我们先前证明奈替尼与培美曲塞协同作用可引发非小细胞肺癌（NSCLC）细胞死亡。通过开发其他药物组合，我们观察到在接触药物后的几天内，细胞活化了生存机制以对抗药物毒性。本研究旨在定义相应的机制，以降低奈替尼和培美曲塞的疗效。奈替尼和培美曲塞协同作用可引发对表达野生型RAS蛋白、突变型KRAS（G12S; Q61H; G12A和G12C）或突变型NRAS（Q61K）或突变型ERBB1（L858R; L858R T790M和exon 19缺失）的NSCLC细胞的杀伤效应。奈替尼和培美曲塞在24小时内、以及在进一步培养24小时且不含药物的情况下，以一种大于加法效应的方式相互作用，产生杀伤效果。突变型KRAS G12V的细胞保护作用比激活的MEK1或激活的AKT更强。突变型KRAS的沉默可降低药物联合治疗的杀伤效果。尽管在不含药物的情况下培养24小时，ERBB1、ERBB2和ERBB4的表达和活性仍明显降低，以及哺乳动物雷帕霉素靶蛋白（mTOR）C1和mTORC2的活性也降低。药物联合治疗可在24小时内降低KRAS和NRAS水平，然而，在不含药物的情况下，RAS水平在48小时内恢复正常。Beclin1和ATG5的表达仍然升高，而MCL1和BCL-XL的表达降低。我们观察未发现ERBB3、c-KIT、c-MET或PDGFRβ的生存信号激活，以及细胞内信号通路的进化激活。这些发现反驳了奈替尼和培美曲塞暴露后发展出的“早期”耐药机制。未来的研究将进一步探究NSCLC细胞如何对奈替尼和培美曲塞产生耐药性。版权所有 © 2023 Wolters Kluwer Health, Inc. 保留所有权利。

We previously demonstrated that neratinib interacted with pemetrexed to kill non-small cell lung cancer (NSCLC) cells. From developing other drug combinations, we observed that several days following exposure, cells activated survival mechanisms to counteract drug toxicity. The present studies attempted to define mechanisms that evolve to reduce the efficacy of neratinib and pemetrexed. Neratinib and pemetrexed synergized to kill NSCLC cells expressing wild-type RAS proteins, mutant KRAS (G12S; Q61H; G12A and G12C) or mutant NRAS (Q61K) or mutant ERBB1 (L858R; L858R T790M and exon 19 deletion). Neratinib and pemetrexed interacted in a greater than additive fashion to kill after 24 h, and after a further 24 h culture in the absence of drugs. Mutant KRAS G12V was more cytoprotective than either activated MEK1 or activated AKT. Knockdown of mutant KRAS reduced drug combination killing at the 48 h timepoint. Despite culture for 24 h in the absence of the drugs, the expression and activities of ERBB1, ERBB2 and ERBB4 remained significantly lower as did the activities of mammalian target of rapamycin (mTOR) C1 and mTORC2. The drug combination reduced KRAS and NRAS levels for 24 h, however, in the absence of the drugs, RAS levels had normalized by 48 h. Expression of Beclin1 and ATG5 remained elevated and of MCL1 and BCL-XL lower. No evolutionary activations of survival signaling by ERBB3, c-KIT, c-MET or PDGFRβ or in intracellular signaling pathways were observed. These findings argue against the development of 'early' resistance mechanisms after neratinib and pemetrexed exposure. Future studies will be required to understand how NSCLC cells become resistant to neratinib and pemetrexed.Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.