Mutation-Specific and Common Phosphotyrosine Signatures of KRAS G12D and G13D Alleles

KRAS is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic KRAS mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D KRAS mutation, we observed both shared as well as unique signaling events induced by the two KRAS mutations. Remarkably, while the G12D mutation led to an increase in membrane proximal and adherens junction signaling, the G13D mutation led to activation of signaling molecules such as nonreceptor tyrosine kinases, MAPK kinases, and regulators of metabolic processes. The importance of one of the cell surface molecules, MPZL1, which was found to be hyperphosphorylated in G12D cells, was confirmed by cellular assays as its knockdown led to a decrease in proliferation of G12D but not G13D expressing cells. Overall, our study reveals important signaling differences across two common KRAS mutations and highlights the utility of our approach to systematically dissect subtle differences between related oncogenic mutants and potentially lead to individualized treatments.

KRAS（Kirsten大鼠肉瘤病毒癌基因同源物）是所有癌症亚型中最常见的突变基因之一。临床中最常见的两类致癌性KRAS突变，均为甘氨酸向天冬氨酸的密码子替换：分别为12号密码子突变（G12D）与13号密码子突变（G13D）。尽管目前尚未完全阐明这两类主流突变的生化差异，但二者诱导的肿瘤具有截然不同的临床特征，提示其信号转导机制存在显著差异。
本研究对分别携带G12D或G13D KRAS突变的同基因结直肠癌细胞系的全磷酸酪氨酸蛋白质组谱进行对比分析，发现两类突变既可诱导共有的信号转导事件，也可引发独特的信号变化。值得注意的是，G12D突变可增强膜近端与黏着连接相关的信号通路活性，而G13D突变则激活了非受体酪氨酸激酶、丝裂原活化蛋白激酶激酶以及代谢过程调控因子等信号分子。
研究发现，在表达G12D KRAS的细胞中，细胞表面分子MPZL1呈高磷酸化状态；通过细胞实验验证了该分子的重要性：敲低MPZL1可显著降低表达G12D KRAS的细胞的增殖能力，但对表达G13D KRAS的细胞无明显影响。
综上，本研究揭示了两类常见KRAS突变间关键的信号转导差异，证明本研究方法可系统解析相关致癌突变体间的细微差异，有望为个体化治疗提供潜在方向。