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

项目摘要/摘要 PIK3CA是人类癌症中最常发生突变的癌基因。PI3Kα抑制剂是一种新的标准 CARE在PIK3CA突变ER+转移性乳腺癌(ER+MBC)和一些患者有持久的反应。 在这项建议中，我利用了我们最近发现的一种新的癌基因激活机制 PIK3CA突变。我们已经证明了双重PIK3CA突变在所有PIK3CA中都是频繁的 突变的癌症，发生在重复的氨基酸位置，并在同一等位基因上顺式。双重突变 在体外和体内激活PI3K信号和生长超过单个热点突变，以及生化 通过破坏P85抑制和增加膜结合来激活PI3K。双重突变增加 细胞和PI3CA突变ER+单核细胞白血病患者对PI3Kα抑制的敏感性。我们的工作揭示了一个 基于突变数的PI3K激活和抑制的分层模型(Vasan等人)。科学 2019年)。我将测试这个分层模型扩展到PI3K监管的假设，并与 雌激素受体，以及新的AKT底物的激活。我将使用重组双突变体PI3K 与RAS和RTK重组的复合体，以剖析调节机制(目标1.1)和 PIP3的细胞生成机制(目标1.2)。我将利用细胞敲门和过度表达 检测双突变ER+乳腺癌激活和抑制的模型(Aim 2.1)及其调控 ER依赖转录(目标2.2)。这些实验将在体外、体内、患者体内进行 样品，在PI3K抑制剂处理下。我将在双突变细胞上使用无偏向的磷酸蛋白质组学 确认新的AKT蛋白激酶底物(AIM 3.1)，包括组蛋白赖氨酸KMT家族 甲基转移酶(目标3.2)。这些目标加在一起将验证我们的癌基因激活分层模型 通过双PIK3CA突变体跨越广泛的细胞和生物过程，并将导致新的 抑制PI3K的策略包括在多个PIK3CA突变患者中测试PI3Kα抑制剂。我是一个 纪念斯隆·凯特琳癌症中心(MSKCC)乳房医学服务助理主治医生， 我已经勾勒出了一个5年的职业计划，这个计划建立在我学习结构生物学和我的 乳房肿瘤学临床培训。我已经组建了一支优秀的刘易斯·坎特利博士的指导团队 (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·莱文和科马尔·贾维里博士。他们是各自领域的国际知名科学家 将为我实现科学独立提供指导和支持。我将有机会访问无与伦比的 在MSKCC和威尔·康奈尔医学院的机构支持。这两家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。总体而言，这种培训环境将 使我能够实现我的目标，最终获得一个终身教职的独立职位。

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.