Peter Hagedorn

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma

The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains elusive. Recent discoveries indicate that the oncogenic microRNA miR-155 is overexpressed in affected skin from CTCL patients. Here, we address what drives the expression of miR-155 and investigate its role in the pathogenesis of CTCL. We show that malignant T cells constitutively express high levels of miR-155 and its host gene BIC (B cell integration cluster). Using ChIP-seq, we identify BIC as a target of transcription factor STAT5, which is aberrantly activated in malignant T cells and induced by IL-2/IL-15 in non-malignant T cells. Incubation with JAK inhibitor or siRNA-mediated knockdown of STAT5 decreases BIC/miR-155 expression, whereas IL-2 and IL-15 increase their expression in cell lines and primary cells. In contrast, knockdown of STAT3 has no effect, and BIC is not a transcriptional target of STAT3, indicating that regulation of BIC/miR-155 expression by STAT5 is highly specific. Malignant proliferation is significantly inhibited by an antisense-miR-155 as well as by knockdown of STAT5 and BIC. In conclusion, we provide the first evidence that STAT5 drives expression of oncogenic BIC/miR-155 in cancer. Moreover, our data indicate that the STAT5/BIC/miR-155 pathway promotes proliferation of malignant T cells, and therefore is a putative target for therapy in CTCL.

Hepatotoxic Potential of Therapeutic Oligonucleotides Can Be Predicted from Their Sequence and Modification Pattern

Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines.

IL-23-Mediated Epidermal Hyperplasia Is Dependent on IL-6

Psoriasis is a chronic inflammatory skin disease primarily driven by Th17 cells. IL-23 facilitates the differentiation and induces complete maturation of Th17 cells. Lesional psoriatic skin has increased levels of IL-23 and recent studies show that intradermal injections of IL-23 induce a psoriasis-like skin phenotype in mice. We have now characterized the IL-23-induced skin inflammation in mice at the molecular level and found a significant correlation with the gene expression profile from lesional psoriatic skin. As observed in psoriasis, the pathogenesis of the IL-23-induced skin inflammation in mice is driven by Th17 cells. We demonstrate a dramatic upregulation of IL-6 mRNA and protein after intradermal injections of IL-23 in mice. Using IL-6(-/-) mice we show that IL-6 is essential for development of the IL-23-elicited responses. Despite producing high levels of IL-22, IL-6(-/-) mice were unable to express the high-affinity IL-22 receptor chain and produced minimal IL-17A in response to intradermal injections of IL-23. In conclusion, we provide evidence for the critical role played by IL-6 in IL-23-induced skin inflammation and show that IL-6 is required for expression of IL-22R1A.

Allergic contact dermatitis induces upregulation of identical microRNAs in humans and mice

Background. MicroRNAs are short, endogenous RNA molecules that can bind to parts of target mRNAs, thus inhibiting their translation and causing accelerated turnover or degradation of transcripts, thereby regulating gene expression. Several microRNAs have been found to be upregulated in atopic dermatitis and psoriasis, indicating a role in inflammatory skin diseases. However, there have been no studies on the expression of microRNAs in allergic contact dermatitis.

A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

Comparison of microRNA expression using different preservation methods of matched psoriatic skin samples

Abstract:  MicroRNAs are non‐coding RNA molecules modulating gene expression post‐transcriptionally. Formalin‐fixed, paraffin‐embedding (FFPE) is a standard preservation method often used in clinical practices, but induces RNA degradation. Extracting high‐quality RNA from human skin can be challenging as skin contains high levels of RNases. As microRNAs are 19‐23 nucleotides long and lack a poly‐A tail, they may be less prone to RNA degradation than mRNAs. We investigated whether microRNAs in psoriatic (FFPE) samples reliably reflect microRNA expression in samples less prone to RNA degradation such as fresh‐frozen (FS) and Tissue‐Tek‐embedding (OCT). We found a strong correlation of the microRNA expression levels between all preservation methods of matched psoriatic skin samples (rs ranging from 0.91 to 0.95 (P < 0.001)). These observations were further confirmed with qRT‐PCR. Our results demonstrate that microRNA detection in human skin is robust irrespective of preservation method; thus, microRNAs offer an appropriate and flexible approach in clinical practices and for diagnostic purposes in skin disorders.