上皮间质转化在非小细胞肺癌EGFR-TKIs获得性耐药中的作用研究

目的 探讨上皮间质转化（EMT）在非小细胞肺癌（NSCLC）对表皮生长因子受体 酪氨酸激酶抑制剂（EGFR TKIs）获得性耐药中的作用及可能机制。方法 选用EGFR基因19号外显子突变型吉非替尼耐药细胞PC9／AB和EGFR野生型厄洛替尼耐药细胞H460/ER,通过基因转染获得E-cadherin稳定过表达细胞PC9／AB-CDH1和H460ER-CDH1。四甲基偶氮唑盐（MTT）法检测细胞的增殖情况,划痕实验及Transwell侵袭实验检测细胞迁移和侵袭能力,实时荧光定量PCR（qRT-PCR）和蛋白印迹法检测EMT相关分子、EGFR信号通路分子的mRNA和蛋白表达水平。结果 PC9／AB和H460/ER细胞未发生T790M突变和c-Met基因扩增,但发生了EMT,表现为E-cadherin表达降低和Vimentin表达增加。通过基因转染提高E-cadherin表达水平能逆转PC9／AB和H460／ER耐药细胞的EMT,使其对EGFR TKIs的敏感性增加, PC9／AB-CDH1细胞较PC9／AB对吉非替尼的敏感性增加约11.4倍,其半数抑制浓度（IC50）分别为（0.70±0.22） μmol／L和（8.68±0.44）μmol／L,差异有统计学意义（P＜0.05）; H460／ER-CDH1较H460／ER对厄洛替尼的敏感性增加约6.1倍,其IC50分别为（7.51±1.12）μmol／L和（53.72±12.95）μmol／L,差异有统计学意义（P＜0.05）。同时,逆转EMT后,细胞的EGFR、p-EGFR的mRNA和蛋白表达量均增加,差异有统计学意义（P＜0.05）。结论 阻断耐药细胞的EMT可逆转NSCLC对EGFR-TKIs的获得性耐药,EMT在NSCLC对EGFR-TKIs的获得性耐药中起重要作用,其机制可能与EGFR磷酸化水平降低有关。     

Targeting c-kit inhibits gefitinib resistant NSCLC cell growth and invasion through attenuations of stemness,EMT and acquired resistance

EGFR tyrosine kinase inhibitors (TKIs) are the first-line drugs for NSCLC. But, the acquired resistance limited their efficacy, so that the patients deteriorate eventually. Therefore, it is necessary to clarify the mechanism of the acquired resistance and overcome it for effective NSCLC therapy. In this experimental study, a stable gefitinib resistant lung adenocarcinoma cell line (PC9/GR) infected with shRNA-c-kit-homo-1386 were established; c-kit siRNA and c-kit inhibitors were used to block c-kit signaling; the acquired resistance of PC9/GR cells and the effects of c-kit siRNA and c-kit inhibitors on the growth and invasion of PC9/GR cells were investigated with CCK-8 assay, colony formation and cell invasion assays in vitro; the tumor growth inhibition effects of c-kit inhibitors on PC9/GR cell generated tumors were tested in vivo; the mechanisms involved in the acquired resistance reverse, growth and invasion inhibition effects of c-kit siRNA and c-kit inhibitors on PC9/GR cells were evaluated with qRT-PCR, Western blot and immunohistochemistry staining. The proliferation, colony formation, and invasion of PC9/GR cells were decreased by c-kit siRNA and inhibitors in vitro significantly; c-kit inhibitors suppressed the tumor growth of PC9/GR cell generated tumors in vivo. In the stable shRNA-c-kit transfected PC9/GR cells, the protein expressions of c-kit signaling and stemness phenotype related proteins, including ALDH1A1, Oct4, Sox2 and ABCG2 were decreased, and EMT phenotype related protein expressions including vimentin, N-cadherin, and Slug, were downregulated and with upregulation of E-cadherin; c-kit inhibitors reduced stemness phenotype related protein expressions, downregulated EMT phenotype related protein expressions including vimentin, N-cadherin, and Slug, with upregulation of E-cadherin, and the stemness related protein expressions of c-kit, ALDH1A1, ABCG2 and EMT-related proteins of vimentin and slug were decreased in the imatinib treated tumor tissues. The findings of this study indicated that c-kit signaling mediated the acquired gefitinib resistance, cell growth, invasion, stemness and EMT phenotype of PC9/GR cells. Targeting c-kit signaling with c-kit siRNA and small molecule c-kit inhibitors might overcome the acquired gefitinib resistance, and inhibit PC9/GR cell growth in vitro and in vivo.     

非小细胞肺癌PC-9细胞厄洛替尼耐药潜在机制的RNA测序分析

目的:通过RNA测序分析比较亲本PC-9细胞和厄洛替尼获得性耐药PC-9细胞(PC-9/ER)表达谱的差异,揭示非小细胞肺癌(NSCLC)表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKI)耐药的潜在机制。方法:采用间歇诱导方法,建立PC-9/ER耐药细胞株,通过MTT实验绘制厄洛替尼药物浓度-细胞存活率曲线。通过RNA测序分析PC-9/ER细胞的差异表达基因并进行GO和KEGG功能富集分析;通过qRT-PCR进一步筛选可能参与EGFR-TKI耐药的潜在基因或可能的靶点。结果:厄洛替尼对PC-9/ER细胞增殖的抑制率显著低于PC-9细胞的抑制率,PC-9/ER细胞的耐药指数为41.92。与PC-9细胞相比,RNA测序PC-9/ER细胞筛选出1 028个差异表达基因,其中720个基因表达上调,308个基因表达下调,而且差异表达基因显著富集在PI3K-AKT通路和癌症通路。qRT-PCR验证差异表达基因的转录水平与测序结果基本一致。结论:ST6GALNAC3、CYP1A1、PAPPA2、INHBE和ACSS3等基因可能参与EGFR-TKI的耐药过程,针对PC-9/ER细胞差异表达基因的后续...     

Epidermal growth factor receptor tyrosine kinase inhibitors for non-small cell lung cancer

First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have proven to be highly effective agents for advanced non-small cell lung cancer (NSCLC) in patients harboring an activating EGFR mutation such as the exon 19 deletion mutation and L858R. Although those reversible small molecular targeted agents provide a significant response and survival benefit, all responders eventually acquire resistance. Second-generation EGFR-targeting agents, such as irreversible EGFR/HER2 tyrosine kinase inhibitors and pan-HER TKIs, may improve survival further and be useful for patients who acquired resistance to first-generation EGFR-TKIs. This review discusses novel therapeutic strategies for EGFR-mutated advanced NSCLC using first- and second-generation EGFR-TKIs.     

MicroRNA-214 regulates the acquired resistance to gefitinib via the PTEN/AKT pathway in EGFR-mutant cell lines

Patients with non-small cell lung cancer (NSCLC) who have activating epidermal growth factor receptor (EGFR) mutations derive clinical benefit from treatment with EGFR-tyrosine kinase inhibitors ((EGFR-TKIs)- namely gefitinib and erlotinib. However, these patients eventually develop resistance to EGFR-TKIs. Despite the fact that this acquired resistance may be the result of a secondary mutation in the EGFR gene, such as T790M or amplification of the MET proto-oncogene, there are other mechanisms which need to be explored. MicroRNAs (miRs) are a class of small non-coding RNAs that play pivotal roles in tumorigenesis, tumor progression and chemo-resistance. In this study, we firstly successfully established a gefitinib resistant cell line-HCC827/GR, by exposing normal HCC827 cells (an NSCLC cell line with a 746E-750A in-frame deletion of EGFR gene) to increasing concentrations of gefitinib. Then, we found that miR-214 was significantly up-regulated in HCC827/ GR. We also showed that miR-214 and PTEN were inversely expressed in HCC827/GR. Knockdown of miR-214 altered the expression of PTEN and p-AKT and re-sensitized HCC827/GR to gefitinib. Taken together, miR-214 may regulate the acquired resistance to gefitinib in HCC827 via PTEN/AKT signaling pathway. Suppression of miR-214 may thus reverse the acquired resistance to EGFR-TKIs therapy.     

MEK inhibitors reverse resistance in epidermal growth factor receptor mutation lung cancer cells with acquired resistance to gefitinib

Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib. Acquired resistance to EGFR-TKIs develops after prolonged treatments. The study was prompt to explore effective strategies against resistance to EGFR-TKIs. We established gefitinib resistant PC-9 cells which harbor EGFR exon 19 deletion. Known mechanisms for intrinsic or acquired EGFR-TKI resistance, including KRAS mutation, HER2 mutation, EGFR T790M mutation and MET gene amplification, were studied, and we did not observe any known mechanisms for intrinsic or acquired resistance to EGFR-TKIs in the resistant cells. In the parental PC-9 cells, labeled as PC-9/wt, gefitinib completely inhibited EGF-induced phosphorylation of EGFR, AKT and ERK. Gefitinib inhibited EGFR phosphorylation, but was unable to block EGF-induced phosphorylation of ERK in resistant cells, labeled as PC-9/gef cells, including PC-9/gefB4, PC-9/gefE3, and PC-9/gefE7 subclones. We detected NRAS Q61K mutation in the PC-9/gef cells but not the PC-9/wt cells. MEK inhibitors, either AZD6244 or CI1040, inhibited ERK phosphorylation and sensitized gefitinib-induced cytotoxicity in PC-9/gef cells. Whereas MEK inhibitors or gefitinib alone did not activate caspases in PC-9/gef cells, combination of gefitinib and AZD6244 or CI1040 induced apoptosis. Our in vivo studies showed that gefitinib inhibited growth of PC-9/wt xenografts but not PC-9/gef xenografts. Furthermore, combination of a MEK inhibitor and gefitinib inhibited growth of both PC-9/wt xenografts and PC-9/gefB4 xenografts. To conclude, persistent activation of ERK pathway contributes to the acquired gefitinib-resistance. Combined treatment of gefitinib and MEK inhibitors may be therapeutically useful for acquired gefitinib-resistance lung adenocarcinoma cells harboring EGFR mutations.     

Epidermal growth factor receptor and K-Ras mutations and resistance of lung cancer to insulin-like growth factor 1 receptor tyrosine kinase inhibitors

BACKGROUND:

Most patients with nonsmall cell lung cancer (NSCLC) have responded poorly to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The authors investigated the involvement of insulinlike growth factor 1 receptor (IGF‐1R) signaling in primary resistance to EGFR TKIs and the molecular determinants of resistance to IGF‐1R TKIs.

METHODS:

Phosphorylated IGF‐1R/insulin receptor (pIGF‐1R/IR) was immunohistochemically evaluated in an NSCLC tissue microarray. The authors analyzed the antitumor effects of an IGF‐1R TKI (PQIP or OSI‐906), either alone or in combination with a small‐molecular inhibitor (PD98059 or U0126) or with siRNA targeting K‐Ras or mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase (MEK), in vitro and in vivo in NSCLC cells with variable histologic features and EGFR or K‐Ras mutations.

RESULTS:

pIGF‐1R/IR expression in NSCLC specimens was associated with a history of tobacco smoking, squamous cell carcinoma histology, mutant K‐Ras, and wild‐type (WT) EGFR, all of which have been strongly associated with poor response to EGFR TKIs. IGF‐1R TKIs exhibited significant antitumor activity in NSCLC cells with WT EGFR and WT K‐Ras but not in those with mutations in these genes. Introduction of mutant K‐Ras attenuated the effects of IGF‐1R TKIs on NSCLC cells expressing WT K‐Ras. Conversely, inactivation of MEK restored sensitivity to IGF‐TKIs in cells carrying mutant K‐Ras.

CONCLUSIONS:

The mutation status of both EGFR and K‐Ras could be a predictive marker of response to IGF‐1R TKIs. Also, MEK antagonism can abrogate primary resistance of NSCLC cells to IGF‐1R TKIs. Cancer 2012. © 2012 American Cancer Society.     

Overexpression of Cathepsin L is associated with gefitinib resistance in non-small cell lung cancer

Lung cancer, the most common malignancy, is still the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for 80 % of all lung cancers. Recent studies showed Cathepsin L (CTSL) is overexpressed in various cancerous tissues; however, the association between CTSL expression and EGFR-TKI resistance remains unknown. In this study, we investigated the expression of CTSL in lung cancer specimens and matched normal tissues by quantitative real-time PCR and IHC. The functional role of CTSL in resistant PC-9/GR cell line was investigated by proliferation and apoptosis analysis compared with control PC-9 cells. Our results found that the level of CTSL expression was higher in NSCLC tissues compared with matched normal adjacent tissue samples, and CTSL was more highly expressed in PC-9/GR cells compared to PC-9 cells. Knocking-down of CTSL in PC-9/GR cells could decrease cell proliferation and potentiate apoptosis induced by gefitinib, suggesting CTSL may contribute to gefitinib resistance in NSCLC. CTSL might be explored as a candidate of therapeutic target for modulating EGFR-TKI sensitivity in NSCLC.     

Notch-1 contributes to epidermal growth factor receptor tyrosine kinase inhibitor acquired resistance in non-small cell lung cancer in vitro and in vivo

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs in non-small cell lung cancer (NSCLC) patients who initially respond to TKI treatment but whose cancer then progresses. Recent studies have shown that Notch signal is associated with drug resistance. However, the exact mechanism of Notch during acquisition of resistance to EGFR-TKI in human lung cancer remains unclear. In the present study, we showed that the expression of Notch-1 was highly upregulated in EGFR-TKI acquired resistant lung cancer cells. More importantly, Notch-1 contributed to the acquisition of the epithelial–mesenchymal transition (EMT) phenotype, which was critically associated with acquired resistance to EGFR-TKI. Silencing of Notch-1 using siRNA resulted in mesenchymal–epithelial transition (MET), which was associated with impaired invasion and anchorage-independent growth of lung cancer and resensitisation to gefitinib in acquired resistant NSCLC cells. Finally, gefitinib treatment of Balb/c nu/nu with acquired resistant lung cancer xenografts in combination with Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) resulted in effective tumour growth retardation, with decreased proliferative activity and increased apoptotic activity. Collectively, these data suggest that Notch-1 might play a novel role in acquired resistance to gefitinib, which could be reversed by inhibiting Notch-1.     

BIM induction of apoptosis triggered by EGFR-sensitive and resistance cell lines of non-small-cell lung cancer

We sought to improve the understanding of oncogene-dependent and independent non-small-cell lung cancer (NSCLC), which could provide insight into mechanism of sensitivity and/or resistance to tyrosine kinase inhibitors or chemotherapeutics. NSCLC cell lines with different EGFR genotypes were used in this study; MTT assay and flow cytometry were applied to study the sensitivities of these cell lines to gefitinib and cisplatin. Western blot was performed to determine the expression levels of BIM and other Bcl-2 family proteins pre- and pro-treatment. Gefitinib provoked apoptosis of caspase activation via the intrinsic pathways and significantly up-regulated expression of BIM protein in drug-sensitive PC-9 cell line, but not resistant PC-9/BB4 cell line. The knockdown of BIM expression by RNA interference virtually eliminated gefitinib-induced cell killing in PC-9 cells in vitro. Cisplatin could induce apoptosis of the cell lines, including H1299, A549, PC-9, and PC-9/BB4 cells, but which was not associated with overexpression of BIM. BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cell line. Down-regulation of BIM and resistance to gefitinib were both seen in the acquired resistant PC-9/BB4 cell line. The induction of BIM may have a role in the treatment of TKI-resistant tumors.     

Combined inhibition of IGFR enhances the effects of gefitinib in H1650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors

Purpose

H1650 non-small cell lung cancer (NSCLC) cells display primary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) although they have a deletion mutation on exon 19 of the EGFR gene. We investigated the effect of inhibition of both insulin-like growth factor receptor (IGFR) and EGFR signaling considering that IGFR signaling pathway has been implicated in the development and progression with therapeutic resistance of various cancers including lung cancer.

Methods

Three human NSCLC cell lines with an EGFR mutation of PC-9, HCC827 and H1650 were used for experiment. Cell viability and proliferative activity were assessed by MTT and three-dimensional culture assay. Combination index was obtained by CalcuSyn software. The change of EGFR- and IGFR-related signals was evaluated by western blots.

Results

H1650 cells were 1,000 times more resistant to gefitinib and erlotinib than HCC827 and PC-9 cells possessing the same EGFR mutation. Phosphatase and tensin homolog loss and sustained phosphorylation of Akt in spite of treatment with gefitinib were evident only in H1650 cells. Interestingly, IGFR phosphorylation was decreased by gefitinib in HCC827 and PC-9 cells while being maintained in H1650 cells. Combined treatment with the IGFR inhibitors α-IR3 and AG1024 enhanced gefitinib-induced growth inhibition and apoptosis, and down-regulated phosphorylation of Akt, EGFR and IGFR.

Conclusion

Combined inhibition of IGFR signaling enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the primary resistance to EGFR-TKIs in lung cancer.     

非小细胞肺癌分子靶向治疗中EGFR-TKI的耐药机制研究进展

随着分子靶向治疗药物的发展,以吉非替尼(gefitinib,iressa)和厄洛替尼(erlotinib,tarceva)为代表的表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinases inhibitors,EGFR-TKIs)在治疗非小细胞肺癌中发挥了重要作用。然而在临床前和临床研究中发现许多患者对此药物存在原发性耐药或获得性耐药,使该类药物的使用受到一定限制。本文就近年来对EGFR-TKIs耐药机制的研究进展进行综述。     

Epithelial to mesenchymal transition derived from repeated exposure to gefitinib determines the sensitivity to EGFR inhibitors in A549, a non-small cell lung cancer cell line

Epithelial to mesenchymal transition (EMT) has been reported to be related with reduced sensitivity to EGFR tyrosine kinase (EGFR-TK) inhibitors. We performed this study to investigate whether this phenomenon would play a role in acquired resistance to gefitinib. In this study, we established a gefitinib-resistant subline (A549/GR), which was derived from the parental A549 cell line by chronic, repeated exposure to gefitinib. Compared with the A549 cells, the A549/GR cells were approximately 7.7-fold more resistant to gefitinib and they showed the cross-resistance against other EGFR-TK inhibitors, including CL-387,758, erlotinib and ZD6478. Phenotypic changes such as a spindle-cell shape and increased pseudopodia formation suggesting EMT was present in the A549/GR cells. These changes were accompanied by a decrease of E-cadherin and an increase of vimentin, which is a mesenchymal marker. In addition, the ability of invasion and migration was increased in the A549/GR cells. TGF-beta1 treatment for 72 h also induced EMT in the A549 cells and this transition led to resistance to gefitinib. Conversely, this was reversed through the removal of TGF-beta1. In conclusion, induction of EMT may contribute to the decreased efficacy of therapy in primary and acquired resistance to gefitinib.     

The insulin‐like growth factor 1 receptor causes acquired resistance to erlotinib in lung cancer cells with the wild‐type epidermal growth factor receptor

Epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor (TKI) therapy often provides a dramatic response in lung cancer patients with EGFR mutations. In addition, moderate clinical efficacy of the EGFR‐TKI, erlotinib, has been shown in lung cancer patients with the wild‐type EGFR. Numerous molecular mechanisms that cause acquired resistance to EGFR‐TKIs have been identified in lung cancers with the EGFR mutations; however, few have been reported in lung cancers with the wild‐type EGFR. We used H358 lung adenocarcinoma cells lacking EGFR mutations that showed modest sensitivity to erlotinib. The H358 cells acquired resistance to erlotinib via chronic exposure to the drug. The H358 erlotinib‐resistant (ER) cells do not have a secondary EGFR mutation, neither MET gene amplification nor PTEN downregulation; these have been identified in lung cancers with the EGFR mutations. From comprehensive screening of receptor tyrosine kinase phosphorylation, we observed increased phosphorylation of insulin‐like growth factor 1 receptor (IGF1R) in H358ER cells compared with parental H358 cells. H358ER cells responded to combined therapy with erlotinib and NVP‐AEW541, an IGF1R‐TKI. Our results indicate that IGF1R activation is a molecular mechanism that confers acquired resistance to erlotinib in lung cancers with the wild‐type EGFR.     

Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation

Afatinib is a second-generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor and has shown a significant clinical benefit in non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. However, the potential therapeutic effects of afatinib combining with other modalities, including ionizing radiation (IR), are not well understood. In this study, we developed a gefitinib-resistant cell subline (PC-9-GR) with a secondary EGFR mutation (T790M) from NSCLC PC-9 cells after chronic exposures to increasing doses of gefitinib. The presence of afatinib significantly increases the cell killing effect of radiation in PC-9-GR cells harboring acquired T790M, but not in H1975 cells with de novo T790M or in H460 cells that express wild-type EGFR. In PC-9-GR cells, afatinib remarkable blocks baseline of EGFR and ERK phosphorylations, and causes delay of IR-induced AKT phosphorylation. Afatinib treatment also leads to increased apoptosis and suppressed DNA damage repair in irradiated PC-9-GR cells, and enhanced tumor growth inhibition when combined with IR in PC-9-GR xenografts. Our findings suggest a potential therapeutic impact of afatinib as a radiation sensitizer in lung cancer cells harboring acquired T790M mutation, providing a rationale for a clinical trial with combination of afatinib and radiation in NSCLCs with EGFR T790M mutation.     

Lamellarin 14, a derivative of marine alkaloids,inhibits the T790M/C797S mutant epidermal growth factor receptor

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.     

Trametinib overcomes KRAS-G12V–induced osimertinib resistance in a leptomeningeal carcinomatosis model of EGFR-mutant lung cancer

Leptomeningeal carcinomatosis (LMC) occurs frequently in non–small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations and is associated with acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, the mechanism by which LMC acquires resistance to osimertinib, a third-generation EGFR-TKI, is unclear. In this study, we elucidated the resistance mechanism and searched for a novel therapeutic strategy. We induced osimertinib resistance in a mouse model of LMC using an EGFR-mutant NSCLC cell line (PC9) via continuous oral osimertinib treatment and administration of established resistant cells and examined the resistance mechanism using next-generation sequencing. We detected the Kirsten rat sarcoma (KRAS)-G12V mutation in resistant cells, which retained the EGFR exon 19 deletion. Experiments involving KRAS knockdown in resistant cells and KRAS-G12V overexpression in parental cells revealed the involvement of KRAS-G12V in osimertinib resistance. Cotreatment with trametinib (a MEK inhibitor) and osimertinib resensitized the cells to osimertinib. Furthermore, in the mouse model of LMC with resistant cells, combined osimertinib and trametinib treatment successfully controlled LMC progression. These findings suggest a potential novel therapy against KRAS-G12V–harboring osimertinib-resistant LMC in EGFR-mutant NSCLC.