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The hematopoietic neoplasm chronic myeloid leukemia (CML) is a rare disease caused by chromosomal reciprocal translocation t(9;22)(q34:q11) with subsequent formation of the BCR-ABL1 fusion gene. This fusion gene encodes a constitutively active tyrosine kinase, which results in malignant transformation of the cells. Since 2001, CML can be effectively treated using tyrosine kinase inhibitors (TKIs) such as imatinib, which prevent phosphorylation of downstream targets by blockade of the BCR-ABL kinase. Due to its tremendous success, this treatment became the role model of targeted therapy in precision oncology. Here, we review the mechanisms of TKI resistance focusing on BCR-ABL1-dependent and -independent mechanisms. These include the genomics of the BCR-ABL1, TKI metabolism and transport and alternative signaling pathways.
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Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Medicina de Precisão , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genéticaRESUMO
Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.
Assuntos
Glioblastoma , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Carmustina/farmacologia , Carmustina/uso terapêutico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Lomustina/uso terapêutico , Lomustina/farmacologia , Sistemas CRISPR-Cas , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismoRESUMO
Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.
Assuntos
Anti-Inflamatórios/farmacologia , Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Mercaptopurina/farmacologia , Neurônios/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Animais , Células Cultivadas , Sistema Nervoso Entérico/citologia , Inflamação/induzido quimicamente , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Lipopolissacarídeos , Camundongos , Ratos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Preparations of Echinacea purpurea (E. purpurea) are widely used for the management of upper respiratory infections, influenza, and common cold, often in combination with other conventional drugs. However, the potential of phytochemical constituents of E. purpurea to cause herb-drug interactions via ABCB1 and ABCG2 efflux transporters remains elusive. The purpose of this study was to investigate the impact of E. purpurea-derived caffeic acid derivatives (cichoric acid and echinacoside) and tetraenes on the mRNA and protein expression levels as well as on transport activity of ABCB1 and ABCG2 in intestinal (Caco-2) and liver (HepG2) cell line models. The safety of these compounds was investigated by estimating EC20 values of cell viability assays in both cell lines. Regulation of ABCB1 and ABCG2 protein in these cell lines were analyzed after 24 h exposure to the compounds at 1, 10, and 50 µg/mL. Bidirectional transport of 0.5 µg/mL Hoechst 33342 and 5 µM rhodamine across Caco-2 monolayer and profiling for intracellular concentrations of the fluorophores in both cell lines were conducted to ascertain inhibition effects of the compounds. Cichoric acid showed no cytotoxic effect, while the EC20 values of tetraenes and echinacoside were 45.0 ± 3.0 and 52.0 ± 4.0 µg/mL in Caco-2 cells and 28.0 ± 4.3 and 62.0 ± 9.9 µg/mL in HepG2 cells, respectively. In general, the compounds showed heterogeneous induction of ABCB1 with the strongest 3.6 ± 1.2-fold increase observed for 10 µg/mL tetraenes in Caco-2 cells (p < 0.001). However, the compounds did not induce ABCG2. None of the phytocompounds inhibited significantly net flux of the fluorophores across Caco-2 monolayers. Overall, tetraenes moderately induced ABCB1 but not ABCG2 in Caco-2 and HepG2 cells while no compound significantly inhibited activity of these transporters at clinically relevant concentration to cause herb-drug interactions.
Assuntos
Ácidos Cafeicos/farmacologia , Echinacea/química , Glicosídeos/farmacologia , Interações Ervas-Drogas , Succinatos/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/agonistas , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/agonistas , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Células Hep G2 , Eliminação Hepatobiliar , Humanos , Eliminação Intestinal , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismoRESUMO
ATP-binding cassette (ABC) transporters represent a large group of efflux pumps that are strongly involved in the pharmacokinetics of various drugs and nutrient distribution. It was recently shown that micro-RNAs (miRNAs) may significantly alter their expression as proven, e.g., for miR-379 and ABCC2 However, alternative mRNA polyadenylation may result in expression of 3'-untranslated regions (3'-UTRs) with varying lengths. Thus, length variants may result in presence or absence of miRNA binding sites for regulatory miRNAs with consequences on posttranscriptional control. In the present study, we report on 3'-UTR variants of ABCC1, ABCC2, and ABCC3 mRNA. Applying in vitro luciferase reporter gene assays, we show that expression of short ABCC2 3'-UTR variants leads to a significant loss of miR-379/ABCC2 interaction and subsequent upregulation of ABCC2 expression. Furthermore, we show that expression of ABCC2 3'-UTR lengths varies significantly between human healthy tissues but is not directly correlated to the respective protein level in vivo. In conclusion, the presence of altered 3'-UTR lengths in ABC transporters could lead to functional consequences regarding posttranscriptional gene expression, potentially regulated by alternative polyadenylation. Hence, 3'-UTR length variability may be considered as a further mechanism contributing to variability of ABCC transporter expression and subsequent drug variation in drug response. SIGNIFICANCE STATEMENT: micro-RNA (miRNA) binding to 3'-untranslated region (3'-UTR) plays an important role in the control of ATP-binding cassette (ABC)-transporter mRNA degradation and translation into proteins. We disclosed various 3'-UTR length variants of ABCC1, C2, and C3 mRNA, with loss of mRNA seed regions partly leading to varying and tissue-dependent interaction with miRNAs, as proven by reporter gene assays. Alternative 3'-UTR lengths may contribute to variable ABCC transporter expression and potentially explains inconsistent findings in miRNA studies.
Assuntos
MicroRNAs/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Idoso , Idoso de 80 Anos ou mais , Células CACO-2 , Colo/metabolismo , Feminino , Vesícula Biliar/metabolismo , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , PoliadenilaçãoRESUMO
1. The aim of this study was to investigate the regulatory effect of Echinacea purpurea (EP) on efflux transporters ABCB1 and ABCG2 and to identify specific microRNAs contributing to their post-transcriptional regulation. 2. ABCB1 and ABCG2 levels were assessed in human hepatoblastoma HepG2 cells treated with 50 µg/mL methanolic extract of commercial EP capsules for different durations. The microRNA expression profile of HepG2 cells after EP treatment was evaluated and in silico target prediction was subsequently conducted to identify specific microRNAs with binding sites in the 3'-UTR of ABCB1 and ABCG2. Luciferase reporter gene assays and site-directed mutagenesis were used to confirm the binding site of identified microRNA within the 3'-UTR of the target gene. 3. EP increased ABCB1 (10-fold ± 3.4, p < 0.001) and ABCG2 (2.7-fold ± 0.5, p < 0.01) mRNA levels after 12 h exposure. Twenty-four microRNAs showed significant expression differences at all durations of exposure to EP. MiR-655-3p showed a 6.79-fold decrease in expression after 12 h exposure compared to 0 h, was predicted in silico to bind ABCG2 3'-UTR and showed a significant negative correlation (p = 0.01) to ABCG2 expression level. The binding of miR-655-3p to ABCG2 3'-UTR was confirmed by reporter gene assays (reduction of reporter gene activity to 60%; p = 0.0001). 4. These results suggest that EP regulates ABCG2 expression via downregulation of miR-655-3p in the liver cells. Thus, miR-655-3p downregulation could be applied to predict EP mediated drug interactions.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Echinacea/química , MicroRNAs/metabolismo , Proteínas de Neoplasias/genética , Regiões 3' não Traduzidas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação da Expressão Gênica , Células Hep G2 , Humanos , MicroRNAs/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The use of tyrosine kinase inhibitors, such as imatinib, against the chronic myeloid leukemia (CML)-causing kinase BCR::ABL1 has become the model for successful targeted therapy. Nevertheless, drug resistance remains a clinical problem. Analysis of genome-wide expression and genetic aberrations of an in vitro imatinib-resistant CML cell line revealed downregulation of Bruton's tyrosine kinase (BTK), predominantly associated with B cell malignancies, and a novel BTK kinase domain variant in imatinib resistance. This raised the question of the role of BTK in imatinib-resistant CML. In the present study, BTK downregulation and the presence of the BTK variant c.1699_1700delinsAG p.(Glu567Arg) were confirmed in imatinib resistance in vitro. Similarly, BTK inhibition or small interfering RNA-mediated BTK knockdown reduced imatinib susceptibility by 84 and 71%, respectively. BTK overexpression was detrimental to CML cells, as proliferation was significantly reduced by 20.5% under imatinib treatment. In addition, BTK rescue in imatinib-resistant cells restored imatinib sensitivity. The presence of the BTK p.(Glu567Arg) variant increased cell numbers (57%) and proliferation (37%) under imatinib exposure. These data demonstrate that BTK is important for the development of imatinib resistance in CML: Its presence increased drug response, while its absence promotes imatinib resistance. Moreover, the BTK p.(Glu567Arg) variant abrogates imatinib sensitivity. These findings demonstrate a context-dependent role for BTK as an oncogene in B cell malignancies, but as a tumor suppressor in other neoplasms.
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Leukemia represents a diverse group of hematopoietic neoplasms that can be classified into different subtypes based on the molecular aberration in the affected cell population. Identification of these molecular classification is required to identify specific targeted therapeutic approaches for each leukemic subtype. In general, targeted therapy approaches achieve good responses in some leukemia subgroups, however, resistance against these targeted therapies is common. In this review, we summarize molecular drug resistance biomarkers in targeted therapies in BCR::ABL1-driven chronic myeloid leukemia (CML) and JAK2-driven myeloproliferative neoplasms (MPNs). While acquisition of secondary mutations in the BCR::ABL1 kinase domain is the a common mechanism associated with TKI resistance in CML, in JAK2-driven MPNs secondary mutations in JAK2 are rare. Due to high prevalence and lack of specific therapy approaches in MPNs compared to CML, identification of crucial pathways leading to inhibitor persistence in MPN model is utterly important. In this review, we focus on different alternative signaling pathways activated in both, BCR::ABL1-mediated CML and JAK2-mediated MPNs, by combining data from in vitro and in vivo-studies that could be used as potential biomarkers of drug resistance. In a nutshell, some common similarities, especially activation of PDGFR, Ras, PI3K/Akt signaling pathways, have been demonstrated in both leukemias. In addition, induction of the nucleoprotein YBX1 was shown to be involved in TKI-resistant JAK2-mediated MPN, as well as TKI-resistant CML highlighting deubiquitinating enzymes as potential biomarkers of TKI resistance. Taken together, whole exome sequencing of cell-based or patients-derived samples are highly beneficial to define specific resistance markers. Additionally, this might be helpful for the development of novel diagnostic tools, e.g., liquid biopsy, and novel therapeutic agents, which could be used to overcome TKI resistance in molecularly distinct leukemia subtypes.
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Although great progress has been made in the fine-tuning of diplotypes, there is still a need to further improve the predictability of individual phenotypes of pharmacogenetically relevant enzymes. The aim of this study was to analyze the additional contribution of sex and variants identified by exome chip analysis to the metabolic ratio of five probe drugs. A cocktail study applying dextromethorphan, losartan, omeprazole, midazolam, and caffeine was conducted on 200 healthy volunteers. CYP2D6, 2C9, 2C19, 3A4/5, and 1A2 genotypes were analyzed and correlated with metabolic ratios. In addition, an exome chip analysis was performed. These SNPs correlating with metabolic ratios were confirmed by individual genotyping. The contribution of various factors to metabolic ratios was assessed by multiple regression analysis. Genotypically predicted phenotypes defined by CPIC discriminated very well the log metabolic ratios with the exception of caffeine. There were minor sex differences in the activity of CYP2C9, 2C19, 1A2, and CYP3A4/5. For dextromethorphan (CYP2D6), IP6K2 (rs61740999) and TCF20 (rs5758651) affected metabolic ratios, but only IP6K2 remained significant after multiple regression analysis. For losartan (CYP2C9), FBXW12 (rs17080138), ZNF703 (rs79707182), and SLC17A4 (rs11754288) together with CYP diplotypes, and sex explained 50% of interindividual variability. For omeprazole (CYP2C19), no significant influence of CYP2C:TG haplotypes was observed, but CYP2C19 rs12777823 improved the predictability. The comprehensive genetic analysis and inclusion of sex in a multiple regression model significantly improved the explanation of variability of metabolic ratios, resulting in further improvement of algorithms for the prediction of individual phenotypes of drug-metabolizing enzymes.
Assuntos
Genótipo , Fenótipo , Polimorfismo de Nucleotídeo Único , Humanos , Masculino , Feminino , Adulto , Exoma/genética , Cafeína/farmacocinética , Cafeína/metabolismo , Dextrometorfano/farmacocinética , Dextrometorfano/metabolismo , Losartan/farmacocinética , Preparações Farmacêuticas/metabolismo , Adulto Jovem , Omeprazol/farmacocinética , Fatores Sexuais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Estudos de Associação Genética/métodos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Thyroid hormones (THs) are important regulators of systemic energy metabolism. In the liver, they stimulate lipid and cholesterol turnover and increase systemic energy bioavailability. It is still unknown how the TH state interacts with the circadian clock, another important regulator of energy metabolism. We addressed this question using a mouse model of hypothyroidism and performed circadian analyses. Low TH levels decreased locomotor activity, food intake, and body temperature mostly in the active phase. Concurrently, liver transcriptome profiling showed only subtle effects compared to elevated TH conditions. Comparative circadian transcriptome profiling revealed alterations in mesor, amplitude, and phase of transcript levels in the livers of low-TH mice. Genes associated with cholesterol uptake, biosynthesis, and bile acid secretion showed reduced mesor. Increased and decreased cholesterol levels in the serum and liver were identified, respectively. Combining data from low- and high-TH conditions allowed the identification of 516 genes with mesor changes as molecular markers of the liver TH state. We explored these genes and created an expression panel that assesses liver TH state in a time-of-day dependent manner. Our findings suggest that the liver has a low TH action under physiological conditions. Circadian profiling reveals genes as potential markers of liver TH state.
Assuntos
Fígado , Transcriptoma , Masculino , Animais , Ritmo Circadiano/genética , Hormônios Tireóideos , ColesterolRESUMO
Background: The development of obesity-associated comorbidities such as type 2 diabetes (T2D) and hepatic steatosis has been linked to selected microRNAs in individual studies; however, an unbiased genome-wide approach to map T2D induced changes in the miRNAs landscape in human liver samples, and a subsequent robust identification and validation of target genes are still missing. Methods: Liver biopsies from age- and gender-matched obese individuals with (n=20) or without (n=20) T2D were used for microRNA microarray analysis. The candidate microRNA and target genes were validated in 85 human liver samples, and subsequently mechanistically characterized in hepatic cells as well as by dietary interventions and hepatic overexpression in mice. Results: Here, we present the human hepatic microRNA transcriptome of type 2 diabetes in liver biopsies and use a novel seed prediction tool to robustly identify microRNA target genes, which were then validated in a unique cohort of 85 human livers. Subsequent mouse studies identified a distinct signature of T2D-associated miRNAs, partly conserved in both species. Of those, human-murine miR-182-5 p was the most associated with whole-body glucose homeostasis and hepatic lipid metabolism. Its target gene LRP6 was consistently lower expressed in livers of obese T2D humans and mice as well as under conditions of miR-182-5 p overexpression. Weight loss in obese mice decreased hepatic miR-182-5 p and restored Lrp6 expression and other miR-182-5 p target genes. Hepatic overexpression of miR-182-5 p in mice rapidly decreased LRP6 protein levels and increased liver triglycerides and fasting insulin under obesogenic conditions after only seven days. Conclusions: By mapping the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, validating conserved miRNAs in diet-induced mice, and establishing a novel miRNA prediction tool, we provide a robust and unique resource that will pave the way for future studies in the field. As proof of concept, we revealed that the repression of LRP6 by miR-182-5 p, which promotes lipogenesis and impairs glucose homeostasis, provides a novel mechanistic link between T2D and non-alcoholic fatty liver disease, and demonstrate in vivo that miR-182-5 p can serve as a future drug target for the treatment of obesity-driven hepatic steatosis. Funding: This work was supported by research funding from the Deutsche Forschungsgemeinschaft (KI 1887/2-1, KI 1887/2-2, KI 1887/3-1 and CRC-TR296), the European Research Council (ERC, CoG Yoyo LepReSens no. 101002247; PTP), the Helmholtz Association (Initiative and Networking Fund International Helmholtz Research School for Diabetes; MB) and the German Center for Diabetes Research (DZD Next Grant 82DZD09D1G).
Assuntos
Diabetes Mellitus Tipo 2 , Fígado , MicroRNAs , Obesidade , Transcriptoma , MicroRNAs/metabolismo , MicroRNAs/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Animais , Humanos , Obesidade/genética , Obesidade/metabolismo , Fígado/metabolismo , Camundongos , Masculino , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Feminino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Perfilação da Expressão GênicaRESUMO
Introduction: Resistance in anti-cancer treatment is a result of clonal evolution and clonal selection. In chronic myeloid leukemia (CML), the hematopoietic neoplasm is predominantly caused by the formation of the BCR::ABL1 kinase. Evidently, treatment with tyrosine kinase inhibitors (TKIs) is tremendously successful. It has become the role model of targeted therapy. However, therapy resistance to TKIs leads to loss of molecular remission in about 25% of CML patients being partially due to BCR::ABL1 kinase mutations, while for the remaining cases, various other mechanisms are discussed. Methods: Here, we established an in vitro-TKI resistance model against the TKIs imatinib and nilotinib and performed exome sequencing. Results: In this model, acquired sequence variants in NRAS, KRAS, PTPN11, and PDGFRB were identified in TKI resistance. The well-known pathogenic NRAS p.(Gln61Lys) variant provided a strong benefit for CML cells under TKI exposure visible by increased cell number (6.2-fold, p < 0.001) and decreased apoptosis (-25%, p < 0.001), proving the functionality of our approach. The transfection of PTPN11 p.(Tyr279Cys) led to increased cell number (1.7-fold, p = 0.03) and proliferation (2.0-fold, p < 0.001) under imatinib treatment. Discussion: Our data demonstrate that our in vitro-model can be used to study the effect of specific variants on TKI resistance and to identify new driver mutations and genes playing a role in TKI resistance. The established pipeline can be used to study candidates acquired in TKI-resistant patients, thereby providing new options for the development of new therapy strategies to overcome resistance.
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Although chronic myeloid leukemia (CML) can be effectively treated using BCRABL1 kinase inhibitors, resistance due to kinase alterations or to BCRABL1 independent mechanisms remain a therapeutic challenge. For the latter, the underlying mechanisms are widely discussed; for instance, gene expression changes, epigenetic factors and alternative signaling pathway activation. In the present study, in vitroCML cell models of resistance against the tyrosine kinase inhibitors (TKIs) imatinib (0.5 and 2 µM) and nilotinib (0.1 µM) with biological replicates were generated to identify novel mechanisms of resistance. Subsequently, genomewide mRNA expression and DNA methylation were analyzed. While mRNA expression patterns differed largely between biological replicates, there was an overlap of 71 genes differentially expressed between cells resistant against imatinib or nilotinib. Moreover, all TKI resistant cell lines demonstrated a slight hypermethylation compared with native cells. In a combined analysis of 151 genes differentially expressed in the biological replicates of imatinib resistance, cell adhesion signaling, in particular the cellular matrix protein fibronectin 1 (FN1), was significantly dysregulated. This gene was also downregulated in nilotinib resistance. Further analyses showed significant FN1downregulation in imatinib resistance on mRNA (P<0.001) and protein level (P<0.001). SiRNAmediated FN1knockdown in native cells reduced cell adhesion (P=0.02), decreased imatinib susceptibility visible by higher Ki67 expression (1.5fold, P=0.04) and increased cell number (1.5fold, P=0.03). Vice versa, recovery of FN1expression in imatinib resistant cells was sufficient to partially restore the response to imatinib. Overall, these results suggested a role of cell adhesion signaling and fibronectin 1 in TKI resistant CML and a potential target for novel strategies in treatment of resistant CML.
Assuntos
Fibronectinas , Leucemia Mielogênica Crônica BCR-ABL Positiva , Adesão Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fibronectinas/genética , Fibronectinas/metabolismo , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Metilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
DNA methylation is dynamically regulated in metabolic diseases, but it remains unclear whether the changes are causal or consequential. Therefore, we used a longitudinal approach to refine the onset of metabolic and DNA methylation changes at high temporal resolution. Male C57BL/6N mice were fed with 60 % high-fat diet (HFD) for up to 12 weeks and metabolically characterized weekly. Liver was collected after 1, 2, 4, 5, 6, 7, 8, and 12 weeks and hepatic DNA methylation and gene expression were analyzed. A subset of obese mice underwent vertical sleeve gastrectomy (VSG) or metformin treatment and livers were studied. Distinct hepatic gene expression patterns developed upon feeding HFD, with genes from the fatty acid metabolism pathway being predominantly altered. When comparing metabolic data with gene expression and DNA methylation, in particular Fgf21 DNA methylation decreased before the onset of increased Fgf21 expression and metabolic changes. Neither weight loss induced by VSG nor improved glucose tolerance by metformin treatment could revert hepatic Fgf21 DNA methylation or expression. Our data emphasize the dynamic induction of DNA methylation upon metabolic stimuli. Reduced Fgf21 DNA methylation established before massive overexpression of Fgf21, which is likely an adaptive effort of the liver to maintain glucose homeostasis despite the developing insulin resistance and steatosis. Fgf21 DNA methylation resisted reversion by intervention strategies, illustrating the long-term effects of unhealthy lifestyle. Our data provide a temporal roadmap to the development of hepatic insulin resistance, comprehensively linking DNA methylation with gene expression and metabolic data.
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Metilação de DNA , Fatores de Crescimento de Fibroblastos/genética , Resistência à Insulina , Fígado/metabolismo , Obesidade/metabolismo , Animais , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Glucose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Transcriptoma , Redução de PesoRESUMO
Diurnal (i.e., 24 hr) physiological rhythms depend on transcriptional programs controlled by a set of circadian clock genes/proteins. Systemic factors like humoral and neuronal signals, oscillations in body temperature, and food intake align physiological circadian rhythms with external time. Thyroid hormones (THs) are major regulators of circadian clock target processes such as energy metabolism, but little is known about how fluctuations in TH levels affect the circadian coordination of tissue physiology. In this study, a high triiodothyronine (T3) state was induced in mice by supplementing T3 in the drinking water, which affected body temperature, and oxygen consumption in a time-of-day-dependent manner. A 24-hr transcriptome profiling of liver tissue identified 37 robustly and time independently T3-associated transcripts as potential TH state markers in the liver. Such genes participated in xenobiotic transport, lipid and xenobiotic metabolism. We also identified 10-15% of the liver transcriptome as rhythmic in control and T3 groups, but only 4% of the liver transcriptome (1033 genes) were rhythmic across both conditions - amongst these, several core clock genes. In-depth rhythm analyses showed that most changes in transcript rhythms were related to mesor (50%), followed by amplitude (10%), and phase (10%). Gene set enrichment analysis revealed TH state-dependent reorganization of metabolic processes such as lipid and glucose metabolism. At high T3 levels, we observed weakening or loss of rhythmicity for transcripts associated with glucose and fatty acid metabolism, suggesting increased hepatic energy turnover. In summary, we provide evidence that tonic changes in T3 levels restructure the diurnal liver metabolic transcriptome independent of local molecular circadian clocks.
Many environmental conditions, including light and temperature, vary with a daily rhythm that affects how animals interact with their surroundings. Indeed, most species have developed so-called circadian clocks: internal molecular timers that cycle approximately every 24 hours and regulate many bodily functions, including digestion, energy metabolism and sleep. The energy metabolism of the liver the chemical reactions that occur in the organ to produce energy from nutrients is controlled both by the circadian clock system, and by the hormones produced by a gland in the neck called the thyroid. However, the interaction between these two regulators is poorly understood. To address this question, de Assis, Harder et al. elevated the levels of thyroid hormones in mice by adding these hormones to their drinking water. Studying these mice showed that, although thyroid hormone levels were good indicators of how much energy mice burn in a day, they do not reflect daily fluctuations in metabolic rate faithfully. Additionally, de Assis, Harder et al. showed that elevating T3, the active form of thyroid hormone, led to a rewiring of the daily rhythms at which genes were turned on and off in the liver, affecting the daily timing of processes including fat and cholesterol metabolism. This occurred without changing the circadian clock of the liver directly. De Assis, Harder et al.'s results indicate that time-of-day critically affects the action of thyroid hormones in the liver. This suggests that patients with hypothyroidism, who produce low levels of thyroid hormones, may benefit from considering time-of-day as a factor in disease diagnosis, therapy and, potentially, prevention. Further data on the rhythmic regulation of thyroid action in humans, including in patients with hypothyroidism, are needed to further develop this approach.
Assuntos
Relógios Circadianos , Ritmo Circadiano , Animais , Relógios Circadianos/genética , Ritmo Circadiano/genética , Suplementos Nutricionais , Regulação da Expressão Gênica , Lipídeos , Fígado/metabolismo , Camundongos , Transcriptoma , Tri-Iodotironina/genética , Tri-Iodotironina/metabolismo , Xenobióticos/metabolismoRESUMO
The use of small molecules became one key cornerstone of targeted anti-cancer therapy. Among them, tyrosine kinase inhibitors (TKIs) are especially important, as they were the first molecules to proof the concept of targeted anti-cancer treatment. Since 2001, TKIs can be successfully used to treat chronic myelogenous leukemia (CML). CML is a hematologic neoplasm, predominantly caused by reciprocal translocation t(9;22)(q34;q11) leading to formation of the so-called BCR-ABL1 fusion gene. By binding to the BCR-ABL1 kinase and inhibition of downstream target phosphorylation, TKIs, such as imatinib or nilotinib, can be used as single agents to treat CML patients resulting in 80 % 10-year survival rates. However, treatment failure can be observed in 20-25 % of CML patients occurring either dependent or independent from the BCR-ABL1 kinase. Here, we review approved TKIs that are indicated for the treatment of CML, their side effects and limitations. We point out mechanisms of TKI resistance focusing either on BCR-ABL1-dependent mechanisms by summarizing the clinically observed BCR-ABL1-mutations and their implications on TKI binding, as well as on BCR-ABL1-independent mechanisms of resistances. For the latter, we discuss potential mechanisms, among them cytochrome P450 implications, drug efflux transporter variants and expression, microRNA deregulation, as well as the role of alternative signaling pathways. Further, we give insights on how TKI resistance could be analyzed and what could be learned from studying TKI resistance in CML in vitro.
RESUMO
The mRNA-destabilizing proteins ZFP36L1 and ZFP36L2 are described as mediators of quiescence and play a pivotal role in hematopoietic malignancies. Both genes are mainly classified as tumor suppressor genes as they posttranscriptionally downregulate the expression of oncogenes and contribute to cellular quiescence. Here, we analyzed the role of ZFP36L1 and ZFP36L2 in chronic myeloid leukemia (CML). We found ZFP36L1 and ZFP36L2 expression to be deregulated in patients with CML. By use of in vitro models of tyrosine kinase inhibitor resistance, an increase in ZFP36L1 and ZFP36L2 expression was detected during the development of imatinib resistance. CRISPR/Cas9-derived knockout of ZFP36L1, but not of ZFP36L2, in imatinib-sensitive cells led to decreased proliferation rates in response to tyrosine kinase inhibitor treatment. This effect was also observed in untreated ZFP36L1 knockout cells, albeit to a lower extent. Genomewide gene expression analyses of ZFP36L1 knockout cells revealed differential expression of cell cycle regulators, in particular upregulation of the cell cycle inhibitor CDKN1A. In addition, the 3' untranslated region of CDKN1A was proven to be a direct target of ZFP36L1. This indicates that tumor suppressor genes can also be targeted by ZFP36L1. Hence, ZFP36L1 cannot unambiguously be regarded as a tumor suppressor gene.
Assuntos
Fator 1 de Resposta a Butirato , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21 , Regulação Leucêmica da Expressão Gênica , Neoplasias Hematológicas , Leucemia Mielogênica Crônica BCR-ABL Positiva , Adulto , Idoso , Idoso de 80 Anos ou mais , Fator 1 de Resposta a Butirato/biossíntese , Fator 1 de Resposta a Butirato/genética , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Although the introduction of tyrosine kinase inhibitors (TKIs) has substantially improved patients' prognosis, drug resistance remains one of the major challenges in CML therapy. MicroRNAs (miRNAs), a class of short non-coding RNAs acting as post-transcriptional regulators, are implicated in CML progression and drug resistance. The aim of the present study was to analyze the miRNA expression profiles of 45 treatment-naïve CML patients in chronic phase (28 peripheral blood and 17 bone marrow samples) with respect to future response to imatinib therapy. METHODS: TaqMan low density arrays were used to analyze the miRNA expression pattern of the patient samples. For selected microRNAs, reporter gene assays were performed to study their ability to regulate CML associated target genes. RESULTS: Significant lower expression levels of miR-142-5p were identified in both, peripheral blood and bone marrow samples of future non-responders suggesting a potential tumor suppressor role of this miRNA. This was supported by reporter gene assays that identified the survival, proliferation and invasion promoting CML related genes ABL2, cKIT, MCL1 and SRI as targets of miR-142-5p and miR-365a-3p, the latter identified as potential biomarker in peripheral blood samples. CONCLUSION: MiR-142-5p and to a certain extend also miR-365a-3p were able to discriminate treatment-naïve CML patients not responding to imatinib in the course of their treatment from patients, who responded to therapy. However, further large-scale studies should clarify if the identified miRNAs have the potential as predictive biomarkers for TKI resistance.
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Cancer pharmacogenetics implies a complex combination of germline variants from the patient and somatic mutations in tumor cells. Somatic mutations meanwhile have become drugable targets or biomarkers, whereas germline mutations potentially predict adverse drug effects or drug response. Here, we evaluate hereditary variants in biotransforming enzymes and drug transporters, such as thiopurine S-methyltransferase, UDP-glucuronosyltransferase (UGT1A1), dihydropyrimidine dehydrogenase (DPD), as well as ABC transporters (ABCB1, ABCG2 and ABCC subfamily) with respect to cytostatics and targeted therapies. Furthermore, gene expression regulation with regards to epigenetics and posttranscriptional modification are discussed.
RESUMO
Pain sensitivity is characterized by interindividual variability, determined by factors including genetic variation of nociceptive receptors and pathways. The sigma-1 receptor (SIGMAR1) is involved in pain modulation especially under pre-sensitized conditions. However, the contribution of SIGMAR1 genetic variants to pain generation and sensitivity is unknown yet. This study aimed to identify effects of 5 SIGMAR1 variants on the somatosensory phenotype of neuropathic pain patients (nâ¯=â¯228) characterized by standardized quantitative sensory testing. Principal component analysis revealed that the SIGMAR1 variants -297G>T (rs10814130) and 5A>C (rs1800866) significantly lowered thermal detection and heat/pressure nociception in particular in neuropathic pain patients with mainly preserved somatosensory function. Compared to wild-type, the variant allele -297T was associated with loss of warm detection (Pâ¯=â¯.049), lower heat-pain sensitivity (Pâ¯=â¯.027) and wind-up ratio (Pâ¯=â¯.023) as well as increased paradoxical heat sensation (Pâ¯=â¯.020). Likewise for 5A>C the strongest genotype-associated differences observed were reduced peripheral (less heat hyperalgesia; Pâ¯=â¯.026) and central sensitization (lower mechanical pain sensitivity; Pâ¯=â¯.026) in variant compared to wild-type carriers. This study indicates lack of association of SIGMAR1 -297G>T and 5A>C genetic variants to susceptibility to develop chronic pain, but significant modulation of somatosensory function in neuropathic pain patients. PERSPECTIVE: This article presents the first study indicating a modulation of somatosensory function in neuropathic pain patients by selected genetic variants in SIGMAR1. As our findings could contribute to the explanation of interindividual differences in drug response they might help to improve the treatment of neuropathic pain.