DYRK1B Inhibition for Prostate Cancer

DYRK1B 抑制前列腺癌

• 批准号: 10665942
• 负责人: Edward P Gelmann
• 金额: $ 15.35万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10665942
• 关键词: Affinity; Age Months; Amino Acids; Animals; Arizona; Biological Assay; Castration; Cell Differentiation process; Cells; Code; Collaborations; Cycloheximide; Development; Disease Resistance; Doctor of Philosophy; Dose; Drug Screening; Drug resistance; Dysplasia; Effectiveness; Enzymes; Epithelial Cell Proliferation; Epithelial Cells; Epithelium; Exposure to; Gatekeeping; Gene Deletion; Gene Expression; Genes; Goals; Growth; Growth Inhibitors; Half-Life; Hand; Heterozygote; Hour; In Vitro; Inhibition of Cell Proliferation; Intraepithelial Neoplasia; LNCaP; Letters; Malignant neoplasm of prostate; Missense Mutation; Mus; Mutation; NKX3-1 gene; Outsourcing; Pathologist; Pathway interactions; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Proliferating; Prostate; Prostate Cancer therapy; Prostatic Neoplasms; Prostatic hypertrophy; Proteins; Publishing; Qualifying; Residual state; Series; Serine; Specific qualifier value; Specificity; Study models; Substrate Interaction; Testing; Therapeutic; Tissues; Triage; Tumor Suppressor Proteins; Universities; advanced prostate cancer; castration resistant prostate cancer; cell growth; design; drug candidate; drug discovery; enzyme substrate; experimental study; first-in-human; growth inhibitory proteins; in vitro Assay; in vivo; inhibitor; morphogens; mouse model; neoplastic; neoplastic cell; novel; pharmacologic; pre-clinical; preclinical development; pressure; prostate cancer cell; prostate cancer cell line; prostate carcinogenesis; protein expression; small molecule; targeted treatment; therapeutic target; transcriptome sequencing

Background: DYRK1B, one of the kinases specified in this RFA, is the key regulator of steady state turnover of the gatekeeper prostate cancer suppressor protein NKX3.1. NKX3.1 loss occurs in the majority of prostate cancers and the NKX3.1 is the most frequently deleted gene in prostate cancer. NKX3.1 is a haploinsufficient protein and reduction of cellular protein levels by as little as 1/3 results in a neoplastic phenotype of prostate epithelial cells. However, some NKX3.1 expression is retained even in advanced prostate cancer cells so that the residual protein expression is exploited by pathologists as a tissue specific marker for prostate cancer. Moreover, NKX3.1 is a potent growth suppressor and differentiation factor. It follows that increasing NKX3.1 levels is a logical therapeutic strategy to reverse the neoplastic phenotype of prostate cancer. To prove the validity of this approach, we showed that whereas Nkx3.1+/- mice developed prostate hyperplasia and dysplasia within 6 months of age, loss of the single Nkx3.1 Dyrk1b phosphorylation site at serine 186 essentially reversed this phenotype in monoallelic Nkx3.1S186A/- mice. This remarkable finding provides preclinical justification to identify DYRK1B inhibitors for the treatment of prostate cancer. We have also demonstrated that short-term administration of a small molecule DYRK1B inhibitor to Nkx3.1+/- mice increased Nkx3.1 levels in prostate epithelial cells. Hypothesis: DYRK1B inhibition will increase intracellular Nkx3.1, resulting in retarded or reversed prostate carcinogenesis, epithelial cell differentiation, and prostate cancer growth inhibition. Experiments: In a collaboration with Chris Hulme, PhD, Director of the University of Arizona BIO5 Translational Drug Discovery Center, we will test a large panel of DYRK inhibitors from his lab. In the one year of this project, we will identify the most potent and highest affinity inhibitors of DYRK1B by in vitro assay in LNCaP cells by assessing inhibition of NKX3.1 degradation. NKX3.1 half-life is ~30 minutes. Therefore, screening drug candidates can be done by treating cycloheximide-exposed cells for up to 3 hours and assaying for NKX3.1 levels. In Aim 2 we will chose up to five of the agents most effective in vitro for administration over one week to Nkx3.1+/- mice at different doses to determine the potency of each to increase Nkx3.1 expression levels in vivo. To assure drug availability in vivo we will conduct, full KinomeScans™ and PK studies of each inhibitor to assure in vivo effectiveness. In addition, we will conduct RNAseq analysis of Nkx3.1+/- prostate tissue after one week of exposure to the most potent DYRK inhibitors to define their target pathways. As controls, the inhibitors will be administered over one week to Nkx3.1+/+ and Nkx3.1-/- mice to carry out RNAseq to identify on-target, off-Nkx3.1 gene expression effects and off-target effects. Comparison will also be made with prostate gene expression from untreated Nkx3.1S186A/- mice that have a missense mutation at the Dyrk1b phosphorylation site.

背景：DYRK 1B是RFA中指定的激酶之一，是稳态转换的关键调节因子， 守门人前列腺癌抑制蛋白NKX3.1。NKX3.1丢失发生在大多数前列腺中， NKX3.1是前列腺癌中最常见的缺失基因。NKX3.1是一个单倍不足 蛋白质和细胞蛋白质水平降低1/3导致前列腺的肿瘤表型 上皮细胞然而，即使在晚期前列腺癌细胞中，也保留了一些NKX3.1表达， 病理学家利用残余蛋白表达作为前列腺癌的组织特异性标记。 此外，NKX3.1是一种有效的生长抑制因子和分化因子。因此，增加NKX3.1 水平是逆转前列腺癌肿瘤表型的合理治疗策略。证明 为了验证这种方法的有效性，我们发现，尽管Nkx3.1+/-小鼠发生了前列腺增生和发育不良， 在6月龄内，Nkx3.1 Dyrk 1b丝氨酸186位磷酸化位点的缺失基本上逆转， 这种表型在单等位基因Nkx3.1S186A/-小鼠中。这一显著的发现为以下方面提供了临床前依据： 识别DYRK 1B抑制剂用于治疗前列腺癌。我们还证明，短期 向Nkx3.1+/-小鼠施用小分子DYRK 1B抑制剂增加前列腺中的Nkx3.1水平 上皮细胞 假设：DYRK 1B抑制将增加细胞内Nkx3.1，导致前列腺增生延迟或逆转。 致癌作用、上皮细胞分化和前列腺癌生长抑制。 实验：与亚利桑那大学生物转化中心主任Chris Hulme博士合作 药物发现中心，我们将测试一个大面板的DYRK抑制剂从他的实验室。在这个项目的一年里， 我们将通过在LNCaP细胞中的体外测定来鉴定最有效和最高亲和力的DYRK 1B抑制剂， 评估NKX3.1降解的抑制。NKX3.1半衰期约为30分钟。因此，筛选药物 通过处理暴露于环己酰亚胺的细胞长达3小时并测定NKX3.1， 程度.在目标2中，我们将选择最多五种体外最有效的药物，在一周内给药， Nkx3.1+/-小鼠以确定每种在体内增加Nkx3.1表达水平的效力。 为了确保药物在体内的可用性，我们将对每种抑制剂进行完整的KinomeScans™和PK研究，以确保 体内有效性。此外，我们将在施用Nkx 3.1 +/-前列腺组织一周后进行Nkx 3.1 +/-前列腺组织的RNAseq分析。 暴露于最有效的DYRK抑制剂，以确定其靶向途径。作为对照，将抑制剂 1 +/+和Nkx3.1-/-小鼠进行RNAseq，以鉴定中靶、脱Nkx3.1 基因表达效应和脱靶效应。还将与来自前列腺的前列腺基因表达进行比较。 在Dyrk 1b磷酸化位点具有错义突变的未处理的Nkx3.1S186A/-小鼠。