azd-6244 and Melanoma

The identification of driver mutations in melanoma has changed the field of cancer treatment. BRAF and NRAS mutations are predominant in melanoma and lead to overactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. Selective inhibitors targeting key effectors of the MAPK pathway have revolutionized the treatment of patients with advanced metastatic BRAF-mutant melanoma. However, resistance to therapy is almost universal and remains a major challenge in clinical care, with the majority of patients progressing within 1 year. Dissecting the mechanisms of resistance to targeted therapies may offer new insights into strategies for overcoming resistance. This review describes the efficacy of therapies targeting the MAPK and PI3K/AKT signaling pathways in melanoma, details the mechanisms contributing to drug resistance, and discusses current approaches to improving outcomes further. Cancer 2017;123:2118-29. © 2017 American Cancer Society.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; CTLA-4 Antigen; Drug Administration Schedule; Drug Resistance, Neoplasm; Humans; Immunotherapy; Indoles; Ipilimumab; MAP Kinase Kinase 1; Melanoma; Molecular Targeted Therapy; Niacinamide; Nivolumab; Phenylurea Compounds; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Programmed Cell Death 1 Receptor; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Pyrimidines; Quinazolines; Signal Transduction; Skin Neoplasms; Sorafenib; Sulfonamides; Vemurafenib

New treatments for metastatic melanoma work through distinct mechanisms: enhancing the immune response and blocking cellular proliferation. Agents that enhance the immune response include ipilimumab, pembrolizumb, and nivolumab; agents that block cellular proliferation include vemurafenib, dabrafenib, trametinib, cobimetinib, binimetinib, and selumetinib. The translational impact of laboratory discoveries has revolutionized management of metastatic melanoma and enhanced the prognosis of affected patients.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Azetidines; Benzimidazoles; Humans; Imidazoles; Immunologic Factors; Indoles; Ipilimumab; Melanoma; Molecular Targeted Therapy; Nivolumab; Oximes; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Skin Neoplasms; Sulfonamides; Vemurafenib

Uveal melanoma is a rare but aggressive subtype of melanoma. Nearly 50% of patients will develop metastatic disease despite primary enucleation or radiation therapy. There is currently no standard of care therapy for metastatic uveal melanoma, and no therapy that has been shown to prolong overall survival. Uveal melanoma is characterized by activation of signaling pathways including the MAPK pathway and the PI3K/AKT pathway, among others, via mutations in the G-α-proteins GNAQ and GNA11. MEK inhibition with selumetinib has been evaluated as a therapeutic strategy in metastatic uveal melanoma. This review will discuss preclinical and clinical studies evaluating selumetinib in metastatic uveal melanoma, as well as potential future perspectives on MEK inhibition in the management of metastatic uveal melanoma.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Humans; Melanoma; Uveal Neoplasms

Melanoma is the fifth most common cancer in men and seventh most common in women in the USA, and prognosis for patients with advanced melanoma is poor. The mitogen-activated protein (MAP) kinase signaling pathway is essential for proliferation and survival of melanoma cells. Effective inhibition of MAP kinase kinase (MEK) protein has been shown to downregulate the MAP kinase pathway, resulting in melanoma cell growth arrest and apoptosis. Selumetinib is an orally available, selective non-ATP-competitive MEK1 and MEK2 inhibitor.. In this review, the authors discuss the rationale for MEK inhibition therapy in melanoma and summarize data from the preclinical and clinical studies of selumetinib for advanced melanoma.. As a majority of advanced melanomas have an activated MAP kinase signal transduction pathway, there is a strong preclinical rationale for investigating selumetinib in patients with metastatic melanoma. The results of early clinical studies of selumetinib suggest that selumetinib may have a role in melanoma therapy, especially in certain subsets of patients, such as those whose tumor harbors a BRAF mutation. Current studies of selumetinib are addressing the efficacy of selumetinib in these patients.

Topics: Animals; Benzimidazoles; Clinical Trials as Topic; Drug Evaluation, Preclinical; Humans; Melanoma; Mitogen-Activated Protein Kinase Kinases; Neoplasm Metastasis; Randomized Controlled Trials as Topic; Signal Transduction

Aiming to improve treatment options for BRAF wild-type melanoma, we previously conducted the DOC-MEK study of docetaxel with MEK inhibitor (MEKi) selumetinib or placebo, revealing trends to prolongation of progression-free survival (hazard ratio 0.75, P = 0.130), and improved response rates (32% vs 14%, P = 0.059) with docetaxel plus selumetinib. NRAS status did not associate with outcome. Here, the aim was to identify novel biomarkers of response to MEKi.. A MEK 6 gene signature was quantified using NanoString and correlated with clinical outcomes. Two components of the gene signature were investigated by gene silencing in BRAF/NRAS wild-type melanoma cells.. In melanomas of patients on the selumetinib but not the placebo arm, two gene signature components, dual-specificity protein phosphatase 4 (DUSP4) and ETS translocation variant 4 (ETV4), were expressed more highly in responders than non-responders. In vitro, ETV4 depletion inhibited cell survival but did not influence sensitivity to MEKi selumetinib or trametinib. In contrast, DUSP4-depleted cells showed enhanced cell survival and increased resistance to both selumetinib and trametinib.. ETV4 and DUSP4 associated with clinical response to docetaxel plus selumetinib. DUSP4 depletion induced MEKi resistance, suggesting that DUSP4 is not only a biomarker but also a mediator of MEKi sensitivity.. DOC-MEK (EudraCT no: 2009-018153-23).

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Docetaxel; Drug Resistance, Neoplasm; Dual-Specificity Phosphatases; Humans; MAP Kinase Kinase Kinases; Melanoma; Mitogen-Activated Protein Kinase Phosphatases; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-ets; Transcriptome

Purpose Uveal melanoma is the most common primary intraocular malignancy in adults with no effective systemic treatment option in the metastatic setting. Selumetinib (AZD6244, ARRY-142886) is an oral, potent, and selective MEK1/2 inhibitor with a short half-life, which demonstrated single-agent activity in patients with metastatic uveal melanoma in a randomized phase II trial. Methods The Selumetinib (AZD6244: ARRY-142886) (Hyd-Sulfate) in Metastatic Uveal Melanoma (SUMIT) study was a phase III, double-blind trial ( ClinicalTrial.gov identifier: NCT01974752) in which patients with metastatic uveal melanoma and no prior systemic therapy were randomly assigned (3:1) to selumetinib (75 mg twice daily) plus dacarbazine (1,000 mg/m

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Dacarbazine; Double-Blind Method; Female; Humans; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Placebos; Progression-Free Survival; Uveal Neoplasms

Clinical trials commonly use physician-adjudicated adverse event (AE) assessment via the common terminology criteria for adverse events (CTCAE) for decision-making. Patient-reported health-related quality of life (HRQoL) data are becoming more frequent in oncology; however, the relationship between physician-adjudicated AE assessment and HRQoL is understudied.. Data from a phase II trial (clinicaltrials.gov identifier: NCT01143402) where patients with metastatic uveal melanoma were randomized to receive selumetinib, an oral MEK inhibitor, or chemotherapy were analyzed. Patients reported HRQoL at baseline, after 1 month, and end of treatment (n = 118), whereas physicians adjudicated AEs via CTCAE. Mean HRQoL scores were compared between patient randomization arms, as well as between those patients who did/did not receive dose modifications.. Ninety-four percent had a CTCAE grade ≥1 for at least one treatment-associated AE, with 18% undergoing dose modification due to toxicity. Mean HRQoL scores did not significantly differ at each of the three time points. Patient and physician-adjudicated reports of nausea were significantly correlated at the start (r = 0.31, p < 0.01) and end of treatment (r = 0.42, p < 0.05). There were no significant correlations between need for dose modification and HRQoL scores.. Despite the high rate of physician-adjudicated AEs and need for dose modifications with selumetinib, patient-reported HRQoL was not impacted by treatment. Since HRQoL did not differ in the subgroup of patients who received dosage reductions due to AEs, patients may be willing to tolerate select AEs without dose modification (if medically appropriate). More research is needed to determine how to best integrate HRQoL data into clinical trial conduct.

Topics: Adult; Aged; Benzimidazoles; Drug-Related Side Effects and Adverse Reactions; Female; Humans; Male; Melanoma; Middle Aged; Patient Reported Outcome Measures; Physicians; Quality of Life; Uveal Neoplasms

Uveal melanoma is characterised by mutations in GNAQ and GNA11, resulting in Ras/Raf/MEK/ERK pathway activation. Treatment with selumetinib (AZD6244, ARRY-142886), a MEK1/2 inhibitor, results in antitumour effects in uveal melanoma pre-clinical models. A randomised phase II trial demonstrated improved progression-free survival (PFS) and response rate (RR) with selumetinib monotherapy versus chemotherapy with temozolomide or dacarbazine in patients with metastatic uveal melanoma. Pre-clinically, selumetinib in combination with alkylating agents enhanced antitumour activity compared with chemotherapy alone. We hypothesise that selumetinib in combination with dacarbazine will result in improved clinical outcomes in patients with metastatic uveal melanoma versus dacarbazine alone.. SUMIT is a randomised, international, double-blind, placebo-controlled, phase III study assessing the efficacy and safety of selumetinib in combination with dacarbazine in patients with metastatic uveal melanoma who have not received prior systemic therapy. Primary endpoint is PFS. Secondary endpoints include objective RR, duration of response, change in tumour size at Week 6, overall survival, safety and tolerability. Exploratory endpoints include efficacy in tumours with GNAQ or GNA11 mutations. Eligible patients must have: ≥1 lesion that can be accurately measured at baseline, and is suitable for accurate repeated measurements; ECOG performance status 0-1; life expectancy>12 weeks. Mutation status for GNAQ/GNA11 will be assessed retrospectively. An estimated 128 patients from approximately 50 sites globally will be randomised (3:1) to selumetinib 75 mg twice daily or placebo in combination with dacarbazine 1000 mg/m(2) on Day 1 of every 21-day cycle until objective disease progression, intolerable toxicity or occurrence of another discontinuation criterion. Randomisation will be stratified by the presence/absence of liver metastases. Tumours will be evaluated by RECIST v1.1 every 6 weeks. All patients have the option of receiving selumetinib with or without dacarbazine at disease progression. Study enrolment began in April 2014 and is expected to complete in early 2015.. Treatment of patients with metastatic uveal melanoma represents an area of high unmet medical need. This study evaluating selumetinib in combination with dacarbazine was designed with input from the US FDA, and is the first potential registration trial to be conducted in patients with metastatic uveal melanoma.. Clinicaltrials.gov (Date of registration, October 10, 2013) REGISTRATION NUMBER: NCT01974752 Trial abbreviation: SUMIT.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Dacarbazine; Disease-Free Survival; Female; Humans; Male; Melanoma; Middle Aged; Neoplasm Metastasis; Uveal Neoplasms

Treatment options for wild-type BRAF melanoma patients remain limited. Selumetinib, a MEK 1/2 inhibitor, suppresses pERK levels independent of BRAF and NRAS mutation status, and combination with docetaxel has demonstrated synergy in xenograft models. The aim of this study was to assess the efficacy and safety of selumetinib plus docetaxel as first-line treatment in patients with wild-type BRAF advanced melanoma.. In this double-blind multicentre phase II trial patients with wild-type BRAF melanoma were randomized (1:1) to docetaxel with selumetinib or placebo. Docetaxel 75 mg/m(2) was administered intravenously every 3 weeks up to six cycles. Selumetinib 75 mg or placebo was given orally twice daily until disease progression or unacceptable toxicity. The primary end point was progression-free survival (PFS). Tumour NRAS mutation status was analysed retrospectively and correlated with treatment outcomes.. Eighty-three patients were randomized to docetaxel plus selumetinib (n = 41) or docetaxel plus placebo (n = 42). The PFS hazard ratio (HR) (selumetinib:placebo) was 0.75 [90% confidence interval (CI) 0.50-1.14; P = 0.130], with a median PFS of 4.23 months (90% CI 3.63-6.90) for docetaxel plus selumetinib and 3.93 months (90% CI 2.07-4.16) for docetaxel alone. There was no significant difference in overall survival. The objective response rate was 32% with selumetinib versus 14% with placebo (P = 0.059). In a retrospective subset analysis, NRAS mutation status did not affect significantly upon clinical outcomes in either arm. The combination of docetaxel and selumetinib could be administered effectively to patients with metastatic melanoma, although the combination was less well tolerated than docetaxel alone.. The combination of docetaxel with selumetinib showed no significant improvement in PFS compared with docetaxel alone, although more patients showed a response to combination therapy. We found no evidence to support using tumour NRAS mutation as a basis for selecting patients for combined MEK inhibitor and chemotherapy.. DOC-MEK (EudraCT no: 2009-018153-23).

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Disease-Free Survival; DNA Mutational Analysis; Docetaxel; Double-Blind Method; GTP Phosphohydrolases; Humans; Kaplan-Meier Estimate; Male; Melanoma; Membrane Proteins; Middle Aged; Proportional Hazards Models; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Taxoids; Treatment Outcome

Uveal melanoma is characterized by mutations in GNAQ and GNA11, resulting in mitogen-activated protein kinase pathway activation.. To assess the efficacy of selumetinib, a selective, non-adenosine triphosphate competitive inhibitor of MEK1 and MEK2, in uveal melanoma.. Randomized, open-label, phase 2 clinical trial comparing selumetinib vs chemotherapy conducted from August 2010 through December 2013 among 120 patients with metastatic uveal melanoma at 15 academic oncology centers in the United States and Canada.. One hundred one patients were randomized in a 1:1 ratio to receive selumetinib, 75 mg orally twice daily on a continual basis (n = 50), or chemotherapy (temozolomide, 150 mg/m2 orally daily for 5 of every 28 days, or dacarbazine, 1000 mg/m2 intravenously every 21 days [investigator choice]; n = 51) until disease progression, death, intolerable adverse effects, or withdrawal of consent. After primary outcome analysis, 19 patients were registered and 18 treated with selumetinib without randomization to complete the planned 120-patient enrollment. Patients in the chemotherapy group could receive selumetinib at the time of radiographic progression.. Progression-free survival, the primary end point, was assessed as of April 22, 2013. Additional end points, including overall survival, response rate, and safety/toxicity, were assessed as of December 31, 2013.. Median progression-free survival among patients randomized to chemotherapy was 7 weeks (95% CI, 4.3-8.4 weeks; median treatment duration, 8 weeks; interquartile range [IQR], 4.3-16 weeks) and among those randomized to selumetinib was 15.9 weeks (95% CI, 8.4-21.1 weeks; median treatment duration, 16.1 weeks; IQR, 8.1-25.3 weeks) (hazard ratio, 0.46; 95% CI, 0.30-0.71; P < .001). Median overall survival time was 9.1 months (95% CI, 6.1-11.1 months) with chemotherapy and 11.8 months (95% CI, 9.8-15.7 months) with selumetinib (hazard ratio, 0.66; 95% CI, 0.41-1.06; P = .09). No objective responses were observed with chemotherapy. Forty-nine percent of patients treated with selumetinib achieved tumor regression, with 14% achieving an objective radiographic response to therapy. Treatment-related adverse events were observed in 97% of patients treated with selumetinib, with 37% requiring at least 1 dose reduction.. In this hypothesis-generating study of patients with advanced uveal melanoma, selumetinib compared with chemotherapy resulted in a modestly improved progression-free survival and response rate; however, no improvement in overall survival was observed. Improvement in clinical outcomes was accompanied by a high rate of adverse events.. clinicaltrials.gov Identifier: NCT01143402.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Alkylating; Benzimidazoles; Dacarbazine; Disease Progression; Female; Humans; Male; Melanoma; Middle Aged; Survival Analysis; Temozolomide; Treatment Outcome; Uveal Neoplasms

Test the hypothesis that in BRAF-mutated melanomas, clinical responses to selumetinib, a MEK inhibitor, will be restricted to tumors in which the PI3K/AKT pathway is not activated.. We conducted a phase II trial in patients with melanoma whose tumors harbored a BRAF mutation. Patients were stratified by phosphorylated-AKT (pAKT) expression (high vs. low) and treated with selumetinib 75 mg per os twice daily. Pretreatment tumors were also analyzed for genetic changes in 230 genes of interest using an exon-capture approach.. The high pAKT cohort was closed after no responses were seen in the first 10 patients. The incidence of low pAKT melanoma tumors was low (∼25% of melanomas tested) and this cohort was eventually closed because of poor accrual. However, among the five patients with melanoma accrued in the low pAKT cohort, there was one partial response (PR). Two other patients had near PRs before undergoing surgical resection of residual disease (one patient) or discontinuation of treatment due to toxicity (one patient). Among the two nonresponding, low pAKT patients with melanoma, co-mutations in MAP2K1, NF1, and/or EGFR were detected.. Tumor regression was seen in three of five patients with BRAF-mutated, low pAKT melanomas; no responses were seen in the high pAKT cohort. These results provide rationale for co-targeting MEK and PI3K/AKT in patients with BRAF mutant melanoma whose tumors express high pAKT. However, the complexity of genetic changes in melanoma indicates that additional genetic information will be needed for optimal selection of patients likely to respond to MEK inhibitors.

Topics: Administration, Oral; Adult; Aged; Benzimidazoles; Cohort Studies; Disease-Free Survival; Drug Administration Schedule; Exanthema; Female; Humans; Lymphopenia; Male; Melanoma; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Mutation; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Signal Transduction; Treatment Outcome; Young Adult

Patients with metastatic melanoma, 50% of whose tumours harbour a BRAF mutation, have a poor prognosis. Selumetinib, a MEK1/2 inhibitor, has shown antitumour activity in patients with BRAF-mutant melanoma and in preclinical models when combined with chemotherapy. This study was designed to look for a signal of improved efficacy by comparing the combination of selumetinib and dacarbazine with dacarbazine alone.. This double-blind, randomised, placebo-controlled phase 2 study investigated selumetinib plus dacarbazine versus placebo plus dacarbazine as first-line treatment in patients older than 18 years with histologically or cytologically confirmed advanced BRAF-mutant cutaneous or unknown primary melanoma. Patients were randomly assigned by central interactive voice response system (1:1 ratio, block size four) to take either oral selumetinib (75 mg twice daily in a 21-day cycle) or placebo; all patients received intravenous dacarbazine (1000 mg/m(2) on day 1 of a 21-day cycle). Patients, investigators, and the study team were masked to the treatment assigned. The primary endpoint was overall survival analysed by intention to treat. This study is registered at ClinicalTrials.gov, NCT00936221.. Between July 20, 2009, and April 8, 2010, 91 patients were randomly assigned to receive dacarbazine in combination with selumetinib (n=45) or placebo (n=46). Overall survival did not differ significantly between groups (median 13·9 months, 80% CI 10·2-15·6, in the selumetinib plus dacarbazine group and 10·5 months, 9·6-14·7, in the placebo plus dacarbazine group; hazard ratio [HR] 0·93, 80% CI 0·67-1·28, one-sided p=0·39). However, progression-free survival was significantly improved in the selumetinib plus dacarbazine group versus the placebo plus dacarbazine group (HR 0·63, 80% CI 0·47-0·84, one-sided p=0·021), with a median of 5·6 months (80% CI 4·9-5·9) versus 3·0 months (2·8-4·6), respectively. The most frequent adverse events included nausea (28 [64%] of 44 patients on selumetinib vs 25 [56%] of 45 on placebo), acneiform dermatitis (23 [52%] vs one [2%]), diarrhoea (21 [48%] vs 13 [29%]), vomiting (21 [48%] vs 15 [33%]), and peripheral oedema (19 [43%] vs three [7%]). The most common grade 3-4 adverse event was neutropenia (six [14%] patients in the selumetinib plus dacarbazine group vs four [9%] in the placebo plus dacarbazine group).. Selumetinib plus dacarbazine showed clinical activity in patients with BRAF-mutant cutaneous or unknown primary melanoma, reflected by a significant benefit in progression-free survival compared with placebo plus dacarbazine group, although no significant change in overall survival was noted. The tolerability of this combination was generally consistent with monotherapy safety profiles.. AstraZeneca.

Topics: Adult; Aged; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Brazil; Dacarbazine; Disease-Free Survival; DNA Mutational Analysis; Double-Blind Method; Drug Administration Schedule; Europe; Female; Genetic Predisposition to Disease; Humans; Kaplan-Meier Estimate; Logistic Models; Male; Melanoma; Middle Aged; Mutation; Neoplasms, Unknown Primary; Phenotype; Proportional Hazards Models; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Time Factors; Treatment Outcome; United States

The high prevalence of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) and neuroblastoma v-ras oncogene homolog (NRAS) mutations in melanoma provides a strong rationale to test the clinical efficacy of mitogen-activated protein kinase kinase (MEK) inhibition in this disease. The authors hypothesized that the presence of BRAF or NRAS mutations would correlate with clinical benefit among patients who received treatment with combination regimens that included the MEK inhibitor selumetinib.. BRAF and NRAS mutation status was determined retrospectively in available tissue specimens from patients with melanoma who were enrolled in a phase 1 trial of selumetinib in combination with 1 of 4 drugs (dacarbazine, docetaxel, temsirolimus, or erlotinib). The clinical response rate and the time to progression (TTP) were assessed as a function of BRAF and NRAS mutation status.. Among 18 patients analyzed, 9 patients (50%) harbored a BRAF mutation (8 had a valine-to-glutamic acid substitution at residue 600 [V600E]; 1 had an arginine nonsense mutation at residue 603 [R603]), 4 patients (22%) harbored an NRAS mutation (2 had a glutamine-to-arginine substitution at residue 61 [Q61R], 1 had a glutamine-to-lysine substitution at residue 61 [Q61K], and 1 had a glycine-to-lysine substitution at residue 12 [G12S]), and 5 patient (28%) had the wild type of both genes. These mutations were mutually exclusive. Among the 9 patients who had BRAF mutations, 5 patients (56%) achieved a partial response, and 4 patients (44%) achieved stable disease for at least 6 weeks. No patient with the wild-type BRAF gene achieved a clinical response (P = .01 vs patients with BRAF mutations). The presence of an NRAS mutation did not correlate with the clinical response rate. The presence of a BRAF mutation was correlated significantly with the TTP in a multivariate model (hazard ratio, 0.22; P = .02 vs wild-type BRAF).. Higher response rates and longer TTP were observed with selumetinib-containing regimens in patients who had tumors that harbored a BRAF mutation compared with patients who had wild-type BRAF.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Enzyme Inhibitors; Female; Humans; Male; MAP Kinase Kinase Kinases; Melanoma; Middle Aged; Multivariate Analysis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Skin Neoplasms; Treatment Outcome

To compare the efficacy and tolerability of the mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) 1/2 inhibitor selumetinib versus temozolomide in chemotherapy-naive patients with unresectable stage III/IV melanoma.. This phase II, open-label, multicenter, randomized, parallel-group study examined the effect of 100 mg oral selumetinib twice daily in 28-day cycles versus oral temozolomide (200 mg/m(2)/d for 5 days, then 23 days off-treatment). The primary endpoint was progression-free survival.. Two hundred patients were randomized. Progression-free survival did not differ significantly between selumetinib and temozolomide (median time to event 78 and 80 days, respectively; hazard ratio, 1.07; 80% confidence interval, 0.86-1.32). Objective response was observed in six (5.8%) patients receiving selumetinib and nine (9.4%) patients in the temozolomide group. Among patients with BRAF mutations, objective responses were similar between selumetinib and temozolomide groups (11.1% and 10.7%, respectively). However, five of the six selumetinib partial responders were BRAF mutated. Frequently reported adverse events with selumetinib were dermatitis acneiform (papular pustular rash; 59.6%), diarrhea (56.6%), nausea (50.5%), and peripheral edema (40.4%), whereas nausea (64.2%), constipation (47.4%), and vomiting (44.2%) were reported with temozolomide.. No significant difference in progression-free survival was observed between patients with unresectable stage III/IV melanoma unselected for BRAF/NRAS mutations, who received therapy with selumetinib or temozolomide. Five of six patients with partial response to selumetinib had BRAF mutant tumors.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Alkylating; Benzimidazoles; Dacarbazine; Disease-Free Survival; DNA Mutational Analysis; Drug Substitution; Female; Humans; Kaplan-Meier Estimate; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Middle Aged; Mutation; Neoplasm Staging; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Temozolomide; Treatment Outcome; Young Adult

Topics: Aged; Benzimidazoles; Female; Humans; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Neuromuscular Diseases; Protein Kinase Inhibitors; Syndrome; Uveal Neoplasms

Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway is constitutively activated in melanoma. AZD6244 blocks MEK1/2, inhibiting ERK phosphorylation. We focus on associated cutaneous toxicity and we attempt to understand the underlying pathophysiology and design treatment strategies.. Dermatologic conditions of 22 patients with unresectable melanoma stage III/IV in a phase II trial were evaluated. Thirteen patients received AZD6244 initially, and nine patients were treated with AZD6244 following tumor progression with temozolomide. Biopsies were compared with matched controls in normal skin. Immunohistochemistry was performed. Half-side treatment of acute skin toxicity compared therapeutic options.. Nineteen of 22 (86%) AZD6244-treated patients presented with cutaneous eruptions. Seventeen patients (77%) developed acute papulopustular rash. Chronic skin changes included xerosis, paronychia, and fissured fingertips, resembling cutaneous toxicity of epidermal growth factor receptor inhibition. In addition, we observed reduced pigmentation of hair and skin. Histology of acute skin lesions revealed a significant increase of apoptotic keratinocytes (P = 0.0008), focal neutrophilic infiltrates, destruction of the adnexal structures by neutrophils, and reduced cytokeratins. A significant proliferation shift from basal to suprabasal keratinocytes was shown in acute and chronic lesions. The number and viability of melanocytes was not affected. Corticosteroids plus antibacterial topical therapy ameliorate acute skin toxicity.. AZD6244-associated skin reactions partly overlap with those observed upon epidermal growth factor receptor inhibition. Additionally, pigmentation of skin and hair is affected. The interruption of the MEK signaling pathway results in an acute keratinocyte stress response with disturbed epidermal homeostasis, inflammation, and tissue damage. Chronic adaptation controls inflammatory tissue damage but leads to cutaneous malfunctions that explain chronic skin toxicity.

Topics: Adult; Apoptosis; Benzimidazoles; Drug Eruptions; Exanthema; Extracellular Signal-Regulated MAP Kinases; Female; Homeostasis; Humans; Keratinocytes; Male; Melanoma; Middle Aged; Mitogen-Activated Protein Kinases; Pigmentation Disorders; Protein Kinase Inhibitors; Skin Neoplasms

This study investigated the potential clinical utility of circulating free DNA (cfDNA) as a source of BRAF mutation detection in patients enrolled into a phase II study of AZD6244, a specific MEK1/2 inhibitor, in patients with advanced melanoma.. BRAF mutations were detected using Amplification Refractory Mutation System allele-specific PCR. BRAF mutation status was assessed in serum-derived cfDNA from 126 patients enrolled into the study and from 94 matched tumour samples.. Of 94 tumour samples, 45 (47.9%) were found to be BRAF mutation positive (BRAF+). Serum-derived cfDNA was BRAF+ in 33 of 126 (26.2%) samples, including in five samples for which tumour data were unavailable. Of BRAF+ tumours, 25 of 45 (55.6%) were BRAF+ in cfDNA. In three cases in which the tumour was negative, cfDNA was BRAF+. Progression-free survival (PFS) of patients with BRAF+ tumour and cfDNA was not significantly different compared with tumour BRAF+ but cfDNA BRAF-negative patients, indicating that cfDNA BRAF detection is not associated with poorer prognosis on PFS in stage III/IV advanced melanoma.. These data demonstrate the feasibility of BRAF mutation detection in cfDNA of patients with advanced melanoma. Future studies should aim to incorporate BRAF mutation testing in cfDNA to further validate this biomarker for patient selection.

Topics: Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Disease-Free Survival; DNA Mutational Analysis; DNA, Neoplasm; HT29 Cells; Humans; Melanoma; Mutation; Prognosis; Proto-Oncogene Proteins B-raf; Skin Neoplasms

To assess the tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancer.. In part A, patients received escalating doses to determine the maximum-tolerated dose (MTD). In both parts, blood samples were collected to assess PK and PD parameters. In part B, patients were stratified by cancer type (melanoma v other) and randomly assigned to receive the MTD or 50% MTD. Biopsies were collected to determine inhibition of ERK phosphorylation, Ki-67 expression, and BRAF, KRAS, and NRAS mutations.. Fifty-seven patients were enrolled. MTD in part A was 200 mg bid, but this dose was discontinued in part B because of toxicity. The 50% MTD (100 mg bid) was well tolerated. Rash was the most frequent and dose-limiting toxicity. Most other adverse events were grade 1 or 2. The PKs were less than dose proportional, with a median half-life of approximately 8 hours and inhibition of ERK phosphorylation in peripheral-blood mononuclear cells at all dose levels. Paired tumor biopsies demonstrated reduced ERK phosphorylation (geometric mean, 79%). Five of 20 patients demonstrated >or= 50% inhibition of Ki-67 expression, and RAF or RAS mutations were detected in 10 of 26 assessable tumor samples. Nine patients had stable disease (SD) for >or= 5 months, including two patients with SD for 19 (thyroid cancer) and 22 (uveal melanoma plus renal cancer) 28-day cycles.. AZD6244 was well tolerated with target inhibition demonstrated at the recommended phase II dose. PK analyses supported twice-daily dosing. Prolonged SD was seen in a variety of advanced cancers. Phase II studies are ongoing.

Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Benzimidazoles; Cell Proliferation; DNA Mutational Analysis; Dose-Response Relationship, Drug; Drug Administration Schedule; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Ki-67 Antigen; Leukocytes, Mononuclear; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Maximum Tolerated Dose; Melanoma; Middle Aged; Mutation; Neoplasm Staging; Neoplasms; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); ras Proteins; Treatment Outcome

Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted MEK inhibition causes qualitative alterations of carcinogenic MAPK signals. Phosphorylation of the MEK1 activation loop at the positions S218 and S222 by RAF kinases triggers the conformational alignment of MEK's catalytic pocket to enable ATP-binding and substrate phosphorylation. We have extended a kinase conformation (KinCon) biosensor platform to record MEK1 activity dynamics. In addition to MEK phosphorylation by BRAF, the integration of the phosphorylation-mimetic mutations S218D/S222D triggered opening of the kinase. Structural rearrangement may involve the flexibility of the N terminal MEK1 A-helix. Application of the allosterically acting MEK inhibitors (MEKi) trametinib, cobimentinib, refametinib, and selumetinib converted activated MEK1 KinCon reporters back into a more closed inactive conformation. We confirmed MEK1 KinCon activity dynamics upon drug engagement using the patient-derived melanoma cell line A2058, which harbors the V600E hotspot BRAF mutation. In order to confirm biosensor dynamics, we simulated structure dynamics of MEK1 kinase in the presence and absence of mutations and/or MEKi binding. We observed increased dynamics for the S218D/S222D double mutant particularly in the region of the distal A-helix and alpha-C helix. These data underline that MEK1 KinCon biosensors have the potential to be subjected to MEKi efficacy validations in an intact cell setting.

Topics: Benzimidazoles; Cell Line, Tumor; Computer Simulation; Drug Evaluation, Preclinical; HEK293 Cells; Humans; MAP Kinase Kinase 1; Melanoma; Molecular Dynamics Simulation; Mutation; Phosphorylation; Protein Conformation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Recombinant Proteins

Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Despite of important progress in the local therapy, high radioresistance in primary tumor and chemoresistance in metastatic disease are the major obstacles for UM therapy. Therefore, strategies to overcome resistance to radiation or chemotherapy in UM are urgently needed. In this study, we found that phosphorylation of DNA-PKcs, which is the key factor of non-homologous end joining (NHEJ) pathway, was remarkably overexpressed in ionizing radiation (IR)- and Selumetinib resistant UM cells. Increased amount of NHEJ events were also observed in resistant UM cells. Inhibition of DNA-PKcs by NU7441 significantly impaired DNA repair and re-sensitized resistant UM cells to radiation and Selumetinib both in vitro and in vivo. The results demonstrate increased DNA double strand break repair as a mechanism of resistance to ionizing radiation and Selumetinib, and identify DNA-PKcs as a promising target for radio-and chemotherapy in UM patients.

Topics: Animals; Benzimidazoles; Cell Line, Tumor; Chromones; DNA-Activated Protein Kinase; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Humans; Melanoma; Mice, Inbred BALB C; Morpholines; Phosphorylation; Radiation-Sensitizing Agents; Uveal Neoplasms

The RAS/RAF and PI3K/Akt pathways play a key regulatory role in cancer and are often hit by oncogenic mutations. Despite molecular targeting, the long-term success of monotherapy is often hampered by de novo or acquired resistance. In the case of concurrent mutations in both pathways, horizontal combination could be a reasonable approach. In our study, we investigated the MEK inhibitor selumetinib and PI3K/mTOR dual inhibitor BEZ235 alone and in combination in BRAF-only mutant and BRAF + PI3K/PTEN double mutant cancer cells using short- and long-term 2D viability assays, spheroid assays, and immunoblots. In the 2D assays, selumetinib was more effective on BRAF-only mutant lines when compared to BRAF + PI3K/PTEN double mutants. Furthermore, combination therapy had an additive effect in most of the lines while synergism was observed in two of the double mutants. Importantly, in the SW1417 BRAF + PI3K double mutant cells, synergism was also confirmed in the spheroid and in the in vivo model. Mechanistically, p-Akt level decreased only in the SW1417 cell line after combination treatment. In conclusion, the presence of concurrent mutations alone did not predict a stronger response to combination treatment. Therefore, additional investigations are warranted to identify predictive factors that can select patients who can benefit from the horizontal combinational inhibition of these two pathways.

Topics: Animals; Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Imidazoles; MAP Kinase Kinase Kinases; Melanoma; Mice; Mice, Nude; Mice, SCID; Mutation; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; PTEN Phosphohydrolase; Quinolines; Signal Transduction; Spheroids, Cellular; TOR Serine-Threonine Kinases

The MAPK/ERK signaling pathway is an essential regulator of numerous cell processes that are crucial for normal development as well as cancer progression. While much is known regarding MAPK/ERK signal conveyance from the cell membrane to the nucleus, the transcriptional and epigenetic mechanisms that govern gene expression downstream of MAPK signaling are not fully elucidated.. This study employed an integrated epigenome analysis approach to interrogate the effects of MAPK/ERK pathway inhibition on the global transcriptome, the active chromatin landscape, and protein-DNA interactions in 501mel melanoma cells. Treatment of these cells with the small-molecule MEK inhibitor AZD6244 induces hyperpigmentation, widespread gene expression changes including alteration of genes linked to pigmentation, and extensive epigenomic reprogramming of transcriptionally distinct regulatory regions associated with the active chromatin mark H3K27ac. Regulatory regions with differentially acetylated H3K27ac regions following AZD6244 treatment are enriched in transcription factor binding motifs of ETV/ETS and ATF family members as well as the lineage-determining factors MITF and SOX10. H3K27ac-dense enhancer clusters known as super-enhancers show similar transcription factor motif enrichment, and furthermore, these super-enhancers are associated with genes encoding MITF, SOX10, and ETV/ETS proteins. Along with genome-wide resetting of the active enhancer landscape, MEK inhibition also results in widespread SOX10 recruitment throughout the genome, including increased SOX10 binding density at H3K27ac-marked enhancers. Importantly, these MEK inhibitor-responsive enhancers marked by H3K27ac and occupied by SOX10 are located near melanocyte lineage-specific and pigmentation genes and overlap numerous human SNPs associated with pigmentation and melanoma phenotypes, highlighting the variants located within these regions for prioritization in future studies.. These results reveal the epigenetic reprogramming underlying the re-activation of melanocyte pigmentation and developmental transcriptional programs in 501mel cells in response to MEK inhibition and suggest extensive involvement of a MEK-SOX10 axis in the regulation of these processes. The dynamic chromatin changes identified here provide a rich genomic resource for further analyses of the molecular mechanisms governing the MAPK pathway in pigmentation- and melanocyte-associated diseases.

Topics: Benzimidazoles; Cell Line, Tumor; Chromatin; Chromatin Assembly and Disassembly; Gene Expression Regulation, Neoplastic; Histone Code; Histones; Humans; MAP Kinase Signaling System; Melanoma; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinase Kinases; Mutation; Pigmentation; Protein Binding; Proto-Oncogene Proteins B-raf; RNA Interference; RNA, Small Interfering; SOXE Transcription Factors

Topics: Acrylonitrile; Aged; Aniline Compounds; Barrett Esophagus; Benzimidazoles; Drug Therapy, Combination; Endoscopy, Digestive System; Female; Humans; Liver; Liver Neoplasms; Magnetic Resonance Imaging; Melanoma; Mutation; Neoplasm Metastasis; Paclitaxel; Prognosis; Stomach; Tomography, X-Ray Computed; Tubulin Modulators; Uveal Neoplasms

To analyze the activity of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinases/mechanistic target of rapamycin (PI3K/mTOR) pathways in benign and malignant conjunctival melanocytic proliferations and explore whether specific inhibitors can suppress growth of conjunctival melanoma (CJM) cells.. The presence of a BRAF V600E mutation and activation of ERK, MEK, S6, and AKT were assessed with immunohistochemistry in 35 conjunctival nevi and 31 melanomas. Three CJM cell lines were used: CRMM1, carrying the BRAF V600E mutation; CRMM2, harboring the NRAS Q61L mutation; and T1527A, with a BRAF G466E mutation. WST-1 assays were performed with a BRAF inhibitor (vemurafenib), two MEK inhibitors (trametinib, selumetinib), a PI3K inhibitor (pictilisib), and a dual PI3K/mTOR inhibitor (dactolisib). The phosphorylation of ERK, MEK, and S6 were tested with western blots and apoptosis with cleaved caspase-3 immunostaining.. A BRAF V600E mutation was detected in 42.6% of nevi and in 35.5% of CJM. MEK and ERK activation were higher in CJM, occurring in 62.9% and 45.7% of the nevi and 90.3% and 96.8% of the CJM, respectively. There was also a significant increase in S6 activation in CJM (90.3%) compared with the nevi (20%). CRMM1 was sensitive to trametinib and the PI3K inhibitors but only marginally to vemurafenib. CRMM2 was moderately sensitive to pictilisib, whereas T1527A was resistant to all drugs tested.. The MAPK pathway activity in CJM is increased, not only as a consequence of the BRAF V600E mutation. Targeted therapy may be useful for patients with CJM, especially those with activating BRAF mutations, whereas NRAS-mutated melanomas are relatively resistant.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Benzimidazoles; Blotting, Western; Conjunctival Neoplasms; Female; Fluorescent Antibody Technique, Indirect; Humans; Imidazoles; Indazoles; Male; Melanoma; Middle Aged; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Phosphatidylinositol 3-Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Quinolines; Sulfonamides; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Genotyping of melanomas is used to identify patients for treatment with BRAF and MEK inhibitors, but clinical responses are highly variable. This study investigated the utility of protein expression phenotyping to provide an integrated assessment of gene expression programs in BRAF/NRAS melanoma which would be useful for prognosis and may predict response to MEK inhibition.. Mass spectrometry profiling of early passage cell lines established from Stage III cutaneous melanomas was conducted. Basal protein expression was correlated with in vitro response to the MEK inhibitor, selumetinib. Protein expression in a cohort of 32 drug naïve BRAF/NRAS metastatic melanoma specimens was examined. The prognostic utility of a subset of these proteins and mRNA transcripts from a separate cohort was determined.. Unsupervised analysis of basal cell line protein abundances delineated response to selumetinib, but BRAF/NRAS genotype did not. Resistance was associated with functions including cell motility, cell adhesion and cytoskeletal organization. Several of these response biomarkers were observed in lymph node biospecimens and correlated with melanoma-specific survival. Loss of ICAM-1 protein and mRNA expression was a strong prognosticator of diminished survival in BRAF/NRAS mutant melanoma.. These results demonstrate the utility of proteomic phenotyping to identify both putative biomarkers of response to MEK inhibition and prognostication associated with metastatic melanoma.

Topics: Benzimidazoles; Cell Line, Tumor; Chromatography, Liquid; Cohort Studies; Female; GTP Phosphohydrolases; Humans; Intercellular Adhesion Molecule-1; Male; Melanoma; Melanoma, Cutaneous Malignant; Membrane Proteins; Mutation; Neoplasm Staging; Prognosis; Protein Kinase Inhibitors; Proteomics; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Survival Analysis; Tandem Mass Spectrometry

Primary or acquired resistance to MEK inhibitors has been a barrier to successful treatment with MEK inhibitors in many tumors. In this study, we analyzed genome-wide gene expression profiling data from 6 sensitive and 6 resistant cell lines to identify candidate genes whose expression changes are associated with responses to a MEK inhibitor, selumetinib (AZD6244). Of 62 identified differentially expressed genes, we selected Immunoglobulin Transcription Factor 2, also known as transcription factor 4 as a potential drug resistance marker for further analysis. This was because the ITF-2 expression increase in resistant cell lines was relatively high and a previous study has suggested that ITF-2 functions as an oncogene in human colon cancers. We also established an AZD6244 resistant cell line (M14/AZD-3) from an AZD6244 sensitive M14 cell line. The expression of the ITF-2 was elevated both in primary AZD6244 resistant cell line, LOX-IMVI and acquired resistant cell line, M14/AZD-3. Targeted silencing of ITF-2 by siRNA significantly enhanced susceptibility to AZD6244 in resistant cells. Wnt/β-catenin pathway was activated through direct interaction of p-ERK and GSK3β. Our results suggest that up-regulation of the ITF-2 gene expression is associated with cellular resistance to MEK inhibitors, and activation of Wnt signaling pathway through interaction of p-ERK and GSK3β seems to be a mechanism for increase of ITF-2.

Topics: Benzimidazoles; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; K562 Cells; Melanoma; Mitogen-Activated Protein Kinase Kinases; Protein Kinase Inhibitors; RNA Interference; Transcription Factor 4; Wnt Signaling Pathway

Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, suggesting that durable control of resistance will be a challenge. By combining statistical modeling of genomic data from matched pre-treatment and post-relapse patient tumors with functional interrogation of >20 in vitro and in vivo resistance models, we discovered that major pathways of resistance converge to activate the transcription factor, c-MYC (MYC). MYC expression and pathway gene signatures were suppressed following drug treatment, and then rebounded during progression. Critically, MYC activation was necessary and sufficient for resistance, and suppression of MYC activity using genetic approaches or BET bromodomain inhibition was sufficient to resensitize cells and delay BRAFi resistance. Finally, MYC-driven, BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, including SRC family and c-KIT tyrosine kinases, as well as glucose, glutamine, and serine metabolic pathways. These insights enable the design of combination therapies that select against resistance evolution.

Topics: Antineoplastic Agents, Hormonal; Benzimidazoles; Cell Line, Tumor; Estradiol; Evolution, Molecular; Female; Fulvestrant; Humans; Imidazoles; Indoles; Male; Melanoma; Oximes; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; Pyridones; Pyrimidinones; Quinolines; Signal Transduction; Sulfonamides

We investigated the therapeutic effects of a mitogen-activated protein (MEK) inhibitor, selumetinib, in a hepatic melanoma metastasis model and studied its possible mechanism of action.. Melanoma cell lines were exposed to selumetinib under different experimental conditions. We established a mouse model of liver metastasis and treated mice orally with vehicle or selumetinib and then evaluated metastasis progress.. Growth inhibition was observed in melanoma cells as a consequence of G. We established a mouse model of hepatic metastasis using a human melanoma cell line, such models are essential in elucidating the therapeutic effects of anti-metastatic drugs. Our data suggest the possibility that selumetinib presents a new strategy to treat liver metastasis in patients with melanoma by suppressing epithelial-to-mesenchymal transition-related genes.

Topics: Administration, Oral; Animals; Apoptosis; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Integrin alpha5; Jagged-1 Protein; Liver Neoplasms; Male; Melanoma; Mice, Inbred BALB C; Mice, Nude; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta1; Xenograft Model Antitumor Assays

The lack of advanced animal models of human cancers is considered a barrier to developing effective therapeutics. Canine and human melanomas are histologically disparate but show similar disease progression and response to therapies. The purpose of these studies was to compare human and canine melanoma tumours and cell lines regarding MAPK and PI3K/AKT signalling dysregulation, and response to select molecularly targeted agents. Pathway activation was investigated via microarray and mutational analysis. Growth inhibition and cell cycle effects were assessed for pathway inhibitors AZD6244 (MAPK) and rapamycin (PI3K/AKT) in human and canine melanoma cells. Human and canine melanoma share similar differential gene expression patterns within the MAPK and PI3K/AKT pathways. Constitutive pathway activation and similar sensitivity to AZD6244 and rapamycin was observed in human and canine cells. These results show that human and canine melanoma share activation and sensitivity to inhibition of cancer-related signalling pathways despite differences in activating mutations.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Benzimidazoles; Cell Line, Tumor; Databases, Genetic; Dog Diseases; Dogs; GTP Phosphohydrolases; Humans; Melanoma; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Mouth Neoplasms; Mutation; Oncogene Protein v-akt; Polymerase Chain Reaction; Proto-Oncogene Proteins B-raf; Signal Transduction; Sirolimus; Tissue Array Analysis

MEK inhibitors are being evaluated in clinical trials for treatment of different malignant neoplasms; trametinib dimethyl sulfoxide was approved by the US Food and Drug Administration for melanoma in 2013. We present 3 cases of patients receiving MEK inhibitors who developed an atypical eruption.. Three patients who were receiving different MEK inhibitors (selumetinib, cobimetinib, and trametinib) developed an eruption, all associated with unique duskiness. Drug hypersensitivity was confirmed by histopathologic testing in 2 of the 3 cases. The skin eruption responded well to corticosteroids and did not recur when treatment with the MEK inhibitor was restarted in 2 of the patients.. The typical skin reaction associated with MEK inhibitors is a papulopustular eruption. To our knowledge, the dusky erythema that occurred in the 3 patients described here has not previously been reported for this drug class.

Topics: Adult; Azetidines; Benzimidazoles; Drug Eruptions; Erythema; Female; Glucocorticoids; Humans; Male; Melanoma; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Piperidines; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Skin Neoplasms

Inhibitors of oncogenic B-RAF(V600E) and MKK1/2 have yielded remarkable responses in B-RAF(V600E)-positive melanoma patients. However, the efficacy of these inhibitors is limited by the inevitable onset of resistance. Despite the fact that these inhibitors target the same pathway, combination treatment with B-RAF(V600E) and MKK1/2 inhibitors has been shown to improve both response rates and progression-free survival in B-RAF(V600E) melanoma patients. To provide insight into the molecular nature of the combinatorial response, we used quantitative mass spectrometry to characterize the inhibitor-dependent phosphoproteome of human melanoma cells treated with the B-RAF(V600E) inhibitor PLX4032 (vemurafenib) or the MKK1/2 inhibitor AZD6244 (selumetinib). In three replicate experiments, we quantified changes at a total of 23,986 phosphosites on 4784 proteins. This included 1317 phosphosites that reproducibly decreased in response to at least one inhibitor. Phosphosites that responded to both inhibitors grouped into networks that included the nuclear pore complex, growth factor signaling, and transcriptional regulators. Although the majority of phosphosites were responsive to both inhibitors, we identified 16 sites that decreased only in response to PLX4032, suggesting rare instances where oncogenic B-RAF signaling occurs in an MKK1/2-independent manner. Only two phosphosites were identified that appeared to be uniquely responsive to AZD6244. When cells were treated with the combination of AZD6244 and PLX4032 at subsaturating concentrations (30 nm), responses at nearly all phosphosites were additive. We conclude that AZD6244 does not substantially widen the range of phosphosites inhibited by PLX4032 and that the benefit of the drug combination is best explained by their additive effects on suppressing ERK1/2 signaling. Comparison of our results to another recent ERK1/2 phosphoproteomics study revealed a surprising degree of variability in the sensitivity of phosphosites to MKK1/2 inhibitors in human cell lines, revealing unexpected cell specificity in the molecular responses to pathway activation.

Topics: Benzimidazoles; Cell Line, Tumor; Humans; Indoles; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Phosphoproteins; Protein Kinase Inhibitors; Proteomics; Proto-Oncogene Proteins B-raf; Sulfonamides; Vemurafenib

BRAF inhibitors have revolutionized treatment of mutant BRAF metastatic melanomas. However, resistance develops rapidly following BRAF inhibitor treatment. We have found that BRAF-mutant melanoma cell lines are more sensitive than wild-type BRAF cells to the small molecule tyrosine kinase inhibitor dovitinib. Sensitivity is associated with inhibition of a series of known dovitinib targets. Dovitinib in combination with several agents inhibits growth more effectively than either agent alone. These combinations inhibit BRAF-mutant melanoma and colorectal carcinoma cell lines, including cell lines with intrinsic or selected BRAF inhibitor resistance. Hence, combinations of dovitinib with second agents are potentially effective therapies for BRAF-mutant melanomas, regardless of their sensitivity to BRAF inhibitors.

Topics: Animals; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Humans; Indoles; Male; Melanoma; Melanoma, Cutaneous Malignant; Mice, Nude; Mutation; Neoplasm Proteins; Protein Kinase Inhibitors; Quinolones; Signal Transduction; Skin Neoplasms; Small Molecule Libraries; Sulfonamides; Vemurafenib

Uveal melanoma patients with metastatic disease usually die within one year, emphasizing an urgent need to develop new treatment strategies for this cancer. MEK inhibitors improve survival in cutaneous melanoma patients but show only modest efficacy in metastatic uveal melanoma patients. In this study, we screened for growth factors that elicited resistance in newly characterized metastatic uveal melanoma cell lines to clinical-grade MEK inhibitors, trametinib and selumetinib. We show that neuregulin 1 (NRG1) and hepatocyte growth factor (HGF) provide resistance to MEK inhibition. Mechanistically, trametinib enhances the responsiveness to NRG1 and sustained HGF-mediated activation of AKT. Individually targeting ERBB3 and cMET, the receptors for NRG1 and HGF, respectively, overcome resistance to trametinib provided by these growth factors and by conditioned medium from fibroblasts that produce NRG1 and HGF. Inhibition of AKT also effectively reverses the protective effect of NRG1 and HGF in trametinib-treated cells. Uveal melanoma xenografts growing in the liver in vivo and a subset of liver metastases of uveal melanoma patients express activated forms of ERBB2 (the coreceptor for ERBB3) and cMET. Together, these results provide preclinical evidence for the use of MEK inhibitors in combination with clinical-grade anti-ERBB3 or anti-cMET monoclonal antibodies in metastatic uveal melanoma.

Topics: Animals; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Hepatocyte Growth Factor; Humans; MAP Kinase Kinase Kinases; Melanoma; Mice; Neuregulin-1; Proto-Oncogene Proteins c-met; Pyridones; Pyrimidinones; Receptor, ErbB-3; Tumor Cells, Cultured; Uveal Neoplasms; Xenograft Model Antitumor Assays

The aim of combination drug treatment in cancer therapy is to improve response rate and to decrease the probability of the development of drug resistance. Preferably, drug combinations are synergistic rather than additive, and, ideally, drug combinations work synergistically only in cancer cells and not in non-malignant cells. We have developed a workflow to identify such targeted synergies, and applied this approach to selectively inhibit the proliferation of cell lines with mutations in genes that are difficult to modulate with small molecules. The approach is based on curve shift analysis, which we demonstrate is a more robust method of determining synergy than combination matrix screening with Bliss-scoring. We show that the MEK inhibitor trametinib is more synergistic in combination with the BRAF inhibitor dabrafenib than with vemurafenib, another BRAF inhibitor. In addition, we show that the combination of MEK and BRAF inhibitors is synergistic in BRAF-mutant melanoma cells, and additive or antagonistic in, respectively, BRAF-wild type melanoma cells and non-malignant fibroblasts. This combination exemplifies that synergistic action of drugs can depend on cancer genotype. Next, we used curve shift analysis to identify new drug combinations that specifically inhibit cancer cell proliferation driven by difficult-to-drug cancer genes. Combination studies were performed with compounds that as single agents showed preference for inhibition of cancer cells with mutations in either the CTNNB1 gene (coding for β-catenin), KRAS, or cancer cells expressing increased copy numbers of MYC. We demonstrate that the Wnt-pathway inhibitor ICG-001 and trametinib acted synergistically in Wnt-pathway-mutant cell lines. The ERBB2 inhibitor TAK-165 was synergistic with trametinib in KRAS-mutant cell lines. The EGFR/ERBB2 inhibitor neratinib acted synergistically with the spindle poison docetaxel and with the Aurora kinase inhibitor GSK-1070916 in cell lines with MYC amplification. Our approach can therefore efficiently discover novel drug combinations that selectively target cancer genes.

Topics: Antineoplastic Combined Chemotherapy Protocols; Aza Compounds; Benzimidazoles; beta Catenin; Cell Line, Tumor; Cell Proliferation; Humans; Imidazoles; Indazoles; Indoles; Melanoma; Molecular Targeted Therapy; Mutation; Oxazoles; Oximes; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins p21(ras); Pyridones; Pyrimidinones; Quinolines; Sulfonamides; Triazoles; Vemurafenib

Development of small molecular inhibitors against BRAF and MEK has been a breakthrough in the treatment of malignant melanoma. However, the long-term effect is foiled in virtually all patients by the emergence of resistant tumor cell populations. Therefore, mechanisms resulting in the acquired resistance against BRAF and MEK inhibitors have gained much attention and several strategies have been proposed to overcome tumor resistance, including interval treatment or withdrawal of these compounds after disease progression.. Using a panel of cell lines with an acquired resistance against MEK inhibitors, we have evaluated the sensitivity of these cells against compounds targeting AKT/mTOR signaling, as well as novel ERK1/2 inhibitors. Furthermore, the effects of withdrawal of MEK inhibitor on migration in resistant cell lines were analyzed.. We demonstrate that withdrawal of BRAF or MEK inhibitors in tumor cells with an acquired resistance results in reactivation of ERK1/2 signaling and upregulation of EMT-inducing transcription factors, leading to a highly migratory and invasive phenotype of cancer cells. Furthermore, we show that migration in these cells is independent from AKT/mTOR signaling. However, combined targeting of AKT/mTOR using MK-2206 and AZD8055 efficiently inhibits proliferation in all resistant tumor cell lines analyzed.. We propose that combined targeting of MEK/AKT/mTOR or treatment with a novel ERK1/2 inhibitor downstream of BRAF/MEK suppresses proliferation as well as migration and invasion in resistant tumor cells. We provide a rationale against the discontinuation of BRAF or MEK inhibitors in patients with an acquired resistance, and provide a rationale for combined targeting of AKT/mTOR and MEK/ERK1/2, or direct targeting of ERK1/2 as an effective treatment strategy.

Topics: Benzimidazoles; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; HCT116 Cells; Heterocyclic Compounds, 3-Ring; HT29 Cells; Humans; Melanoma; Melanoma, Cutaneous Malignant; Mitogen-Activated Protein Kinase Kinases; Morpholines; Neoplasm Invasiveness; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6; Signal Transduction; Skin Neoplasms; TOR Serine-Threonine Kinases

We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis.

Topics: Antineoplastic Agents; Benzimidazoles; DNA Mutational Analysis; Drug Resistance, Neoplasm; Exome; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Inhibitory Concentration 50; Kaplan-Meier Estimate; Loss of Heterozygosity; Male; Melanoma; Mutation, Missense; Neurofibromin 1; ras Proteins; Sequence Analysis, RNA; Skin Neoplasms; Sunlight; Tumor Cells, Cultured

BRAF-mutant melanoma can be successfully treated by BRAF kinase inhibitors (BRAFi) and MEK kinase inhibitors (MEKi). However, the administration of BRAFi followed by MEKi did not generate promising response rate (RR). The purpose of this investigation was to evaluate the time to progression (TTP) with a mitogen-activated protein kinase (MAPK) pathway upstream inhibition strategy in BRAF mutated melanoma patients. BRAF mutation positive metastatic melanoma patients were identified within the Dermatology Cooperative Oncology Group (DeCOG) network and were treated first with a MEKi and upon progression with a selective BRAFi. A total of 23 melanoma patients (six females, 17 males, aged 47-80 years) were retrospectively analysed for TTP. The total median TTP was 8.9 months. The median TTP for MEKi was 4.8 (1.2-23.2) and subsequent for BRAFi 4.5 (1.2-15.7) months, respectively. A higher RR for MEKi (39%, nine partial responses and 0 complete responses) than previously reported was observed. Our analysis suggests that the reversed inhibition of the MAPK pathway is feasible in BRAF mutated melanoma. The median TTP (8.9 months) is close to the promising BRAF- and MEKi combination therapy (median progression-free survival (PFS) 9.4 months). The total treatment duration of the MAPK inhibition when a MEKi is administered first is similar compared to the reversed sequence, but TTP shifts in favour to the MEKi. This approach is feasible with reasonable tolerability. This clinical investigation encourages further studies in prospective clinical trials to define the optimal treatment schedule for the MAPK pathway inhibition and should be accompanied by molecular monitoring using repeated biopsies.

Topics: Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Disease Progression; Disease-Free Survival; Feasibility Studies; Female; Humans; Imidazoles; Indoles; Male; MAP Kinase Signaling System; Melanoma; Middle Aged; Mitogen-Activated Protein Kinases; Mutation; Outcome Assessment, Health Care; Oximes; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Retrospective Studies; Sulfonamides; Time Factors; Vemurafenib

Drug resistance is a major obstacle in the targeted therapy of melanoma using BRAF/MEK inhibitors. This study was to identify BRAF V600E-associated oncogenic pathways that predict resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors. We took in silico approaches to analyze the activities of 24 cancer-related pathways in melanoma cells and identify those whose activation was associated with BRAF V600E and used the support vector machine (SVM) algorithm to predict the resistance of BRAF-mutated melanoma cells to BRAF/MEK inhibitors. We then experimentally confirmed the in silico findings. In a microarray gene expression dataset of 63 melanoma cell lines, we found that activation of multiple oncogenic pathways preferentially occurred in BRAF-mutated melanoma cells. This finding was reproduced in 5 additional independent melanoma datasets. Further analysis of 46 melanoma cell lines that harbored BRAF mutation showed that 7 pathways, including TNFα, EGFR, IFNα, hypoxia, IFNγ, STAT3, and MYC, were significantly differently expressed in AZD6244-resistant compared with responsive melanoma cells. A SVM classifier built on this 7-pathway activation pattern correctly predicted the response of 10 BRAF-mutated melanoma cell lines to the MEK inhibitor AZD6244 in our experiments. We experimentally showed that TNFα, EGFR, IFNα, and IFNγ pathway activities were also upregulated in melanoma cell A375 compared with its sub-line DRO, while DRO was much more sensitive to AZD6244 than A375. In conclusion, we have identified specific oncogenic pathways preferentially activated in BRAF-mutated melanoma cells and a pathway pattern that predicts resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors, providing novel clinical implications for melanoma therapy.

Topics: Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Cytokines; Drug Resistance, Neoplasm; Gene Expression Profiling; Humans; Melanoma; Mitogen-Activated Protein Kinases; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; PTEN Phosphohydrolase; Signal Transduction

Activating BRAF kinase mutations serve as oncogenic drivers in over half of all melanomas, a feature that has been exploited in the development of new molecularly targeted approaches to treat this disease. Selective BRAF(V600E) inhibitors, such as vemurafenib, typically induce initial, profound tumor regressions within this group of patients; however, durable responses have been hampered by the emergence of drug resistance. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90, in melanoma lines harboring the BRAF(V600E) mutation. Ganetespib exposure resulted in the loss of mutant BRAF expression and depletion of mitogen-activated protein kinase and AKT signaling, resulting in greater in vitro potency and antitumor efficacy compared with targeted BRAF and MAP-ERK kinase (MEK) inhibitors. Dual targeting of Hsp90 and BRAF(V600E) provided combinatorial benefit in vemurafenib-sensitive melanoma cells in vitro and in vivo. Importantly, ganetespib overcame mechanisms of intrinsic and acquired resistance to vemurafenib, the latter of which was characterized by reactivation of extracellular signal-regulated kinase (ERK) signaling. Continued suppression of BRAF(V600E) by vemurafenib potentiated sensitivity to MEK inhibitors after acquired resistance had been established. Ganetespib treatment reduced, but not abolished, elevations in steady-state ERK activity. Profiling studies revealed that the addition of a MEK inhibitor could completely abrogate ERK reactivation in the resistant phenotype, with ganetespib displaying superior combinatorial activity over vemurafenib. Moreover, ganetespib plus the MEK inhibitor TAK-733 induced tumor regressions in vemurafenib-resistant xenografts. Overall these data highlight the potential of ganetespib as a single-agent or combination treatment in BRAF(V600E)-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Blotting, Western; Cell Line, Tumor; Cell Survival; Cells, Cultured; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; HSP90 Heat-Shock Proteins; Humans; Indoles; Melanoma; Mice, Nude; Mice, SCID; Mitogen-Activated Protein Kinase Kinases; Mutation; Proto-Oncogene Proteins B-raf; Pyridones; Pyrimidinones; Sulfonamides; Triazoles; Vemurafenib; Xenograft Model Antitumor Assays

Many clinical cases of acquired resistance to the BRAF inhibitor vemurafenib have recently been reported. One of the causes of this acquired resistance is the BRAF downstream kinase point mutation MEK1-C121S. This mutation confers resistance to not only vemurafenib, but also to the allosteric MEK inhibitor selumetinib (AZD6244). Here, we investigated the pharmacologic activities and effectiveness of the novel MEK inhibitor E6201 against BRAF (v-raf murine sarcoma viral oncogene homolog B1)-V600E mutant melanoma harboring the MEK1-C121S mutation. A cell-free assay confirmed that E6201 is an ATP-competitive MEK inhibitor, meaning it has a different binding mode with MEK compared with allosteric MEK inhibitors. E6201 is more effective against BRAF-V600E mutant melanoma compared with BRAF wild-type melanoma based on MEK inhibition. We found that the acquired MEK1-C121S mutation in BRAF-V600E mutant melanoma conferred resistance to both vemurafenib and selumetinib but not E6201. The effectiveness of E6201 in this preclinical study is a result of its binding with MEK1 far from the C121S point mutation so the mutation is unable to influence the MAPK pathway inhibitory activity. These results support further clinical investigation of E6201.

Topics: Benzimidazoles; Drug Resistance, Neoplasm; Humans; Indoles; Lactones; MAP Kinase Kinase 1; MAP Kinase Signaling System; Melanoma; Mutagenesis, Site-Directed; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Sulfonamides; Vemurafenib

The evolution of cancer therapy into complex regimens with multiple drugs requires novel approaches for the development and evaluation of companion biomarkers. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) is a versatile platform for biomarker measurement. In this study, we describe the development and use of the LC-MRM platform to study the adaptive signaling responses of melanoma cells to inhibitors of HSP90 (XL888) and MEK (AZD6244). XL888 had good anti-tumor activity against NRAS mutant melanoma cell lines as well as BRAF mutant cells with acquired resistance to BRAF inhibitors both in vitro and in vivo. LC-MRM analysis showed HSP90 inhibition to be associated with decreased expression of multiple receptor tyrosine kinases, modules in the PI3K/AKT/mammalian target of rapamycin pathway, and the MAPK/CDK4 signaling axis in NRAS mutant melanoma cell lines and the inhibition of PI3K/AKT signaling in BRAF mutant melanoma xenografts with acquired vemurafenib resistance. The LC-MRM approach targeting more than 80 cancer signaling proteins was highly sensitive and could be applied to fine needle aspirates from xenografts and clinical melanoma specimens (using 50 μg of total protein). We further showed MEK inhibition to be associated with signaling through the NFκB and WNT signaling pathways, as well as increased receptor tyrosine kinase expression and activation. Validation studies identified PDGF receptor β signaling as a potential escape mechanism from MEK inhibition, which could be overcome through combined use of AZD6244 and the PDGF receptor inhibitor, crenolanib. Together, our studies show LC-MRM to have unique value as a platform for the systems level understanding of the molecular mechanisms of drug response and therapeutic escape. This work provides the proof-of-principle for the future development of LC-MRM assays for monitoring drug responses in the clinic.

Topics: Animals; Azabicyclo Compounds; Benzimidazoles; beta Catenin; Biomarkers, Tumor; Cell Line, Tumor; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; GTP Phosphohydrolases; HSP90 Heat-Shock Proteins; Humans; Indoles; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mass Spectrometry; Melanoma; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, SCID; Neoplasm Transplantation; NF-kappa B; Phosphoinositide-3 Kinase Inhibitors; Phthalic Acids; Piperidines; Proteomics; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Receptor, Platelet-Derived Growth Factor beta; RNA Interference; RNA, Small Interfering; Sulfonamides; Transplantation, Heterologous; Vemurafenib; Wnt Signaling Pathway

Most melanomas harbor oncogenic BRAF(V600) mutations, which constitutively activate the MAPK pathway. Although MAPK pathway inhibitors show clinical benefit in BRAF(V600)-mutant melanoma, it remains incompletely understood why 10% to 20% of patients fail to respond. Here, we show that RAF inhibitor-sensitive and inhibitor-resistant BRAF(V600)-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAF(V600)-mutant melanoma.. Although most BRAF(V600)-mutant melanomas are sensitive to RAF and/or MEK inhibitors, a subset fails to respond to such treatment. This study characterizes a transcriptional cell state distinction linked to MITF and NF-κB that may modulate intrinsic sensitivity of melanomas to MAPK pathway inhibitors.

Topics: Anilides; Benzimidazoles; Benzocycloheptenes; Cell Line, Tumor; Cells, Cultured; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Hepatocyte Growth Factor; Humans; Indoles; MAP Kinase Signaling System; Melanocytes; Melanoma; Microphthalmia-Associated Transcription Factor; NF-kappa B p50 Subunit; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-met; Pyridines; Quinolines; Sulfonamides; Triazoles

Activating mutations in the MAPK pathway are prevalent drivers of several cancers. The chief consequence of these mutations is a hyperactive ERK1/2 MAPK able to promote cell proliferation, producing a critical hallmark of metastatic disease. The biochemistry of the ERK pathway is well characterized; however, how the pathway achieves different outcomes in the face of genetic aberrations of cancer and subsequent treatment with chemical inhibitors is not clear. To investigate this, we used mass spectrometry to complete a global phosphoproteomic analysis of a BRAFV600E thyroid cancer cell line (SW1736) after treatment with the mutation-selective inhibitor vemurafenib (PLX4032) and MEK1/2 inhibitor selumetinib (AZD6244). We identified thousands of phosphorylation events orchestrated in BRAFV600E cells and performed kinase landscape analysis to identify putative kinases regulated in response to MAPK blockade. The abundance of phosphopeptides containing consensus motifs for acidophilic kinases increased after short-term inhibition with these compounds. We showed that coinhibition of the pleiotropic acidophilic protein kinase CK2 (CK2) and BRAFV600E synergistically reduced proliferation in patient-derived melanomas and thyroid cancer cells harboring the BRAF lesion. We investigated this mechanism and show a role for CK2 in controlling AKT activation that was not reliant on changes to PTEN or PDK1 phosphorylation. These findings highlight a role for CK2 blockade in potentiating the antiproliferative effects of BRAF and MEK inhibition in BRAF cancers.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Casein Kinase II; Cell Line, Tumor; Drug Synergism; Humans; Indoles; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Protein Kinase Inhibitors; Proteomics; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Sulfonamides; Thyroid Neoplasms; Vemurafenib

The majority of uveal melanomas carry oncogenic mutations in the G proteins GNAQ and GNA11, with consequent activation of the MAPK pathway. Selective MEK inhibitors, such as selumetinib, have shown clinical benefit in uveal melanoma. However, mechanisms of drug resistance limit their efficacy in some patients. Analysis of MEK inhibitor-resistant uveal melanoma cell lines revealed the induction of RAS protein expression and activity. This effect was mediated by the RNA helicase DDX43, which was remarkably overexpressed in these cells. Depletion of DDX43 in MEK inhibitor-resistant cells decreased RAS proteins and inhibited ERK and AKT pathways. On the contrary, ectopic expression of DDX43 in parental uveal melanoma cells induced RAS protein levels and rendered cells resistant to MEK inhibition. Similar to DDX43 depletion, downregulation of KRAS, HRAS, and NRAS inhibited downstream pathways in the resistant cells, overcoming mutant GNAQ signaling. We also analyzed the expression of DDX43 in liver metastases of patients with uveal melanoma by RT-PCR, and found a significant overexpression of DDX43 in patients who did not benefit from selumetinib therapy. In conclusion, DDX43 induces RAS protein expression and signaling, mediating a novel mechanism of MEK inhibitor resistance. The detection of DDX43 in patients with uveal melanoma could lead to more targeted therapies for this disease.

Topics: Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Cell Survival; DEAD-box RNA Helicases; Drug Resistance, Neoplasm; Gene Expression; Humans; Liver Neoplasms; MAP Kinase Kinase Kinases; Melanoma; Neoplasm Proteins; Proto-Oncogene Proteins c-akt; ras Proteins; Up-Regulation; Uveal Neoplasms

Melanoma is a devastating form of skin cancer with limited therapeutic options. Fifteen to 20% of patients with melanoma have an activating mutation in the GTPase, NRAS. The major downstream effectors of RAS are RAFs (ARAF, BRAF, and CRAF), phosphoinositide 3-kinase (PI3K), and the Ral guanine exchange factors (RalGEF). TANK-binding kinase 1 (TBK1) is an atypical IκB kinase family member that acts downstream of RalGEFs. Whereas many studies have analyzed RAF and PI3K signaling in mutant NRAS melanoma, the role of RalGEF/Ral is understudied and TBK1 has not been examined. To address this, TBK1 was modulated with knockdown approaches and targeted therapies to determine the role of TBK1 in motility, apoptosis, and signaling. In melanoma, NRAS overexpression increased TBK1 phosphorylation. TBK1 depletion inhibited migration and invasion, whereas its constitutive overexpression led to an increase in invasion. In three-dimensional systems that mimic the dermal microenvironment, TBK1 depletion or inhibition cooperated with MEK inhibitors to promote apoptosis, particularly in the context of MEK-insensitive mutant NRAS. This effect was absent in melanoma cells that are wild-type for NRAS. These results suggest the utility of TBK1 inhibitors as part of a treatment regimen for patients with mutant NRAS melanoma, for whom there are no current effective therapies.. TBK1 promotes the malignant properties of NRAS-mutant melanoma and its targeting, in combination with MEK, promotes apoptosis, thus providing a potential novel targeted therapeutic option.

Topics: Apoptosis; Benzimidazoles; Cell Line, Tumor; Cell Movement; Drug Resistance, Neoplasm; Gene Knockdown Techniques; GTP Phosphohydrolases; Humans; Melanoma; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Molecular Targeted Therapy; Mutation; Neoplasm Invasiveness; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrimidines; Skin Neoplasms; Thiophenes

Improving treatment of advanced melanoma may require the development of effective strategies to overcome resistance to different anti-tumor agents and to counteract relevant pro-tumoral mechanisms in the microenvironment. Here we provide preclinical evidence that these goals can be achieved in most melanomas, by co-targeting of oncogenic and death receptor pathways, and independently of their BRAF, NRAS, p53 and PTEN status. In 49 melanoma cell lines, we found independent susceptibility profiles for response to the MEK1/2 inhibitor AZD6244, the PI3K/mTOR inhibitor BEZ235 and the death receptor ligand TRAIL, supporting the rationale for their association. Drug interaction analysis indicated that a strong synergistic anti-tumor activity could be achieved by the three agents and the AZD6244-TRAIL association on 20/21 melanomas, including cell lines resistant to the inhibitors or to TRAIL. Mechanistically, synergy was explained by enhanced induction of caspase-dependent apoptosis, mitochondrial depolarization and modulation of key regulators of extrinsic and intrinsic cell death pathways, including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members. Moreover, silencing experiments confirmed the central role of Apollon downmodulation in promoting the apoptotic response of melanoma cells to the combinatorial treatments. In SCID mice, the AZD6244-TRAIL association induced significant growth inhibition of a tumor resistant to TRAIL and poorly responsive to AZD6244, with no detectable adverse events on body weight and tissue histology. Reduction in tumor volume was associated not only with promotion of tumor apoptosis but also with suppression of the pro-angiogenic molecules HIF1α, VEGFα, IL-8 and TGFβ1 and with inhibition of tumor angiogenesis. These results suggest that synergistic co-targeting of oncogenic and death receptor pathways can not only overcome melanoma resistance to different anti-tumor agents in vitro but can also promote pro-apoptotic effects and inhibition of tumor angiogenesis in vivo.

Topics: Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; Mice, SCID; Neovascularization, Pathologic; Receptors, Death Domain; TNF-Related Apoptosis-Inducing Ligand

Stimulation of the c-Kit receptor tyrosine kinase has a critical role in the development and migration of melanocytes, and oncogenic c-Kit mutants contribute to the progression of some melanomas. c-Kit signalling activates the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways and their relative contribution to the activities of oncogenic and ligand-dependent c-Kit remains uncertain. We show that PI3K is a major regulator of MAPK activation in response to c-Kit activity and the dominant effector of c-Kit-driven melanocyte proliferation and melanoma survival. Nevertheless, inhibition of the PI3K pathway in c-Kit mutant melanoma cells did not replicate the apoptotic efficacy of the c-Kit inhibitor, imatinib mesylate. Instead, the simultaneous suppression of the PI3K and MAPK pathways promoted a strong synergistic apoptotic effect. These data indicate that MAPK functions as a redundant survival signal that reinforces the PI3K cascade in c-Kit mutant melanoma. Thus, the concurrent inhibition of PI3K and MAPK signalling is required to suppress oncogenic c-Kit activity and may provide an effective therapeutic strategy in c-Kit mutant melanomas.

Topics: Benzimidazoles; Cell Proliferation; Cell Survival; Cells, Cultured; Humans; MAP Kinase Signaling System; Melanoma; Mutation; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-kit

Oncogenic mutations in GNAQ and GNA11 genes are found in 80% of uveal melanoma. These mutations result in the activation of the RAF/MEK signaling pathway culminating in the stimulation of ERK1/2 mitogen-activated protein kinases. In this study, using a siRNA strategy, we show that mutant GNAQ signals to both MEK and AKT, and that combined inhibition of these pathways with the MEK inhibitor selumetinib (AZD6244) and the AKT inhibitor MK2206 induced a synergistic decrease in cell viability. This effect was genotype dependent as autophagic markers like beclin1 and LC3 were induced in GNAQ-mutant cells, whereas apoptosis was the mechanism of cell death of BRAF-mutant cells, and cells without either mutation underwent cell-cycle arrest. The inhibition of MEK/ATK pathways induced activation of AMP-activated protein kinase (AMPK) in the GNAQ-mutant cells. The downregulation of AMPK by siRNA or its inhibition with compound C did not rescue the cells from autophagy, rather they died by apoptosis, defining AMPK as a key regulator of mutant GNAQ signaling and a switch between autophagy and apoptosis. Furthermore, this combination treatment was effective in inhibiting tumor growth in xenograft mouse models. These findings suggest that inhibition of MEK and AKT may represent a promising approach for targeted therapy of patients with uveal melanoma.

Topics: Animals; Autophagy; Benzimidazoles; Cell Line, Tumor; Cell Survival; Gene Knockdown Techniques; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Heterocyclic Compounds, 3-Ring; Humans; Male; Melanoma; Mice; Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Tumor Burden; Uveal Neoplasms; Xenograft Model Antitumor Assays

The RAS/BRAF/MEK/ERK signaling pathway is a central driver in cancer with many BRAF and MEK inhibitors being evaluated in clinical trials. Identifying noninvasive biomarkers of early pharmacodynamic responses is important for development of these targeted drugs. As increased aerobic glycolysis is often observed in cancer, we hypothesized that MEK1/2 (MAP2K1/MAP2K2) inhibitors may reduce lactate levels as detected by magnetic resonance spectroscopy (MRS), as a metabolic biomarker for the pharmacodynamic response. MRS was used to monitor intracellular and extracellular levels of lactate in human cancer cells in vitro and in melanoma tumors ex vivo. In addition, we used (1)H MRS and a fluorescent glucose analog to evaluate the effect of MEK inhibition on glucose uptake. MEK1/2 signaling inhibition reduced extracellular lactate levels in BRAF-dependent cells but not BRAF-independent cells. The reduction in extracellular lactate in BRAF-driven melanoma cells was time-dependent and associated with reduced expression of hexokinase-II driven by c-Myc depletion. Taken together, these results reveal how MEK1/2 inhibition affects cancer cell metabolism in the context of BRAF oncogene addiction. Furthermore, they offer a preclinical proof-of-concept for the use of MRS to measure lactate as a noninvasive metabolic biomarker for pharmacodynamic response to MEK1/2 inhibition in BRAF-driven cancers.

Topics: Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Deoxyglucose; Diphenylamine; Female; Gene Knockdown Techniques; Glucose; Hexokinase; Humans; Lactic Acid; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Mice; Mutation, Missense; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-myc; RNA, Small Interfering; Tumor Burden; Xenograft Model Antitumor Assays

Selumetinib plus dacarbazine prolongs progression-free survival in BRAF-mutant melanoma.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Dacarbazine; Disease-Free Survival; Humans; Melanoma; Mutation; Proto-Oncogene Proteins B-raf; Skin Neoplasms

Compared with temozolomide chemotherapy, the MEK inhibitor selumetinib extended progression-free survival by nearly 9 weeks in patients with melanoma of the eye participating in a phase II trial, making it the first effective drug for the rare disease.

Topics: Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Clinical Trials, Phase II as Topic; Dacarbazine; Disease-Free Survival; Eye Neoplasms; Humans; Melanoma; Neoplasm Metastasis; Temozolomide; Uveal Neoplasms

Non-invasive imaging biomarkers underpin the development of molecularly targeted anti-cancer drugs. This study evaluates tumour apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (DW-MRI), as a biomarker of response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in human tumour xenografts.. Nude mice bearing human BRAF(V600D) WM266.4 melanoma or BRAF(V600E) Colo205 colon carcinoma xenografts were treated for 4 days with vehicle or selumetinib. DW-MRI was performed before and 2 h after the last dose and excised tumours analysed for levels of phospho-ERK1/2, cleaved caspase 3 (CC3) and necrosis.. Selumetinib treatment induced tumour stasis and reduced ERK1/2 phosphorylation in both WM266.4 and Colo205 tumour xenografts. Relative to day 0, mean tumour ADC was unchanged in the control groups but was significantly increased by up to 1.6-fold in selumetinib-treated WM266.4 and Colo205 tumours. Histological analysis revealed a significant increase in necrosis in selumetinib-treated WM266.4 and Colo205 xenografts and CC3 staining in selumetinib-treated Colo205 tumours relative to controls.. Changes in ADC following treatment with the MEK1/2 inhibitor selumetinib in responsive human tumour xenografts were concomitant with induction of tumour cell death. ADC may provide a useful non-invasive pharmacodynamic biomarker for early clinical assessment of response to selumetinib and other MEK-ERK1/2 signalling-targeted therapies.

Topics: Animals; Benzimidazoles; Cell Line, Tumor; Colonic Neoplasms; Diffusion Magnetic Resonance Imaging; Female; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Mice; Mice, Nude; Molecular Targeted Therapy; Protein Kinase Inhibitors; Xenograft Model Antitumor Assays

The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas, and targeting MEK as a central kinase of this pathway is currently tested in clinical trials. However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response. Apart from dose limitations, a reason for the low response to MEK targeting drugs is thought to be the upregulation of counteracting signalling cascades as a direct response to MEK inhibition. Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of the drug being used. We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352, while efficiently suppressing proliferation, stimulates increased invasiveness. Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion. Most importantly, and surprisingly, MEK inhibition results in a significant increase in matrix metalloproteases (MMP)-2 and membrane-type 1-MMP expression. All together, MEK inhibition in melanoma cells induces a 'mesenchymal' phenotype that is characterised by protease-driven invasion. This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are the main regulators of this process, the SRC kinase inhibitor, saracatinib (AZD0530), completely abolished the MEK inhibitor-induced invasion. Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in melanoma.

Topics: Benzimidazoles; Benzodioxoles; Cell Adhesion; Cell Division; Collagen; Humans; Integrin beta1; MAP Kinase Kinase Kinases; Melanoma; Neoplasm Invasiveness; Protein Kinase Inhibitors; Quinazolines; src-Family Kinases

Topics: Animals; Antineoplastic Agents; Benzamides; Benzimidazoles; Humans; Melanoma; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Ubiquitin-Protein Ligases

The mitogen-activated protein-kinase pathway consisting of the kinases RAF, MEK, and ERK is central to cell proliferation and survival and is deregulated in more than 90% of melanomas. MEK inhibitors are currently trialled in the clinic, but despite efficient target inhibition, cytostatic rather than cytotoxic activity limits their efficacy.. We assessed the cytotoxicity to MEK inhibitors (PD184352 and selumetinib) in melanoma cells by toluidine-blue staining, caspase 3 cleavage, and melanoma-sphere growth. Western blotting and quantitative real-time polymerase chain reaction were applied to determine SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2), PAX3, and MITF expression. Human melanoma samples (n = 77) from various stages were analyzed for SMURF2 and PAX3 expression. RNA interference was performed to target SMURF2 during MEK inhibition in vivo in melanoma xenografts in mice and zebrafish. All statistical tests were two-sided.. Activation of transforming growth factor β (TGF-β) signalling sensitized melanoma cells to the cytotoxic effects of MEK inhibition. Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signalling through overexpression of the E3 ubiquitin ligase SMURF2, which resulted in increased expression of the transcription factors PAX3 and MITF. High MITF expression protected melanoma cells against MEK inhibitor cytotoxicity. Depleting SMURF2 reduced MITF expression and substantially lowered the threshold for MEK inhibitor-induced apoptosis. Moreover, SMURF2 depletion sensitized melanoma cells to the cytotoxic effects of selumetinib, leading to cell death at concentrations approximately 100-fold lower than the concentration required to induce cell death in SMURF2-expressing cells. Mice treated with selumetinib alone at a dosage of 10mg/kg body weight once daily produced no response, but in combination with SMURF2 depletion, selumetinib suppressed tumor growth by 97.9% (95% confidence interval = 38.65% to 155.50%, P = .005).. Targeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Benzimidazoles; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Mice; Mice, Nude; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Paired Box Transcription Factors; PAX3 Transcription Factor; Real-Time Polymerase Chain Reaction; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays; Zebrafish

In melanoma, the RAS/RAF/MEK/ERK pathway is frequently activated by mutations in BRAF and NRAS. Selumetinib (AZD6244) is an oral, selective, non-ATP-competitive inhibitor of MEK1/2. Here, we describe a patient with metastatic melanoma (T1N2cM1a) with a BRAF V600E mutation. She is currently being treated with selumetinib 75 mg twice daily in a phase I trial and has shown complete response for the past 4 years. This case report raises questions regarding treatment schedule, treatment duration and management of adverse events.

Topics: Adult; Antineoplastic Agents; Benzimidazoles; Disease-Free Survival; Female; Head and Neck Neoplasms; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Thoracic Neoplasms

The development of acquired drug resistance hampers the long-term success of B-RAF inhibitor therapy for melanoma patients. Here we show (V600E)B-RAF copy-number gain as a mechanism of acquired B-RAF inhibitor resistance in 4 out of 20 (20%) patients treated with B-RAF inhibitor. In cell lines, (V600E)B-RAF overexpression and knockdown conferred B-RAF inhibitor resistance and sensitivity, respectively. In (V600E)B-RAF amplification-driven (versus mutant N-RAS-driven) B-RAF inhibitor resistance, extracellular signal-regulated kinase reactivation is saturable, with higher doses of vemurafenib down-regulating phosho-extracellular signal-regulated kinase and re-sensitizing melanoma cells to B-RAF inhibitor. These two mechanisms of extracellular signal-regulated kinase reactivation are sensitive to the MEK1/2 inhibitor AZD6244/selumetinib or its combination with the B-RAF inhibitor vemurafenib. In contrast to mutant N-RAS-mediated (V600E)B-RAF bypass, which is sensitive to C-RAF knockdown, (V600E)B-RAF amplification-mediated resistance functions largely independently of C-RAF. Thus, alternative clinical strategies may potentially overcome distinct modes of extracellular signal-regulated kinase reactivation underlying acquired B-RAF inhibitor resistance in melanoma.

Topics: Adult; Aged; Antineoplastic Agents; Base Sequence; Benzimidazoles; Cell Line; Cell Line, Tumor; DNA Copy Number Variations; Drug Resistance, Neoplasm; Exome; Extracellular Signal-Regulated MAP Kinases; Female; Gene Amplification; HEK293 Cells; Humans; Indoles; Male; MAP Kinase Kinase 1; Melanoma; Middle Aged; Proto-Oncogene Proteins B-raf; Sequence Analysis, DNA; Sulfonamides; Vemurafenib

Metastatic uveal melanoma represents the most common intraocular malignancy with very poor prognosis and no effective treatments. Oncogenic mutations in the G-protein α-subunit q and 11 have been described in about 85% of uveal melanomas and confer constitutive activation. Multiple signaling pathways are induced as a consequence of GNAQ/11 activation, which include the MEK/ERK kinase cascade. We analyzed the transcriptional profile of cell lines treated with a mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibitor to identify gene targets of activated GNAQ and to evaluate the biologic importance of these genes in uveal melanoma.. We conducted microarray analysis of uveal melanoma cell lines with GNAQ mutations treated with the MEK inhibitor selumetinib. For comparison, we used cells carrying BRAF(V600E) and cells without either mutation. Changes in the expression of selected genes were then confirmed by quantitative real-time PCR and immunoblotting.. We found that GNAQ mutant cells have a MEK-dependent transcriptional output and identified a unique set of genes that are downregulated by MEK inhibition, including the RNA helicase DDX21 and the cyclin-dependent kinase regulator CDK5R1 whereas Jun was induced. We provide evidence that these genes are involved in cell proliferation, tumor cell invasion, and drug resistance, respectively. Furthermore, we show that selumetinib treatment regulates the expression of these genes in tumor tissues of patients with metastatic GNAQ/11 mutant uveal melanoma.. Our findings define a subset of transcriptionally regulated genes by selumetinib in GNAQ mutant cells and provide new insights into understanding the biologic effect of MEK inhibition in this disease.

Topics: Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Clinical Trials, Phase II as Topic; Drug Resistance, Neoplasm; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Melanoma; Mutation, Missense; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins B-raf; Transcription, Genetic; Uveal Neoplasms

Uveal melanomas possess activation of the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT/mammalian Target of Rapamycin (mTOR) pathways. MAPK activation occurs via somatic mutations in the heterotrimeric G protein subunits GNAQ and GNA11 for over 70% of tumors and less frequently via V600E BRAF mutations. In this report, we describe the impact of dual pathway inhibition upon uveal melanoma cell lines with the MEK inhibitor selumetinib (AZD6244/ARRY-142886) and the ATP-competitive mTOR kinase inhibitor AZD8055. While synergistic reductions in cell viability were observed with AZD8055/selumetinib in both BRAF and GNAQ mutant cell lines, apoptosis was preferentially induced in BRAF mutant cells only. In vitro apoptosis assay results were predictive of in vivo drug efficacy as tumor regressions were observed only in a BRAF mutant xenograft model, but not GNAQ mutant model. We went on to discover that GNAQ promotes relative resistance to AZD8055/selumetinib-induced apoptosis in GNAQ mutant cells. For BRAF mutant cells, both AKT and 4E-BP1 phosphorylation were modulated by the combination; however, decreasing AKT phosphorylation alone was not sufficient and decreasing 4E-BP1 phosphorylation was not required for apoptosis. Instead, cooperative mTOR complex 2 (mTORC2) and MEK inhibition resulting in downregulation of the pro-survival protein MCL-1 was found to be critical for combination-induced apoptosis. These results suggest that the clinical efficacy of combined MEK and mTOR kinase inhibition will be determined by tumor genotype, and that BRAF mutant malignancies will be particularly susceptible to this strategy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Benzimidazoles; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Drug Synergism; Genotype; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Mechanistic Target of Rapamycin Complex 2; Melanoma; Mice; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Morpholines; Multiprotein Complexes; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptor, IGF Type 1; TOR Serine-Threonine Kinases; Uveal Neoplasms; Xenograft Model Antitumor Assays

Anticancer drug development is inefficient, but genetically engineered murine models (GEMM) and orthotopic, syngeneic transplants (OST) of cancer may offer advantages to in vitro and xenograft systems.. We assessed the activity of 16 treatment regimens in a RAS-driven, Ink4a/Arf-deficient melanoma GEMM. In addition, we tested a subset of treatment regimens in three breast cancer models representing distinct breast cancer subtypes: claudin-low (T11 OST), basal-like (C3-TAg GEMM), and luminal B (MMTV-Neu GEMM).. Like human RAS-mutant melanoma, the melanoma GEMM was refractory to chemotherapy and single-agent small molecule therapies. Combined treatment with AZD6244 [mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor] and BEZ235 [dual phosphoinositide-3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor] was the only treatment regimen to exhibit significant antitumor activity, showed by marked tumor regression and improved survival. Given the surprising activity of the "AZD/BEZ" combination in the melanoma GEMM, we next tested this regimen in the "claudin-low" breast cancer model that shares gene expression features with melanoma. The AZD/BEZ regimen also exhibited significant activity in this model, leading us to testing in even more diverse GEMMs of basal-like and luminal breast cancer. The AZD/BEZ combination was highly active in these distinct breast cancer models, showing equal or greater efficacy compared with any other regimen tested in studies of over 700 tumor-bearing mice. This regimen even exhibited activity in lapatinib-resistant HER2(+) tumors.. These results show the use of credentialed murine models for large-scale efficacy testing of diverse anticancer regimens and predict that combinations of PI3K/mTOR and MEK inhibitors will show antitumor activity in a wide range of human malignancies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Breast Neoplasms; Female; Humans; Imidazoles; Mammary Neoplasms, Animal; MAP Kinase Kinase Kinases; Melanoma; Mice; Neoplasms, Experimental; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases

The limitations of revolutionary new mutation-specific inhibitors of BRAF(V600E) include the universal recurrence seen in melanoma patients treated with this novel class of drugs. Recently, our lab showed that simultaneous activation of the Wnt/β-catenin signaling pathway and targeted inhibition of BRAF(V600E) by PLX4720 synergistically induces apoptosis across a spectrum of BRAF(V600E) melanoma cell lines. As a follow-up to that study, treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 also induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlates with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1. Apoptosis-resistant NRAS-mutant lines can sensitize to AZD6244 by pretreatment with AXIN1 siRNA, similar to what we previously reported in BRAF-mutant cell lines. Taken together, these findings indicate that NRAS-mutant melanoma share with BRAF-mutant melanoma the potential to regulate apoptosis upon MEK inhibition through WNT3A and dynamic regulation of cellular AXIN1. Understanding the cellular context that makes melanoma cells susceptible to this combination treatment will contribute to the study and development of novel therapeutic combinations that may lead to more durable responses.

Topics: Antineoplastic Agents; Apoptosis; Axin Protein; Benzimidazoles; beta Catenin; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Humans; Indoles; Melanoma; Mutation; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); RNA, Small Interfering; Signal Transduction; Sulfonamides; Wnt3A Protein

The discovery of potent inhibitors of the BRAF proto-oncogene has revolutionized therapy for melanoma harboring mutations in BRAF, yet NRAS-mutant melanoma remains without an effective therapy. Because direct pharmacological inhibition of the RAS proto-oncogene has thus far been unsuccessful, we explored systems biology approaches to identify synergistic drug combination(s) that can mimic RAS inhibition. Here, leveraging an inducible mouse model of NRAS-mutant melanoma, we show that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activates apoptosis but not cell-cycle arrest, which is in contrast to complete genetic neuroblastoma RAS homolog (NRAS) extinction, which triggers both of these effects. Network modeling pinpointed cyclin-dependent kinase 4 (CDK4) as a key driver of this differential phenotype. Accordingly, combined pharmacological inhibition of MEK and CDK4 in vivo led to substantial synergy in therapeutic efficacy. We suggest a gradient model of oncogenic NRAS signaling in which the output is gated, resulting in the decoupling of discrete downstream biological phenotypes as a result of incomplete inhibition. Such a gated signaling model offers a new framework to identify nonobvious coextinction target(s) for combined pharmacological inhibition in NRAS-mutant melanomas.

Topics: Animals; Apoptosis; Benzimidazoles; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase 4; Doxycycline; Female; Genes, ras; Humans; Melanoma; Mice; Mice, Knockout; Mice, Nude; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins p21(ras); Signal Transduction

AZD6244 (ARRY-142886) (AstraZeneca, Macclesfield, UK) is a novel small molecule MEK1/2 inhibitor that is currently being tested in Phase II trials. With the recent publication of human pharmacokinetic data from clinical studies, we now know the achievable levels and range of AZD6244 exposure in humans. This study aimed to describe the pharmacokinetic profile of AZD6244 in mice in order to design preclinical studies that recapitulate exposure levels in humans.. Male athymic, nude mice received subcutaneous inoculation of A375 human melanoma cells. Once tumors reached 400-700 mm(3), mice were given a single dose of either 5 or 10 mg/kg AZD6244 via oral gavage. Additionally, a subset of mice was dosed once daily for 1 week (10 mg/kg). Mice were killed and plasma and tissues were collected at various time points after the last dose. Samples were analyzed by LC/MS/MS for AZD6244 concentration. Additionally, pharmacodynamic endpoints such as tumor proliferation and ERK phosphorylation were analyzed at various time points after the last dose.. After either a single dose or at steady state, at clinically equivalent exposures, AZD6244 effectively inhibits ERK phosphorylation and suppresses proliferation. Furthermore, we describe a hysteretic relationship between the pharmacokinetics and the pharmacodynamics of AZD6244 and both target and pharmacologic responses.. The information presented herein will drive the rational design of pre-clinical studies that are not only relevant to the clinical setting, but also pave the way to understand the biological response to AZD6244 treatment.

Topics: Animal Structures; Animals; Area Under Curve; Benzimidazoles; Bromodeoxyuridine; Cell Death; Cell Line, Tumor; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Melanoma; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Protein Kinase Inhibitors; Tissue Distribution; Xenograft Model Antitumor Assays

Inhibitors of B-Raf and MEK kinases hold promise for the management of cutaneous melanomas harboring BRAF mutations. BRAF mutations are rare in uveal melanomas (UMs), but somatic mutations in the G protein α subunits Gαq and Gα11 (encoded by GNAQ and GNA11, respectively) occur in a mutually exclusive pattern in ∼80% of UMs. The impact of B-Raf and MEK inhibitors on Gα-mutant UMs remains unknown.. The impact of the B-Raf inhibitor PLX4720, the MEK inhibitor AZD6244, and the Akt inhibitor MK2206 on UM cell lines was assessed with the use of cell viability, proliferation, and apoptosis assays and immunoblot analysis.. BRAF-mutant UM cells were sensitive to both PLX4720 and AZD6244, undergoing cell cycle arrest but not apoptosis. UM cells with a Gα-protein mutation (GNAQ or GNA11) were mildly sensitive to AZD6244 but completely resistant to PLX4720. In fact, PLX4720 paradoxically increased ERK phosphorylation in Gα-mutant UM cells. The combination of AZD6244 with PLX4720 had synergistic anticancer activity in BRAF-mutant cells but not in Gα-mutant cells. The Akt inhibitor MK2206 sensitized BRAF-mutant cells to both PLX4720 and AZD6244 and sensitized Gα-mutant cells to AZD6244 but did not overcome the resistance of the Gα-mutant cells to PLX4720.. The response of UM cells to inhibition of B-Raf, MEK, and Akt depends on their genotype. Future use of such targeted therapies in clinical trials of UM patients will require careful design and patient selection based on genotype to provide personalized and effective therapy.

Topics: Apoptosis; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Cell Survival; Genotype; Heterocyclic Compounds, 3-Ring; Humans; Immunoblotting; In Situ Nick-End Labeling; Indoles; MAP Kinase Kinase Kinases; Melanoma; Precision Medicine; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Sulfonamides; Uveal Neoplasms

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF(V600) mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.. The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.. Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.

Topics: Benzimidazoles; Carrier Proteins; Cell Line, Tumor; Drug Resistance, Neoplasm; Gene Silencing; Humans; Indoles; Inhibitory Concentration 50; MAP Kinase Signaling System; Melanoma; Mitogen-Activated Protein Kinase Kinases; Mutation; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Rapamycin-Insensitive Companion of mTOR Protein; Ribosomal Protein S6 Kinases; RNA, Small Interfering; Sirolimus; Sulfonamides; TOR Serine-Threonine Kinases; Vemurafenib

Drug resistance is a central challenge of cancer therapy that ultimately leads to treatment failure. In this study, we characterized a mechanism of drug resistance that arises to AZD6244, an established mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 inhibitor currently being evaluated in cancer clinical trials. AZD6244 enhanced the expression of transcription factor FOXO3a, which suppressed cancer cell proliferation. In AZD6244-resistant cancer cells, we observed the impaired nuclear localization of FOXO3a, reduced FOXO3a-mediated transcriptional activity, and decreased the expression of FOXO3a target gene Bim after cell treatment with AZD6244. Resistant cells could be sensitized by phosphoinositide 3-kinase (PI3K)/AKT inhibitors, which are known to enhance FOXO3a nuclear translocation. Our findings define FOXO3a as candidate marker to predict the clinical efficacy of AZD6244. Furthermore, they suggest a mechanism of resistance to MEK inhibitors that may arise in the clinic yet can be overcome by cotreatment with PI3K/AKT inhibitors.

Topics: Animals; Apoptosis; Benzimidazoles; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Chlorpropamide; Chromones; Colonic Neoplasms; Drug Resistance, Neoplasm; Drug Synergism; Forkhead Box Protein O3; Forkhead Transcription Factors; HCT116 Cells; HT29 Cells; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Melanoma; Mice; Morpholines; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt

The majority of melanomas show constitutive activation of the RAS-RAF-MAP/ERK kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway. AZD6244 is a selective MEK1/2 inhibitor that markedly reduces tumor P-MAPK levels, but it produces few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the phosphoinositide 3-kinase (PI3K)-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines showed AZD6244-induced activation of AKT. In contrast, sensitive cell lines showed AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or insulin-like growth factor I receptor blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells and the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients.

Topics: Apoptosis; Benzimidazoles; Blotting, Western; Cell Proliferation; Extracellular Signal-Regulated MAP Kinases; Humans; Melanoma; Mutation; Phosphatidylinositol 3-Kinase; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Skin Neoplasms; Tumor Cells, Cultured

Melanoma is relatively resistant to chemotherapy; improved targeting of molecules critical for cell proliferation and survival are needed. Phosphatidylinositol-3 kinase (PI3K) is an important target in melanoma; however, activity of PI3K inhibitors (PI3KI) is limited. Our purpose was to assess mTOR as a cotarget for PI3K.. Using a method of quantitative immunofluorescence to measure mTOR expression in a large melanoma cohort, we studied associations with PI3K subunits, p85 and p110α. We assessed addition of the mTOR inhibitor rapamycin to 2 PI3KIs, NVP-BKM120 and LY294002. We studied in vitro activity of a novel dual PI3K/mTOR inhibitor NVP-BEZ235 and activity of the combination of NVP-BEZ235 and the MAP/ERK kinase (MEK) inhibitor AZD6244.. Strong coexpression of mTOR and p110α was observed (ρ = 0.658; P < 0.0001). Less coexpression was seen with p85 (ρ = 0.239; P < 0.0001). Strong synergism was shown between rapamycin and both PI3KIs. Activity of both PI3KIs was similarly enhanced with all rapamycin concentrations used. The dual PI3K/mTOR inhibitor effectively inhibited viability in 23 melanoma cell lines (IC(50) values in the nanomolar range), regardless of B-Raf mutation status, with resultant reduction in clonogenicity and downregulation of pAkt and pP70S6K. Synergism was seen when combining NVP-BEZ235 and AZD6244, with resultant increases in poly(ADP-ribose) polymerase and caspase-2 cleavage.. mTOR and p110α are coexpressed in melanoma. Rapamycin concentrations as low as 1 nmol/L enhance activity of PI3KIs. The dual PI3K/mTOR inhibitor NVP-BEZ235 is highly active in melanoma cells in vitro, suggesting that concurrent PI3K and mTOR targeting in melanoma warrants further investigation, both alone and in combination with MEK inhibitors.

Topics: Benzimidazoles; Cell Line, Tumor; Drug Evaluation, Preclinical; Drug Synergism; Humans; Imidazoles; Melanoma; Molecular Targeted Therapy; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Quinolines; Signal Transduction; Skin Neoplasms; Tissue Array Analysis; TOR Serine-Threonine Kinases

Genetic alterations that activate the mitogen-activated protein kinase (MAP kinase) pathway occur commonly in cancer. For example, the majority of melanomas harbor mutations in the BRAF oncogene, which are predicted to confer enhanced sensitivity to pharmacologic MAP kinase inhibition (e.g., RAF or MEK inhibitors). We investigated the clinical relevance of MEK dependency in melanoma by massively parallel sequencing of resistant clones generated from a MEK1 random mutagenesis screen in vitro, as well as tumors obtained from relapsed patients following treatment with AZD6244, an allosteric MEK inhibitor. Most mutations conferring resistance to MEK inhibition in vitro populated the allosteric drug binding pocket or alpha-helix C and showed robust ( approximately 100-fold) resistance to allosteric MEK inhibition. Other mutations affected MEK1 codons located within or abutting the N-terminal negative regulatory helix (helix A), which also undergo gain-of-function germline mutations in cardio-facio-cutaneous (CFC) syndrome. One such mutation, MEK1(P124L), was identified in a resistant metastatic focus that emerged in a melanoma patient treated with AZD6244. Both MEK1(P124L) and MEK1(Q56P), which disrupts helix A, conferred cross-resistance to PLX4720, a selective B-RAF inhibitor. However, exposing BRAF-mutant melanoma cells to AZD6244 and PLX4720 in combination prevented emergence of resistant clones. These results affirm the importance of MEK dependency in BRAF-mutant melanoma and suggest novel mechanisms of resistance to MEK and B-RAF inhibitors that may have important clinical implications.

Topics: Base Sequence; Benzimidazoles; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; MAP Kinase Kinase 1; Melanoma; Molecular Sequence Data; Mutagenesis; Mutation, Missense; Protein Binding; Protein Conformation; Proto-Oncogene Proteins B-raf; Sequence Analysis, DNA; Tumor Stem Cell Assay

Disseminated melanoma is highly therapy resistant. The finding that 66% of melanomas harbor the activating BRAF(V600E) mutation has raised expectations for targeting the Ras/RAF/mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway in melanoma. This study addresses the anti-melanoma activity of the MEK inhibitor AZD6244 (ARRY-142886).. We recently have shown that growing melanoma cells as three-dimensional collagen-implanted spheroids enhances resistance to the MEK inhibitor U0126. Here, we investigated the anti-melanoma activity of AZD6244 in two-dimensional cell culture, the three-dimensional spheroid model, and an in vivo model.. In two-dimensional cell culture, AZD6244 was cytostatic and reduced the growth of melanoma cells in a concentration-dependent fashion through the induction of G(1)-phase cell cycle arrest. In our three-dimensional spheroid model, the effects of AZD6244 were largely cytostatic and reversible, with drug washout leading to spheroid regrowth. Finally, 1205Lu cells were grown as tumor xenografts in severe combined immunodeficient mice. After tumor establishment, mice were dosed twice daily with 0, 10, or 30 mg/kg AZD6244 p.o. AZD6244 treatment decreased phospho-ERK in the tumors and significantly suppressed tumor growth. The original tumors remained viable, suggesting that AZD6244 monotherapy was largely cytostatic, and not proapoptotic in this model. Further studies showed that co-administration of AZD6244 (30 mg/kg) with docetaxel (15 mg/kg) led to tumor regression, indicating the potential for MEK inhibitor/chemotherapy drug combinations.. Inhibition of MEK is cytostatic as a monotherapy in melanoma, but cytotoxic when combined with docetaxel.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytostatic Agents; Cytotoxins; Docetaxel; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fluorescent Antibody Technique; Humans; In Situ Nick-End Labeling; Melanoma; Mice; Mice, SCID; Nucleic Acid Hybridization; Taxoids; Xenograft Model Antitumor Assays