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Searching for the Chokehold of NRAS Mutant Melanoma

  • Christian Posch
    Correspondence
    Correspondence: Christian Posch, Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Juchgasse 25, 1030 Vienna, Austria.
    Affiliations
    Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA

    Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Vienna, Austria
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  • Igor Vujic
    Affiliations
    Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA

    Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Vienna, Austria
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  • Babak Monshi
    Affiliations
    Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Vienna, Austria
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  • Martina Sanlorenzo
    Affiliations
    Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA

    Department of Medical Sciences, Section of Dermatology, University of Turin, Turin, Italy
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  • Felix Weihsengruber
    Affiliations
    Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Vienna, Austria
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  • Klemens Rappersberger
    Affiliations
    Department of Dermatology, The Rudolfstiftung Hospital, Academic Teaching Hospital, Medical University Vienna, Vienna, Austria
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  • Susana Ortiz-Urda
    Affiliations
    Department of Dermatology, Mt. Zion Cancer Research Center, University of California San Francisco, San Francisco, California, USA
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Open ArchivePublished:May 07, 2016DOI:https://doi.org/10.1016/j.jid.2016.03.006
      Up to 18% of melanomas harbor mutations in the neuroblastoma rat-sarcoma homolog (NRAS). Yet, decades of research aimed to interfere with oncogenic RAS signaling have been largely disappointing and have not resulted in meaningful clinical outputs. Recent advances in disease modeling, structural biology, and an improved understanding of RAS cycling as well as RAS signaling networks have renewed hope for developing strategies to selectively block hyperactive RAS function. This review discusses direct and indirect blocking of activated RAS with a focus on current and potential future therapeutic approaches for NRAS mutant melanoma.

      Abbreviations:

      ERK (extracellular signal-regulated kinase), GEF (guanine nucleotide exchange factor), HRAS (Harvey-rat-sarcoma homolog), KRAS (Kirsten-rat-sarcoma homolog), MAPK (mitogen-activated protein kinase), NRAS (neuroblastoma rat-sarcoma homolog), RAL (retinaldehyde), RAS (rat-sarcoma homolog), RTK (receptor tyrosine kinase)

      Introduction

      A central regulator of prosurvival signaling in the mitogen-activated protein kinase (MAPK) pathway is rat-sarcoma homolog (RAS). The four highly homologous members of the RAS protein family neuroblastoma-, Harvey-, and Kirsten-RAS (NRAS, HRAS, KRAS4A, KRAS4B) are ubiquitously expressed and control cellular proliferation, differentiation, and survival (
      • Cox A.D.
      • Fesik S.W.
      • Kimmelman A.C.
      • Luo J.
      • Der C.J.
      Drugging the undruggable RAS: mission possible?.
      ,
      • Malumbres M.
      • Barbacid M.
      RAS oncogenes: the first 30 years.
      ). Dysregulated RAS signaling, mainly mediated by oncogenic mutations, is found in about one-third of all cancers with NRAS being most frequently mutant in melanoma (18%) (
      • Lee J.-H.
      • Choi J.-W.
      • Kim Y.-S.
      Frequencies of BRAF and NRAS mutations are different in histological types and sites of origin of cutaneous melanoma: a meta-analysis.
      ,
      • Roberts P.J.
      • Der C.J.
      Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer.
      ). Hence, the NRAS protein would be an attractive target for melanoma therapy; however, decades of research aimed toward direct blocking of the mutant oncogene in vivo have been largely unsuccessful. To date, indirect inhibition of mutant RAS by downstream pathway interference is one of the clinically most promising strategies (
      • Ascierto P.A.
      • Schadendorf D.
      • Berking C.
      • Agarwala S.S.
      • van Herpen C.M.
      • Queirolo P.
      • et al.
      MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study.
      ,
      • Carlino M.S.
      • Todd J.R.
      • Gowrishankar K.
      • Mijatov B.
      • Pupo G.M.
      • Fung C.
      • et al.
      Differential activity of MEK and ERK inhibitors in BRAF inhibitor resistant melanoma.
      ,
      • Kwong L.N.
      • Costello J.C.
      • Liu H.
      • Jiang S.
      • Helms T.L.
      • Langsdorf A.E.
      • et al.
      Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma.
      ,
      • Tolcher A.W.
      • Patnaik A.
      • Papadopoulos K.P.
      • Rasco D.W.
      • Becerra C.R.
      • Allred A.J.
      • et al.
      Phase I study of the MEK inhibitor trametinib in combination with the AKT inhibitor afuresertib in patients with solid tumors and multiple myeloma.
      ,
      • Vu H.L.
      • Aplin A.E.
      Targeting TBK1 inhibits migration and resistance to MEK inhibitors in mutant NRAS melanoma.
      ). In addition, recent discoveries indicate that RAS activity is also increased by loss of function mutations in the negative regulator of RAS, NF1, which is mutated in approximately 14% of patients with melanoma (
      Cancer Genome Atlas Network
      Genomic Classification of Cutaneous Melanoma.
      ,
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ). Such discoveries not only highlight the importance of RAS signaling in melanoma but also have the potential to open up new avenues for indirect targeting of NRAS activity.
      Latest developments in systems and molecular biology highlight that targeting of mutant RAS is not a dead end; instead, it needs to be revisited systematically. Recently launched programs, such as “The RAS Initiative” by the National Cancer Institute (http://RasCentral.org), reflect researchers’ optimism to advance therapy for RAS mutant malignancies (
      • Thompson H.
      US National Cancer Institute’s new Ras project targets an old foe.
      ).
      This review aims to give an overview of recent discoveries in direct and indirect targeting of mutant RAS proteins with a focus on NRAS mutant melanoma and discusses opportunities and limitations for their future clinical application.

      Background—NRAS Function

      RAS proteins function as small GTPases with low intrinsic catalytic activity, transducing signals from membrane localized receptor tyrosine kinases (RTKs) to the nucleus. Cycling of the RAS protein between a GTP-bound active state and a GDP-bound inactive state is catalyzed by guanine nucleotide exchange factors (GEFs), such as SOS1, and GTPase activating proteins (GAPs), such as NF1 (
      • Bos J.L.
      • Rehmann H.
      • Wittinghofer A.
      GEFs and GAPs: critical elements in the control of small G proteins.
      ,
      • Plowman S.J.
      • Hancock J.F.
      Ras signaling from plasma membrane and endomembrane microdomains.
      ,
      • Rajalingam K.
      • Schreck R.
      • Rapp U.R.
      • Albert S.
      Ras oncogenes and their downstream targets.
      ). The docking sites for effector proteins, such as rapidly accelerated fibrosarcoma (RAF) and phosphatidylinositol-3-kinases (PI3K), are located in the “effector lobe,” whereas the “allosteric lobe” contains affinity hot spots for direct interaction with membrane components. Hotspots for membrane localization and the C-terminal hypervariable region distinguish the structures of the otherwise highly homologous RAS family members and are important loci for posttranslational (lipid) modifications (
      • Buhrman G.
      • Holzapfel G.
      • Fetics S.
      • Mattos C.
      Allosteric modulation of Ras positions Q61 for a direct role in catalysis.
      ,
      • Gorfe A.A.
      • Grant B.J.
      • McCammon J.A.
      Mapping the nucleotide and isoform-dependent structural and dynamical features of Ras proteins.
      ) (Figure 1).
      Figure 1
      Figure 1Conceptual schema of NRAS cycling and mutation hotspots. (a) Wild-type NRAS cycles between an inactive GDP bound and an active GTP bound state. Upstream signals trigger the activation of NRAS which is regulated by GEFs (such as SOS1). NRAS deactivation is catalyzed by GAPs (such as NF1). (b) NRAS mutations are located at codon 12/13, affecting the P-loop (Walker A motif) of the protein. Mutations at codon 61 impair the intrinsic catalytic activity of NRAS located in the switch II domain of the protein. (c) Mutant NRAS impairs normal protein cycling, locking the protein in its active GTP bound state. GAP, GTPase activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factors; GTP, guanosine triphosphate; HVR, hypervariable region; NRAS, neuroblastoma rat-sarcoma homolog; RTK, receptor tyrosine kinases; SWI/II, switch I/II.
      The mutational hotspots of NRAS are codon 61 (approximately 85% of cases) and to a much smaller extent codons 12 and 13 (
      • Hodis E.
      • Watson I.R.
      • Kryukov G.V.
      • Arold S.T.
      • Imielinski M.
      • Theurillat J.-P.
      • et al.
      A landscape of driver mutations in melanoma.
      ). While mutant NRAS(Q61) disrupts the GTPase activity of RAS, locking it in its active conformation, NRAS(G12) and NRAS(G13) mutations affect the Walker A-motif (p-loop) of the protein, thus decreasing its sensitivity to GAPs (
      • Curtin J.A.
      • Fridlyand J.
      • Kageshita T.
      • Patel H.N.
      • Busam K.J.
      • Kutzner H.
      • et al.
      Distinct sets of genetic alterations in melanoma.
      ,
      • Fedorenko I.V.
      • Gibney G.T.
      • Smalley K.S.M.
      NRAS mutant melanoma: biological behavior and future strategies for therapeutic management.
      ,
      • Smith M.J.
      • Neel B.G.
      • Ikura M.
      NMR-based functional profiling of RASopathies and oncogenic RAS mutations.
      ) (Figure 1). Mutations in G12/13 and Q61 can all be described as activating, yet, they affect the NRAS protein in distinct ways. Small differences in intrinsic hydrolysis rate and minimal conformational changes, which have for the most part been disregarded as biologically irrelevant, impact (mutation specific) changes in downstream pathway activation and RAS cycling (
      • Burd C.E.
      • Liu W.
      • Huynh M.V.
      • Waqas M.A.
      • Gillahan J.E.
      • Clark K.S.
      • et al.
      Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma.
      ,
      • Smith M.J.
      • Neel B.G.
      • Ikura M.
      NMR-based functional profiling of RASopathies and oncogenic RAS mutations.
      ). These differences in RAS-structure and RAS-dependent signaling are central elements of recent research projects (
      • Buhrman G.
      • Kumar V.S.S.
      • Cirit M.
      • Haugh J.M.
      • Mattos C.
      Allosteric modulation of Ras-GTP is linked to signal transduction through RAF kinase.
      ,
      • Nussinov R.
      • Tsai C.-J.
      • Mattos C.
      “Pathway drug cocktail”: targeting Ras signaling based on structural pathways.
      ).

      Direct targeting of mutant NRAS

      Mutations in NRAS favor the formation of GTP-bound, active RAS proteins (
      • Bos J.L.
      • Rehmann H.
      • Wittinghofer A.
      GEFs and GAPs: critical elements in the control of small G proteins.
      ). Similar to kinase inhibitors, which compete with ATP, attempts to directly target RAS have been designed to compete for GTP binding; however, because of the picomolar affinity of GTP toward RAS (in contrast to micromolar affinity of ATP toward kinases) and the high abundance of intracellular GTP, it has been challenging to discover such inhibitors with broad anti-RAS activity.
      We understand that more defined approaches are needed. Ideally, inhibitors would target the activating substitution itself to achieve high selectivity and specificity. This is possible as recently exemplified in preclinical models of codon 12 mutant KRAS: Because the intrinsic catalytic capacity of mutant RAS(G12) is not affected, the mutant protein displays a balanced equilibrium of GDP and GTP bound states. This presents an opportunity to use GDP analogs that slowly but steadily trap RAS in its inactive state. These compounds covalently and specifically bind to cysteine-12 in G12C mutants and block the activation of downstream signaling mediators of RAS including v-akt murine thymoma viral oncogene homolog (AKT) and extracellular signal-regulated kinase (ERK) (
      • Fetics S.K.
      • Guterres H.
      • Kearney B.M.
      • Buhrman G.
      • Ma B.
      • Nussinov R.
      • et al.
      Allosteric effects of the oncogenic RasQ61L mutant on Raf-RBD.
      ,
      • Lim S.M.
      • Westover K.D.
      • Ficarro S.B.
      • Harrison R.A.
      • Choi H.G.
      • Pacold M.E.
      • et al.
      Therapeutic targeting of oncogenic K-Ras by a covalent catalytic site inhibitor.
      ,
      • Ostrem J.M.
      • Peters U.
      • Sos M.L.
      • Wells J.A.
      • Shokat K.M.
      K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions.
      ). Even though this research has been carried out in models of oncogenic KRAS, it is likely that, such compounds will also specifically block codon G12C mutant NRAS given the high structural similarities in the effector lobe of RAS proteins. In melanoma, NRAS(G12) mutants are rare and comparable strategies for inhibiting the majority of codon 61 mutants have not been described to date; however, these findings remind us that mutation-specific direct targeting of RAS can be achieved, which is a concept that has largely been abandoned after decades of unsuccessful attempts.
      Another approach to specifically block RAS family members is targeting the allosteric lobe and hypervariable region in which K-, N-, and H-RAS differ the most. Such inhibitors would thus not distinguish between the wild-type and the mutant protein; instead, they would functionally deplete one isoform, which appears to be biologically tolerable, at least in animal models (
      • Stephen A.G.
      • Esposito D.
      • Bagni R.K.
      • McCormick F.
      Dragging ras back in the ring.
      ).
      Interfering with the membrane association of RAS also reduces protein activity. Blocking farnesylation, a posttranslational modification step essential for RAS binding to the cell membrane, showed promising results in preclinical models of mutant HRAS (
      • Liu M.
      • Bryant M.S.
      • Chen J.
      • Lee S.
      • Yaremko B.
      • Lipari P.
      • et al.
      Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice.
      ); however, clinical testing of farnesyltransferase inhibitors in K- and N-RAS mutant malignancies failed to provide a benefit for patients. This was later discovered to be due to alternative lipid modifications mediated by the related enzyme geranylgeranyltransferase that functionally substitutes for farnesylation (
      • Whyte D.B.
      • Kirschmeier P.
      • Hockenberry T.N.
      • Nunez-Oliva I.
      • James L.
      • Catino J.J.
      • et al.
      K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors.
      ). Similarly, inhibitors of posttranslational palmitoylation, such as palmostatin B, which also aim to disrupt the membrane localization of RAS, were effective in preclinical models of NRAS mutant cells but have not (yet) translated into clinical applications (
      • Vujic I.
      • Sanlorenzo M.
      • Esteve-Puig R.
      • Vujic M.
      • Kwong A.
      • Tsumura A.
      • et al.
      Acyl protein thioesterase 1 and 2 (APT-1, APT-2) inhibitors palmostatin B, ML348 and ML349 have different effects on NRAS mutant melanoma cells.
      ,
      • Xu J.
      • Hedberg C.
      • Dekker F.J.
      • Li Q.
      • Haigis K.M.
      • Hwang E.
      • et al.
      Inhibiting the palmitoylation/depalmitoylation cycle selectively reduces the growth of hematopoietic cells expressing oncogenic Nras.
      ). The clinical value of targeting other posttranslational modifications of RAS such as phosphorylation, nitrosylation, monoubiquitination, and acetylation, which mainly affect the subcellular localization of the protein, is currently unknown.
      Activated RAS proteins form complexes with regulators controlling RAS cycling (GEFs, GAPs) and effectors mediating downstream signaling (RAF, RAS related GTP binding protein [RAL], PI3K, and others). Peptide library screens identified compounds that interfere with the binding of RAS to son of sevenless homolog 1 (SOS1), one of the most prominent RAS-GEFs. These molecules reduce RAS activation and subsequent MAPK pathway signaling (
      • Patgiri A.
      • Yadav K.K.
      • Arora P.S.
      • Bar-Sagi D.
      An orthosteric inhibitor of the Ras-Sos interaction.
      ,
      • Sun Q.
      • Burke J.P.
      • Phan J.
      • Burns M.C.
      • Olejniczak E.T.
      • Waterson A.G.
      • et al.
      Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation.
      ). Yet, it remains unknown to which extent GEFs contribute to mutant RAS activation and if compounds targeting RAS-GEF interactions might be beneficial in the context of the mutant protein. Another study identified compounds that block RAS-GTP binding to the downstream mediator CRAF, using a computer-based screen (
      • Shima F.
      • Yoshikawa Y.
      • Ye M.
      • Araki M.
      • Matsumoto S.
      • Liao J.
      • et al.
      In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction.
      ). The design of molecules that selectively occupy these RAS binding sites has been challenging, and small molecules reported thus far only bind weakly to RAS. Similar to studies mentioned above, most research was carried out in models of mutant HRAS and KRAS, and it remains unclear whether such findings hold promise for NRAS mutant melanoma.
      Direct targeting of mutant NRAS can also be achieved by genetic silencing using small interfering RNAs, which prevent translation of the NRAS protein. This technique is widely used to abolish mutant NRAS signaling in experimental models (
      • Eskandarpour M.
      • Kiaii S.
      • Zhu C.
      • Castro J.
      • Sakko A.J.
      • Hansson J.
      Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells.
      ,
      • Jaiswal B.S.
      • Janakiraman V.
      • Kljavin N.M.
      • Eastham-Anderson J.
      • Cupp J.E.
      • Liang Y.
      • et al.
      Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.
      ). The overarching problem with using small interfering RNAs clinically is their fast degradation in vivo, a limitation that might be solved in the future using nanotechnology-based small interfering RNAs-delivery systems (
      • Davis M.E.
      • Zuckerman J.E.
      • Choi C.H.J.
      • Seligson D.
      • Tolcher A.
      • Alabi C.A.
      • et al.
      Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles.
      ).

      Indirect NRAS targeting—pathway interference

      Monotherapy

      Assuming that it is impossible to design clinically effective, direct inhibitors of RAS, research has explored concepts of indirect targeting by blocking downstream mediators of RAS. The most investigated signaling cascades in NRAS mutant melanoma are the MAPK, PI3K/mammalian target of rapamycin (mTOR), and cell-cycle pathways (Figure 2) (
      • Hodis E.
      • Watson I.R.
      • Kryukov G.V.
      • Arold S.T.
      • Imielinski M.
      • Theurillat J.-P.
      • et al.
      A landscape of driver mutations in melanoma.
      ,
      • Kwong L.N.
      • Costello J.C.
      • Liu H.
      • Jiang S.
      • Helms T.L.
      • Langsdorf A.E.
      • et al.
      Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma.
      ,
      • Posch C.
      • Moslehi H.
      • Feeney L.
      • Green G.A.
      • Ebaee A.
      • Feichtenschlager V.
      • et al.
      Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo.
      ).
      Figure 2
      Figure 2Conceptual schema highlighting some of the most studied pathways involved in signaling of NRAS mutant melanoma. Horizontal inhibition refers to targeting of signaling members within different pathways, such as combinations of MEK/AKT, MEK/PI3K, MEK/CDK4, and MEK/Plk1. Vertical inhibition refers to targeting of signaling members within one pathway. This approach is currently investigated by combinatorial targeting of MEK/ERK in the MAPK pathway. MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; NRAS, neuroblastoma rat-sarcoma homolog.
      RAF kinases (A-, B-, and C-RAF) are validated effectors of RAS signaling. Several multikinase inhibitors with modest pan-RAF activity were developed but only showed minimal effects in patients with NRAS mutant melanoma (
      • Eisen T.
      • Ahmad T.
      • Flaherty K.T.
      • Gore M.
      • Kaye S.
      • Marais R.
      • et al.
      Sorafenib in advanced melanoma: a Phase II randomised discontinuation trial analysis.
      ). More specific RAF inhibitors, such as vemurafenib and dabrafenib, which preferentially target the mutant BRAF(V600) protein, even caused worsening of NRAS mutant disease. This was later found to be due to paradoxical activation of ERK signaling in BRAF wild-type cells caused by enhanced dimerization, conformational changes, and transactivation of RAF induced by these drugs, as well as the switch from BRAF to CRAF signaling when NRAS is mutant (
      • Dumaz N.
      • Hayward R.
      • Martin J.
      • Ogilvie L.
      • Hedley D.
      • Curtin J.A.
      • et al.
      In melanoma, RAS mutations are accompanied by switching signaling from BRAF to CRAF and disrupted cyclic AMP signaling.
      ,
      • Hatzivassiliou G.
      • Song K.
      • Yen I.
      • Brandhuber B.J.
      • Anderson D.J.
      • Alvarado R.
      • et al.
      RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.
      ,
      • Poulikakos P.I.
      • Zhang C.
      • Bollag G.
      • Shokat K.M.
      • Rosen N.
      RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF.
      ). Yet, RAF inhibition in RAS mutant cells is not considered obsolete and compounds with increased anti-CRAF activity or inhibitors blocking RAF-dimerization are currently tested (
      • Su Y.
      • Vilgelm A.E.
      • Kelley M.C.
      • Hawkins O.E.
      • Liu Y.
      • Boyd K.L.
      • et al.
      RAF265 inhibits the growth of advanced human melanoma tumors.
      ).
      Direct substrates of RAF are the highly homologous kinases MEK1 and MEK2. Inhibition of MAPK/ERK kinase (MEK) with specific, small molecules suppresses MAPK signaling and has shown promising results in patients with NRAS mutant melanoma. Notably, treatment with binimetinib, a selective MEK1/2 inhibitor, has resulted in a 20% objective response rate and prolonged overall survival for 3.7 months (
      • Ascierto P.A.
      • Schadendorf D.
      • Berking C.
      • Agarwala S.S.
      • van Herpen C.M.
      • Queirolo P.
      • et al.
      MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study.
      ) (Supplementary Table S1 online). Still, the majority of patients did not benefit from MEK inhibitor therapy or rapidly developed resistance.
      Exclusively blocking further downstream by targeting ERK1/2, the only well-validated substrate of MEK, is also ineffective in RAS mutant malignancies. Similar to MEK inhibitors, ERK inhibition releases feedback loops that activate RTKs, such as ERBB3, EGFR, and c-MET upstream of RAS (
      • Carlino M.S.
      • Todd J.R.
      • Gowrishankar K.
      • Mijatov B.
      • Pupo G.M.
      • Fung C.
      • et al.
      Differential activity of MEK and ERK inhibitors in BRAF inhibitor resistant melanoma.
      ,
      • Duncan J.S.
      • Whittle M.C.
      • Nakamura K.
      • Abell A.N.
      • Midland A.A.
      • Zawistowski J.S.
      • et al.
      Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.
      ,
      • Fattore L.
      • Marra E.
      • Pisanu M.E.
      • Noto A.
      • de Vitis C.
      • Belleudi F.
      • et al.
      Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies.
      ,
      • Rebecca V.W.
      • Alicea G.M.
      • Paraiso K.H.T.
      • Lawrence H.
      • Gibney G.T.
      • Smalley K.S.M.
      Vertical inhibition of the MAPK pathway enhances therapeutic responses in NRAS-mutant melanoma.
      ). Activated RTKs enhance the flux through the MAPK pathway and activate additional prosurvival cascades such as the PI3K, RAL, and PLC cascades among others.
      It is now clear that the MAPK pathway is a complex network with multiple inputs and outputs. Even though RAF, MEK, and ERK comprise the core of MAPK signaling, we now understand that a one-directional picture of the pathway is insufficient to account for the diverse regulatory mechanisms that ensure mutant NRAS cell homeostasis.
      Research also provides compelling evidence for the importance of PI3K signaling in NRAS-driven melanoma. PI3K is a substrate of RAS and can also be activated through non-RAS-mutant mechanisms, most importantly through a variety of RTKs (
      • Pacold M.E.
      • Suire S.
      • Perisic O.
      • Lara-Gonzalez S.
      • Davis C.T.
      • Walker E.H.
      • et al.
      Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma.
      ). The efficacy of available PI3K/mTOR inhibitors is mainly limited by a lack of a clear therapeutic window. Nonetheless, their value as anti-RAS drugs may be revisited, particularly in combination with inhibitors of other pathways (
      • Greger J.G.
      • Eastman S.D.
      • Zhang V.
      • Bleam M.R.
      • Hughes A.M.
      • Smitheman K.N.
      • et al.
      Combinations of BRAF, MEK, and PI3K/mTOR inhibitors overcome acquired resistance to the BRAF inhibitor GSK2118436 dabrafenib, mediated by NRAS or MEK mutations.
      ,
      • Posch C.
      • Moslehi H.
      • Feeney L.
      • Green G.A.
      • Ebaee A.
      • Feichtenschlager V.
      • et al.
      Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo.
      ).
      Even though a role of RAL-GDS in NRAS melanomagenesis has been established, this pathway has not been studied extensively (
      • Mishra P.J.
      • Ha L.
      • Rieker J.
      • Sviderskaya E.V.
      • Bennett D.C.
      • Oberst M.D.
      • et al.
      Dissection of RAS downstream pathways in melanomagenesis: a role for Ral in transformation.
      ,
      • Zipfel P.A.
      • Brady D.C.
      • Kashatus D.F.
      • Ancrile B.D.
      • Tyler D.S.
      • Counter C.M.
      Ral activation promotes melanomagenesis.
      ). Like RAS, RAL-GTPases are similarly difficult targets for direct inhibition. Yet, indirect blockades of RAL-downstream molecules, such as TBK1, might have therapeutic value for NRAS-driven melanoma (
      • Vu H.L.
      • Aplin A.E.
      Targeting TBK1 inhibits migration and resistance to MEK inhibitors in mutant NRAS melanoma.
      ).

      Combinatorial therapy

      The search for NRAS coextinction targets has become central in the development of RAS treatment regimens. The important role of MAPK signaling in NRAS mutant cancers and the fact that to date only MEK inhibitors have shown clinical activity in patients with NRAS mutant melanoma are the rationale for using MEK inhibitors as a cornerstone for combinational treatments (Supplementary Table S1). Two strategies can be defined for targeted combinatorial therapy: horizontal and vertical inhibition (Figure 2).

      Horizontal inhibition

      Horizontal inhibition refers to parallel targeting of multiple pathways. The most advanced horizontal combination for NRAS mutant melanoma is simultaneous MEK and CDK4 inhibition (
      • Kwong L.N.
      • Costello J.C.
      • Liu H.
      • Jiang S.
      • Helms T.L.
      • Langsdorf A.E.
      • et al.
      Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma.
      ). CDK4 is a serine/threonine kinase important for G1 cell-cycle progression that is controlled by the CDK inhibitor p16 (INK4a). Clinical testing of MEK/CDK4 inhibition showed activity in NRAS mutant melanoma; however, the majority of patients still did not respond. Current research suggests that the efficacy of this combination might be increased when tumor cells also have genetic aberrations increasing CDK4 activity such as loss of p16(INK4a) or amplifications of cyclin D1 (
      • VanArsdale T.
      • Boshoff C.
      • Arndt K.T.
      • Abraham R.T.
      Molecular pathways: targeting the cyclin D–CDK4/6 axis for cancer Treatment.
      ,
      • Young R.J.
      • Waldeck K.
      • Martin C.
      • Foo J.H.
      • Cameron D.P.
      • Kirby L.
      • et al.
      Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines.
      ). Another, yet experimental, combination blocking the MAPK and cell-cycle pathways targets MEK and the cell-cycle regulator Plk1, which effectively reduces growth of NRAS mutant melanoma xenografts independent of p16(INK4a) mutation status (
      • Posch C.
      • Cholewa B.D.
      • Vujic I.
      • Sanlorenzo M.
      • Ma J.
      • Kim S.T.
      • et al.
      Combined inhibition of MEK and Plk1 has synergistic antitumor activity in NRAS mutant melanoma.
      ).
      The rationale for dual inhibition of the MAPK and PI3K/mTOR pathways is supported by effective growth reduction of NRAS mutant melanoma in mouse models and the upregulation of PI3K/mTOR pathway members due to the release of negative feedback to RTKs after single MEK inhibitor treatment (
      • Fattore L.
      • Marra E.
      • Pisanu M.E.
      • Noto A.
      • de Vitis C.
      • Belleudi F.
      • et al.
      Activation of an early feedback survival loop involving phospho-ErbB3 is a general response of melanoma cells to RAF/MEK inhibition and is abrogated by anti-ErbB3 antibodies.
      ,
      • Jaiswal B.S.
      • Janakiraman V.
      • Kljavin N.M.
      • Eastham-Anderson J.
      • Cupp J.E.
      • Liang Y.
      • et al.
      Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.
      ,
      • Posch C.
      • Moslehi H.
      • Feeney L.
      • Green G.A.
      • Ebaee A.
      • Feichtenschlager V.
      • et al.
      Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo.
      ). However, with only a few patients experiencing stable disease as best response, combined targeting of MEK/AKT or MEK/PI3K did not meet preclinical expectations and was overall poorly tolerated (
      • Greger J.G.
      • Eastman S.D.
      • Zhang V.
      • Bleam M.R.
      • Hughes A.M.
      • Smitheman K.N.
      • et al.
      Combinations of BRAF, MEK, and PI3K/mTOR inhibitors overcome acquired resistance to the BRAF inhibitor GSK2118436 dabrafenib, mediated by NRAS or MEK mutations.
      ,
      • Juric D.
      • Soria J.-C.
      • Sharma S.
      • Banerji U.
      • Azaro A.
      • Desai J.
      • et al.
      A phase 1b dose-escalation study of BYL719 plus binimetinib (MEK162) in patients with selected advanced solid tumors.
      ,
      • Tolcher A.W.
      • Patnaik A.
      • Papadopoulos K.P.
      • Rasco D.W.
      • Becerra C.R.
      • Allred A.J.
      • et al.
      Phase I study of the MEK inhibitor trametinib in combination with the AKT inhibitor afuresertib in patients with solid tumors and multiple myeloma.
      ). In addition, cotargeting of both PI3K and mTOR signaling molecules in combination with MEK inhibition might be necessary for effective NRAS mutant tumor shrinkage (
      • Posch C.
      • Moslehi H.
      • Feeney L.
      • Green G.A.
      • Ebaee A.
      • Feichtenschlager V.
      • et al.
      Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo.
      ). An early phase clinical trial testing dactolisib, a dual PI3K/mTOR inhibitor, in combination with the MEK inhibitor binimetinib will offer insights into the clinical profile and efficacy of such a combination (Supplementary Table S1).
      Inhibition of PI3K/mTOR pathway activity can potentially also be achieved by using high doses of metformin. Because metformin has been used for decades as an antidiabetic drug, has a known side effect profile, is cheap and generically available, this combination might be an interesting addition to current treatment modalities (
      • Vujic I.
      • Sanlorenzo M.
      • Posch C.
      • Esteve-Puig R.
      • Yen A.J.
      • Kwong A.
      • et al.
      Metformin and trametinib have synergistic effects on cell viability and tumor growth in NRAS mutant cancer.
      ).
      The overactivation of the hepatocyte growth factor receptor (c-MET) in NRAS mutant melanoma prompted testing of the RAF kinase inhibitor sorafenib in combination with the c-MET inhibitor tivantinib. In a first clinical trial, 2 of 8 patients with NRAS mutant melanoma experienced complete or partial response; 2 additional patients had stable disease (
      • Puzanov I.
      • Sosman J.
      • Santoro A.
      • Saif M.W.
      • Goff L.
      • Dy G.K.
      • et al.
      Phase 1 trial of tivantinib in combination with sorafenib in adult patients with advanced solid tumors.
      ). Interestingly, indirect c-Met inhibition was reported in cancer cells treated with the cardiac glycoside digitoxin (
      • Yang Q.F.
      • Dalgard C.L.
      • Eidelman O.
      • Jozwik C.
      • Pollard B.S.
      • Srivastava M.
      • et al.
      Digitoxin induces apoptosis in cancer cells by inhibiting nuclear factor of activated T-cells-driven c-MYC expression.
      ). Thus, it is possible that in the context of NRAS mutant melanoma, digitoxin (or derivates with comparable characteristics) could be used therapeutically.
      Other inhibitor combinations using analogs of the mTORC1 inhibitor rapamycin (rapalogs) together with compounds affecting DNA repair/replication (carboplatin, cisplatin, gemcitabine), mitosis (paclitaxel), or angiogenesis (bevacizumab) are additional therapeutic approaches for patients with solid tumors including NRAS mutant melanoma (
      • Algazi A.P.
      • Cha E.
      • Ortiz-Urda S.M.
      • McCalmont T.
      • Bastian B.C.
      • Hwang J.
      • et al.
      The combination of axitinib followed by paclitaxel/carboplatin yields extended survival in advanced BRAF wild-type melanoma: results of a clinical/correlative prospective phase II clinical trial.
      ,
      • Hauke R.J.
      • Infante J.R.
      • Rubin M.S.
      • Shih K.C.
      • Arrowsmith E.R.
      • Hainsworth J.D.
      Everolimus in combination with paclitaxel and carboplatin in patients with metastatic melanoma: a phase II trial of the Sarah Cannon Research Institute Oncology Research Consortium.
      ,
      • Mudigonda T.V.
      • Wyman K.
      • Spigel D.R.
      • Dahlman K.B.
      • Greco F.A.
      • Puzanov I.
      • et al.
      A phase II trial of erlotinib and bevacizumab for patients with metastatic melanoma.
      ,
      • Puzanov I.
      • Sosman J.
      • Santoro A.
      • Saif M.W.
      • Goff L.
      • Dy G.K.
      • et al.
      Phase 1 trial of tivantinib in combination with sorafenib in adult patients with advanced solid tumors.
      ). The clinical activity of such combinations is yet to be fully explored.

      Vertical inhibition

      Vertical inhibition of a signaling pathway refers to the blockade of more than one signaling member within a single cascade. This concept has proved effective in BRAF mutant melanoma, where combined BRAF/MEK inhibition significantly prolongs overall survival compared with BRAF or MEK inhibition alone (
      • Flaherty K.T.
      • Infante J.R.
      • Daud A.
      • Gonzalez R.
      • Kefford R.F.
      • Sosman J.
      • et al.
      Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations.
      ,
      • Larkin J.
      • Ascierto P.A.
      • Dréno B.
      • Atkinson V.
      • Liszkay G.
      • Maio M.
      • et al.
      Combined vemurafenib and cobimetinib in BRAF-mutated melanoma.
      ). A similar concept might also be effective in NRAS mutant melanoma: As discussed, one of the mechanisms of resistance using MEK inhibitors in NRAS mutant melanoma is the reactivation or inability to fully suppress ERK signaling (
      • Fedorenko I.V.
      • Gibney G.T.
      • Smalley K.S.M.
      NRAS mutant melanoma: biological behavior and future strategies for therapeutic management.
      ). In preclinical studies, targeting of MEK and ERK in combination potently suppresses levels of phospho-ERK and induces apoptosis in NRAS mutant cells (
      • Morris E.J.
      • Jha S.
      • Restaino C.R.
      • Dayananth P.
      • Zhu H.
      • Cooper A.
      • et al.
      Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors.
      ,
      • Rebecca V.W.
      • Alicea G.M.
      • Paraiso K.H.T.
      • Lawrence H.
      • Gibney G.T.
      • Smalley K.S.M.
      Vertical inhibition of the MAPK pathway enhances therapeutic responses in NRAS-mutant melanoma.
      ). Another approach for vertical pathway inhibition uses combined pan-RAF and MEK inhibition (
      • Atefi M.
      • Titz B.
      • Avramis E.
      • Ng C.
      • Wong D.J.L.
      • Lassen A.
      • et al.
      Combination of pan-RAF and MEK inhibitors in NRAS mutant melanoma.
      ). Similarly, single compound, dual RAF/MEK inhibitors are currently investigated for their potential therapeutic activity in RAS mutated malignancies, including melanoma (
      • Wada M.
      • Horinaka M.
      • Yamazaki T.
      • Katoh N.
      • Sakai T.
      The dual RAF/MEK inhibitor CH5126766/RO5126766 may be a potential therapy for RAS-mutated tumor cells.
      ).
      To date, combined vertical inhibition of the PI3K/mTOR pathway in melanoma has only been investigated in BRAF mutant cells (
      • Werzowa J.
      • Koehrer S.
      • Strommer S.
      • Cejka D.
      • Fuereder T.
      • Zebedin E.
      • et al.
      Vertical inhibition of the mTORC1/mTORC2/PI3K pathway shows synergistic effects against melanoma in vitro and in vivo.
      ). Still, research suggests that dual PI3K/mTOR inhibition might also show activity in NRAS mutant melanoma (
      • Posch C.
      • Moslehi H.
      • Feeney L.
      • Green G.A.
      • Ebaee A.
      • Feichtenschlager V.
      • et al.
      Combined targeting of MEK and PI3K/mTOR effector pathways is necessary to effectively inhibit NRAS mutant melanoma in vitro and in vivo.
      ; personal communication Schatton T).

      Immunity and targeted therapy

      Systemic therapy with targeted molecules also affects cells other than tumor cells, including immune cells. MEK inhibitors suppress MAPK signaling in all exposed cells, potentially dampening immune cell activity, too. Still, the successful use of single MEK inhibitors in NRAS mutant melanoma makes it appealing to test the effects of combined MEK and immune-checkpoint inhibition in patients with NRAS mutant melanoma. In contrast, compounds designed to target mutant BRAF(V600) cause paradox activation of ERK signaling in non-BRAF(V600) mutant cells. This can potentially be harnessed to augment MAPK activity and thus boost immune cell function in treatment combinations with immune-checkpoint inhibitors or adoptive cell immunotherapy (
      • Koya R.C.
      • Mok S.
      • Otte N.
      • Blacketor K.J.
      • Comin-Anduix B.
      • Tumeh P.C.
      • et al.
      BRAF inhibitor vemurafenib improves the antitumor activity of adoptive cell immunotherapy.
      ).

      Conclusion

      NRAS mutations are common in melanoma and are associated with clinical features of poor biological behavior (
      • Devitt B.
      • Liu W.
      • Salemi R.
      • Wolfe R.
      • Kelly J.
      • Tzen C.-Y.
      • et al.
      Clinical outcome and pathological features associated with NRAS mutation in cutaneous melanoma.
      ,
      • Jakob J.A.
      • Bassett Jr., R.L.
      • Ng C.S.
      • Curry J.L.
      • Joseph R.W.
      • Alvarado G.C.
      • et al.
      NRAS mutation status is an independent prognostic factor in metastatic melanoma.
      ). Because of difficulties of direct NRAS targeting, interference with downstream signaling cascades is to date the most promising targeted therapeutic modality. It is likely that the complexity of NRAS signaling events and the genetic heterogeneity, such as low-activating BRAF mutations, loss of p16(INK4a) and MITF amplification, will require a diverse set of therapeutics for optimized patient care. Hence, studies investigating other than the discussed combinatorial regimens target ROCK, HSP90, NFkB, and Wnt signaling members and are essential to advance our understanding of mutant NRAS biology (
      • Conrad W.H.
      • Swift R.D.
      • Biechele T.L.
      • Kulikauskas R.M.
      • Moon R.T.
      • Chien A.J.
      Regulating the response to targeted MEK inhibition in melanoma: enhancing apoptosis in NRAS- and BRAF-mutant melanoma cells with Wnt/β-catenin activation.
      ,
      • Feng Y.
      • Lau E.
      • Scortegagna M.
      • Ruller C.
      • De S.K.
      • Barile E.
      • et al.
      Inhibition of melanoma development in the Nras((Q61K)) ::Ink4a(-/-) mouse model by the small molecule BI-69A11.
      ,
      • Haarberg H.E.
      • Paraiso K.H.T.
      • Wood E.
      • Rebecca V.W.
      • Sondak V.K.
      • Koomen J.M.
      • et al.
      Inhibition of Wee1, AKT, and CDK4 underlies the efficacy of the HSP90 inhibitor XL888 in an in vivo model of NRAS-mutant melanoma.
      ,
      • Vogel C.J.
      • Smit M.A.
      • Maddalo G.
      • Possik P.A.
      • Sparidans R.W.
      • van der Burg S.H.
      • et al.
      Cooperative induction of apoptosis in NRAS mutant melanoma by inhibition of MEK and ROCK.
      ). To further complete our picture of RAS-driven melanoma growth, it will be necessary to also take into account other forms of RAS pathway activation, such as loss of function mutations in NF1, or copy number alterations of RAS itself.
      To date most RAS research has been performed in models of mutant KRAS. The slight but distinct functional differences of RAS family members mandate dedicated NRAS research to further advance melanoma treatment. Recent developments in targeting oncogenic RAS function allow for an optimistic yet cautious outlook that effective targeted treatment of NRAS mutant melanoma can be achieved.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This study was supported by the National Cancer Institute of the National Institutes of Health under award number K08CA155035 and the Melanoma Research Alliance Young Investigator Award. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors are grateful to Timothy Dattels, the Max Kade Foundation, and the René Touraine Foundation for their generous support. They are also grateful to the Society for Dermatology and Venerology KA Rudolfstiftung. Further, the authors thank K. Hölbling, G. Posch, and R. Panzer-Grümayer for their support.

      Supplementary Material

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