Targeting NRAS Mutations in Advanced Melanoma

Mutations in the small GTPase NRAS are the second most common oncogenic driver in cutaneous melanoma, being found in 15%-20% of cases.¹ To date, little progress has been made in developing targeted therapy strategies for NRAS-mutant melanoma and most patients are treated with immune checkpoint inhibitors (ICI) such as anti–programmed cell death protein 1.^2-4 Individuals with NRAS-mutant melanoma who do not respond to ICI therapy or subsequently develop resistance have few other therapeutic options. Ras proteins have proven difficult to target, and although inhibitors of KRAS have recently been developed with promising clinical activity reported,^5-7 equivalent inhibitors of mutant NRAS are lacking. In the accompanying article, de Braud et al⁸ report encouraging results from the expansion arm of a phase Ib dose escalation trial of the BRAF/CRAF kinase inhibitor naporafenib (LXH254) in combination with the MEK inhibitor trametinib in NRAS-mutant melanoma. In this understanding the pathway article, we review the biology of mutant NRAS in melanoma and discuss strategies for targeting this pathway.

NRAS was first identified as a melanoma oncogene in 1984, predating the discovery of activating BRAF mutations by over 17 years.⁹ The most common NRAS mutations in melanoma (accounting for > 80%) are at Codon 61 (Q61R, Q61L, and Q61K) and serve to impair GTPase activity, locking the gene in a constitutively ON position.¹⁰ Melanoma is somewhat unusual in favoring NRAS mutations over KRAS or HRAS mutations (which are more common in other cancers). Recent work has suggested that the NRAS mutations associated with melanoma development (NRAS Q61R, Q61K, or Q61L) have an enhanced capacity to activate the mitogen-activated protein kinase (MAPK) pathway than the rarer NRAS mutations (G12D, G13D, G13R, Q61H, and Q61P).¹¹ Melanomas are uniquely addicted to the MAPK signaling pathway, a highly amplified signaling cascade comprising multiple kinases (RAF, MEK, and ERK) that link external growth signals (from growth factors and receptor tyrosine kinases [RTKs]) to the transcriptional machinery of the cell (Fig 1). Virtually, all melanomas have constitutive activity in the MAPK pathway that plays a key role in malignant transformation through its effects on cell proliferation (through the maintenance of cyclin D1 expression and downregulation of cell cycle inhibitors), the suppression of apoptosis (decreased BIM expression and BAD phosphorylation), immune escape (decreased major histocompatibility complex (MHC) class 1 expression), and invasion (modulation of the actin cytoskeleton).^1,12-14

FIG 1.

Overview of strategies to target mutant NRAS signaling in melanoma. NRAS is a small GTPase that serves to link extracellular growth factor signals through RTKs to cell growth and survival. In its mutated state, NRAS can signal in the absence of upstream growth factor signaling resulting in constitutive activation of (1) the MAPK pathway consisting of RAF/MEK/ERK, (2) the PI3K/AKT pathway consisting of PI3K/AKT/mTOR, (3) RAL-GDS, Ral A and Ral B, and (4) other signaling proteins, including AF6, RIN1, and PLCE. In NRAS-mutant melanoma, activation of MEK is dependent on deregulated adenylate cyclase and PKA signaling that relieves the inhibitory phosphorylation of CRAF at S43 and S233. Various strategies are being explored to target mutant NRAS in melanoma including single MEK inhibition and multiple combinations that target MEK in combination with components of the MAPK pathway (MEK + RAFi), PI3K/AKT (MEKi + AKTi, MEKi + mTORi), and growth pathways (MEKi + CDK4/6i). The combination of naporafenib + trametinib is predicted to work by limiting the relief of feedback inhibition and increased RTK signaling that results from single-agent MEKi therapy. This figure was created with BioRender. AMP, adenosine monophosphate; cAMP, cyclic AMP; MAPK, mitogen-activated protein kinase; MSH, melanocyte stimulating hormone; PDE, phosphodiesterase; PI3K, phospho-inositide-3 kinase; PKA, protein kinase A; RTK, receptor tyrosine kinase.

The majority of oncogenic drivers in melanoma strongly activate MAPK signaling, and mutant NRAS is no exception. NRAS-mutant melanomas and BRAF-mutant melanomas use different mechanisms to activate the MAPK pathways with NRAS-mutant melanomas using CRAF rather than BRAF. Mechanistically, NRAS-mediated MAPK activation involves the inhibition of BRAF activity (through phosphorylation of RAF at its inhibitory sites), and the disruption of the protein kinase-A–mediated inhibition of CRAF via increased phosphodiesterase IV expression and decreased accumulation of cyclic adenosine monophosphate (AMP).¹⁵ NRAS mutations activate multiple other signaling pathways in addition to MAPK (Fig 1). These include the phospho-inositide 3-kinase/AKT pathway,¹ which contributes to melanoma development through effects upon metabolism (via regulation of mTOR and ribosomal protein S6 kinase beta-1: S6K) and cell cycle entry (through inhibition of glycogen synthase-3 kinase: GSK3beta).¹⁶ Additional oncogenic pathways activated through mutant NRAS include the Ral-guanine nucleotide exchange factors (the Ral-GEFs), such as Ral-GDS, RalA and RalB, which drive cell survival, invasion, and cell cycle entry,^17,18 as well as other less characterized effectors, such as AF6, Rin1, and PLCE.¹

As selective NRAS inhibitors do not yet exist, therapeutic strategies have focused on the targeting of downstream signaling pathways. One of the first approaches to be explored both clinically and preclinically was single-agent MEKi (including selumetinib, trametinib, and binimetinib), with mixed results. In an open-label, phase III, clinical trial of the MEKi binimetinib, the overall response rate was 15% with a progression-free survival (PFS) of 2.8 months.¹⁹ Greater benefit was seen in patients who had received prior ICI therapy (PFS of 5.5 months).¹⁹ The lack of single-agent MEKi activity led to the preclinical exploration of MEK inhibitor–based combination therapies. These studies were motivated by the observation that MAPK inhibition in melanoma cells (eg, BRAFi in BRAF-mutant melanoma or MEKi in NRAS-mutant melanoma) relieves feedback inhibition within the RAF/MEK/ERK signaling pathway, leading to activation of RTKs (eg, EGFR, c-MET, PDGFR, IGF1R), signaling through BRAF and CRAF and increased output through the MAPK pathway, and other parallel pathways (such as PI3K/AKT).^20-22 This rebound signaling can be attenuated through vertical pathway inhibition, in which inhibitors of two pathways components are combined (eg, BRAF + MEK, RTK + MEK or MEK + ERK, etc). In NRAS-mutant melanoma, preclinical studies suggested that inhibition of adaptive growth/survival signaling could be achieved through the combination of MEKi + ERKi, MEKi + PI3K/AKTi, MEKi + mTORi, MEKi + RTKi, MEKi + CDK4i, or MEKi + HSP90i (to diminish RTK, CRAF, and CDK4 expression).^23-28 Some of these combinations have been evaluated clinically. In an open-label, phase II, clinical trial, the combination of trametinib with the AKT inhibitor GSK2141795 did not lead to any objective responses in a cohort of patients with NRAS-mutant melanoma.²⁹ By contrast, the MEKi + CDK4/6i combination of binimetinib + ribociclib did show clinical activity and was associated with a response rate of 19% and a PFS of 3.7 months.³⁰ The response rate in patients whose melanomas had NRAS mutations in addition to concurrent CDK2NA, CDK4, or cyclin D1 alterations was > 32%.³⁰

A more promising rational strategy to target MEK and the adaptive signaling response is the combination of a MEKi with a pan-RAFi³¹ (Fig 1). In preclinical studies, the MEKi + Pan-RAFi combination led to increased apoptosis and cell cycle arrest in melanoma cell lines with NRAS mutations.³¹ Similar findings were seen in other Ras-mutant tumors when the BRAF/CRAF inhibitor naporafenib was combined with the MEKi trametinib.³² In the phase Ib study of naporafenib + trametinib, reported here by de Braud et al,⁸ the evidence of antitumor activity was seen in > 46% of patients with heavily pretreated NRAS-mutant melanoma. These encouraging findings suggest that targeted therapies could be developed for NRAS-mutant melanoma. Heading forwards, the sequencing of the naporafenib + trametinib combination could prove critical. There is already evidence that patients who receive ICI as their first-line therapy have better outcomes with subsequent single-agent MEKi therapy.¹⁹ Other studies have demonstrated that upfront anti–programmed cell death protein 1 therapy can improve responses to the BRAF-MEKi combination by potentially priming the immune response.^25,33 Although much remains to be done, the results of de Braud et al⁸ offer the possibility of developing effective targeted therapies for patients with NRAS-mutant melanoma.

AUTHOR CONTRIBUTIONS

Conception and design: Keiran S.M. Smalley

Collection and assembly of data: Manali S. Phadke

Manuscript writing: All authors

Final approval of manuscript: All authors

Accountable for all aspects of the work: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Targeting NRAS Mutations in Advanced Melanoma

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