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

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，历史上一直被认为因此，治疗方法集中于下游效应器。丝裂原激活蛋白激酶 (MAPK) 通路，包括 RAF、MEK 和 ERK——尽管目前尚无方法导致了一种治疗 KRAS 驱动疾病的有效药物，然而，遗传学研究强烈支持 MAPK。信号通路是 KRAS 突变癌症的关键依赖性；那么为什么当前的 MAPK 药物会失败呢？最近的研究表明，这些药物是 MAPK 酶活性的有效抑制剂，但由于以下原因而失败依赖蛋白质-蛋白质相互作用 (PPI) 的反馈机制即使在我的总体假设是，目前的 MAPK 抑制剂无法发挥作用。有效调节 MAPK 成分中的关键 PPI 为了检验这一假设，我将通过以下方式改变关键 PPI。调节称为 Ras 激酶抑制剂 (KSR) 的 MAPK 支架与之前的 MAPK 靶标相比， KSR是一种假激酶，缺乏催化活性，但作为支架蛋白促进RAF和MEK 我们的小组表明，一种名为 APS-2-79 的先导化合物在 ATP 结合位点与 KSR2 结合。并通过阻止 KSR 与 RAF 的相互作用，与 KRAS 驱动的细胞系中的 MEK 抑制剂 (MEKi) 产生协同作用。虽然 APS-2-79 作为生化研究的工具很有用，但它有一些局限性，包括亲和力适中和此外，KSR 抑制剂 (KSRi) 与 MEKi 在 KRAS 突变细胞中协同作用的机制。线是未知的，并且可能是脱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，我的目的是测试使用遗传工具在 KRAS 突变细胞中对 KSR 产生 KSRi 协同作用。 KRAS 突变细胞中 MEKi 和 KSRi 之间的差异取决于 KSR ATP 结合袋的可用性。为了检验这个假设，我将使用慢病毒载体过表达 KSR，ATP 结合袋中的 +/- 突变已知可防止化合物结合，以进一步探讨 KSR 在介导 RAS-MAPK 中的重要性。信号传导，我还旨在通过小分子蛋白质水解靶向诱导 KSR 降解嵌合体 (PROTAC) 这些小分子工具同时结合其蛋白质靶点和 E3 泛素连接酶，允许靶标泛素化和下游蛋白水解。介导的 KSR1 降解将模仿基因缺失研究，支持 KSR1 对我的目标概述了研究 KSR 失活的遗传和药理学方法：在 KRAS 驱动的 MAPK 信号通路中利用关键 PPI 的机制对本研究至关重要。开发有效且特异的下一代 KSRi 类似物和针对目标的精确遗传工具最终，该培训计划培养了合成化学、分析开发、药物方面的技能。发现和目标验证，这将广泛适用于我作为一名医师科学家的未来目标。