Validation and Development of WEE1 as a therapeutic target in AML

WEE1 作为 AML 治疗靶点的验证和开发

基本信息

批准号：
8688970
负责人：
CHRISTOPHER C PORTER
金额：
$ 30.56万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8688970
关键词：
Acute Myelocytic Leukemia Animal Model Anthracyclines Antimetabolites Antineoplastic Agents Biological Assay Biological Markers Biopsy Specimen Blast Cell Bone marrow biopsy CDC2 Protein Kinase Cell Cycle Checkpoint Cell Death Cell Line Cell Survival Cells Clinic Clinical Trials Combination Drug Therapy Combined Modality Therapy Cytarabine Cytotoxic Chemotherapy DNA Damage DNA Repair DNA biosynthesis Data Development Disease remission Dose Drug Targeting Ensure Event FLT3 gene Failure Genetic Goals Heterogeneity Immunohistochemistry Knowledge Lead Mediator of activation protein Methodology Methods Mitosis Modeling Molecular Molecular Abnormality Mutation Myeloid Progenitor Cells Oncogenic Outcome Patients Pharmaceutical Preparations Pharmacodynamics Phase Proteins Public Health Recovery Relapse Remission Induction Research Resistance Role S Phase Stress Surveys Survival Rate Testing Therapeutic Time Translating Translations Treatment Protocols Validation Work Xenograft procedure cancer diagnosis cancer therapy cell killing chemotherapy combinatorial cytotoxic design ds-DNA genome-wide improved in vivo in vivo Model inhibitor/antagonist innovation leukemia leukemogenesis mouse model new therapeutic target novel novel therapeutics pre-clinical public health relevance response small hairpin RNA therapeutic target treatment strategy

项目摘要

DESCRIPTION (provided by applicant): Survival rates for patients with acute myeloid leukemia (AML) remain low, despite the fact that knowledge of the molecular events that cause AML has evolved rapidly in the last decade. This is in part due to a distinct lack of understanding of the response to chemotherapy at the cellular and molecular levels. Previous work has been directed toward the long term goal of developing novel, rational combination-therapies to improve the outcomes of patients with AML. The objective of this specific project is to provide pre-clinical validation of one such therapeutic strategy, combining antimetabolite chemotherapeutics with inhibition of Wee1, a cell cycle checkpoint protein. The central hypothesis of this project is that Wee1 is a critical mediator of leukemia cell survival, acting inS phase to ensure accurate DNA replication, and that combining inhibition of Wee1 with antimetabolites will be more effective in eliminating leukemia cells than antimetabolites alone. This hypothesis emanates from our preliminary data from a genome-wide shRNA screen, in which we identified Wee1 as a critical mediator of AML cell death after cytarabine treatment. We have validated these findings in a broad panel of AML cell lines and found that the effect of inhibiting Wee1 in combination with cytarabine involves abrogation of the S phase checkpoint induced by cytarabine. Notably, preliminary data show that cells with Class I leukemogenic mutations are quite sensitive to Wee1 inhibition alone and suggest that Wee1 may contribute to leukemogenesis. The hypothesis will be tested with three specific aims: 1) Determine whether Wee1 contributes to leukemogenesis, 2) Confirm the efficacy and tolerability of cytarabine and Wee1 inhibition in vivo and 3) Identify the mechanism of enhanced AML cell killing with cytarabine and Wee1 inhibition. To achieve Aim 1, we will examine the consequences of Class I and II mutation expression and Wee1 inhibition as they relate to oncogenic stresses in myeloid progenitor cells and perform immunohistochemistry for Wee1 expression in AML bone marrow biopsy specimens. To achieve Aim 2, mouse models of AML, including direct patient xenografts will be used to test the pharmacodynamics, tolerability and efficacy of combining cytarabine with Wee1 inhibition. To achieve Aim 3, pharmacologic and genetic inhibition will be used to test the functional significance of proteins upstream, downstream and in parallel with Wee1 in cell cycle checkpoint cascades to identify the key molecules involved; complementary cellular and molecular assays will be used to further define the mechanism of combinatorial cell killing. The project is innovative in 1) the study of Wee1 contributing to leukemogenesis, 2) the study of non-oncogene vulnerabilities of FLT3-ITD+ cells, 3) the development of a novel therapeutic target in AML, 4) translational modeling of leukemia with direct patient xenografts, and 5) high-throughput methodology to survey the role of DNA damage repair proteins in specific contexts. The proposed research is significant because it is expected to advance the understanding of a novel therapeutic target in AML that can be rapidly translated into clinical trials combining standard and targeted therapy.

描述（由申请人提供）：尽管对导致 AML 的分子事件的认识在过去十年中迅速发展，但急性髓系白血病 (AML) 患者的生存率仍然很低。这部分是由于对细胞和分子水平上化疗的反应明显缺乏了解。先前的工作致力于开发新颖、合理的联合疗法以改善 AML 患者的预后的长期目标。该特定项目的目标是为此类治疗策略提供临床前验证，将抗代谢化疗药物与细胞周期检查点蛋白 Wee1 的抑制相结合。该项目的中心假设是，Wee1 是白血病细胞存活的关键介质，在 S 期发挥作用以确保 DNA 的准确复制，并且将 Wee1 抑制与抗代谢药物相结合将比单独使用抗代谢药物更有效地消除白血病细胞。这一假设源自我们全基因组 shRNA 筛选的初步数据，其中我们确定 Wee1 是阿糖胞苷治疗后 AML 细胞死亡的关键介质。我们在大量 AML 细胞系中验证了这些发现，并发现联合阿糖胞苷抑制 Wee1 的效果涉及废除阿糖胞苷诱导的 S 期检查点。值得注意的是，初步数据表明，具有 I 类致白血病突变的细胞对单独抑制 Wee1 非常敏感，这表明 Wee1 可能有助于白血病的发生。该假设将通过三个具体目标进行测试：1) 确定 Wee1 是否有助于白血病发生，2) 确认阿糖胞苷和 Wee1 抑制在体内的功效和耐受性，以及 3) 确定阿糖胞苷和 Wee1 抑制增强 AML 细胞杀伤作用的机制。为了实现目标 1，我们将检查 I 类和 II 类突变表达以及 Wee1 抑制的后果，因为它们与骨髓祖细胞中的致癌应激有关，并对 AML 骨髓活检标本中的 Wee1 表达进行免疫组织化学分析。为了实现目标 2，AML 小鼠模型（包括直接患者异种移植物）将用于测试阿糖胞苷与 Wee1 抑制相结合的药效学、耐受性和功效。为了实现目标3，将利用药理学和遗传抑制来测试细胞周期检查点级联中上游、下游以及与Wee1平行的蛋白质的功能意义，以识别所涉及的关键分子；互补的细胞和分子测定将用于进一步确定组合细胞杀伤机制。该项目的创新之处在于：1) Wee1 促进白血病发生的研究，2) FLT3-ITD+ 细胞的非癌基因脆弱性研究，3) AML 中新治疗靶点的开发，4) 直接用于白血病的转化模型。患者异种移植物，以及 5) 高通量方法来调查 DNA 损伤修复蛋白在特定情况下的作用。拟议的研究意义重大，因为它有望促进对 AML 新型治疗靶点的理解，该靶点可以快速转化为标准治疗和靶向治疗相结合的临床试验。