Department of Gastrointestinal Surgery IV, Key Laboratory of Carcinogenesis and Translational Research, Peking University Cancer Hospital and Institute, Beijing, China.
BGI-Shenzhen, Shenzhen, China.
Cancer Sci. 2021 Sep;112(9):3895-3910. doi: 10.1111/cas.15049. Epub 2021 Jul 27.
Mutated KRAS promotes the activation of the MAPK pathway and the progression of colorectal cancer (CRC) cells. Aberrant activation of the PI3K pathway strongly attenuates the efficacy of MAPK suppression in KRAS-mutated CRC. The development of a novel strategy targeting a dual pathway is therefore highly essential for the therapy of KRAS-mutated CRC. In this study, a quadruple-depleting system for the KRAS, MEK1, PIK3CA, and MTOR genes based on CRISPR/SaCas9 was developed. Adenovirus serotype 5 (ADV5) was integrated with two engineered proteins, an adaptor and a protector, to form ADV-protein complex (APC) for systemic delivery of the CRISPR system. Quadruple-editing could significantly inhibit the MAPK and PI3K pathways in CRC cells with oncogenic mutations of KRAS and PIK3CA or with KRAS mutation and compensated PI3K activation. Compared with MEK and PI3K/MTOR inhibitors, quadruple-editing induced more significant survival inhibition on primary CRC cells with oncogenic mutations of KRAS and PIK3CA. The adaptor specifically targeting EpCAM and the hexon-shielding protector could dramatically enhance ADV5 infection efficiency to CRC cells and significantly reduce off-targeting tropisms to many organs except the colon. Moreover, quadruple-editing intravenously delivered by APC significantly blocked the dual pathway and tumor growth of KRAS-mutated CRC cells, without influencing normal tissues in cell- and patient-derived xenograft models. Therefore, APC-delivered quadruple-editing of the MAPK and PI3K pathways shows a promising therapeutic potential for KRAS-mutated CRC.
突变型 KRAS 促进 MAPK 通路的激活和结直肠癌（CRC）细胞的进展。PI3K 通路的异常激活强烈削弱了 KRAS 突变型 CRC 中 MAPK 抑制的疗效。因此，开发针对双重通路的新策略对于 KRAS 突变型 CRC 的治疗至关重要。在这项研究中，开发了一种基于 CRISPR/SaCas9 的 KRAS、MEK1、PIK3CA 和 MTOR 基因的四重缺失系统。腺病毒血清型 5（ADV5）与两种工程蛋白（衔接子和保护蛋白）整合，形成 ADV-蛋白复合物（APC），用于 CRISPR 系统的全身递送。四重编辑可显著抑制 KRAS 和 PIK3CA 致癌突变或 KRAS 突变和补偿性 PI3K 激活的 CRC 细胞中的 MAPK 和 PI3K 通路。与 MEK 和 PI3K/MTOR 抑制剂相比，四重编辑对具有 KRAS 和 PIK3CA 致癌突变的原发性 CRC 细胞的生存抑制更为显著。特异性靶向 EpCAM 的衔接子和六聚体屏蔽保护蛋白可显著增强 ADV5 对 CRC 细胞的感染效率，并显著降低除结肠以外的许多器官的非靶向趋向性。此外，通过 APC 静脉内递送的四重编辑可显著阻断 KRAS 突变型 CRC 细胞的双重通路和肿瘤生长，而不会影响细胞和患者衍生异种移植模型中的正常组织。因此，APC 递送的 MAPK 和 PI3K 通路的四重编辑为 KRAS 突变型 CRC 显示出有前途的治疗潜力。
