Structural and molecular determinants of protein phosphatase 2A function in lung cancer

项目摘要/摘要 肺癌的进展涉及癌基因和肿瘤抑制功能的协调变化。蛋白 磷酸酶2A(PP2A)是一种在肺癌中调节失调和失活的肿瘤抑制因子。PP2A是 通过几种机制失活，包括体细胞突变，抑制个别亚基， 内源性PP2A抑制剂的表达增加和C蛋白翻译后修饰的变化 亚单位。虽然已经开发了几种治疗方法来间接针对PP2A，但最近的研究已经 发现了直接激活PP2A的小分子，如吩噻嗪。然而，因为他们的 明显的锥体外系和抗胆碱能作用是严重的剂量限制，进一步追求 吩噻嗪和相关的二苯并氮卓类药物似乎不太可能用于癌症的治疗。我们的实验室已经重新设计了 将中枢神经系统药理与该药物的抗增殖特性脱钩的三环抗神经药- 班级。我们已经开发了这些PP2A的小分子激活剂(SMAP)，并将其表征为 探索PP2A调节和治疗PP2A依赖型疾病的工具。我们的组合结构- 功能研究表明，SMAP以一种保护PP2A调控C末端尾巴的方式与PP2A结合 催化亚基和稳定的全酶。我们的目标是定义SMAP诱导的PP2A改变 促进PP2A抑瘤性能的全酶组合物。我们的目标是确定肿瘤 SMAP增强的抑制PP2A调节亚基与临床疗效的相关性 和遗传特征，并帮助设计更具选择性的下一代SMAP。在这个项目中， 设计了三个可靠的目标来评估使用SMAP激活PP2A如何代表有效的治疗 诊断为非小细胞肺癌患者的选择。目标1将从结构和功能上描述生理学 以及SMAP对PP2A的翻译后调控。我们将调查特定于SMAP的 不同的肿瘤复发突变通过偶联对PP2A全酶的结构调控 基于甲基-PP2A-C抗体和免疫沉淀-质谱法的后遗症定量方法 翻译修饰和PP2A活性。目标2将弄清PP2Aα突变如何调节生物 SMAP的活动。我们将评估常见错义突变在肺癌中的功能作用 等基因CRISPR/Cas9细胞系鉴定细胞表型、亚单位表达、SMAP反应和 裸鼠的生物学反应。目标3将确定促进消退的联合治疗策略 在KRAS突变肺癌中的肿瘤。我们将基于以下方面研究SMAP的合理治疗组合 我们的研究表明，蛋白磷酸酶2A(PP2A)活性决定了KRAS突变体的反应 跨越&gt；200激酶抑制剂库的肺癌细胞。总而言之，这些研究将揭示对 肺内PP2A调控的分子基础及PP2A激活/失活的功能后果 癌症。

Project Summary/Abstract Lung cancer progression involves coordinate changes in both oncogene and tumor suppressor function. Protein Phosphatase 2A (PP2A) is a tumor suppressor that is dysregulated and deactivated in lung cancer. PP2A is inactivated through several mechanisms including somatic mutations, suppression of individual subunits, increased expression of endogenous PP2A inhibitors and changes in post-translational modifications of the C subunit. While several therapeutics have been developed to indirectly target PP2A, recent studies have uncovered small molecules such as phenothiazines that directly activate PP2A. Nevertheless, because their pronounced extrapyramidal and anti-cholinergic effects were severely dose limiting, further pursuit of phenothiazines and related dibenzazepines for treatment of cancer seems unlikely. Our lab has reengineered the tricyclic neuroleptics to decouple the CNS pharmacology from the anti-proliferative properties of this drug- class. We have developed and characterized these small molecule activators of PP2A (SMAPs) as both chemical tools to probe PP2A regulation and for the treatment of PP2A dependent diseases. Our combined structure- function studies have revealed that SMAPs bind to PP2A in a way that protects the regulatory C-terminal tail of the catalytic subunit and stabilizes the holoenzyme. Our goal is to define the SMAP induced alterations on PP2A holoenzyme composition that promote the tumor suppressive properties of PP2A. We aim to identify the tumor suppressive PP2A regulatory subunits enhanced by SMAPs in order to correlate treatment response to clinical and genetic characteristics, and to aid in the design of more selective, next-generation SMAPs. In this project, three robust aims are designed to assess how activation of PP2A using SMAPs represents an effective treatment option for patients diagnosed with NSCLC. Aim 1 will structurally and functionally characterize the physiologic and cancer-relevant posttranslational regulation of PP2A by SMAPs. We will investigate the SMAPs-specific structural modulation of the PP2A holoenzyme by various, recurrent tumor derived mutations through coupling methyl-PP2A-C antibody and immunoprecipitation-mass spectrometry based approaches to quantify post- translational modification and PP2A activity. Aim 2 will discern how PP2A Aα mutations modulate the biological activity of SMAPs. We will assess the functional role of common missense mutations in lung cancer by using isogenic CRISPR/Cas9 cell lines to identify cellular phenotypes, subunit expression, SMAPs response, and biological responses in nude mice. Aim 3 will determine combination treatment strategies to promote regression of tumors in KRAS mutant lung cancer. We will examine rational treatment combinations with SMAPs based on our studies demonstrating that protein phosphatase 2A (PP2A) activity defines the response of KRAS mutant lung cancer cells across library of >200 kinase inhibitors. Collectively, these studies will reveal novel insights into the molecular basis of regulation of PP2A and functional consequences of PP2A activation/inactivation in lung cancer.