Validation and Development of WEE1 as a therapeutic target in AML

描述(由申请人提供)：急性髓系白血病(AML)患者的存活率仍然很低，尽管关于导致AML的分子事件的知识在过去十年中发展迅速。这在一定程度上是因为在细胞和分子水平上对化疗的反应缺乏明显的了解。以前的工作是针对开发新的、合理的联合疗法来改善急性髓细胞白血病患者的预后的长期目标。这个特定项目的目标是提供一种这样的治疗策略的临床前验证，将抗代谢化疗药物与抑制细胞周期检查点蛋白Wee1结合起来。该项目的中心假设是Wee1是白血病细胞存活的关键介质，作用于INS期以确保准确的DNA复制，并且联合抑制Wee1和抗代谢药物将比单独使用抗代谢药物更有效地清除白血病细胞。这一假说源于我们来自全基因组shRNA筛查的初步数据，在该筛查中，我们确定Wee1是阿糖胞苷治疗后AML细胞死亡的关键介质。我们已经在一系列急性髓细胞白血病细胞系中验证了这些发现，并发现抑制Wee1与阿糖胞苷联合使用的效果涉及消除阿糖胞苷诱导的S期检查点。值得注意的是，初步数据显示，具有I类白血病突变的细胞对Wee1单独抑制非常敏感，表明Wee1可能有助于白血病的发生。这一假设将以三个具体目标进行验证：1)确定Wee1是否有助于白血病的发生；2)确认阿糖胞苷和Wee1抑制在体内的有效性和耐受性；3)确定阿糖胞苷和Wee1抑制增强AML细胞杀伤的机制。为了实现目标1，我们将研究I类和II类突变的表达和Wee1抑制的后果，因为它们与骨髓前体细胞的致癌压力有关，并对AML骨髓活检标本中Wee1的表达进行免疫组织化学检测。为了实现目标2，将使用包括直接患者异种移植在内的AML小鼠模型来测试阿糖胞苷与Wee1抑制联合应用的药效学、耐受性和有效性。为了实现目标3，将使用药理学和遗传抑制来测试细胞周期检查点级联中上游、下游和与Wee1平行的蛋白质的功能意义，以确定涉及的关键分子；将使用互补的细胞和分子分析来进一步确定组合细胞杀伤的机制。该项目在1)Wee1促进白血病发生的研究，2)Flt3-ITD+细胞非癌基因脆弱性的研究，3)AML治疗新靶点的开发，4)白血病患者直接异种移植的翻译模型的建立，以及5)高通量方法学研究DNA损伤修复蛋白在特定环境中的作用方面具有创新性。这项拟议的研究具有重要意义，因为它有望促进对急性髓细胞白血病新的治疗靶点的理解，该靶点可以迅速转化为结合标准和靶向治疗的临床试验。

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.