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MEK inhibitor resistance mechanisms and recent developments in combination trials

  • E. Kun
    Affiliations
    Department of Gynecologic Oncology & Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
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  • Y.T.M. Tsang
    Affiliations
    Department of Gynecologic Oncology & Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
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  • C.W. Ng
    Affiliations
    Department of Gynecologic Oncology & Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
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  • D.M. Gershenson
    Affiliations
    Department of Gynecologic Oncology & Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
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  • K.K. Wong
    Correspondence
    Corresponding author at: Department of Gynecologic Oncology & Reproductive Medicine, Room T4-3900, Clinical Research Building, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
    Affiliations
    Department of Gynecologic Oncology & Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences, Houston, TX, USA
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Open AccessPublished:December 15, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.102137

      Highlights

      • A variety of MEK inhibitors have been tested as a single agent in clinical trials and shown to be limited to certain types of cancers.
      • Side effects from MEK inhibition can be significant but are more tolerable than effects from conventional cancer therapies.
      • Novel mechanisms of adaptive resistance to MEK inhibitor have been discovered in different cancers.
      • Combination clinical trials are being developed to overcome MEK inhibitor resistance.

      Abstract

      The mitogen-activated protein kinase (MAPK) pathway plays a vital role in cellular processes such as gene expression, cell proliferation, cell survival, and apoptosis. Also known as the RAS-RAF-MEK-ERK pathway, the MAPK pathway has been implicated in approximately one-third of all cancers. Mutations in RAS or RAF genes such as KRAS and BRAF are common, and these mutations typically promote malignancies by over-activating MEK and ERK downstream, which drives sustained cell proliferation and uninhibited cell growth. Development of drugs targeting this pathway has been a research area of great interest, especially drugs targeting the inhibition of MEK. In vitro and clinical studies have shown promise for certain MEK inhibitors (MEKi) , and MEKi have become the first treatment option for certain cancers. Despite promising results, not all patients have a response to MEKi, and mechanisms of resistance typically arise in patients who do have a positive initial response. This paper summarizes recent developments regarding MEKi, the mechanisms of adaptive resistance to MEKi, and the potential solutions to the issue of adaptive MEKi resistance.

      Keywords

      Introduction

      MAPK/ERK pathway

      A simple overview of the MAPK pathway begins with the binding of various growth factors, cytokines, and hormones to cell surface receptors, which increases the level of RAS guanosine triphosphate (GTP) in cells (Fig. 1). GTP binds to and activates RAS, which has four forms, HRAS, NRAS, KRAS-A, and KRAS-B [
      • Lowy D.R.
      • Willumsen B.M.
      Function and regulation of ras.
      ]. Once activated, RAS binds to RAF, which has three isoforms, C-RAF-1, A-RAF, and B-RAF [
      • Peyssonnaux C.
      • Eychene A.
      The Raf/MEK/ERK pathway: new concepts of activation.
      ,
      • Wong K.K.
      Recent developments in anti-cancer agents targeting the Ras/Raf/ MEK/ERK pathway.
      ,
      • Hagemann C.
      • Rapp U.R.
      Isotype-specific functions of Raf kinases.
      ]. Activation of RAF by phosphorylation in this state activates MEK1/2, which then activate ERK [
      • Hilger R.A.
      • Scheulen M.E.
      • Strumberg D.
      The Ras-Raf-MEK-ERK pathway in the treatment of cancer.
      ]. ERK is responsible for activating several downstream molecules, many of which directly influence the proliferation, survival, and differentiation of cells [
      • Chang L.
      • Karin M.
      Mammalian MAP kinase signalling cascades.
      ,
      • Cheng Y.
      • Tian H.
      Current Development Status of MEK Inhibitors.
      ]. ERK is also directly responsible for activation of Cyclin D and CDK4/6, which allow the cell cycle to progress from the G1 to S phase [
      • Chambard J.C.
      • Lefloch R.
      • Pouyssegur J.
      • Lenormand P.
      ERK implication in cell cycle regulation.
      ].
      Figure thumbnail gr1
      Fig. 1Mechanisms of Resistance to MEK inhibitors. 1) Reactivation of multiple RTKs upstream of MAPK pathways such as Growth factors bind to RTKs such as ERBB3, EGFR and ERBB2, which phosphorylate and recruit GRB2-SOS complex which promotes removal of GDP from RAS allowing it to bind GTP and become active. SHP2 acts upstream of SOS and also plays a vital role in RAS activation. 2) Activation of parallel pathways such as the PI3K, STAT3, and Hippo signaling pathways. PTEN is a negative regulator of PI3K. PP2A is a phosphatase which is a negative regulator of various key proteins involved in the cell proliferation pathways. 3) Activation of transcription factors that control epithelial and mesenchymal transition.
      Dysregulation and often overactivation of the MAPK pathway occurs in multiple cancers and possibly more than one-third of all malignancies, leading to downregulation of antiproliferative genes and uninhibited cell growth [
      • Cheng Y.
      • Tian H.
      Current Development Status of MEK Inhibitors.
      ,
      • Chambard J.C.
      • Lefloch R.
      • Pouyssegur J.
      • Lenormand P.
      ERK implication in cell cycle regulation.
      ]. Mutations responsible for upregulation of this pathway often occur upstream of ERK, involving either RAS or RAF. RAS is a crucial player in the promotion of tumorigenesis because it is responsible for activation of not only ERK but also other cell signaling pathways such as the PI3K/AKT pathway, which also promotes cell growth [
      • Mendoza M.C.
      • Er E.E.
      • Blenis J.
      The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation.
      ,
      • Mahapatra D.K.
      • Asati V.
      • Bharti S.K.
      MEK inhibitors in oncology: a patent review (2015-Present).
      ]. For example, mutations in KRAS are found in around 30% of all cancers, and mutations in BRAF are associated with biliary tract, colorectal, lung (non-small cell), ovarian, thyroid, and skin cancers (in particular, melanoma) [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ,
      • Wan P.T.
      • Garnett M.J.
      • Roe S.M.
      • Lee S.
      • Niculescu-Duvaz D.
      • Good V.M.
      • et al.
      Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.
      ,
      • Adjei A.A.
      Blocking oncogenic Ras signaling for cancer therapy.
      ,
      • Subbiah V.
      • Kreitman R.J.
      • Wainberg Z.A.
      • Cho J.Y.
      • Schellens J.H.M.
      • Soria J.C.
      • et al.
      Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600-Mutant Anaplastic Thyroid Cancer.
      ,
      • Planchard D.
      • Smit E.F.
      • Groen H.J.M.
      • Mazieres J.
      • Besse B.
      • Helland A.
      • et al.
      Dabrafenib plus trametinib in patients with previously untreated BRAF(V600E)-mutant metastatic non-small-cell lung cancer: an open-label, phase 2 trial.
      ]. In a typical RAS oncogenic mutation, RAS remains in an activated state with GTP bound to the protein. Typically, GTPase-activating proteins (GAPs) accelerate RAS-mediated GTP hydrolysis, but mutations in RAS can invalidate GAPs from functioning correctly, thus leading to constant downstream activation of RAF, leading to MEK activation, and so on [
      • Pylayeva-Gupta Y.
      • Grabocka E.
      • Bar-Sagi D.
      RAS oncogenes: weaving a tumorigenic web.
      ]. Similarly, the most common BRAF mutation, BRAFV600E, can activate independently of RAS-GTP and evade repression from SPRY2, a known MAPK pathway inhibitor, leading to increased downstream activation [
      • Wong K.K.
      Recent developments in anti-cancer agents targeting the Ras/Raf/ MEK/ERK pathway.
      ,
      • Tsavachidou D.
      • Coleman M.L.
      • Athanasiadis G.
      • Li S.
      • Licht J.D.
      • Olson M.F.
      • et al.
      SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant.
      ]. Studies have shown that BRAF and RAS mutations are typically mutually exclusive in the same tumor, which suggests that RAS acts to activate BRAF in these tumors in the same pathway [
      • Wan P.T.
      • Garnett M.J.
      • Roe S.M.
      • Lee S.
      • Niculescu-Duvaz D.
      • Good V.M.
      • et al.
      Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.
      ].
      Ever since the discovery of the MAPK pathway and its importance in cancer cell growth and the cell cycle, researchers have sought to target the pathway for therapeutic means. The first MAPK pathway inhibitor was discovered almost 30 years ago and functioned as an apparent allosteric inhibitor of MEK1 and MEK2 in an ATP-independent manner [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ,
      • Dudley D.T.
      • Pang L.
      • Decker S.J.
      • Bridges A.J.
      • Saltiel A.R.
      A synthetic inhibitor of the mitogen-activated protein kinase cascade.
      ]. It quickly became clear that MEK was a possible target for inhibition of the ERK pathway for several reasons: MEKi are not ATP competitive, MEK has relatively few phosphorylation sites involved in activation and inactivation, and MEK has a high specificity for binding to ERK [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ,
      • McCubrey J.A.
      • Steelman L.S.
      • Chappell W.H.
      • Abrams S.L.
      • Wong E.W.
      • Chang F.
      • et al.
      Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance.
      ,
      • Sebolt-Leopold J.S.
      Advances in the development of cancer therapeutics directed against the RAS-mitogen-activated protein kinase pathway.
      ]. Furthermore, only activation of MEK will lead to activation of ERK and subsequent downstream activity such as cell growth and proliferation. This signaling funnel makes MEK an especially prime target for inhibition of this particular cell signaling pathway [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ]. MEKi were shown to inhibit ERK activation and its downstream processes leading to inhibition of the proliferation, survival, and motility of some tumor cells both in vitro and in vivo [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ,
      • Downward J.
      Targeting RAS signalling pathways in cancer therapy.
      ]. The importance of the MAPK pathway in cancer has not gone unnoticed, and efforts to target this pathway have focused on the major proteins in this pathway (RAS, RAF, MEK, and ERK) [
      • Sebolt-Leopold J.S.
      Advances in the development of cancer therapeutics directed against the RAS-mitogen-activated protein kinase pathway.
      ]. BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) have had particular success in eliciting patient response in various tumors, with BRAFi having a significant impact on melanoma patients [
      • Arozarena I.
      • Wellbrock C.
      Overcoming resistance to BRAF inhibitors.
      ]. However, a portion of patients will initially respond, only to have their tumor return or resume growth after a short duration of time because drug resistance mechanisms have restored the MAPK pathway [
      • Arozarena I.
      • Wellbrock C.
      Overcoming resistance to BRAF inhibitors.
      ]. Various mechanisms of resistance have been discovered, from activation of various receptor tyrosine kinases (RTKs) to activation of other cell signaling pathways such as the PI3K/AKT pathway [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ,
      • Turke A.B.
      • Song Y.
      • Costa C.
      • Cook R.
      • Arteaga C.L.
      • Asara J.M.
      • et al.
      MEK inhibition leads to PI3K/AKT activation by relieving a negative feedback on ERBB receptors.
      ]. In response to this, researchers have begun to investigate possible solutions regarding acquired resistance such as use of additional pathway inhibitors or immunotherapy in combination with MAPK inhibitors [
      • Kozar I.
      • Margue C.
      • Rothengatter S.
      • Haan C.
      • Kreis S.
      Many ways to resistance: How melanoma cells evade targeted therapies.
      ]. We will review recent developments regarding MEKi before discussing the various mechanisms of resistance to MAPK inhibition that have been discovered in various cancers. Lastly, we will discuss various solutions to circumvent the issue of acquired resistance and how cancers can possibly be re-sensitized to inhibition of the MAPK pathway.

      MEK recent developments

      Inhibition of MEK was presumed to be particularly efficacious in cancers harboring a RAS or RAF mutation, leading to overactivation of the MAPK pathway [
      • Mahapatra D.K.
      • Asati V.
      • Bharti S.K.
      MEK inhibitors in oncology: a patent review (2015-Present).
      ]. Over time, clinical studies have shown this hypothesis to be true in some cancers but far from a universal truth. Cancers in which MEK inhibition has seen recent human trial success include low-grade serous ovarian cancer (LGSOC). A recent phase II clinical trial of selumetinib showed an overall response rate of 15% and stable disease in 60% of patients with relapsed LGSOC [
      • Farley J.
      • Brady W.E.
      • Vathipadiekal V.
      • Lankes H.A.
      • Coleman R.
      • Morgan M.A.
      • et al.
      Selumetinib in women with recurrent low-grade serous carcinoma of the ovary or peritoneum: an open-label, single-arm, phase 2 study.
      ]. In another subsequent phase II/III clinical trial, trametinib was shown to improve PFS compared with the current standard of care. Thus, trametinib represents a new standard-of-care treatment option for women with recurrent low-grade serous carcinoma [

      D.M. Gershenson AM, W. Brady,J. Paul,K. Carty,W. Rodgers,D. Millan,R.L. Coleman,K.N. Moore,S. Banerjee,K. Connolly,A.A. Secord,D.M. O’Malley,O. Dorigo,S. Gaillard,H. Gabra,P. Hanjani,H. Huang,L. Wenzel,C. Gourley. A randomized phase II/III study to assess the efficacy of trametinib in patients with recurrent or progressive low-grade serous ovarian or peritoneal cancer. Annals of Oncology. 2019;30:2.

      ]. Other clinical successes for MEKi include a phase I and subsequent phase II trial testing the efficacy of selumetinib in children with neurofibromatosis type 1 and inoperable plexiform neurofibromas which had a partial response rate of 68%, leading to tumor shrinkage and reduced pain in patients [
      • Dombi E.
      • Baldwin A.
      • Marcus L.J.
      • Fisher M.J.
      • Weiss B.
      • Kim A.
      • et al.
      Activity of Selumetinib in Neurofibromatosis Type 1-Related Plexiform Neurofibromas.
      ,
      • Gross A.M.
      • Wolters P.L.
      • Dombi E.
      • Baldwin A.
      • Whitcomb P.
      • Fisher M.J.
      • et al.
      Selumetinib in Children with Inoperable Plexiform Neurofibromas.
      ]. Subsequently, selumetinib was approved by FDA as a single agent to treat pediatric patients two years of age and older with symptomatic, inoperable plexiform neurofibromas in April 2020. Furthermore, a study involving the use of cobimetinib to treat histiocytic neoplasms had an overall response rate of 89% with 72% of the patients experiencing a complete response [
      • Diamond E.L.
      • Durham B.H.
      • Ulaner G.A.
      • Drill E.
      • Buthorn J.
      • Ki M.
      • et al.
      Efficacy of MEK inhibition in patients with histiocytic neoplasms.
      ]. Apart from single agent treatment applications where success has been limited, MEKi has seen better success when combined with other treatments, especially when combined with BRAF inhibitors. In BRAF V600E–mutant melanoma, MEK inhibition was shown to activate caspase-3 through BIM-EL and BMF-mediated mitochondrial depolarization, which leads to apoptosis in the tumor cells [

      Beck D, Niessner H, Smalley KS, Flaherty K, Paraiso KH, Busch C, et al. Vemurafenib potently induces endoplasmic reticulum stress-mediated apoptosis in BRAFV600E melanoma cells. Sci Signal. 2013;6:ra7.

      ]. Trametinib was the first MEKi to be approved for use in melanoma by FDA as a single agent in 2013, and then it was approved for use in combination with dabrafenib for treatment of patients with unresectable or metastatic melanoma with BRAF V600E or V600K mutations in 2018. This was followed soon after by cobimetinib in combination with vemurafenib and binimetinib in combination with encorafenib. The combination of these MEKi with a BRAFi has become standard for melanoma patients with a V600E mutation [
      • Cheng Y.
      • Tian H.
      Current Development Status of MEK Inhibitors.
      ,

      Array BioPharma. Binimetinib FDA Approval. 2018.

      ,

      Genentech. Cobimetinib FDA Approval. 2015.

      ,

      GlaxoSmithKline. Trametinib FDA Approval. 2013.

      ]. This combination prolonged positive response, delaying acquired resistance and increasing progression-free survival (PFS) as well as overall survival [
      • Mahapatra D.K.
      • Asati V.
      • Bharti S.K.
      MEK inhibitors in oncology: a patent review (2015-Present).
      ,
      • Kakadia S.
      • Yarlagadda N.
      • Awad R.
      • Kundranda M.
      • Niu J.
      • Naraev B.
      • et al.
      Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of US Food and Drug Administration-approved targeted therapy in advanced melanoma.
      ]. Response rates to combination therapy in melanoma patients with a V600E BRAF mutation were high, with one clinical study showing a 64% response rate and another showing 68% response, and many patients either have tumor shrinkage or stable disease [
      • Kakadia S.
      • Yarlagadda N.
      • Awad R.
      • Kundranda M.
      • Niu J.
      • Naraev B.
      • et al.
      Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of US Food and Drug Administration-approved targeted therapy in advanced melanoma.
      ,
      • Larkin J.
      • Ascierto P.A.
      • Dréno B.
      • Atkinson V.
      • Liszkay G.
      • Maio M.
      • et al.
      Combined Vemurafenib and Cobimetinib in BRAF-Mutated Melanoma.
      ,
      • Robert C.
      • Karaszewska B.
      • Schachter J.
      • Rutkowski P.
      • Mackiewicz A.
      • Stroiakovski D.
      • et al.
      Improved Overall Survival in Melanoma with Combined Dabrafenib and Trametinib.
      ]. The latest treatment approved by the US Food and Drug Administration (FDA) involves use of a BRAFi in conjunction with a MEKi, which was shown to be more efficacious than single-drug treatment or previous conventional chemotherapy [
      • Kakadia S.
      • Yarlagadda N.
      • Awad R.
      • Kundranda M.
      • Niu J.
      • Naraev B.
      • et al.
      Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of US Food and Drug Administration-approved targeted therapy in advanced melanoma.
      ]. Other studies where BRAFi in conjunction with a MEKi have proven to be more efficacious than single-drug treatment involve a phase II trial combining dabrafenib, a BRAFi, and trametinib to treat BRAF V600E-mutant anaplastic thyroid cancer which had an overall response rate of 69% as well as another phase II trial again using dabrafenib in combination with trametinib to treat BRAF V600E-mutant NSCLC which had an overall response rate of 64%. The combination of dabrafenib with trametinib was also able to achieve an overall response rate of 47% for biliary tract cancer [
      • Subbiah V.
      • Kreitman R.J.
      • Wainberg Z.A.
      • Cho J.Y.
      • Schellens J.H.M.
      • Soria J.C.
      • et al.
      Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600-Mutant Anaplastic Thyroid Cancer.
      ,
      • Planchard D.
      • Smit E.F.
      • Groen H.J.M.
      • Mazieres J.
      • Besse B.
      • Helland A.
      • et al.
      Dabrafenib plus trametinib in patients with previously untreated BRAF(V600E)-mutant metastatic non-small-cell lung cancer: an open-label, phase 2 trial.
      ,
      • Subbiah V.
      • Lassen U.
      • Elez E.
      • Italiano A.
      • Curigliano G.
      • Javle M.
      • et al.
      Dabrafenib plus trametinib in patients with BRAF(V600E)-mutated biliary tract cancer (ROAR): a phase 2, open-label, single-arm, multicentre basket trial.
      ].
      Though the mechanism by which MEK inhibition causes cell death has been explored, another factor contributing to its success in treating tumors seems to lie in how MEK inhibition affects the tumor immune profile. Studies involving metastatic melanoma patients and cell lines have shown that the success and efficacy of BRAF and MEK inhibition may depend on an intact immune system, as MEKi were associated with increased T-cell infiltration of tumors and increased major histocompatibility complex expression on tumor cells [
      • Erkes D.A.
      • Cai W.
      • Sanchez I.M.
      • Purwin T.J.
      • Rogers C.
      • Field C.O.
      • et al.
      Mutant BRAF and MEK Inhibitors Regulate the Tumor Immune Microenvironment via Pyroptosis.
      ,
      • Wilmott J.S.
      • Long G.V.
      • Howle J.R.
      • Haydu L.E.
      • Sharma R.N.
      • Thompson J.F.
      • et al.
      Selective BRAF inhibitors induce marked T-cell infiltration into human metastatic melanoma.
      ,
      • Frederick D.T.
      • Piris A.
      • Cogdill A.P.
      • Cooper Z.A.
      • Lezcano C.
      • Ferrone C.R.
      • et al.
      BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma.
      ]. This increased T-cell infiltration was confirmed in a mouse model study of NSCLC showing that MEK inhibition alters the tumor immune environment regardless of cancer type [
      • Lee J.W.
      • Zhang Y.
      • Eoh K.J.
      • Sharma R.
      • Sanmamed M.F.
      • Wu J.
      • et al.
      The Combination of MEK Inhibitor With Immunomodulatory Antibodies Targeting Programmed Death 1 and Programmed Death Ligand 1 Results in Prolonged Survival in Kras/p53-Driven Lung Cancer.
      ]. Deeper analysis of the melanoma study revealed that T-cell activation and tumor regression are dependent on gasdermin E (GSDME)-mediated pyroptosis of tumor cells, revealing an immune-driven therapeutic response in tumors after treatment with MAPK inhibitors [
      • Erkes D.A.
      • Cai W.
      • Sanchez I.M.
      • Purwin T.J.
      • Rogers C.
      • Field C.O.
      • et al.
      Mutant BRAF and MEK Inhibitors Regulate the Tumor Immune Microenvironment via Pyroptosis.
      ]. These findings are also opening the door to new studies combining MEKi with immunotherapy agents such as anti–PD-L1 antibodies, which allow T-cells to recognize and kill tumors cells, and some early results have shown that MEK inhibition sensitized previously unresponsive KRAS-mutated tumors to immunotherapy [
      • Lee J.W.
      • Zhang Y.
      • Eoh K.J.
      • Sharma R.
      • Sanmamed M.F.
      • Wu J.
      • et al.
      The Combination of MEK Inhibitor With Immunomodulatory Antibodies Targeting Programmed Death 1 and Programmed Death Ligand 1 Results in Prolonged Survival in Kras/p53-Driven Lung Cancer.
      ,
      • Alsaab H.O.
      • Sau S.
      • Alzhrani R.
      • Tatiparti K.
      • Bhise K.
      • Kashaw S.K.
      • et al.
      PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunotherapy: Mechanism, Combinations, and Clinical Outcome.
      ].
      As use of MEKi has become more widespread, hospitals are getting a clear picture of the adverse effects of its use on patients. Side effects from MEK inhibition can be significant but are proving more tolerable than effects from other conventional cancer therapies [
      • Wong K.K.
      Recent developments in anti-cancer agents targeting the Ras/Raf/ MEK/ERK pathway.
      ]. The most common adverse effects of MEKi are blurred vision, fatigue, nausea, vomiting, diarrhea, asthenia, and skin- and gastrointestinal-related toxicities [
      • Mahapatra D.K.
      • Asati V.
      • Bharti S.K.
      MEK inhibitors in oncology: a patent review (2015-Present).
      ]. Other, more toxic effects are generally managed with dose discontinuation for a period of time or dose modification [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ]. Table 1 lists the toxicities observed in various clinical studies.
      Table 1Toxicities of MEK inhibitors when used as a single agent.
      Drug (NCT number)Trial PhasePopulationToxicitiesReference
      Binimetinib (NCT02094872)2Patients with melanoma that has spread to other parts of the bodySerious adverse events: Hospitalized for anemia, thrombocytopenia, nausea and vomiting, anemia due to gastric hemorrhage, abdominal and back pain, generalized weakness, fatigue, weakness and fatigue, death, anaphylactic reaction to paclitaxel, UTI, infectious enterocolitis, fever, strep B sepsis, hyponatremia, hypochloridemia, dropped head syndrome, ocular muscle weakness, back pain, chronic intractable pain, headache, brain metastasis, pneumonitis, acneiform rash, elevated alkaline phosphatase, elevated aspartate aminotransferase, elevated alanine aminotransferase, transaminitis, creatine phosphokinase elevation, uric acid elevations, radical resection of malignant soft tissue tumor (melanoma) on back, hypotension related to adrenal insufficiency, hypotension, hypertension, pulmonary embolism Total: 48/49 (97.96%) Other adverse events: 32/49 (65.31%)LoRusso et al., 2020
      • LoRusso P.
      Molecularly Targeted Therapy in Treating Patients With BRAF Wild-type Melanoma That is Metastatic.
      Selumetinib (NCT00559949)2Patients with papillary thyroid cancer that did not respond to radioactive iodineSerious adverse events: Atrial fibrillation, death NOS, dehydration, memory impairment, syncope, confusion, pneumonitis, hypotension Total: 7/39 (17.95%) Other adverse events: 39/39Hayes et al., 2012
      • Hayes D.N.
      • Lucas A.S.
      • Tanvetyanon T.
      • Krzyzanowska M.K.
      • Chung C.H.
      • Murphy B.A.
      • et al.
      Phase II efficacy and pharmacogenomic study of Selumetinib (AZD6244; ARRY-142886) in iodine-131 refractory papillary thyroid carcinoma with or without follicular elements.
      Selumetinib (NCT01011933)2recurrent or persistent endometrial cancerSerious adverse events: Leukopenia, anemia, edema: limb, atrial fibrillation, hypotension, ocular/visual disorder, dysphagia, oral cavity, small bowel NOS, dehydration, constipation, disease progression NOS, death NOS, chest/thorax pain NOS, abdominal pain NOS, liver dysfunction, skin infection, hyponatremia, dizziness, neuropathy, ureter obstruction, vaginitis, dyspnea, rash, gastrointestinal hemorrhage, thrombosis/thrombus/embolism Total:32/50 (64%) Other adverse events: 38/50 (76%)Coleman et al., 2015
      • Coleman R.L.
      • Sill M.W.
      • Thaker P.H.
      • Bender D.P.
      • Street D.
      • McGuire W.P.
      • et al.
      A phase II evaluation of selumetinib (AZD6244, ARRY-142886), a selective MEK-1/2 inhibitor in the treatment of recurrent or persistent endometrial cancer: an NRG Oncology/Gynecologic Oncology Group study.
      Trametinib (NCT01553851)2oral cavity squamous cell carcinomaSerious adverse events: Anemia, colitis, mucositis oral, vomiting, duodenal perforation, sepsis, skin infection, shingles, lung infection, activated partial thromboplastin time prolonged, alanine aminotransferase increased, aspartate aminotransferase increased, blood bilirubin increased, neutrophil count increased, platelet count decreased, white cell count decreased, lymphocyte count decreased, hyperglycemia, hypoalbuminemia, hypocalcemia, hyponatremia, hypophosphatemia, neck soft tissue necrosis, hypertension, hypotension Total: 6/20 (30%) Other adverse events: 14/20 (70%)Atkins, 2016
      • Atkins D.
      GSK1120212 in Surgically Resectable Oral Cavity Squamous Cell Cancer.
      Trametinib (NCT01362296)2Locally Advanced or Metastatic Non-small Cell Lung Cancer (NSCLC Stage IV)Serious adverse events (trametinib randomized phase): Diarrhea, vomiting, abdominal pain, gastric ulcer perforation, gastrointestinal hemorrhage, pyrexia, edema, death, pneumonia, sepsis, bronchitis, bronchitis bacterial, gastroenteritis, lung infection, radiation pneumonitis, spinal compression fracture, dehydration, hypoalbuminemia, transient ischemic attack, ischemic stroke, renal failure acute, hematuria, dyspnea, acute respiratory failure, bronchospasm, hemoptysis, pneumonitis, pulmonary hypertension, respiratory failure, rash, dermatitis exfoliative, palmar-plantar erythrodysesthesia syndrome, generalized rash, angioedema, thrombosis, embolism, venous thrombosis Total: 32/87 (36.78%) Serious adverse events (trametinib cross-over phase [patients who experienced disease progression in the randomized phase under docetaxel]): Atrial fibrillation, diarrhea, vomiting, general physical health deterioration, pneumonia, sepsis, pneumocystis jiroveci infection, septic shock, pubis fracture, hepatic enzyme increased, hyponatremia, loss of consciousness, somnolence, dyspnea, hemoptysis, pleural effusion, thrombosis, venous thrombosis Total: 12/23 (52.17%)Blumenschein et al., 2015
      • Blumenschein Jr., G.R.
      • Smit E.F.
      • Planchard D.
      • Kim D.W.
      • Cadranel J.
      • De Pas T.
      • et al.
      A randomized phase II study of the MEK1/MEK2 inhibitor trametinib (GSK1120212) compared with docetaxel in KRAS-mutant advanced non-small-cell lung cancer (NSCLC)dagger.
      Abbreviations: NOS, not otherwise specified; UTI, urinary tract infection.
      Despite promising preclinical data and its successes in various types of cancers, MEK inhibition has failed to be the panacea it once was hypothesized to become. In uveal melanoma studies expected to have positive results due to the success of BRAFi and MEKi in BRAF-mutant cutaneous melanoma, the MEKi selumetinib passed phase I and phase II trials only to fail a phase III trial when a combination of selumetinib plus dacarbazine did not improve PFS compared with dacarbazine alone [
      • Faiao-Flores F.
      • Emmons M.F.
      • Durante M.A.
      • Kinose F.
      • Saha B.
      • Fang B.
      • et al.
      HDAC Inhibition Enhances the In Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma.
      ]. Failure of MEK inhibition to show positive results in some cancers and loss of efficacy in cancers that do respond well necessitates a closer look at exactly how MEK inhibition alters cell signaling pathways in tumors and how tumors can find mechanisms to bypass MAPK inhibition. For example, MEKi have had little success in treating colorectal cancer (CRC), despite the presence of RAS and RAF mutations, and the mechanisms behind this poor response remain unclear [
      • Zhan T.
      • Ambrosi G.
      • Wandmacher A.M.
      • Rauscher B.
      • Betge J.
      • Rindtorff N.
      • et al.
      MEK inhibitors activate Wnt signalling and induce stem cell plasticity in colorectal cancer.
      ]. A recent study regarding the failure of MEK inhibition in CRC revealed some insight into how MEKi were actually promoting Wnt signaling, which is crucial for tumorigenesis and maintenance of cancer stem cells in CRC [
      • Zhan T.
      • Ambrosi G.
      • Wandmacher A.M.
      • Rauscher B.
      • Betge J.
      • Rindtorff N.
      • et al.
      MEK inhibitors activate Wnt signalling and induce stem cell plasticity in colorectal cancer.
      ]. Furthermore, this study also showed that MEKi were promoting stem cell plasticity and cancer relapse in patient-derived CRC organoids, revealing previously undocumented effects of MAPK inhibition [
      • Zhan T.
      • Ambrosi G.
      • Wandmacher A.M.
      • Rauscher B.
      • Betge J.
      • Rindtorff N.
      • et al.
      MEK inhibitors activate Wnt signalling and induce stem cell plasticity in colorectal cancer.
      ].
      Another issue regarding MEK inhibition resides with the fact that positive patient response is often short-lived. For example, despite combination therapy of BRAF and MEK inhibition’s improving patient survival in BRAF-mutant melanoma, tumors often recur around 13 months of treatment [
      • Hartsough E.
      • Shao Y.
      • Aplin A.E.
      Resistance to RAF inhibitors revisited.
      ]. Studies are beginning to show a plethora of ways that tumor cells come to evade MEK inhibition, and this often involves a re-wiring of the MAPK pathway to reactivate ERK signaling, showing that many cancer cells have a dependence on this pathway for survival and proliferation [
      • Arozarena I.
      • Wellbrock C.
      Overcoming resistance to BRAF inhibitors.
      ,
      • Sulahian R.
      • Kwon J.J.
      • Walsh K.H.
      • Pailler E.
      • Bosse T.L.
      • Thaker M.
      • et al.
      Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.
      ]. In addition, other cell signaling pathways, such as the PI3K/AKT pathway, may show upregulation following MEKi treatment, revealing other mechanisms by which tumor cells confer resistance to MAPK inhibition [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ,
      • Turke A.B.
      • Song Y.
      • Costa C.
      • Cook R.
      • Arteaga C.L.
      • Asara J.M.
      • et al.
      MEK inhibition leads to PI3K/AKT activation by relieving a negative feedback on ERBB receptors.
      ]. These mechanisms of resistance to MEKi therapy have been described in various tumors originating from different tissues, which would suggest tissue-specific differences in MEK resistance pathways. In the next section, we will go into greater detail about recently discovered mechanisms of resistance and how solutions are being presented to this issue surrounding MEK inhibition.

      Mechanisms of resistance

      Reactivation of the MAPK pathway

      Most cancers that acquire resistance to MEKi and continue to proliferate do so through reactivation of the MAPK pathway and subsequent reactivation of ERK. ERK reactivation can occur through alterations or mutations to molecules upstream of ERK in the MAPK pathway such as RAS, RAF, NF1, or MEK [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ,
      • Kozar I.
      • Margue C.
      • Rothengatter S.
      • Haan C.
      • Kreis S.
      Many ways to resistance: How melanoma cells evade targeted therapies.
      ]. Other mechanisms of resistance occur when MEK mutates after MEKi treatment, resulting in overactivation of MEK or rendering inhibitors unable to bind to MEK [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ,
      • Wagle N.
      • Van Allen E.M.
      • Treacy D.J.
      • Frederick D.T.
      • Cooper Z.A.
      • Taylor-Weiner A.
      • et al.
      MAP kinase pathway alterations in BRAF-mutant melanoma patients with acquired resistance to combined RAF/MEK inhibition.
      ,
      • Emery C.M.
      • Vijayendran K.G.
      • Zipser M.C.
      • Sawyer A.M.
      • Niu L.
      • Kim J.J.
      • et al.
      MEK1 mutations confer resistance to MEK and B-RAF inhibition.
      ]. Another major effect of MEKi is the reactivation of multiple RTKs upstream of the MAPK pathway responsible for beginning the signaling cascade that eventually leads to cellular growth and proliferation which leads to adaptive resistance [
      • Schlessinger J.
      Cell signaling by receptor tyrosine kinases.
      ]. RTKs have multiple pathways by which they can promote cell signaling, and reactivation of RTKs following MEK inhibition stimulates cellular growth through these various pathways [
      • Kauko O.
      • O'Connor C.M.
      • Kulesskiy E.
      • Sangodkar J.
      • Aakula A.
      • Izadmehr S.
      • et al.
      PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells.
      ,
      • Nazarian R.
      • Shi H.
      • Wang Q.
      • Kong X.
      • Koya R.C.
      • Lee H.
      • et al.
      Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation.
      ,
      • Duncan J.S.
      • Whittle M.C.
      • Nakamura K.
      • Abell A.N.
      • Midland A.A.
      • Zawistowski J.S.
      • et al.
      Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.
      ]. This reactivation occurs because RTKs are typically repressed by ERK1/2. When ERK is suppressed by MEK inhibition, RTKs such as EGFR, ERBB3, IGF-1R, or PDGFR are rapidly reactivated in adaptive response [
      • Caunt C.J.
      • Sale M.J.
      • Smith P.D.
      • Cook S.J.
      MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
      ,
      • Arozarena I.
      • Wellbrock C.
      Overcoming resistance to BRAF inhibitors.
      ,
      • Villanueva J.
      • Vultur A.
      • Lee J.T.
      • Somasundaram R.
      • Fukunaga-Kalabis M.
      • Cipolla A.K.
      • et al.
      Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K.
      ]. In particular, ERBB3, which is negatively repressed by MYC that can be stabilized by MEK-ERK signaling, has been implicated by a number of studies in various cancers as a mechanism of resistance, including a recent case study regarding metastatic reoccurrence of LGSOC localized to the brain [
      • Colombo I.
      • Garg S.
      • Danesh A.
      • Bruce J.
      • Shaw P.
      • Tan Q.
      • et al.
      Heterogeneous alteration of the ERBB3-MYC axis associated with MEK inhibitor resistance in a KRAS-mutated low-grade serous ovarian cancer patient.
      ,
      • Sun C.
      • Hobor S.
      • Bertotti A.
      • Zecchin D.
      • Huang S.
      • Galimi F.
      • et al.
      Intrinsic resistance to MEK inhibition in KRAS mutant lung and colon cancer through transcriptional induction of ERBB3.
      ]. Previous studies had already shown that MYC-dependent transcriptional upregulation of ERBB3 was a potential driver of resistance to MEK inhibition in KRAS-mutated lung and colon cancers [
      • Sun C.
      • Hobor S.
      • Bertotti A.
      • Zecchin D.
      • Huang S.
      • Galimi F.
      • et al.
      Intrinsic resistance to MEK inhibition in KRAS mutant lung and colon cancer through transcriptional induction of ERBB3.
      ]. One particular patient had low levels of MYC in the brain lesion compared with other areas such as the lung and colon, thus allowing ERBB3 to be highly expressed, leading to MEKi adaptive resistance along with rapid disease progression exclusively in the brain [
      • Colombo I.
      • Garg S.
      • Danesh A.
      • Bruce J.
      • Shaw P.
      • Tan Q.
      • et al.
      Heterogeneous alteration of the ERBB3-MYC axis associated with MEK inhibitor resistance in a KRAS-mutated low-grade serous ovarian cancer patient.
      ].

      Activation of parallel signaling pathways

      Apart from the MAPK pathway, parallel pathways exist for driving cell proliferation and growth. These pathways include the PI3K, STAT, and Hippo signaling pathways [
      • Balmanno K.
      • Chell S.D.
      • Gillings A.S.
      • Hayat S.
      • Cook S.J.
      Intrinsic resistance to the MEK1/2 inhibitor AZD6244 (ARRY-142886) is associated with weak ERK1/2 signalling and/or strong PI3K signalling in colorectal cancer cell lines.
      ,
      • Dai B.
      • Meng J.
      • Peyton M.
      • Girard L.
      • Bornmann W.G.
      • Ji L.
      • et al.
      STAT3 mediates resistance to MEK inhibitor through microRNA miR-17.
      ,
      • Lin L.
      • Sabnis A.J.
      • Chan E.
      • Olivas V.
      • Cade L.
      • Pazarentzos E.
      • et al.
      The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies.
      ]. When the MAPK pathway is inhibited, cancer cells may turn to these other pathways for the necessary signals to promote growth, resulting in MEKi adaptive resistance.
      In particular, the PI3K pathway stands out as a major mechanism of resistance to MEK inhibition. This pathway has been identified as a driving mechanism of tumorigenesis in various cancers already and is also a promising target for various therapies [
      • Fruman D.A.
      • Rommel C.
      PI3K and cancer: lessons, challenges and opportunities.
      ]. Multiple studies in a variety of cancers have shown upregulation in this pathway after the induction of MEKi treatment, painting a clear picture of how tumor cells are responding [
      • Turke A.B.
      • Song Y.
      • Costa C.
      • Cook R.
      • Arteaga C.L.
      • Asara J.M.
      • et al.
      MEK inhibition leads to PI3K/AKT activation by relieving a negative feedback on ERBB receptors.
      ,
      • Irvine M.
      • Stewart A.
      • Pedersen B.
      • Boyd S.
      • Kefford R.
      • Rizos H.
      Oncogenic PI3K/AKT promotes the step-wise evolution of combination BRAF/MEK inhibitor resistance in melanoma.
      ]. This activation of PI3K in the presence of MEKi comes from multiple possible sources including RTK reactivation. A study involving KRAS-mutated CRC cell lines resistant to EGFR and MEKi showed that activation of PI3K/AKT through upregulated RTKs such as HER2, HER3, and IGF1R was the major mechanism of survival for those cells [
      • Vitiello P.P.
      • Cardone C.
      • Martini G.
      • Ciardiello D.
      • Belli V.
      • Matrone N.
      • et al.
      Receptor tyrosine kinase-dependent PI3K activation is an escape mechanism to vertical suppression of the EGFR/RAS/MAPK pathway in KRAS-mutated human colorectal cancer cell lines.
      ]. Another major reason that this pathway contributes so heavily to oncogenesis and MEKi resistance lies in the many possible mutations that lead this pathway astray. Firstly, this pathway is activated by RAS itself, so oncogenic mutations in RAS can easily turn this pathway on in the face of MEK inhibition [
      • Gille H.
      • Downward J.
      Multiple ras effector pathways contribute to G(1) cell cycle progression.
      ]. Additionally, mutations farther down the pathway such as activating mutations in PIK3CA or the loss of PTEN, a tumor suppressor gene, can also over activate this pathway when MEK inhibition is active [
      • Shi H.
      • Hugo W.
      • Kong X.
      • Hong A.
      • Koya R.C.
      • Moriceau G.
      • et al.
      Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy.
      ]. For example, a study of MEKi resistance in CRC cell lines indicated that mutated PIK3CA contributed to MEKi intrinsic resistance by activating AKT to regulate expression of BCL-2 and BCL-XL, two anti-apoptotic factors, as well as expression of BAX and BIM, two pro-apoptotic factors [
      • Tsubaki M.
      • Takeda T.
      • Noguchi M.
      • Jinushi M.
      • Seki S.
      • Morii Y.
      • et al.
      Overactivation of Akt Contributes to MEK Inhibitor Primary and Acquired Resistance in Colorectal Cancer Cells.
      ]. Additionally, a recent study directly implicated PTEN loss as a major mechanism of intrinsic resistance to MEK inhibition in AML and went on to show that deletion of PTEN in AML cell lines was enough to confer MEKi resistance [
      • Smith A.M.
      • Zhang C.R.C.
      • Cristino A.S.
      • Grady J.P.
      • Fink J.L.
      • Moore A.S.
      PTEN deletion drives acute myeloid leukemia resistance to MEK inhibitors.
      ]. PTEN loss has also been associated with MEKi intrinsic resistance in a variety of cancers [
      • Milella M.
      • Falcone I.
      • Conciatori F.
      • Matteoni S.
      • Sacconi A.
      • De Luca T.
      • et al.
      PTEN status is a crucial determinant of the functional outcome of combined MEK and mTOR inhibition in cancer.
      ,
      • Wee S.
      • Jagani Z.
      • Xiang K.X.
      • Loo A.
      • Dorsch M.
      • Yao Y.M.
      • et al.
      PI3K pathway activation mediates resistance to MEK inhibitors in KRAS mutant cancers.
      ]. Furthermore, loss of another tumor suppressor gene, PP2A, in KRAS-mutant lung cancer was found to confer MEKi resistance through the PI3K/AKT pathway. PP2A is a serine/threonine phosphatase responsible for downregulating kinases such as MEK and ERK as well as AKT [
      • Millward T.A.
      • Zolnierowicz S.
      • Hemmings B.A.
      Regulation of protein kinase cascades by protein phosphatase 2A.
      ]. Rather than direct reactivation of the MAPK pathway, PP2A was found to instead collaterally activate AKT/mTOR as well as MYC, which are downstream of PI3K [
      • Kauko O.
      • O'Connor C.M.
      • Kulesskiy E.
      • Sangodkar J.
      • Aakula A.
      • Izadmehr S.
      • et al.
      PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells.
      ]. This study discovered that only a combination of an activator of PP2A with a MEKi was able to suppress both p-AKT and MYC in two KRAS-driven lung cancer mouse models, resulting in tumor regression [
      • Kauko O.
      • O'Connor C.M.
      • Kulesskiy E.
      • Sangodkar J.
      • Aakula A.
      • Izadmehr S.
      • et al.
      PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells.
      ].
      Another signaling pathway, STAT, was confirmed to be a mechanism of adaptive resistance to MEKi through a study combining a STAT3 inhibitor with selumetinib, a MEKi, which resulted in overcoming resistance and cancer cell death in an astrocytoma xenograft model [
      • Bid H.K.
      • Kibler A.
      • Phelps D.A.
      • Manap S.
      • Xiao L.
      • Lin J.
      • et al.
      Development, characterization, and reversal of acquired resistance to the MEK1 inhibitor selumetinib (AZD6244) in an in vivo model of childhood astrocytoma.
      ]. In a similar fashion, the Hippo pathway effector YAP1 was shown to promote adaptive resistance to MEKi in a study involving human NSCLC cells that combined YAP1 inhibitors and MEKi, resulting in overcoming adaptive resistance and leading to death in tumor cells [
      • Lin L.
      • Sabnis A.J.
      • Chan E.
      • Olivas V.
      • Cade L.
      • Pazarentzos E.
      • et al.
      The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies.
      ]. Another in vivo study of uveal melanoma further confirmed both AKT and YAP1 upregulation following MEKi treatment, resulting in improved tumor cell survival and a return to oncogenic activity [
      • Faiao-Flores F.
      • Emmons M.F.
      • Durante M.A.
      • Kinose F.
      • Saha B.
      • Fang B.
      • et al.
      HDAC Inhibition Enhances the In Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma.
      ].

      Transcriptional factors and cellular transformation

      Another possible mechanism of resistance involves the ability of tumor cells to switch phenotypes and rewire metabolic pathways. Studies have shown that melanoma cell lines with a higher expression of MITF, a transcription factor and regulator of melanocyte development, were more sensitive to MEK inhibition, whereas cell lines with low MITF were resistant [
      • Kemper K.
      • de Goeje P.L.
      • Peeper D.S.
      • van Amerongen R.
      Phenotype switching: tumor cell plasticity as a resistance mechanism and target for therapy.
      ]. Previous studies had also shown that melanoma cells with high MITF expression were associated with a more “proliferative” signature, and cells with low MITF expression had a more “invasive” signature [
      • Kemper K.
      • de Goeje P.L.
      • Peeper D.S.
      • van Amerongen R.
      Phenotype switching: tumor cell plasticity as a resistance mechanism and target for therapy.
      ]. Cells of the invasive phenotype had increased motility and migration with more metastatic potential, and this phenotype can be induced by MAPK inhibitor treatment [
      • Zipser M.C.
      • Eichhoff O.M.
      • Widmer D.S.
      • Schlegel N.C.
      • Schoenewolf N.L.
      • Stuart D.
      • et al.
      A proliferative melanoma cell phenotype is responsive to RAF/MEK inhibition independent of BRAF mutation status.
      ]. Melanoma cells have been observed to have the ability to switch back and forth between an invasive and proliferative cell type, but an additional consequence of this ability appears to be inductive resistance to MEK or BRAF inhibition [
      • Kozar I.
      • Margue C.
      • Rothengatter S.
      • Haan C.
      • Kreis S.
      Many ways to resistance: How melanoma cells evade targeted therapies.
      ]. Another study involving KRAS-mutant lung cancer cell lines described a similar phenomenon to melanoma phenotype switching known as epithelial-mesenchymal transition (EMT), which also correlated with MEKi resistance. Previous lung cancer studies showed that EMT was able to rewire cells, leading to a different feedback mechanism and MAPK activation following MEKi treatment [
      • Kitai H.
      • Ebi H.
      • Tomida S.
      • Floros K.V.
      • Kotani H.
      • Adachi Y.
      • et al.
      Epithelial-to-Mesenchymal Transition Defines Feedback Activation of Receptor Tyrosine Kinase Signaling Induced by MEK Inhibition in KRAS-Mutant Lung Cancer.
      ]. Epithelial phenotype cells were reactivated by ERBB3 after MEKi, while mesenchymal-like KRAS-mutant cells were reactivated through fibroblast growth factor receptor 1 (FGFR1) [
      • Kitai H.
      • Ebi H.
      • Tomida S.
      • Floros K.V.
      • Kotani H.
      • Adachi Y.
      • et al.
      Epithelial-to-Mesenchymal Transition Defines Feedback Activation of Receptor Tyrosine Kinase Signaling Induced by MEK Inhibition in KRAS-Mutant Lung Cancer.
      ]. Mouse models demonstrated that EMT in KRAS-mutated lung cancer cells is regulated by ZEB1 and miR-200, where higher ZEB1 expression drives EMT while increased miR-200 reverts cells to an epithelial phenotype [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ]. ZEB1, also known as zinc finger E-box binding homeobox 1, is a transcription factor that is regulated by the microRNA-200 family to promote EMT [
      • Gregory P.A.
      • Bert A.G.
      • Paterson E.L.
      • Barry S.C.
      • Tsykin A.
      • Farshid G.
      • et al.
      The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1.
      ]. High expression of ZEB1 in cells led to MAPK-independent tumor growth and increased MEKi resistance [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ]. Further studies showed that suppressing ZEB1 in mesenchymal cells increased MAPK signaling while inducing ZEB1 in epithelial cells decreased MAPK signaling [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ]. High expression of ZEB1 is able to suppress MAPK activity through suppressing scaffold protein interleukin-17 receptor D (IL17RD), which is crucial for MAPK signaling activity in epithelial lung cancer cells [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ].

      Future directions

      Targeting parallel pathways

      Many mechanisms of resistance to MEKi involve activation of other cellular signaling pathways such as the PI3K/AKT/mTOR pathway or the STAT pathway. In order to bypass these mechanisms, many studies have theorized and experimented with combination therapy inhibiting multiple pathways at once. In BRAF-mutant cutaneous melanoma, combining a BRAFi (encorafenib) with a MEKi (binimetinib) has already been established as a more potent treatment than single-inhibitor treatment and has been approved by the FDA [
      • Kozar I.
      • Margue C.
      • Rothengatter S.
      • Haan C.
      • Kreis S.
      Many ways to resistance: How melanoma cells evade targeted therapies.
      ,
      • Kakadia S.
      • Yarlagadda N.
      • Awad R.
      • Kundranda M.
      • Niu J.
      • Naraev B.
      • et al.
      Mechanisms of resistance to BRAF and MEK inhibitors and clinical update of US Food and Drug Administration-approved targeted therapy in advanced melanoma.
      ]. Combination therapy in melanoma has resulted in delayed acquired resistance and has improved the overall survival of patients [
      • Shirley M.
      Encorafenib and Binimetinib: First Global Approvals.
      ]. These positive results have bolstered hopes that combination therapy will work in other cancers in which different signaling pathways confer resistance to MEKi. Initial studies involving inhibiting the PI3K pathway through either AKT inhibitors, PI3K inhibitors, or dual PI3K/mTOR inhibitors in conjunction with MEKi and/or BRAFi have shown promising preclinical data [
      • Arozarena I.
      • Wellbrock C.
      Overcoming resistance to BRAF inhibitors.
      ]. Studies conducted on multiple BRAFi/MEKi-resistant melanoma cell lines showed that combining inhibition of the PI3K/AKT pathway with MAPK inhibitors could reverse acquired resistance and lead to tumor cell death [
      • Tsubaki M.
      • Takeda T.
      • Noguchi M.
      • Jinushi M.
      • Seki S.
      • Morii Y.
      • et al.
      Overactivation of Akt Contributes to MEK Inhibitor Primary and Acquired Resistance in Colorectal Cancer Cells.
      ,
      • Greger J.G.
      • Eastman S.D.
      • Zhang V.
      • Bleam M.R.
      • Hughes A.M.
      • Smitheman K.N.
      • et al.
      Combinations of BRAF, MEK, and PI3K/mTOR inhibitors overcome acquired resistance to the BRAF inhibitor GSK2118436 dabrafenib, mediated by NRAS or MEK mutations.
      ,
      • Atefi M.
      • von Euw E.
      • Attar N.
      • Ng C.
      • Chu C.
      • Guo D.
      • et al.
      Reversing melanoma cross-resistance to BRAF and MEK inhibitors by co-targeting the AKT/mTOR pathway.
      ]. Other studies, including one involving a KRAS-mutant lung cancer mouse model, showed that inhibition of MAPK and PI3K signaling pathways had a synergistic effect on tumor cells [
      • Engelman J.A.
      • Chen L.
      • Tan X.
      • Crosby K.
      • Guimaraes A.R.
      • Upadhyay R.
      • et al.
      Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers.
      ]. These results have led to a number of clinical trials combining PI3K pathway inhibitors with MEKi across a variety of cancers. Many of these studies have found an initial positive tumor response with cancers responding better than monotherapy alone, but these studies have run into issues regarding dose toxicity for longer-term treatment [
      • Bardia A.
      • Gounder M.
      • Rodon J.
      • Janku F.
      • Lolkema M.P.
      • Stephenson J.J.
      • et al.
      Phase Ib Study of Combination Therapy with MEK Inhibitor Binimetinib and Phosphatidylinositol 3-Kinase Inhibitor Buparlisib in Patients with Advanced Solid Tumors with RAS/RAF Alterations.
      ,
      • Arend R.C.
      • Davis A.M.
      • Chimiczewski P.
      • O'Malley D.M.
      • Provencher D.
      • Vergote I.
      • et al.
      EMR 20006–012: A phase II randomized double-blind placebo controlled trial comparing the combination of pimasertib (MEK inhibitor) with SAR245409 (PI3K inhibitor) to pimasertib alone in patients with previously treated unresectable borderline or low grade ovarian cancer.
      ,
      • Bedard P.L.
      • Tabernero J.
      • Janku F.
      • Wainberg Z.A.
      • Paz-Ares L.
      • Vansteenkiste J.
      • et al.
      A phase Ib dose-escalation study of the oral pan-PI3K inhibitor buparlisib (BKM120) in combination with the oral MEK1/2 inhibitor trametinib (GSK1120212) in patients with selected advanced solid tumors.
      ]. Similarly, studies targeting the Hippo pathway through YAP inhibition after MEKi treatment showed positive results in BRAF-mutant tumor cell lines, and combination treatment was able to overcome acquired resistance to MEKi [
      • Lin L.
      • Sabnis A.J.
      • Chan E.
      • Olivas V.
      • Cade L.
      • Pazarentzos E.
      • et al.
      The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies.
      ]. Further building upon this idea of multiple pathway inhibition, a recent study involving uveal melanoma cell lines showed that use of a histone deacetylase (HDAC) inhibitor in conjunction with a MEKi was able to suppress the adaptive YAP and AKT signaling that arose after MEKi treatment, leading to better antitumor results than single-drug treatment [
      • Faiao-Flores F.
      • Emmons M.F.
      • Durante M.A.
      • Kinose F.
      • Saha B.
      • Fang B.
      • et al.
      HDAC Inhibition Enhances the In Vivo Efficacy of MEK Inhibitor Therapy in Uveal Melanoma.
      ].

      RTK inhibition

      Other common mechanisms of resistance include upregulation of RTKs, which are often re-activated following MEKi treatment. Nonetheless, some specific RTKs have higher correlation with MEKi resistance, and studies have been conducted to test whether combination therapy inhibiting these RTKs alongside MEK inhibition can bypass adaptive mechanisms of resistance. Some of the RTKs studied include ERBB2, EGFR, and FGFR1, all of which were found to have a synergistic effect when inhibited alongside MEK [
      • Sun C.
      • Hobor S.
      • Bertotti A.
      • Zecchin D.
      • Huang S.
      • Galimi F.
      • et al.
      Intrinsic resistance to MEK inhibition in KRAS mutant lung and colon cancer through transcriptional induction of ERBB3.
      ,
      • Fernandez M.L.
      • Dawson A.
      • Hoenisch J.
      • Kim H.
      • Bamford S.
      • Salamanca C.
      • et al.
      Markers of MEK inhibitor resistance in low-grade serous ovarian cancer: EGFR is a potential therapeutic target.
      ,

      Manchado E, Weissmueller S, Morris JPt, Chen CC, Wullenkord R, Lujambio A, et al. A combinatorial strategy for treating KRAS-mutant lung cancer. Nature. 2016;534:647-51.

      ]. Other efforts tackling the issue of RTK upregulation after MEKi treatment have focused on nodes downstream of RTKs such as SHOC2 and SHP2, thus allowing a single inhibitor to block multiple possible mechanisms of resistance. SHP2 is a protein-tyrosine phosphatase that acts between RTKs and RAS and subsequently allows for full activation of MEK/ERK [
      • Ran H.
      • Tsutsumi R.
      • Araki T.
      • Neel B.G.
      Sticking It to Cancer with Molecular Glue for SHP2.
      ]. Inhibition of SHP2 was able to block ERK reactivation following MEK inhibition and prevent adaptive resistance in multiple cancer models [
      • Fedele C.
      • Ran H.
      • Diskin B.
      • Wei W.
      • Jen J.
      • Geer M.J.
      • et al.
      SHP2 Inhibition Prevents Adaptive Resistance to MEK Inhibitors in Multiple Cancer Models.
      ]. Similarly, depletion of SHOC2, a scaffolding protein responsible for allowing the activation of RAF through MRAS, was found to play a critical role in disrupting survival pathways activated by RTKs following MEK inhibition [
      • Sulahian R.
      • Kwon J.J.
      • Walsh K.H.
      • Pailler E.
      • Bosse T.L.
      • Thaker M.
      • et al.
      Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.
      ,
      • Simanshu D.K.
      • Nissley D.V.
      • McCormick F.
      RAS Proteins and Their Regulators in Human Disease.
      ]. In particular, SHOC2 inhibition was found to sensitize RAS-driven cancers in vitro to MEK inhibition and was found to play a remarkably similar role to SHP2 in RTK signaling [
      • Sulahian R.
      • Kwon J.J.
      • Walsh K.H.
      • Pailler E.
      • Bosse T.L.
      • Thaker M.
      • et al.
      Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.
      ]. In a study of combined EGFR/MEK inhibition in EGFR-mutated non-small cell lung cancer cell lines, it has been shown that such combination could only promote the cancer cells into a senescence-like dormant state but without going into apoptosis [
      • Kurppa K.J.
      • Liu Y.
      • To C.
      • Zhang T.
      • Fan M.
      • Vajdi A.
      • et al.
      Treatment-Induced Tumor Dormancy through YAP-Mediated Transcriptional Reprogramming of the Apoptotic Pathway.
      ]. It was shown that up-regulation of YAP/TEAD by combined EGFR/MEK inhibition could repress pro-apoptotic BMF and limited drug induced apoptosis [
      • Kurppa K.J.
      • Liu Y.
      • To C.
      • Zhang T.
      • Fan M.
      • Vajdi A.
      • et al.
      Treatment-Induced Tumor Dormancy through YAP-Mediated Transcriptional Reprogramming of the Apoptotic Pathway.
      ]. Pharmacological co-inhibition of YAP and TEAD might enhance EGFR/MEK inhibition-induced apoptosis.

      ZEB1

      Another mechanism of resistance to target involves EMT. As reported above, KRAS-mutant lung cancer cells resistant to MEKi showed higher expression of ZEB1, and suppression of this transcription factor through either induced miR-200 expression or mocetinostat, an HDAC inhibitor, can sensitize these resistant cells to MEKi treatment [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ]. Combination of MEKi with mocetinostat also showed a synergistic effect in reducing the number and size of metastatic mouse lung cancer tumors [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ]. This combination therapy may prove more useful than RTK inhibition when targeting resistant cancer cells that are driven by MAPK-independent pathways and express high levels of ZEB1 [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ].

      BCL-Xl

      Using synthetic lethal shRNA screening in KRAS mutant cancer models, BCL-XL was found to be a potential target for combination with MEKi [
      • Corcoran R.B.
      • Cheng K.A.
      • Hata A.N.
      • Faber A.C.
      • Ebi H.
      • Coffee E.M.
      • et al.
      Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models.
      ]. BCL-XL binds and inhibits the key pro-apoptotic protein BIM, which was induced by MEKi [
      • Meng J.
      • Fang B.
      • Liao Y.
      • Chresta C.M.
      • Smith P.D.
      • Roth J.A.
      Apoptosis induction by MEK inhibition in human lung cancer cells is mediated by Bim.
      ]. BCL-2/BCL-XL inhibition increases the efficacy of MEK inhibition in lung and pancreatic tumor cell lines [
      • Tan N.
      • Wong M.
      • Nannini M.A.
      • Hong R.
      • Lee L.B.
      • Price S.
      • et al.
      Bcl-2/Bcl-xL inhibition increases the efficacy of MEK inhibition alone and in combination with PI3 kinase inhibition in lung and pancreatic tumor models.
      ]. Similarly, combined MEK and BCL-2/XL inhibition has also been shown to be effective in high-grade serous ovarian cancer patient–derived xenograft models [
      • Iavarone C.
      • Zervantonakis I.K.
      • Selfors L.M.
      • Palakurthi S.
      • Liu J.F.
      • Drapkin R.
      • et al.
      Combined MEK and BCL-2/XL Inhibition Is Effective in High-Grade Serous Ovarian Cancer Patient-Derived Xenograft Models and BIM Levels Are Predictive of Responsiveness.
      ]. Currently, a phase I/II clinical trial combining BCL-XL and MEKi has shown initial signs of efficacy, with a favorable disease control rate and durable partial response in patients with RAS-mutant gynecologic cancer [
      • Corcoran R.B.
      • Do K.T.
      • Cleary J.M.
      • Parikh A.R.
      • Yeku O.O.
      • Weekes C.D.
      • et al.
      Phase I/II study of combined BCL-XL and MEK inhibition with navitoclax (N) and trametinib (T) in KRAS or NRAS mutant advanced solid tumours.
      ].

      Immunotherapy and MEK inhibition

      Apart from combining MEKi with other pathway inhibitors, studies are investigating how MEK inhibition affects the immune environment of tumors. MEKi have been shown to enhance T-cell infiltration in the tumor microenvironment, as a study showed that low-dose trametinib was able to “degrade tumor burden by apoptosis and be followed by an increase of tumor antigens available” that are recognizable by T-cells [
      • Lee J.W.
      • Zhang Y.
      • Eoh K.J.
      • Sharma R.
      • Sanmamed M.F.
      • Wu J.
      • et al.
      The Combination of MEK Inhibitor With Immunomodulatory Antibodies Targeting Programmed Death 1 and Programmed Death Ligand 1 Results in Prolonged Survival in Kras/p53-Driven Lung Cancer.
      ]. This may in part due to the fact that MEK inhibition was shown to decrease the number of myeloid-derived suppressor cells in the tumor, which are known for suppressing T-cell responses [
      • Lee J.W.
      • Zhang Y.
      • Eoh K.J.
      • Sharma R.
      • Sanmamed M.F.
      • Wu J.
      • et al.
      The Combination of MEK Inhibitor With Immunomodulatory Antibodies Targeting Programmed Death 1 and Programmed Death Ligand 1 Results in Prolonged Survival in Kras/p53-Driven Lung Cancer.
      ,
      • Gabrilovich D.I.
      • Nagaraj S.
      Myeloid-derived suppressor cells as regulators of the immune system.
      ]. In this study of KRAS-mutant lung cancer models, combining MEK inhibition with immunotherapies such as anti-PD-1 or anti-PD-L1 antibodies showed a synergistic effect and increased antitumor response and survival outcome in vivo compared with single-agent treatment [
      • Lee J.W.
      • Zhang Y.
      • Eoh K.J.
      • Sharma R.
      • Sanmamed M.F.
      • Wu J.
      • et al.
      The Combination of MEK Inhibitor With Immunomodulatory Antibodies Targeting Programmed Death 1 and Programmed Death Ligand 1 Results in Prolonged Survival in Kras/p53-Driven Lung Cancer.
      ]. Furthermore, a clinical trial combining BRAFi, MEKi, and PD-1 blockade therapy in BRAF-mutant melanoma showed favorable patient objective response (11 of 15 patients), with six patients continuing to have a response at a median follow-up of 27 months [
      • Ribas A.
      • Lawrence D.
      • Atkinson V.
      • Agarwal S.
      • Miller Jr., W.H.
      • Carlino M.S.
      • et al.
      Combined BRAF and MEK inhibition with PD-1 blockade immunotherapy in BRAF-mutant melanoma.
      ]. Though these therapies complement each other well, resistance to MAPK inhibition often leads to loss of efficacy for immunotherapy as well, so the order of treatment plays a critical role in improving patient survival [
      • Kozar I.
      • Margue C.
      • Rothengatter S.
      • Haan C.
      • Kreis S.
      Many ways to resistance: How melanoma cells evade targeted therapies.
      ]. This may be a result of reduced tumor antigen presentation and loss of intra-tumoral T cells [
      • Hugo W.
      • Shi H.
      • Sun L.
      • Piva M.
      • Song C.
      • Kong X.
      • et al.
      Non-genomic and Immune Evolution of Melanoma Acquiring MAPKi Resistance.
      ]. Table 2 lists some current drug combination clinical trials with MEKi, including the combination of immunotherapy and MEKi in various cancers with primary outcomes. The primary outcomes indicate that these combination trials can improve clinical benefit to some extent but further development will be needed.
      Table 2List of MEK inhibitor drug combination clinical trials with primary outcomes.
      DrugCombination DrugTrial PhasePopulationPrimary Outcomes
      Trametinib
      • Algazi A.P.
      • Esteve-Puig R.
      • Nosrati A.
      • Hinds B.
      • Hobbs-Muthukumar A.
      • Nandoskar P.
      • et al.
      Dual MEK/AKT inhibition with trametinib and GSK2141795 does not yield clinical benefit in metastatic NRAS-mutant and wild-type melanoma.
      GSK21417952Patients with BRAF wild-type mutant melanomaObjective response rate/stable disease (without NRAS Exon 1 and 2 mutations): 5/10 (50%); objective response rate/stable disease (with NRAS Exon 1 and 2 mutations): 4/10 (40%)
      Trametinib
      • Bhuvaneswari Ramaswamy M.
      Trametinib and Akt Inhibitor GSK2141795 in Treating Patients With Metastatic Triple-Negative Breast Cancer.
      GSK21417952Estrogen receptor–negative, HER2/neu–negative, invasive breast carcinoma; progesterone receptor–negative recurrent breast carcinoma; stage IV breast cancer; triple-negative breast carcinomaClinical benefit rate (CR + PR + SD): trametinib only: 8/37 (21.6%); trametinib + GSK2141795: 6/19 (31.6%)
      Trametinib
      • Davies M.A.
      • Saiag P.
      • Robert C.
      • Grob J.J.
      • Flaherty K.T.
      • Arance A.
      • et al.
      Dabrafenib plus trametinib in patients with BRAF(V600)-mutant melanoma brain metastases (COMBI-MB): a multicentre, multicohort, open-label, phase 2 trial.
      Dabrafenib2BRAF mutation–positive melanoma that has metastasized to the brainIntracranial response rate (the percentage of patients achieving a confirmed intracranial CR or PR): 59/76 (77.63%)
      Selumetinib
      • Carvajal R.D.
      • Piperno-Neumann S.
      • Kapiteijn E.
      • Chapman P.B.
      • Frank S.
      • Joshua A.M.
      • et al.
      Selumetinib in Combination With Dacarbazine in Patients With Metastatic Uveal Melanoma: A Phase III, Multicenter, Randomized Trial (SUMIT).
      Dacarbazine3Patients with metastatic uveal melanomaSelumetinib + dacarbazine PFS: 82 progression events/97 patients ; Placebo + Dacarbazine PFS: 24 progression events/32 patients
      Cobimetinib
      • Ascierto P.A.
      • McArthur G.A.
      • Dreno B.
      • Atkinson V.
      • Liszkay G.
      • Di Giacomo A.M.
      • et al.
      Cobimetinib combined with vemurafenib in advanced BRAF(V600)-mutant melanoma (coBRIM): updated efficacy results from a randomised, double-blind, phase 3 trial.
      Vemurafenib3Untreated BRAF V600 mutation–positive patients with unresectable locally advanced or metastatic melanomaPlacebo + vemurafenib PFS: median 6.21 months; cobimetinib + vemurafenib PFS: median 9.89 months; placebo + vemurafenib objective response: 44.8%; cobimetinib + vemurafenib objective response: 67.6%; placebo + vemurafenib OS: median 17.38 months; cobimetinib + vemurafenib OS: median 22.28 months
      Cobimetinib
      • Eng C.
      • Kim T.W.
      • Bendell J.
      • Argiles G.
      • Tebbutt N.C.
      • Di Bartolomeo M.
      • et al.
      Atezolizumab with or without cobimetinib versus regorafenib in previously treated metastatic colorectal cancer (IMblaze370): a multicentre, open-label, phase 3, randomised, controlled trial.
      Atezolizumab3Patients with unresectable locally advanced or metastatic colorectal cancer who have received at least two prior regimens of cytotoxic chemotherapy for metastatic diseaseRegorafenib OS: median 8.51 months; cobimetinib + atezolizumab OS: median 8.87 months; atezolizumab OS: median 7.10 months; regorafenib PFS: median 2.00 months; cobimetinib + atezolizumab PFS: median 1.91 months; atezolizumab PFS: median 1.94 months
      Pimasertib
      • Van Cutsem E.
      • Hidalgo M.
      • Canon J.L.
      • Macarulla T.
      • Bazin I.
      • Poddubskaya E.
      • et al.
      Phase I/II trial of pimasertib plus gemcitabine in patients with metastatic pancreatic cancer.
      Gemcitabine2Patients with metastatic pancreatic adenocarcinomaGemcitabine + placebo PFS: median 2.83 months; gemcitabine + pimasertib PFS: median 3.75 months
      Abbreviations: CR, complete response; PR, partial response; PFS, progression-free survival; OS, overall survival; SD, stable disease.
      The following tables will cover all currently existing ongoing clinical trials involving MEK inhibitors in combination with other drugs.
      Table 3 lists some of the principal ongoing clinical trials that will evaluate the efficacy of combining MEKi with other targeted inhibitors. Some of these targets exist directly upstream of the MAPK pathway such as SHP2, which was mentioned as a target of interest earlier in this review, and SOS1, which partakes in the exchange of RAS from RAS-GDP to RAS-GTP thus allowing RAS to signal further downstream [

      Gort E, Johnson ML, Hwang JJ, Pant S, Dünzinger U, Riemann K, et al. A phase I, open-label, dose-escalation trial of BI 1701963 as monotherapy and in combination with trametinib in patients with KRAS mutated advanced or metastatic solid tumors. Journal of Clinical Oncology. 2020;38:TPS3651-TPS.

      ]. Another target directly downstream of the MAPK pathway, CDK, is associated with CDK4 and CDK6, which are responsible for promoting cell proliferation [
      • Kato J.
      • Matsushime H.
      • Hiebert S.W.
      • Ewen M.E.
      • Sherr C.J.
      Direct binding of cyclin D to the retinoblastoma gene product (pRb) and pRb phosphorylation by the cyclin D-dependent kinase CDK4.
      ]. Other targets for inhibition in combination with MEK include RTKs such as EGFR and ALK as well as kinases in alternate signaling pathways such as PIK3CA and mTOR. Two additional targets of interest in this table include PKC and FAK. PKC is a kinase that plays a role in cell proliferation, apoptosis, and angiogenesis, and when PKC is over-activated, it can promote activation of the MAPK pathway as well as upregulate BCL-XL, an anti-apoptotic factor [
      • Corcoran R.B.
      • Do K.T.
      • Cleary J.M.
      • Parikh A.R.
      • Yeku O.O.
      • Weekes C.D.
      • et al.
      Phase I/II study of combined BCL-XL and MEK inhibition with navitoclax (N) and trametinib (T) in KRAS or NRAS mutant advanced solid tumours.
      ,
      • Griner E.M.
      • Kazanietz M.G.
      Protein kinase C and other diacylglycerol effectors in cancer.
      ]. Pre-clinical data has also shown increased efficacy when combining PKC and MEK inhibitors in melanoma xenograft models [
      • Chen X.
      • Wu Q.
      • Tan L.
      • Porter D.
      • Jager M.J.
      • Emery C.
      • et al.
      Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations.
      ]. Similarly, FAK is a tyrosine kinase overexpressed in cancer cells that promotes tumor development but also has had promising clinical results in LGSOC when combined with a RAF/MEK inhibitor [

      Shinde R, Terbuch A, Little M, Caldwell R, Kurup R, Riisnaes R, et al. Phase I study of the combination of a RAF-MEK inhibitor CH5126766 and FAK inhibitor defactinib in an intermittent dosing schedule with expansions in KRAS mutant cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT143. 2020.

      ,
      • Lee B.Y.
      • Timpson P.
      • Horvath L.G.
      • Daly R.J.
      FAK signaling in human cancer as a target for therapeutics.
      ].
      Table 3Current clinical trials combining MEKi and other targeting agents.
      StatusStudy TitleInterventionsDrug TargetPhaseNCT Number
      RecruitingA Phase IB/II Study of Alectinib Combined With Cobimetinib in Advanced ALK-Rearranged (ALK + ) NSCLCCobimetinibMEK1, 2NCT03202940
      AlectinibALK
      RecruitingSARC031: MEK Inhibitor Selumetinib (AZD6244) in Combination With the mTOR Inhibitor Sirolimus for Patients With Malignant Peripheral Nerve Sheath TumorsSelumetinibMEK2NCT03433183
      SirolimusmTOR
      RecruitingCombination of Alpelisib and Trametinib in Progressive Refractory MeningiomasTrametinibMEK1NCT03631953
      AlpelisibPIK3CA
      RecruitingStudy of MK-8353 + Selumetinib in Advanced/Metastatic Solid Tumors (MK-8353–014)SelumetinibMEK1NCT03745989
      MK-8353ERK
      RecruitingStudy of IDE196 in Patients With Solid Tumors Harboring GNAQ/11 Mutations or PRKC FusionsBinimetinibMEK1, 2NCT03947385
      IDE196PKC
      RecruitingBinimetinib and Palbociclib or TAS-102 in Treating Patients With KRAS and NRAS Mutant Metastatic or Unresectable Colorectal CancerBinimetinibMEK2NCT03981614
      PalbociclibCDK
      RecruitingIN10018 Monotherapy and Combination Therapy for Metastatic MelanomaCobimetinibMEK1NCT04109456
      IN10018FAK
      RecruitingA Study to Test Different Doses of BI 1,701,963 Alone and Combined With Trametinib in Patients With Different Types of Advanced Cancer (Solid Tumours With KRAS Mutation)TrametinibMEK1NCT04111458
      BI 1,701,963SOS1
      RecruitingDose-Escalation/Expansion of RMC-4630 and Cobimetinib in Relapsed/Refractory Solid Tumors and RMC-4630 and Osimertinib in EGFR Positive Locally Advanced/Metastatic NSCLCCobimetinibMEK1, 2NCT03989115
      OsimertinibEGFR
      RMC-4630SHP2
      Table 4 lists ongoing clinical trials that will evaluate the efficacy of combination treatment combining MEKi, BRAFi, and other targets of interest. JAK1 is a kinase responsible for activating cell growth and proliferation through the STAT pathway [
      • Rawlings J.S.
      • Rosler K.M.
      • Harrison D.A.
      The JAK/STAT signaling pathway.
      ]. Studies conducted on mice with myeloproliferative neoplasms combining JAK inhibition with MEKi showed increased therapeutic efficacy when compared to single drug treatment [
      • Stivala S.
      • Codilupi T.
      • Brkic S.
      • Baerenwaldt A.
      • Ghosh N.
      • Hao-Shen H.
      • et al.
      Targeting compensatory MEK/ERK activation increases JAK inhibitor efficacy in myeloproliferative neoplasms.
      ]. CSF-1 is a growth factor that binds to an RTK and promotes proliferation and differentiation of myeloid cells through various pathways including the MAPK pathway[
      • Lee A.W.
      Synergistic activation of mitogen-activated protein kinase by cyclic AMP and myeloid growth factors opposes cyclic AMP's growth-inhibitory effects.
      ,
      • Pixley F.J.
      • Stanley E.R.
      CSF-1 regulation of the wandering macrophage: complexity in action.
      ]. Thus, small molecule inhibitors targeting the CSF-1 pathway could reprogram the tumor microenvironment to enhance T-cell-mediated tumor eradication. Additionally, Table 4, Table 5 have trials involving hydroxychloroquine (HCQ) in combination with MEKi. This drug affects cancer in a variety of ways, the effects of which are still being studied, but the main mechanism by which HCQ is thought to affect cancer is through inhibiting autophagy, which is a process where a cell degrades old cellular components into products such as amino acids and fatty acids that can be recycled by the cell to promote cell survival in certain conditions [
      • Verbaanderd C.
      • Maes H.
      • Schaaf M.B.
      • Sukhatme V.P.
      • Pantziarka P.
      • Sukhatme V.
      • et al.
      Repurposing Drugs in Oncology (ReDO)-chloroquine and hydroxychloroquine as anti-cancer agents.
      ]. This process can be used by cancer cells to promote tumor growth [
      • Janku F.
      • McConkey D.J.
      • Hong D.S.
      • Kurzrock R.
      Autophagy as a target for anticancer therapy.
      ]. HCQ has been used in combination with other anti-cancer agents in pre-clinical studies and has shown to increase the efficacy of said drugs as well as limit acquired drug resistance [
      • Verbaanderd C.
      • Maes H.
      • Schaaf M.B.
      • Sukhatme V.P.
      • Pantziarka P.
      • Sukhatme V.
      • et al.
      Repurposing Drugs in Oncology (ReDO)-chloroquine and hydroxychloroquine as anti-cancer agents.
      ].
      Table 4Current clinical trials combining MEKi, BRF1 and additional targeting agents.
      StatusStudy TitleInterventionsDrug TargetPhaseNCT Number
      RecruitingINCB039110 in Combination With Dabrafenib and Trametinib in Patients With BRAF-mutant Melanoma and Other Solid Tumors.TrametinibMEK1NCT03272464
      DabrafenibBRAF V600E
      INCB039110JAK1
      RecruitingMCS110 With BRAF/MEK Inhibition in Patients With MelanomaTrametinibMEK1,2NCT03455764
      DabrafenibBRAF V600E
      MCS110CSF-1
      RecruitingDabrafenib + Trametinib + PDR001 In Colorectal CancerTrametinibMEK2NCT03668431
      DabrafenibBRAF V600E
      PDR001PD1
      Active, not recruitingEncorafenib, Binimetinib and Cetuximab in Subjects With Previously Untreated BRAF-mutant ColoRectal CancerBinimetinibMEK2NCT03693170
      encorafenibBRAF V600E
      CetuximabEGFR
      RecruitingA Trial of Dabrafenib, Trametinib and Hydroxychloroquine for Patients With Recurrent LGG or HGG With a BRAF AberrationTrametinibMEK1, 2NCT04201457
      DabrafenibBRAF V600E
      HydroxychloroquineAutophagy
      Table 5Current clinical trials combining MEKi, Checkpoint inhibitors and additional targeting agents.
      StatusStudy TitleInterventionsDrug TargetPhaseNCT Number
      Active, not recruitingA Study of Cobimetinib Administered as Single Agent and in Combination With Venetoclax, With or Without Atezolizumab, in Participants With Relapsed and Refractory Multiple MyelomaCobimetinibMEK1, 2NCT03312530
      AtezolizumabPD-L1
      VenetoclaxBCL-2
      RecruitingBEACON - ABC in Recurrent Platinum Resistant HGSOCCobimetinibMEK2NCT03363867
      AtezolizumabPD-L1
      BevacizumabVEGF
      RecruitingDurvalumab, Tremelimumab, and Selumetinib in Treating Participants With Recurrent or Stage IV Non-small Cell Lung CancerSelumetinibMEK1, 2NCT03581487
      Biological: DurvalumabPD-L1
      Biological: TremelimumabCTLA-4
      RecruitingA Clinical Study of Cobimetinib Administered in Combination With Niraparib, With or Without Atezolizumab to Patients With Advanced Platinum-sensitive Ovarian CancerCobimetinibMEK1NCT03695380
      AtezolizumabPD-L1
      NiraparibPARP
      RecruitingStudy of Combination Therapy With the MEK Inhibitor, Cobimetinib, Immune Checkpoint Blockade, Atezolizumab, and the AUTOphagy Inhibitor, Hydroxychloroquine in KRAS-mutated Advanced MalignanciesCobimetinibMEK1, 2NCT04214418
      AtezolizumabPD-L1
      HydroxychloroquineAutophagy
      Table 5 lists ongoing clinical trials combining MEKi with immunotherapy as well as other inhibitors targeting other various hallmarks of cancer cells. Both PD-L1 and CTLA4 inhibitors have been proven and approved for treatment of certain cancers as individual single therapeutical agents or as a combo agent, but combination of these drugs with MEKi have not been fully explored [
      • Vaddepally R.K.
      • Kharel P.
      • Pandey R.
      • Garje R.
      • Chandra A.B.
      Review of Indications of FDA-Approved Immune Checkpoint Inhibitors per NCCN Guidelines with the Level of Evidence.
      ]. Nonetheless, initial in vivo studies seem promising as they have shown that MEKi in combination with these immunotherapies can be beneficial due to how MEK inhibitors alter the TME [
      • Lee J.W.
      • Zhang Y.
      • Eoh K.J.
      • Sharma R.
      • Sanmamed M.F.
      • Wu J.
      • et al.
      The Combination of MEK Inhibitor With Immunomodulatory Antibodies Targeting Programmed Death 1 and Programmed Death Ligand 1 Results in Prolonged Survival in Kras/p53-Driven Lung Cancer.
      ,
      • Poon E.
      • Mullins S.
      • Watkins A.
      • Williams G.S.
      • Koopmann J.O.
      • Di Genova G.
      • et al.
      The MEK inhibitor selumetinib complements CTLA-4 blockade by reprogramming the tumor immune microenvironment.
      ]. Other targets in these trials include VEGF, an angiogenic cytokine that plays a role in the angiogenesis of cancers, and PARP, a protein involved in DNA repair [
      • Herceg Z.
      • Wang Z.Q.
      Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death.
      ,
      • Kieran M.W.
      • Kalluri R.
      • Cho Y.J.
      The VEGF pathway in cancer and disease: responses, resistance, and the path forward.
      ] A study combining MEKi with antiangiogenic therapy targeting VEGFR in human lung cancer models showed improvement in suppression of lung cancer progression and angiogenesis, and additionally, studies treating RAS mutant cancer cell lines by combining MEKi with PARP inhibitors also showed a synergistic effect [
      • Sun C.
      • Fang Y.
      • Yin J.
      • Chen J.
      • Ju Z.
      • Zhang D.
      • et al.
      Rational combination therapy with PARP and MEK inhibitors capitalizes on therapeutic liabilities in.
      ,
      • Takahashi O.
      • Komaki R.
      • Smith P.D.
      • Jürgensmeier J.M.
      • Ryan A.
      • Bekele B.N.
      • et al.
      Combined MEK and VEGFR inhibition in orthotopic human lung cancer models results in enhanced inhibition of tumor angiogenesis, growth, and metastasis.
      ].

      Biomarkers

      Continuing forward, many different avenues exist to tackle the issues of MEKi resistance in cancer cells. However, if patients could be prescreened for biomarkers that would definitively tell whether they would respond to MAPK inhibition, research in this field could be expedited. Some predictive biomarkers previously found in solid tumor cell lines include activating mutations in RAS or RAF, which usually indicated better tumor response to MEK inhibition [
      • Jing J.
      • Greshock J.
      • Holbrook J.D.
      • Gilmartin A.
      • Zhang X.
      • McNeil E.
      • et al.
      Comprehensive predictive biomarker analysis for MEK inhibitor GSK1120212.
      ]. However, within cancers with these activating mutations, response to MEKi treatment remains far from universal. For example, cell lines with an activating KRAS mutation and expression signatures of EMT were less responsive to treatment than cells that did not express EMT properties [
      • Corcoran R.B.
      • Cheng K.A.
      • Hata A.N.
      • Faber A.C.
      • Ebi H.
      • Coffee E.M.
      • et al.
      Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models.
      ,
      • Jing J.
      • Greshock J.
      • Holbrook J.D.
      • Gilmartin A.
      • Zhang X.
      • McNeil E.
      • et al.
      Comprehensive predictive biomarker analysis for MEK inhibitor GSK1120212.
      ]. Biomarkers that may help predict higher EMT signatures and therefore lower MEKi sensitivity include high ZEB1 and low IL7RD expression [
      • Peng D.H.
      • Kundu S.T.
      • Fradette J.J.
      • Diao L.
      • Tong P.
      • Byers L.A.
      • et al.
      ZEB1 suppression sensitizes KRAS mutant cancers to MEK inhibition by an IL17RD-dependent mechanism.
      ]. Other potential biomarkers that denote higher levels of resistance to MEKi treatment include EGFR and PKC-alpha protein expression [
      • Fernandez M.L.
      • Dawson A.
      • Hoenisch J.
      • Kim H.
      • Bamford S.
      • Salamanca C.
      • et al.
      Markers of MEK inhibitor resistance in low-grade serous ovarian cancer: EGFR is a potential therapeutic target.
      ]. These proteins were discovered to be overexpressed in resistant LGSOC cell lines, and both play a role in MAPK signaling [
      • Fernandez M.L.
      • Dawson A.
      • Hoenisch J.
      • Kim H.
      • Bamford S.
      • Salamanca C.
      • et al.
      Markers of MEK inhibitor resistance in low-grade serous ovarian cancer: EGFR is a potential therapeutic target.
      ]. Mutations in other signaling pathways such as PI3KA, PTEN, or YAP also signaled higher resistance to MEK inhibition, due to tumors’ lowered reliance on the MAPK signaling pathway for sustained growth [
      • Lin L.
      • Sabnis A.J.
      • Chan E.
      • Olivas V.
      • Cade L.
      • Pazarentzos E.
      • et al.
      The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies.
      ,
      • Milella M.
      • Falcone I.
      • Conciatori F.
      • Matteoni S.
      • Sacconi A.
      • De Luca T.
      • et al.
      PTEN status is a crucial determinant of the functional outcome of combined MEK and mTOR inhibition in cancer.
      ,
      • Jing J.
      • Greshock J.
      • Holbrook J.D.
      • Gilmartin A.
      • Zhang X.
      • McNeil E.
      • et al.
      Comprehensive predictive biomarker analysis for MEK inhibitor GSK1120212.
      ]. More recently, a clinical trial involving dabrafenib and trametinib in melanoma patients has generated some potential biomarkers associated with progression free survival. Patients who exhibited low tumor mutational burden had greater benefit from the combination treatment, and patients who had high tumor mutation burden with low type II interferon signatures derived the least benefit [
      • Dummer R.
      • Brase J.C.
      • Garrett J.
      • Campbell C.D.
      • Gasal E.
      • Squires M.
      • et al.
      Adjuvant dabrafenib plus trametinib versus placebo in patients with resected, BRAF(V600)-mutant, stage III melanoma (COMBI-AD): exploratory biomarker analyses from a randomised, phase 3 trial.
      ]. Furthermore, in another recent phase 2 clinical trial evaluating the efficacy of BRAF, MEK and PD-1 inhibition, Dummer et al. showed that patients responding to treatment had a characteristic low T-cell-inflamed gene expression signature compared to poor responders[
      • Dummer R.
      • Lebbe C.
      • Atkinson V.
      • Mandala M.
      • Nathan P.D.
      • Arance A.
      • et al.
      Combined PD-1, BRAF and MEK inhibition in advanced BRAF-mutant melanoma: safety run-in and biomarker cohorts of COMBI-i.
      ].

      Conclusion

      The field of signaling pathway therapeutics continues to shift and expand, and many advancements toward improving patient outcomes have occurred in the past decade. Though acquired resistance to MEKi treatment continues to be a pressing issue, advances in combination therapies present a new avenue to explore and have shown promising results. Many clinical trials combining MEKi with either other pathway inhibitors or immunotherapy are currently ongoing, but such treatments have shown promising preclinical data. Combination therapy seems to be the current best answer for tackling the issue of MEKi resistance, but increased toxicity in patients has slowed progress. Possible solutions to this issue may lie in discovery of new inhibitors that are more tolerable for the human body or in treating patients intermittently at doses that can be safely burdened. Furthermore, the discovery of new potential biomarkers for detecting MEKi resistance or sensitivity in cancers has presented clinicians with new diagnostic tools for improving patient response. Nonetheless, continued research in discovering new biomarkers remains an essential need, as no universal biomarker for prediction of patient outcomes to MEKi treatment has been discovered. The discovery of such a predictor would vastly improve our knowledge of the MAPK signaling pathway in cancers and be helpful in expediting treatment for patients.

      Funding

      This work was supported by the Ovarian Cancer Moon Shot Program at The University of Texas MD Anderson Cancer Center, Texas, USA and the U.S. Department of Defense Ovarian Cancer Research Program [grant number W81XWH-19–1-0169].

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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