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

EGFR-RAS-ERK信号通路与结直肠癌(CRC)生物学和治疗反应密切相关。然而，没有靶向治疗方案可用于KRAS突变的结直肠癌患者。这是因为这些癌症本质上对EGFR抑制以及下游信号转导节点的治疗性抑制具有抵抗力。针对RAS-ERK网络的治疗通常会通过信号网络的动态变化导致ERK重新激活。我们以前已经证明，ERK信号网络的活性在本质上与CRC中的细胞分化有关。在这个应用中，我们假设ERK活性是检测靶向治疗过程中出现的耐药CRC细胞状态的合适读数。为了验证这一假说，我们将用我们实验室建立的单细胞方法：细胞TOF和单细胞RNA测序来分析异源结直肠癌有机物。第一步，我们将把这些单细胞技术与荧光记者结合起来，读出KRAS突变的ERK活性和亚细胞定位，并控制有机类细胞。第二步，我们将评估靶向治疗对结直肠癌有机物的影响。结合CyTOF和单细胞RNA-seq，我们将记录细胞分化状态，汇聚在ERK上的信号网络关键节点的活动，以及不同水平的ERK调控。对于计划中的实验，我们列出了几种目前正在进行临床研究的结直肠癌单一药物和联合治疗方法，主要包括RAF、MEK、ERK和EGFR抑制剂的联合治疗。我们还将在KRAS(G12C)突变的有机系中测试新的直接KRAS(G12C)抑制剂AMG510。在第三步，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之前的实验中收集的关于ERK活性的细粒度数据来指导和促进。与NILS Blüthgen小组合作，我们将使用收敛于ERK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脆弱性，这些脆弱性赋予靶向治疗抵抗力。在一种平行的方法中，我们将寻求对有机物进行预处理，以便在实验治疗之前将细胞带到更均匀的(细胞分化)状态，即对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重新激活和靶向抑制剂耐药性之间的联系，以测试如何为许多KRAS突变的CRC患者提供靶向治疗方案的新想法，这些患者目前没有靶向治疗方案。

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.