Project description:CDK8 (cyclin-dependent kinase 8) is a kinase component of a multi - protein Mediator complex, involved in transcription control. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer (CRC). Herewith we present molecular insights into mechanism of action of SEL120-34A - a selective small molecule inhibitor of CDK8 kinase. Biochemical and binding studies indicated that SEL120-34A selectively binds and inhibits enzymatic activity of CDK8 in the low nM range. Recently CDK8 has been described as a regulator of STAT1 activity in NK cells where by phosphorylating STAT1 serine 727 (Ser727) influences a possible immunoescape mechanism in various cancers. Consistently, SEL120-34A and other recently reported selective CDK8 inhibitors could repress phosphorylation of STAT1 at a Ser727 at low nM concentrations in cancer cells without any significant changes on tyrosine sites directly regulated by JAK kinases. SEL120-34A inhibited expression of several STAT1 dependent genes in CRC cell lines, stimulated by various cytokines and growth factors. These results were further corroborated with specific CDK8 siRNA knockdown experiments and chromatin immunoprecipitation studies showing CDK8 occupancy on promoters of SEL120-34A regulated genes. In order to better characterize in vivo mechanism of action, mice bearing HCT116 and Colo205 xenograft tumors were treated with SEL120-34A and gene expression changes were measured with microarrays in excised tumors. In animals treated with the CDK8 inhibitor a dose dependent repression of STAT1 Ser727 was observed. The functional analyses of significantly (adj. p. value < 0.05) altered genes with Gene Ontology revealed that those with reduced expression belong to interferon I pathway and type I interferon-mediated signaling pathway terms. This subset of STAT regulated genes was further characterized as an interferon-related DNA damage resistance signature (IRDS) - a prosurvival pathway which correlated strongly with resistance to radiation and chemotherapy in various tumors. Consistently, SEL120-34A has shown very potent cytotoxic synergy with standard of care drugs in CRC, particularly in cells stimulated with interferons. Taken together, for the first time we have shown that selective CDK8 inhibitors are potent regulators of STAT related - IRDS signaling pathway in vitro and in vivo. In addition to previously reported stand-alone efficacy of CDK8 inhibitors in vivo, we provide also a combination treatment rationale for CRC. Female mice, SCID beige , 7-8 weeks old, were maintained according to the standards of the specific pathogen free conditions. Colo205 or HCT116 cells were centrifuged at 1000 rpm for 5 min and counted using Trypan blue method. A 5M cells were suspended in PBS and MatrigelÂ® (BD Biosciences) (3:1, v:v) and were injected subcutaneously (SC), on the right hind limb. As the tumor was maintained until it reached ~100-150 mm3. Mice were randomized into uniform groups and subjected to the compound administrations. Prior to use, compound was dissolved freshly in water and administered 30mg/kg BIDx3 per os using cannula in a volume of 10 ul per 1 g of body weight. Tumor samples were collected 4 and 16 hours after last compound dose and preserved in RNAlater.

Project description:CDK8 (cyclin-dependent kinase 8) is a kinase component of a multi - protein Mediator complex, involved in transcription control. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer (CRC). Herewith we present molecular insights into mechanism of action of SEL120-34A - a selective small molecule inhibitor of CDK8 kinase. Biochemical and binding studies indicated that SEL120-34A selectively binds and inhibits enzymatic activity of CDK8 in the low nM range. Recently CDK8 has been described as a regulator of STAT1 activity in NK cells where by phosphorylating STAT1 serine 727 (Ser727) influences a possible immunoescape mechanism in various cancers. Consistently, SEL120-34A and other recently reported selective CDK8 inhibitors could repress phosphorylation of STAT1 at a Ser727 at low nM concentrations in cancer cells without any significant changes on tyrosine sites directly regulated by JAK kinases. SEL120-34A inhibited expression of several STAT1 dependent genes in CRC cell lines, stimulated by various cytokines and growth factors. These results were further corroborated with specific CDK8 siRNA knockdown experiments and chromatin immunoprecipitation studies showing CDK8 occupancy on promoters of SEL120-34A regulated genes. In order to better characterize in vivo mechanism of action, mice bearing HCT116 and Colo205 xenograft tumors were treated with SEL120-34A and gene expression changes were measured with microarrays in excised tumors. In animals treated with the CDK8 inhibitor a dose dependent repression of STAT1 Ser727 was observed. The functional analyses of significantly (adj. p. value < 0.05) altered genes with Gene Ontology revealed that those with reduced expression belong to interferon I pathway and type I interferon-mediated signaling pathway terms. This subset of STAT regulated genes was further characterized as an interferon-related DNA damage resistance signature (IRDS) - a prosurvival pathway which correlated strongly with resistance to radiation and chemotherapy in various tumors. Consistently, SEL120-34A has shown very potent cytotoxic synergy with standard of care drugs in CRC, particularly in cells stimulated with interferons. Taken together, for the first time we have shown that selective CDK8 inhibitors are potent regulators of STAT related - IRDS signaling pathway in vitro and in vivo. In addition to previously reported stand-alone efficacy of CDK8 inhibitors in vivo, we provide also a combination treatment rationale for CRC.

Project description:ObjectiveThe paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input-output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding.MethodsUsing a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding.ResultsWe found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior.ConclusionsOur results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation.

Dataset Information

Functional Interfaces, Biological Pathways, and Regulations of Interferon-Related DNA Damage Resistance Signature (IRDS) Genes.

Publications

Functional Interfaces, Biological Pathways, and Regulations of Interferon-Related DNA Damage Resistance Signature (IRDS) Genes.

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