Investigating a hierarchical model for PI3K activation and inhibition in breast cancer by double PIK3CA mutations in cis

项目总结/摘要 PIK 3CA是人类癌症中最常见的突变癌基因。PI 3 K α抑制剂是一种新的标准， 在PIK 3CA突变型ER+转移性乳腺癌（ER+ MBC）中的护理中，一些患者具有持久的反应。 在这个提议中，我利用了我们最近发现的一种新的癌基因激活机制， PIK 3CA突变。我们已经证明，在所有PIK 3CA中，双PIK 3CA突变是常见的。 突变型癌症发生在重复氨基酸位置，并且在相同等位基因上顺式。双突变 在体外和体内激活PI 3 K信号传导并生长超过单个热点突变， 通过破坏p85抑制和增加膜结合来激活PI 3 K。双突变增加 细胞和PIK 3CA突变型ER+ MBC患者中对PI 3 K α抑制的敏感性。我们的工作揭示了 基于突变数的PI 3 K激活和抑制的分级模型（Vasan等人，Science 2019年）。我将测试这个分层模型扩展到PI 3 K调节的假设， 雌激素受体和新的AKT底物的活化。我将使用重组双突变PI 3 K 用Ras和RTK重建复合物，以剖析调控机制（目的1.1）， PIP 3的细胞生成机制（目的1.2）。我会利用基因敲入和细胞过度表达 用于测量双突变ER+乳腺癌的激活和抑制的模型（目标2.1）和 ER依赖性转录（目的2.2）。这些实验将在体外、体内、患者体内进行。 样品，并且在PI 3 K抑制剂处理下。我将在双突变细胞上使用无偏磷酸蛋白质组学 确认新的AKT蛋白激酶底物（Aim 3.1），包括组蛋白赖氨酸的KMT家族 甲基转移酶（目标3.2）。这些目标将共同验证我们的致癌基因激活的层次模型 通过双PIK 3CA突变体在各种各样的细胞和生物学过程，并将导致新的 抑制PI 3 K的策略，包括在多个PIK 3CA突变患者中测试PI 3 K α抑制剂。我是一个 纪念斯隆凯特琳癌症中心（MSKCC）乳腺医学服务助理主治医师， 我已经概述了一个5年的职业计划，该计划建立在我学习结构生物学的背景和我的 乳腺肿瘤临床培训。我召集了一个优秀的导师团队刘易斯坎特利博士 （主要导师）和Maurizio Scaltriti博士（共同导师）。我的顾问委员会将包括尼尔罗森博士， 罗斯·莱文和科玛尔·贾韦里博士他们是各自领域的国际知名科学家， 将为我提供指导和支持，以实现科学独立。我将有机会获得无与伦比的 MSKCC和威尔康奈尔医学院的机构支持。这两个机构都处于领先地位， 癌症研究，并在职业发展方面投入巨资。总的来说，这种培训环境将 使我能够实现我的目标，最终确保终身制轨道独立的立场。

PROJECT SUMMARY/ABSTRACT PIK3CA is the most frequently mutated oncogene in human cancer. PI3Kα inhibitors are a new standard of care in PIK3CA mutant ER+ metastatic breast cancer (ER+ MBC) and some patients have durable responses. In this proposal, I capitalize on our recent discovery of a novel mechanism of oncogene activation by double PIK3CA mutations. We have demonstrated that double PIK3CA mutations are frequent across all PIK3CA mutant cancers, occur at recurrent amino acid positions, and are in cis on the same allele. Double mutations activate PI3K signaling and growth more than single hotspot mutations in vitro and in vivo, and biochemically activate PI3K through disruption of p85 inhibition and increased membrane binding. Double mutations increase sensitivity to PI3Kα inhibition in cells and in PIK3CA mutant ER+ MBC patients. Our work has uncovered a hierarchical model for the activation and inhibition of PI3K based on mutation number (Vasan, et al. Science 2019). I will test the hypotheses that this hierarchical model extends to PI3K regulation, crosstalk with the estrogen receptor, and activation of novel AKT substrates. I will use recombinant double mutant PI3K complexes reconstituted with Ras and RTK to dissect the mechanisms of regulation (Aim 1.1) and the mechanisms of cellular generation of PIP3 (Aim 1.2). I will leverage knockin and overexpression cellular models to measure activation and inhibition of double mutant ER+ breast cancer (Aim 2.1) and modulation of ER-dependent transcription (Aim 2.2). These experiments will be performed in vitro, in vivo, in patient samples, and under PI3K inhibitor treatment. I will utilize unbiased phosphoproteomics on double mutant cells to credential new AKT protein kinase substrates (Aim 3.1) including the KMT family of histone lysine methyltransferases (Aim 3.2). Together these aims will validate our hierarchical model of oncogene activation by double PIK3CA mutants across a wide variety of cellular and biological processes and will lead to new strategies to inhibit PI3K including testing PI3Kα inhibitors in multiple PIK3CA mutant patients. I am an Assistant Attending with the Breast Medicine Service at Memorial Sloan Kettering Cancer Center (MSKCC), and I have outlined a 5-year career plan that builds upon my background studying structural biology and my clinical training in breast oncology. I have assembled an outstanding mentoring team of Dr. Lewis Cantley (primary mentor) and Dr. Maurizio Scaltriti (co-mentor). My advisory committee will include Dr. Neal Rosen, Dr. Ross Levine, and Dr. Komal Jhaveri. They are internationally renowned scientists in their respective fields who will provide me the mentorship and support to attain scientific independence. I will have access to unparalleled institutional support at MSKCC and Weill Cornell Medical College. Both institutions are at the leading edge of cancer research and are heavily invested in career development. Collectively, this training environment will enable me to achieve my goals of ultimately securing a tenure-track independent position.