Genetic and Pharmacological Manipulation of KSR in KRAS-driven Cancer

项目摘要：KRAS突变是肿瘤发生的驱动因素，历史上一直被认为是 “无法下药。”因此，治疗方法将重点放在血管紧张素转换酶的下游效应器。 丝裂原活化蛋白激酶(MAPK)途径，包括RAF、MEK和ERK-尽管没有方法 导致了一种治疗KRAS引发的疾病的有效药物。然而，基因研究强烈支持MAPK 信号通路是KRAS突变癌症的关键依赖因素；那么为什么目前的MAPK药物失败了？ 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 反馈机制依赖于蛋白质-蛋白质相互作用(PPI)来维持MAPK信号，即使在 毒品的存在。我的主要假设是，目前的MAPK抑制剂受限于它们无法 有效监管MAPK各组成部分之间的关键PPI。为了检验这个假设，我将通过以下方式更改关键PPI 调节一种称为RAS的激酶抑制因子(KSR)的MAPK支架。与之前的MAPK目标不同， KSR是一种缺乏催化活性的假激酶，但可作为促进RAF和MEK的支架蛋白 有约束力的。我们的研究小组发现，一种名为APS-2-79的先导化合物与KSR2在三磷酸腺苷结合部位结合 并通过阻止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。 虽然APS-2-79作为生化研究的工具很有用，但也有几个局限性，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突变细胞中的协同作用机制 LINES尚不清楚，可能是脱靶蛋白激酶抑制的结果，而不是直接的KSR靶向。 为了研究KSRi与MEK抑制剂的协同作用机制，我旨在测试KSRi与MEK抑制剂之间的依赖性。 利用基因工具在KRAS突变细胞中的KSRi协同作用。我假设这种协同效应观察到 KRAS突变细胞中Meki和KSRi之间的关系取决于KSR的ATP结合口袋的可用性。 为了验证这一假设，我将使用慢病毒载体在ATP结合口袋中过表达KSR，+/-突变 已知可防止化合物结合。进一步探讨KSR在调节RAS-MAPK中的重要性 信号，我的目标也是通过小分子蛋白水解靶向诱导KSR的靶向降解 嵌合体(PROTAC)这些小分子工具同时结合它们的蛋白质靶标和E3泛素 连接酶，允许目标泛素化和下游蛋白分解。我假设PROTAC 介导的KSR1降解将模拟基因缺失研究，支持KSR1对 致癌的KRAS。我的目标概述了研究KSR失活的遗传和药理学方法，如 一种利用KRAS驱动的MAPK信号通路中关键的PPI的机制。对这项研究至关重要的是 针对靶点的有效和特异的下一代KSRi类似物和精确遗传工具的开发 验证。最终，这项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内科科学家的未来目标。

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.