Genetic and Pharmacological Manipulation of KSR in KRAS-driven Cancer

KRAS突变是肿瘤发生的驱动因素，历史上一直被认为是 "无法抗拒"因此，治疗方法集中在肿瘤的下游效应物上。 丝裂原活化蛋白激酶（MAPK）通路，包括RAF，MEK和ERK-尽管没有方法 导致了一种治疗KRAS驱动的疾病的有效药物。然而，遗传学研究强烈支持MAPK 信号通路是KRAS突变型癌症的关键依赖;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），以维持MAPK信号，即使在 药物的存在。我的总体假设是，目前的MAPK抑制剂是有限的，他们不能 有效地调节MAPK组分中的关键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。与以前的MAPK靶相反， KSR是一种缺乏催化活性的假激酶，但作为支架蛋白促进RAF和MEK 约束力我们的研究小组发现，一种名为APS-2 - 79的先导化合物在ATP结合位点与KSR 2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。 虽然APS-2 - 79可用作生物化学研究的工具，但它有几个局限性，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也有待进一步研究。 细胞系未知，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KSRi与MEK抑制剂的依赖性。 使用遗传工具在KRAS突变细胞中对KSR的KSRi协同作用。我假设观察到的协同作用 KRAS突变细胞中MEKi和KSRi之间的相互作用依赖于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过表达KSR，在ATP结合口袋中+/-突变， 已知可防止化合物结合。进一步探讨KSR在介导RAS-MAPK中的作用 信号传导，我还旨在用小分子蛋白水解诱导靶向KSR降解 嵌合体（PROTAC）这些小分子工具同时结合其蛋白质靶标和E3泛素 连接酶，允许靶的泛素化和下游蛋白水解。我假设PROTAC 介导的KSR1降解将模拟遗传缺失研究，支持KSR1对 致癌KRAS我的目的是概述遗传和药理学方法来研究KSR失活， 利用KRAS驱动的MAPK信号通路中的关键PPI的机制。这项研究的关键是 开发有效和特异性下一代KSRi类似物和用于靶向的精确遗传工具 验证。最终，该培训计划培养了合成化学，分析开发，药物 发现，和目标验证，这将广泛适用于我未来的目标，作为一个医生科学家。

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.