Mechanisms of caspase-2 activation by nucleophosmin in AML cell death and survival

项目摘要 儿童急性髓性白血病（AML）在治疗前的高复发率和治疗相关死亡率 对开发靶向治疗的迫切需求进行了评分。作为细胞遗传学中最常发生突变的基因之一， 对于遗传学正常的AML，核磷蛋白（NPM 1）代表了有希望的靶点。通常是一种核仁蛋白， NPM 1的定位导致在细胞质中的错误定位。NPM 1具有多种功能，可以预防癌症， 而突变形式与增加的化学敏感性有关。申请人先前报告说， NPM 1激活细胞核中的促凋亡蛋白酶和肿瘤抑制因子caspase-2。初步数据 来自申请人实验室的结果表明，核仁NPM 1依赖性的细胞凋亡不是诱导细胞凋亡， 半胱天冬酶-2激活复合物阻碍细胞分裂，甚至可能促进细胞存活。相反， 在NPM 1突变型AML细胞中，依赖于caspase-2。这表明，长期以来一直被控制的半胱天冬酶- 被认为是促凋亡的NPM 1具有促凋亡和促存活功能，这取决于NPM 1的位置。 定位在细胞中。核心假设是NPM 1介导的caspase-2调节是一个关键节点， AML细胞死亡和存活的调节，提供了化学敏感性的关键决定因素。这一假设 将通过追求三个具体目标进行测试：1）确定NPM 1如何激活caspase-2来调节感觉神经元的功能， AML中细胞凋亡和细胞周期阻滞的活性; 2）确定如何改变caspase-2活化的定位 影响AML对化疗的耐药性; 3）确定靶向caspase-3的治疗潜力。 AML中的2-NPM 1通路。在第一个目标下，CRISPR/Cas9将用于删除caspase-2，以确定 阻断caspase-2对NPM 1野生型DNA损伤诱导的细胞凋亡和细胞周期阻滞的影响 和突变亚型。在第二个目标下，细胞将被工程化以切换caspase-2的定位 从核仁到细胞质或从细胞质到核仁的激活。化疗的影响- 将测量灵敏度和电阻。在第三个目标下，抑制该途径对白血病的影响 将测量AML异种移植物的植入、进展和化学敏感性。这些研究将提供 NPM 1在调节AML化学敏感性中的作用的重要机制见解。最后，这些人-- 研究人员希望能够确定这一途径中降低白血病负荷的靶点，为合理治疗白血病提供候选药物。 用于AML治疗的药物设计。

PROJECT SUMMARY The high rate of recurrence and treatment-related mortalities in pediatric acute myeloid leukemia (AML) under- scores the critical need to develop targeted therapies. As one of the most frequently mutated genes in cytoge- netically normal AML, nucleophosmin (NPM1) represents a promising target. Normally a nucleolar protein, mu- tation of NPM1 leads to mislocalization in the cytoplasm. NPM1 has multiple functions that safeguard from cancer, while the mutant form is associated with increased chemosensitivity. The applicant previously reported that NPM1 activates the pro-apoptotic protease and tumor suppressor, caspase-2 in the nucleolus. Preliminary data from the applicant’s laboratory indicates rather than inducing apoptosis, the nucleolar NPM1-dependent caspase-2-activating complex impedes cell division and may even facilitate cell survival. In contrast, apoptosis in NPM1 mutant AML cells is dependent on caspase-2. This suggests that a caspase that has long been con- sidered to be pro-apoptotic has both pro-apoptotic and pro-survival functions depending on where NPM1 is localized in the cell. The central hypothesis is that NPM1-mediated regulation of caspase-2 is a critical node in the regulation of AML cell death and survival, providing a key determinant of chemosensitivity. This hypothesis will be tested by pursuing three specific aims: 1) Determine how NPM1 activates caspase-2 to modulate sensi- tivity to apoptosis and cell cycle arrest in AML; 2) Determine how altering the localization of caspase-2 activation impacts resistance to chemotherapy in AML; and 3) Determine the therapeutic potential of targeting the caspase- 2-NPM1 pathway in AML. Under the first aim, CRISPR/Cas9 will be used to delete caspase-2 to determine the effects of blocking caspase-2 on DNA damage-induced apoptosis and cell cycle arrest in the NPM1 wild type and mutant subtypes. Under the second aim, cells will be engineered to switch the localization of caspase-2 activation from the nucleolus to the cytoplasm or from the cytoplasm to the nucleolus. The impacts of chemo- sensitivity and resistance will be measured. Under the third aim, the impact of inhibiting this pathway on leukemia engraftment, progression and chemosensitivity of AML xenografts will be measured. These studies will provide important mechanistic insights into the role of NPM1 in modulating AML chemosensitivity. Ultimately, these stud- ies are expected to identify targets in this pathway that reduce leukemia burden, providing candidates for rational drug design for AML therapies.