Data_Sheet_1_Cancer Cells Expressing Oncogenic Rat Sarcoma Show Drug-Addiction Toward Epidermal Growth Factor Receptor Antibodies Mediated by Sustained MAPK Signaling.docx

Epidermal growth factor receptor (EGFR) antibodies may have detrimental effects in patients with metastatic colorectal cancer expressing oncogenic Rat sarcoma (RAS). Since a significant number of patients acquire RAS-mediated resistance during EGFR-directed treatment, understanding the molecular mechanism underlying these antibody-mediated tumor-promoting effects is of relevance to design more resistance-preventive treatment approaches. To test this, we set up a Ba/F3 cellular model system transformed to EGFR/RAS dependency to be able to study proliferation, RAS activity as well as MAPK signaling upon inhibition of wild-type RAS isoforms by therapeutic EGFR antibodies. Here, we show that the EGFR antibodies cetuximab and panitumumab induce paradoxical stimulation and enhance proliferation in cells expressing oncogenic RAS (KRAS G12V). These experiments clearly showed that the stimulatory effect is a direct result of the antibody-EGFR interaction leading to prolonged mitogen-activated protein-Kinase (MAPK) signaling. The effect was also induced by antibody-chemotherapy combinations but always depended on simultaneous low-level ligand-dependent EGFR pathway activation. Moreover, we observed significant growth retardation of RAS mutant cells after antibody withdrawal compatible with a drug-addiction phenotype. Our data suggests that EGFR antibodies paradoxically sustain MAPK signaling downstream of oncogenic RAS thereby driving proliferation of RAS mutant tumors or tumor subclones. The observed drug-addiction encourages fixed-duration or liquid-biopsy-guided drug holiday concepts to preventively clear RAS mutant subclones selected under EGFR-directed therapeutic pressure.

表皮生长因子受体（EGFR）抗体可能对表达致癌性鼠肉瘤（RAS）的转移性结直肠癌患者产生有害影响。鉴于大量病人在EGFR靶向治疗过程中获得了RAS介导的耐药性，理解这些抗体介导的肿瘤促进作用背后的分子机制对于设计更有效的耐药性预防治疗方案具有重要意义。为此，我们构建了一个Ba/F3细胞模型系统，该系统被转化为EGFR/RAS依赖性，以便研究在通过治疗性EGFR抗体抑制野生型RAS同种型后，细胞的增殖、RAS活性和MAPK信号传导。在本研究中，我们发现EGFR抗体西妥昔单抗和帕尼单抗在表达致癌RAS（KRAS G12V）的细胞中诱导了矛盾性的刺激并增强了细胞的增殖。这些实验明确显示，刺激效应是抗体与EGFR相互作用导致有丝分裂原激活蛋白激酶（MAPK）信号传导延长的直接结果。这种效应也由抗体-化疗联合诱导，但始终依赖于同时低水平的配体依赖性EGFR通路激活。此外，我们还观察到抗体撤回后RAS突变细胞的显著生长抑制，这与药物成瘾表型相符。我们的数据表明，EGFR抗体通过矛盾性地维持致癌RAS下游的MAPK信号传导，从而驱动RAS突变肿瘤或肿瘤亚克隆的增殖。观察到的药物成瘾现象鼓励采用固定期限或液体活检引导的药物假期概念，以预防性清除在EGFR靶向治疗压力下选出的RAS突变亚克隆。