背景:致癌KRAS突变,非小细胞肺癌(NSCLC)中最常见的突变,是一种侵袭性风险因素,并通过促进葡萄糖导致癌细胞的代谢重编程,谷氨酰胺,脂肪酸吸收和糖酵解。最近,sotorasib被FDA批准为一流的KRAS-G12C抑制剂。然而,Sotorasib仍然有衍生障碍,这对其他KRAS突变类型无效,除了G12C。此外,对索托拉西的抗性可能会发展,要求需要替代治疗策略。
方法:KRAS突变体,和野生型NSCLC细胞用于体外细胞分析。细胞活力,扩散,通过MTT测量死亡,细胞计数,菌落分析,和用于FACS的膜联蛋白V染色。细胞跟踪器染料用于研究细胞形态,通过全息检查,和共聚焦显微镜。进行RNA测序以鉴定关键靶分子或途径,通过qRT-PCR证实,西方印迹,和代谢产物的UHPLC-MS分析将斑马鱼和小鼠异种移植模型用于体内分析。
结果:在这项研究中,我们发现nutlin-3a,MDM2拮抗剂,抑制KRAS-PI3K/Akt-mTOR通路并破坏自噬小体和巨噬小体与溶酶体的融合。这进一步阐明了非凋亡性和灾难性巨细胞胞吞作用相关的类甲醇样细胞死亡。被发现依赖于己糖胺生物合成途径的GFPT2,特别是在KRAS突变体/p53野生型NSCLC细胞中。
结论:这些结果表明nutlin-3a作为治疗KRAS突变/p53野生型NSCLC细胞的替代药物的潜力。
BACKGROUND: Oncogenic KRAS mutation, the most frequent mutation in non-small cell lung cancer (NSCLC), is an aggressiveness risk factor and leads to the metabolic reprogramming of cancer cells by promoting glucose, glutamine, and fatty acid absorption and glycolysis. Lately, sotorasib was approved by the FDA as a first-in-class KRAS-G12C inhibitor. However, sotorasib still has a derivative barrier, which is not effective for other KRAS mutation types, except for G12C. Additionally, resistance to sotorasib is likely to develop, demanding the need for alternative therapeutic strategies.
METHODS: KRAS mutant, and wildtype NSCLC cells were used in vitro cell analyses. Cell viability, proliferation, and death were measured by MTT, cell counting, colony analyses, and annexin V staining for FACS. Cell tracker dyes were used to investigate cell morphology, which was examined by holotomograpy, and confocal microscopes. RNA sequencing was performed to identify key target molecule or pathway, which was confirmed by qRT-PCR, western blotting, and metabolite analyses by UHPLC-MS/MS. Zebrafish and mouse xenograft model were used for in vivo analysis.
RESULTS: In this study, we found that nutlin-3a, an MDM2 antagonist, inhibited the KRAS-PI3K/Akt-mTOR pathway and disrupted the fusion of both autophagosomes and macropinosomes with lysosomes. This further elucidated non-apoptotic and catastrophic macropinocytosis associated methuosis-like cell death, which was found to be dependent on GFPT2 of the hexosamine biosynthetic pathway, specifically in KRAS mutant /p53 wild type NSCLC cells.
CONCLUSIONS: These results indicate the potential of nutlin-3a as an alternative agent for treating KRAS mutant/p53 wild type NSCLC cells.