A small molecule targeting NMHC IIA phosphorylation exerts promising anti-tumor effects on colorectal cancer

Colorectal cancer (CRC) is one of the most lethal cancers when it progresses to the advanced/metastatic stage. Treatment options for refractory metastatic colorectal cancer (mCRC) are limited. Therefore, there is an urgent need to develop effective treatment methods for metastatic colorectal cancer. In this study, we screened a library of small molecules for inhibitors of CRC recurrence using CRC cell lines and patient-derived organoids (PDOs) from metastatic, heavily pretreated CRC. The in vivo efficacy of LS-1-2 was evaluated in the HCT116 and the resistant HCT8 cell lines, patient-derived xenografts(PDXs) including refractory mCRC, and a liver metastasis mouse model. Biotin pull-down and liquid chromatography tandem-mass spectrometry (LC-MS/MS) were performed to identify proteins that interact with LS-1-2. The genome-wide gene expression and quantitative phosphoproteomic analyses were performed to determine the signaling pathway involved in LS-1-2. Molecular docking, molecular dynamics analysis and cellular thermal shift assays were used to predict and validate the binding sites of LS-1-2 on non-muscle Myosin IIA (NMHC IIA). Our results showed that LS-1-2 exhibited broad antiproliferative effects on a series of CRC cell lines and PDOs. The in vivo anti-tumor efficacy of LS-1-2 was demonstrated across cell line- and patient-derived xenografts and in the liver metastatic CRC model. NMHC IIA was identified as a direct target of LS-1-2, and LS-1-2 competitively inhibited the phosphorylation of NMHC IIA at S1943 and S1714 by CK2. NMHC IIA phosphorylation promoted CRC cell proliferation and invasion, which was reduced by LS-1-2. NMHC IIA phosphorylation-mediated YAP activity induced activation of AKT and inactivation of FOXO3a and was suppressed by LS-1-2. In conclusion, our findings suggest that NMHC IIA phosphorylation can be used as a potential molecular target in CRC metastasis, and that targeting NMHC IIA phosphorylation by LS-1-2 may be a promising strategy for the treatment and prevention of CRC metastasis.

结直肠癌（Colorectal cancer, CRC）进展至晚期/转移阶段时，属于致死性最高的癌症之一。难治性转移性结直肠癌（metastatic colorectal cancer, mCRC）的治疗选择十分有限，因此亟须开发针对转移性结直肠癌的有效治疗手段。本研究利用来自经多线治疗的转移性CRC细胞系及患者来源类器官（patient-derived organoids, PDOs），筛选小分子化合物库以寻找CRC复发抑制剂。通过HCT116、耐药株HCT8细胞系、包含难治性mCRC的患者来源异种移植瘤（patient-derived xenografts, PDXs）以及肝转移小鼠模型，评估LS-1-2的体内抗肿瘤活性。采用生物素pull-down实验与液相色谱-串联质谱（liquid chromatography tandem-mass spectrometry, LC-MS/MS）技术，鉴定与LS-1-2相互作用的蛋白。通过全基因组基因表达分析与定量磷酸化蛋白质组学分析，明确LS-1-2所调控的信号通路。利用分子对接、分子动力学分析及细胞热位移实验，预测并验证LS-1-2在非肌肌球蛋白IIA（non-muscle Myosin IIA, NMHC IIA）上的结合位点。研究结果显示，LS-1-2对一系列CRC细胞系及PDOs展现出广谱的抗增殖活性。在细胞系来源及患者来源异种移植瘤模型，以及肝转移性CRC模型中，均证实了LS-1-2的体内抗肿瘤功效。鉴定出NMHC IIA为LS-1-2的直接作用靶点，且LS-1-2可竞争性抑制CK2介导的NMHC IIA在S1943与S1714位点的磷酸化。NMHC IIA磷酸化可促进CRC细胞增殖与侵袭，而LS-1-2可削弱这一效应。NMHC IIA磷酸化介导的YAP活性激活可诱导AKT活化并抑制FOXO3a活性，该过程亦可被LS-1-2阻断。综上，本研究结果表明，NMHC IIA磷酸化可作为CRC转移的潜在分子靶点，而通过LS-1-2靶向NMHC IIA磷酸化，有望成为治疗与预防CRC转移的有效策略。