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MOLECULAR MODEL OF MELANOMA

E. Shtivelman  ((Cancer Commons, Palo Alto CA 94301)) , K.T. Flaherty[1] ,  and D.E. Fisher[1]

INTRODUCTION
MOLECULAR SUBTYPES
INTRINSIC RESISTANCE TO BRAF AND MEK INHIBITORS
ACQUIRED RESISTANCE TO BRAF AND MEK INHIBITORS
COMBINATORIAL THERAPIES
IMMUNOTHERAPY
Molecular targets for immunotherapy of melanoma
Adoptive cell transfer
Influence of targeted therapies on the responses to immune therapy
METABOLISM AND AUTOPHAGY AS TARGETS IN MELANOMA THERAPY
NEW PROGNOSTIC MARKERS
CONCLUSIONS

TABLES
Table 1. Pathways involved in melanoma
Table 2. List of gene products known to be altered in melanoma
Table 3. Molecular events involved in the intrinsic resistance to BRAF and MEK inhibitors
Table 4. Molecular events involved in acquired resistance to BRAF and MEK inhibitors

Supplemental Table 1. Targeted Drugs for Treatment of Melanoma
Supplemental Table 2. Clinical Trials for Targeted Therapies in Melanoma

 

 

INTRODUCTION

The revolutionary discovery of a striking if temporary effect that targeted inhibition of BRAF has on the clinical course of metastatic melanoma has spiked a new wave of research into molecular targets.  In addition, it has raised a number of new questions:  what are the mechanisms of both inherent and acquired resistance to BRAF inhibitors and the possible ways to overcome this resistance; how is the activating effect of BRAF inhibition on the MAPK pathway in cells with nonmutated BRAF avoided; how should melanoma tumors that have no activating mutations in BRAF, such as tumors with mutated NRAS or tumors that are wild type for both BRAF and NRAS, be targeted; which targeted or nontargeted drug combinations should be pursued as determined by the molecular profile of each and every tumor; what is the future of combination targeted therapy and immunotherapy; and many more.
The mutational landscape of melanoma was examined in several large studies employing NGS (next-generation sequencing) and large-scale expression analyses of tumors.  The mutation rate of melanoma exceeds those reported for other aggressive tumors, probably due to the heavy involvement of ultraviolet (UV) radiation in the genesis of superficial cutaneous melanomas.  Indeed, the rate of transversions characteristic of UV-induced lesions is much higher in melanoma than the rates of other nucleotide substitutions.  The high rate of mutations in melanoma makes it particularly difficult to distinguish between causative (“driver”) mutations and bystander (“passenger”) mutations.
In one recent study [2] a wide range of point mutation rates was observed:  mutation rates were lowest in melanomas in which primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3/ and 14/megabase [Mb] of genome); intermediate in those originating from hair-bearing skin of the trunk (5-55/Mb); and highest in a patient with a documented history of chronic sun exposure (111/ Mb).
This paper describes genetic alterations that are known to occur in melanoma and information about their role in melanomagenesis, as well as their suitability as targets for therapeutic intervention.  The latest information on molecular events underlying inherent or acquired resistance to targeted therapies is also included.
Multiple cellular pathways have been implicated in melanomagenesis, ranging from signal transduction to developmental and transcriptional pathways and cell cycle deregulation (Table 1).  The search for driver mutations in melanoma continues, with the previously identified subtypes involving BRAF, NRAS, KIT, GNAQ, and GNA11 expanded just recently to include NF1 and telomerase (Table 2).  Together, these driver mutations are very likely to define the vast majority of molecular subtypes in melanoma and, eventually, the identified mutations in drivers will be guiding targeted therapy choices.  However, for a number of reasons, such as oncogene-induced senescence (OIS, discussed below), driver mutations result in a frank tumorigenic phenotype only in the presence of “supporting mutations” that are also listed in Table 2 and described below.  It is clear now that these supporting mutations have to be targeted along with driver mutations to achieve durable responses. Finally, immunotherapies continue to play an important role in melanoma treatment strategies and are also described here.

 

 

MOLECULAR SUBTYPES

Table 1 summarizes the molecular pathways involved in melanomagenesis, and Table 2 lists know driver and additional mutations/alterations.

 

BRAF

BRAF mutations (V600E in 90% of BRAF mutations in all cancers) occur early in melanomagenesis and are found in a high percentage of melanocytic nevi.  The latter rarely progress to melanoma; this is associated with the phenomenon known as OIS  ((Michaloglou, C., Vredeveld, L. C., Soengas, M. S., Denoyelle, C., Kuilman, T., van der Horst, C. M., Majoor, D. M., Shay, J. W., Mooi, W. J., and Peeper, D. S. (2005). BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720-724. http://www.ncbi.nlm.nih.gov/pubmed/16079850)) , which induces a cell cycle arrest in BRAFV600E-expressing cells.  Additional genetic events in BRAF-mutant cells, such as deletion of CDKN2A or PTEN, are necessary to elicit a fully cancerous phenotype (reviewed in Flaherty, et al, 2012  ((Flaherty, K. T., Hodi, F. S., and Fisher, D. E. (2012a).  From genes to drugs: targeted strategies for melanoma. Nature reviews Cancer 12, 349-361.  http://www.nature.com/nrc/journal/v12/n5/full/nrc3218.html)) .
About 50% of melanomas of all clinical types have mutations in BRAF. BRAF mutations are more frequent in melanomas that develop in intermittently sun-exposed skin and less so in acral and mucosal melanomas.  BRAF mutations are not found in uveal melanomas. Mutations in BRAF and NRAS (mutated in about 0.20 of melanomas of the same origin/location as BRAF-mutated melanomas) are mutually exclusive.  The V600E mutation in BRAF confers to this kinase the ability to activate MEK (the only known downstream target of BRAF) independent of RAS.
The reported results of trials with selective oral BRAF inhibitors showed efficacy in melanoma patients with BRAFV600E mutations  ((Flaherty, K. T., Puzanov, I., Kim, K. B., Ribas, A., McArthur, G. A., Sosman, J. A., O’Dwyer, P. J., Lee, R. J., Grippo, J. F., Nolop, K., and Chapman, P. B. (2010). Inhibition of mutated, activated BRAF in metastatic melanoma. The New England journal of medicine 363, 809-819. http://www.nejm.org/doi/full/10.1056/NEJMoa1002011#t=articleTop)) .  In addition, inhibition of MEK (the only known phosphorylation substrate of BRAF) is considered to be a valid therapeutic intervention for both BRAF- and NRAS-mutated melanoma.  However, the success of BRAF inhibitors has not come without a slew of problems:
• Almost all patients initially responding to BRAF inhibitors have experienced a relapse, after developing resistance to BRAF inhibitors.
• BRAF inhibitor-treated patients developed cutaneous squamous cell carcinomas  ((Jordan, E. J., and Kelly, C. M. (2012). Vemurafenib for the treatment of melanoma. Expert opinion on pharmacotherapy 13, 2533-2543. http://www.ncbi.nlm.nih.gov/pubmed/23094782)) . Photodynamic therapy (PTD) was reported to be effective in treatment of these usually numerous tumors  ((Alloo, A., Garibyan, L., LeBoeuf, N., Lin, G., Werchniak, A., Hodi, F. S., Jr., Flaherty, K. T., Lawrence, D. P., and Lin, J. Y. (2012). Photodynamic therapy for multiple eruptive keratoacanthomas associated with vemurafenib treatment for metastatic melanoma. Archives of dermatology 148, 363-366. http://archderm.jamanetwork.com/article.aspx?articleid=1105231)) .
• Many patients with mutant BRAF show no response to BRAF inhibition[3] . Preclinical results suggest that this group has a variety of additional genetic somatic alterations that confer the resistance phenotype (discussed below).
• Paradoxical adverse effects of BRAFV600 inhibitors in activating wild-type BRAF; the mechanism of this is thought to be understood now[4] . This effect might trigger growth of new malignant melanoma tumors that have wild-type BRAF, but high expression of Akt and cyclin D1  ((Dalle, S., Poulalhon, N., Debarbieux, S., and Thomas, L. (2012). Second primary melanomas under vemurafenib. The British journal of dermatology. http://www.ncbi.nlm.nih.gov/m/pubmed/23066856/)) ;  ((Zimmer, L., Hillen, U., Livingstone, E., Lacouture, M. E., Busam, K., Carvajal, R. D., Egberts, F., Hauschild, A., Kashani-Sabet, M., Goldinger, S. M., et al. (2012). Atypical melanocytic proliferations and new primary melanomas in patients with advanced melanoma undergoing selective BRAF inhibition. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 2375-2383)) .A recent study provided a likely mechanism of activation of wild-type BRAF by inhibitors through a relief of inhibitory autophosphorylation (Holderfield et al., 2013)

• Accelerated development of RAS-mutant leukemia in patients treated with vemurafenib was described (Callahan, M. K., Rampal, R., Harding, J. J., Klimek, V. M., Chung, Y. R., Merghoub, T., Wolchok, J. D., Solit, D. B., Rosen, N., Abdel-Wahab, O., et al. (2012). Progression of RAS-mutant leukemia during RAF inhibitor treatment. The New England journal of medicine 367, 2316-2321. http://www.nejm.org/doi/full/10.1056/NEJMoa1208958)) .There is a significant concern about the development of SCC and other malignancies as a consequence of BRAF inhibition(Gibney et al., 2013).

 

Potential treatment approaches

The U.S. Food and Drug Administration (FDA) approved the mutant BRAF inhibitor vemurafenib in 2011.  Another inhibitor, dabrafenib/Tafinlar was approved in May 2013; LGX818 is in early stages of testing (Supplemental Table 1).  New inhibitors are in development to eliminate the side effects associated with the paradoxical activation by Vemurafenib of the ERK1/2 pathway in wild-type BRAF cells. PLX7904, a new “paradox-breaking” BRAF inhibitor appears to inhibit ERK1/2 in BRAF mutant cells but not in BRAF wild type cells  ((Le, K., Blomain, E., Rodeck, U., and Aplin, A. E. (2013).  Selective RAF inhibitor impairs ERK1/2 phosphorylation and growth in mutant NRAS, vemurafenib-resistant melanoma cells.  Pigment cell & melanoma research. http://onlinelibrary.wiley.com/doi/10.1111/pcmr.12092/abstract)) . Dozing schedules with BRAF inhibitors were explored in a genetically engineered mouse model) GEMM, where intermittent inhibition of BRAF by Vemurafenib works better than continuous treatment  ((Das Thakur, M., Salangsang, F., Landman, A. S., Sellers, W. R., Pryer, N. K., Levesque, M. P., Dummer, R., McMahon, M., and Stuart, D. D. (2013) .  Modelling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance. Nature. http://www.nature.com/nature/journal/v494/n7436/full/nature11814.html)) . With some limitations, the dosing of Vemurafenib could be considered in patients.
Most importantly, it is increasingly clear that combination treatments, such as the BRAF and MEK inhibition investigated in a recently reported clinical study [133], have a potential for a robust and lasting clinical response in the treatment of BRAF-mutant melanoma.  BRAF inhibitors are explored in a number of clinical trials in combination with inhibitors of MEK, PI3K, AKT, and HSP90 (Supplemental Table 2).  New combinatorial treatments are emerging from high throughput analyses in preclinical studies  ((Held, M. A., Langdon, C. G., Platt, J. T., Graham-Steed, T., Liu, Z., Chakraborty, A., Bacchiocchi, A., Koo, A., Haskins, J. W., Bosenberg, M. W., and Stern, D. F. (2013). Genotype-selective combination therapies for melanoma identified by high-throughput drug screening. Cancer discovery 3, 52-67. http://cancerdiscovery.aacrjournals.org/content/3/1/52.short)) . Inhibition of MEK was and is explored in multiple clinical trials for melanoma with mutant BRAF  ((Falchook, G. S., Lewis, K. D., Infante, J. R., Gordon, M. S., Vogelzang, N. J., DeMarini, D. J., Sun, P., Moy, C., Szabo, S. A., Roadcap, L. T., et al. (2012). Activity of the oral MEK inhibitor trametinib in patients with advanced melanoma: a phase 1 dose-escalation trial. The lancet oncology 13, 782-789. http://www.sciencedirect.com/science/article/pii/S1470204512702693)) ; so far the responses produced are less durable than those seen with BRAF inhibitors. MEK inhibitor trametinib showed some clinical responses in BRAF mutant patients that had underwent previous immune- or chemotherapy, but had no effect in patients who were previously treated with Vemurafenib  ((Kim, K. B., Kefford, R., Pavlick, A. C., Infante, J. R., Ribas, A., Sosman, J. A., Fecher, L. A., Millward, M., McArthur, G. A., Hwu, P., et al. (2013). Phase II study of the MEK1/MEK2 inhibitor Trametinib in patients with metastatic BRAF-mutant cutaneous melanoma previously treated with or without a BRAF inhibitor. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 31, 482-489. http://jco.ascopubs.org/content/31/4/482.abstract)) .  This strongly indicates that mechanisms involved in development of resistance to mutant BRAF confer resistance to MEK inhibition as well. However, combination of BRAF and MEK inhibitors has a better clinical activity(Flaherty et al., 2012b). Trametinib/Mekinist was approved by FDA for melanoma in May 2013, but not as a part of combination therapy with BRAF inhibitor.

 

BRAF mutations other than V600

The BRAF mutations G469E/D594G were identified as low activity mutations in a panel of melanoma cell lines[5] that signal through CRAF. Analysis of BRAF exon 15 in 49 tumors negative for BRAFV600 mutations, as well as other known driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that two (4%) harbored L597 mutations and another two involved BRAF D594 and K601 mutations.

Potential treatment approaches

Melanomas with BRAF mutations other than V600 are not specifically targeted in clinical trials. Data from in vitro studies of G469E/D594G (low-activity mutants) indicated signaling through CRAF. CRAF responds to sorafenib better than mutant BRAF; sorafenib induced apoptosis in vitro and in a xenograft model in vivo in tumors with G469E/D594G[5] .  In vitro signaling induced by L597R/S/Q mutants was suppressed by inhibition of MEK. A patient with BRAF(L597S)-mutant metastatic Melanoma Responded significantly to treatment with the MEK inhibitor TAK-733[6] .

 

NRAS

Approximately 20% of melanomas have mutations in the GTPase NRAS. NRAS and BRAF mutations are mutually exclusive.  Therapeutic approaches targeting mutant NRAS directly have not been successful. Combination treatments targeting the downstream effectors of NRAS remain a viable option.

 

Potential treatment approaches

Potential treatment approaches
The pathways that could be targeted simultaneously in NRAS-mutant melanoma include, but are not limited to, MEK, PI3K/mTOR, and cell cycle-related targets.  PTEN abnormalities are not found in NRAS-mutant tumors, but additional PI3K pathway abnormalities are found[7] and could be potentially targeted.  Monotherapy with the MEK inhibitor MEK162/trametinib showed limited partial responses (20%) in NRAS-mutant patients[8] .

 

Preclinical studies indicate several potential points of intervention:

  •  NRAS-driven melanoma in genetically engineered mice responded only to the combination of MEK and PI3K/mTOR dual inhibitors out of 16 treatment combinations tested  ((Roberts, P. J., Usary, J. E., Darr, D. B., Dillon, P. M., Pfefferle, A. D., Whittle, M. C., Duncan, J. S., Johnson, S. M., Combest, A. J., Jin, J., et al. (2012). Combined PI3K/mTOR and MEK inhibition provides broad antitumor activity in faithful murine cancer models. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 5290-5303. http://www.ncbi.nlm.nih.gov/pubmed/22872574)) .  Combined targeting of MEK and PI3K was superior to MEK and mTOR inhibition in NRAS-mutant melanoma cell lines and xenografts[9] .
  •   In an inducible model of NRAS-mutant melanoma, genetic ablation of NRAS triggered cell cycle arrest and apoptosis, while pharmacological inhibition of MEK activated apoptosis, but not cell cycle arrest.  CDK4 was implicated as a key driver of these differences and combined pharmacological inhibition of MEK and CDK4 in vivo led to substantial synergy in therapeutic efficacy in a mouse model  ((Kwong, L. N., Costello, J. C., Liu, H., Jiang, S., Helms, T. L., Langsdorf, A. E., Jakubosky, D., Genovese, G., Muller, F. L., Jeong, J. H., et al. (2012). Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma. Nature medicine 18, 1503-1510. http://www.bu.edu/abl/files/naturemedicine_costello.pdf)) .
  • Sensitivity of NRAS-mutant cell lines to MEK inhibitors was shown to be associated with expression of AHR (aryl hydrocarbon receptor) in vitro[10] .
  •  A study of combinatorial drug interactions in vitro pinpointed the combination of simvastatin with a CDK inhibitor as the only fairly effective cytotoxic treatment for NRAS-mutated melanoma cell lines  ((Held, M. A., Langdon, C. G., Platt, J. T., Graham-Steed, T., Liu, Z., Chakraborty, A., Bacchiocchi, A., Koo, A., Haskins, J. W., Bosenberg, M. W., and Stern, D. F. (2013). Genotype-selective combination therapies for melanoma identified by high-throughput drug screening. Cancer discovery 3, 52-67. http://www.ncbi.nlm.nih.gov/pubmed/23239741)) .

The combinations of inhibitors to target NRAS activated signaling through MEK and PI3K, MEK and AKT, MEK and PI3K/mTOR, as well as MEK and VEGF receptor inhibition, are now in early phase clinical trials. Only a few trials specifically target melanomas with NRAS mutations, but a number of trials use combinations of agents or single agents that could have therapeutic benefits in this subgroup of melanoma.  Single agents in phase I or early phase II trials include inhibitors of CDK (PD0332991, dinaciclib, LY2835219, BAY1000394, LEE011), the Notch pathway (RO4929097), and Aurora kinase A (MLN8237/alisertib, GSK1070916A) (Supplemental Table 2).

 

GNAQ and GNA11

Activating mutations in GNAQ and GNA11, encoding members of the Gα(q) family of G protein α subunits, are the driver oncogenes in uveal melanoma[11] .  Mutations in GNAQ and GNA11 are mutually exclusive and are present in a vast majority of uveal melanomas[12].  GNA11 has a stronger association with metastatic than with nonmetastatic uveal melanoma.  Mutations in these GTP-binding proteins activate the MAPK pathway.

 

Potential treatment approaches

These include inhibitors of the MAPK pathway and one ongoing clinical trial with the MEK inhibitor selumetinib (AZD6244).  GNAQ mutation promotes resistance in vitro to selumetinib, but the combination of selumetinib with the ATP-competitive mTOR inhibitor AZD8055 might be more promising  ((Ho, A. L., Musi, E., Ambrosini, G., Nair, J. S., Deraje Vasudeva, S., de Stanchina, E., and Schwartz, G. K. (2012). Impact of combined mTOR and MEK inhibition in uveal melanoma is driven by tumor genotype. PloS one 7, e40439. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0040439)) .  A trial of the MTOR inhibitor everolimus and the somatostatin receptor activating peptide pasireotide/SOM232 is recruiting patients (NCT01252251).
Inhibition of both PI3K and MAPK, but neither of them singly, might also work in uveal melanoma as seen from in vitro experiments  ((Khalili, J. S., Yu, X., Wang, J., Hayes, B. C., Davies, M. A., Lizee, G., Esmaeli, B., and Woodman, S. E. (2012b).  Combination small molecule MEK and PI3K inhibition enhances uveal melanoma cell death in a mutant GNAQ- and GNA11-dependent manner.  Clinical cancer research : an official journal of the American Association for Cancer Research 18, 4345-4355. http://clincancerres.aacrjournals.org/content/18/16/4345.short)) .  No trials involving this combination are ongoing.
Enzastaurin and AEB071, PKC inhibitors, have shown some activity against uveal melanoma cell lines in vitro  ((Wu, X., Li, J., Zhu, M., Fletcher, J. A., and Hodi, F. S. (2012a). Protein kinase C inhibitor AEB071 targets ocular melanoma harboring GNAQ mutations via effects on the PKC/Erk1/2 and PKC/NF-kappaB pathways. Molecular cancer therapeutics 11, 1905-1914. http://www.ncbi.nlm.nih.gov/pubmed/22653968)) .  PKC is involved in signal transduction from GNAQ to MEK; an inhibitor of PKC is also being tested in a phase I clinical study.
Recently, it was shown that the guanine nucleotide exchange factor Trio is involved in mitogenic signaling through GNAQ and GNA11.  Trio is essential for activating Rho- and Rac-regulated signaling pathways acting on JNK and p38, thereby transducing proliferative signals from Gα(q) to the nucleus independently of phospholipase C-β  ((Vaque, J. P., Dorsam, R. T., Feng, X., Iglesias-Bartolome, R., Forsthoefel, D. J., Chen, Q., Debant, A., Seeger, M. A., Ksander, B. R., Teramoto, H., and Gutkind, J. S. (2012). A Genome-wide RNAi Screen Reveals a Trio-Regulated Rho GTPase Circuitry Transducing Mitogenic Signals Initiated by G Protein-Coupled Receptors. Molecular cell. http://www.sciencedirect.com/science/article/pii/S1097276512008957)) . These findings might open new avenues for treatment of uveal melanoma.

 

MITF

Microphthalmia-associated transcription factor MITF is a lineage survival oncogene, amplified in 20% of melanoma  ((Garraway, L. A., Widlund, H. R., Rubin, M. A., Getz, G., Berger, A. J., Ramaswamy, S., Beroukhim, R., Milner, D. A., Granter, S. R., Du, J., et al. (2005). Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature 436, 117-122. http://www.broadinstitute.org/mpr/publications/projects/SNP_Analysis/Garraway.pdf)) ; amplification of MITF is associated with a reduced 5-year survival. MITF is mutated in some familial melanomas  ((Yokoyama, S., Feige, E., Poling, L. L., Levy, C., Widlund, H. R., Khaled, M., Kung, A. L., and Fisher, D. E. (2008). Pharmacologic suppression of MITF expression via HDAC inhibitors in the melanocyte lineage. Pigment cell & melanoma research 21, 457-463. http://www.mendeley.com/catalog/pharmacologic-suppression-mitf-expression-via-hdac-inhibitors-melanocyte-lineage/)) .  The variant MITF(E318K) co-segregates with affected individuals in familial melanoma and is likely to be a gain-of-function (GOF) mutation.  It abolishes a SUMO-ilation site on MITF that reportedly acts to inhibit transcriptional activity of MITF.  Transcriptional targets of MITF have been identified and, besides a large number of lineage-specific transcripts, include genes related to regulation of cell cycle, among them CDK2  ((Du, J., Widlund, H. R., Horstmann, M. A., Ramaswamy, S., Ross, K., Huber, W. E., Nishimura, E. K., Golub, T. R., and Fisher, D. E. (2004). Critical role of CDK2 for melanoma growth linked to its melanocyte-specific transcriptional regulation by MITF. Cancer cell 6, 565-576. http://www.sciencedirect.com/science/article/pii/S1535610804003095)) .  Considering that MITF itself is currently not a druggable target, inhibition of CDK2 is a plausible aim in melanoma with MITF aberrations.
Other candidate targets in the MITF program include receptor tyrosine kinase TYRO3, which regulates expression of MITF in a SOX-10-dependent manner  ((Zhu, S., Wurdak, H., Wang, Y., Galkin, A., Tao, H., Li, J., Lyssiotis, C. A., Yan, F., Tu, B. P., Miraglia, L., et al. (2009). A genomic screen identifies TYRO3 as a MITF regulator in melanoma. Proceedings of the National Academy of Sciences of the United States of America 106, 17025-17030. http://www.pnas.org/content/106/40/17025.short)) . Ubiquitin-specific protease 13 (USP13) was shown to be responsible for MITF deubiquitination and stabilization.  USP13 is essential for melanoma growth in vitro and in vivo and might be another target in MITF-mutated melanoma  ((Zhao, X., Fiske, B., Kawakami, A., Li, J., and Fisher, D. E. (2011). Regulation of MITF stability by the USP13 deubiquitinase. Nature communications 2, 414. http://www.nature.com/ncomms/journal/v2/n8/full/ncomms1421.html)) . Hypoxia-inducible factor HIF1 was also reported to be a transcriptional target of MITF(Busca et al., 2005), and its expression is apparently stimulated by MITF with the well know consequences of stimulating tumor survival, angiogenesis and metastases. Paradoxically, expression ofMITF itself is reduced under hypoxic conditions in normal melanocytes and melanoma via direct binding of transcription repressor DEC1, which is activated by HIF1(Feige et al., 2011). This suggests a negative feedback loop. A recent study implicated HIF1 factors in promoting melanoma invasion and metastases without affecting proliferation of the primary tumors (Hanna et al., 2013). MITF expression is reduced under hypoxic conditions in normal melanocytes and melanoma via direct binding of transcription repressor DEC1, which is activated by HIF1  ((Feige, E., Yokoyama, S., Levy, C., Khaled, M., Igras, V., Lin, R. J., Lee, S., Widlund, H. R., Granter, S. R., Kung, A. L., and Fisher, D. E. (2011). Hypoxia-induced transcriptional repression of the melanoma-associated oncogene MITF. Proceedings of the National Academy of Sciences of the United States of America 108, E924-933. www.pnas.org/content/108/43/E924.full)) .  This suggests another mechanism whereby amplification of MITF in melanomas provides a survival advantages under hypoxic conditions.  This also suggests a counterintuitive strategy of stabilizing HIF1 expression in melanoma to target MITV expression  ((Feige, E., Yokoyama, S., Levy, C., Khaled, M., Igras, V., Lin, R. J., Lee, S., Widlund, H. R., Granter, S. R., Kung, A. L., and Fisher, D. E. (2011). Hypoxia-induced transcriptional repression of the melanoma-associated oncogene MITF. Proceedings of the National Academy of Sciences of the United States of America 108, E924-933. www.pnas.org/content/108/43/E924.full)) .

MITF is regulated by the transcription factor ATF2. Primary melanoma specimens that exhibit a high nuclear ATF2:MITF ratio were found to be associated with metastatic disease and poor prognosis  ((Shah, M., Bhoumik, A., Goel, V., Dewing, A., Breitwieser, W., Kluger, H., Krajewski, S., Krajewska, M., Dehart, J., Lau, E., et al. (2010). A role for ATF2 in regulating MITF and melanoma development. PLoS genetics 6, e1001258. http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1001258)) .  ATF2 control of melanoma development is mediated, in part, through its negative regulation of SOX10 and consequently, of MITF transcription. In human patients, virtually all congenital nevi and melanomas are SOX10 positive.  SOX10 silencing in human melanoma cells suppresses neural crest stem cell properties, counteracts proliferation and cell survival, and completely abolishes in vivo tumor formation  ((Shakhova, O., Zingg, D., Schaefer, S. M., Hari, L., Civenni, G., Blunschi, J., Claudinot, S., Okoniewski, M., Beermann, F., Mihic-Probst, D., et al. (2012). Sox10 promotes the formation and maintenance of giant congenital naevi and melanoma. Nature cell biology 14, 882-890. http://www.nature.com/ncb/journal/v14/n8/full/ncb2535.html)) .
SOX10, PAX3, and MITF participate in regulation of MET (HGF receptor), which is expressed at high levels in human melanoma  ((Puri, N., Ahmed, S., Janamanchi, V., Tretiakova, M., Zumba, O., Krausz, T., Jagadeeswaran, R., and Salgia, R. (2007). c-Met is a potentially new therapeutic target for treatment of human melanoma. Clinical cancer research : an official journal of the American Association for Cancer Research 13, 2246-2253. http://clincancerres.aacrjournals.org/content/13/7/2246.short)) . MITF and PAX3 bind directly to the MET promoter; coexpression of these three proteins is found melanoma biopsies  ((Mascarenhas, J. B., Littlejohn, E. L., Wolsky, R. J., Young, K. P., Nelson, M., Salgia, R., and Lang, D. (2010). PAX3 and SOX10 activate MET receptor expression in melanoma. Pigment cell & melanoma research 23, 225-237. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2979310/)) .
PPAR-γ coactivators PGC-1α and PGC-1β are critical components of the melanogenic system governed by MITF. Melanomas with high expression of PGC-1α exhibit increased expression of mitochondrial respiration complexes and increased oxidative phosphorylation  ((Vazquez, F., Lim, J. H., Chim, H., Bhalla, K., Girnun, G., Pierce, K., Clish, C. B., Granter, S. R., Widlund, H. R., Spiegelman, B. M., and Puigserver, P. (2013). PGC1alpha Expression Defines a Subset of Human Melanoma Tumors with Increased Mitochondrial Capacity and Resistance to Oxidative Stress. Cancer cell. http://www.sciencedirect.com/science/article/pii/S1535610813000342)) .  The high MITF-high PGC-1α expressing cells have an increased capacity to withstand oxidative stress, and, unlike PGC-1α low cells, respond poorly to ROS-inducing agents such as PEITC or piperlongumine[13] .  In addition, polymorphism studies reveal expression quantitative trait loci in the PGC-1β gene that correlate with protection from melanoma in humans  ((Shoag, J., Haq, R., Zhang, M., Liu, L., Rowe, G. C., Jiang, A., Koulisis, N., Farrel, C., Amos, C. I., Wei, Q., et al. (2012). PGC-1 Coactivators Regulate MITF and the Tanning Response. Molecular cell. http://www.sciencedirect.com/science/article/pii/S1097276512009082)) .
The crystal structure of MITF was resolved recently, paving the way for the future targeting of MITF in melanoma and other cancers  ((Pogenberg, V., Ogmundsdottir, M. H., Bergsteinsdottir, K., Schepsky, A., Phung, B., Deineko, V., Milewski, M., Steingrimsson, E., and Wilmanns, M. (2012). Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF. Genes & development 26, 2647-2658. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521630/)) .

 

Potential treatment approaches

The findings on the MITF-mediated pathways listed above remain in the domain of preclinical research, although targeting MET receptor is feasible because MET inhibitors (cabozantinib, et al) are in clinical trials for cancers other than melanoma. For now, panobinostat/ LBH589 (HDAC inhibitor) is the only drug in clinical development for MITF-amplified melanoma (NCT01065467); the rationale behind its use supported by a study demonstrating efficacy of the HDAC inhibitor in suppressing MITF expression in vitro and in vivo  ((Yokoyama, S., Feige, E., Poling, L. L., Levy, C., Widlund, H. R., Khaled, M., Kung, A. L., and Fisher, D. E. (2008). Pharmacologic suppression of MITF expression via HDAC inhibitors in the melanocyte lineage. Pigment cell & melanoma research 21, 457-463. http://www.mendeley.com/catalog/pharmacologic-suppression-mitf-expression-via-hdac-inhibitors-melanocyte-lineage/)) . A new study opened a possibility of targeting the nuclear translocation of ATF2 by inhibiting PKCε which phosphorylates ATF2 and induces its transport to the nucleus. The two compounds found to promote cytoplasmic localization of ATF2 were identified in an image-based screen (Varsano et al., 2013).

 

KIT

KIT is a receptor tyrosine kinase activated by binding of stem cell factor (SCF). KIT mutations or amplifications activate a signal transduction pathway that ultimately leads to cell proliferation.  Mutations in KIT are found in mucosal, acral, and permanently exposed skin melanomas.  KIT mutations are rare in melanoma, but the availability of selective inhibitors for KIT has prompted interest in targeting this oncogene.  These inhibitors, imatinib, sunitinib, nilotinib, and dasatanib, were developed for different cancers (chronic myeloid leukemia, gastrointestinal stromal tumors) and even different kinases, but they show activity against KIT.  Targeting KIT with imatinib has demonstrated remarkable efficacy in patients with gastrointestinal stromal tumors, but initial trials in melanoma were unsuccessful, most likely due to the absence of selection of patients with aberrations in KIT.  The Src inhibitor dasatinib was tested as a single agent in unselected melanoma patients and showed poor response and high toxicity  ((Kluger, H. M., Dudek, A. Z., McCann, C., Ritacco, J., Southard, N., Jilaveanu, L. B., Molinaro, A., and Sznol, M. (2011). A phase 2 trial of dasatinib in advanced melanoma. Cancer 117, 2202-2208. http://meeting.ascopubs.org/cgi/content/abstract/27/15S/9010)) . Nevertheless, case reports continued to surface that demonstrated the efficacy of imatinib for patients with specific KIT genetic aberrations (listed in  ((Vidwans, S. J., Flaherty, K. T., Fisher, D. E., Tenenbaum, J. M., Travers, M. D., and Shrager, J. (2011). A melanoma molecular disease model. PloS one 6, e18257. A melanoma molecular disease model. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0018257)) .  Recently, trials of imatinib and nilotinib have shown promising results in selected KIT mutant cancers.

 

Treatment approaches

Two phase II trials with imatinib produced only modest responses to treatment.  Sunitinib malate was tested in another trial, and, again, no correlation was observed between the degree of KIT expression and longer PFS or OS  ((Mahipal, A., Tijani, L., Chan, K., Laudadio, M., Mastrangelo, M. J., and Sato, T. (2012). A pilot study of sunitinib malate in patients with metastatic uveal melanoma. Melanoma research 22, 440-446. http://www.ncbi.nlm.nih.gov/pubmed/23114504)) .  From another study it appears that patients with overexpressed or amplified KIT do not respond to sunitinib, while patients with mutated KIT (in exons 11 and 13) show a marked increase in PFS  ((Minor, D. R., Kashani-Sabet, M., Garrido, M., O’Day, S. J., Hamid, O., and Bastian, B. C. (2012). Sunitinib therapy for melanoma patients with KIT mutations. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 1457-1463. http://clincancerres.aacrjournals.org/content/early/2012/01/18/1078-0432.CCR-11-1987)) .  Preliminary results of a trial with nilotinib were published when it showed durable responses in patients with KIT mutations  ((Cho, J. H., Kim, K. M., Kwon, M., Kim, J. H., and Lee, J. (2012). Nilotinib in patients with metastatic melanoma harboring KIT gene aberration. Investigational new drugs 30, 2008-2014. http://www.ncbi.nlm.nih.gov/pubmed/22068222)) .
At least 10 ongoing trials test the activity of the aforementioned inhibitors in KIT-mutated melanoma as single agents (Supplemental Table 2).  It is conceivable that future trials will include combinations of KIT inhibitors with other targeted drugs and immunotherapy (one such trial, NCT01738139, might begin to recruit patients in the near future).

 

NF1/neurofibromin

NF1 is a tumor suppressor; germline mutations in NF1 deregulate both PI3K and MAPK pathways and result in familial neurofibromatosis. Some neurofibromatosis patients with inactivation of NF1 develop melanomas. Moreover, NF1 expression is low in 47% of uveal melanomas  ((Johnson, M. R., Look, A. T., DeClue, J. E., Valentine, M. B., and Lowy, D. R. (1993). Inactivation of the NF1 gene in human melanoma and neuroblastoma cell lines without impaired regulation of GTP.Ras. Proceedings of the National Academy of Sciences of the United States of America 90, 5539-5543. http://www.ncbi.nlm.nih.gov/pubmed/8516298)) and allelic losses are seen in other types of melanoma  ((Gutzmer, R., Herbst, R. A., Mommert, S., Kiehl, P., Matiaske, F., Rutten, A., Kapp, A., and Weiss, J. (2000). Allelic loss at the neurofibromatosis type 1 (NF1) gene locus is frequent in desmoplastic neurotropic melanoma. Human genetics 107, 357-361. http://www.ncbi.nlm.nih.gov/pubmed/11129335)) .  Mutations in NF1 are enriched in melanoma that have wild-type BRAF and NRAS (25% of those), suggesting that they could be considered as driver mutations in this subset that was, until now, not amenable for targeted therapy approaches.  In addition, NF1 mutations or suppression occur also in human melanomas that harbor concurrent BRAF mutations  ((Hodis, E., Watson, I. R., Kryukov, G. V., Arold, S. T., Imielinski, M., Theurillat, J. P., Nickerson, E., Auclair, D., Li, L., Place, C., et al. (2012). A landscape of driver mutations in melanoma. Cell 150, 251-263. http://211.144.68.84:9998/91keshi/Public/File/42/150-2/pdf/1-s2.0-S0092867412007787-main.pdf)) ,  ((Maertens, O., Johnson, B., Hollstein, P., Frederick, D. T., Cooper, Z. A., Messaien, L., Bronson, R. T., McMahon, M., Granter, S., Flaherty, K. T., et al. (2012). Elucidating distinct roles for NF1 in melanomagenesis. Cancer discovery. http://www.ncbi.nlm.nih.gov/pubmed/23171796)) .  Mutations in the NF1 cooperate with BRAF mutations in a mouse model of melanomagenesis by suppressing BRAF-induced senescence (OIS), promoting melanocyte hyperproliferation, and enhancing melanoma development.  Knockdown of NF1 in vitro promotes activation of both KRAS and CRAF.

 

Potential treatment approaches

In recent studies NF1 could be successfully targeted in mouse models with a combination of MEK and PI3K/mTOR inhibitors[14] ; a combination of irreversible RAF inhibition and MEK inhibition was also effective in vivo[15] .

 

TELOMERASE

Two very recent studies—one based on analysis of genomic alterations in a melanoma-prone family, the other based on analysis of the genomic sequence data from melanoma tumors—have revealed new, frequent, and unexpected mutations in the regulatory regions of the catalytic subunit of telomerase[16] ;[17]. Mutations were found in 33% primary and 85% metastatic in the first study versus overall 71% of tumors in the second study. Mutations create a new binding motif for TCF/ETS transcription factors and result in an increased transcription from TERT promoter. Mutations in the promoter region of TERT are considered to be driver mutations because of their association with familial melanoma and high frequency in sporadic melanoma. TERT promoter mutations are not limited to melanoma, and were found in 16% of tumor cell lines from diverse cancers[17].

 

Potential treatment options

Telomerase inhibitor imetelstat sodium/GRN163L (antisense oligonucleotide) is trialed in breast cancer and a telomerase vaccine GV1001 in non-small cell lung cancer (NSCLC). It remains to be seen if telomerase targeted drugs would be tested in patients with tumors with mutations in the telomerase promoter region.

 

Receptor tyrosine kinases and PI3K pathway

Activation of the PI3K pathway serves to overcome OIS that is associated with mutant BRAF. In support of this notion, while BRAF mutations are present in both nevi and melanoma sections of contiguous nevi-melanoma biopsies, activation of PI3K (through loss of PTEN expression or activation of AKT3) was detected in the melanoma portions only[18]. This indicates that the AKT3/PI3K pathway is activated during progression to malignant melanoma, most likely in order to overcome OIS. PI3K pathway activation serves as a rate-limiting event and dual inhibition of PI3K/mutant BRAF eliminated cells resistant to BRAF inhibition in vitro[19]. Several mutations in different components of the PI3K pathway, from receptor tyrosine kinases (RTK) to PTEN and to AKT3 have been described.

     ERBB4. Mutations in this tyrosine kinase receptor are found in 19% of melanomas, based on the results of targeted sequencing of the tyrosine kinase family in seven melanoma tumors[20]. These might respond to lapatinib, which is currently in a clinical trial for advanced melanoma (NCT01264081).

     MET. Mutations in MET have not been described in melanoma, but there is strong evidence that RTK is involved in melanoma growth and metastases (see below: WNT pathway and HGF expression in stroma). Copy number gains involving MET locus in melanomas were documented[21]. SU11274, an inhibitor of MET, has shown significant activity in a xenograft model[22]. Cabozantinib, an inhibitor of MET and other RTKs is in clinical trials for a variety of cancers with deregulated RTK signaling.

MERTK. MER, a receptor tyrosine kinase sharing a family relationship with TYRO3 and AXL, was found to be expressed at increasing levels during progression from nevi to metastatic melanoma.Stimulation of MER with its ligand GAS6 leads to activation ofMAPK, PI3K and JAK/STAT pathways. Inhibition of MERTK with a synthetic compound UNC1062 inhibits invasion and induces apoptosis in melanoma cells in vitro (Schlegel et al., 2013). MERTK and AXL are expressed alternatively in melanoma (Tworkoski et al., 2013). MERTK expressing melanoma cells are more proliferative that AXL expressing cells, though the latter are more invasive (Tworkoski et al., 2013).

     AKT. Deregulated Akt3 activity was shown to promote development of malignant melanoma; amplifications of Akt3 were detected in melanoma[23]. Alterations of Akt1 and Akt2 are rare, but genetic gain of Akt3 is seen in 25% of melanoma. High activity of Akt3 promotes progression of BRAFV600E-positive nevi to melanoma[24]. A trial combining MK2206 (Akt inhibitor) in combination with AZD6244 (the MEK inhibitor selumetinib) is in progress (NCT01519427).

     PTEN is a well-known tumor suppressor affected in 50% to 60% of melanomas, most commonly via allelic loss and focal deletions[25];[26] ;[27]. PTEN is also deregulated in melanoma via loss of ZEB2, a competitive endogenous RNA (ceRNA)[28]. In addition, disruptions of MAGI2, a protein that associates with and stabilizes PTEN, occur in melanoma[25]. PTEN aberrations are often associated with the presence of BRAFV600E, and cooperate with mutant BRAF in a GEMM[29], most likely providing an OIS inhibitory function by activating the PI3K pathway. In this model, growth of melanoma could be inhibited by combined treatment with PD325901 (MEK inhibitor) and rapamycin (mTOR inhibitor). PTEN inactivating aberrations are not associated with NRAS mutations, perhaps because the latter lead to activation of PI3K in the absence of this pathway’s mutations.

    Other PI3K pathway mutations. Mutations in other PI3K pathways genes MTOR, IRS4, PIK3R1, PIK3R4, and PIK3R5 were detected in 17% of BRAFV600 and in 9% of NRAS-mutant tumors[7]. It will be interesting to determine the status of these genes in melanoma tumors that show inherent or acquired resistance to BRAF inhibitors.

 

MAPK pathway downstream of BRAF

 

MEK1 and MEK2. Sequencing of seven melanoma cell lines and donor-matched germline cells found MAP2K1 and MAP2K2 (MEK1 and MEK2, respectively) mutations, resulting in constitutive ERK phosphorylation and higher resistance to MEK inhibitors. Screening of a larger cohort of melanoma tumors revealed the presence of recurring somatic MAP2K1 and MAP2K2 mutations at an overall frequency of 8%[30].

Scaffold protein in MAPK pathway as a possible target. There are new data indicating that it might be possible to target the MAPK pathway without direct inhibition of enzymatic activity of kinases in this pathway. It is well known that kinases usually depend on scaffold proteins that assemble signaling complexes. One of this scaffold proteins, IQGAP1 is essential for activity of ERK1/2, and disruption of IQGAP interaction with ERK using a small peptide inhibited RAS or BRAF driven tumorigenesis and even overcame resistance to Vemurafenib(Jameson et al., 2013).

RAC pathway. RAC1 is a Rho GTPase, a GTP exchange protein known to affect the cell cytoskeleton and motility. Its role in conveying oncogenic signaling from mutant NRAS in melanoma was described[31] and references therein). Recently P29S mutations in the conserved switch domain were described in melanoma, in 5% of tumors of the experimental set[32], and their functional role in vitro was confirmed. Mutations were also found in other Rho family members: RAC2 (P29L), RHOT1 (P30L), and in CDC42 (G12D).

MAP3K5 and MAP3K9. These MAP3 kinases are directly downstream of Rac and Rho signaling activated by various stress signals. Mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, suggest inactivation of these kinases. Indeed, mutants MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity in vitro. Overexpression of these mutants reduced the phosphorylation of downstream MAP kinases, while siRNA-mediated depletion of MAP3K9 in melanoma cells led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K activity acts as a pro-survival adaptation[33].

PREX2. Mutations of phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2 were found in 14% of a cohort of 107 tumors[25]. Functional studies in vitro have confirmed a role for PREX2 in melanomagenesis. All three types of mutations indicate involvement of RAC pathway in pathogenesis of melanoma.

 

Cell cycle and apoptosis related genes

CDK4, a cell cycle G1/S kinase, and Cyclin D1 (CCND1) were shown to be amplified in melanoma (Muthusamy et al., 2006). In a clinical study of FOLFIRI (chemotherapeutic agent) with the nonselective CDK inhibitor flavopirodol in solid tumors, one melanoma patient had a complete response[34]. Targeted CDK inhibitors are in clinical development (PD0332991, Dinaciclib, LY2835219, BAY1000394, LEE011), and at least four of them are currently tested in trials for melanoma and other tumors (Supplemental Tables 1 and 2).

P53 mutations are rare in melanoma, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Indeed, several alterations in genes affecting p53 activity have been discovered in melanoma. Among them are the long known mutations in p14ARF, the recent identification of MDM4 as a target of frequent amplification in melanoma, and the finding of the elevated expression and the anti-apoptotic role of p53 related protein p63. This means that the rare “p53 mutant subtype” could be now expanded to a “p53 pathway aberrations” subtype.

CDKN2A locus is frequently deleted in melanoma of all primary subtypes[35] ;[36] ;[37]. Two tumor suppressors are encoded within this locus: p14ARF, which activates p53 through inhibition of its major negative regulator MDM2; and p16INK4a, a cyclin-dependent kinase inhibitor that activates retinoblastoma (RB) through negative regulation of CDK4. Loss of CDKN2A was reported to occur in 16% to 41% of sporadic melanoma and with a high frequency in familial melanoma[38].

Potential treatment of ARF deficiency would involve inhibition of MDM2-p53 interaction. This interaction has attracted serious efforts to develop specific inhibitors, of which Nutlin is the more advanced. The new generations of Nutlin-based drugs are in clinical studies for various solid tumors. Encouraging results were reported for one of them, RG7112, in early clinical testing for liposarcoma and other tumors[39].

MDM4. In a recent study it was shown that MDM4, a negative regulator of p53, is upregulated in about 65% of melanomas, and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by Nras[40]. Inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAFV600E. MDM4 could be a key determinant of impaired p53 function in human melanoma and a promising target for anti-melanoma combination therapy[40].

P63, a protein related to p53, has been reported to have an anti-apoptotic role in melanoma, which is mediated through its interaction with p53 in melanoma cells. P63 is expressed at high levels in melanoma cell lines and clinical samples and prevents translocation of p53 to the nucleus[41]. This study further expands the role of the aberrations of the p53 pathway in melanomagenesis.

iASPP . is a conserved ankyrin repeat protein that shuttles between nuclear and cytoplasmic compartments, and nuclear iASPP is found in proliferating cells. The nuclear iASPP was shown to inhibit the pro-apoptotic function of p53 (Bergamaschi et al., 2006). High levels of nuclear iASPP were observed in metastatic melanoma versus primary melanoma Lu and Breyssens. The authors found that iASPP is phosphorylated by cyclinB/CDK1 which promotes its nuclear localization, and binding to p53, inhibiting its pro-apoptotic function. Prevention of iASPP phosphorylation by CDK1 inhibitor or knock-down of iASPP induced apoptotic death in melanoma cell lines which is further enhanced by Nutlin3 (inhibitor of p53-MDM2 interaction and degradation of p53). Furthermore, inhibition of BRAF with vemurafenib, or MEK with UO126, potentiated effects of Nutlin3 and cyclinB/CDK1 inhibition, inducing apoptosis of melanoma in vitro and in vivo (Lu et al., 2013). These findings indicate an alternative strategy for combining therapies targeting MAPK pathway and cyclinB/CDK1 in wild-type p53 melanoma.

     NFkB. As in many, many other tumors, the NFkB pathway is activated in melanoma, but systemic inhibition of NFkB might have catastrophic general adverse effects/toxicity. A new faucet of NFkB activation was described recently in drug-treated melanoma cells that might acquire a senescent secretory phenotype; the latter results in a pro-inflammatory and pro-metastatic phenotype characterized by production of CCL-2. Activated NFkB and PARP-1 contribute to this phenomenon and could be involved in the therapeutic failure[42].

    BCL2. There is abundant literature that documents elevated BCL2 expression in melanoma and its contribution to melanoma and melanocyte cell survival. A particular member of this family, BCL2A1, was shown to be amplified in 30% of melanoma and contribute to resistance to BRAF inhibition[43] (see under “Intrinsic resistance”). Attempts to target BCL2 with antisense RNA in melanoma patients have not been successful. Obatoclax, a drug inhibiting interaction of BCL2 with proapoptotic proteins Bax and Bak is in clinical trials for other malignancies. BH3 mimetics to inhibit function of anti-apoptotic proteins, such as BCL2 and BCL-xL, are still subjects of significant interest. One of them, ABT-737 resensitized both melanoma cell lines in vitro and tumors in the in vivo model to common chemotherapeutics (including the only FDA-approved chemotherapeutic for melanoma: dacarbazine), leading to marked BIM (Bcl-2 interacting mediator of cell death) -mediated apoptosis. ABT-737 may be a beneficial adjuvant therapy to improve Melanoma Response rates when conventional chemotherapy is the only option[44].

β-catenin and WNT pathway
A number of reports have heavily implicated WNT signaling in melanoma progression and metastases. Rare mutations in -catenin and in other members of the WNT signaling family were identified in malignant melanoma 10 years ago[45]. -catenin suppresses expression of p16INK and cooperates with NRAS in transformation to a frank melanoma[46].WNT5a, in particular, by binding to the Frizzled4- LRP6 complex activates ARF6 (guanosine triphosphatase adenosine diphosphate ribosylation factor 6), leading to displacement of -catenin from N-cadherin in melanoma. This stimulates signaling from -catenin and increases invasiveness (Grossmann et al., 2013).
In a mouse melanoma model based on PTEN loss and BRAFV600E mutation, -catenin was shown to be a central mediator of metastases as well as a regulator of both MAPK and PI3K pathways. Recent findings established Wnt signaling as a metastasis regulator in melanoma[47]. Mutant BRAF signaling is thought to inhibit WNT/-catenin signaling. Endogenous -catenin was apparently required for the efficacy of PLX4720 in vitro; activation of WNT/-catenin signaling was found to enhance the anticancer activity of PLX4720 in vitro and in vivo[48].
Negative regulation of WNT/-catenin signaling by MAPK pathway was confirmed in an additional study (Conrad, W. H., Swift, R. D., Biechele, T. L., Kulikauskas, R. M., Moon, R. T., and Chien, A. J. (2012). Regulating the response to targeted MEK inhibition in melanoma: enhancing apoptosis in NRAS- and BRAF-mutant melanoma cells with Wnt/beta-catenin activation. Cell cycle 11, 3724-3730. http://www.landesbioscience.com/journals/cc/article/21645/?nocache=858365738). Treatment of BRAF-mutant and NRAS-mutant melanoma lines with WNT3A and the MEK inhibitor AZD6244 induces apoptosis. The susceptibility of BRAF-mutant lines and NRAS-mutant lines to apoptosis correlated with negative regulation of Wnt/β-catenin signaling by ERK/MAPK signaling and dynamic decreases in abundance of the downstream scaffolding protein, AXIN1[49]. WNT inhibitors such as PRI-724 (inhibitor of interaction between -catenin and CBP) and OMP-54F28 (a fusion protein antagonistic to Fzd8) are starting to enter clinical testing in tumors other than melanoma.

Transcriptional factors in melanoma

     MYC. This universal oncogene and transcriptional master regulator is overexpressed or present at increased copy numbers in 41% or more of melanoma tumors (Moore, S. R., Persons, D. L., Sosman, J. A., Bobadilla, D., Bedell, V., Smith, D. D., Wolman, S. R., Tuthill, R. J., Moon, J., Sondak, V. K., and Slovak, M. L. (2008). Detection of copy number alterations in metastatic melanoma by a DNA fluorescence in situ hybridization probe panel and array comparative genomic hybridization: a southwest oncology group study (S9431). Clinical cancer research : an official journal of the American Association for Cancer Research 14, 2927-2935. http://clincancerres.aacrjournals.org/content/14/10/2927.full.pdf+html). It is currently considered not druggable.

     ETV1. Transcription factor from the ETS family was implicated as an oncogene in melanoma and copy gain numbers were found in 40% of cases examined, with amplification of ETV1 in 13% to18% of cases[50].

 

Other significant genetic abnormalities in melanoma

     NEDD9, an integrin adaptor protein related to P130CAS, and a member of a family implicated in pathogenesis of a variety of cancers, was identified as a bona fide melanoma metastasis gene in melanoma. NEDD9 enhanced invasion in vitro and metastasis in vivo of both normal and transformed melanocytes, and was frequently overexpressed in metastatic melanoma relative to primary melanoma[51]. Fifty-seven percent of melanomas were found to have amplification of NEDD9[21].

     PPP6C is a serine-threonine phosphatase, mutated in 12% of sun-exposed melanomas exclusively with BRAF or NRAS mutations[52][53]. PPP6C is the catalytic unit of a phosphatase complex that negatively regulates activity of the mitotic Aurora kinase, a known oncogene. Most mutations map in conserved domain involved in interaction with the regulatory subunit of the complex.

     TACC1 (transforming acidic coiled-coil containing protein 1) is mutated in 5% of an experimental set of 121. TACC1 is known to stimulate the PI3K and Ras pathways and interact with Aurora kinase, which is notable considering the PPP6C mutations that inactivate Aurora kinase[52]. At least 16 Aurora kinase inhibitors are in clinical studies, of them two inhibitors (MLN8237/alisertib, GSK1070916A in melanoma (Supplemental Tables 1 and 2).

     BAP1 (BRCA1-associated protein-1/ubiquitin carboxy-terminal hydrolase) is involved in metastatic progression of ocular and cutaneous melanoma. BAP1 is a known tumor suppressor gene. BAP1 mutations are frequently found in uveal melanoma[54]. Germline BAP1 mutations have recently been associated with an increased risk of several cancers, including atypical melanocytic tumors[55] and uveal melanoma[56]. Uveal melanoma might be sensitive to HDAC inhibitors[57].

     SF3B1. Codon 625 of the SF3B1 gene, encoding splicing factor 3B subunit 1, is consistently mutated in low-grade uveal melanomas with good prognosis[58].

     SNX31 was identified as one of the 11 new genes mutated in melanoma[52]. It encodes a poorly characterized sorting nexin 31 protein. It could be a Ras effector protein that selectively binds GTP loaded H-RAS[59].

     STK19. A five percent mutation rate of this kinase gene with unknown functions is seen in melanoma[52].

     LKB1/STK11. LKB1 might be a central kinase that integrates energy metabolism and tumor growth, in part through activation of the family of AMPK kinases. Germline mutations in LKB1 (STK11) are associated with the Peutz-Jeghers syndrome (PJS), which includes aberrant mucocutaneous pigmentation, and somatic LKB1 mutations, which occur in 10% of cutaneous melanoma[60]. Somatic inactivation of LKB1 with K-Ras activation in murine melanocytes led to highly metastatic melanoma with 100% penetrance. Downstream events of LKB1 inactivation, in addition to AMPK-related effects, included increased phosphorylation of the SRC family kinase YES, increased expression of WNT target genes, and expansion of a CD24(+) cell population in melanoma with increased metastatic behavior in vitro and in vivo[61]. Dasatinib, an SRC inhibitor, was shown previously to exhibit a higher activity towards YES rather than SRC, and could be a promising treatment for LKB1-mutated melanoma[61]. Metformin, an indirect activator of AMPK, the downstream target of LKB1, is currently in a clinical trial in combination with vemurafenib (NCT01638676).

     ARID2 is a component of the SWI/SNF chromatin remodeling complex. Loss of function mutations were found in 7% of melanomas. Targeted search identified mutations in other members of the ARID family (ARID1B, ARID1A, SMARCA4), all together amounting to 13% of the experimental set[52].

     TRRAP2. Identified mutations occur in 4% of the melanoma set examined. TRRAP functions as part of a multiprotein coactivator complex possessing histone acetyltranferase activity that is central to the transcriptional activity of p53, c-Myc, and E2F1[62].

     GRIN2A encodes the glutamate N-methyl-(D)-aspartic acid (NMDA) receptor subunit ε-1 that is part of the class of ionotropic glutamate receptors and bears the agonist binding site for glutamate. GRIN2A was found to be mutated in 25% of melanomas (Wei et al., 2011); this was confirmed in another study[33]. Many mutations are missense or nonsense; therefore, it is unlikely to behave as a canonical oncogene.

     GRM3 is a metabotropic glutamate receptor, GPCRs that activate phospholipase C upon ligand binding. It was found to be mutated in melanoma through exome capture analysis of GPCR genes[20]. Mutated GRM3 was shown to contribute to the proliferation and invasiveness of melanoma cells in vitro and induce an increased phosphorylation of MEK. AZD-6244, an inhibitor of MEK, was able to reduce cell proliferation by inducing apoptosis in vitro. There is some interest in using available inhibitors of glutamate release for treatment of melanoma, since one of them, riluzole, was shown to inhibit growth of melanoma cells in vitro and in vivo[63]. Even though riluzole was shown to inhibit growth of cell lines expressing GRM1, a clinical trial is ongoing to explore the antitumor activity of riluzole in melanoma without prior analysis of GRM3 status.

    Phosphoglycerate dehydrogenase PHGDH serves to divert glycolytic carbon into serine and glycine metabolism in some cancer cells to supply the increased biosynthetic needs of transformed phenotype[64]. The same study found that PHGDH is recurrently amplified in a genomic region of a focal copy number gain most commonly found in melanoma. Melanoma cell lines with amplified PHGDH had increased flux through the serine pathway. This pathway, as well as proliferation of cells with high PHGDH, was sensitive to short hairpin RNA (shRNA)-mediated knockdown of PHGDH[64].

     WEE1. Cell cycle regulatory kinase Wee1 is upregulated in melanoma and is associated with poor prognosis[65]. Selective inhibitor of Wee1 MK-1775 showed somewhat promising results as a single agent in previously treated patients with metastatic melanoma, but additional trials are not being conducted at this time.

     NUAK2 (AMPK-related kinase). High levels of expression of NUAK2 were found in patients with acral melanoma and are associated with increased risk of relapse[66]. NUAK2 knockdown suppresses melanoma cell growth in vitro and tumorigenicity in vivo and has been proposed as a new oncogene in acral melanoma.

     Exosomes are small membrane vesicles with an endosome origin that are released by cells into the extracellular environment. Tumor-derived exosomes are emerging mediators of tumorigenesis; there is a growing interest in exploring treatment options targeting exosomes. A recent exciting study analyzed exosomes from highly metastatic melanomas. It found that these “metastatic” exosomes increased the metastatic behavior of primary tumors by permanently “educating” bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at premetastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype. Reducing MET expression in exosomes diminished the prometastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating bone marrow progenitors from individuals with metastatic melanoma. The study identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70 (HSP90) isoform, and the MET oncoprotein[67].

 

Various non-coding RNAs and other epigenetic alterations


A number of reports found significant roles for miRNA, ncRNA, or ceRNA in pathogenesis of melanoma; for example, ZEB2, a ceRNA for PTEN, upregulates expression of this tumor suppressor in melanoma. Abrogated ZEB2 cooperates with BRAFV600E to promote melanomagenesis.[28]. ADAR1, a protein of the family known as adenosine deaminase acting on RNA, is substantially downregulated during metastatic progression of melanoma. ADAR1 was found to regulate expression of numerous miRNAs, as well as of the key miRNA processing protein DICER. Two miRNAS were implicated in silencing of ADAR1 itself (http://www.jci.org/articles/view/62980). These findings reaffirm the significant contribution of epigenetic miRNA regulation to melanoma pathogenesis. Strategies to inhibit or increase expression of ncRNAs in clinical setting are only beginning to emerge. A growing interest in epigenetic alterations such as chromatin remodeling, DNA methylation, and histone modification regarding their role in melanomagenesis, might lead to the identification of novel therapeutic targets (reviewed in van den Hurk, et al, 2012[68].

 

INTRINSIC RESISTANCE TO BRAF AND MEK INHIBITORS (Table 3)
Resistance to BRAF inhibitors could be intrinsic (as in lack of response to selective BRAF inhibitors in patients with BRAF-mutated tumors) or acquired (development of resistance after treatment with BRAF or MEK inhibitors). Both are of the utmost concern. The current understanding of the origins of resistance, as well as possible approaches to overcoming it, are addressed below (and Table 3).
BRAF mutations play a well-established role in melanomagenesis; however, without additional genetic alterations, tumor development is restricted by OIS. As listed above and in Table 1, additional genetic alterations are present in BRAF-mutant tumors, some of which serve to overcome OIS, and could play a role in inherent resistance to mutant BRAF and MEK inhibitors. These two groups overlap, as could be expected. The necessity of targeting multiple signaling pathways to overcome drug resistance of aggressive melanoma was demonstrated in vitro (Smalley et al., 2006). Genomic analyses and the informed choice of combinatorial approaches analyzed in preclinical models are critical in selecting the right combination of targeted therapies in a personalized approach to melanoma treatment. In general, in addition to the MAPK pathway that is deregulated in most melanomas, the other targets might include any of the ones listed in Table 1. Experimental evidence indicating involvement of several pathways/genes in the inherent resistance to BRAF inhibition is cited below.

     PI3K/AKT. Mutations of the PI3K pathway are frequent in the BRAF-mutant setting. These have been shown to overcome BRAVV600E-induced OIS and contribute to inherent resistance to BRAF inhibitors[69]. In particular, loss of PTEN and consequent loss of expression of the pro-apoptotic BIM that is regulated by PTEN were implicated in inherent resistance to BRAF inhibitor in vitro[70]. PTEN- or AKT3-overexpressing melanomas do not undergo apoptosis in response to BRAF inhibition and do not upregulate pro-apoptotic protein BIM. PLX4720 was found to stimulate AKT signaling in the PTEN-, but not the PTEN+, cell lines. A clinical trial with inhibitors of both MAPK and PI3K showed promise in patients with various solid tumors[71].
Recent study demonstrated an essential role for ERK-phosphorylated MEK1 (pT292) in membrane recruitment of PTEN and consequent negative regulation of AKT (Zmajkovicova et al., 2013). Inhibition of BRAFV600-MEK1-ERK therefore might lead to the inhibition of the restraining role that this pathway has on activity of PI3K/AKT pathway via PTEN.

     CDK4 pathway. Increased cyclin D expression mediates inherent resistance to mutant BRAF inhibition[72].

      NF1 mutations. In a mouse model, NF1 ablation decreases the sensitivity of NF1 wild-type melanoma cell lines to BRAF inhibitors, and NF1 is lost in tumors from patients following treatment with these agents. Nf1/BRAF-mutant tumors are resistant to BRAF inhibitors, but are sensitive to combined MEK/mTOR inhibition[73]. In another study, NF1 mutations were documented in BRAF-mutant tumor cells that were intrinsically resistant to BRAF inhibition, and in melanoma tumors obtained from patients exhibiting resistance to vemurafenib, thus demonstrating the clinical significance for NF1-driven resistance to RAF/MEK-targeted therapies[15].

     MET and SRC. The activation of MET and SRC signaling was detected in two patient-derived melanoma cell lines with BRAFV600E that were resistant to BRAF inhibitor PLX4032. MET or SRC, respectively, was targeted with siRNA or drugs in combination with PLX4032. This was effective in inhibiting cell growth and reducing cell invasion and migration, indicating a functional role for MET and SRC signaling in primary resistance to PLX4032[74].

     Metabolic signature of melanoma sensitive to mutant BRAF inhibition.  An interesting study could not find correlation between sensitivity to PLX4032 and genetic profiles in a panel of BRAF-mutant cell lines. However, the sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. This indicates that melanoma with a higher dependence on glycolysis might be more sensitive to mutant BRAF inhibition. This also indicates that FDG-PET could be useful in assessing sensitivity to BRAF inhibitors[75]. Indeed, a clinical study that incorporated FDG-PET monitoring of responses to vemurafenib strongly indicated that there is a positive correlation between responses to therapy (PFS) and reduction in the uptake of FDG[76].

MITF-PGC1a axis in resistance to BRAF inhibition. Inhibition of BRAFV600 and, to a lesser extent,of MEK were found to induce expression of genes involved in citric acid cycle and oxidative phosphorylation (OXPHOS) in melanoma. Search for factors regulating OXPHOS revealed that PGC1a is upregulated in resistant melanoma lines(Haq et al., 2013a). PGC1a, in turn, was found to be a direct transcriptional target of MITF(Haq et al., 2013a; Vazquez et al., 2013). MITF expression and, as a consequence, PGC1a levels are upregulated in melanoma lines and in tumors of patients treated with Vemurafenib. Inhibition of BRAF leads to inhibition of glycolytic pathway for ATP production, butMITF-expressing melanomas can undergo a bioenergetics adaptation via MITF-PGC1a-OXPHOS upregulation(Haq et al., 2013a). The authors suggest targeting OXPHOS in this group of patients prior to use of MAPK inhibitors because lines selected to resistance to Vemurafenib have elevated levels of PGC1a.

     Tumor stroma influence. An important study demonstrated production of HGF by stromal cells in patients with melanoma, which resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K pathways, and resistance to BRAF. High production of HGF was observed in the stroma samples from patients who had a poor response to the inhibition of mutant BRAF. In a cellular model, cotreatment with BRAF and HGF or MET inhibitors reversed drug resistance[77].

     BCL-2. Inhibition of anti-apoptotic protein Bcl-2 might have a potential role in the future studies aimed to prevent the development of resistance to BRAF inhibition. The BH3 mimetic ABT-737 (inhibiting both Bcl2 and Bcl-xL) sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors, but does not reverse acquired resistance in vitro[78].

     BCL2A1. Amplification of one family member, BCL2A1 in 30% of melanoma was shown to contribute to resistance to BRAF inhibition[43]. BCL2A1 expression is apparently restricted to melanocytic lineage as it is indirectly controlled by MITF, and because of this its expression is limited to high MITF expression melanoma. Obatoclax, inhibitor of BCL family, helps to overcome the resistance of cell lines with amplified BCL2A1 to BRAF inhibition.

FOXD3-ERBB3. Transcription factor FOXD3 was shown to be upregulated when mutant BRAF is inhibited in melanoma cell lines PGC1a(Basile et al., 2012). Subsequent work revealed that FOXD3 directly activates expression of ERBB3 that contributes to resistance to Vemurafenib via activation of PI3K pathway, with involvement of ERBB2. The latter finding indicates a possibility of targeting ERBB2 alongside BRAF to overcome resistance(Abel et al., 2013).

     Chaperone Hsp90 is required for the stability of several of the oncoproteins that mediate RAF inhibitor resistance. Inhibitors of Hsp90 may be effective in patients with intrinsic or acquired resistance to BRAF inhibition[79]. In lab studies, treatment of melanoma cells with XL888, the inhibitor of Hsp90, induced apoptosis more effectively than dual MEK/PI3K inhibition in several different models of resistance[70]. Multiple proteins including PDGFRβ, COT, IGFR1, CRAF, ARAF, S6, cyclin D1, and AKT were degraded as a result of inhibition of Hsp90, which led to the nuclear accumulation of FOXO3a, an increase in BIM expression, and the downregulation of Mcl-1. XL888 is now in clinical studies in combination with vemurafenib (NCT01657591). Another non-geldanamycin Hsp90 inhibitor, STA-9090 (ganetispib), is also in clinical trials.

 

Intrinsic resistance to MEK inhibitors

Signaling through TGF-β/SMURF2/PAX3 and MITF. In an in vitro study, cells sensitive to MEK inhibition demonstrated increased transforming growth factor β (TGF-β) signaling. Melanoma cells resistant to the cytotoxic effects of MEK inhibitors counteracted TGF-β signaling 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. The study also found increased SMURF2 expression in advanced stages of melanoma[80].
Activation of the PI3K pathway was seen as a factor in resistance of a panel of melanoma cell lines to novel MEK inhibitor E6201. The sensitivity of cell lines to MEK inhibition correlated with wild-type PTEN and mutant BRAF[81].

 

ACQUIRED RESISTANCE TO BRAF AND MEK INHIBITORS (Table 4)

Mechanistic studies have provided insights into the development of resistance in two major ways: new mutations in the RAF-MAPK pathway itself and changes in other oncogenic pathways that relieve melanoma cells from reliance on BRAF signaling[82].
The identification of mechanisms of acquired resistance to BRAF inhibitors usually involves selection of surviving cells/clones from BRAF-mutant cells lines in vitro after treatment with a BRAF inhibitor and identification of new acquired mutations or other somatic changes. Biopsies from relapsed patients are then analyzed for the presence of these in vitro-identified changes. Alternatively, biopsies are subjected to massive sequencing for identification of newly acquired mutations/aberrations. Not all of the possible mechanisms of acquired resistance listed below were confirmed in post-relapsed biopsies (Table 4).

New aberrations in BRAF. Unlike the experiences with other molecularly targeted therapies of oncogenic kinases, treatment with BRAFV600E inhibitors did not lead to the emergence of gatekeeper mutations in BRAF itself. However, amplification of mutant BRAF was detected in 4 of 20 patients who developed resistance to vemurafenib[83].
Analysis of a subset of cells resistant to vemurafenib (PLX4032) in vitro detected the 61-kDa variant form of BRAFV600E, p61BRAFV600E, which lacks exons 4 to 8, a region that encompasses the RAS-binding domain. p61BRAFV600E shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAFV600E that acts as a monomer. The p61BRAFV600E splicing variants lacking the RAS-binding domain were identified in the tumors of 6 of 19 patients with acquired resistance to vemurafenib. These data determined a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAFV600E that dimerize in a RAS-independent manner[84].

Activation of signaling through RTKs

     EGFR/SFK/STAT3. BRAF inhibitor-mediated activation of EGFR/SRC family kinase/STAT3 signaling was shown to mediate resistance in BRAF-mutant melanoma cell lines and was demonstrated in patient biopsies. In vitro treatments with an EGFR inhibitor in combination with a BRAF inhibitor, or monotherapy with dasatinib, appeared to overcome this resistance and could deliver therapeutic efficacy in drug-resistant BRAF-mutant melanoma patients[85].
Upregulation of FGFR3 signaling in selected vemurafenib-resistant cells was described in vitro. Signaling through FGFR3 activated the MAPK pathway through RAS, but these cells were still sensitive to inhibition of MEK or pan-RAF inhibition[86].

     IGF-1R/PI3K pathway. Mutations in the IGF-1R/PI3K pathway were identified as another way for developing resistance to the BRAF inhibitor SB590885. IGF-1R/PI3K signaling was enhanced in resistant melanomas; combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample were consistent with the proposed role for IGF-1R/PI3K-dependent resistance to BRAF inhibitors[87].
Activation of PDGFR signaling was shown to occur during selection for BRAF inhibitor resistance in vitro. The clones with PDGFR activation were also resistant to MEK inhibition implying that activation of PDGFR bypasses the tumor dependence on RAF signaling entirely[88].

     NF1 expression is lost in tumors from patients following treatment with BRAF and MEK inhibitors[73]. NF1 mutations were observed in melanoma tumors obtained from patients exhibiting resistance to vemurafenib, as already mentioned above. However, cells lacking NF1 retained sensitivity to the irreversible RAF inhibitor AZ628 and an ERK inhibitor[15].

     NRAS. Secondary mutations in NRAS were shown to confer resistance to PLX4032 in vitro[88]. These acquired changes were mutually exclusive with PDGFR activation observed in the same study. In contrast to PDGFR-activated resistant cells, NRAS-mutated cells were sensitive to MEK inhibitors. Mutations of NRAS were also found in 4 out of 19 patients that developed resistance to PLX4032[88]. New mutant BRAF inhibitors, such as PLX7904 have been explored in vitro. PLX7904 inhibits ERK1/2 activation in cells with mutant BRAF, but not in cells with wild type BRAF[89]. Promisingly, PLX 7904 inhibited ERK1/2 phosphorylation in mutant BRAF melanoma cells with acquired resistance to vemurafenib/PLX4720 that is mediated by a secondary mutation in NRAS.

     AKT3. Activation of AKT3 in response to PLX4720 or BRAF siRNA was implicated in the resistance of primary three-dimensional cultures of melanoma cells to BRAFV600E inhibitors[90].

     ERK activation, independent of classical RAF/MEK/ERK pathways, was shown to occur in resistant lines with mutant BRAF. PI3K pathway activation could be responsible for ERK activity. Both inhibition of PIK3CA and ERK inhibited growth of these lines selected in vitro for resistance to PLX4720[91].

     MAP3K8/COT. In a massive functional kinome screening study, a kinase known as MAP3K8/COT was implicated in both inherent and acquired resistance to BRAFV600E inhibitors. COT is a MAPK agonist-independent of RAF and is expressed highly in inherently resistant melanoma or in cells from patients with acquired resistance. Its expression drives resistance to BRAF inhibitors in vitro and occurs in relapsing patient tumors[92]. COT-mediated resistance to a RAF inhibitor cannot be overcome by a MEK inhibitor, suggesting that activation of ERK in these tumors occurs outside of the classical RAF-MAPK-ERK pathway. COT might be a direct ERK phosphorylating kinase.

     MEK1 mutation C121S was identified in a patient treated with vemurafenib[93]. This mutation was shown to increase kinase activity of MEK1 and confer resistance to both BRAF and MEK inhibition.

     RND3. An in vitro study with a single BRAF-mutant melanoma line showed that BRAF-inhibitor treatments were associated with reduced expression of RND3, an antagonist of RHOA activation, and elevated RHOA-dependent signaling. Restoration of RND3 expression or RHOA knockdown attenuated the migratory ability of residual cells without affecting overall cell survival[94].

Factors that mediate acquired resistance to MEK inhibitors (Table 4)
Similar to the adaptation of BRAF-mutant melanoma cells to BRAF inhibition, resistance to MEK inhibitors could involve multiple pathways. In a study of triple-negative breast cancer treated with a MEK inhibitor, it was found that inhibition of MEK, similar to inhibition of BRAFV600E in melanoma, induces a reprogramming of RTK activation[95]. It is possible that activation of RTK when MEK is acutely inhibited is also involved in responses of melanoma to MEK inhibition, though this has not been shown yet.

     Mutations in MEK1 and MEK2. De novo mutations in MEK1 were strongly implicated in resistance to MEK inhibitor AZD6244, both in treated cell lines and in a metastatic tumor from a relapsed patient[96]. Mutations in selected lines resulted in constitutive ERK phosphorylation and higher resistance to MEK inhibitors, but also conferred cross-resistance to PLX4023. Variant MEK1(P124L) was identified in a resistant metastatic focus that emerged in a melanoma patient treated with AZD6244. However, cotreatment of mutant BRAF melanoma with both inhibitors prevented emergence of resistant clones in vitro.

PI3K-AKT pathway. A study in vitro has identified basal and treatment-induced activation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in BRAF-mutant cutaneous melanoma cells. Sensitive, but not resistant lines showed upregulation of PTEN expression by AZD6244. Combination of a MEK inhibitor with inhibitors of Akt, mTOR, or IGF1R was able to overcome resistance to MEK inhibitors[97].

 

COMBINATORIAL THERAPIES

Inherent resistance, partial and low durability responses, and acquired resistance are major problems in the clinical development of targeted therapies. Recent results of clinical studies combining two targeted therapies have confirmed the long held belief that combinatorial approaches have significant advantages over monotherapy[98].
Two recent publications examined possible synergistic interactions between targeted therapies. The first study[99] looked at synthetic lethal pairwise interactions of a library of 300 compounds in a panel of nine established melanoma cell lines. This study did not find synergistic interactions that were genotype-specific (BRAF, NRAS-mutant or wild-type cell lines were screened). A synergistic interaction was found for just one combination, which was not genotype-related: sorafenib and the nonsteroidal antiinflammatory drug (NSAID) diclofenac. Drug substitution experiments confirmed that the synergy is due to the combined effects on MAPK and cyclooxygenase (COX).
The second study[100] examined drug interactions between 150 small molecule inhibitors in a panel of 28 early passage cell lines from melanomas with mutations either in BRAF, NRAS, or neither. This study produced results different from those of the first study, perhaps due to the choice of early passage lines versus established ones. The authors first selected inhibitors that showed some activity in at least one group and proceeded to analyze the antiproliferative effects of all drugs pairwise (over 7,000 combinations). The single drug screen produced several drugs with activity in BRAF-mutant melanoma, some of them known (vemurafenib and MEK inhibitors), but also non-BRAF-targeted kinase inhibitors of SRC/ABL (bosutinib), FGFR (dovitinib), and EGFR (gefitinib). There were almost no drugs effective against NRAS-mutant lines, except 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) inhibitor simvastatin, which indirectly blocked prenylation and membrane localization of RAS. Drug combination analysis showed that the most effective combination for BRAF-mutant lines was that of vemurafenib, EGFR, and AKT inhibitors, which was cytotoxic even in lines with primary resistance to vemurafenib or in lines selected for resistance to vemurafenib. NRAS-mutant lines were most sensitive to combination of the HMGCR inhibitor simvastatin and a nonspecific CDK inhibitor flavopiridol; simvastatin was able to displace NRAS from the plasma membrane, thus exhibiting an inhibitory effect on its activity. The importance of these results for mapping out future preclinical and clinical studies is extremely high.

 

IMMUNOTHERAPY

Melanoma is considered to be highly immunogenic and responsive to immunotherapy. In addition to IL-2 approval in 1998, pegylated interferon-α2b as an adjuvant therapy and ipilimumab (anti-CTLA-4 antibody) as therapy for advanced disease were approved by the U.S. Food and Drug Administration (FDA) for melanoma in March 2011. The response rates to either are not impressive. In recent years, investigational agents have included adoptive T cell transfer, blocking antibodies against inhibitory immune molecules, stimulatory antibodies for immune cells, and immunization with cancer cell antigenes. A rapidly developing field is identification and validation of biomarkers predictive of responses to immunotherapy (reviewed in[8]. Table 5 shows some of the clinical trials with the immune system targeting antibodies that are described below.

 

Molecular targets for immunotherapy of melanoma

CTLA4 is an inhibitory molecule expressed on T cells that is involved in the negative regulation of the T cell interaction with antigen presenting dendritic cells (APCs). CTLA4 inhibits binding of CD28 on T cells to B7 proteins on APCs, thus weakening the costimulation of T cells. CTLA4 is also expressed on tumor cell lines[101] and in human melanoma[102]; blockade of CTLA4 in vitro induces apoptosis of melanoma cells, indicating that CTLA4 might have nonimmune functions. Available results from clinical trials indicate that the response rates to CTLA4 blockade with human monoclonal antibodies ipilimumab and tremelimumab are, at most, at 18%, but responses tend to be more durable than those seen with cytotoxic therapies. Significant autoimmune toxicities were reported in a number of trials, and, interestingly, strong association of immune-related toxicities and responses were observed (reviewed in Flaherty, et al, 2012; Sapoznik, et al, 2012[103] ;[104].
Potential markers that could predict response to ipilimumab are of obvious importance. Clinical trial NCT00261365 incorporated investigation of a number of parameters in tumor biopsies pre- and post-treatment with ipilimumab. Significant associations were detected between clinical activity and high-baseline expression of FoxP3 and indoleamine 2,3-dioxygenase and between clinical activity and increase in tumor-infiltrating lymphocytes (TILs)[105].
Trials of ipilimumab and gp100 peptide vaccine, each as a single agent or in combination, showed limited responses, but the responses were long lasting. Combination trials with ipilimumab, ongoing or posed to start, include the following second agents: bevacizumab, high doze interferon -2b, IL-2, GM-CSF, anti-PD-1 antibody (see below), and even an oncolytic herpes simplex 1 virus (talimogene laherparepvec) designed to replicate selectively in tumor cells and to express GM-CSF (the virus preparation is injectable directly into tumors), as well as a variety of chemotherapeutic agents and surgical/radiotherapy interventions. Targeted BRAF inhibitors with ipilimumab are also tested in several trials (Supplementary Table 2).

PD-1, like CTLA-4, is an inhibitory receptor; however, its expression is not limited to T cells and is found in B cells and some myeloid cells. PD-1 ligands, PD-L1 and PD-L2, have different expression patterns, with PD-L1 found on multiple normal and cancerous cells including melanoma tumors, where it provides, once bound by PD-1, peripheral tolerance to “self” antigens (Iwai, Y., Ishida, M., Tanaka, Y., Okazaki, T., Honjo, T., and Minato, N. (2002). Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade. Proceedings of the National Academy of Sciences of the United States of America 99, 12293-12297. http://www.pnas.org/content/99/19/12293.full). PD-L2 is expressed on APC cells, providing tolerance to orally administered antigens. Interactions between PD-1 and its ligands attenuate immune responses[106], and, in the context of cancer, serve to protect tumor cells from cytotoxic T cells. At the same time, PD-1 is expressed on CD8+ T cells in patients with metastatic melanoma, which suggests that the immune response to melanoma is inhibited under these circumstances.
Humanized antibodies to PD-1 and PD-L1 have been developed and tested in clinical trials for several cancers including melanoma. Anti-PD1 antibodies were well tolerated and the maximum tolerated dose has not been reached[107]. This study also found a strong correlation between pretreatment tumor expression of PD-L1 and responses, with a 36% responses rate in patients whose tumors expressed PD-L1, and 0% in PD-L1 – negative group. These findings highlighted the importance of the identification of predictive markers in the future design of immunotherapy trials. The better safety profile of PD-1 antibody versus CTLA-4 antibody is most likely due to the fact that the latter targets a peripheral interaction between T cells and APCs. Inhibition of PD-1 probably inhibits peripheral interactions as well, through PD-L2 on APCs, but it could be expected to act more locally at tumor sites by preventing inhibition of tumor-infiltrating PD-1 expressing T cells through interaction with PD-L1 on tumor cells. Anti-PD-L1 antibody was also tested in several cancers, including melanoma, and objective responses were observed in 9 of 52 melanoma patients; the responses were durable[108].

Clinical trials targeting PD-1/ PD-L1 interaction
Results from an early-phase clinical trial with anti-PD1 antibody BMS-936558 (MDX-1106, ONO-4538: Bristol-Myers Squibb) showed durable responses in 28% of patients with melanoma, among others[109]. It was noted that expression of PD-L1 on tumor cells was a strong predictive marker of response to anti-PD-1 therapy. Currently there are eight trials ongoing with MDX-1106 for melanoma. Two other anti-PD-1 antibodies (CT-011: CureTech Ltd.; MK-3475: Merck) are also in clinical trials and one anti-PD-L1 (BMS-936559: Bristol-Myers Squibb) is being tested as well. PD-1 and CTLA-4 antibodies are being trialed together with the hope of synergistic effects (NCT01024231). Dosing of anti PD-L1 antibody MDX-1105-01-1 is tested in cancers other than melanoma.

Other targets for immunotherapy include several proteins that are involved in stimulating T cell responses; relevant therapies aim to elicit agonistic responses.

     4-1BB. Also known as CD137, 4-1BB is a member of the TNF receptor superfamily, and is an activation-induced costimulatory molecule. Binding of 4-1BB by its ligand or antibody induces powerful CD8+ T-cell activation, IFN-γ production, and cytolytic activity. An agonistic antibody, BMS-663513 (Bristol-Myers Squibb) was in clinical trials for melanoma and other tumors. Safety concerns caused suspension of the trial after several patients developed liver problems, including high-grade hepatitis.

    GITR is a costimulatory receptor expressed after T cell activation that enhances T cell function and survival. GITR also negatively affects regulatory T cells (Tregs), a specialized T-cell lineage that downregulates cellular immunity. A trial with anti-GITR antibody TRX-518 is ongoing.

    OX40 is not involved in effector T cell activation, but rather, promotes T cell survival and expansion. In a clinical study, based at the Portland Providence Medical Center in Oregon, patients received three infusions of the agonistic mouse anti-OX40 antibody within a week. The nature of the antibody precluded further treatments. Nine of 27 patients experienced minor tumor shrinkage, although none met RECIST (response evaluation criteria in solid tumors) criteria for objective responses.

    CD40. Unlike the costimulatory targets above, CD40 is expressed on APCs, while its ligand is expressed on T cells. Binding of the two acts as a powerful enhancer of APCs’ ability to present antigens and activate T cells against foreign targets[110]. A large number of cancer patients received infusions of agonistic antibody CP870,893 and some responses were observed. A surprising finding was that treatments did not increase numbers of TILs in the tumors. In a mouse model, antibody treatments induced an influx of macrophages into tumors, presumably with enhanced cytotoxic activities.

    CEACAM1 is a potential new target for the development of targeting antibodies. CEACAM1 is a carcinoembryonic, antigen-related adhesion molecule 1 from the IG superfamily whose expression is absent from normal melanocytes, but often found in melanomas, particularly those that are metastatic. CEACAM1 was shown to protect melanoma cells from T cells in vitro, and, moreover, its expression was found on T cells and NK cells from melanoma patients, presumably enabling a homotypic inhibitory interaction. A mouse antibody to CEACAM has no apparent effect on CEACAM1-expressing melanoma cells in vitro, but renders them susceptible to elimination by T cells in vitro and in an in vivo xenograft model. These findings provide a strong rationale for developing CEACAM1-based therapeutics for the treatment of metastatic melanoma[104].

Adoptive cell transfer
Adoptive cell transfer (ACT) involves the selection of autologous lymphocytes with antitumor activity, their expansion/activation ex vivo, and their reinfusion into the patient, often in the context of lymphodepleting regimens to minimize endogenous immunosuppression (reviewed in Galluzzi, et al, 2007; Itzhaki, et al, 2013[111] ;[112]. ACT was and remains a subject of intense clinical interest in melanoma treatment. A number of clinical trials were completed and are ongoing, but no versions of ACT have been approved by the FDA so far.
Different aspects of ACT are being examined, among them the role of cytokines such as T cell-produced IL-9 in T cell-based therapy[113]; concomitant low-dose IL-2 in patients treated with ACT[114]; facilitation of the long-term engraftment of T cells by using the memory T cell population[115] ;[116]; the basis for the relapse frequently observed after ACT immunotherapy, such as inflammation-induced reversible loss of melanocytic antigens mediated by TNF-a[117]; the immunosuppressive role of Tregs and myeloid-derived suppressor cells (MDSC)[118], the significance of the rare Vδ1 T cells in recognition of tumor cell antigens[119] ; and many more.
A recent phase II clinical trial reported objective clinical responses in almost half of the 31 patients enrolled, with two complete remission and a significant increase in PFS[120]. The study also analyzed characteristic of infused T cells associated with significant responses, and found significant correlations with: higher number of TIL infused, a higher proportion of CD8(+) T cells in the infusion product, a more differentiated effector phenotype of the CD8(+) population, and, unexpectedly, a higher frequency of CD8(+) T cells coexpressing the negative costimulation molecule “B- and T-lymphocyte attenuator” (BTLA). A detailed analyses of the functional activity and dynamics of the infused genetically engineered T cells in patients highlighted the need to develop approaches to maintain antitumor T-cell functionality (Ma et al., 2013).
Some remarkable responses were observed in individual patients treated with CD4+T cells. For example, autologous transfer of expanded CD4+ T cells recognizing melanoma antigen NY-ESO-1 produced a complete and durable response in one patient trial[121]. Recent findings indicate that CD4+ cells induce tumor senescence by producing high levels of IFN- and TNF[122].

Influence of targeted therapies on the responses to immune therapy

     BRAF/MAPK signaling and TIL. Activated BRAF/MAPK signaling was shown to be essential in the development of immune evasion in melanoma[123]. Prior to clinical development of BRAF and MEK inhibitors, it was shown that inhibition of the MAPK pathway leads to increased expression of melanocytic antigen expression, including MART-1, gp100, and tyrosinase; whereas enforced expression of mutant RAF downregulated expression of these antigens[124]. Mutant BRAF induces expression of IL1 in stroma, also leading to immune suppression[125]. In a mouse model, BRAFV600E induced expression of Ccl2, an immunosuppressive cytokine, while PLX4720 treatment downregulated tumor Ccl2 gene expression and increased numbers of CD8 TILs[126].
There were concerns that treatment with BRAF/MEK inhibitors might have a direct inhibitory effect on T cell function and it was indeed shown later that treatment with MEK inhibitors, but not BRAFV600E inhibitors, impairs T lymphocyte function[127].
A number of subsequent reports have confirmed that inhibition of mutant BRAF could potentiate immune responses in melanoma, suggesting that blockade of immune checkpoints in combination of BRAF inhibitors could have a great clinical value. Treatment with BRAF inhibitors vemurafenib and GSK2118436 resulted in markedly increased numbers of TIL (tumor infiltrating lymphocytes) in tumor biopsies obtained pre- and post-treatment in 15 patients[128]. Another study found that GSK2118436 has no detectable negative impact on existing systemic immunity or the de novo generation of tumor-specific T cells in patients[129].
A single report claimed that selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse melanoma model; moreover, treatment with ipilimumab has not restored the numbers of TIL[130].
The latest reported analysis of biopsies taken before and after treatment with BRAF or BRAF+MEK inhibitors showed the following: enhancement of melanoma antigen expression, increase in CD8+ TILs, and decrease in immunosuppressive cytokines IL6 and IL8. Interestingly, inhibitory PD-1 was increased on T cells and its immunosuppressive ligand PDL1 was also increased with BRAF inhibition. This supports the hypothesis that targeting inhibitory immune interactions may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma[131].

Adoptive cell transfer therapy and targeted therapies. Adoptive cell transfer therapy could also be of more benefit when combined with vemurafenib, at least in a mouse model[132]. In a recent study, vemurafenib increased MAPK signaling, in vivo cytotoxic activity, and intratumoral cytokine secretion by adoptively transferred cells (T cells genetically engineered to recognize tumor-expressing antigens). Another study reported that administration of PLX4720 significantly increased tumor infiltration of adoptively transferred T cells in vivo and enhanced their antitumor activity in a mouse model. Apparently, PLX4720 negatively affected expression of VEGF by tumor cells. Importantly, analysis of human melanoma biopsies before and during BRAF inhibitor treatment also showed downregulation of VEGF consistent with the preclinical murine model[133].
An in vitro study analyzed effects of BRAF and MEK inhibition on function of dendritic cells (DC) in vitro and found that BRAF-mutant melanomas suppress immune function of dendritic cells. Inhibition of BRAF, but not MEK could reverse suppression of DC function. As has been reported for the effects of MEK inhibition on T cells[127], inhibition of MEK did negatively affect DC function and viability. Vemurafenib, but not MEK inhibitors, was therefore suggested as a preferable candidate for combination immunotherapy approaches in BRAF-mutant melanoma.[134].
Another in vitro study examined how vemurafenib affects the ability of the clinical grade tumor-infiltrating lymphocytes to recognize autologous BRAF(V600) mutant melanoma cell lines in vitro[135]. The results showed a significant increase in recognition of the inhibitor-treated melanoma cells that was attributed to increased expression of MHC class I-associated proteins and heat-shock proteins.
These studies in general provide a strong rationale for combination therapies involving BRAF inhibition with immunotherapies involving either antibodies to immunosuppressive molecules or adoptive cell transfer. Clinical trials are underway that combine vemurafenib and ipilimumab (NCT01400451), vemurafenib and anti-PD-L1 antibody MPDL3280A (NCT01656642), vemurafenib and HD-IL2 (NCT01683188), adoptive cell transfer (NCT01585415), and HD-IL2 + adoptive cell transfer + vemurafenib (NCT01659151) and more. Sequencing of BRAF inhibition and ipilimumab treatments present a serious clinical problem for patients who experience rapid disease progression on BRAF inhibitors[136].
Mutant BRAF and/or its inhibition are not expected to influence outcomes of all immune therapies. For example, a recent trial showed that treatment with IL21 produced an overall response rate of 22.5% in melanoma and responses were not related to the status of BRAF[137]. Treatment with high-dose IL-2 was more effective in patients in patients with NRAS mutations versus BRAF mutations on WT/WT tumors[138].
Other melanoma-related pathways might also play a role in responses to immune therapy. Wnt/-catenin signaling might be involved in immunosuppression in melanoma, as high levels of nuclear -catenin in melanoma cells impaired maturation of DCs at least in part through increased production of IL-10 and inhibited IFN- production by melanoma-specific CTLs[139]. It is entirely possible and even likely that other oncogenic pathways in melanoma also mediate immune suppression in melanoma.

Tumor microenvironment and immune responses

The role of B cells in the immune responses in melanoma. An extensive study measured humoral B cell responses in melanoma patients, concluding that melanoma patients, on average, have highly increased antibody responses against melanoma cells compared to healthy volunteeres. Interestingly, the B cell mediated immune responses were significantly diminished in patients with metastatic melanoma versus primary disease (Gilbert et al., 2011). The impaired B cell functionality with loss of CD27+ (memory) cells was previously reported in metastatic melanoma (Carpenter et al., 2009).
Intratumoral IgG producing B cells have been reported in melanoma, but their functional significance was largely unknown. A study published in March of 2013 found a skewing of B cell repertoire in melanoma tumors, with highly increased numbers of IgG4 producing B cells relative to normal ratios. High IgG4/IgGtotal ratios are considered a limiting response by the immune system to contain immune activation. In melanoma, production of B cells secreting inhibitory IgG4, is stimulated by tumor secreted IL-10 and IL-4. The tumor-specific IgG4 even inhibited IgG1-dependent tumor cell killing through engagement of the FcγRI effector mechanisms. Moreover, high circulating levels of IgG4 in serum of patients were associated with poor prognosis (Karagiannis et al., 2013).

Immune cell infiltrates in melanoma. The presence of lymph node-like structures in solid tumors has been recognized relatively recently, and is considered to have a prognostic value, in particular as related to immune therapy. (Jochems and Schlom, 2011). It was suggested that the presence of these lymphoid structures could be due to locally tumor-produced chemokines, and might be useful in selection of patients for whom immunotherapies might work best. Moreover, a particular 12 chemokine signature produced by tumors, was identified by expression profiling, and was shown to be predictive of the formation of these lymph node-like structures. The hope is that this expression signature might be used for selection of patients for whom long-lasting responses to immunotherapy are possible (Messina et al., 2012).

 

METABOLISM AND AUTOPHAGY AS TARGETS IN MELANOMA THERAPY

There have been few attempts to target melanoma metabolism with the exception of several known metabolic preferences listed below. However, recent discoveries, such as high levels of OXPHOS (oxidative phosphorylation) in highly aggressive melanoma mediated by elevated expression of PGC-1a[13] might prompt more interest in targeting melanoma metabolism. For many years, serum lactate dehydrogenase (LDH) levels were used as a predictive factor to identify melanoma patients with worsened prognosis, and they presumably are indicative of the high glycolytic character of tumors, though the energy requirements of melanoma might be fulfilled through a more complex relationship between glycolysis and OXPHOS[140]. An OXPHOS inhibitor elesclamol in combination with paclitaxel was clinically tested in a randomized trial in unselected patients. The study was terminated due to increased death in patients with high LDH receiving paclitaxel alone[141]. However, the study revealed a statistically significant increase in PFS in patients with normal serum levels of LDH receiving elesclamol, that is, a group of patients with tumors that rely on OXPHOS.
Regarding the possible effects of metabolic preferences of melanoma tumors on the efficacy of targeted therapy, it is of interest that mutant BRAF inhibition might be more efficacious in tumors with higher glycolytic index[76] ;[75]. As mentioned above, this metabolic trait could be useful as a predictive marker of response to BRAF inhibition. In addition, inhibition of BRAF(V600E) appears to induce a switch from glycolytic to OXPHOS metabolism, via induction of MITF, followed by MITF’s induction of PGC1alpha (Haq et al., 2013a). A consequence of this shift was an apparent dependency of BRAF-inhibited cells upon OXPHOS, thus suggesting new BRAF-combination strategies for patients.
A most interesting study implicated enzyme PDH (pyruvate dehydrogenase) that links glycolysis to OXPHOS in the abrogation of mutant BRAF induced senescence (OIS). Oxygen consumption was increased in senescent cells modified to express mutant BRAF, which was mediated by activated PDH. Two key PDH regulating enzymes were involved inthe observed activation of PDH: inhibitory PDK1 was suppressed, while activating PDP2 was upregulated. Ectopic expression of PDK1 was able to overcome BRAF induced senescence of melanocytes and promote robust tumor growth in vivo. Moreover, PDK1 depletion in melanoma tumors dramatically increased their sensitivity to vemurafenib in vivo by eliminating subpopulation of cells resistance to the drug(Kaplon et al., 2013). These results established a direct role for metabolic axis in the development and drug resistance of melanomas.

The role of autophagy in tumor development and responses to various treatments remains controversial, most likely due to the fact that autophagy can contribute either to death or survival of cancer cells depending on the nature of the death signal and cellular context. Deprivation of amino acids is considered to be of potential benefit in melanoma treatment due to specific metabolic preferences/dependency of melanoma tumors, but induction of “starvation-induced” autophagy could be detrimental.

Arginine deprivation. Melanoma tumors, along with hepatocellular cancer (HCC) and prostate cancer, frequently show deficiency of the enzyme argininosuccinate synthetase (ASS)[142], cannot synthesize arginine from citrulline, and depend on exogenous arginine for survival. Arginine degradation using arginine deiminase (ADI) leads to growth inhibition and eventually cell death, while normal cells that express ASS can survive (reviewed in[143]. Pegylated ADI (ADI-PEG20) has shown antitumor activity in melanoma [183]. Another arginine degrading enzyme, arginase, in a recombinant pegylated and cobalt-substituted form, Co-ArgI-PEG, is being developed for clinical trials. It is already clear that treatment with ADI-PEG20 induces resistance in patients by at least two identified mechanisms: induction of protective autophagy and reexpression of ASS and activation of the MAPK pathway (reviewed in Yoon, et al, 2013[143]. If arginine deprivation therapy with arginine-degrading enzymes is to become a valid therapeutic option, concurrent therapies targeting autophagy or the MAPK pathway should be considered.

Leucine deprivation. Leucine deprivation was also shown to induce apoptotic death in melanoma cells; moreover, the dependence of melanoma cells on leucine was functionally linked to the activated RAS-MAPK pathway, in particular BRAFV600E mutation. The latter rendered the mTOR pathway resistant to leucine deprivation, but inhibition of autophagy was able to restore sensitivity of these cells to leucine deprivation. Dietary leucine deprivation combined with an inhibitor of autophagy suppressed melanoma xenograft growth in vivo[144].

Metformin, an inhibitor of mitochondrial complex I, activates AMPK as a result of decreased AMP/ATP ratio, and inhibits mTOR. Long known for its antihyperglycemic properties, it is being explored for antineoplastic activities. Metformin was shown to induce suicidal autophagy in melanoma cells in vitro and in vivo[145]. A trial of metformin with vemurafenib is ongoing (NCT01638676).
Because autophagy is known to be involved in both innate and adaptive immunity, there is an interest in exploring manipulation of autophagy for immunotherapy. Autophagy was reported to enhance antigen presentation by dendritic cells, therefore potentiating T cell activation, and could be enhanced by nanoparticle-based antigen presentation[146]; autophagosome preparations enriched for tumor antigens were used to pulse dendritic cells for successful vaccination of tumor-bearing mice[147]. On the other hand, inhibition of autophagy was shown to promote antitumor responses to systemic IL-2 immunotherapy[148] ;[149]. Apparently, the specific roles of autophagy in different immune responses would have to be explored individually.

 

NEW PROGNOSTIC MARKERS

Circulating Melanoma Cells

Detection of circulating tumor cells (CTC) in melanoma was explored for at least last twenty years.  CTC have been shown to serve as seeds for metastatic lesions, and therefore their presence could be interpreted as a prognostic marker.  In addition, monitoring of CTC levels could serve as a marker of disease progression and treatment success or failure (reviewed in[150] ;[151]. Molecular profiling of CTC could become a “liquid biopsy”, and could reflect genetic heterogeneity, both intratumoral or between multiple metastatic tumors.

The earlier attempts were directed mostly towards developing a multimarker RT-PCR to detect and monitor the presence of circulating tumor cells (CTC) in the bloodstream.  The first detection of melanoma CTC was based on RT-PCR of tyrosinase[152]. Numerous studies have employed RT-PCR of a variety of melanoma markers to analyze peripheral blood preparations and examine prognostic significance of these assays. For example, a serial analysis of two melanoma-associated markers (tyrosinase and MART-1) was shown to be an independent predictor of disease recurrence for stage IV melanoma patients after surgery and adjuvant therapy[153], possibly due to thechoice markers.  Overall, the many studies performed indicate that a validated set of CTC biomarkers as well as standardized methodology is necessary to make RT-PCR analysis of CTC a useful tool in assessment of melanoma prognosis.For now, RT-PCR of peripheral blood is not considered to be highly promising.

A different approach involves physical separation of CTC prior to their analyses. This methodology is somewhat limited by its most often used approach, which relies on expression of certain surface proteins on circulating tumor cells for their identification and separation.  CellSearchVeridex technology, widely used for the detection of circulating tumor cells (CTC) in blood was designed for breast, prostate, colorectal, and lung cancer, which express EpCAM markers. Continuing expression of these proteins on CTC could not be counted upon when de-differentiation (such as epithelial to mesenchyme transition in epithelial cancers)occurs during tumor progression.   Nevertheless, use of CellSearch platform was reported to detect CTC in 40% of patients with advanced melanoma, and the numbers of CTC were prognostic in themselves for overall survival and reflect treatments’ outcomes[154]. The CellSearchVeridex platform was recently adapted for melanoma markers, and was reported to be successful in detection and quantification of CTC in cerebrospinal fluid in two melanoma patients with leptomeningial metastases[155].

New techniques are being developed, such as isolation by size of epithelial tumor cells (ISET) which was able to detect CTC in 29% of patients with primary invasive melanoma and in 62.5% of metastatic melanoma patients, with an excellent correlation for detection of CTC by RT-PCR of tyrosinase[156]. A new technique named photoacoustic blood cancer testing was successfully applied to melanoma CTC in mice and spiked human blood and could be used to capture CTC[157] ;[158].A recent report described a method to detect and isolate single circulating melanoma cells that integrates a polymer-nanofiber-embedded nanovelcro cell-affinity assay with a laser microdissection (LMD) technique. This method not only separates melanoma CTC from peripheral blood, but also allows sequencing of individual cells for specific mutations[159].

The inertial focusing-enhanced microfluidic CTC capture platform, termed “CTC-iChip” is capable of rapid sorting of rare CTCs from whole blood. The iChip technology is capable of isolating CTCs using strategies that involve recognition of extracellular epitopes, but could be used independently of tumor-specific membrane protein recognition[160]. The methodology was successfully tested in epithelial cancer and melanoma, and enables RNA profiling of single cells.  Isolation of CTC using the ScreenCell filtration technique with quantitative analysis of CTC telomeres by TeloView was described recently[161].

Molecular profiling of CTC might provide a very valuable representation of a tumor(s) genotype because CTCpresumably reflect tumor heterogeneity better than a single tumor biopsy. Indeed, several studies have discovered discordant mutations in CTC versus biopsies. Inconsistencies of BRAF and KIT mutations between tumor biopsies and CTC were described[162] ;[163]. Presumably, CTCs also represent tumor cells with higher metastatic potential, and thus could be more relevant to the molecular profiling of aggressive tumor variants or metastases.

Several clinical trials are ongoing that incorporate CTC detection and analyses prior to and after treatments to investigate the prognostic value of CTC. The CellSearchVeridex technique will be evaluated in terms of its predictive and prognostic ability (NCT01573494). CellSearch and Epispot (another platform based on immunomagnetic separation) are compared in NCT01558349. TrialNCT01528774addresses the possibility of long term culture of melanoma CTCs isolated using TrueCells technology. A future trial NCT01776905 will evaluate use of photoacoustic flow cytometry for in vivo, real time detection of CTC.Treatment with ipilimumab and sterotactic ablative radiation therapy (NCT01565837) or with Sargamostim/GM-CSF will include analysis of CTC as a predictive/prognostic markers (NCT01489423). Multiple parameters including CTC are evaluated in an ongoing trial of Imatininb (NCT00470470).
Recent studies have addressed the utility of yet another method of “liquid biopsy”, i.e. analysis of circulating tumor DNA (ctDNA) by NGS techniques rather than RT-PCR of defined biomarkers. A side-by-side comparison was performed of the analyses for a serum biomarker versus analysis of CTC versus analysis of ctDNA by targeted or whole genomic sequence in breast cancer patients. This study reported the superiority of ctDNA analysis, as having a greater dynamic range, a greater correlation with changes in tumor burden, and better measure of treatment response (Dawson et al., 2013). The next publication from the same group reported monitoring of ctDNA by exomic sequencing in breast cancer patients prior to and during treatments (Murtaza et al., 2013). Serial analysis of ctDNA had successfully detected a number of prognostic genomic alterationsthat were either increased or appeared de novo as a consequence of treatments. It remains to be seen if detection of ctDNA will be a useful prognostic companion or even a substitute for tumor biopsies.
Recent studies have addressed the utility of yet another method of “liquid biopsy”, i.e. analysis of circulating tumor DNA (ctDNA) by NGS techniques rather than RT-PCR of defined biomarkers. A side-by-side comparison was performed of the analyses for a serum biomarker versus analysis of CTC versus analysis of ctDNA by targeted or whole genomic sequence in breast cancer patients. This study reported the superiority of ctDNA analysis, as having a greater dynamic range, a greater correlation with changes in tumor burden, and better measure of treatment response (Dawson et al., 2013). The next publication from the same group reported monitoring of ctDNA by exomic sequencing in breast cancer patients prior to and during treatments (Murtaza et al., 2013). Serial analysis of ctDNA had successfully detected a number of prognostic genomic alterationsthat were either increased or appeared de novo as a consequence of treatments. It remains to be seen if detection of ctDNA will be a useful prognostic companion or even a substitute for tumor biopsies.

 

Exosomes.

Exosomes are small membrane vesicles with an endosome origin that are released by cells into the extracellular environment. They could fuse with other cells thereby transferring the RNAs and proteins they carry. Tumor-derived exosomes are emerging mediators of tumorigenesis; there is a growing interest in exploring treatment options targeting exosomes. Exosomes are thought to participate in the formation of pre-metastatic niche, i.e. specific microenvironment that is re-programmed to support survival and growth of metastatic cells. There has been significant interest in exploring the role of exosomes as prognostic markers (reviewed in[164]. A recent exciting study analyzed exosomes from highly metastatic melanomas. It found that these metastatic exosomes increased the invasive behavior of primary tumors by permanently “educating” bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype. Reducing MET expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating bone marrow progenitors from individuals with metastatic melanoma. The study identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70 (HSP90) isoform, and the MET oncoprotein[165].

 

Conclusions

It is very likely, considering the most recent findings, that the majority of driver mutations in melanoma have been identified. The study most quoted in this paper[166] reported that, outside of the most frequent mutations in BRAF and NRAS, driver mutations were identified in 83% of remaining BRAF and NRAS wild-type tumors. These results will be confirmed in much larger cohorts of melanoma tumors; some changes (identification of new rare driver or helper mutations) could be anticipated.
However, the accumulated knowledge is already sufficient to concentrate on the biological significance of genetic aberrations identified in melanoma and to evaluate their suitability as targets. Combination therapies are obviously the way of the future and might include two or more targeted therapies in combination with immunotherapies. One can also envision combinations that include novel therapies that target basic processes (eg, metabolism, autophagy) that are sufficiently deranged in melanoma compared to normal cells.

 

Table 1. Pathways Involved In Melanomagenesis

Table 1. Pathways Involved In Melanomagenesis

 

PATHWAY* COMPONENTS MUTATED/ACTIVATED TYPE OF ALTERATION
Receptor tyrosine kinases KIT Mutation/amplification
EGFR Activation
MET Activation; high level of ligand in stroma
ERBB4 Mutation
FGFR Activation; high levels of ligands
Integrin adaptors/ECM signaling NEDD9/HEF Amplification
RAS/RAF/MEK/ERK NRAS Mutation
BRAF Mutation
MEK1 Mutation
RAS/PI3K/PTEN/AKT/mTOR PIK3CA Mutation
PTEN Mutation
AKT1, AKT2 Rare mutation
AKT3 Amplification
NF1 (PI3K + MAPK pathways) NF1 Mutation
RHO/RAC/other MAPKs RAC Mutation
MAP3K5 & MAP3K9 Mutation
PREX Mutation
Glutamate receptors GRIN2A Mutation
GRM3 Mutation
G proteins other than RAS, effectors of MAPK GNAQ Mutation
GNA11 Mutation
Apoptosis BCL2A1 Amplification
WNT/b-catenin CTNNB1 Mutation
CDK CDK4 Mutation/amplification
CCND1 Amplification
P53 P14ARF(CDKN2A) Mutation/deletion
MDM4 Amplification
RB1 P116INK4A(CDKN2A) Mutation/deletion
MITF transcriptional program MITF Mutation/amplification
MYC transcriptional program MYC Amplification/overexpression
ETV1 transcriptional program ETV1 Amplification
TERT Promoter region of catalytic subunit Mutation

 

* The order of pathways in this Table has no relationship to their significance in melanoma; it is simply from the cell periphery to the nucleus.

 

TABLE 2. List of Gene Products Known To Be Altered in Melanoma

PRIMARY SUBTYPES PATHWAY ABERRATION FOUND IN TUMORS WITH… FREQUENCY POSSIBLE THERAPIES
BRAF MAPK Point mutation NRAS wild type 50-60% BRAFi + MEKiBRAFi + EGFRi + AKT
NRAS MAPK, PI3K, RALGDS BRAF wild type 20-25% HMGCRi + CDKi
KIT MAPK, PI3K Point mutation, amplification NRAS BRAF wild type mostly 1% overall; 10% in in mucosal; 10% in acral Sunitinib, nilotinib

GNAQ/GNA11

Gα(q) family of G protein α subunits; MAPK activators Point mutation NRAS BRAF wild type 1%; 40-50% each in uveal MEKi + PI3Ki, enzastaurin

MITF*

Transcription, lineage, cell cycle Amplification ALL???[JS1] 20% HDACi

NF1*

MAPK, PI3K negative regulator of RAS Mutations, loss of expression BRAF, NRAS wild type, but also in mutated 4% overall; 25% of BRAF, NRAS wild type MEKi + mTORi or PI3Ki

TERT*

Telomerase Mutations in the promoter of catalytic subunit  ND 70-80% overall;33% primary; 85% metastatic TERT inhibitors in preclinical
ERBB4 PI3K, MAPK Point mutation ALL types 15-20% Lapatinib (ERBBi) + PI3Ki
MET PI3K, MAPK Activation by stromal HGF All types ND Cabozantinib?
AKT3 PI3K Amplification All types 25% AKTi, PI3Ki, mTORi
PTEN PI3K Point mutation or deletions BRAF mutated; BRAF and NRAS wild type 40-60% PI3Ki
MAGI PI3K; stabilizes PTEN All types PI3Ki
TACC Possibly stimulates PI3K AURKA signaling BRAF and NRAS mutated 5% PI3Ki, AURKAi
PREX2 RHO/RAC/MAPK; Rac exchange factor Point mutations BRAF or NRAS mutated 14%
RAC1 RHO/RAC/MAPK; Regulator of cell adhesion, invasion, migration Point mutations BRAF or NRAS mutated 9% of sun exposed
MAP2K1, MAP2K2 MAPK (MEK1/2) Mutations BRAF mutated; BRAF, NRAF wild type 5% ERKi
MAP3K5, MAP3K9 RHO/RAC/MAPK Mutations, loss of heterozygocity All types 85% and 67% MEKi, ERKi
MYC Transcription Amplification All types 20-40% mTORi?
ETV1 Transcription Amplification All types 15%
TP53 Cell cycle, apoptosis Point mutation All types 10-20%
MDM4 Negative regulator of p53 Overexpression All types 65%
CDKN2A(P16INK4a, p14ARF)* Negative regulator of TP53 and RB Point mutation, deletion BRAF and NRAS mutated, KIT amplified 30-40%
BCL2, BCL2A1 Suppression of apoptosis Elevated expression,Amplification (BCL2A1) All types ND30% (BCL2A1) BH3 mimetics?
CCND1 Cell cycle, G1/S cyclin Amplifications More frequent in BRAF, NRAS wild type 11% CDKi?
CDK4* Cell cycle, G1/S cyclin-dependent kinase Amplifications More frequent in BRAF, NRAS wild type 3% Flavopiridol, selective CDKi
PPP6C Catalytic unit of phosphatase, negative regulator of CCND1, Aurora Point mutations BRAF and NRAS mutated 12% sun exposed AURKAi?CDKi?
STK19 Kinase; unknown function Point mutations BRAF and NRAS mutated 5%
SNX3 Endosome protein sorting Point mutations BRAF mutated; BRAF,NRAS wild type 7%
GRIN2A Ionotropic glutamate-gated ion channel, NMDA binding 25.%
GRM3 Possibly accessory MAPK signaling Point mutations ND
TRRAP Part of histone acetyltransferase complex Point mutations ND
ARID2 SWI/SNF Chromatin remodeling, SWI/SNF complex Inactivating mutations BRAF, NRAS mutated 7-9%
BAP1 BRCA1 DNA repair Inactivating mutations BRAF, NRAS wild type—uveal 1% overall, 84% uveal
NEDD9 Integrin adaptor, promotes EMT and migration; metastasis Amplification Probably all 50-60%

Bold font of gene names indicates that these mutations are considered to be driver mutations based on presence in familial melanoma and/or high frequency of mutations is sporadic, even though experimental evidence of their precise role in melanomagenesi is not always available* germline mutations in high-risk melanoma families. MC1R mutation is not listed

Not listed: rare mutations in HRAS, RAF1 and other oncogenes

Many of the data on the mutation frequency are based on relative sample tumor sizes and should be considered with caution

Table 3. Molecular Events Involved in the Intrinsic Resistance to BRAF and MEK Inhibitors

MOLECULAR CHANGE CONFIRMED IN PATIENT BIOPSIES? DRUGS TO ADD TO OVERCOME RESISTANCE REFERENCE
Resistance to Vemurafenib      
Loss of PTEN and consequent loss of BIM expression In vitro and in biopsies PI3Ki + MEKi Paraiso, K. H., Xiang, Y., Rebecca, V. W., Abel, E. V., Chen, Y. A., Munko, A. C., Wood, E., Fedorenko, I. V., Sondak, V. K., Anderson, A. R., et al. (2011). PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression. Cancer research 71, 2750-2760. http://cancerres.aacrjournals.org/content/71/7/2750.full
NF1 mutation or loss of expression Yes MEKi/mTORi Maertens, O., Johnson, B., Hollstein, P., Frederick, D. T., Cooper, Z. A., Messaien, L., Bronson, R. T., McMahon, M., Granter, S., Flaherty, K. T., et al. (2012). Elucidating distinct roles for NF1 in melanomagenesis. Cancer discovery. http://cancerdiscovery.aacrjournals.org/content/3/3/338.abstract, Whittaker, S. R., Theurillat, J. P., Van Allen, E., Wagle, N., Hsiao, J., Cowley, G. S., Schadendorf, D., Root, D. E., and Garraway, L. A. (2013). A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition. Cancer discovery. http://www.ncbi.nlm.nih.gov/pubmed/23288408
Increased levels of cyclin D No, in vitro only CDKi Smalley, K. S., Lioni, M., Dalla Palma, M., Xiao, M., Desai, B., Egyhazi, S., Hansson, J., Wu, H., King, A. J., Van Belle, P., et al. (2008). Increased cyclin D1 expression can mediate BRAF inhibitor resistance in BRAF V600E-mutated melanomas. Molecular cancer therapeutics 7, 2876-2883. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2651569/
Expression of Hsp90 Inhibition of Hsp90 induces death of vemurafenib resistant cells and xenografts XL888, Hsp90i Paraiso, K. H., Haarberg, H. E., Wood, E., Rebecca, V. W., Chen, Y. A., Xiang, Y., Ribas, A., Lo, R. S., Weber, J. S., Sondak, V. K., et al. (2012). The HSP90 inhibitor XL888 overcomes BRAF inhibitor resistance mediated through diverse mechanisms. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 2502-2514. http://clincancerres.aacrjournals.org/content/18/9/2502.short
NFkB pathway activation No, in vitro only METi? Wood, K. C., Konieczkowski, D. J., Johannessen, C. M., Boehm, J. S., Tamayo, P., Botvinnik, O. B., Mesirov, J. P., Hahn, W. C., Root, D. E., Garraway, L. A., and Sabatini, D. M. (2012). MicroSCALE screening reveals genetic modifiers of therapeutic response in melanoma. Science signaling 5, rs4. http://stke.sciencemag.org/cgi/content/abstract/sigtrans;5/224/rs4
MET and SRC signaling METi? Vergani, E., Vallacchi, V., Frigerio, S., Deho, P., Mondellini, P., Perego, P., Cassinelli, G., Lanzi, C., Testi, M. A., Rivoltini, L., et al. (2011). Identification of MET and SRC activation in melanoma cell lines showing primary resistance to PLX4032. Neoplasia 13, 1132-1142. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257188/
Production of HGF by stroma Observed in patients’ tumors; functionally confirmed in vitro METi? Straussman, R., Morikawa, T., Shee, K., Barzily-Rokni, M., Qian, Z. R., Du, J., Davis, A., Mongare, M. M., Gould, J., Frederick, D. T., et al. (2012). Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487, 500-504. http://www.nature.com/nature/journal/v487/n7408/full/nature11183.html?WT.ec_id=NATURE-20120726
Elevated CRAF expression (resistance to AZ628) No BRAFi + MEKi Montagut, C., Sharma, S. V., Shioda, T., McDermott, U., Ulman, M., Ulkus, L. E., Dias-Santagata, D., Stubbs, H., Lee, D. Y., Singh, A., et al. (2008). Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer research 68, 4853-4861. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692356/
Expression of antiapoptotic proteins, BCL2 and BCL2A1 Observed in tumors BH3 mimetics Wroblewski, D., Mijatov, B., Mohana-Kumaran, N., Lai, F., Gallagher, S. J., Haass, N. K., Zhang, X. D., and Hersey, P. (2012). The BH3-mimetic ABT-737 sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors but does not reverse acquired resistance. Carcinogenesis. http://carcin.oxfordjournals.org/content/34/2/237.short
Elevated expression of FOXD3 No ? Basile, K. J., Abel, E. V., and Aplin, A. E. (2012). Adaptive upregulation of FOXD3 and resistance to PLX4032/4720-induced cell death in mutant B-RAF melanoma cells. Oncogene 31, 2471-2479.
Resistance to MEK Inhibitors
Signaling through TGF-b/SMURF2/PAX3 and MITF No Smith, M. P., Ferguson, J., Arozarena, I., Hayward, R., Marais, R., Chapman, A., Hurlstone, A., and Wellbrock, C. (2013). Effect of SMURF2 Targeting on Susceptibility to MEK Inhibitors in Melanoma. Journal of the National Cancer Institute 105, 33-46. http://jnci.oxfordjournals.org/content/105/1/33.full
Downregulation of PTEN, activation of PI3K No PI3Ki + MEKi Byron, S. A., Loch, D. C., Wellens, C. L., Wortmann, A., Wu, J., Wang, J., Nomoto, K., and Pollock, P. M. (2012). Sensitivity to the MEK inhibitor E6201 in melanoma cells is associated with mutant BRAF and wildtype PTEN status. Molecular cancer 11, 75. http://www.biomedcentral.com/content/pdf/1476-4598-11-75.pdf

Table 4. Molecular Events Involved in Acquired Resistance to BRAF and MEK Inhibitors

MOLECULAR CHANGE CONFIRMED IN PATIENT BIOPSIES DRUGS TO ADD TO OVERCOME RESISTANCE REFERENCE
Resistance to Vemurafenib      
Expression of splicing variant of BRAF lacking exons 4-8 Yes, in 6 of 19 relapsed patients Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M., and Rosen, N. (2010). RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427-430. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3266695/
Amplification of BRAF Yes, in 4 of 20 relapsed patients Shi, H., Moriceau, G., Kong, X., Lee, M. K., Lee, H., Koya, R. C., Ng, C., Chodon, T., Scolyer, R. A., Dahlman, K. B., et al. (2012). Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nature communications 3, 724. http://www.ncbi.nlm.nih.gov/pubmed/22395615
Activation of EGFR/SFK/STAT3 Yes EGFRi, SRKi Girotti, M. R., Pedersen, M., Sanchez-Laorden, B., Viros, A., Turajlic, S., Niculescu-Duvaz, D., Zambon, A., Sinclair, J., Hayes, A., Gore, M., et al. (2012). Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. Cancer discovery. http://www.ncbi.nlm.nih.gov/pubmed/23242808
Activation of FGFR3-RAS pathway In vitro only FGFRi Yadav, V., Zhang, X., Liu, J., Estrem, S., Li, S., Gong, X. Q., Buchanan, S., Henry, J. R., Starling, J. J., and Peng, S. B. (2012). Reactivation of mitogen-activated protein kinase (MAPK) pathway by FGF receptor 3 (FGFR3)/Ras mediates resistance to vemurafenib in human B-RAF V600E mutant melanoma. The Journal of biological chemistry 287, 28087-28098. http://www.ncbi.nlm.nih.gov/pubmed/22730329
Activation of IGFR1-PIK3 pathway Yes IGFRi Villanueva, J., Vultur, A., Lee, J. T., Somasundaram, R., Fukunaga-Kalabis, M., Cipolla, A. K., Wubbenhorst, B., Xu, X., Gimotty, P. A., Kee, D., et al. (2010). Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer cell 18, 683-695. http://www.nchu.edu.tw/~ibms/index2.files/seminar/7098059004.pdf
Activation of PDGFRb- NRAS Yes RTKi Nazarian, R., Shi, H., Wang, Q., Kong, X., Koya, R. C., Lee, H., Chen, Z., Lee, M. K., Attar, N., Sazegar, H., et al. (2010). Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468, 973-977. http://www.nature.com/nature/journal/v468/n7326/full/nature09626.html
Loss or mutations NF1 Yes MEKi + PI3Ki + RAFi Maertens, O., Johnson, B., Hollstein, P., Frederick, D. T., Cooper, Z. A., Messaien, L., Bronson, R. T., McMahon, M., Granter, S., Flaherty, K. T., et al. (2012). Elucidating distinct roles for NF1 in melanomagenesis. Cancer discovery. http://cancerdiscovery.aacrjournals.org/content/3/3/338.abstract , Whittaker, S. R., Theurillat, J. P., Van Allen, E., Wagle, N., Hsiao, J., Cowley, G. S., Schadendorf, D., Root, D. E., and Garraway, L. A. (2013). A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition. Cancer discovery. http://www.ncbi.nlm.nih.gov/pubmed/23288408
Mutation of NRAS Yes, in relapsed patients Nazarian, R., Shi, H., Wang, Q., Kong, X., Koya, R. C., Lee, H., Chen, Z., Lee, M. K., Attar, N., Sazegar, H., et al. (2010). Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468, 973-977. http://www.nature.com/nature/journal/v468/n7326/full/nature09626.html
AKT3 In vitro only AKTi Shao, Y., and Aplin, A. E. (2010). Akt3-mediated resistance to apoptosis in B-RAF-targeted melanoma cells. Cancer research 70, 6670-6681. http://www.ncbi.nlm.nih.gov/pubmed/20647317
Activation of ERK independently of MEK through PI3K In vitro only Jiang, C. C., Lai, F., Thorne, R. F., Yang, F., Liu, H., Hersey, P., and Zhang, X. D. (2011). MEK-independent survival of B-RAFV600E melanoma cells selected for resistance to apoptosis induced by the RAF inhibitor PLX4720. Clinical cancer research : an official journal of the American Association for Cancer Research 17, 721-730. http://www.ncbi.nlm.nih.gov/pubmed/21088259
Activation of MAP3K8/COT, ERK activating kinase independent of MEK Yes Johannessen, C. M., Boehm, J. S., Kim, S. Y., Thomas, S. R., Wardwell, L., Johnson, L. A., Emery, C. M., Stransky, N., Cogdill, A. P., Barretina, J., et al. (2010). COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968-972. http://ccsb.dfci.harvard.edu/web/export/sites/default/ccsb/publications/papers/2010/Johannessen-etal_Nature_Dec2010.pdf
MEK mutations In vitro, and in 2 relapsed patients Wagle, N., Emery, C., Berger, M. F., Davis, M. J., Sawyer, A., Pochanard, P., Kehoe, S. M., Johannessen, C. M., Macconaill, L. E., Hahn, W. C., et al. (2011). Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 3085-3096. http://www.ncbi.nlm.nih.gov/pubmed/21383288
Loss of expression of RND3 In vitro only Klein, R. M., and Higgins, P. J. (2011). A switch in RND3-RHOA signaling is critical for melanoma cell invasion following mutant-BRAF inhibition. Molecular cancer 10, 114. http://www.ncbi.nlm.nih.gov/pubmed/21917148
Resistance to MEK Inhibitors
Mutations in MEK In vitro only BRAFi + MEKi Emery, C. M., Vijayendran, K. G., Zipser, M. C., Sawyer, A. M., Niu, L., Kim, J. J., Hatton, C., Chopra, R., Oberholzer, P. A., Karpova, M. B., et al. (2009). MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proceedings of the National Academy of Sciences of the United States of America 106, 20411-20416. http://www.pnas.org/content/106/48/20411.full
Activation of PI3K/AKT In vitro only IGFRi, AKTi, mTORi Gopal, Y. N., Deng, W., Woodman, S. E., Komurov, K., Ram, P., Smith, P. D., and Davies, M. A. (2010). Basal and treatment-induced activation of AKT mediates resistance to cell death by AZD6244 (ARRY-142886) in Braf-mutant human cutaneous melanoma cells. Cancer research 70, 8736-8747. http://www.ncbi.nlm.nih.gov/pubmed/20959481

 

 

Table 5. Current targets and trials in antibody-based immunotherapy of melanoma

Target Desired effects Antibodies Trials
CTLA-4 Relieve the immune checkpoint Ipilimumab/Yervoy Approved
PD-1 Relieve the immune checkpoint MDX-1106, CT-011, MK-3475 NCT01024231, NCT01176461, NCT01721746, NCT01621490, NCT01721772, NCT01783938, NCT01714739, NCT01435369, NCT01295827,NCT01704287
PD-L1/B7-H1 Relieve the immune checkpoint MDX-1105-01, MPDL3280A, MEDI4736 NCT00729664, NCT01633970, NCT01375842NCT01693562
4-1BB/CD137 Stimulate T cells BMS-663513 NCT01471210
CD40 Stimulate T cells CP870,893 NCT01103635
GITR Inhibit T regs TRX518 NCT01239134
OX40/CD134 Stimulate T cells Anti-OX40 NCT01689870
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