Analytical Sciences, BioPharmaceuticals R&D, AstraZeneca, Gaithersburg, MD, USA.
NCI RAS Initiative, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
Methods Mol Biol. 2024;2823:27-46. doi: 10.1007/978-1-0716-3922-1_3.
With the advent of promising lung cancer immunotherapies targeting proteins at the cell surface of RAS-driven human cancers, the mass spectrometry (MS)-based surfaceomics remains a feasible strategy for therapeutic target discovery. This chapter describes a protocol for discovery of druggable protein targets at the surface of RAS-driven human cancer cells. This method relies on bottom-up MS-based quantitative surfaceomics that employs in parallel, targeted hydrazide-based cell-surface glycoproteomics and global shotgun membrane proteomics to enable unbiased quantitative profiling of thousands of cell surface membrane proteins. A large-scale molecular map of the KRAS surface was attained, resulting in confident detection and quantitation of more than 500 cell surface membrane proteins that were found to be unique or upregulated at the surface of cells harboring the KRAS mutant. A multistep bioinformatic progression revealed a subset of unique and/or significantly upregulated proteins as priority drug targets selected for orthogonal cross-validation using immunofluorescence, structured illumination microscopy, and western blotting. Among cross-validated targets, CUB domain containing protein 1 (CDCP1) and basigin (BSG-CD147) were selected as leading targets due to their involvement in cell adhesion and migration, consistent with the KRAS malignant phenotype as revealed by scanning electron microscopy and phenotypic cancer cell assays. Follow-up studies confirmed CDCP1 as an actionable therapeutic target, resulting in development of recombinant antibodies capable of killing KRAS-transformed cancer cells in preclinical setting. The present MS-based surfaceomics workflow represents a powerful drug target discovery platform that enables development of innovative immunotherapeutics (e.g., antibody drug conjugate against CDCP1) for attacking oncogenic RAS-driven cancers at the cell surface.
随着针对 RAS 驱动型人类癌症细胞表面蛋白的有前途的肺癌免疫疗法的出现，基于质谱（MS）的表面组学仍然是一种可行的治疗靶点发现策略。本章描述了一种用于发现 RAS 驱动型人类癌细胞表面可成药蛋白靶标的方案。该方法依赖于基于 MS 的定量表面组学，该方法并行采用靶向酰肼基细胞表面糖蛋白组学和全局鸟枪法膜蛋白质组学，实现了数千种细胞膜表面蛋白的无偏定量分析。获得了 KRAS 表面的大规模分子图谱，从而能够可靠地检测和定量超过 500 种细胞膜表面蛋白，这些蛋白被发现是携带 KRAS 突变的细胞表面上的独特或上调蛋白。经过多步生物信息学进展，选择了一组独特和/或显著上调的蛋白作为优先药物靶点，这些蛋白通过免疫荧光、结构光照明显微镜和 Western blot 进行正交验证。在经过验证的靶点中，CUB 结构域蛋白 1（CDCP1）和基底膜糖蛋白 1（BSG-CD147）被选为主要靶点，因为它们参与细胞黏附和迁移，与扫描电子显微镜和表型癌细胞测定揭示的 KRAS 恶性表型一致。后续研究证实 CDCP1 是一个可操作的治疗靶点，导致能够在临床前环境中杀死 KRAS 转化癌细胞的重组抗体的开发。本基于 MS 的表面组学工作流程代表了一种强大的药物靶点发现平台，能够为针对致癌性 RAS 驱动型癌症的细胞表面开发创新的免疫疗法（例如针对 CDCP1 的抗体药物偶联物）。
