MEK Inhibitors In Melanoma Research Articles

Biguanides and targeted anti-cancer treatments.

Drug repositioning and molecularly targeted novel drug development are two contrasting strategies in cancer research. Yet, certain combinations of the two may bring together the best of both worlds. Drug repositioning, i.e. the use of a drug for treating diseases other than the drug-specified, is a strategy to advance patient care more quickly and efficiently. The idea was promoted in 2012 through the Discovering New Therapeutic Uses for Existing Molecules program, initiated by NIH. Several drugs used for other purposes have proved to be effective anti-cancer agents, including thalidomide, aspirin, and celecoxib. In contrast, novel drug development is a lengthy process. Molecularly tailored drugs fail for various reasons including inadequate bioavailability and adverse side effects, and are often very expensive. Despite drawbacks to targeted drug development, there are also successes in this area of research. Cancer therapeutics designed to inhibit kinases involved in cell growth and survival pathways are potent and selective in the short term. By directly blocking cell signaling, often at the source of over-activation, these drugs kill cancer cells. One example of success in targeted therapy is the development of BRAFV600E inhibitors, vemurafenib and dabrafenib, and their ability to prolong progression-free survival time in melanoma patients. However, a major problem with monotherapy is the inevitable development of resistance. Even with combination treatment using specific inhibitors, such as BRAFV600E and MEK inhibitors in melanoma, improvement in patient outcome is limited [1,2] and resistance emerges. There is increasing interest in combining targeted inhibitors with common metabolic regulators to hinder resistance. This unique combination reaps both the intrinsic benefits of repurposing drugs and the selectivity of targeted therapy. The major biguanides, metformin and phenformin, have known pharmacokinetics, high safety profiles, and are relatively inexpensive. Metformin in particular is widely used for treatment of patients with type 2 diabetes. Although there is evidence in literature for both pro-cancer and anti-cancer effects of metformin on cancer cells, a clear association between metformin therapy and reduced risk of cancer in diabetic patients exists. Recent studies point out direct cellular benefits of combining biguanides with current targeted therapy.

Game of isoforms: PI3K β-sparing inhibitor is coming.

Cutaneous melanoma has been at the forefront of targeted therapy breakthroughs over the last 5 years. The FDA approval of BRAF and MEK inhibitors for the treatment of late-stage, mutant V600E/K BRAF-harboring melanoma patients has been heralded due to high response rates and improvements in progression-free survival, but the durable effects of these inhibitors are limited. Alterations in the phosphoinositide 3-kinase (PI3K) pathway including inactivation of PTEN (a tumor suppressor that normally down-regulates PI3K signaling) and, less frequently, mutations in PIK3CA (the gene encoding the α isoform of the PI3K catalytic subunit) have been identified in mutant BRAF melanomas. Furthermore, PI3K pathway signaling is often up-regulated in association with resistance to mutant BRAF and/or MEK targeting agents. Despite the evidence highlighting the importance of the PI3K pathway in melanoma, initial testing of class I PI3K inhibitors in patients has not produced dramatic results. An issue for combinatorial studies is the overlapping toxicities of MAPK pathway inhibitors and PI3K-targeting agents that limits their effective dosing. A recent study from Deuker and colleagues provides important preclinical advances in this area by examining the PI3K isoform dependencies in mutant BRAF melanomas. PI3K functions as a heterodimer with a p110 catalytic subunit and a regulatory subunit. Four isoforms of p110 subunits exist: α, β, δ and γ (Jean and Kiger, 2014; Yuan and Cantley, 2008). p110α and p110β are ubiquitously expressed, whereas δ and γ isoforms display highest expression in immune cells. Importantly, isoform-selective PI3K inhibitors have been developed to target tumor dependencies and minimize toxicities in non-tumor cells. Deuker et al. utilized these selective inhibitors to determine the PI3K isoform dependency of mutationally active PI3Kα and PTEN-deficient subgroups of mutant BRAF melanomas using cell lines derived from mouse models and human patients. In vitro, PTEN-deficient lines were less sensitive to the α-isoform PI3K inhibitor, BYL719, compared to mutant H1047R PI3Kα cells. In contrast to other cancer types, in which PTEN-deficient tumors are often PI3Kβ-dependent (Wee et al., 2008), PTEN-null melanomas did not rely on PI3Kβ. Growth of PTEN-null mutant BRAF melanomas was blocked by a PI3Kα/δ/γ-targeting but β-sparing inhibitor, GDC-0032. In addition, the δ/γ inhibitor, IPI145, enhanced the effects of BYL719 suggesting involvement of the δ/γ isoforms in melanoma. PI3Kα-dependency is also suggested to be present in BRAF V600E/WT PTEN melanomas, which represent a substantial proportion of mutant BRAF melanomas, although confirmation is warranted in a larger panel of BRAF V600E/WT PTEN melanoma models. “More dramatic tumor regressions were observed in vivo with the combination of BRAF-MEK pathway and PI3K inhibitors. ” A long-term limitation to both BRAF and MEK inhibitors in melanoma is acquired drug resistance and patients who develop resistance have few second-line treatment options. In addition to up-front combination, treatment of MEK inhibitor-resistant tumors with the PI3K β-sparing inhibitor resulted in a cytostatic response. These data were generated from a single MEK inhibitor-resistant tumor. As multiple mechanisms of resistance are likely to occur, it will be important to further explore effects in cultured- and patient-derived BRAF and MEK inhibitor-resistant models. Overall, the study presented by Deuker et al., provides an important preclinical advance in targeting the PI3K pathway in both first-line combinations as well as a possible second-line salvage option for BRAF-MEK pathway resistant melanomas. These findings should propel investigation of methods to quantitatively and non-invasively measure PI3K signaling in preclinical in vivo melanoma models and to determine the most effective dosing regimens. Given the high degree of immune reactivity of melanomas and remarkable advances in immune-based therapies, other key issues will be to determine the effect of targeting PI3K on infiltration of immune cells into melanomas and the extent to which PI3K-targeting agents can be combined with immune-based therapies. Advances in mutant BRAF melanomas will lead to studies addressing the importance of PI3K isoform specific targeting in wild-type BRAF subsets of melanoma for which there is an unmet need for new therapeutic strategies.

Highlights of This Issue

The epidermal growth factor receptor (EGFR) is activated through corresponding gene amplification, and/or mutation in approximately 40% of glioblastomas, and the large subgroup of glioblastoma patients whose tumors have this molecular characteristic are thought to be excellent candidates for benefitting from EGFR-targeted therapy. However, available EGFR inhibitors have shown poor activity in treating glioblastoma patients, in large part due to poor CNS penetration. In this report, Yoshida and colleagues show that NT113, a novel pan-ERBB inhibitor, has excellent CNS penetration and activity against glioblastoma xenografts with activated EGFR, thereby supporting the clinical development of NT113 for treating this cancer.Clinical resistance to BRAF and MEK inhibitors demonstrates the need for further drug combination approaches in melanoma. The use of AT13387, an HSP90 inhibitor currently in clinical trials, to tackle resistance was investigated. AT13387 was effective in melanoma models with acquired resistance to BRAF and MEK inhibitors, and also delayed the emergence of resistance when combined upfront with vemurafenib in sensitive models. These data suggest HSP90 inhibition could be used to combat clinical resistance to BRAF and MEK inhibitors in melanoma. Significantly, frontline combinations could delay the emergence of resistance and extend progression-free survival, supporting clinical investigation in BRAF-mutant melanoma.Effective lactate transport is crucial for tumor cell homeostasis. AZD3965 inhibits the function of the lactate transporter MCT1, and in small cell lung cancer cells (SCLC) treatment with AZD3965 causes an increase in glycolysis via inhibition of lactate uptake. The treatment of SCLC xenografts with a combination of AZD3965 and fractionated radiation resulted in a significantly greater therapeutic effect than the use of either modality alone. These data support the notion of combining MCT1 inhibition with radiotherapy in the treatment of SCLC and other solid tumors.Triple-negative breast cancer (TNBC) patients have poor treatment outcomes and there are no validated targets for therapy. Of the 106 patients with metastatic TNBC treated in our phase I clinic, patients treated on matched therapies had higher response rates, and improved progression-free survival (PFS) than patients on nonmatched therapies. Patients on combinations of chemotherapy and targeted agents had improved PFS compared with those on either chemotherapy or targeted therapy alone. In particular, combinations that included chemotherapy with antiangiogenic therapy and/or PI3K/AKT/mTOR pathway inhibitors showed improved PFS. Currently, next-generation sequencing in a large prospective study is ongoing.

The next battle line against melanoma

Boston and Los Angeles researchers have reported mechanisms of resistance to BRAF and MEK inhibitors in melanoma. The work points to targets downstream of BRAF that could lead to combination therapies to forestall resistance to marketed melanoma therapeutics.

Concurrent MEK2 Mutation and BRAF Amplification Confer Resistance to BRAF and MEK Inhibitors in Melanoma

Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.

Abstract 3405: Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines.

Abstract Background: Resistance to targeted therapies represents a key hurdle in the treatment of cancer. Preclinical data suggest that resistance is frequently driven by redundant growth factor signaling. For example, HGF has been reported to mediate resistance to BRAF inhibitors in V600E BRAF mutant melanoma. Furthermore, clinical data support these findings as circulating and tumor HGF levels have been shown to correlate with outcome in BRAF mutant melanoma patients treated with vemurafenib. In this study, we evaluated the ability of HGF / MET signaling to attenuate the effects of BRAF and MEK inhibitors in the context of V600E BRAF and NRAS mutant cell lines. Our data suggest that combined blockade of MET and MAPK pathway signaling may be required for a robust and durable clinical response in melanoma. Methods: To confirm the ability of HGF to rescue V600E BRAF mutant melanoma cell lines from the effects of vemurafenib, G361 and SKMEL5 cells were treated with vemurafenib alone or in combination with HGF. Time lapse imaging, viability assays and cell cycle analysis were used to measure treatment effects. Analogous experiments were performed with the selective MEK inhibitor PD0325901. Combinations of a potent and selective MET inhibitor (Compound A) with vemurafenib or PD0325901 were tested to evaluate their ability to attenuate the HGF-mediated resistance. To identify other cell lines rescued by HGF, thirteen V600E BRAF mutant melanoma cell lines were treated with a dose response matrix of vemurafenib and HGF. The ability of six additional growth factors to rescue cells from the effects of vemurafenib was also tested. Three NRAS mutant melanoma cell lines were also screened for HGF-mediated resistance to PD0325901. Results: Nine of thirteen V600E BRAF mutant melanoma cell lines were rescued from the effects of vemurafenib by co-treatment with HGF. Detailed analysis of G361 and SKMEL5 cells revealed that HGF was also capable of rescuing the cells from the effects of PD0325901. This HGF-mediated rescue was attenuated by treatment with combinations of vemurafenib + compound A or PD0325901 + compound A. Mechanistic cell cycle analysis of the HGF-mediated rescue of vemurafenib in G361 cells revealed an increase in proliferating cells and a reduction in growth arrested cells, whereas rescue of PD0325901 treatment occurred through a reduction in the percentage of dead or dying cells. Of the seven growth factors tested, HGF was the predominate growth factor capable of conferring resistance to vemurafenib. Furthermore, HGF rescued one of three NRAS mutant melanoma cell lines from the effects of PD0325901, highlighting the breadth of HGF-mediated resistance in melanoma cell lines. Conclusion: These findings demonstrate the potential role for HGF / MET signaling in mediating resistance to BRAF and MEK inhibitors in melanoma, and support the clinical evaluation of MET kinase inhibitors and neutralizing antibodies to HGF in this setting. Citation Format: Sean Caenepeel, Elaina Cajulis, Rick Kendall, Angela Coxon, Paul Hughes. Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2013-3405

Abstract 2284: Phase II trial of MEK inhibitor selumetinib (AZD6244) in patients with BRAFV600E/K- or NRAS-mutated melanoma.

Abstract Our pre-clinical in vitro data suggested that all BRAF-mutated cell lines, and some NRAS-mutated melanoma are sensitive to MEK inhibition. BRAFwt/NRASwt were uniformly resistant. Among the sensitive cell lines, those with enhanced activation of the PI3K/AKT pathway did not undergo apoptosis. This is consistent with a recent phase II trial with Selumetinib, an allosteric MEK inhibitor, which showed a 6% response rate in unselected melanoma patients. Retrospectively, 5 of 6 of the responders were found to harbor BRAFV600E mutations. We hypothesized that treatment of BRAF-mutated or NRAS-mutated melanomas with Selumetinib will induce clinical responses only in the subset of tumors in which the PI3K/AKT pathway is not activated. In this phase II trial, melanoma patients with either BRAF or NRAS mutation were stratified based on phospho-AKT (pAKT) expression (high vs. low), as measured by immunohistochemistry staining of pre-treatment tumor specimens. Patients were treated with Selumetinib 75 mg p.o. daily in 28-day cycles. We found that the incidence of high pAKT melanoma tumors was about 4 times higher than low pAKT tumors. None of the 10 patients in the high pAKT cohort responded, although 4 patients showed stable disease for ≥4 months. This cohort was closed to further accrual. In contrast, in the low pAKT group, 1 patient had a true partial response (PR) and 2 others had near PRs. One of these patients had to discontinue treatment due to toxicity; the other had his remaining tumor resected. This cohort was closed due to slow patient accrual. We used an exon capture, next-generation sequencing assay to define the mutational status of 230 cancer associated genes in pre-treatment tumors, derived from patients belonging to both cohorts. The assay detects point mutations, small indels and copy number alterations. Among the two low pAKT patients who were resistant to MEK inhibition, one had a mutation in MAP2K1 that encodes for a K57N mutation in helix A of MEK1. We speculate that this is an activating mutation, since a missense mutation in the amino acid just proximal (Q56P) was previously shown to be highly activating. The other non-responding patient in the low pAKT cohort had an activating mutation in EGFR. Both of these could drive increased activation of the MAPK pathway, thus explaining the resistance to the drug. We conclude that future trials with MEK inhibitors in melanoma should exclude patients with high pAKT tumors. The occurrence of complex genetic changes in melanoma requires studies to better stratify the patients according the prediction of response to MEK inhibitors. Citation Format: Federica Catalanotti, David B. Solit, Melissa P. Pulitzer, Michael F. Berger, Sasinya N. Scott, Tunc Iyriboz, Mario E. Lacouture, Katherine S. Panageas, Jedd D. Wolchok, Richard D. Carvajal, Gary K. Schwartz, Neal Rosen, Paul B. Chapman. Phase II trial of MEK inhibitor selumetinib (AZD6244) in patients with BRAFV600E/K- or NRAS-mutated melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2284. doi:10.1158/1538-7445.AM2013-2284

Melanoma adapts to RAF/MEK inhibitors through FOXD3-mediated upregulation of ERBB3

The mechanisms underlying adaptive resistance of melanoma to targeted therapies remain unclear. By combining ChIP sequencing with microarray-based gene profiling, we determined that ERBB3 is upregulated by FOXD3, a transcription factor that promotes resistance to RAF inhibitors in melanoma. Enhanced ERBB3 signaling promoted resistance to RAF pathway inhibitors in cultured melanoma cell lines and in mouse xenograft models. ERBB3 signaling was dependent on ERBB2; targeting ERBB2 with lapatinib in combination with the RAF inhibitor PLX4720 reduced tumor burden and extended latency of tumor regrowth in vivo versus PLX4720 alone. These results suggest that enhanced ERBB3 signaling may serve as a mechanism of adaptive resistance to RAF and MEK inhibitors in melanoma and that cotargeting this pathway may enhance the clinical efficacy and extend the therapeutic duration of RAF inhibitors.

Effect of SMURF2 Targeting on Susceptibility to MEK Inhibitors in Melanoma

BackgroundThe 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.MethodsWe 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.ResultsActivation 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).ConclusionsTargeting SMURF2 may be a novel therapeutic approach for increasing the antitumor efficacy of MEK inhibitors.

Targeted drug development in melanoma and nonsmall cell lung cancer: BRAF, MEK, and ALK inhibitors

In the past few decades, many advances have been witnessed in the development of personalized molecular therapies, especially in the areas of melanoma and nonsmall cell lung cancer (NSCLC) among all other medical oncology fields. These therapies can be roughly divided into three categories at present: (1) targeting membrane-bound receptor tyrosine kinase (RTK), using either monoclonal antibodies or tyrosine kinase inhibitor (TKI), (2) targeting intracellular mitogen-activated protein kinase (MAPK) pathway, and (3) targeting intracellular phosphoinositide 3-kinase (PI3K) pathway. Currently, melanoma research focuses on MAPK pathway, spending efforts to clarify the intricate interactions in RAS–MEK–ERK-signaling cascades, whereas in the field of NSCLC, most achievements have been made targeting RTKs. This review will discuss the recent movements of drug development in these two areas, specifically BRAF and MEK inhibitors in melanoma, and ELM4-ALK inhibitors in lung cancer, along with the mechanisms of drug resistance and potential future strategies.