Henrike Bruckmueller

Down-Regulation of ATP-Binding Cassette C2 Protein Expression in HepG2 Cells after Rifampicin Treatment Is Mediated by MicroRNA-379

microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3′-untranslated region-interference, have been shown to be involved in the regulation of ATP-binding cassette (ABC) membrane transporters. The aim of this study was to investigate whether ABCC2, an important efflux transporter for various endogenous and exogenous compounds at several compartment barriers, is subject to miRNA-mediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 μM rifampicin [a pregnane X receptor (PXR) ligand] or vehicle using reverse transcription-polymerase chain reaction-based low-density arrays. Specific miRNA, ABCC2 mRNA, and protein expression were monitored in HepG2 cells undergoing rifampicin treatment for 72 h. Loss- and gain-of-function experiments and reporter gene assays were performed for further confirmation. Highly deregulated miRNAs compared with in silico data revealed miRNA (miR) 379 as candidate miRNA targeting ABCC2 mRNA. Under rifampicin treatment, ABCC2 mRNA increased significantly, with a maximal fold change of 1.56 ± 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 ± 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 ± 0.08 after 72 h. In contrast, transfection of miR-379 inhibitor led to an elevation of ABCC2 protein expression after rifampicin incubation for 48 h. We identify a miRNA negatively regulating ABCC2 on the post-transcriptional level and provide evidence that this miRNA impedes overexpression of ABCC2 protein after a PXR-mediated external transcriptional stimulus in HepG2 cells.

A European spectrum of pharmacogenomic biomarkers: Implications for clinical pharmacogenomics

Pharmacogenomics aims to correlate inter-individual differences of drug efficacy and/or toxicity with the underlying genetic composition, particularly in genes encoding for protein factors and enzymes involved in drug metabolism and transport. In several European populations, particularly in countries with lower income, information related to the prevalence of pharmacogenomic biomarkers is incomplete or lacking. Here, we have implemented the microattribution approach to assess the pharmacogenomic biomarkers allelic spectrum in 18 European populations, mostly from developing European countries, by analyzing 1,931 pharmacogenomics biomarkers in 231 genes. Our data show significant inter-population pharmacogenomic biomarker allele frequency differences, particularly in 7 clinically actionable pharmacogenomic biomarkers in 7 European populations, affecting drug efficacy and/or toxicity of 51 medication treatment modalities. These data also reflect on the differences observed in the prevalence of high-risk genotypes in these populations, as far as common markers in the CYP2C9, CYP2C19, CYP3A5, VKORC1, SLCO1B1 and TPMT pharmacogenes are concerned. Also, our data demonstrate notable differences in predicted genotype-based warfarin dosing among these populations. Our findings can be exploited not only to develop guidelines for medical prioritization, but most importantly to facilitate integration of pharmacogenomics and to support pre-emptive pharmacogenomic testing. This may subsequently contribute towards significant cost-savings in the overall healthcare expenditure in the participating countries, where pharmacogenomics implementation proves to be cost-effective.

Which Genetic Determinants Should be Considered for Tacrolimus Dose Optimization in Kidney Transplantation? A Combined Analysis of Genes Affecting the CYP3A Locus

Background: Tacrolimus is established as immunosuppressant after kidney transplantation. Polymorphism of the cytochrome P450 3A5 (CYP3A5) gene contributes significantly to tacrolimus dose requirements. Recently, CYP3A4*22 was reported to additionally affect tacrolimus pharmacokinetics (PK). In addition, there are further polymorphic genes, possibly influencing CYP3A activity [pregnane x receptor NR1I2, P450 oxidoreductase (POR), and peroxisome proliferator-activator receptor alpha (PPARA)]. We aimed to investigate combined effects of these gene variants on tacrolimus maintenance dose and PK in patients with stable kidney transplantation of 2 study centers. Methods: A total of 223 white patients (German cohort, 136; Danish cohort, 87) was included and genotyped for CYP3A5 (rs776746), CYP3A4 (rs35599367), NR1I2 (rs2276707), POR (rs1057868), and PPARA (rs4253728). Dosage and trough concentration/dose ratios were considered separately. A subset was investigated for comprehensive PK parameters. Results: Tacrolimus dose, trough concentration, and trough concentration/dose ratio did not differ between the German and Danish cohort. CYP3A5*3 and CYP3A4*22 contributed to dose requirements only in the German and in the total cohort. Homozygous carriers of both variants required 4.8 ± 3.1 mg, whereas carriers of the wild types required 165% higher mean tacrolimus doses (12.5 ± 7.7 mg, P = 1.4 × 10−5). The PK investigation revealed only nonsignificant impact of CYP3A4 genotypes on AUC12h in CYP3A5 nonexpressers (P = 0.079, power = 57%). For the entire sample, the final multiple linear regression model for trough concentration/dose ratio included CYP3A5, CYP3A4, and age. It explained 18.3% of the interindividual variability of tacrolimus trough concentration/dose ratios (P = 8.8 × 10−10). Conclusions: Therapeutic drug monitoring remains essential in clinical care of patients with kidney transplantation. Genotyping of CYP3A5 and CYP3A4, however, could facilitate rapid dose finding to adapt the appropriate immunosuppressant dose, whereas other genetic factors had only little or no effect.

Genetic variants may play an important role in mRNA-miRNA interaction: evidence for haplotype-dependent downregulation of ABCC2 (MRP2) by miRNA-379

Background The functional influence of single-nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCC2 (MRP2) has been characterized in numerous studies. The aim of this study was to address the question of whether distinct ABCC2 haplotypes, which differ in their mRNA secondary structures, show an influence on the degree of mRNA and protein downregulation through miRNA interaction. Methods A model using human peripheral blood monocytic cells (PBMCs) isolated from healthy Caucasian volunteers, with three defined ABCC2 haplotypes comprising the 5′-UTR SNP −24C>T, the 1249G>A SNP (V417I), and the silent 3972C>T SNP, was outlined. Cells were transiently transfected with miRNA-379, already known to target ABCC2 in HepG2 cells. Results ABCC2 was downregulated through miR-379 in a haplotype-dependent manner: the wild-type CGC/CGC was modestly affected (mRNA: −12.7±4.2%, protein: −9.9±0.1%), whereas variant haplotypes were more strongly suppressed: CGT/CGT (mRNA: −36.7±2.4%, protein: −21.6±0.4%) and TGT/TGT (mRNA: −55.7±1.2%, protein: −46.3±4.0%). In addition, glutathione–methylfluorescein efflux was significantly reduced in miR-379-transfected peripheral blood monocytic cells corresponding to ABCC2 protein expression. Conclusion This observation may suggest a differential suppression of ABCC2 by miR-379 caused by haplotype-dependent differences in mRNA secondary structures, resulting in changes in mRNA target accessibility or mRNA stability.

Hematopoietic stem cell involvement in BCR-ABL1–positive ALL as a potential mechanism of resistance to blinatumomab therapy

The bispecific T-cell engager blinatumomab targeting CD19 can induce complete remission in relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, some patients ultimately relapse with loss of CD19 antigen on leukemic cells, which has been established as a novel mechanism to escape CD19-specific immunotherapies. Here, we provide evidence that CD19-negative (CD19-) relapse after CD19-directed therapy in BCP-ALL may be a result of the selection of preexisting CD19- malignant progenitor cells. We present 2 BCR-ABL1 fusion-positive BCP-ALL patients with CD19- myeloid lineage relapse after blinatumomab therapy and show BCR-ABL1 positivity in their hematopoietic stem cell (HSC)/progenitor/myeloid compartments at initial diagnosis by fluorescence in situ hybridization after cell sorting. By using the same approach with 25 additional diagnostic samples from patients with BCR-ABL1-positive BCP-ALL, we identified HSC involvement in 40% of the patients. Patients (6 of 8) with major BCR-ABL1 transcript encoding P210BCR-ABL1 mainly showed HSC involvement, whereas in most of the patients (9 of 12) with minor BCR-ABL1 transcript encoding P190BCR-ABL1, only the CD19+ leukemia compartments were BCR-ABL1 positive (P = .02). Our data are of clinical importance, because they indicate that both CD19+ cells and CD19- precursors should be targeted to avoid CD19- relapses in patients with BCR-ABL1-positive ALL.

MicroRNA-655-3p regulates Echinacea purpurea mediated activation of ABCG2

Abstract 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.

SIGMA-1 receptor gene variants affect the somatosensory phenotype in neuropathic pain patients

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 (n = 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 (P = .049), lower heat-pain sensitivity (P = .027) and wind-up ratio (P = .023) as well as increased paradoxical heat sensation (P = .020). Likewise for 5A>C the strongest genotype-associated differences observed were reduced peripheral (less heat hyperalgesia; P = .026) and central sensitization (lower mechanical pain sensitivity; P = .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.

Retinoic acid-induced survival effects in SH-SY5Y neuroblastoma cells: WAETZIG et al.

Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high‐risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA‐resistant cells substantially lowers 5‐year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH‐SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal‐regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c‐Jun N‐terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt‐mediated phosphorylation of the cell‐cycle regulator p21 stimulated complex formation with caspase‐3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH‐SY5Y cells, which increased cell viability.

Abstract 5846: Drug resistance in chronic myeloid leukemia: Impact of methylation on gene expression in imatinib and nilotinib resistance

Background: Chronic myeloid leukemia (CML) is one of the most extensively studied hematopoietic disorders. Caused by reciprocal translocation t(9;22) and formation of the Philadelphia chromosome, it leads to constitutive expression of the BCR-ABL kinase and malignant transformation. Targeted therapy using tyrosine kinase inhibitors (TKI), such as imatinib or nilotinib, led to tremendous success in treatment. However, resistances are an uprising problem in patient cure. Besides BCR-ABL-dependent mechanisms (i.e. BCR-ABL amplification, mutations), cellular alterations of gene expression and its regulation are involved. Therefore, we performed an in vitro-study in K-562 CML cells to investigate changes of gene expression and epigenetics, as well as occurrence of mutations during the course of imatinib or nilotinib resistance. Methods: We analyzed mRNA expression, DNA-methylation and mutation status of treatment-naive and TKI-resistant sublines (0.5 µM, 2 µM imatinib; 100 nM nilotinib) of the in vitro-cell line model (K-562). mRNA expression was analyzed using Affymetrix Human Gene 2.0 ST arrays and DNA-methylation by Infinium MethylationEPIC BeadChips (Illumina); exome sequencing was performed at GATC, Konstanz, Germany. Target verification was performed using qRT-PCR, pyrosequencing or Sanger sequencing. Effects on TKI-susceptibility were analyzed using transfection experiments with analyses of cell viability (WST-1 assay) and apoptosis (Caspase Glo 9 assay). Results: Cell resistant to low imatinib doses exhibited differentially expression of 485 mRNAs compared to treatment-naive cells, whereas at high dose-resistance 166 mRNAs differed. 151 genes showed expression changes independent from the imatinib dose. In addition, we found 45 genes with expression changes during the course of imatinib resistance, i.e. the drug efflux transporter ABCG2, which was significantly upregulated at low and downregulated at high imatinib doses. A total of 254 genes was associated with nilotinib resistance, while 71 genes were differentially expressed in all resistant sublines. Regarding methylation and occurrence of mutations in imatinib resistance, we observed distinct variations pointing to genetic changes, especially at high-dose resistance, i.e. in the promoter region of microRNA-212, a proven regulator of ABCG2. In nilotinib resistance, we found 125 genes differentially methylated during development of resistance. Conclusions: Our data indicate that development of resistance to tyrosine kinase inhibitors is based on complex expression profile changes that can in part be explained by epigenetic alteration e.g. in drug efflux transporters. Besides, the mechanisms underlying resistances to imatinib or nilotinib seem to be dissimilar. After verification in clinical studies, these in vitro findings could be relevant in attempts to overcome TKI-resistances. Citation Format: Meike Kaehler, Inga Nagel, Henrike Bruckmueller, Ruwen Boehm, Ole Ammerpohl, Ingolf Cascorbi. Drug resistance in chronic myeloid leukemia: Impact of methylation on gene expression in imatinib and nilotinib resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5846.

Correction: A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

[This corrects the article DOI: 10.1371/journal.pone.0162866.].