Henrik Seidel

Automated image analysis for array hybridization experiments

MOTIVATION Image analysis is a major part of data evaluation for array hybridization experiments in molecular biology. The program presented here is designed to analyze automatically images from hybridization experiments with various arrangements: different kinds of probes (oligonucleotides or complex probes), different supports (nylon filters or glass slides), different labeling of probes (radioactively or fluorescently). The program is currently applied to oligonucleotide fingerprinting projects and complex hybridizations. The only precondition for the use of the program is that the targets are arrayed in a grid, which can be approximately transformed to an orthogonal equidistant grid by a projective mapping. RESULTS We demonstrate that our program can cope with the following problems: global distortion of the grid, missing of grid nodes, local deviation of the spot from its specified grid position. This is checked by different quality measures. The image analysis of oligonucleotide fingerprint experiments on an entire genetic library is used, in clustering procedures, to group related clones together. The results show that the program yields automatically generated high quality input data for follow up analysis such as clustering procedures. AVAILABILITY The executable files will be available upon request for academics.

Expression profiling of IL-10-regulated genes in human monocytes and peripheral blood mononuclear cells from psoriatic patients during IL-10 therapy.

Interleukin‐10 (IL‐10), originally identified as an inhibitor of pro‐inflammatory cytokine production, exerts multiple immunomodulatory functions. Its ability to inhibit a Th1 response has been used in clinical trials for the treatment of inflammatory diseases including psoriasis. However, little is known about the molecular mechanisms of IL‐10 functions. We aimed at identifying possiblemediators of in vitro IL‐10 treatment in monocytes by gene chip technology using Hu95a Affymetrix mRNA arrays with 12,000 genes. To prove relevance of the identified genes for the clinicalsituation we compared these in vitro results with genes being regulated by IL‐10 in peripheral blood mononuclear cells from psoriatic patients undergoing IL‐10 therapy. A high proportion of the 1,600 genes up‐regulated and 1,300 genes down‐regulated in vitro was found to be similarly regulated in vivo. Some genes, which were previously unknown to be regulated by IL‐10, can be assigned to known IL‐10 functions like e.g. the increase of pathogen clearance. Other new potentially immunomodulating genes have been identified to be regulated by IL‐10, but their impact needs to be experimentally evaluated. We could confirm a recently reported up‐regulation of heme oxygenase‐1 (HO‐1). However, we demonstrate that the anti‐inflammatory mechanisms of IL‐10 remain functional even when HO‐1 is irreversibly inhibited.

Abstract 3086: Inhibitors of the enzyme dihydroorotate dehydrogenase, overcome the differentiation blockade in acute myeloid leukemia

The prognosis for adults diagnosed with acute myeloid leukemia (AML) remains poor, with a five-year survival of only 25%. This prognosis is even more dismal in older patients who are not well enough to receive standard induction chemotherapy. Speaking to the need for new therapies is the fact that our therapeutic backbone - a combination of cytarabine and an anthracycline - remains unchanged since 1973. The promise of differentiation therapy was realized in the small subset of patients diagnosed with acute promyelocytic leukemia (APL). Here, treatment in the form of all-trans retinoic acid (ATRA) and arsenic trioxide inverted the survival curve; where APL was once the worst form of myeloid leukemia, it now carries the best prognosis, with a five-year survival exceeding 85%. The goal of this study was thus to develop differentiation therapy for patients with non-promyelocytic AML with the question: “Can we identify small molecules that overcome myeloid differentiation arrest?” A phenotypic differentiation screen in a HOXA9 driven leukemia model followed by target deconvolution, identified DHODH as an unexpected target for overcoming differentiation arrest in AML. We used 2 potent small molecule inhibitors of DHODH to validate this initial finding: Brequinar, a known DHODH inhibitor and an in house compound BAY DHODHi. In several in vitro experiments we demonstrated induction of AML differentiation in a dose dependent fashion. Interestingly, these effects could be completely rescued by addition of uridine, confirming target specificity. Treating mice in multiple genetically diverse AML in vivo models with a DHODH inhibitor led to tumor growth reduction and AML differentiation. Expression analysis of leukemia cells explanted from mice xenografts treated with a DHODH inhibitor demonstrate an early onset of differentiation markers indicating a direct role of DHODH with the onset of differentiation in vivo. The mechanism for selective vulnerability of leukemia cells to DHODH inhibition remains under investigation. Despite the observation that DHODH is expressed in all cells, normal and malignant, mice can tolerate DHODH inhibitor therapy for more than 100 days without weight-loss or other concerning side-effects. Thus, our pre-clinical studies point towards DHODH as a new metabolic target in the differentiation treatment of AML. Hopefully, small molecule DHODH inhibitors will provide a much-needed differentiation therapy for patients with acute myeloid leukemia. Citation Format: Andreas Janzer, David Sykes, Stefan Gradl, Steven Ferrara, Sven Christian, Claudia Merz, Henrik Seidel, Andreas Bernthaler, Ralf Lesche, Mathias Wawer, David T. Scadden. Inhibitors of the enzyme dihydroorotate dehydrogenase, overcome the differentiation blockade in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3086. doi:10.1158/1538-7445.AM2017-3086

Abstract 5036: Correlation of preclinical antitumor activity of regorafenib in CRC-PDX xenografts with gene expression and clinical parameters of the primary tumor

Regorafenib is a small molecule inhibitor of multiple transmembrane and intracellular kinases involved in normal cellular functions and in pathologic processes such as oncogenesis, tumor angiogenesis, metastasis, and tumor immunity. Regorafenib is approved for the treatment of patients with metastatic colorectal cancer (CRC) who have been previously treated with fluoropyrimidine-, oxaliplatin- and irinotecan-based chemotherapy, an anti-VEGF therapy, and, if RAS wild-type, an anti-EGFR therapy or with locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) who have been previously treated with imatinib mesylate and sunitinib malate. Recently an overall survival benefit has been shown in patients with hepatocellular carcinoma (HCC) who had previously been treated with sorafenib (RESORCE). OncoTrack is an Innovative Medicines Initiative (IMI) sponsored project with the goal to improve the basis for identification of biomarkers based on the mechanisms of action of therapies approved for this indication. For this purpose, a panel of CRC-PDX xenografts was generated by the OncoTrack project. At the time of the analysis reported here fifty xenografts had been treated with regorafenib at a dose of 10 mg/kg/d or with vehicle for 24 days. The analysis of tumor growth rates (TGR) showed pronounced differences between different tumors and between vehicle and regorafenib treated models. The relative antitumor activity (relative TGR) of regorafenib varied between -0,13 (good response) and 0.0 (no response). Investigations of relative TGR in relationship to (non-) clinical parameters of the primary tumor such as age, gender, sidedness and tumor histology identified a marginally significant (p= 0.04) better response in tumors from younger patients. No other correlations were detected to this end, which may be due to the small sample number. To correlate antitumor activity of regorafenib with gene expression, RNA was isolated from sections of selected vehicle and regorafenib treated xenografts and hybrized on Affymetrix HuGene-2.1_st human transcriptome arrays. Expression profiles were subsequently analyzed using the Random Forests algorithm to identify gene expression signatures predicting response to regorafenib. The best signatures did not perform better than signatures derived after randomizing responses, i.e. no predictive signature could be identified. Further studies with larger samples sizes are necessary to improve the outcome of such an approach; however one should acknowledge that to date no predictive gene signatures could be identified for multikinase inhibitors, which may be intrinsic to their complex mechanism of action. The research reported here received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement 115234 (OncoTrack). Citation Format: Henrik Seidel, Jens Hoffmann, Ralf Lesche, Sylvia Grunewald, David Henderson, Dieter Zopf. Correlation of preclinical antitumor activity of regorafenib in CRC-PDX xenografts with gene expression and clinical parameters of the primary tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5036. doi:10.1158/1538-7445.AM2017-5036