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

项目概要/摘要 肺癌的进展涉及癌基因和抑癌基因功能的协调变化。蛋白 磷酸酶2A（PP 2A）是一种肿瘤抑制因子，在肺癌中失调和失活。PP 2A是 通过几种机制失活，包括体细胞突变，抑制单个亚基， 内源性PP 2A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间接靶向PP 2A的治疗剂，但最近的研究表明， 发现了一些小分子，如吩噻嗪，可以直接激活PP 2A。然而，因为他们的 明显的锥体外系和抗胆碱能作用严重限制剂量，进一步研究 吩噻嗪类和相关的二苯并氮杂类用于治疗癌症似乎不太可能。我们的实验室重新设计了 三环类神经抑制剂，使CNS药理学与该药物的抗增殖特性脱钩- 课我们已经开发并表征了PP 2A的这些小分子活化剂（SMAPs）， 研究PP 2A调节和治疗PP 2A依赖性疾病的工具。我们的组合结构- 功能研究表明，SMAPs与PP 2A结合的方式可以保护PP 2A的调节C末端尾， 催化亚基并稳定全酶。我们的目标是确定SMAP诱导的PP 2A的改变 促进PP 2A肿瘤抑制特性的全酶组合物。我们的目标是找出肿瘤 抑制性PP 2A调节亚基由SMAPs增强，以便将治疗反应与临床 和遗传特征，并帮助设计更具选择性的下一代SMAPs。在这个项目中， 设计了三个强有力的目标来评估使用SMAPs激活PP 2A如何代表有效的治疗方法 诊断为NSCLC的患者的选择。目的1将在结构和功能上表征生理 以及SMAPs对PP 2A的癌症相关翻译后调节。我们将调查特定于SMAP的 PP 2A全酶通过偶联的各种复发性肿瘤衍生突变的结构调节 甲基-PP 2A-C抗体和基于免疫沉淀-质谱法的方法来定量 翻译修饰和PP 2A活性。目的2将阐明PP 2AA α突变如何调节生物学特性。 SMAPS的活动。我们将通过以下方法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，以鉴定细胞表型、亚基表达、SMAPs应答和 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 KRAS突变型肺癌中的肿瘤。我们将根据以下因素检查SMAPs的合理治疗组合： 我们的研究表明，蛋白磷酸酶2A（PP 2A）活性决定了KRAS突变体的反应， 肺癌细胞中的多200激酶抑制剂的文库。总的来说，这些研究将揭示新的见解， PP 2A调节的分子基础和肺中PP 2A活化/失活的功能后果 癌

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.