MEK inhibitors for the treatment of non-small cell lung cancer

BRAF and KRAS are two key oncogenes in the RAS/RAF/MEK/MAPK signaling pathway. Concomitant mutations in both KRAS and BRAF genes have been identified in non-small cell lung cancer (NSCLC). They lead to the proliferation, differentiation, and apoptosis of tumor cells by activating the RAS/RAF/MEK/ERK signaling pathway. To date, agents that target RAS/RAF/MEK/ERK signaling pathway have been investigated in NSCLC patients harboring BRAF mutations. BRAF and MEK inhibitors have gained approval for the treatment of patients with NSCLC. According to the reported findings, the combination of MEK inhibitors with chemotherapy, immune checkpoint inhibitors, epidermal growth factor receptor-tyrosine kinase inhibitors or BRAF inhibitors is highly significant for improving clinical efficacy and causing delay in the occurrence of drug resistance. This review summarized the existing experimental results and presented ongoing clinical studies as well. However, further researches need to be conducted to indicate how we can combine other drugs with MEK inhibitors to significantly increase therapeutic effects on patients with lung cancer.


Introduction
Lung cancer is the most common cause of cancer-related death worldwide, with over 1.8 million lung cancer deaths annually [1]. Over the past decades, the treatment of nonsmall cell lung cancer (NSCLC) has changed dramatically with the development of molecular profiling, targeted therapeutic agents, and precision medicine, while the overall prognosis of lung cancer is still poor with a 5-year overall survival (OS) rate of 18% across all stages [2]. NSCLC accounts for about 80-85% of lung cancer cases and almost 70% of NSCLC patients presenting with locally advanced or metastatic disease at initial diagnosis [3]. NSCLC comprises several histologic subtypes, such as squamous cell carcinoma, adenocarcinoma, large cell or undifferentiated carcinoma. Non-squamous carcinoma (70-75%) and squamous cell carcinoma (25-30%) are two major subtypes [4]. In NSCLC somatic mutations in epidermal growth factor receptor (EGFR) and rearrangements in anaplastic lymphoma kinase gene (ALK) and ROS proto-oncogene1 (ROS1) have been validated as strong predictive biomarkers and attractive drug targets. However, the mitogen-activated protein kinase (MAPK) pathway, comprising the kinases RAS, RAF, MEK, and ERK, is also implicated in the tumorigenesis of NSCLC. Thus, MEK inhibitors' monotherapy or combination with other targeted drugs harboring MAPK pathway become a promising strategy for NSCLC patients with B-Raf protooncogene (BRAF) or Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. Currently, the prevalence of BRAF mutations is 3-5% in NSCLC patients, of which BRAF V600E mutations constitute approximately 50% [5]. To date, BRAF plus MEK inhibitors have shown a remarkable survival and response rate in advanced and unresectable melanoma patients, compared with singleagent BRAF inhibition [6,7]. Moreover, concomitant inhibition of both BRAF and MEK has been validated to overcome acquired resistance to BRAF inhibitors alone [8,9]. Besides, the prevalence of KRAS mutations is ~ 25% and ~ 15% in Western and Asian populations with lung adenocarcinoma, respectively [10]. Although the unprecedented challenge of effective KRAS targeting is evidenced by disappointing results to date, MEK inhibitors plus other targeted agents are actively exploring the potential effect in some clinical trials right now.
The present study aimed to review researches concentrated on the effects of MEK inhibitors on NSCLC patients to facilitate the clinical management of such patients.

Structures and functions of MEK proteins
MEK proteins are mitogen-activated protein kinase kinase, a dual specificity Tyr/Thr protein kinase that selectively phosphorylates serine/threonine and tyrosine residues in the activation loop of ERK1 and ERK2. MEK proteins are coded by 7 different genes, among which MEK1 and MEK2 are of significance. MEK1 gene exists in human chromosome 15q22.31, and MEK2 gene exists in chromosome 9q13.3 [11]. The MEK1/2 proteins have three crucial domains (Fig. 1): a core protein kinase domain, an N-terminal domain (approximately 80 amino acids), and a shorter C-terminal region (within 30 amino acids) [11,12]. The protein kinase domain contains the ATP site and catalytic segment; besides, a pocket structure near the ATP-binding site is an ideal target for small target agents that can change the molecule to an inactive state. The N-terminal region plays a regulatory role in signal transduction, including the D-domain (docking site) binding to the ERK substrate. Additionally, mitogen-activated protein kinase (MAPK) is localized to the cytoplasm through its specific association with the N-terminal 1-32 residues of MAPKK in unstimulated cells [13]. The C-terminal region contains the domain for versatile docking (DVD), a critical binding site for the upstream apparatus of the MAPK signaling pathway [14].

Molecular pathways and MEK inhibitors
MEK is the downstream of RAS/RAF/MEK/ERK signaling pathway, highly regulating and playing an important role in cell proliferation, differentiation, apoptosis, and stress responses [15]. It transmits mitogenic signals from outside the cell to the nucleus through multistage phosphorylation [16]. In tumor cells, certain growth factors are combined with transmembrane receptors on the cell surface, leading to the increase in RAS guanosine triphosphate-binding protein in the cell [17]. Once RAS is activated, the plasma membrane of the cell secretes and activates the downstream molecule RAF kinase, stimulates a series of protein kinases, and forms the RAS/ RAF/MEK/ERK signaling pathway [18] (Fig. 2).

Evidence for MEK monotherapy for NSCLC patients
Several trials have explored the function of single-agent MEK inhibition in early clinical development. An initial phase II study evaluated the efficacy and safety of AZD6244 versus pemetrexed as second-or third-line treatment in patients with advanced NSCLC. In this trial, 84 patients were enrolled, and 5% and 4.5% of patients achieved an objective response in AZD6244 group and pemetrexed group, respectively. However, there was no significant difference in median progression-free survival (PFS) between the two groups (90 days vs 67 days, HR:1.08, P = 0.79). The incidence of treatment-related serious adverse events appeared more commonly in the pemetrexed group (6.8% vs 2.5%) than in the AZD6244 group. Most frequently, toxicities were primarily dermatitis acneiform (43%), diarrhea (30%), nausea (18%), and vomiting (18%) with AZD6244 [36]. Another single-arm phase II study was conducted to test PD-0325901 in two administration schedules. This study enrolled 34 patients. Thirteen patients were administered intermittently (3 weeks on/1 week off ), while 21 patients were administered adjusting schedule (5 days on/2 days off for 3 weeks, followed by 1 week off ). No objective responses were observed in two schedules, while 7 patients had stable disease. Median PFS was 1.8 months (95% CI 1.5-1.9), and overall survival was 7.8 months (95% CI 4.5-13.9). The most common treatment-related toxicities (incidence in schedule A/incidence in schedule B) were diarrhea (54%/76%), fatigue (31%/48%), rash (46%/33%), vomiting (38%/33%), and nausea (38%/29%) [37]. Another phase II study evaluated the safety and efficacy of trametinib versus docetaxel for patients with KRAS-mutant NSCLC patients. In this trial, 129 were enrolled. However, there was no significant difference in median PFS in trametinib and docetaxel arm (12 weeks vs 11 weeks, HR:1.14, P = 0.5197) and in median OS (8 months vs not reached, HR:0.97, P = 0.934). Partial responses (PRs) for these two groups were 12% and 12% (P = 1.0000). The most frequent grade 3 or higher toxicities were primarily hypertension (9%), rash (9%), diarrhea (5%), sepsis (5%), and asthenia (5%) vs. neutropenia (35%) in trametinib and docetaxel arms, respectively. One treatment-related death occurred with trametinib and none with docetaxel [38]. An initial phase II basket trial evaluated the efficacy of selumetinib in NSCLC patients with molecular profiling. In this trial, 110 patients presented RAS/RAF mutations with KRAS mutations (24.9%), BRAF mutations (2%), HRAS and NRAS mutations (0.7%), and 10 patients were enrolled onto the selumetinib arm. However, 9 patients failed to achieve selumetinib monotherapy primary end point, with only one partial response (ORR 11%, 95% CI 0-48%), a median PFS time of 2.3 months, and median OS time of 6.5 months [39]. The results of these phase II studies indicated that MEK inhibitors' monotherapy seemed to have poor clinical outcomes and more toxicities for NSCLC patients compared with chemotherapy alone.

Evidences for combination of BRAF and MEK inhibitors for NSCLC patients
The combination of BRAF and MEK inhibitors has been proved to be clinically effective for NSCLC patients to date. An initial phase II trial evaluated the combination of dabrafenib and trametinib in previously treated BRAF(V600E)-mutant NSCLC patients. Fifty-seven patients were enrolled in this study. The overall response MEK: mitogen-activated protein kinase kinase; ERK: extracellular signal-regulated kinase; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase B; mTOR: mammalian target of rapamycin; NF-kB: nuclear factor-kB was 63.2% (95% CI 49.3-75.6%), the median PFS was 9.7 months (95% CI 6.9-19.6), and median duration of response (DOR) was 9.0 months (95% CI 6.9-18.3). Common grade 3/4 AEs were neutropenia (9%), hyponatremia (7%), and anemia (5%) [40]. Besides, the same research team developed another phase II study to assess the efficacy and safety of dabrafenib plus trametinib treatment in previously untreated patients with BRAF(V600E)mutant metastatic NSCLC. In this study, 36 patients were enrolled and treated with first-line dabrafenib plus trametinib. The ORR was 64% (95% CI 46-79%), median DOR was 10.4 months (95%CI 8.3-17.9), and PFS was 10.9 months (95% CI 7.0-16.6). Grade 3 or 4 AEs were pyrexia (11%), alanine aminotransferase increase (11%), hypertension (11%), and vomiting (8%) [41]. The NCI-MATCH Trial Subprotocol H evaluated the combination of dabrafenib and trametinib in solid tumor patients, 5 lung adenocarcinoma patients included. One patient was progression-free at 32.5 months, and 1 patient who was considered unevaluable, with an 81% reduction in the sum of measured lesions, had a PFS of 12.7 months. Three patients had SD for 15.6, 6.6, and 3.6 months which is sought to investigate the selective BRAF inhibitor [42]. The clinical data showed the efficacy of combination of MEK and BRAF inhibitors with untreated or treated BRAF V600E-mutant metastatic NSCLC, indicating that physicians can flexibly treat patients with this targeted therapy combination in either the first-line or following chemotherapy and provide strategies to accommodate the individual patient needs.

Evidence for combination of chemotherapy and MEK inhibitors for NSCLC patients
Chemotherapy is no longer the most efficacious treatment, and targeted agents have been rationally designed to inhibit particular mutations, leading to a more streamlined clinical trial process. Ten years ago, numerous clinical trials have concentrated on exploration of the  (Table 3) [50]. Another phase I study evaluated the safety and tolerability of selumetinib as a monotherapy, or in combination with docetaxel as a second-line therapy for Japanese patients with advanced NSCLC. Thirty-three patients were enrolled and 25 assigned to treatment. Grade 3 dose-limiting toxicities were febrile neutropenia and pneumonitis (Table 3) [51]. Current clinical data showed that MEK inhibitor combined with chemotherapy can improve the outcomes while some not. One possibility is that clinical benefit may occur in a specific subset of tumors that exhibits a favorable genetic of signaling environment. So effective drug candidates of MEK inhibitors and proper special patients should be detected for this combination therapy.

Evidence for combination of immune checkpoint inhibitors and MEK inhibitors for NSCLC patients
Immune checkpoint inhibitors (ICIs) have opened up a new era for lung cancer treatment in recent years. However, even when patients with 50% or higher positivity for PD-L1 expression are selected, overall response rates still do not exceed 31% [52,53]. Thus, different combination treatments have been proposed. Preclinical data suggested an improved T cell activation and increased CTLA-4 expression for selumetinib and trametinib. Besides, pulsatile MEKi treatment combined with CTLA-4 blockade prolonged survival in mice-bearing tumors with mutant KRAS [54,55]. An initial phase Ib  [56]. Another phase I/II trial evaluated immunotherapy with durvalumab and tremelimumab with continuous or intermittent administration of selumetinib in NSCLC patients. The trial is actively screening and enrolling patients, and the estimated study completion is scheduled for April 2021 [57]. Currently, the clinical data about ICI-combined MEK inhibitors are still not efficient enough to validate the most proper way to treat NSCLC. More clinical outcomes are worthy being awaited furthermore. Similarly, OS was 21.8 and 10.5 months, respectively. In terms of safety, grade 3 and 4 toxicities were also increased in combination therapy, with diarrhea, dehydration, and fatigue all occurring in > 20% of patients [59]. TATTON was initially designed as a phase Ib trial to assess the safety and tolerability of osimertinib in combination with selumetinib, savolitinib, or durvalumab for EGFR-mutant lung cancer patients. Seventy-seven patients were enrolled in this study. The ORR was 42%, 44%, and 43% in selumetinib + osimertinib, savolitinib + osimertinib, and durvalumab + osimertinib arms, respectively. The most common AEs in selumetinib plus osimertinib group were diarrhea (75%), skin rash (58%), nausea (47%) [60]. Another phase I study evaluated the efficacy of afatinib plus selumetinib in patients with KRAS-mutant-positive solid tumors, 6 NSCLC patients included. Dose-limiting toxicities (DLTs) consisted of grade 3 diarrhea, decreased appetite, nausea/vomiting, dehydration, and mucositis. Stable disease for 221 days in a NSCLC patient was the best response [61]. In ESMO 2019 Congress, a phase I study evaluated the combination of lapatinib and trametinib for patients with KRASmutant solid tumors, 15 NSCLC patients included. One patient was confirmed partial response. Grade 3 AEs were diarrhea, rash, and nausea [62]. The clinical data showed that a number of trials were focused on detecting the strong rationale supporting combination therapy with MEK inhibitors for overcoming or delaying drug resistance in EGFR-mutant NSCLC. However, there are no EGFR-based combination therapies with global adoption, and therapies for patients with acquired resistance to EGFR-TKIs remain to be detected.

Mechanisms of resistance to MEK inhibitors
RAS/RAF/MEK/ERK signaling pathway-associated inhibitors have proven to be effective in treatment of various types of cancer, but have presented drug resistance in clinical application and MEK inhibitors

Other combined therapies and ongoing studies
As acquired resistance becomes a frequent problem for all the target agents, a number of clinical trials have been designed to evaluate the efficacy and safety of combination of two different types of targets plus MEK inhibitors, according to the probable resistance mechanisms in the former part. A preclinical experiment revealed that selumetinib combined with BEZ235 (PI3K/mTOR inhibitor) markedly enhanced their antitumor effects and inhibited the tumor growth of NCI-H1993 in gefitinib-resistant NSCLC xenograft models [71]. Other ongoing clinical trials on administration of MEK inhibitors for NSCLC patients have been summarized (Table 4). To date, a variety of MEK1/2 inhibitors have been applied for different types of cancer, including NSCLC at various stages of clinical testing. The publication of the final results of these studies is still awaited.

Conclusions/expectations
The functions of EGFR-TKIs, checkpoint inhibitors, and traditional chemotherapy have been widely studied in NSCLC patients, while the role of MEK inhibitors in the  Table 5. At the early stage, MEK inhibitors' monotherapy had been detected a lot but seemed not to be effective for NSCLC patients for its poor efficacy and higher toxicities. No matter compared with pemetrexed or docetaxel, no significant difference in median PFS or OS was observed and dermatitis acneiform, hypertension, and diarrhea toxicities were more common [36][37][38][39].
MEK inhibitors in combination with BRAF inhibitors as a treatment demonstrated an improved efficacy for NSCLC patients. Currently, trametinib combined with dabrafenib has been the only therapy approved by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of BRAF V600E-mutant NSCLC patients, which has been written into the National Comprehensive Cancer Network (NCCN) guidelines as well. The phase II trials in previously treated and untreated BRAF V600E-mutant NSCLC demonstrated that PFS and OS were longer and ORR was higher, which were much better than the outcomes of single-agent BRAF inhibitor in the previous study [40,41,72]. According to these trials, NCCN guidelines recommend that dabrafenib combined with trametinib be the first-line and subsequent therapy for BRAF V600E mutationpositive NSCLC patients. However, the challenge with this combination is the emergence of drug resistance and no effective treatment strategy to overcome it yet. Another challenge in targeted therapy for non-V600E mutation patients is still lacking.
Chemotherapy plus MEK inhibitors have showed obscure clinical outcomes to date. Some trials demonstrated that this combination therapy had the trend of longer PFS, OS, and higher ORR, but with no significant difference, especially in the SELECT series trials [47,49,50]. Other trails [44][45][46] tried to do some exploration in subgroup NSCLC patients, such as KRAS-mutant and KRAS-WT patients. Regrettably, different chemotherapy drugs seemed to influence the outcomes as well. A phase 1/1b study showed that ORR was 18% versus 24% in KRAS-WT and KRAS-mutant patients in trametinib plus docetaxel group, while ORR was 17% versus 11% KRAS-WT and KRAS-mutant patients in trametinib plus pemetrexed group [46]. Chemotherapy applied concurrently with MEK inhibitors requires further specific validation including the different chemotherapy agents, KRAS or other gene mutations and different MEK inhibitors before this combination strategy can become a standard treatment option for NSCLC patients.
Based on the preclinical studies, MEK inhibitors could improved T cell activation, conditioned the tumor microenvironment to facilitate improved response to anti-CTLA-4 treatment and prolonged survival in KRASmutant mice in combination with CTLA-4 blockade [54,55]. However, the current relevant clinical trials of ICIs plus MEK inhibitors were not sufficient to draw the conclusion yet. Since only a phase Ib study [56] investigated the safety and efficacy of cobimetinib plus atezolizumab in a single arm for few NSCLC patients and several PD-1/ L1 inhibitors plus MEK inhibitors clinical trials [57] ( Table 4) are still ongoing, the finial clinical outcomes are worthy being looking forward to furthermore.
Although targeted therapy has dramatically changed our approach to treating NSCLC, the emergency of drug resistance and the lack of effective treatments to some special target such as KRAS still affect the prognosis of NSCLC patients. Preclinical data showed that MEK inhibitors plus EGFR-TKIs could inhibit cell proliferation significantly of EGFR-TKIs-resistant cells, while similar clinical trials have not been designed yet [58]. Current clinical trials [59][60][61][62] focused on EGFR-TKIs, including erlotinib, osimertinib, and afatinib, in combination with MEK inhibitors appearing somewhat illusory for OS, PFS or ORR. These outcomes seemed not to be improved under this strategy and more obvious toxicities were revealed. Further researches should be designed more on the administration way to combine two or three drugs together to optimize the therapeutic effect in appropriate subset patients. In addition to the emerging drugs and clinical studies mentioned above, there are still many more new treatment combinations that have conducted in early stages of clinical development. Novel combination drugs can be broadly classified as BRAF inhibitors, EGFR-TKIs, multitarget tyrosine kinase inhibitors, CDK4/6 inhibitors, ALK inhibitors, platin-based chemotherapy, and ICIs. Additionally, many treatment combinations being explored in early-stage clinical studies, such as PI3K and AKT inhibitors should be further detected in a more rational way with MEK inhibitors in human bodies [73][74][75][76][77] (Fig. 2). The preclinical data indicated that the combined therapy of MEK and PI3K inhibitors has presented promising outcomes for NSCLC patients with the acquired resistance to EGFR-TKIs [78], but more clinical effects should be validated in the future. Overall, there seems to be hope on the horizon for NSCLC patients administrated with MEK inhibitors combined with other promising agents to improve patient outcomes finally.