Investigation of ERK Activity as a Proxy for Targeted Therapy Resistance in KRAS-mutant Colorectal Cancer

ERK 活性作为 KRAS 突变结直肠癌靶向治疗耐药性代理的研究

• 批准号: 444691702
• 负责人: Dr. Markus Morkel
• 金额: --
• 依托单位: Institut für Pathologie (CCM)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/444691702?language=en
• 关键词: Investigation; ERK; Activity; Proxy; Targeted

The EGFR-RAS-ERK signaling cascade has fundamental relevance for colorectal cancer (CRC) biology and therapy response. However, no targeted therapy options are available for patients with KRAS-mutant CRC. This is because these cancers are intrinsically resistant to EGFR inhibition, as well as therapeutic inhibition of downstream signaling nodes. Therapies targeting the RAS-ERK network generally result in ERK reactivation through dynamic changes in the signaling network. We have shown previously that activity of the signaling network converging on ERK is intrinsically coupled to cell differentiation in CRC.In this application, we hypothesize that ERK activity is a suitable read-out to detect resistant CRC cell states emerging during targeted therapy. To test this hypothesis, we will analyze heterocellular CRC organoids with single-cell methodologies established in our lab: CyTOF and single-cell RNA sequencing.In a first step, we will combine these single-cell technologies with fluorescent reporters to read-out ERK activity and subcellular localization in KRAS-mutant and control organoid cells.In a second step, we will evaluate the impact of targeted therapies on the CRC organoids. Combining CyTOF and single-cell RNA-seq, we will record cell differentiation states, activities of key nodes of the signaling network converging on ERK, and the different levels of ERK regulation. For the planned experiments, we have shortlisted several single agent and combinatorial therapies that are currently under clinical investigation in CRC, mainly comprising combinations of RAF, MEK, ERK, and EGFR inhibitors. We will also test the new direct KRAS(G12C) inhibitor AMG510 in KRAS(G12C)-mutant organoid lines.In a third step, we will use the data to improve the therapeutic approaches, and these experiments will be guided and facilitated by the fine-grained data on ERK activity that we have gathered in the previous experiments. In collaboration with the group of Nils Blüthgen, we will employ the single cell-resolved network activity data converging on ERK for quantitative modeling. This approach aims at identifying and exploiting novel vulnerabilities of signaling network states that confer resistance to targeted therapy. In a parallel approach, we will seek to pre-treat the organoids in order to bring the cells to a more uniform (cell differentiation) state that is drug-sensitive rather than resistant before experimental therapy. In summary, the planned experiments aim at unraveling the connections between cancer cell differentiation, ERK re-activation, and resistance to targeted inhibitors, in order to test new ideas on how to provide targeted treatment options to the many KRAS-mutant CRC patients that are currently without targeted treatment options.

EGFR-RAS-ERK信号级联与结直肠癌（CRC）生物学和治疗反应具有根本相关性。然而，对于KRAS突变型CRC患者，没有靶向治疗选择。这是因为这些癌症对EGFR抑制以及下游信号传导节点的治疗性抑制具有内在抗性。靶向RAS-ERK网络的治疗通常通过信号网络中的动态变化导致ERK再激活。我们以前已经表明，集中在ERK上的信号网络的活性与CRC中的细胞分化内在地偶联。在本申请中，我们假设ERK活性是检测靶向治疗期间出现的耐药CRC细胞状态的合适读数。为了验证这一假设，我们将使用我们实验室建立的单细胞方法：CyTOF和单细胞RNA测序来分析异细胞CRC类器官。第一步，我们将联合收割机这些单细胞技术与荧光报告分子结合，以读取KRAS突变体和对照类器官细胞中的ERK活性和亚细胞定位。第二步，我们将评估靶向治疗对CRC类器官的影响。结合CyTOF和单细胞RNA-seq，我们将记录细胞分化状态、汇聚ERK的信号网络关键节点的活动以及ERK调节的不同水平。对于计划的实验，我们已经入围了目前在CRC中进行临床研究的几种单一药物和组合疗法，主要包括RAF，MEK，ERK和EGFR抑制剂的组合。我们还将在KRAS（G12 C）突变的类器官系中测试新的直接KRAS（G12 C）抑制剂AMG 510。在第三步中，我们将使用这些数据来改进治疗方法，这些实验将由我们在先前实验中收集的ERK活性的细粒度数据指导和促进。 在与Nils Blüthgen小组的合作中，我们将采用汇聚在ERK上的单细胞分辨网络活动数据进行定量建模。这种方法旨在识别和利用信号网络状态的新漏洞，从而对靶向治疗产生抗性。在一种平行的方法中，我们将寻求预处理类器官，以使细胞在实验治疗之前达到更均匀的（细胞分化）状态，即药物敏感而不是耐药。总之，计划中的实验旨在解开癌细胞分化，ERK再激活和对靶向抑制剂的抗性之间的联系，以测试如何为目前没有靶向治疗选择的许多KRAS突变CRC患者提供靶向治疗选择的新思路。