We present data suggesting a function of ␣ 2 -HS glycoproteins/fetuins in serum and in mineralization, namely interference with calcium salt precipitation. Fetuins occur in high serum concentration during fetal life. They accumulate in bones and teeth as a major fraction of noncollagenous bone proteins. The expression pattern in fetal mice confirms that fetuin is predominantly made in the liver and is accumulated in the mineralized matrix of bones. We arrived at a hypothesis on the molecular basis of fetuin function in bones using primary rat calvaria osteoblast cultures and salt precipitation assays. Our results indicate that fetuins inhibit apatite formation both in cell culture and in the test tube. This inhibitory effect is mediated by acidic amino acids clustering in cystatin-like domain D1. Fetuins account for roughly half of the capacity of serum to inhibit salt precipitation. We propose that fetuins inhibit phase separation in serum and modulate apatite formation during mineralization.

Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor.

The mediator complex-associated cyclin dependent kinase CDK8 regulates β-catenin-dependent transcription following activation of WNT signaling. Multiple lines of evidence suggest CDK8 may act as an oncogene in the development of colorectal cancer. Here we describe the successful optimization of an imidazo-thiadiazole series of CDK8 inhibitors that was identified in a high-throughput screening campaign and further progressed by structure-based design. In several optimization cycles, we improved the microsomal stability, potency, and kinase selectivity. The initial imidazo-thiadiazole scaffold was replaced by a 3-methyl-1H-pyrazolo[3,4-b]-pyridine which resulted in compound 25 (MSC2530818) that displayed excellent kinase selectivity, biochemical and cellular potency, microsomal stability, and is orally bioavailable. Furthermore, we demonstrated modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8 activity, and tumor growth inhibition in an APC mutant SW620 human colorectal carcinoma xenograft model after oral administration. Compound 25 demonstrated suitable potency and selectivity to progress into preclinical in vivo efficacy and safety studies.

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Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with immunosuppressive function, which inhibit the antitumor activity of T-cells and natural killer (NK) cells. MDSC number is greatly increased in tumor-bearing mice and in cancer patients, and in the clinic, MDSC accumulation is associated with cancer progression, recurrence, and poor response to chemo-, radio- and immunotherapies. The increasing evidence for the clinical significance of MDSC has triggered a strong interest in the therapeutic modulation of their function. To date, however, limited progress has been made in this direction, as a major challenge in the field remains the identification of suitable therapeutic targets for the development of novel drugs. Here, we describe a systematic approach in which a small-molecule high-throughput phenotypic screen was used to identify MDSC targets and pathways of therapeutic relevance. This screen was based on a validated in vitro mouse mononuclear MDSC (M-MDSC) model, in which hematopoietic progenitors, immortalized using a NUP98/HOXB4 transgene, were differentiated into immunosuppressive MDSC. Using this model, we developed a 384-well-based phenotypic screening assay, in which the suppressive effect of mouse M-MDSC on CD8+ T-cell proliferation and cytokine secretion was monitored. We screened a small molecule library, comprising 5000+ biologically active compounds with known target(s), and identified 116 compounds that potently disrupted MDSC suppression of T-cell function. With the help of chemoinformatics methods, reported target activities associated with the compounds were annotated, and a set of targets and pathways of potential significance for MDSC-driven immunosuppression was identified. Altogether, this work provides insight into the signaling nodes that could be of relevance for MDSC function, and offers a path forward for the therapeutic targeting of MDSC. Citation Format: Elissaveta Petrova, Sandra Schäffner, Jan-Carsten Pieck, Christian Herhaus, Friedrich Rippmann, Oliver Pöschke, Laura Helming. Using high-throughput phenotypic screening to identify therapeutic targets for the inhibition of myeloid-derived suppressor cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A099.