Abstract

EGFR-TKI targeted therapy is one of the most effective treatments for lung cancer patients harboring EGFR activating mutations. However, inhibition response is easily attenuated by drug resistance, which is mainly due to bypass activation or downstream activation. Herein, we established osimertinib-resistant cells by stepwise dose-escalation in vitro and an osimertinib-resistant patient-derived xenograft model through persistent treatment in vivo. Phosphorylated proteomics identified that MEK1 and AKT1/2 were abnormally activated in resistant cells compared with parental cells. Likewise, EGFR inhibition by osimertinib induced activation of MEK1 and AKT1/2, which weakened osimertinib sensitivity in NSCLC cells. Consequently, this study aimed to identify a novel inhibitor which could suppress resistant cell growth by dual targeting of MEK1 and AKT1/2. Based on computational screening, we identified that costunolide could interact with MEK1 and AKT1/2. Further exploration using in vitro kinase assays validated that costunolide inhibited the kinase activity of MEK1 and AKT1/2, which restrained downstream ERK-RSK2 and GSK3β signal transduction and significantly induced cell apoptosis. Remarkably, the combination of osimertinib and costunolide showed synergistic or additive inhibitory effects on tumor growth in osimertinib-resistant cell lines and PDX model. Hence, this study highlights a potential therapeutic strategy for osimertinib-resistant patients through targeting of MEK1 and AKT1/2 by costunolide.

Background

Based on the Global Cancer Statistics of 2020, lung cancer ranks the second most frequently diagnosed cancer (11.4% of total cases) and is the leading cause of cancer-related death (18% of total cancer deaths)[1]. At present, therapeutic regimens for lung cancer include surgery, chemotherapy, immunotherapy, and targeted therapy[2]. Despite the continuous refinement of treatment options, the 5-year survival rate still remains below 20%[3]. Therefore, further investigation is needed to optimize therapeutic strategies.

Of the diverse therapeutic schemes, targeted therapy showed significant preponderance with lower side effects, stronger pertinence, and more convenience for patients[4]. Epidermal Growth Factor Receptor (EGFR)-focused targeted therapy is one of the most widely used treatments for non-small cell lung cancer (NSCLC) patients that harbor EGFR mutations, with more than 60% object response rate[5]. Osimertinib is a third generation EGFR- tyrosine kinase inhibitor (TKI) that has been approved by the FDA as a second-line treatment of EGFR acquired mutant(T790M) NSCLC patient, a first-line treatment for EGFR activating mutant (L858R or exon 19 deletion) NSCLC patients, and as a postoperative adjuvant therapy approved by National Medical Products Administration in China[6]. However, drug resistance is an inevitable issue. Due to tumor heterogeneity, mechanisms of drug resistance vary among different populations and are mainly caused by acquired EGFR mutations, activation or tetraploidization of bypass signal molecules, or phenotypic transformation[5]. Bypass activation, such as Erb-B2 receptor tyrosine kinase 2 (HER2) activation could abnormally activate the mitogen-activated protein kinase (MAPK) or protein-serine-threonine kinase- glycogen synthase kinase 3 beta (AKT-GSK3β) pathways, leading to increased cell proliferation and drug resistance[7]. Currently, EGFR-TKI combined with other drugs are popular regimens for managing drug resistance.

To further explore strategies that could overcome osimertinib resistance, we established osimertinib-resistant cells through a stepwise dose-escalation method and performed phosphorylated proteomics analysis to identify the aberrant activated pathways in resistant cells. In the present study, we identified that mitogen-activated protein kinase kinase 1 (MEK1) and AKT1/2 were abnormally activated in resistant cells. Knockdown of MEK1 and AKT1/2 inhibited the growth of osimertinib-resistant cells and partially restored osimertinib sensitivity. Moreover, we found that costunolide functions as a dual inhibitor of MEK1 and AKT1/2 that significantly induces cell apoptosis in the osimertinib-resistant cell pool. Combination of costunolide with osimertinib showed synergistic or additive inhibitory effect on osimertinib-resistant cells and a resistant patient-derived xenograft (PDX) model. These data demonstrated that costunolide may be considered as a promising strategy for osimertinib-resistant patients with activated MEK1 and AKT1/2.

Discussion

EGFR targeted therapy has achieved prominent performance for NSCLC treatment; however, acquired drug resistance inevitably limits long-term effects[7]. An appropriate drug resistant model is rather important for preclinical studies. Consequently, we generated osmertinib-resistance in cell lines harboring EFGR mutations through a step-wise dose escalation method, which showed a remarkably higher IC₅₀ of osimertinib compared with parental cells. The lower drug susceptibility was further confirmed by foci formation and cell apoptosis assays in the resistant cells. To establish a more comprehensive resistance mechanism in vivo, we also generated an osimertinib-resistant PDX model through continuous induction using lung cancer tissue harboring an EGFR mutation. These long-term inducted resistant models are effective tools to realistically simulate the process of drug resistance in a laboratory setting.

Due to tumor heterogeneity, the reported mechanisms of osimertinib resistance may vary depending on the terms of different regimens. Acquired EGFR mutation, c-MET amplification, HER2 amplification or mutation, PIK3CA mutation, BRAF and KRAS mutation have been reported as the dominant factors contributing to osimertinib resistance in response to first-line treatment. Acquired EGFR mutation, c-MET amplification, cell cycle gene alteration, HER2 amplification, PIK3CA amplification or mutation have been reported as contributors to osimertinib resistance in response to second-line treatment. Obviously, most of the dysregulated proteins highlighted above can activate PI3K/AKT and MAPK-ERK pathways. As reported, AKT is a key modulator in regulating multi-drug resistance[12]. One mechanism occurs through AKT-triggered activation of NFκB, which can inhibit cell apoptosis and promote tumor growth. Furthermore, activated AKT also modulates cell proliferation through the phosphorylation of GSK3β, which can facilitate resistance by promoting the evasion of EGFR-targeted therapy. Besides, MEK also plays a profound role in regulating drug resistance. The paradoxical activation of MEK stimulates ERK to promote cell proliferation and drug resistance[13]. Most often, activation of MEK or AKT also play crucial roles during the drug resistance process. As reported, combination of gefitinib with MEK1/2 inhibitor synergistically inhibited gefitinib-resistant NSCLC cell growth[14]. Dual blockade of PI3K/AKT and MEK/ERK pathways potentiated gefitinib sensitivity in gefitinib resistant NSCLC and breast cancer cells. Accordingly, AKT/GSK and MEK/ERK are the most frequently dysregulated signaling pathways in acquired drug resistance. However, individually targeting AKT or MEK may facilitate active bypass or downstream signaling which will limit the success of therapies. Thus, the rational to inhibit PI3K/AKT and MAPK pathways simultaneously seems logical to produce a more robust inhibitory response that may prevent further resistance. In present study, we identified that costunolide is an effective inhibitor capable of suppressing the kinase activity of MEK1 and AKT1/2, thereby inducing significant cell apoptosis and inhibition of cell growth. Costunolide is a natural bioactive sesquiterpene lactone with antioxidant, anti-inflammatory and anticancer effects that is extracted from the roots of Saussurea lappa. Recent studies have shown that costunolide can inhibit the proliferation of various cancer cells. In ovarian cancer cells, costunolide promotes the expression of apoptosis signals, such as caspase 3, caspase 8 and caspase 9 by enhancing the production of ROS, thereby inhibiting the growth of cisplatin-resistant cells[15]. In addition, costunolide can inhibit the growth of colorectal cancer and melanoma cells by inhibiting the kinase activity of AKT[10]. Costunolide also showed a similar inhibitory effect compared with the combination of AKTi and MEKi, but at a higher dose. Our data suggested that, costunolide could act as a safe and effective inhibitor to suppress osimertinib-resistant cell growth.

Another critical finding of our study is that costunolide reversed osimertinib resistance in vivo. Due to the stable biological characteristics of patient derived tissues, we used an EGFR mutant PDX model to further evaluate the combination effects of costunolide and osimertinib. Based on the data, costunolide inhibited tumor growth and a significant synergistic effect was observed in the model. Moreover, downstream signaling effectors of MEK and AKT were markedly inhibited in the combination treatment group. Additionally, we did not observe obvious changes in total body weight, ALT or AST level between the different groups, indicating a well-tolerated dose of costunolide plus osimertinib. However, it should be noted that costunolide did not show a growth inhibitory effect in the HLG57-DMSO model. This observation is mainly because p-MEK and p-AKT protein expression levels are lower in the HLG57 relative to other lung tumor tissues. Based on this in vivo study, we concluded that the efficiency of costunolide is dependent on the levels of activated MEK1 and AKT1/2. Additional studies are required to further characterize suitable strategies for managing osimertinib-resistant cell populations deficient in active MEK and AKT.

Conclusion

Our study demonstrated that MEK1 and AKT1/2 are critical for the development of osimertinib resistance. Moreover, costunolide reversed osimertinib resistance through direct targeting of MEK1 and AKT1/2. A synergistic or additive effect was observed with the combination treatment of costunolide and osimertinb both in vitro and in vivo, which might offer a candidate strategy in the clinic.

Abbreviations

EGFR:

epidermal growth factor receptorNSCLC:

non-small cell lung cancerTKI:

tyrosine kinase inhibitorHER2:

Erb-B2 receptor tyrosine kinase 2BRAF:

B-Raf Proto-OncogenePIK3CA:

phosphatidylinositol-4,5-Bisphosphate 3-kinase catalytic subunit alphaAKT:

protein-serine-threonine kinaseGSK3β:

glycogen synthase kinase 3 betaMEK1:

mitogen-activated protein kinase kinase 1PDX:

patient-derived xenograft

source: Molecular Cancer