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RASopathy Gene Mutations in Melanoma

  • Author Footnotes
    4 These authors contributed equally to this work.
    Ruth Halaban
    Correspondence
    Correspondence: Ruth Halaban, Department of Dermatology, Yale University School of Medicine, 15 York Street, New Haven, Connecticut, 06520, USA.
    Footnotes
    4 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, Yale University School of Medicine, New Haven, Connecticut, USA
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  • Author Footnotes
    4 These authors contributed equally to this work.
    Michael Krauthammer
    Footnotes
    4 These authors contributed equally to this work.
    Affiliations
    Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA

    Program in Computational Biology and Bioinformatics, Yale University School of Medicine, New Haven, Connecticut, USA
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  • Author Footnotes
    4 These authors contributed equally to this work.
Open ArchivePublished:May 25, 2016DOI:https://doi.org/10.1016/j.jid.2016.05.095
      Next-generation sequencing of melanomas has unraveled critical driver genes and genomic abnormalities, mostly defined as occurring at high frequency. In addition, less abundant mutations are present that link melanoma to a set of disorders, commonly called RASopathies. These disorders, which include neurofibromatosis and Noonan and Legius syndromes, harbor germline mutations in various RAS/mitogen-activated protein kinase signaling pathway genes. We highlight shared amino acid substitutions between this set of RASopathy mutations and those observed in large-scale melanoma sequencing data, uncovering a significant overlap. We review the evidence that these mutations activate the RAS/mitogen-activated protein kinase pathway in melanoma and are involved in melanomagenesis. Furthermore, we discuss the observations that two or more RASopathy mutations often co-occur in melanoma and may act synergistically on activating the pathway.

      Abbreviations:

      ERK (extracellular signal-regulated kinase), GTP (guanosine triphosphate), MAPK (mitogen-activated protein kinase), NF1 (neurofibromatosis type 1), WT (wild type)
      Exome and genome sequencing have unraveled a large number of genetic and genomic changes in melanoma (
      • Hodis E.
      • Watson I.R.
      • Kryukov G.V.
      • Arold S.T.
      • Imielinski M.
      • Theurillat J.P.
      • et al.
      A landscape of driver mutations in melanoma.
      ,
      • Krauthammer M.
      • Kong Y.
      • Ha B.H.
      • Evans P.
      • Bacchiocchi A.
      • McCusker J.P.
      • et al.
      Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma.
      ,
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ,
      The Cancer Genome Atlas Network
      Genomic classification of cutaneous melanoma.
      ). The results confirmed the presence of frequent activating mutations in BRAF and NRAS and inactivating mutations in CDKN2A and TP53, and they unraveled additional lower frequency “drivers,” including the recurrent RAC1P29S and IDHR132C and the frequently modified PPP6C, ARID1, and ARID2. The most recent findings highlight the numerous NF1 (neurofibromin 1) mutations affecting up to approximately 12% of all melanomas, with higher frequency (45%) in melanomas that are wild type (WT) for BRAF and RAS, with abundant inactivating mutations, such as early termination, insertions/deletions, and splice variants (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ,
      The Cancer Genome Atlas Network
      Genomic classification of cutaneous melanoma.
      ). Consequently, the consensus is that melanomas can be subdivided into four categories: BRAFmut, RASmut, NF1mut, and triple WT (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ,
      The Cancer Genome Atlas Network
      Genomic classification of cutaneous melanoma.
      ). Other cancers with large number of NF1 mutations include glioblastoma (14%) (
      The Cancer Genome Atlas Network
      Comprehensive genomic characterization defines human glioblastoma genes and core pathways.
      ) and squamous cell carcinoma (11%) (
      The Cancer Genome Atlas Network
      Comprehensive genomic characterization of squamous cell lung cancers.
      ).
      The “NF1 discovery” draws attention to the autosomal-dominant genetic disorder neurofibromatosis type 1 (NF1), caused by haploinsufficiency of neurofibromin, a RAS guanosine triphosphate (GTP)ase-activating protein that affects 1 in 2,500 to 1 in 3,500 individuals (
      • Aoki Y.
      • Niihori T.
      • Inoue S.
      • Matsubara Y.
      Recent advances in RASopathies.
      ,
      • Ratner N.
      • Miller S.J.
      A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor.
      ,
      • Smpokou P.
      • Zand D.J.
      • Rosenbaum K.N.
      • Summar M.L.
      Malignancy in Noonan syndrome and related disorders.
      ;). The classic manifestations of NF1 include café-au-lait macules (observed in 95% of patients), skinfold freckling, neurofibromas, brain tumors, iris hamartomas, and characteristic bony lesions. NF1 early-termination mutations in patients’ germlines are frequent (∼80%), leading to release of constraints on RAS, followed by mitogen-activated protein kinase (MAPK) activation (
      • Ratner N.
      • Miller S.J.
      A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor.
      ), recapitulating the observations in melanoma (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ).
      Neurofibromatosis is one of many autosomal-dominant genetic disorders with overlapping sets of symptoms, currently termed RASopathies (including Noonan and Legius syndromes), that have germline nonsynonymous mutations in genes encoding proteins in the RAS/MAPK signaling cascade. In addition to NF1, the list includes BRAF, RAF1, NRAS, KRAS, HRAS, RASA2, PTPN11, SPRED1, SOS1, CBL, SHOC2, MAP2K1, MAP2K2, and RIT1 (
      • Ratner N.
      • Miller S.J.
      A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor.
      ,
      • Aoki Y.
      • Niihori T.
      • Inoue S.
      • Matsubara Y.
      Recent advances in RASopathies.
      ) (Figure 1 and Table 1). Somatic mutations in these genes are also observed in cancer, where they may be functionally relevant, as assessed by their ability to activate the RAS/MAPK pathway and/or enhance cell proliferation. Often, these somatic mutations alter the very same amino acid present in the germline of RASopathy patients (Table 1). In melanoma, this relationship and functional consequences are most clearly established for changes in BRAF, NRAS, MAP2K1, and RASA2.
      Figure 1
      Figure 1Mitogen-activated protein kinase pathway indicating RASopathy mutant genes and those shared with melanoma. The green and yellow bars are RASopathy genes with (presumably) activating and inactivating mutations, respectively. Those with amino acid changes shared with melanoma are marked with a red dot. CFCS, cardio-facio-cutaneous syndrome; GDP, guanosine diphosphate; GTP, guanosine triphosphate Noonan-LS, Noonan-like syndrome; RTK, receptor tyrosine kinase.
      Table 1Melanoma and RASopathy Shared Gene Mutations
      The melanoma mutations are from Yale, Broad Institute, and The Cancer Genome Atlas data; the RASopathy syndrome mutations are from the Human Gene Mutation Database (Stenson et al., 2012) and ClinVar (Landrum et al., 2014). The additional alternative amino acid substitutions in RASopathy genes not shared with melanomas are indicated in parenthesis. An asterisk indicates early termination.
      Gene SymbolShared Amino Acid ChangeRASopathy Syndrome Type
      NF1R1241*, R1362*, R1870Q, and other nonsense mutations causing premature truncationNeurofibromatosis 1
      BRAFL245F, F468S, G469R, L485F, N581H (K/D), V600GCardio-facio-cutaneous syndrome, Noonan syndrome
      NRASG12D/R/V, G13D, T50INoonan syndrome
      KRASG12A/I/D/R (S), Q22K (E/R/L), Q61RCardio-facio-cutaneous syndrome, Noonan syndrome
      HRASG13R/D (C), Q61K (R)Costello syndrome
      RAF1S257L, P261L (H/T/A/S), T491I (R)Noonan syndrome, LEOPARD syndrome
      MAP2K1P124L (D)Cardio-facio-cutaneous syndrome
      MAP2K2F57L/V (C)Cardio-facio-cutaneous syndrome, Noonan syndrome
      RASA2R511CNoonan syndrome
      SPRED1R117Q (*) and other nonsense mutations causing premature truncationNeurofibromatosis 1-like syndrome, Legius syndrome
      PTPN11F71L, Y279C, A461T, T468M, P491L, Q506P, Q510HNoonan syndrome, LEOPARD syndrome
      SOS1P102S (R), M269K (T/R), G434R, R552K (T/S/M/G), D1200ENoonan syndrome
      CBLL493FNoonan-like syndrome
      1 The melanoma mutations are from Yale, Broad Institute, and The Cancer Genome Atlas data; the RASopathy syndrome mutations are from the Human Gene Mutation Database (
      • Stenson P.D.
      • Ball E.V.
      • Mort M.
      • Phillips A.D.
      • Shaw K.
      • Cooper D.N.
      The Human Gene Mutation Database (HGMD) and its exploitation in the fields of personalized genomics and molecular evolution.
      ) and ClinVar (
      • Landrum M.J.
      • Lee J.M.
      • Riley G.R.
      • Jang W.
      • Rubinstein W.S.
      • Church D.M.
      • et al.
      ClinVar: public archive of relationships among sequence variation and human phenotype.
      ). The additional alternative amino acid substitutions in RASopathy genes not shared with melanomas are indicated in parenthesis. An asterisk indicates early termination.

      Comparisons of Specific Genes

      BRAF

      The canonical V600E/K substitutions lead to BRAF-kinase activation, the first to be targeted by specific inhibitors (
      • Bollag G.
      • Tsai J.
      • Zhang J.
      • Zhang C.
      • Ibrahim P.
      • Nolop K.
      • et al.
      Vemurafenib: the first drug approved for BRAF-mutant cancer.
      ). Other changes in BRAF (L245F, F468S, G469R, L485F, N581S/T, K601E) are shared between melanoma and the RASopathies cardio-facio-cutaneous and Noonan syndromes (
      • Rodriguez-Viciana P.
      • Rauen K.A.
      Biochemical characterization of novel germline BRAF and MEK mutations in cardio-facio-cutaneous syndrome.
      ) (Table 1). Many of these noncanonical alterations are located within the kinase domain (amino acids 457–713) and are activating mutations that lead to increased kinase activity over BRAFWT and extracellular signal-regulated kinase (ERK) activation in transfected COS cells (
      • Rodriguez-Viciana P.
      • Rauen K.A.
      Biochemical characterization of novel germline BRAF and MEK mutations in cardio-facio-cutaneous syndrome.
      ,
      • Wan P.T.
      • Garnett M.J.
      • Roe S.M.
      • Lee S.
      • Niculescu-Duvaz D.
      • Good V.M.
      • et al.
      Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.
      ). Furthermore, BRAF G469E, D594G, and K601E mutant melanomas display increased ERK phosphorylation over nonmutant control cell lines (
      • Smalley K.S.
      • Xiao M.
      • Villanueva J.
      • Nguyen T.K.
      • Flaherty K.T.
      • Letrero R.
      • et al.
      CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations.
      ).

      NRAS

      Melanomas typically harbor changes in the Q61 position of NRAS and, to a much lesser degree, in G12 and G13. Mice knock-in studies showed that expression of NrasQ61R but not NrasG12D promoted melanoma formation in vivo in p16INK4A-deficient mice (
      • Burd C.E.
      • Liu W.
      • Huynh M.V.
      • Waqas M.A.
      • Gillahan J.E.
      • Clark K.S.
      • et al.
      Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma.
      ). Functional studies showed that the basis for these differences is NrasQ61R enhanced GTP binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D (
      • Burd C.E.
      • Liu W.
      • Huynh M.V.
      • Waqas M.A.
      • Gillahan J.E.
      • Clark K.S.
      • et al.
      Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma.
      ). Germline mutations in Q61 were not reported, but Noonan syndrome patients and those with melanomas share the very same G12 and G13 NRAS amino acid substitutions (Table 1).

      MAP2K1

      Recurrent MAP2K1P124L/S mutations are present in melanoma tumors (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ,
      • Nikolaev S.I.
      • Rimoldi D.
      • Iseli C.
      • Valsesia A.
      • Robyr D.
      • Gehrig C.
      • et al.
      Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma.
      ), and MAP2K1P124L is also observed in the RASopathy cardio-facio-cutaneous syndrome. The mutation confers increased kinase activity (
      • Carlino M.S.
      • Fung C.
      • Shahheydari H.
      • Todd J.R.
      • Boyd S.C.
      • Irvine M.
      • et al.
      Preexisting MEK1P124 mutations diminish response to BRAF inhibitors in metastatic melanoma patients.
      ,
      • Emery C.M.
      • Vijayendran K.G.
      • Zipser M.C.
      • Sawyer A.M.
      • Niu L.
      • Kim J.J.
      • et al.
      MEK1 mutations confer resistance to MEK and B-RAF inhibition.
      ). The effect of the mutation on drug response is likely to be cell specific. The MAP2K1P124L appeared in the tumor of patient who relapsed after treatment with the MEK inhibitor selumetinib (
      • Emery C.M.
      • Vijayendran K.G.
      • Zipser M.C.
      • Sawyer A.M.
      • Niu L.
      • Kim J.J.
      • et al.
      MEK1 mutations confer resistance to MEK and B-RAF inhibition.
      ). In addition, pre-existing MAP2K1P124L diminished, but did not preclude, the clinical response to BRAF inhibitors of BRAFmut melanomas (
      • Carlino M.S.
      • Fung C.
      • Shahheydari H.
      • Todd J.R.
      • Boyd S.C.
      • Irvine M.
      • et al.
      Preexisting MEK1P124 mutations diminish response to BRAF inhibitors in metastatic melanoma patients.
      ,
      • Johnson D.B.
      • Menzies A.M.
      • Zimmer L.
      • Eroglu Z.
      • Ye F.
      • Zhao S.
      • et al.
      Acquired BRAF inhibitor resistance: A multicenter meta-analysis of the spectrum and frequencies, clinical behaviour, and phenotypic associations of resistance mechanisms.
      ). In culture, two double mutant melanoma cells lines showed intermediate sensitivity to dabrafenib but were exquisitely sensitive to the downstream MAPK/ERK kinsase and ERK inhibitors trametinib and VX-11e (
      • Carlino M.S.
      • Fung C.
      • Shahheydari H.
      • Todd J.R.
      • Boyd S.C.
      • Irvine M.
      • et al.
      Preexisting MEK1P124 mutations diminish response to BRAF inhibitors in metastatic melanoma patients.
      ). Likewise, in our studies, treatments with the MAPK/ERK kinsase inhibitor selumetinib showed that one patient-derived melanoma cell line carrying both BRAFV600K and MAP2K1P124L mutations was relatively resistant (YUKSI melanoma line, half maximal inhibitory concentration = 374 nmol/L), whereas another one with BRAFV600R and MAP2K1P124L was highly sensitive (YUZEAL melanoma line, half maximal inhibitory concentration = 15 nmol/L) (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ).

      RASA2

      Melanomas carry 25 different RASA2 substitutions, five of which are of the early-termination type present also in other cancers (cervical, head and neck cancers), suggesting a tumor suppressor function (Figure 2a). The recurrent RASA2R511C somatic mutation is one of three RASA2 variants described in patients with Noonan syndrome (
      • Chen P.C.
      • Yin J.
      • Yu H.W.
      • Yuan T.
      • Fernandez M.
      • Yung C.K.
      • et al.
      Next-generation sequencing identifies rare variants associated with Noonan syndrome.
      ) (Figure 2a). The R511C amino acid change located in the RAS-GTPase activating protein (GAP) domain abolishes RASA2 activity and increases the activity levels of RAS-GTP and ERK (
      • Chen P.C.
      • Yin J.
      • Yu H.W.
      • Yuan T.
      • Fernandez M.
      • Yung C.K.
      • et al.
      Next-generation sequencing identifies rare variants associated with Noonan syndrome.
      ). The protein, like NF1, functions as a suppressor by activating RAS GTPase and converting RAS-GTP to RAS (Figure 1). Similarly, suppression of RASA2 in melanoma cells by small interfering RNAs increased the levels of activated RAS (
      • Arafeh R.
      • Qutob N.
      • Emmanuel R.
      • Keren-Paz A.
      • Madore J.
      • Elkahloun A.
      • et al.
      Recurrent inactivating RASA2 mutations in melanoma.
      ). The impact of RASA2 mutants is likely to be synergistic with NF1, because they occur mostly in NF1-mutant tumors that are BRAF/RAS WT (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ).
      Figure 2
      Figure 2Schematic representation of RASA2 and PTPN11 mutations in melanomas and RASopathies. (a) RASA2. (b) PTPN11. The mutations indicated above and below the bar are those identified in melanoma and those shared with RASopathy syndromes, respectively. In blue are mutations shared with other cancers. The bars indicate conserved domains. Numbers below the bars indicate the amino acid positions. BTK, Bruton’s tyrosine kinase Cys-rich motif; C2, protein kinase C conserved region 2; LP, LEOPARD syndrome; LS, Legius syndrome; NS, Noonan syndrome; PH, pleckstrin homology-like domain; RAS-GAP, GTPase-activator protein for Ras-like GTPases; SB, substrate binding site; SH2, Src homology 2 domain.

      SPRED1

      SPRED1 acts as a tumor suppressor because it enhances NF1 inhibitory activity by recruiting the protein to the plasma membrane and to RAS (
      • Hirata Y.
      • Brems H.
      • Suzuki M.
      • Kanamori M.
      • Okada M.
      • Morita R.
      • et al.
      Interaction between a domain of a negative regulator of the RAS-ERK pathway, SPRED1, and the GTPase-Activating Protein-Related Domain of neurofibromin is implicated in Legius Syndrome and Neurofibromatosis Type 1.
      ,
      • Stowe I.B.
      • Mercado E.L.
      • Stowe T.R.
      • Bell E.L.
      • Oses-Prieto J.A.
      • Hernandez H.
      • et al.
      A shared molecular mechanism underlies the human rasopathies Legius syndrome and neurofibromatosis-1.
      ) (Figure 1). Mutations in SPRED1 act in an autosomal-dominant manner in Legius and NF1-like syndromes, mild forms of NF1 carrying skin features such as multiple café-au-lait macules, but no neurofibromas (
      • Brems H.
      • Pasmant E.
      • Van Minkelen R.
      • Wimmer K.
      • Upadhyaya M.
      • Legius E.
      • et al.
      Review and update of SPRED1 mutations causing Legius syndrome.
      ). Like NF1, this is another RASopathy gene that carries high frequency of early-termination mutations in both melanoma (71%) and the germline of patients (65%). In addition, NF1-like syndrome and melanomas share the SPRED1 R117Q substitution (Table 1). Interestingly, mutational analysis of melanocytes isolated from café-au-lait lesions of a patient with germline SPRED1-R24* carried another SPRED1 mutation, T102fsX6. The two SPRED1 mutations were located on different alleles, suggesting that SPRED1 function was completely absent, allowing increased MAPK activity and enhancing the rate of melanocyte proliferation, providing in vivo confirmation of its importance in melanocyte biology (
      • Brems H.
      • Chmara M.
      • Sahbatou M.
      • Denayer E.
      • Taniguchi K.
      • Kato R.
      • et al.
      Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype.
      ).

      PTPN11

      The gene product, also known as Src homology phosphatase-2 (SHP2), is a nonreceptor protein tyrosine-phosphatase with multiple positive functions in signal transduction (
      • Chan G.
      • Kalaitzidis D.
      • Neel B.G.
      The tyrosine phosphatase Shp2 (PTPN11) in cancer.
      ). It is a docking protein for the growth factor receptor-bound protein-2 (GRB2)/son of sevenless (SOS) complex, thereby promoting MAPK activation and cell division (Figure 1). The protein tyrosine phosphatase, non-receptor type 11 gene, PTPN11, is frequently altered in Noonan and Leopard syndromes and cancer cells (
      • Zhang J.
      • Zhang F.
      • Niu R.
      Functions of Shp2 in cancer.
      ). Analyses of the D61G mutation, frequent in RASopathies, showed that it is gain-function-change, activating the proto-oncogene SRC tyrosine kinase, which in turn activates the serine/threonine kinase polo-like kinase-1 (PLK1), inducing chromosomal instability and disruption of mitosis (
      • Liu X.
      • Zheng H.
      • Li X.
      • Wang S.
      • Meyerson H.J.
      • Yang W.
      • et al.
      Gain-of-function mutations of Ptpn11 (Shp2) cause aberrant mitosis and increase susceptibility to DNA damage-induced malignancies.
      ). Among the 20 mutation sites in melanoma, seven are shared with Noonan and Leopard syndromes (F71L, Y279C, A461T, T468M, P491L, Q506P, Q510H). Except for one that is located in the N-terminal SH2 domain (F71L), the rest are in the tyrosine-protein phosphatase (PTP) domain (Figure 2b). Other cancers, such as acute myeloid leukemia, sarcoma, and glioblastoma, also share PTPN11 mutations with melanomas and RASopathies, the most common being Q510H (Figure 2b, marked with blue). The somatic, like the germline, mutations are likely of the gain-of-function type because of disruption of the autoinhibitory interaction between the N-SH2 and PTP domains of the protein (
      • Chan G.
      • Kalaitzidis D.
      • Neel B.G.
      The tyrosine phosphatase Shp2 (PTPN11) in cancer.
      ,
      • Yu Z.H.
      • Zhang R.Y.
      • Walls C.D.
      • Chen L.
      • Zhang S.
      • Wu L.
      • et al.
      Molecular basis of gain-of-function LEOPARD syndrome-associated SHP2 mutations.
      ). Indeed, a change in the N-SH2 domain (D61G) activates SHP2 and enhances tumor adhesion, proliferation, migration, and invasion of breast cancer cells (
      • Hu Z.
      • Wang X.
      • Fang H.
      • Liu Y.
      • Chen D.
      • Zhang Q.
      • et al.
      A tyrosine phosphatase SHP2 gain-of-function mutation enhances malignancy of breast carcinoma.
      ).

      SOS1

      SOS1 is a guanine nucleotide exchange factor for RAS protein (RasGEF), catalyzing the transition of RAS-guanosine diphosphate to RAS-GTP, that is usually activated downstream of growth factor receptors (Figure 1). In melanomas, this gene carries five shared mutations with Noonan syndrome, two of them present in NF1-mutant/BRAF/RAS WT lesions (G434R, R552K) (Table 1). As with PTPN11, it is expected that the mutations are of the gain-of-function type, promoting an activated RAS-GTP status.
      In total, melanomas share disease-causing, nonsilent amino changes in 13 out of 16 known RASopathy genes (Figure 1 and Table 1).

      Evidence for Functional Cooperation of Rasopathy Genes

      Looking at all mutations in RASopathy genes (other than NF1), we find that they are significantly enriched in NF1-mutant melanomas, that is, 57.7% of NF1-mutant, 15.6% of NRAS-mutant, 6.6% of triple WT, and 4.3% of BRAF-mutant melanomas harbor concurrent RASopathy mutations (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ). The observation that NF1 and other RASopathy gene mutations co-occur likely suggests that they act in a synergistic manner. Next-generation sequencing of DNA from 27 patients with NF1 showed additional variants in more than one gene in the RAS-MAPK pathway, likely contributing to the observed neurofibromatosis features (
      • Chen P.C.
      • Yin J.
      • Yu H.W.
      • Yuan T.
      • Fernandez M.
      • Yung C.K.
      • et al.
      Next-generation sequencing identifies rare variants associated with Noonan syndrome.
      ). Noonan syndrome patients with atypical severe symptoms harbored coexisting mutations in NF1 and PTPN11, in one patient leading to death during early infancy (
      • Nystrom A.M.
      • Ekvall S.
      • Stromberg B.
      • Holmstrom G.
      • Thuresson A.C.
      • Anneren G.
      • et al.
      A severe form of Noonan syndrome and autosomal dominant cafe-au-lait spots - evidence for different genetic origins.
      ,
      • Prada C.E.
      • Zarate Y.A.
      • Hagenbuch S.
      • Lovell A.
      • Schorry E.K.
      • Hopkin R.J.
      Lethal presentation of neurofibromatosis and Noonan syndrome.
      ). In another case, a child with double genetic defects in NF1 and PTPN11 developed bilateral optic nerve gliomas, with other family members who carried only the NF1 mutation displaying mild neurofibromatosis symptoms (café-au-lait spots) (
      • Thiel C.
      • Wilken M.
      • Zenker M.
      • Sticht H.
      • Fahsold R.
      • Gusek-Schneider G.C.
      • et al.
      Independent NF1 and PTPN11 mutations in a family with neurofibromatosis-Noonan syndrome.
      ).
      A similar situation exists in cancer cells. Bioinformatics analyses of over 900 cell lines from the Cancer Cell Line Encyclopedia showed that 31% of cells containing noncanonical KRAS mutations also had an NF1 mutation (P < 0.005) (
      • Stites E.C.
      • Trampont P.C.
      • Haney L.B.
      • Walk S.F.
      • Ravichandran K.S.
      Cooperation between noncanonical Ras network mutations.
      ). A mathematical model based on RAS signaling reactions applied to a neurofibromin-deficient condition predicted, and then was experimentally supported, that loss of NF1 enhances the activity of the noncanonical RASF28L (
      • Stites E.C.
      • Trampont P.C.
      • Haney L.B.
      • Walk S.F.
      • Ravichandran K.S.
      Cooperation between noncanonical Ras network mutations.
      ). Likewise, we identified two cases of NF1-mutant melanomas, one with KRASA146T and the other with KRASQ22K, suggesting a similar situation. Our observation that RASA2 and NF1 significantly co-occur in melanoma (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ) is mirrored by a report that double-loss of Nf1 and Rasa1 in mice is required to enhance the development of T-cell acute lymphoblastic leukemia/lymphoma, supporting a synergistic effect on dysregulation of RAS signaling (
      • Lubeck B.A.
      • Lapinski P.E.
      • Oliver J.A.
      • Ksionda O.
      • Parada L.F.
      • Zhu Y.
      • et al.
      Cutting edge: codeletion of the Ras GTPase-activating proteins (RasGAPs) neurofibromin 1 and p120 RasGAP in T cells results in the development of T cell acute lymphoblastic leukemia.
      ). Similarly, our data showing co-occurring PTPN11 and NF1 mutations in melanoma (
      • Krauthammer M.
      • Kong Y.
      • Bacchiocchi A.
      • Evans P.
      • Pornputtapong N.
      • Wu C.
      • et al.
      Exome sequencing identifies recurrent mutations in NF1 and RASopathy genes in sun-exposed melanomas.
      ) is strengthened by a recent report of the presence of PTPN11 mutations in NF1-mutant desmoplastic melanomas (
      • Shain A.H.
      • Garrido M.
      • Botton T.
      • Talevich E.
      • Yeh I.
      • Sanborn J.Z.
      • et al.
      Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway.
      ).
      Co-occurring pairs of RASopathy mutations are present in melanoma lacking NF1 variants. For example, noncanonical RASopathy BRAF changes G469E and D594G coincide with NRAS G12D (
      • Lin W.M.
      • Baker A.C.
      • Beroukhim R.
      • Winckler W.
      • Feng W.
      • Marmion J.M.
      • et al.
      Modeling genomic diversity and tumor dependency in malignant melanoma.
      ). However, the significance of these double mutations requires further studies because of the observations mentioned that knock-in NrasG12DS transformed normal melanocytes to melanoma much less efficiently when compared with NrasQ61R in p16INK4a knockout mice (
      • Burd C.E.
      • Liu W.
      • Huynh M.V.
      • Waqas M.A.
      • Gillahan J.E.
      • Clark K.S.
      • et al.
      Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma.
      ).
      Functional cooperation was also observed in a subtype of KRAS/NRAS WT acute myeloid leukemia that is characterized by down-regulation of sprouty RTK signaling antagonist 4 (SPRY4), a SPRED1-related gene product that negatively regulates RAS-GTP, and co-occurring heterozygous deletions in TP53 and/or in other negative regulators of RAS signaling, such as NF1, RASA1, DUSP1, and DUSP14 (
      • Geiger O.
      • Hatzl S.
      • Kashofer K.
      • Hoefler G.
      • Wolfler A.
      • Sill H.
      • et al.
      Deletion of SPRY4 is a frequent event in secondary acute myeloid leukemia.
      ,
      • Zhao Z.
      • Chen C.C.
      • Rillahan C.D.
      • Shen R.
      • Kitzing T.
      • McNerney M.E.
      • et al.
      Cooperative loss of RAS feedback regulation drives myeloid leukemogenesis.
      ). Altogether, the presence of more than one RASopathy gene mutation is likely to enhance RAS function and to induce growth advantage by enhancing the MAPK pathway.
      An important question relates to the incidence of cancer in neurofibromatosis and other RASopathy patients. Individuals with germline NF1 alterations are at increased risk of developing various tumors, including malignant peripheral nerve sheath tumor, pheochromocytoma, leukemia, glioma, rhabdomyosarcoma, breast and ovary tumors, and rarely melanomas (
      • Ratner N.
      • Miller S.J.
      A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor.
      ,
      • Smpokou P.
      • Zand D.J.
      • Rosenbaum K.N.
      • Summar M.L.
      Malignancy in Noonan syndrome and related disorders.
      ). Studies show that additional mutations are present in these tumors such as second hits in NF1 and TP53, multiple copy number alterations, and deletion of CDKN2A (
      • Ratner N.
      • Miller S.J.
      A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor.
      ). Whole-exome sequencing of tumors from NF1 patients with NF1L847P showed that each of the lesions (dermal neurofibromas, breast cancer, malignant peripheral nerve sheath tumor) harbored another mutation in NF1; the breast cancer and malignant peripheral nerve sheath tumor presented with additional mutations unique for each tumor (
      • McPherson J.R.
      • Ong C.K.
      • Ng C.C.
      • Rajasegaran V.
      • Heng H.L.
      • Yu W.S.
      • et al.
      Whole-exome sequencing of breast cancer, malignant peripheral nerve sheath tumor and neurofibroma from a patient with neurofibromatosis type 1.
      ).
      These results and our observations that NF1 mutations are more frequent in melanomas that carry a high number of mutations, suggest that suppression of NF1 alone is not sufficient to confer malignancy and that combined loss of multiple negative regulators of the RAS pathway are required for melanomagenesis.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by the Yale Special Program of Research Excellence in Skin Cancer funded by the National Cancer Institute, US National Institutes of Health, under award number 1 P50 CA121974 (RH).

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