Cyclin C-CDK8/19 kinases in development and in cancer

发育和癌症中的细胞周期蛋白 C-CDK8/19 激酶

• 批准号: 10415467
• 负责人: Peter Sicinski
• 金额: $ 52.6万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10415467
• 关键词: Ablation; Address; Affect; Alanine; Allografting; Alpha Interleukin 2 Receptor; Amino Acids; Animals; Antitumor Response; Binding; Biochemical; CD8-Positive T-Lymphocytes; CD8B1 gene; CLN1 gene; CLN2 gene; CLN3 gene; Cancer Model; Cancer Patient; Catalytic Domain; Cell Culture Techniques; Cell Lineage; Cells; Clinical; Clinical Trials; Complementary DNA; Complex; Cultured Cells; Cyclin-Dependent Kinases; Cyclins; Cytotoxic T-Lymphocytes; Development; Endothelial Cells; Enterobacteria phage P1 Cre recombinase; Gatekeeping; Genetic Transcription; Growth; Half-Life; Human; IL2RA gene; IL2RB gene; Immune; Immune response; Immune system; Immunocompromised Host; In Vitro; Interleukin 2 Receptor; Interleukin-2; Interleukin-4; Knockout Mice; Laboratories; Link; LoxP-flanked allele; Lymphoid Cell; Malignant Neoplasms; Maps; Mediating; Mediator of activation protein; Memory; Modality; Molecular; Mus; Mutation; NKTR gene; Oncogenes; Outcome; Patients; Peripheral; Phosphorylation; Phosphotransferases; Play; Polyubiquitination; Proteins; RNA Polymerase II; Regulatory T-Lymphocyte; Research; Role; Saccharomyces cerevisiae; Signal Transduction; Site; T cell differentiation; T-Cell Development; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Thymus Gland; Transcript; Tumor Immunity; Tumor stage; Up-Regulation; Wild Type Mouse; Work; Xenograft procedure; Yeasts; ZNF145 gene; adaptive immune response; adaptive immunity; anti-CTLA4 antibodies; anti-PD-1; anti-cancer; anti-tumor immune response; antitumor effect; cancer cell; cancer therapy; cyclin C; cyclin G1; effector T cell; experimental study; genetic approach; immune checkpoint blockade; in vivo; inhibitor; mouse genetics; novel; overexpression; paralogous gene; peripheral lymphoid organ; protein complex; small molecule; small molecule inhibitor; systemic toxicity; therapeutic target; thymocyte; transcription factor; tumor; tumorigenesis; ubiquitin-protein ligase

Project Summary/Abstract Cyclin C was cloned, along with other G1 cyclins, as a cDNA which can rescue the proliferative block in yeast Saccharomyces cerevisiae lacking CLN1, CLN2 and CLN3 genes. Subsequent work revealed that cyclin C, acting together with its kinase partners, the cyclin-dependent kinase 8 (CDK8) and CDK19, plays a role in regulating gene transcription. Cyclin C serves as a regulatory partner of CDK8 and CDK19, and activates the kinase activity of these proteins. The two paralog kinases show substantial overall amino acid identity, particularly within their catalytic domains. Cyclin C, CDK8 and CDK19 represent parts of the mediator complex, a large multisubunit protein complex that regulates gene transcription by linking RNA polymerase II to sequence-specific transcription factors. In addition, cyclin C-CDK8/19 was shown to phosphorylate various transcription factors and to regulate their stability and activity. Growing evidence indicates that cyclin C-CDK8 and C-CDK19 kinases may represent potential anti-cancer targets. CDK8 has been identified as an oncogene in several human cancers. Cyclin C, CDK8 and CDK19 are overexpressed in a wide range of tumor types. Importantly, higher expression of these three proteins was found to be associated with poor clinical outcome. Consistent with tumor-promoting roles for cyclin C-CDK8/19, treatment of mice bearing xenografts of several human tumor types with small molecule CDK8/19 inhibitors resulted in a potent anti-tumor effect without apparent systemic toxicity. To understand the molecular function of cyclin C in vivo, we generated conditional cyclin C knockout mice (cyclin CF/F). To test the impact of cyclin C-CDK8/19 inhibition on T cell development and adaptive anti-tumor immunity, we crossed cyclin CF/F mice with CD4-Cre animals. The latter strain expresses Cre recombinase at an early stage of T cell development, and drives deletion of the ‘floxed’ sequences in all T cell subsets. In our preliminary analyses we established that cyclin C functions as a gate- keeper of T cell differentiation, likely by directly phosphorylating a lineage-specific transcription factor. By doing so, cyclin C affects signaling networks that regulate the immune response. Hence, this novel function of cyclin C may have a profound effect on the anti-tumor immune response and on tumorigenesis. In the proposed work we will extend these studies. In Aim 1, we will study the exact molecular function of cyclin C- CDK8 and C-CDK19 in the T cell lineage using a combination of biochemical in vitro studies, cell culture experiments, analyses of ex vivo cultured T cells as well as mouse genetic approaches. In Aim 2, we will study how ablation of cyclin C impacts tumorigenesis in vivo, using mouse cancer models and combination treatments with other anti-cancer modalities. In Aim 3, we will extend these cancer studies to cyclin C catalytic partners, using conditional Cdk19 knockout mice (Cdk19F/F), that my laboratory generated for this purpose. The expected overall impact of this proposal is that it will reveal a novel and important function of cyclin C- CDK8 and C-CDK19 in tumorigenesis, and will validate these kinases as attractive therapeutic targets.

项目摘要/摘要 Cyclin C与其他G1期细胞周期蛋白一起被克隆出来，作为一种能够挽救酵母细胞增殖障碍的基因 缺乏CLN1、CLN2和CLN3基因的酿酒酵母。随后的研究发现，细胞周期蛋白C， 细胞周期蛋白依赖性激酶8(CDK8)和CDK19与其蛋白激酶伙伴共同作用，在细胞周期中发挥作用。 调节基因转录。细胞周期蛋白C是CDK8和CDK19的调控伙伴，并激活 这些蛋白的激酶活性。这两个Paralog激酶显示出基本的整体氨基酸同源性， 特别是在它们的催化区域内。细胞周期蛋白C、CDK8和CDK19代表部分介体 复合体，一种大的多亚单位蛋白质复合体，通过将RNA聚合酶II连接到 序列特异性转录因子。此外，细胞周期蛋白C-CDK8/19被证明能够磷酸化各种 转录因子并调节其稳定性和活性。越来越多的证据表明，细胞周期蛋白C-CDK8 C-CDK19可能是潜在的抗癌靶点。CDK8已被确定为癌基因 在几种人类癌症中。细胞周期蛋白C、CDK8和CDK19在多种肿瘤类型中均有过表达。 重要的是，这三种蛋白的高表达被发现与较差的临床结果相关。 与细胞周期蛋白C-CDK8/19的促瘤作用一致，治疗荷瘤鼠的几种 使用小分子CDK8/19抑制剂的人类肿瘤类型在没有抑制的情况下具有强大的抗肿瘤作用 明显的全身毒性。为了了解细胞周期蛋白C在体内的分子功能，我们产生了条件 Cyclin C基因敲除小鼠(Cyclin Cf/F)。检测细胞周期蛋白C-CDK8/19抑制对T细胞发育的影响 和适应性抗肿瘤免疫，我们将Cyclin CF/F小鼠与CD4-Cre小鼠杂交。后一种菌株 在T细胞发育的早期阶段表达Cre重组酶，并驱动‘小花’的缺失 所有T细胞亚群中的序列。在我们的初步分析中，我们确定了细胞周期蛋白C的功能是一个门-- T细胞分化的保持者，可能是通过直接磷酸化一种谱系特异的转录因子。通过 这样一来，细胞周期蛋白C就会影响调节免疫反应的信号网络。因此，这一新的功能 细胞周期蛋白C可能在抗肿瘤免疫反应和肿瘤发生中发挥重要作用。在 建议的工作我们将延长这些研究。在目标1中，我们将研究细胞周期蛋白C-的确切分子功能。 CDK8和C-CDK19在T细胞谱系中的应用结合生化体外研究、细胞培养 实验，体外培养的T细胞的分析以及小鼠的遗传方法。在目标2中，我们将研究 用小鼠肿瘤模型及其组合研究细胞周期蛋白C的消融对体内肿瘤形成的影响 用其他抗癌方法进行治疗。在目标3中，我们将把这些癌症研究扩展到周期蛋白C催化 合作伙伴，使用我的实验室为此目的而培育的有条件的CDK19基因敲除小鼠(CDK19F/F)。 这一提议的预期总体影响是，它将揭示细胞周期蛋白C的一个新颖而重要的功能-- CDK8和C-CDK19在肿瘤发生中的作用，并将验证这些激酶作为有吸引力的治疗靶点。