNA polymerase II (RNAP II) C-terminal domain (CTD). Through inhibition of CDK9, CYC065 suppresses CDK9-dependent gene expression and reduces the level of MCL1, a key anti-apoptotic protein. In the first-in-human study, CYC065 was administered by 4-hour infusion every 3 weeks in patients with advanced solid cancers. Biomarkers related to CYC065 target inhibition, e.g. phosphorylation of RNAP II CTD Ser2, a direct substrate of CDK9, and protein levels of downstream targets, such as MCL1, were determined in patient peripheral blood mononuclear cells (PBMCs). Durable MCL1 suppression was observed after a single dose in 11 out of 13 patients treated at the recommended phase 2 dose (RP2D) of 192 mg/m2. Five of these 13 patients achieved stable disease lasting ≥6 cycles (Do, KT et al, AACR Annual Meeting 2018 Abs CT037). Chronic lymphocytic leukemia (CLL) is a disease that depends upon the overexpression of anti-apoptotic proteins (MCL1, BCL2) for survival. Venetoclax, a BCL2 inhibitor, is one of the most active agents against CLL. However upregulation of MCL1 is associated with resistance to venetoclax (Oppermann S et al., Blood, 2016). Preclinical studies have demonstrated that CYC065 and venetoclax are synergistic in primary CLL cells, inducing apoptosis (Chen R et al, AACR Annual Meeting 2018 Abs 3905). This synergy may arise from targeting parallel anti-apoptotic mechanisms in the intrinsic apoptotic pathway that promote survival in CLL cells. The above findings support the conduct of an ongoing clinical study (NCT03739554) investigating a combination of CYC065 with venetoclax in CLL. CYC065 will be administered intravenously via 4-hour infusion on Day 1 and Day 15 in combination with daily venetoclax. One cycle is 28 days. Initial dose escalation is 33% and then 25% upon occurrence of the first dose limiting toxicity (DLT). RP2D is the highest dose level at which less than one-third of at least 6 patients experience a DLT during the first treatment cycle. Eligible patients are ≥18 years with CLL relapsed or refractory to standard treatment(s) including those with Richter transformation; adequate bone marrow, renal and liver functions are required. All patients will be asked to participate in the pharmacokinetic and pharmacodynamic studies. Plasma levels of CYC065 and its metabolites as well as venetoclax will be determined. PBMCs will be collected to assess MCL1 levels and phosphorylation and protein levels of other downstream targets of CDK9 inhibition. Treatment will continue until progression of disease, unacceptable toxicity or changes in patient condition that renders patients ineligible for further treatment. Laboratory tests and CT scans will be performed regularly to assess response according to standard criteria. Disclosures Wierda: Pharmacyclics LLC: Research Funding; AbbVie: Research Funding; GSK/Novartis: Research Funding; Gilead Sciences: Research Funding; KITE pharma: Research Funding; Sunesis: Research Funding; Genentech: Research Funding; Xencor: Research Funding; Janssen: Research Funding; Loxo Oncology Inc.: Research Funding; Cyclcel: Research Funding; Oncternal Therapeutics Inc.: Research Funding; Miragen: Research Funding; Juno Therapeutics: Research Funding; Acerta Pharma Inc: Research Funding. Chen:Cyclacel Ltd: Research Funding. Zheleva:Cyclacel Ltd: Employment, Equity Ownership, Patents &amp; Royalties. Blake:Cyclacel Ltd: Employment, Equity Ownership, Patents &amp; Royalties. Chiao:Cyclacel Ltd: Employment, Equity Ownership, Patents &amp; Royalties. Plunkett:Cyclacel Ltd: Research Funding.

Bioavailable Dual-Protein Degraders of CK1α and Transcriptional Kinase CDK9 As Potential Therapeutics for Hematological Malignancies

BioTheryx's small-molecule kinase inhibitor, BTX-A51 (the ditosylated salt of A51), has recently received FDA approval of its IND application to initiate a Phase I clinical trial in relapsed or refractory acute myeloid leukemia (AML). A51 (API of BTX-A51) is a multi-kinase inhibitor that blocks the leukemic stem cell target, Casein Kinase 1α (CK1α), as well as the super-enhancer regulator, Cyclin-Dependent Kinase 9 (CDK9), thus preventing the transcription of key oncogenic genes. This molecule has demonstrated remarkable preclinical animal efficacy inferring the eradication of AML stem cells and its potential for use in treating multiple malignancies. To exploit the unique properties of this multi-kinase inhibitor in the context of kinase protein degradation, Proteolysis-Targeting Chimeras (PROTACs) of A51 were investigated. PROTACs utilize the cell's natural ubiquitin-proteasome system to induce the selective and sustained degradation of unwanted disease-causing proteins. PROTACs are heterobifunctional molecules which are comprised of an E3-ubiquitin-ligase ligand which is covalently linked to a target-protein ligand. Through concomitant binding to an E3 ligase and to a target protein, a PROTAC promotes ubiquitination and ultimately degradation of the target protein via the proteasome. As a reversible kinase inhibitor, A51 binds stoichiometrically to CK1α and CDK9 and requires continuous occupancy of these proteins to sustain its unique inhibitory activity. A PROTAC of A51, however, would act catalytically and ablate the target proteins. In this study, PROTACs were assembled through chemically linking A51 to BioTheryx's proprietary Protein Homeostatic Modulators (PHMs™). PHMs™ are a new class of small molecules which bind to the E3 ubiquitin ligase, Cereblon (CRBN), and promote proteasomal degradation of known as well as unreported clinically-relevant CRBN neo-substrates. For the discovery of PHM®-A51 PROTACs, CDK9 crystal structure (6GZD.pdb) and CRBN crystal structure (5FQD.pdb) were used independently for computational drug design. Upon testing in AML and lymphoma cell lines, the PHM®-A51 PROTACs rapidly induced the degradation of both CK1α and CDK9. Additionally, these PROTACs had diminished effect on the protein levels of other A51 targets while inducing the degradation of other desirable targets unaffected by A51. This distinct selectivity arises from the PROTACs' ability to facilitate formation of a ternary complex that enables subsequent ubiquitination of the bound target protein - a selectivity not inherent to any kinase inhibitor. Further, the PHM®-A51 PROTACs have demonstrated low-nanomolar inhibition of cell proliferation in both AML and lymphoma cell lines yet have significantly less toxicity in fibroblast cells and PBMCs. In addition, this class of PROTACs has good pharmaceutical properties, as demonstrated by pharmacokinetic studies in mice, and will advance into AML and lymphoma in vivo studies. The targeted degradation of CK1α and CDK9 using safe, bioavailable drugs designed using strategically-selected PHMs™ with unique biological properties of their own is a very promising approach to treating hematological malignancies and other devasting cancers. Disclosures No relevant conflicts of interest to declare.

CARF, As An Oncogene, Promotes Colorectal Cancer Stemness By Activating ERBB Signaling Pathway.

IntroductionThe role of CARF, a calcium-responsive transcription factor, in colorectal cancer initiation and development is still unknown. Here, we report that CARF promotes colorectal cancer stemness through ERBB signaling pathway.Materials and methodsBoth colorectal cancer cell lines and primary cells were used in this study. The levels of target mRNA and protein in the cells were examined by qRT-PCR and Western blot. Gene manipulation was achieved by the lentivirus delivery system. Luciferase reporter gene assay was employed to analyze the transcriptional activity of the promoter. ChIP assay was performed for the examination of the binding between CARF and the promoters of MAPK8 and JUN. Kaplan-Meier survival curve was generated by the R2 program. Correlation analysis was performed using Spearman correlation analysis.ResultsAberrant upregulation of CARF has been found in tumor tissues of colorectal cancer patients and associated with poor prognosis. Ectopic expression of CARF promoted the sphere-formation activities, as well as the expression of stem cell markers in colorectal cancer cells and knockdown of CARF, inhibited these activities. The mechanistic analysis showed that CARF directly binds to the promoter of MAPK8 and JUN, promotes the expression of MAPK8 and JUN, activates the ERBB signaling pathway, and thereby promotes the maintenance of the stemness in colorectal cancer cells.ConclusionCARF, as an oncogene, promotes colorectal cancer stemness by activating ERBB signaling pathway. The ERBB signaling pathway that serves as the main downstream effector of CARF could be an efficient drug target for colorectal cancer caused by aberrant expression of CARF.

Pseudoprotodioscin inhibits SREBPs and microRNA 33a/b levels and reduces the gene expression regarding the synthesis of cholesterol and triglycerides

The extract of Dioscorea zingiberensis C.H. Wright rhizomes is found to be effective in the therapy of cardiovascular disease. Steroidal saponins make substantial contribution. Previous study has proposed that methylprotodioscin (MP) may promote cholesterol efflux by increasing ABCA1 expression. But the other main saponins ingredients are not referred to. The aim of the present work was to reveal the effect and mechanism of protodioscin (PD), MP and pseudoprotodioscin (PPD) on the synthesis-related gene expression of cholesterol and triglycerides. MTT assay apoptosis assay with annexin AV-APC and 7-AAD double staining were performed. MicroRNA assay and qRT-PCR were used to analyze the gene expression which regulates synthesis of cholesterol and triglycerides. Western blot was to demonstrate the levels of target proteins. Cholesterol efflux assay was executed to study the stimulative effect of saponins on cholesterol efflux. In Hep G2 cells, PPD increased ABCA1 protein and mRNA levels, and promoted the effluxion of ApoA-1-mediated cholesterol. The underlying mechanisms involved that PPD inhibited SREBP1c and SREBP2 transcription by decreasing microRNA 33a/b levels. This procedure reciprocally led to the increase of ABCA1 levels. In THP-1 macrophages, PPD showed the similar effect, which reduced HMGCR, FAS and ACC mRNA levels and promoted low density lipoprotein receptor by decreasing the PCSK9 levels. These studies demonstrated that PPD is a potential agent for cholesterol efflux, SREBPs and microRNA 33a/b inhibition, which related to the gene expression for the synthesis of cholesterol and triglycerides.

RNF20 Functions as a Transcriptional Coactivator for PPARγ by Promoting NCoR1 Degradation in Adipocytes.

Adipose tissue is the key organ coordinating whole-body energy homeostasis. Although it has been reported that ring finger protein 20 (RNF20) regulates lipid metabolism in the liver and kidney, the roles of RNF20 in adipose tissue have not been explored. Here, we demonstrate that RNF20 promotes adipogenesis by potentiating the transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ). Under normal chow diet feeding, Rnf20 defective (Rnf20 +/- ) mice exhibited reduced fat mass with smaller adipocytes compared with wild-type littermates. In addition, high-fat diet-fed Rnf20 +/- mice alleviated systemic insulin resistance accompanied by a reduced expansion of fat tissue. Quantitative proteomic analyses revealed significantly decreased levels of PPARγ target proteins in adipose tissue of Rnf20 +/- mice. Mechanistically, RNF20 promoted proteasomal degradation of nuclear corepressor 1 (NCoR1), which led to stimulation of the transcriptional activity of PPARγ. Collectively, these data suggest that RNF20-NCoR1 is a novel axis in adipocyte biology through fine-tuning the transcriptional activity of PPARγ.

MicroRNA-145 Protects against Myocardial Ischemia Reperfusion Injury via CaMKII-Mediated Antiapoptotic and Anti-Inflammatory Pathways.

MicroRNA-145 (miR-145) has been shown to play an important role in cardiovascular system disorders; however, the underlying mechanism is not completely understood. The purpose of this study was aimed at elucidating the cardioprotective effects of miR-145 against myocardial ischemia/reperfusion (I/R) injury. We established a rat myocardial I/R model with 45 min left anterior descending coronary artery (LAD) occlusion and 2 h reperfusion. The levels of myocardial enzymes, apoptotic, inflammatory, and oxidative indices were determined. The arrhythmia score was assessed by programmed electrical stimulation (PES). Quantitative real-time PCR and western blot were applied to evaluate the expression levels of miR-145 and related target proteins, respectively. I/R injury decreased the expression of miR-145; however, upregulated miR-145 markedly reduced the elevation of ST segment, decreased corrected QT (QTc) intervals, and attenuated I/R-induced electrophysiological instability. Furthermore, miR-145 suppressed myocardium apoptotic, inflammatory, and oxidative response as well as the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), ryanodine receptor2 (RyR2 Ser2814), apoptosis signal-regulating kinase 1 (ASK1), c-Jun NH2-terminal kinases (JNK), and nuclear translocation of nuclear factor kappa-B (NF-κB) p65. In summary, overexpression of miR-145 alleviates I/R-induced myocardial electrophysiological instability and apoptotic and inflammatory response via inhibition of the CaMKII-mediated ASK1 antiapoptotic pathway and NF-κB p65 anti-inflammatory pathways.

Small RNA driven feed-forward loop: critical role of sRNA in noise filtering

Gene regulatory networks are often partitioned into different types of recurring network motifs. A feed-forward loop (FFL) is a common motif in which an upstream regulator is a protein, typically a transcription factor, that regulates the expression of the target protein in two ways—first, directly by regulating the mRNA levels of the target protein and second, indirectly via an intermediate molecule that in turn regulates the target protein level. Investigations on two variants of FFL—purely transcriptional FFL (tFFL) and sRNA-mediated FFL (smFFL) reveal several advantages of using such motifs. Here, we study a distinct sRNA-driven FFL (sFFL) that was discovered recently in Salmonella enterica: the distinction being the upstream regulator here is not a protein but an sRNA that translationally activates the target protein expression directly; and also indirectly via regulation of the transcriptional activator of the target protein. This variant, i.e. sFFL has not been subjected to rigorous analysis. We, therefore, set out to understand two aspects. First is a quantitative comparison of the regulatory response of sFFL with tFFL and smFFL using a differential equation framework. Since the process of gene expression is inherently stochastic, the second objective is to find how noise in gene expression affects the functionality of the sFFL. We find that unlike for tFFL and smFFL, the response of sFFL is stronger and faster: the change in target protein concentration is rapid and depends critically on the initial concentration of sRNA. Further, our analysis based on generating function approach and stochastic simulations leads to a non-trivial prediction that an optimal noise filtration can be attained depending on the synthesis rate of the upstream sRNA and the degradation rate of the intermediate transcriptional activator. A comparison with a simpler process involving only translational activation by sRNA indicates that the design of sFFL is crucial for optimal noise filtration. These observations prompt us to conclude that sFFL has distinct advantages where the master regulator, sRNA, plays a critical role not only in driving a rapid and strong response, but also a reliable response that depends critically on its concentration.

Knockdown of spliceosome U2AF1 significantly inhibits the development of human erythroid cells.

U2AF1 (U2AF35) is the small subunit of the U2 auxiliary factor (U2AF) that constitutes the U2 snRNP (small nuclear ribonucleoproteins) of the spliceosome. Here, we examined the function of U2AF1 in human erythropoiesis. First, we examined the expression of U2AF1 during in vitro human erythropoiesis and showed that U2AF1 was highly expressed in the erythroid progenitor burst‐forming‐unit erythroid (BFU‐E) cell stage. A colony assay revealed that U2AF1 knockdown cells failed to form BFU‐E and colony‐forming‐unit erythroid (CFU‐E) colonies. Our results further showed that knockdown of U2AF1 significantly inhibited cell growth and induced apoptosis in erythropoiesis. Additionally, knockdown of U2AF1 also delayed terminal erythroid differentiation. To explore the molecular basis of the impaired function of erythroid development, RNA‐seq was performed and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results showed that several biological pathways, including the p53 signalling pathway, MAPK signalling pathway and haematopoietic cell lineage, were involved, with the p53 signalling pathway showing the greatest involvement. Western blot analysis revealed an increase in the protein levels of downstream targets of p53 following U2AF1 knockdown. The data further showed that depletion of U2AF1 altered alternatively spliced apoptosis‐associated gene transcripts in CFU‐E cells. Our findings elucidate the role of U2AF1 in human erythropoiesis and reveal the underlying mechanisms.

Matrix Metalloproteinases System and Types of Fibrosis in Rat Heart during Late Pregnancy and Postpartum.

Background and objectives: Cardiac remodeling in pregnancy and postpartum is poorly understood. The aim of this study was to evaluate changes in cardiac fibrosis (pericardial, perivascular, and interstitial), as well as the expression of matrix metalloproteinases (MMP-1, MMP-2, and MMP-9) and their inhibitors (Tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-4) during late pregnancy and postpartum in rat left ventricle. Materials and Methods: Female Sprague–Dawley rats were used for this study. Rats were divided three groups: non-pregnant, late pregnancy, and postpartum. The heart was weighed and cardiac fibrosis was studied by conventional histological procedures. The expression and transcript level of target proteins were evaluated using immunoblot techniques and quantitative PCR. Results: The experiments showed an increase of perivascular, pericardial, and interstitial fibrosis in heart during pregnancy and its reversion in postpartum. Moreover, in late pregnancy, MMP-1, MMP-2, and MMP-9 metalloproteinases were downregulated and TIMP-1 and TIMP-4 were upregulated in left ventricle. Conclusions: Our data suggest that the metalloproteinases system is involved in the cardiac extracellular matrix remodeling during pregnancy and its reversion in postpartum, this improves the knowledge of the adaptive cardiac remodeling in response to a blood volume overload present during pregnancy.

ADRB2 suppresses IL-13-induced allergic rhinitis inflammatory cytokine regulated by miR-15a-5p.

Allergic rhinitis (AR) is a common hypersensitive disease that troubles patients a lot. Nasal epithelial cells (NECs), as the outmost protection of inhalation, play an important role in AR allergic response. Adrenoceptor beta 2 (ADRB2) is an important gene in inflammatory response, which has become the hot spot for AR development and treatment in recent years. MiR-15a-5p has been proved to be involved in AR immune response as the upstream regulator of ADRB2. Human primary NECs were isolated and stimulated by IL-13. qRT-PCR assay was used to detect the RNA level of target genes. ELISA and Western blotting were applied to detect target protein levels. Luciferase reporter assay and biotin pull-down assay were performed to test molecules interaction. ADRB2 was highly expressed in nasal mucosa of AR patients and was positively correlated with IL-13 stimulation, and knockdown of ADRB2 inhibited IL-13-induced expression of GM-CSF, eotaxin, and MUC5AC in NECs. ADRB2 was directly targeted by miR-15a-5p, and miR-15a-5p inhibited IL-13-induced expression of GM-CSF, eotaxin, and MUC5AC in NECs. ADRB2 mediated the effect of miR-15a-5p on the regulation of nasal epithelial immune responses. ADRB2 is negatively regulated by miR-15a-5p, which inhibits IL-13-induced nasal epithelial inflammatory responses.

MicroRNA-124 regulates TRAF6 expression and functions as an independent prognostic factor in colorectal cancer.

An increasing number of studies have confirmed that miR-124 exhibits a suppressive role in glioblastoma, cervical cancer and breast cancer; however, the function of miR-124 in colorectal cancer (CRC) has not been completely elucidated. In the present study, miR-124 expression was confirmed by reverse transcription-quantitative PCR in 80 colorectal tissues and para-cancerous tissues. The influence of altered miR-124 expression was analyzed by statistical approaches including Cox multivariate regression analysis and the Kaplan-Meier method, and the target genes of miR-124 were confirmed by luciferase reporter assays. Immunohistochemical techniques were also performed in order to measure the expression levels of target proteins. miR-124 expression was observed to be decreased in colorectal tissue samples, and this phenomenon was correlated with adverse clinical indicators and poor patient survival time. Luciferase reporter assays indicated that miR-124 directly regulated TNF receptor associated factor 6 (TRAF6) 3′-untranslated region (UTR). Hence, it was proposed that miR-124 dysregulation may negatively influence the expression of TRAF6 and therefore serve as a biomarker of epithelial-mesenchymal transition in CRC tissues. In summary, the present study demonstrated that miR-124 regulates the expression of TRAF6, and may potentially function as an independent prognostic factor and therapeutic target in patients with CRC.

A novel flow cytometry-based method to characterize transfection efficiency in genetically engineered mesenchymal stem cells

Background & Aim Introduction Mesenchymal stem cells (MSCs) are a major type of cell used in regenerative medicine, and benefit of MSC therapy may be enhanced via delivery of a nucleic acid-based vehicle into MSC to overexpress a given therapeutic protein. A typical method to determine the efficiency of gene delivery/protein expression involves transfection of a separate plasmid encoding for a fluorescent protein (such as GFP), as the percentage of cells expressing the GFP can subsequently be quantified using fluorescence microscopy or flow cytometry. Herein, we describe a novel method to assess transfection efficiency without the use of a different plasmid, while allowing for simultaneous quantification on the levels of (1) introduction of the actual plasmid into cells, and (2) target proteins expressed. Methods, Results & Conclusion Methods & Results Two different DNA vectors (plasmid A vs. B), both encoding the same protein of interest, were chemically labeled with fluorescent probes prior to being transfected into MSCs. Twenty-four hours post-transfection, MSCs were stained with a target protein-specific antibody. We analyzed both the expression levels of the protein and labeled vector content by traditional and imaging flow cytometry. Optimization was performed to determine the ideal fluorescent label to vector DNA ratio to be 1:10, allowing the labeled plasmid-transfected MSCs to express similar target protein levels as with unlabeled vector. Assessed by traditional flow cytometry (Fortessa), the transfection efficiencies were different between plasmid A (labeled plasmid: 36.5%, intracellular target protein: 1.46%) and B (labeled plasmid: 49.6%, intracellular target protein: 12.5%). These differences corresponded with secreted target protein production measured by ELISA at 818 or 55472 pg/ml, respectively. Visualization of the transfected MSCs that contain vector and successfully produce the target protein can be further analyzed in single-cell and population levels via an imaging flow cytometer (Amnis). Interestingly, a population of cells appeared to be positive for the labeled vector, but negative for protein expression (35.3-57.7%), suggesting a mismatch between plasmid transfected into the cells vs. actual protein expressed. Conclusion The technique described has several advantages over the conventional method for assessing transfection efficiency and could be used as a quality control protocol for evaluating any genetically enhanced cell therapies.

Re-introducing non-optimal synonymous codons into codon-optimized constructs enhances soluble recovery of recombinant proteins from Escherichia coli.

Gene synthesis services have largely superseded traditional PCR methods for the generation of cDNAs destined for bacterial expression vectors. This, in turn, has increased the application of codon-optimized cDNAs where codons rarely used by Escherchia coli are replaced with common synonymous codons to accelerate translation of the target. A markedly accelerated rate of expression often results in a significant uplift in the levels of target protein but a substantial proportion of the enhanced yield can partition to the insoluble fraction rendering a significant portion of the gains unavailable for native purification. We have assessed several expression attenuation strategies for their utility in the manipulation of the soluble fraction towards higher levels of soluble target recovery from codon optimized systems. Using a set of human small GTPases as a case study, we compare the degeneration of the T7 promoter sequence, the use of alternative translational start codons and the manipulation of synonymous codon usage. Degeneration of both the T7 promoter and the translational start codon merely depressed overall expression and did not increase the percentage of product recovered in native purification of the soluble fraction. However, the selective introduction of rare non-optimal codons back into the codon-optimized sequence resulted in significantly elevated recovery of soluble targets. We propose that slowing the rate of extension during translation using a small number of rare codons allows more time for the co-translational folding of the nascent polypeptide. This increases the proportion of the target recovered in the soluble fraction by immobilized metal affinity chromatography (IMAC). Thus, a “de-optimization” of codon-optimized cDNAs, to attenuate or pause the translation process, may prove a useful strategy for improved recombinant protein production.

Altered expression patterns of complement factor H and miR-146a genes in acute-chronic phases in experimental autoimmune encephalomyelitis mouse.

Considerable advances have been made in identification of the involvement of immune modulators in diseases. There is growing evidence on the role of complement pathway in pathogenesis and course of multiple sclerosis (MS). Moreover, it has been recognized that microRNAs (miRNAs) play an essential role in modulation and development of immune response in the central nervous system. We aimed to investigate the expression profile of complement factor H (CFH) and miR-146a genes in experimental autoimmune encephalomyelitis (EAE) mouse model of MS to detect the possible roles of CFH and miR-146a as biomarkers of MS disease stats. Expression of CFH and miR-146a genes in liver and brain tissues of EAE mice was measured in acute and chronic phases of disease compared to matched controls using real-time polymerase chain reaction. In the liver, increased expression of CFH gene was observed in the chronic phase compared to the acute phase. However, no significant difference was observed between acute and chronic phase mice with normal mice, while miR-146a expression was significantly decreased in livers of EAE mice in chronic group compared to acute and control groups. The expression of CFH gene in brain had a significant decrease in acute and chronic phases compared to healthy mice. Taken together, these observations indicate probable implication of complement system and miR-146a in course of immune-related diseases and reveal more facts about the pathogenesis of MS. However, further work is needed to determine protein levels of CFH and other possible targets of miR-146a in serum and cerebrospinal fluid of MS patients.

Mir‐96 Expression in non‐IPF Fibroblasts Changes their Phenotype from Normal to Fibrotic via FoxO3a Suppression

RationaleIdiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease that the precise etiology is unknown. IPF is caused by the hyper‐activation of apoptosis‐resistant (myo)fibroblasts that produce excessive amounts of collagen rich extracellular matrix (ECM). When normal lung fibroblasts interact with a collagen matrix, they undergo apoptosis, which prevents excessive fibro‐proliferation after tissue injury. However, fibroblasts from IPF patients (IPF fibroblasts) elude this negative regulatory property of collagen, promoting lung fibrosis. However, the crucial mechanism that protects IPF fibroblasts from collagen matrix‐driven apoptosis has not been understood.Study objectiveWe found that Forkhead box O3a (FoxO3a) that promotes apoptosis was suppressed as a result of abnormally high non‐coding RNA miR‐ 96 in IPF fibroblasts, and that this deregulation caused them to maintain a highly viable phenotype in response to a collagen rich matrix. Based on this finding, our study objective was to examine whether miR‐96‐dependent FoxO3a alteration can reprogram non‐IPF lung fibroblasts to be fibrotic, thereby subsequently participating in promoting lung fibrosis. For this, we altered miR‐96 levels in non‐ IPF fibroblasts and investigated FoxO3a levels, fibroblast viability and proliferation on an apoptosis‐inducing 3D collagen rich ECM.ResultsForced expression of miR‐96 in non‐IPF fibroblasts by miR‐96 mimic suppressed FoxO3a and its target proteins Bim, p27 and p21, which caused them to acquire apoptosis‐resistant and proliferative properties that were mainly seen in IPF fibroblasts on collagen. Unlike these, expression of miR‐96 in IPF fibroblasts had no significant effects on fibroblast viability and FoxO3a target protein levels potentially due to pre‐existing enhanced miR‐96. In contrast, miR‐ 96 inhibition greatly sensitized IPF fibroblasts to collagen matrix‐mediated apoptosis. To further test the pathological role of miR‐96‐dependent FoxO3a alteration in the development of lung fibrosis, miR‐96 and FoxO3a levels were also examined in C57/BL6 mice (n=8) with lung fibrosis. Real time PCR and Western analysis showed that miR‐96 levels were significantly increased while FoxO3a levels were reduced in fibrotic mouse lungs compared with that of normal mice. In accordance with this finding, miR‐96 levels were also increased while FoxO3a levels were low in lung tissues (n=4) from IPF patients.ConclusionsOur findings suggest that miR‐96 modulation reprograms non‐IPF fibroblast phenotype from normal to fibrotic via FoxO3a alteration, and that the presence of miR‐96 activated fibroblasts plays a role in promoting lung fibrosis via FoxO3a suppression. These results indicate that targeting miR‐96 can be an effective approach to mitigate the development of lung fibrosis.Support or Funding InformationThis abstract is funded by the National Heart, Lung and Blood Institute Grant R01 HL114662 (Richard Nho).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.