Several studies have shown that NSCLC cells treated with EGFR-TKIs adopt multiple mechanisms to reactivate ERK signaling. The upregulation of phosphorylated ERK following EGFR-TKI resistance was a ubiquitous event in EGFR-TKI-resistant NSCLC tissues and cell lines. Our study suggested that targeting ERK is an effective strategy to overcome acquired resistance to EGFR-TKIs in NSCLC. Furthermore, co-targeting EGFR and ERK will be an even more effective combination strategy.
ERK (ERK1/2) controls several downstream cytoplasmic and nuclear targets by phosphorylating and regulating cell cycle and negative feedback mechanisms [15]. Although specific ERK alterations have not been identified as actionable mechanisms of acquired resistance to TKIs, alterations upstream of ERK are common in acquired resistance to various TKIs including EGFR-TKI [1, 18]. Previous studies reported that acquired resistance to EGFR-TKIs converged on the activation of the MAPK pathway, especially ERK, albeit through different mechanisms such as MET amplification and RAS alteration [9, 2, 19]. Recently, reactivation of ERK signaling through chemokine receptor CXCR7 has been identified as a resistance mechanism to EGFR-TKI in patients with NSCLC [3]. Therefore, targeting ERK would be an attractive strategy for the treatment of a variety of tumor types harboring acquired resistance to TKIs.
Several ERK inhibitors including ASN007, ulixertinib, and LY3214996 are being developed in clinical trials as a treatment for advanced solid tumors with RAS-RAF-MAPK pathway alterations [15]. Some have demonstrated preliminary antitumor activity in preclinical and clinical trials. However, the most common or dose-limiting toxicities observed to date with ERK inhibitors include diarrhea, nausea, fatigue, and rash. Given that ASN007 is a highly potent and selective ERK1/2 inhibitor in the nanomolar range with a long target residence time, it can be hypothesized that toxicities related to AN007 might be reduced compared to other ERK inhibitors. In our study, ASN007 shows promising antitumor activity as a single agent and in combination with EGFR-TKI. ASN007 alone did not cause any dose-limiting toxicities including loss of body weight or skin rash in a xenograft model. Although mice treated with a combination of ASN007 and erlotinib showed body weight loss 1 week after treatment, this loss was recovered after 2 weeks of treatment. These observations suggest that our proposed combination of ASN007 plus EGFR-TKI was well tolerated in a mouse xenograft model.
Intriguingly, we found that decreased FRA1 expression is the main mechanism of ERK inhibition by ASN007 in EGFR-TKI-resistant NSCLC. In contrast, previous studies using other ERK inhibitors such as ulixertinib and LY3214996 demonstrated that the main downstream target of ERK in KRAS-driven tumors is RSK, not FRA1 [20, 21]. Although phosphorylation of RSK was also inhibited by ASN007 treatment, FRA1 protein expression is more abundant than that of RSK1 in our resistant cells. These results suggest that ERK inhibitor could exert antitumor effects through various mechanisms depending on tumor context. FRA1 is a member of the FOS protein family and can form an AP-1 transcription factor. FRA1 is mainly regulated by post-translational phosphorylation by a mitogen-activated protein kinase (MAPK) signaling pathway, especially ERK. Because phosphorylation of FRA1 prevents degradation by ubiquitin-independent proteasome, ERK activation is required for FRA1 accumulation. Many studies have shown that FRA1 is overexpressed in many tumors such as lung cancer, breast cancer, colorectal cancer and other tumors. The abnormal expression of FRA1 in tumor has important roles during tumor progression, promoting cell proliferation and invasion, inhibiting apoptosis, and enhancing tumor angiogenesis and heterogeneity [16, 22, 23]. Previous studies reported that FRA1 promotes KRAS-induced lung cancer progression and metastasis [17, 24, 25]. In addition, FRA1 contributed to oncogenic KRAS-driven PD-L1 expression in high risk, premalignant human bronchial epithelial cells, suggesting that FRA1 may promote cancer progression by facilitating immune evasion [14]. Altogether, FRA1 may be a prognostic marker and potential target for lung cancer with oncogenic mutations or drug resistance.
Our study has several limitations. First, the EGFR-TKI resistance models in this study do not represent the variety of clinical situations patients with acquired resistance can demonstrate. Although our in vitro models have different alterations in ERK upstream, it converged ERK activation regardless of EGFR-TKIs. Second, the regulation of acquired resistance by ERK has not been fully elucidated. Further mechanistic studies are needed to investigate how increased ERK signaling exclusively activates the development of drug resistance.
In summary, our findings suggest that concomitant EGFR and ERK blockade is a promising strategy to overcome acquired resistance in EGFR-mutated NSCLC regardless of whether the acquired resistance arises from first- or third-generation EGFR-TKIs. Further research is needed to determine whether these combinations can also prevent or delay the development of acquired resistance.
Declarations