Genetic and Pharmacological Manipulation of KSR in KRAS-driven Cancer

KSR 在 KRAS 驱动的癌症中的遗传和药理学操作

基本信息

批准号：
10207543
负责人：
Alexander Real
金额：
$ 5.1万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-02 至 2022-07-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10207543
关键词：
A549 Adenocarcinoma Affect Affinity Aftercare BRAF gene Binding Binding Sites Biochemical Biological Assay Cancer Patient Cancer cell line Cell Line Cell Survival Cells Cessation of life Chemicals Clinic Consequentialism Data Dependence Development Disease Exhibits Feedback Future Gatekeeping Genetic Genetic study Goals Growth Homologous Gene Investigation KRAS oncogenesis KRAS2 gene KSR gene Lead Length Lentivirus Vector Libraries Link MEKs Malignant Neoplasms Mediating Medicine Methods Mitogen-Activated Protein Kinase Inhibitor Mitogen-Activated Protein Kinases Mitogens Mutation Oncogenic Pathway interactions Patients Pharmaceutical Preparations Pharmacology Phosphorylation Phosphotransferases Physicians Polyubiquitination Positive Test Result Protac Protein Family Proteins Proteolysis Ras Inhibitor Ras Signaling Pathway Resistance Scaffolding Protein Scientist Serine Signal Pathway Signal Transduction Structure Synthesis Chemistry Testing Therapeutic Training Transfection Ubiquitination Validation Work analog assay development biochemical tools cancer cell design driver mutation drug discovery enzyme activity experimental study improved in vivo inducible gene expression inhibitor/antagonist knock-down multiplex assay mutant new therapeutic target next generation novel novel strategies overexpression prevent protein protein interaction recruit scaffold screening skills small molecule success synergism therapeutic development tool tumorigenesis ubiquitin-protein ligase

A549 Adenocarcinoma Affect Affinity Aftercare BRAF gene Binding Binding Sites Biochemical Biological Assay Cancer Patient Cancer cell line Cell Line Cell Survival Cells Cessation of life Chemicals Clinic Consequentialism Data Dependence Development Disease Exhibits Feedback Future Gatekeeping Genetic Genetic study Goals Growth Homologous Gene Investigation KRAS oncogenesis KRAS2 gene KSR gene Lead Length Lentivirus Vector Libraries Link MEKs Malignant Neoplasms Mediating Medicine Methods Mitogen-Activated Protein Kinase Inhibitor Mitogen-Activated Protein Kinases Mitogens Mutation Oncogenic Pathway interactions Patients Pharmaceutical Preparations Pharmacology Phosphorylation Phosphotransferases Physicians Polyubiquitination Positive Test Result Protac Protein Family Proteins Proteolysis Ras Inhibitor Ras Signaling Pathway Resistance Scaffolding Protein Scientist Serine Signal Pathway Signal Transduction Structure Synthesis Chemistry Testing Therapeutic Training Transfection Ubiquitination Validation Work analog assay development biochemical tools cancer cell design driver mutation drug discovery enzyme activity experimental study improved in vivo inducible gene expression inhibitor/antagonist knock-down multiplex assay mutant new therapeutic target next generation novel novel strategies overexpression prevent protein protein interaction recruit scaffold screening skills small molecule success synergism therapeutic development tool tumorigenesis ubiquitin-protein ligase

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项目摘要

Project Summary: KRAS mutations are drivers of oncogenesis, and historically have been considered “undruggable.” Consequentially, therapeutic approaches have focused on downstream effectors of the mitogen-activated protein kinase (MAPK) pathway, including RAF, MEK, and ERK–though no approach has led to an effective drug for KRAS-driven disease. However, genetic studies strongly support that the MAPK signaling pathway is a critical dependency in KRAS-mutant cancers; so why do current MAPK drugs fail? Recent studies suggest that these drugs are effective inhibitors of MAPK enzyme activity, yet fail due to feedback mechanisms that rely on protein-protein interactions (PPIs) to maintain MAPK signaling even in the presence of drug. My overarching hypothesis is that current MAPK inhibitors are limited by their inability to effectively regulate critical PPIs among MAPK components. To test this hypothesis, I will alter critical PPIs by modulating a MAPK scaffold termed Kinase Suppresor of Ras (KSR). In contrast to previous MAPK targets, KSR is a pseudokinase that lacks catalytic activity, but serves as a scaffold protein to promote RAF and MEK binding. Our group showed that a lead compound termed APS-2-79 binds to KSR2 at the ATP binding site and synergizes with MEK inhibitors (MEKi) in KRAS-driven cell lines by impeding KSR’s interaction with RAF. While useful as a tool for biochemical studies, APS-2-79 has several limitations including modest affinity and selectivity for KSR. Moreover, the mechanism of KSR inhibitor (KSRi) synergy with MEKi in KRAS mutant cell lines is not known, and may be the consequence of off target kinase inhibition instead of direct KSR targeting. To investigate the mechanism of KSRi synergy with MEK inhibitors, I aim to test the dependence of KSRi synergy in KRAS mutant cells on KSR using genetic tools. I hypothesize that the synergy observed between MEKi and KSRi in KRAS mutant cells is dependent on the availability of KSR’s ATP-binding pocket. To test this hypothesis, I will use lentiviral vectors to overexpress KSR, +/- mutations in the ATP binding pocket known to prevent compound binding. To further explore the importance of KSR in mediating RAS-MAPK signaling, I also aim to induce targeted KSR degradation with small-molecule PROteolysis TArgeting Chimeras (PROTACs) These small molecule tools simultaneously bind their protein targets and E3 ubiquitin ligases, allowing for ubiquitination of the target and downstream proteolysis. I hypothesize that PROTAC mediated KSR1 degradation will mimic genetic deletion studies supporting the importance of KSR1 for oncogenic KRAS. My aims outline genetic and pharmacological approaches to investigate KSR inactivation as a mechanism to exploit crucial PPIs within the KRAS-driven MAPK signaling pathway. Critical to this study are the development of potent and specific next-generation KSRi analogs and precise genetic tools for target validation. Ultimately this training proposal nurtures skills in synthetic chemistry, assay development, drug discovery, and target validation, which will be broadly applicable to my future goals as a physician scientist.

项目摘要：KRAS突变是肿瘤发生的驱动因素，从历史上看 “不可能。”因此，治疗方法的重点是有丝分裂原激活的蛋白激酶（MAPK）途径，包括RAF，MEK和ERK - 尽管没有方法导致有效的KRAS驱动疾病药物。但是，遗传研究强烈支持MAPK 信号通路是KRAS突变癌的关键依赖性。那么为什么当前的MAPK药物失败呢？最近的研究表明，这些药物是MAPK酶活性的有效抑制剂，但由于依赖蛋白质 - 蛋白质相互作用（PPI）的反馈机制即使在存在药物。我的总体假设是当前的MAPK抑制剂受到其无法有效地调节MAPK组件之间的关键PPI。为了检验这一假设，我将通过调节称为RAS（KSR）激酶的MAPK支架。与以前的MAPK目标相反， KSR是一种缺乏催化活性的假子酶，但作为脚手架蛋白来促进RAF和MEK 结合。我们的小组表明，称为APS-2-79的铅化合物在ATP结合位点与KSR2结合并通过阻碍KSR与RAF的相互作用在KRAS驱动的细胞系中与MEK抑制剂（MEKI）协同作用。 APS-2-79虽然作为生化研究的工具有用，但有几个局限性，包括适度的亲和力和 KSR的选择性。此外，KSR抑制剂（KSRI）与MEKI在KRAS突变细胞中的机制线尚不清楚，可能是OFF靶激酶抑制而不是直接KSR靶向的结果。为了研究KSRI与MEK抑制剂协同作用的机制，我旨在测试 KSRI使用遗传工具在KSR上的KRAS突变细胞中的协同作用。我假设观察到协同作用 KRAS突变细胞中的Meki和KSRI之间取决于KSR的ATP结合口袋的可用性。为了检验该假设，我将使用慢病毒向量过表达KSR，+/-突变ATP结合袋中已知可以防止复合结合。进一步探讨KSR在介导Ras-Mapk中的重要性信号传导，我还旨在用小分子蛋白水解靶向靶向的KSR降解嵌合体（Protac）这些小分子工具简单地结合其蛋白质靶标和E3泛素连接酶，允许靶标和下游蛋白水解的泛素化。我假设Protac 介导的KSR1降解将模仿遗传缺失研究，以支持KSR1的重要性致癌kras。我的目的概述了研究KSR失活的遗传和药物方法利用KRAS驱动的MAPK信号通路中关键PPI的机制。这项研究至关重要的是潜力和特定的下一代KSRI类似物的开发以及目标的精确遗传工具验证。最终，该培训建议护士在合成化学，测定开发，药物方面的技能发现和目标验证将广泛适用于我作为物理科学家的未来目标。