多靶点酪氨酸激酶抑制剂（mTKIs）如Regorafenib和Sorafenib已经被批准用于多种实体瘤的治疗。然而，在结直肠癌（CRC）中，mTKIs的疗效有限，并且其潜在机制仍然不明确。我们的研究旨在发现CRC中mTKIs的耐药机制。我们使用RNA测序技术确定了mTKIs治疗下Activin A受体样蛋白1（ACVRL1）的表达情况。通过增强/减弱功能实验评估ACVRL1在耐药性中的生物学功能。通过液相色谱-质谱分析（LC-MS）、共免疫沉淀实验（Co-IP）、染色质免疫沉淀实验、泛素化实验、双荧光素酶报告实验等方法探究了ACVRL1介导的mTKIs耐药性的潜在机制。结果: RNA测序发现在CRC细胞中，mTKIs治疗下ACVRL1活化。ACVRL1的缺失和过度表达均显著影响CRC细胞对mTKIs的敏感性，无论是在体外还是体内。从机制上讲，我们发现β-连环蛋白/TCF-1-KCNQ1OT1/miR-7-5p轴介导了ACVRL1的活化。此外，LC-MS实验表明ACVRL1与谷胱甘肽过氧化物酶2（GPX2）蛋白之间存在相互作用。IP实验确定ACVRL1截短（282-503aa）可能负责与GPX2的相互作用，而通过ACVRL1截短的救援实验则证实了这种相互作用在推动mTKI耐药性中的重要性。Co-IP实验证明ACVRL1与泛素特异性蛋白酶15（USP15）结合，后者直接在K187（K，赖氨酸）位点解去泛素化GPX2，导致GPX2蛋白的积累。在GPX2 CRISPR基因敲除细胞模型中进行的修复实验证实了GPX2 K187突变体的重要性。因此，增加ROS清除能力和降低细胞凋亡最终导致CRC中mTKI耐药性的发生。我们的结果表明，Wnt/β-连环蛋白/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2生物轴在CRC中起到了重要作用，瞄准该轴可能是克服mTKI耐药性的有效途径。 © 2023. BioMed Central Ltd., Springer Nature的一部分。

Multitarget tyrosine kinase inhibitors (mTKIs) such as Regorafenib and Sorafenib have already been approved for the treatment of many solid tumours. However, the efficacy of mTKIs in colorectal cancer (CRC) is limited; the underlined mechanism remains largely elusive. Our study was aimed to find out the resistance mechanism of mTKIs in CRC.RNA sequencing was used to identify the expression of Activin A receptor-like type 1 (ACVRL1) under the treatment of mTKIs. Gain/loss-of-function experiments were performed to assess the biological function of ACVRL1 in resistance to mTKIs. The underlying mechanisms of ACVRL1-mediated mTKI resistance were investigated by using liquid chromatography-mass spectrometry assays (LC-MS), co-immunoprecipitation assays (Co-IP), chromatin immunoprecipitation assays, ubiquitination assays, dual luciferase reporter assays, etc. RESULTS: RNA sequencing identified the activation of ACVRL1 under the treatment of mTKIs in CRC cells. ACVRL1 knockdown and overexpression significantly affects the sensitivity of CRC cells to mTKIs both in vitro and vivo. Mechanistically, we found the β-catenin/TCF-1-KCNQ1OT1/miR-7-5p axis mediated the activation of ACVRL1. Furthermore, LC-MS assays indicated the interaction between ACVRL1 and glutathione peroxidase 2(GPX2) protein. IP assay defined ACVRL1 truncation (282-503aa) could be responsible for interacting with GPX2, and rescue experiments with ACVRL1 truncations confirmed the importance of this interaction in driving mTKI resistance. Co-IP assays confirmed that ACVRL1 associates with ubiquitin-specific peptidase 15(USP15) which directly deubiquinates GPX2 at the K187(K, lysine) site, leading to the accumulation of GPX2 protein. Rescue experiments performed with the lysine mutants in GPX2 CRISPR knockout cell model confirmed the importance of GPX2 K187 mutant. As a result, the increased ROS clearance and decreased cell apoptosis eventually lead to mTKI resistance in CRC.Our results demonstrate that the Wnt/β-catenin/KCNQ1OT1/miR-7-5p/ACVRL1/GPX2 biological axis plays a vital role in CRC, targeting which may be an effective approach for overcoming mTKI resistance.© 2023. BioMed Central Ltd., part of Springer Nature.