Current Advances in the Treatment of BRAF-Mutant Melanoma

Melanoma is the most lethal form of skin cancer. Melanoma is usually curable with surgery if detected early, however, treatment options for patients with metastatic melanoma are limited and the five-year survival rate for metastatic melanoma had been 15–20% before the advent of immunotherapy. Treatment with immune checkpoint inhibitors has increased long-term survival outcomes in patients with advanced melanoma to as high as 50% although individual response can vary greatly. A mutation within the MAPK pathway leads to uncontrollable growth and ultimately develops into cancer. The most common driver mutation that leads to this characteristic overactivation in the MAPK pathway is the B-RAF mutation. Current combinations of BRAF and MEK inhibitors that have demonstrated improved patient outcomes include dabrafenib with trametinib, vemurafenib with cobimetinib or encorafenib with binimetinib. Treatment with BRAF and MEK inhibitors has met challenges as patient responses began to drop due to the development of resistance to these inhibitors which paved the way for development of immunotherapies and other small molecule inhibitor approaches to address this. Resistance to these inhibitors continues to push the need to expand our understanding of novel mechanisms of resistance associated with treatment therapies. This review focuses on the current landscape of how resistance occurs with the chronic use of BRAF and MEK inhibitors in BRAF-mutant melanoma and progress made in the fields of immunotherapies and other small molecules when used alone or in combination with BRAF and MEK inhibitors to delay or circumvent the onset of resistance for patients with stage III/IV BRAF mutant melanoma.


Introduction
Melanoma is the uncontrollable division of melanocytes located within the deep layer of the epidermis [1]. Although invasive melanoma is the third most common type of skin cancer, it is the most serious compared to its other two counterparts-basal cell carcinoma and squamous cell carcinoma. The American Cancer Society estimates that in 2019 there will be 96,480 new cases of melanoma diagnosed accompanied by 7,230 expected deaths. The five-year survival rate for metastatic melanoma has been 15-20% [2], although these statistics are rapidly improving with the success of immune checkpoint inhibitors. Treatment with immune checkpoint inhibitors demonstrated substantial clinical efficacy along with long-term survival outcomes in patients with advanced melanoma [3][4][5]. There are several clinical trials such as KEYNOTE-002, CheckMate067 and CheckMate064, which validate these findings, as detailed in Table 1. treatment regimen alone or in combination with BRAF and MEK inhibitors for treatment of patients with BRAF mutant melanoma [13][14][15][16]. New avenues exploring the possible combination therapies of BRAF/MEK inhibitors with immunotherapy drugs are being tested. Combination therapies are not only limited to MAPK pathway targeted therapies plus immunotherapy but have expanded to include other molecules such as AXL and ROS that have a role in the development of drug resistance. These have emerged as alternative treatment options for treating metastatic melanoma patients. Preclinical and clinical trials evaluating the efficacy of various PI3K and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are also initiated [17][18][19][20][21][22].
This review focuses on the current landscape of resistance with the chronic use of BRAF and MEK inhibitors in BRAF -mutant melanoma and progress made in the fields of immunotherapies and other small molecules when used alone or in combination with BRAF and MEK inhibitors to delay or circumvent the onset of resistance for patients with stage III/IV melanoma.

Mechanisms of Resistance
The development and use of the BRAF targeted inhibitors, Vemurafenib and dabrafenib, has improved the treatment arena for patients with metastatic melanoma. However, over half of patients treated with these single agent inhibitors begin to show signs of tumor recurrence within 6 to 7 months of treatment [23,24]. Several mechanisms of drug resistance have been proposed. There are two general types of resistance-primary resistance/intrinsic resistance and secondary or acquired resistance. Intrinsic resistance refers to those patients who do not respond to any type of BRAF inhibitor therapy and accounts for approximately 15% of patients [25]. Acquired resistance, refers to those patients who show tumor regression after an initial positive response to therapy and this is commonly observed in most melanoma patients [26].

Intrinsic Resistance
PTEN is a negative regulator of phosphoinositde 3-kinase (PI3K) and loss of PTEN can lead to an upregulation of the PI3K/Akt pathway whose activation can explain tumor resistance [27,28]. Loss of PTEN alone does not confer a resistance state; it is typically accompanied with Akt phosphorylation and activation [26,29]. Cyclin D1 amplification (CCND1) is observed in about 15-20% of all BRAF-mutant melanoma [30,31]. CCND1 alone can accelerate the resistance in BRAF-mutant melanoma and is intensified when there is both cyclin D1 overexpression along with a cyclin dependent kinase-4 (CDK4) mutation [32].
Neurofibromin-1 (NF1) is a tumor suppressor and a negative regulator of RAS. Loss of NF1 is typically seen in 14% of melanoma cases and leads to activation of RAS and other downstream pathways including the MAPK and PI3K-Akt [26,33]. Loss of NF1 can also mediate resistance to RAF and MEK inhibitors [34]. RAC1 is part of the Rho family of small GTP binding proteins. Mutations in RAC1 are found in 4-9% of patients and is the third "hotspot" mutation in melanoma, following BRAF and NRAS [35][36][37]. RAC1 mutation status is being considered a biomarker for resistance to RAF inhibitor therapy [35]. It has been shown that pre-existing mutations in MEK1 can confer resistance to RAF inhibitor therapy [38].

Acquired Resistance
Starting from the cell surface, several receptor tyrosine kinases (RTK) converge onto parallel pathways such as the MAPK and the PI3K-Akt pathway [39]. Upregulation of RTKs has been shown to directly activate the MAPK pathway via RAS activation [30]. Additionally, upregulation of specific RTKs such as the insulin like growth factor 1 receptor (IGFR1), platelet derived growth factor receptor β (PDGFRβ) can activate the PI3K-Akt pathway in a non-ERK dependent manner [40,41]. Epigenetic changes affecting epidermal growth factor receptor (EGFR) have been shown to also induce the PI3K-Akt pathway in melanoma resistant cells [42]. Dual activation of these pathways strongly contributes to drug resistance, as these pathways promote cell survival and proliferation.
NRAS activating mutations are present in approximately 15-20% of melanomas [43]. Q61 mutations in NRAS keeps it constitutively active in the 'RAS-GTP' state [44]. The activated mutant NRAS can activate the MAPK pathway via induction of dimerization of CRAF and BRAF [45].
Treatment with BRAF inhibitors will only inhibit the mutant monomer in BRAF mutant melanoma cells [46]. This plays an important role in the ability for these cells to maintain RAF dimerization which in turn keeps MAPK signaling active. A phenomenon known as the 'BRAF-inhibitor Paradox' describes the event in which the BRAF inhibitor blocks MAPK signaling in mutant cells but activates the MAPK pathway in non-mutant cells by allowing the drug-free RAF protein to be transactivated and dimerize [47]. RAF dimerization can be fulfilled through a variety of mechanisms including alternative BRAF splicing, amplification of BRAF, and expression of different RAF isoforms such as CRAF overexpression [48][49][50][51]. Resistance to RAF inhibition include activation of HGF and its receptor MET which lead to the reactivation of the MAPK and PI3K-AKT pathways [52]. Screening for the presence of RAF inhibitor resistance genes, found in a high percentage of patients can help improve treatment outcomes especially when used in conjunction with appropriate therapeutic combinations [53].
Secondary mutations in both MEK1 and MEK2 have also been linked to acquired resistance in melanoma cell lines [54]. The resistance to MEK inhibitors is attributed to mutations in MEK1/2 which lead to constitutive activation of MEK1/2 or a mutation in the drug binding site [55]. MAPK reactivation occurs via secondary mutations in MEK1 (Q56P or E203K) which help reactive the MAPK pathway downstream [56]. Additionally, BRAF amplification along with KRAS mutations can be contributing factors to MEK1/2 inhibitor resistance [57].
The RTK AXL has also been identified as a player in both intrinsic and acquired resistance. Patients relapsed of BRAF and MEK inhibitors overexpress AXL as an adaptive response [58]. Non-genomic mechanism of acquired resistance include high expression of transcriptomic alterations and intra-tumoral immunity which involves cytolytic T-cell inflammation [59].
Combination therapy using BRAF and MEK inhibitors has also shown signs of resistance. Proposed mechanisms of resistance include BRAF gene amplification, BRAF splice-variants and mutations in MEK2 [60]. Resistance to BRAF and MEK inhibitors exists by combining or augmenting the mechanisms related to single agent BRAF inhibitor resistance. The overexpressed BRAFV600E and MEK mutants interact via the regulatory interface of BRAFV600E, R662 [61]. Acquired resistance to combination targeted therapy has other factors contributing to it, including whether the patient had received inhibitor monotherapy prior to the combination therapy or is 'monotherapy naïve' [62].
Many recent advancements in the treatment of metastatic melanoma have been in the field of immunotherapy. There are proposed mechanisms of resistance to BRAF and MEK inhibitor therapy involving immune system molecules. Cancer cells, in general, aim to avoid immune recognition by downregulating surface receptors that participate in co-activation of T cells as well as upregulating negative feedback pathways, such as the immune checkpoint inhibitor receptors programmed cell death protein (PD-1) and T-lymphocyte associated protein-4 (CTLA-4). PD-1 and CTLA-4 are localized on the T-cells [63]. It is documented that after 2 weeks of treatment with BRAF and MEK inhibitors, melanoma cells have been able to downregulate melanoma differentiation antigens (MDA) surface expression, decrease T cell activity, and surface display of increase immune checkpoint inhibitory receptors [64,65]. This manipulation of key immune system regulators gives melanoma cells yet another way to bypass drug resistance. The rationale to combine an immune checkpoint inhibitor therapy with targeted therapy is that the treatment with a BRAF and MEK inhibitor renders the tumor microenvironment more immunoresponsive [66].
The sequence of treatment for a patient with targeted therapy and immune therapy is not well established. Starting a patient on a BRAF inhibitor or anti-PD1 inhibitor is effective regardless of the treatment order, but more randomized controlled trials are required to address and establish the superiority and sequencing of one therapy over the other [67,68]. Studies have also shown that the efficacy of immunotherapy is improved in previously untreated patients compared to patients who have single agent immunotherapy failure or failure to targeted therapy [69]. Despite showing exceptional clinical efficacy, treatment with immune checkpoint inhibitors has met some difficulties with respect to development of innate and acquired resistance [70,71]. Various clinical trials evaluating immune therapies such as Toll-like receptor 9 (TRL9) agonists, neoantigen vaccines and oncoloytic viruses alone or in combination with immune checkpoint inhibitors are underway. Combination of these therapies may help combat resistance to immune checkpoint inhibitors [72][73][74][75][76][77]. Figure 1 summarizes the therapies in pre-clinical and clinical phases, described in this review to treat patients with metastatic melanoma. have single agent immunotherapy failure or failure to targeted therapy [69]. Despite showing exceptional clinical efficacy, treatment with immune checkpoint inhibitors has met some difficulties with respect to development of innate and acquired resistance [70,71]. Various clinical trials evaluating immune therapies such as Toll-like receptor 9 (TRL9) agonists, neoantigen vaccines and oncoloytic viruses alone or in combination with immune checkpoint inhibitors are underway. Combination of these therapies may help combat resistance to immune checkpoint inhibitors [72][73][74][75][76][77]. Figure 1 summarizes the therapies in pre-clinical and clinical phases, described in this review to treat patients with metastatic melanoma.

Anti-PD-1/PD-L1
The immunogenic nature of melanoma was utilized to develop several immunotherapeutic treatment strategies especially with regards to the programmed cell death (PD-1) receptor and its ligand, PD-L1. Antibodies targeting the PD-1 axis has shown significant promise in the clinic for treatment of metastatic melanoma either as a monotherapy or in combination with Ipilimumab. There are several ongoing clinical trials using anti-PD1 and anti-PD-L1 antibodies. Programmed cell death protein 1 (PD-1) is an inhibitory receptor expressed on the surface of the cancer cells that inhibits the immune system via suppressing the T-cell activity. Anti-PD-1 monoclonal antibodies block the PD-1 receptor which maintains T-cells in activated state to suppress the tumor growth [78]. There are several anti-PD-1/PD-L1 monoclonal antibodies including pembrolizumab (Keytruda ® ), nivolumab (Opdivo ® ), avelumab (Bavencio ® ), durvalumab (Imfinzi ® ), cemiplimab (Libtayo ® ), atezolizumab (Tecentriq ® ), cosibelimab and INBRX-105 in several stages of clinical trial in melanoma. Pembrolizumab, nivolumab and nivolumab in combination with iIpilimumab (anti-CTLA-4 inhibitor) have been approved by FDA for treatment of melanoma.

Anti-PD-1/PD-L1
The immunogenic nature of melanoma was utilized to develop several immunotherapeutic treatment strategies especially with regards to the programmed cell death (PD-1) receptor and its ligand, PD-L1. Antibodies targeting the PD-1 axis has shown significant promise in the clinic for treatment of metastatic melanoma either as a monotherapy or in combination with Ipilimumab. There are several ongoing clinical trials using anti-PD1 and anti-PD-L1 antibodies. Programmed cell death protein 1 (PD-1) is an inhibitory receptor expressed on the surface of the cancer cells that inhibits the immune system via suppressing the T-cell activity. Anti-PD-1 monoclonal antibodies block the PD-1 receptor which maintains T-cells in activated state to suppress the tumor growth [78]. There are several anti-PD-1/PD-L1 monoclonal antibodies including pembrolizumab (Keytruda ® ), nivolumab (Opdivo ® ), avelumab (Bavencio ® ), durvalumab (Imfinzi ® ), cemiplimab (Libtayo ® ), atezolizumab (Tecentriq ® ), cosibelimab and INBRX-105 in several stages of clinical trial in melanoma. Pembrolizumab, nivolumab and nivolumab in combination with iIpilimumab (anti-CTLA-4 inhibitor) have been approved by FDA for treatment of melanoma.

Pembrolizumab/Lambrolizumab/MK-3475/SCH 900475/Keytruda
This is a humanized monoclonal antibody targeting the PD1 receptor in the lymphocytes. It was developed by Merck and approved for treatment of metastatic melanoma in 2017 [79].

Avelumab/MSB0010718C/Bavencio
This is a humanized monoclonal antibody developed by Merck and Pfizer that targets the PD-L1. It has been approved by FDA for treatment Merkel-cell carcinoma, an aggressive type of skin cancer [83]. It blocks the PD-1/PD-L1 receptor/ligand complex formation leading to suppression of CD8+ T cells action [84]. There is a current clinical trial (NCT01772004) investigating the safety, tolerability, pharmacokinetics and clinical activity of avelumab in melanoma [85].

Atezolizumab/MPDL3280A/Tecentriq
This is a fully humanized engineered monoclonal antibody of IgG1 isotype against PD-L1 developed by Genentech [89,90]. There is an active ongoing phase II trial (NCT02303951) which includes the combination of vemurafenib, cobimetinib and atezolizumab in stage III/IV advanced melanoma patients [91]. Another phase III study (NCT02908672) compares the efficacy of atezolizumab in combination with cobimetinib and vemurafenib versus placebo control plus cobimetinib and vemurafenib in unresectable and advanced melanoma patients with BRAFV600 mutation [92].

Anti-CTLA-4
In addition to PD-1, another immune checkpoint inhibitor, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is important in melanoma. It is found on the surface of regulatory T cells (Treg) and activated T cells [97]. CTLA-4 competes with CD28, another receptor expressed on the surface of T cells, to interact with its two ligands CD80 and CD86, collectively known as the B7 ligands. When CTLA-4 binds with the B7 ligands, commonly found on antigen presenting cells (APC), it results in an immunosuppressive response, which is the inhibition of T cell activation via transendocytosis of CD80 and CD86 from their surfaces [98,99]. Typically, T cell activation requires co-stimulation from the CD28-B7 ligand interaction and the TCR-MHC interaction [100]. However, CTLA-4 has a stronger affinity for the B7 ligands, making it a good immune checkpoint inhibitor that keeps the immune response from turning into an autoimmune one [97]. CTLA-4 is expressed on tumor cells, infiltrating Tregs, and exhausted, activated T cells [101]. Tumor cells, therefore, take advantage of this natural immunosuppressive system in order to prevent an immune response against them. This provides a therapeutic approach which involves anti-CTLA-4 therapy. There are currently three main anti-CTLA-4 antibodies under preclinical and clinical trials for the treatment of melanoma: Tremelimumab, Ipilimumab (Yervoy), and BCD-145.

Ipilimumab/MDX010/BMS-734016
This human monoclonal antibody against CTLA-4 was developed by YERVOY Medarex/BMS. It was approved by the FDA in 2011 for the treatment of unresectable or metastatic melanoma [105]. There are current, active clinical trials devoted to assess the efficacy of ipilimumab in combination with other immunotherapies or targeted therapies for metastatic melanoma. A phase I clinical trial (NCT02115243) that assessed ipilimumab as a neoadjuvant followed by melphalan (chemotherapeutic) via isolated limb perfusion in patients with unresectable in-transit extremity melanoma is completed [106]. A phase Ib clinical trial (NCT02117362) evaluating ipilimumab in combination with GR-MD-02 (galnectin inhibitor) in metastatic melanoma patients has been completed [107]. A phase II clinical trial (NCT03153085) examining ipilimumab in combination with TBI-1401(HF10) in Japanese patients with Stage IIIb, IIIc, IV unresectable or metastatic malignant melanoma has been completed [108]. A phase II clinical trial (NCT01970527) looking at SBRT followed by Ipilimumab in patients with stage IV and recurrent melanoma has been completed [109].

BCD-145
This human monoclonal antibody against CTLA-4 is developed by BIOCAD [110]. A completed phase I clinical trial (NCT03472027) studied the efficacy of BCD-145 in unresectable/metastatic melanoma [111]. The combination of anti-PD-1/PD-L1 and anti-CTLA-4 are also being tested in the clinic for stage III/IV melanoma patients. A phase I clinical trial (NCT02935790) evaluating ipilimumab and nivolumab in combination with ACY-241 (selective HDAC inhibitor) is completed [112]. Current clinical trials, outcomes and adverse events investigating the efficacy of anti-CTLA-4, anti-PD-1/PD-L1 therapies and their combinations used to treat metastatic melanoma patients are listed in Table 1  . Active, not recruiting Genentech, Inc.
Ib, NCT01656642 The triple combination was safe, tolerable and had a promising anti-tumor activity. Atezolimumab + Vemurafenib (n = 17): The best objective response rate and complete response rate was 76.5% (95% CI: 50.1-93.2%) and 17.6% respectively. All the patients demonstrated a reduction in the sum of the longest diameter of the target lesion. The median duration of response, PFS and OS was 10.6 months (95% CI: 9.1-37.6 months), 10.9 months (95% CI: 5.7-22 months) and 46.2 months (95% CI: 24.1-not reached) respectively.
Estimated OS rates for 1 year were 82%. Atezolimumab + Vemurafenib + Cobimetinib (n = 39): The best objective response rate and complete response rate was 71.8% (95% CI: 55.1-85%) and 20.5% respectively. All the patients demonstrated a reduction in the sum of the longest diameter of the target lesion. The median duration of response and PFS was 17.4 months (95% CI: 10.6-25.3 months), 12.9 months (95% CI: 8.7-21.4 months) respectively. The median OS was not reached. Estimated OS rates for 1 year were 83%. Treatment with vemurafenib alone or in combination with cobimetinib exhibited an increase in the proliferating CD4+ T-helper cells and addition of atezolizumab led to further escalation in these cells. CD8+ cytotoxic T cells were augmented on addition of atezolizumab.
Ipilimumab alone or in combination with dacarbazine, paclitaxel and carboplatin [120] Completed BMS in collaboration with Medarex I, NCT00796991 Ipilimumab could be combined safely with these chemotherapies with no major pharmacokinetic/pharmacodynamic interactions being observed in these patients. The combinations exhibited a good anti-tumor activity. Ipilumumab alone (n = 20): Estimated geometric mean for Area Under the Curve (AUC) (0-infinity) and maximum serum concentration (C max ) for Ipilimumab metabolite (AIC) in presence of ipilimumab was changed by 0.970 (90% CI: 0.891-1.056) and 1.058 (90% CI: 0.974-1.150) fold respectively. Based on World Health Organization (WHO) and immune-related criteria ORR were 29.4% and 33.3% respectively and disease control rates b were 59.2% and 73.3% respectively. Ipilimumab + Decarbazine (n = 19): Estimated geometric mean for AUC (0-infinity) and C max for dacarbazine in presence of ipilimumab was changed by 0.912 (90% CI: 0.757-1.099) and 1.027 (90% CI: 0.848-1.243) folds respectively. Based on WHO and immune-related criteria ORR were 27.8% and 33.3% respectively, and disease control rates were 55.6% and 61.1% respectively. Ipilimumab + paclitaxel + carboplatin (n = 20): Estimated geometric mean for AUC (0-infinity) and C max for carboplatin/paclitaxel in presence of ipilimumab was changed by 0.970 (90% CI: 0.891-1.056) and 1.058(90% CI: 0.974-1.150) folds respectively. ORR based on WHO and immune related criteria were 11.1% and 27.8% respectively. Disease control rate based on WHO and immune related criteria were 44.4% and 55.6% respectively. There was a significant increase in the mean relative frequency and counts of HLA-DR+ CD4+ and CD8+ T cells after treatment initiation in all the three groups.
Rash, fatigue, diarrhea, pruritus, nausea, increase in ALT and AST, decreased neutrophil count. The combination was well tolerated and safe and the MTD and recommended phase 2 dose for intravenous ipilimumab was 3 mg/kg every 3 weeks and imatinib mesylate at 400 mg orally twice daily. Twenty six patients were enrolled in dose escalation cohort Expression of ICOS and OX40 was increased on the CD4 + T cells upon ipilimumab treatment.
Ipilimumab and high dose IFN-α2B as a neoadjuvant combination for locally/regionally advanced/recurrent melanoma [122] Completed Diwakar Davar, University of Pittsburgh The combination was well tolerated and exhibited promising durable clinical response rates. 30 patients were enrolled. The median follow-up was 32 months and the pathologic complete response rate was 32% (95% CI: 18-51%). The radiologic response rate was 36% (95% CI: 21-54%). The median PFS was not reached and the probability of PFS at 12 and 6 months was 0.79 (95%CI: 0.65-0.95) and 0.86 (95% CI: 0.74-1) respectively. The probability of OS at 2 years and 1 year was 0.89 (95% CI: 0.79-1) and 0.93 (95% Ci: 0.84-1) respectively. The peripheral blood mononuclear cell was significantly lower at 12 weeks (p = 0.025). The tumor-infiltrating lymphocyte (TIL) was significantly higher in primary melanoma tumors for patients with pathologic complete response (p = 0.033). There was an increase in the number of tumor associated clones following the neoadjuvant treatment and it showed a strong correlation with TIL fraction (ρ = 0.7299; p = 0.0003) and TIL clone diversity (ρ= 0.882; p = 2.7-7). The increase in the tumor T-cell clonality in the primary tumor and a further increase in the clonality after neoadjuvant therapy was statistically significant with relapse-free survival. (p = 0.048 for tumor clonality and p = 0.018 for post treatment metastatic clonality).  [124,125] Completed University of Utah I, NCT01672450 The combination was well tolerated and generated an enhanced systemic immune response at injected and non-injected lesions in these patients. No dose limiting toxicities were observed in the 12 enrolled patients. Immune-related response criteria: Clinical benefit rate was 50% (95% CI: 19-81%). The PR and overall ORR was 30% and 40% (95% CI: 10-70%) respectively. 67% of the subjects (95% CI: 40-94%) had local response on the injected lesion which was assessed by pathology and/or measurement. 88 The combination of ipilimumab (3 mg/kg; n = 7 or 10 mg/kg; n = 9) and SRS was safe without any dose limiting toxicities. 1-37 months) 2.1 months respectively. The median OS was not reached. Immune-related PR was 7%.
Rash, pruritus Completed BMS II, NCT01673854 The study was divided into two parts: one where patients received vemurafenib followed by ipilimumab and the other where subjects who progressed after ipilimumab received vemurafenib. The sequential treatment was efficacious and had a manageable safety profile. The use of targeted therapy followed by immune modulation therapy has helped to understand the optimum regimen of these therapies. VEM1-IPI: 46 patients were treated with vemurafenib followed by 46 patients on ipilimumab induction and eight patients on ipilimumab maintenance. The median duration of response and follow-up was 23. The combination was well tolerated, beneficial and elicited anti-tumor activity. The combination induced an immune-cell infiltration in the TME. 46 patienrs were enrolled. The best overall response rate was at 24 weeks. Immune-related response criteria: 18% and 23% of the patients had a CR and PR respectively. The median PFS and OS were 19 months and 26 months respectively. There was increase om the total tumor infiltrating CD8+ T-cells and lymphocytes along with a decrease in the CD4+ T-cells. The combination exhibited an enhanced antitumor activity. 64 patients were enrolled. The median duration of response and median follow-up was 35 months (Range: 2-57 months) and 20 months (Range: 2-60 months). 15.6% of the subjects had a CR and PR. The median PFS and OS was 5 months and 24.5 months respectively. 6 months PFS was 45%. The PFS for patients with bone metastasis was significantly decreased (p = 0.014) but not for subjects with liver metastasis. 21% and 7% of the subjects with liver metastasis had a CR and PR respectively while no ORR was observed in subjects with bone metastasis Pruritus, skin rash, nausea, constipation, diarrhea, colitis, increase in ALT and AST, hematologic toxicities. No drug related grade 5 toxicities were observed. The combination was well tolerated and demonstrated a durable anti-tumor response. 39 patients were enrolled in the trial. The median follow-up time, estimated median PFS and OS were 36 months (Range: 22-43 months), 27 weeks (95% CI: 9-44 weeks) and 59 weeks (Range: 72-185 weeks) respectively. PFS and OS rates at year one-33% (95% CI: 18-48%), 59% (95% CI: 43-74%); year two-22%(95% CI: 9-36%), 38%(95% CI: 23-53%) and year three-18% (95% CI: 5-31%), 34% (95% CI: 19-50%). Immune-related response criteria: 6 months disease control rate was 51% (95% CI: 36-67%). The ORR was 38%. 20% and 18% of the subjects had a CR and PR respectively. The outcome was poor in patients with brain metastasis. There was a significant increase in the eosinophils, peripheral blood lymphocytes and monocytes after from the baseline treatment with the combination. A significant increase in the CD8+ (p < 0.001), CD4+ (p < 0.001), HLA-DR+ activation marker on CD4+ cells (p < 0.001), CD3+ (p < 0.001), ratio of CD4+ to CD8+ (p = 0.003) and Tregs (p = 0.016) was observed after the combination treatment. Colitis, hepatitis, hypophiisitis, headache. One subject showed radionecrosis.
Ipilumumab alone or in combination with decarbazine in previously untreated metastatic melanoma patients [143] Completed BMS II, NCT00050102 The combination was well tolerated and a durable, clinically meaningful responses were observed. Decarbazine did not affect the PK of ipilimumab. Ipilimumab alone (n = 37): The median follow up time and median OS were 16.4 months and 11.4 months (95% CI: 6.1-15.6 months) respectively. The objective response rate and disease control rate were 5.4% (95% CI: 0.7-18.2%) and 21.6% (95% CI: 9.8-38.2%), respectively. 5.4% of the subjects had a PR. The survival rates for 1 year, 2 year and 36 months were 45%, 21% and 9%, respectively. Combination (n = 35): The median follow up time and the OS were 20.9 months and 14.3 months (95% CI: 10.2-18.8 months) respectively. The objective response rate and disease control rate was 14.3% (95% CI: 4.8-30.3%) and 37.1% (95% CI: 21.5-55.1%) respectively. 5.7% and 8.6% of the patients had a CR and PR for more than 24 weeks respectively. The survival rates for 1 year, 2 year and 36 months were 62%, 24% and 20% respectively. CD4+ and CD8+ expressing HLA-DR T cells were increased in both the groups.
Ipilimumab in combination with HF10 for unresectable Stage IIIb/c/IV or metastatic malignant melanoma [144] Completed Takara Bio Inc. in collaboration with Theradex II, NCT02272855 The combination was well tolerated with positive antitumor activity and there were no dose limiting toxicities. 46 patients were enrolled. The median PFS and OS was 19 months and 21.8 months respectively. Immune-related response criteria: The best overall response rate and disease stability rate was 41% and 68%, respectively. 16% and 25% of the patients had CR and PR, respectively.
Embolism, lymphedema, diarrhea, hypoglycemia, groin pain, immune related events. The combination of ipilimumab and decarbazine was well tolerated and had a long-term durable overall survival. Ipilimumab and decarbazine group (n = 250): The median survival follow-up time and OS was 11 months (Range: 0.4-71.9 months) and 11.2 months (95% CI: 9.5-13.8 months) respectively. At 5 years, 18.2% of the patients were alive which was significantly higher than that in the other group (p = 0.002). 7 Overall ipilimumab resulted in survival of 20% of the patients for more than 2 years. 45% of the patients who survived for more than 2 years survived for more than 3 years. Ipilimumab + placebo (n = 137): 25% of the patients survived for more than 2 years and 3 years. The disease control rate for on-study response and for patients surviving more than 2 years was 28.5% (1.5% CR and 9.5% PR) and 83.3% (8.3% CR and 41.7% PR) respectively. Gp100 vaccine alone (n = 136): 17% and 10% of the patients survived for more than 2 year and 3 years respectively.
The disease control rate for on-study response and for patients surviving more than 2 years was 11% (1.5% PR) and 43.8%, respectively. Combination (n = 403): 19% and 15% of the patients survived for more than 2 years and 3 years respectively. The disease control rate for on-study response and for patients surviving more than 2 years was 20.1% (0.2% CR and 5.5% PR) and 66.7% (1.9% CR and 22.2% PR), respectively.
No grade 5 toxicities were observed.
Ipilimumab alone or in combination with talimogene laherparepvec (T-VEC) in patients with previously untreated unresected, Stage IIIb-IV melanoma [148] Active, not recruiting
Fatigue, chills, GI disorders, pruritus, rash and nausea. Immune related adverse events-GI, hepatic, endocrine, skin and neurologic. Combination of immune checkpoint inhibitors with LTX-315 was safe and tolerable and demonstrated a potent anti-tumor activity. Of 6 melanoma patients received LTX-315 in combination with Ipilimumab, stable disease was observed in 33% of the patients. LTX-315 when administered to patients with solid tumors resulted in increase in number of CD8+ T cells at the site of treated lesions along with tumor infiltrating lymphocyte population. Clonal expansion of T-cells in blood was observed after treatment with LTX-315 as revealed by T-cell receptor sequencing.
Nivolumab and Ipilimumab alone or in combination in patients with previously untreated unresectable or metastatic melanoma (CheckMate067) [155] Active, not recruiting BMS III, NCT01844505 Combination of ipilimumab and nivolumab or nivolumab alone was superior over monotherapy with ipilimumab. No new toxic effects associated with chronic use of these therapies were observed. Nivolumab plus Ipilimumab group (n = 314): Median overall survival was more than 60 months. Hazard ratio for death versus Ipilimumab group was 0.52. 5 year overall survival rate was 52%.
Grade 1 and 2 treatment related adverse events were commonly observed such as skin, GI, endocrine, musculoskeletal, respiratory related and fatigue.
Grade 3/4 related adverse events were infrequent. While the combination of Pembrolizumab and ipilimumab had good antitumor activity and manageable safety profile, the combination of pembrolizumab and PEF-INF did not. Pembrolizumab and Ipilimumab (n = 12): The median follow-up was 25.1 months (Range: 0.8-38.7 months). The median duration of response was not reached. The objective response rate as per independent central review was found to be 42% (95% CI: 15-72%). The CR and PR rates were 8.33% and 33.33% respectively. As per investigator review, the objective response rate and PR were 33% (95% CI: 10-655) and 33.33% respectively. Pembrolizumab and PEG-IFN (n = 17): The median follow up was 22.2 months (Range-25-377 months). As per central and investigator review, the objective response rate was 20% and the partial response rate was 20%.

AXL Inhibitors
The TAM family of receptor tyrosine kinases (RTKs) is comprised of Tyro-3, Axl and Mer (TAM). These TAMS regulate cell proliferation, survival, adhesion, migration, invasion and metastasis of neoplasms [161]. The AXL gene is located on chromosome 19q13.2; encoded by 20 exons. The protein structure consists of an extracellular domain consisting of a combination of two IgG like domains and two fibronectin type III repeats; a conserved intracellular kinase domain and a transmembrane domain [162,163]. Even though all three TAMS have transforming potential, the aberrant overexpression of Axl is associated with cancer progression, drug resistance and supports tumor immune escape in several cancers including melanoma [164][165][166][167][168][169][170][171][172]. In primary and acquired resistance in melanoma, Axl levels inversely correlate with levels of melanocyte lineage factor-Microphthalmia-associated transcription factor (MITF). The high Axl, low MITF drug resistance phenotype is found frequently among BRAF mutant melanoma cell lines. This is associated with a phenotype switch of cells form proliferative to an invasive phenotype and promotes metastasis [165,173,174]. The Axl inhibitors can be classified into 2 types. Type I encompasses inhibitors that compete with ATP and bind to the active conformation of the receptor, DGF-in (constitutes of the aspartate-phenylalanine-glycine (DFG) motif oriented towards the active site). Type II inhibitors interact with the DFG residues of the activation loop which open up an allosteric region, adopt an extended conformation and prefer binding to the inactive DFG-out conformation. [175] 6.1. BGB-324/Bemcentinib (Type I) This highly selective orally bioavailable inhibitor was developed by BerGenBio [176]. Upregulation of Axl leads to drug resistance of BRAF directed therapies in the context of melanoma and also reduces response to PD-1 blockade. A Phase Ib/II trial is ongoing (NCT02872259) evaluating BGB324 in combination with dabrafenib/tramatenib or pembrolizumab in advanced non-resectable Stage IIIc/IV melanoma. The interim results for this study indicate that the combination was well tolerated at the recommended phase 2 dose of 200 mg daily of Bemcentinib. The common adverse events were diarrhea, fatigue, rash and pyrexia [177].

TP-0903 (Type I)
This oral Axl kinase inhibitor was developed by Tolero Pharmaceuticals, Inc. A first-in-human phase Ia/Ib trial (NCT02729298) evaluating TP-0903 in patients with advanced solid tumors encompassing BRAF mutated melanoma patients who haven't responded to BRAF/MEK inhibitor combination or immunotherapy is currently recruiting patients [178,179].

Cabozantinib/XL184/BMS-907351 (Type II)
This inhibitor, developed by Exelixis [180], is an orally bioavailable small molecule inhibitor against various tyrosine kinases which include Axl, MET and VEGF. A phase II trial (NCT00940225) evaluating cabozantinib in patients with metastatic melanoma was discontinued as the study was underpowered to detect statistical significance [181]. A phase I/II trial (NCT03957551) evaluating the combination of cabozantinib and pembrolizumab as a front-line therapy has been initiated for patients with advanced metastatic melanoma [182].

LDC1267 (Type II)
This inhibitor preferentially inhibits Axl, Mer and Tyro3. In an in vivo model, it was observed that treatment with LDC1267 unleashes natural killer (NK) cells to target and kill tumor cells. Treatment with LDC1267 reduced the metastatic spreading of melanoma in an in vivo B16F10 melanoma model [183]. Further pre-clinical and clinical trials need to be initiated to test the efficacy of this drug in melanoma.

AXL-1047-MMAE
This is an antibody-drug conjugate (ADC) in which the Axl targeting human antibody is conjugated to monomethyl auristatin E (MMAE), a microtubule disrupting agent by a valine citrulline linker which is protease-cleavable. This ADC induces cytotoxicity in vitro and in vivo in melanoma models. Treatment with the ADC prevents the emergence of BRAF-inhibitor resistant clones and potentiates the efficacy of BRAF and MEK inhibitors and co-operatively targets the growth of resistant cells. This ADC along with BRAF and MEK inhibitors has shown efficacy in treatment naïve and MAPK pathway inhibitor resistant melanoma. A phase I/II trial (NCT02988817) evaluating enapotamab vendotin (HuMax-AXL-ADC) has been initiated in patients with solid tumors, including melanoma [184,185]

BRAF Inhibitors
BRAF inhibitors are small molecule inhibitors that selectively target mutant BRAF isoforms, preferentially V600E but also other isoforms such as V600K or V600D [186]. BRAF inhibitors are typically used in combination with inhibitors of MEK, the downstream target of BRAF, in order to delay the development of resistance to BRAF inhibitor monotherapy as in the current standard of care for late-stage BRAF V600E melanoma, dabrafenib and trametinib. Vemurafenib/PLX4032/RG7204, a serine/threonine kinase inhibitor, was the first selective BRAF inhibitor that was approved by the FDA. It binds to the ATP-binding domain of BRAF mutants such as V600E, V600R and V600D [187]. 960 mg twice daily was established as the recommended phase 2 dose in the phase 1 (NCT00405587) dose escalation clinical trial [186]. The FDA approval was granted based on the Phase 3 trial (BRIM-3) results (NCT01006980) which exhibited improved overall survival and progression-free survival rate in patients with BRAFV600E mutant melanoma [188]. Dabrafenib, a type I-kinase inhibitor was the second BRAF inhibitor that was approved by the FDA. This reversible ATP-competitive inhibitor, inhibits BRAFV600E, V600D and V600K proteins [189]. The Phase 2 trial (BREAK-2; NCT01153763) trial established a dose of 150 mg twice daily which can either be used as a single agent or in combination with trametinib [190,191]. It was granted FDA approval on the basis of the outcomes of Phase 3 trial (NCT01227889) in which it exhibited improved progression-free survival vs. decarbazine [24].

Encorafenib/LGX818
This molecule is an oral BRAF inhibitor selective for BRAF V600E that was approved by the FDA in June 2018 for use in combination with the MEK inhibitor binimetinib (MEK162) in treating metastatic melanoma patients with the BRAF V600E mutation [192]. A phase II trial (NCT02631447) to determine the optimal sequencing of BRAFi + MEKi (encorafenib + binimetinib) therapy and immunomodulatory antibody (ipilimumab + nivolumab) therapy in stage III-IV metastatic BRAF V600 melanoma is ongoing [193]. A phase II trial (NCT02159066) evaluating the use of third agent in encorafenib + binimetinib therapy in stage III-IV metastatic BRAF V600 melanoma [194].

MEK Inhibitors
MEK inhibitors are small molecule inhibitors targeting MEK1/2 proteins in the MAPK pathway. The addition of a MEK inhibitor in combination with a BRAF inhibitor delayed the development of resistance and decreased the toxicities associated with BRAF inhibitor monotherapy [195]. Trametinib/GSK1120212, a reversible, non-ATP-competitive inhibitor of MEK1/2 was the first MEK inhibitor approved by the FDA. The phase 1 study (NCT00687622) identified 2 mg daily dose of trametinib, which could be safety, administered to the patients [196]. The phase 3 COMBI-D trial (NCT01584648) provided evidence of combining dabrafenib and trametinib in patients with metastatic BRAFV600E/K mutant melanoma as compared to monotherapy with dabrefenib [197]. Cobimetinib/GDC-0973 is used in combination with vemurafenib and is approved for patients with BRAFV600E/K mutant metastatic melanoma. The FDA approval was granted based on the efficacy results of combination of vemurafenib and cobimetinib in Phase-3 co-BRM trial (NCT01689519) [198].
Binimetinib/MEK162 is used in combination with encorafenib and is used in patients harbouring BRAFV600E/K mutation.

KZ-001
This selective MEK1/2 inhibitor is a benzoxazole compound with high potency and exhibits anti-tumor activity in BRAF-and NRAS-mutant tumor cell lines. It presented a synergistic effect in in vitro and in vivo xenograft models when used in combination with docetaxel (microtubule-stabilizing chemotherapeutic agent) and vemurafenib [199].

E6201
This MEK1 inhibitor was developed by Eisai Inc. This ATP-competitive MEK inhibitor is a synthetic analog of a natural product f152A1 occuring from the fungus Curvularia verruculosa [200]. NCT00794781 was a Phase I trial, evaluating the efficacy and safety of E6201 in patients with BRAF mutant advanced melanoma was terminated early due to futility based on response data [201].

TAK-733
This selective, oral, potent, non-ATP competitive allosteric site MEK inhibitor was developed by Millenium Pharmaceuticals, Inc. It demonstrated anti-tumor effects in vitro in melanoma cell lines and in vivo in patients-derived xenograft models [202]. NCT00948467 was a Phase 1 dose escalation trial evaluating TAK-733 in advanced solid tumors including patients with advanced metastatic melanoma had manageable toxicity profile but had limited antitumor activity and based on this result the further investigations are not planned [203].

PD-0325901/Mirdametinib
This selective, potent, oral, noncompetitive MEK inhibitor was developed by Pfizer. It inhibited ERK phosphorylation in in vitro model. It inhibited growth of melanoma cell lines in vitro and in xenograft models. This molecule also inhibited angiogenesis by inhibiting VEGF production and induced apoptosis in in vitro models [204]. NCT00147550, a phase I/II clinical trial evaluating the efficacy of PD-0325901 in advanced melanoma has been terminated due to ocular, neurological and musculoskeletal toxicities at higher doses (> 15 mg twice a day) [205].

ERK Inhibitors
ERK plays a unique role in the MAPK/ERK pathway; it has more than 100 substrates, some of which are involved in MAPK/ERK activating/de-activating feedback loops, yet it has only one upstream effector, MEK1/2 [206]. Due to this role, ERK inhibitors may show promise as a method of overcoming the development of resistance and re-activation of BRAF and MEK in BRAF V600E melanoma. While presently far behind BRAF and MEK inhibitors in development, there has been a recent increase in the development and evaluation of ERK inhibitors for treating BRAF V600E melanoma.

Ulixertinib/BVD-523
Ulixertinib is a novel, selective ERK1/2 inhibitor developed by BioMed Valley Discoveries, that inhibits ERK1/2 in a reversible and competitive manner. Importantly, ulixertinib presented equivalent efficacy in BRAF mutant cells and BRAF + MEK double mutant cells, while the efficacy of BRAF and MEK inhibitors decreased in the double mutant line [207]. Currently, it has been designated for fast track status by the FDA in the treatment of metastatic BRAFV600E-mutant melanoma [208].

LY3214996
A selective ERK1/2 inhibitor developed by Eli Lilly currently in phase I clinical trials [209,210]. Further details of this pre-clinical characterization have not been made publicly available, and Phase I trials are on-going.

MK8353
An orally dosed, selective inhibitor of activated ERK1/2, and non-activated ERK2 developed by Merck & Co. currently recruiting for phase I trials [211,212]. A phase I clinical trial was initiated following these results in healthy volunteers (NCT01358331); however, the study was terminated after phase Ia MTD determination (400 mg orally once daily) for strategic reasons [213].

LTT462
An oral ERK inhibitor developed by Novartis. LTT-462 has completed a phase I clinical trial for use in advanced cancers, including melanoma. A phase I clinical trial (NCT02711345) evaluating the use of LTT462 in advanced melanoma and other advanced cancer has concluded; however, the results are not yet publicly available [214].

GDC0994
An orally-dosed, selective ERK1/2 inhibitor developed by Genentech [216]. A phase Ia trial (NCT01875705) was conducted on MAPK-dysregulated cancers not including melanoma. The phase Ia trial found a safety profile consistent with MAPK inhibition with tolerable adverse events [217]. A following phase Ib trial (NCT02457793) investigating the use of GDC0994 in combination with cobimetinib (MEKi) in advanced cancers including advanced melanoma has completed; however, the comprehensive results have not yet been released [218].

SCH772984
This is a selective, ATP-competitive ERK inhibitor developed by Merck. It exhibits antitumor activity against BRAFV600E mutant and NRAS mutant melanoma. It blocks proliferation of melanoma cell lines in BRAF and MEK inhibitor resistant cell lines in vitro [219]. The synergistic combination of SCH772984 with Vemurafenib delayed the onset of acquired resistance in in vitro models [220]. Intermitent dosing with RAF inhibitor, MEK inhibitor and ERK inhibitor (SCH772984) inhibited tumor growth in low-level BRAF amplification patient derived xenograft model of melanoma [221]. Table 2 summarizes the clinical trials, outcomes and adverse effects of novel BRAF and ERK inhibitors that are under investigation in the clinic to treat metastatic melanoma patients [211,[222][223][224]. 25 BRAF-naïve and 29 BRAF inhibitor pretreated patients were enrolled in the study. The treatment was tolerable up to the MTD of 450 mg once daily, however the RP2D was declared as 300 mg once daily due to the increased risk of adverse events at 450 mg. BRAFi-naïve patients treated with 300-450 mg once daily saw an RR of 60% and PFS of 12.4 months (95% CI: 7.4-Not Reached), while for BRAFi-pretreated patients the RR was 22% and the PFS was 1.9 months (95% CI: 0.9-3.7 months).
Nausea, myalgia, PPED LGX818 in combination with MEK162 in patients with advanced solid tumors [223] Active, not recruiting

Array BioPharma
Ib/II, NCT01543698 The combination was safe with no substantial adverse evets observed. Nine BRAF naïve and 14 BRAF inhibitor pretreated patients were enrolled. The MTD was unable to be determined and the RP2D was 450-600 mg LGX818 + 45 mg MEK162 orally once daily. CR and PR for BRAF-naïve patients were 11% and 78% respectively. The PR for BRAF inhibitor pretreated patient groups was 21%.

Encorafenib in combination with
Binimetinib or Vemurafenib in patients with BRAF-mutant melanoma (COLUMBUS) [224] Active, not recruiting Array BioPharma

ROS Activated Prodrugs
Redox homeostasis is essential for cell transcription, proliferation and survival. Failure of regulating redox homeostasis can cause DNA damage and cell apoptosis [225]. Cancer cells are known to express increased reactive oxygen species (ROS) such as superoxide, H 2 O 2 and the hydroxyl radicals [226][227][228][229]. Evidence has shown a significant increase in ROS levels after B-RAF inhibition in melanoma cells [230]. ROS-activated prodrugs can be potentially utilized in combination with B-RAF inhibitors to target metastatic melanoma cells.

Protein Ribonuclease A (RNase A)/SN-38
These ROS activated drugs usually contain two separate functional domains-a ROS-accepting moiety, "Trigger", and an "Effector". In the presence of H 2 O 2 , the B-C bond within the aryl Byronic acid or esters will become oxidized, releasing the phenol group and activating the pro-drug. Protein ribonuclease A (RNase A) and SN-38 are being studied in the B16F10 murine melanoma cell line which mimics primary tumor growth [231,232]. SN-38 significantly decreases the proliferation of B16F10 murine melanoma cell line [231]. The enzymatic activity of RNase A will be reduced and cytotoxicity is improved when being activated via high ROS levels against skin melanoma cancer cells (B16F10) [232]. The success of these prodrugs in reducing tumor proliferation in murine melanoma cells gives the potential to study these drugs in human melanoma cell lines and potentially into clinical trials.

A100/RAC1
A100 is a quinone derivative. Quinones have substituents on the activated alkene, which are also called Michael acceptors. Cell damage and cytotoxicity occurs through the alkylation of DNA or cellular proteins. A100 sensitizes dabrafenib-resistant melanoma cells to BRAF protein kinase inhibitors [233]. A100, in the presence of high ROS levels, can self-cyclize into a bicyclic ring and cause DNA double strand breaks in cancer cells [234]. This compound and related ROS activated pro-drugs could be useful therapeutic agents where a BRAF inhibition has failed as the first line of treatment in melanoma patients harboring BRAF V600E mutation.

Conclusions
Resistance to therapies continues to push the need to expand our understanding of melanoma treatment. This has led to exploring new treatments that utilizes combination therapies in order to achieve maximum anti-tumor efficacy over long durations of treatment avoiding resistance. Advances in the treatment of metastatic melanoma are on the rise with progress in targeted molecular therapy and immunotherapy. Targeted therapies are now expanding to include new BRAF and MEK inhibitors together and in combination with other therapies. Progress is being made in the field for targeting Axl and with ROS activated prodrugs. Immunotherapies are a new area of interest focusing on manipulation of checkpoint inhibition with durable clinical responses in patients. Melanoma has a strong molecular and genetic basis of pathogenicity, which allows for the development of personalized medicine. Selecting unique and individual treatments for melanoma patient makes it more likely to achieve high success rates in the clinic. Continual research and clinical trials are ongoing to further elucidate and expand knowledge on mechanisms of resistance and novel treatment strategies such as immunotherapies, new small molecule inhibitors and ROS-activated prodrugs to provide effective care to patients with metastatic melanoma.

Conflicts of Interest:
The authors declare no conflict of interest.