Summary BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAFV600E melanoma cells with the BRAF inhibitor SB-590885; these cells are cross resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGFR-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.

Summary Despite success with BRAFV600E–inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multi-drug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (OXPHOS) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1Bhigh subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anti-cancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.

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The expression of cyclin D1 in mid-G1 phase is associated with sustained ERK activity, and we show here that Rho is required for the sustained ERK signal. However, we also report that Rho inhibits an alternative Rac/Cdc42-dependent pathway, which results in a strikingly early G1-phase expression of cyclin D1. Thus, cyclin D1 is induced in mid-G1 phase because a Rho switch couples its expression to sustained ERK activity rather than Rac and Cdc42. Our results show that Rho is crucial for maintaining the correct timing of cyclin D1 expression in G1 phase and describe a new role for cytoskeletal integrity in the regulation of cell cycle progression.

An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the non-canonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Further, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In melanoma patients treated with the BRAF inhibitor, Vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance.

Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E-and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a timedependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.

Summary 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.

Melanoma arises through the accrual of mutations in genes critical for proliferation and survival. Although melanoma had been traditionally conceptualized as a cell-autonomous event, increasing evidence supports the notion that these tumors are not isolated entities but rather depend, interact with, and react to the adjacent microenvironment. Melanoma is composed of not only the malignant cells but also the supporting stroma, which includes fibroblasts, endothelial cells, immune cells, soluble molecules, and the extracellular matrix (ECM). Tumor cells actively interact with the microenvironment in a bidirectional manner through molecular signals that modulate the malignant phenotype. This article briefly reviews the molecular basis of melanomagenesis as well as the interplay of melanoma with other cells of the tumor microenvironment and components of the ECM. It also discusses the influence of the microenvironment on therapeutic targeting of melanoma, highlighting recent studies that propose novel strategies to target tumor–microenvironment interactions.