Targeting the RAS pathway in melanoma

Abstract

Metastatic melanoma is a highly lethal type of skin cancer and is often refractory to all traditional chemotherapeutic agents. Key insights into the genetic makeup of melanoma tumors have led to the development of promising targeted agents. An activated RAS pathway, anchored by oncogenic BRAF, appears to be the central motor driving melanoma proliferation. Although recent clinical trials have brought enormous hope to patients with melanoma, adverse effects and novel escape mechanisms of these inhibitors have already emerged. Definition of the limits of the first successful targeted therapies will provide the basis for further advances in management of disseminated melanoma. In this review, the current state of targeted therapy for melanoma is discussed, including the potent BRAF(V600E) inhibitor vemurafenib.

Figure 1

Key mutational and therapeutic targets in melanoma. The RAS signaling network is rife with cancer-associated mutations. BRAF is the most commonly activated oncogene in cutaneous melanomas (cut mels), followed by NRAS. Upstream of RAS, the c-KIT receptor tyrosine kinase (RTK) is amplified or activated in a substantial fraction of acral and mucosal melanomas. It thus seems that the RTK–NRAS–BRAF–MEK–ERK cascade represents a central axis (highlighted in green) that is activated in nearly all melanomas. Parallel to this axis is the PI3-K pathway, which is also activated in melanomas either through loss of PTEN or activation of AKT3. In addition, it has recently been shown that GNAQ and GNA11, which are transducers of the endothelin receptor signal (ETR), are mutated in ocular melanomas. These heightened signaling events lead to both increased transcription of survival genes (such as MITF) and enhanced pro-survival factors in mitochondria through the regulation of BCL2 family proteins (red, pro-apoptotic; green, pro-survival). MITF, which is a master regulator of melanocyte survival, is also amplified in melanomas and a target of the EWS-ATF1 fusion protein described in clear cell sarcomas (so-called melanoma of the soft parts). Drugs known to inhibit the central axis and with a potential therapeutic impact on melanoma are listed in the purple boxes.

Figure 2

BRAF inhibition and development of vemurafenib. (a) Domain structure of BRAF showing the CR domains and the location of the most common mutations in exons 11 and 15. (b) Chemical structure of PLX4720 and PLX4032 (vemurafenib). These compounds are competitive ATP protein kinase inhibitors (class I) selective for the BRAF^V600E mutant protein. Also shown is co-structure of PLX4720 with the BRAF kinase. It has been postulated that the key to the specificity of PLX4720 lies in the differential interaction of the sulfonamide moiety (red circle) with the Asp and Phe residues of the conserved kinase Asp-Phe-Gly (DFG) sequence. In the active conformation, the nitrogen atom of the sulfonamide moiety is deprotonated and interacts with the main-chain NH group of Asp-594, whereas the oxygen atoms of the sulfonamide form hydrogen bonds to the NH group of Phe-595 and the side chain of Lys-483. Crystallographic data support a selectivity model in which the deprotonated sulfonamide interacts more favorably with the active kinase conformation that is characteristic of BRAF^V600E. (c) In tumor xenografts, a BRAF^V600E mutated melanoma line (1205Lu) showed significantly greater response to PLX4720 (100 mg/kg by oral gavage) compared to a BRAF^WT line (C8161). (d) Dramatic tumor shrinkage after treatment with PLX4032 in a patient with metastatic BRAF^V600E melanoma. Panels b and c and panel d are reproduced with permission from [56] and [83], respectively.

Figure 3

Models of RAF activation and inhibition, (a) In cells with mutated BRAF (Br^V600E), RAS is inactive, cytoplasmic and unable to induce RAF dimerization. MEK phosphorylation and activation occur almost exclusively via BRAF^V600E and thus are effectively silenced by a selective BRAF inhibitor (blue circle). This would explain the dramatic tumor shrinkage early in treatment with vemurafenib. In some cells with wild-type BRAF, RAS is activated (RAS*-GTP), presumably by mutagenesis (e.g. NRAS mutations) or other upstream receptor events. There are at least two models of signal transduction in these RAS-stimulated tumors. In the transactivation model (b), active RAS*-GTP mobilizes to the membrane and induces homodimerization or heterodimerization of wild-type BRAF and CRAF proteins (Br/Cr), which in turn initiates signaling. At low concentrations, an ATP-competitive RAF inhibitor may occupy the active site of one of the RAF protomers. Inhibitor binding to the active site of one RAF kinase induces a conformational change that facilitates transactivation of the other RAF molecule in the dimer; therefore, there is paradoxical enhancement of MEK/ERK signaling through the uninhibited partner. At higher inhibitor concentrations, both RAF molecules are inhibited and MEK/ERK signaling is completely abolished. In the translocation model (c), wild-type BRAF (Br) remains largely inactive in the cytoplasm owing to autoinhibitory signals. Activated RAS*-GTP triggers MAPK signaling through CRAF (Cr). When wild-type BRAF binds a selective ATP-competitive BRAF inhibitor, the BRAF molecule is recruited to the membrane, thereby enhancing the RAS*-GTP/CRAF interaction and signaling (as in b). With pan-RAF inhibitors, wild-type BRAF may still enhance the RAS*-GTP/CRAF interaction, but MEK/ERK signaling is suppressed because CRAF activity itself is concurrently abrogated by inhibitor binding. In situations in which CRAF harbors a gatekeeper mutation, such as p.Thr421Asn (Cr^T421N), a pan-RAF agent inhibits BRAF, which then transactivates CRAF^T421N; however, CRAF^T421N is not inhibited by the pan-RAF agent because the mutation prevents drug binding. Both models suggest mechanisms by which BRAF inhibition may lead to increased stimulation of CRAF in RAS-mutated cells or growth-factor-stimulated cells, (d) Mechanisms of BRAF inhibitor resistance include activation of receptor tyrosine kinases (RTKs) such as PDGFR and IGF1R, CRAF and other protein kinases (e.g. COT), along with mutational activation of NRAS and MEK (MEK*).