DYRK1B Inhibition for Prostate Cancer

DYRK1B 抑制前列腺癌

基本信息

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
项目类别：
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

项目摘要

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：DYRK1B 是本 RFA 中指定的激酶之一，是稳态周转的关键调节因子。大多数前列腺中都会出现前列腺癌抑制蛋白 NKX3.1 的缺失。 NKX3.1 是前列腺癌中最常缺失的基因，NKX3.1 是单倍体不足。蛋白质和细胞蛋白质水平减少 1/3 会导致前列腺肿瘤表型然而，即使在晚期前列腺癌细胞中也保留了一些 NKX3.1 表达，因此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志物。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。为了验证这种方法的有效性，我们发现，尽管 Nkx3.1+/- 小鼠出现了前列腺增生和发育不良在 6 个月龄内，丝氨酸 186 处单个 Nkx3.1 Dyrk1b 磷酸化位点的丢失基本逆转单等位基因 Nkx3.1S186A/- 小鼠中的这种表型这一显着的发现为临床前的合理性提供了依据。我们还证明了 DYRK1B 抑制剂可以短期治疗前列腺癌。对 Nkx3.1+/- 小鼠施用小分子 DYRK1B 抑制剂可增加前列腺中的 Nkx3.1 水平上皮细胞。假设：DYRK1B 抑制会增加细胞内 Nkx3.1，导致前列腺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 BIO5 转化中心主任 Chris Hulme 博士合作药物发现中心，我们将在该项目的一年内测试他实验室的大量 DYRK 抑制剂。我们将通过 LNCaP 细胞的体外测定来鉴定最有效和最高亲和力的 DYRK1B 抑制剂评估NKX3.1降解的抑制作用，NKX3.1半衰期约为30分钟，因此，筛选药物。候选者可以通过处理暴露于放线菌酮的细胞长达 3 小时并检测 NKX3.1 来完成在目标 2 中，我们将选择最多 5 种体外最有效的药物，在一周内给药以达到最佳效果。使用不同剂量的 Nkx3.1+/- 小鼠来确定每种剂量在体内增加 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，以确保此外，我们将在一周后对Nkx3.1+/-前列腺组织进行RNAseq分析。暴露于最有效的 DYRK 抑制剂以确定其目标途径作为对照，抑制剂将是。对 Nkx3.1+/+ 和 Nkx3.1-/- 小鼠施用超过一周的时间，以进行 RNAseq 来鉴定中靶、脱靶 Nkx3.1 还将与前列腺基因表达进行比较。未经治疗的 Nkx3.1S186A/- 小鼠在 Dyrk1b 磷酸化位点存在错义突变。