A Landscape of Driver Mutations in Melanoma

PMID: 22817889 · DOI: 10.1016/j.cell.2012.06.024 · Journal: Cell (2012)

TL;DR

Whole-exome sequencing of 121 melanoma tumor/normal pairs identified six novel significantly mutated melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2) using a permutation-based statistical framework (InVEx) that leverages intronic mutation rates to control for the high UV-induced passenger mutation load in melanoma. The RAC1 P29S hotspot mutation was shown to be an activating, gain-of-function event directly attributable to UVB mutagenesis, providing definitive genomic evidence linking UV-induced DNA damage to oncogenic driver mutations in melanoma.

Cohort & data

• 121 qualified tumor/normal pairs from 135 melanoma patients (15 primary tumors, 30 metastatic samples, 76 short-term cultures from metastatic tissue).
• Subtypes: 95 cutaneous, 5 acral, 2 mucosal, 1 uveal, 18 unknown primary.
• Dataset: skcm_broad.
• Assay: whole-exome sequencing with solution-phase hybrid capture; 103-fold mean target coverage.
• Copy number: Affymetrix SNP 6.0 arrays analyzed with GISTIC.
• Validation: Sequenom mass spectrometric genotyping in extension sets (n=59, n=63, n=175).

Key findings

• Median somatic mutation rate was 14.4 coding mutations/Mb (higher than any other tumor type at the time); 82.2% were UV-signature C>T transitions.
• The InVEx permutation framework identified 11 genes with significant functional mutation burden (q<=0.2): BRAF, NRAS, PTEN, TP53, CDKN2A, MAP2K1, PPP6C, RAC1, SNX31, TACC1, and STK19.
• RAC1 P29S was the third most frequent hotspot after BRAF and NRAS mutations, validated at 3.9% prevalence (14/355 patients across discovery + extension sets).
• RAC1 P29S is a gain-of-function mutation that shifts RAC1 toward the active GTP-bound state (demonstrated by PAK1 PBD pull-down assays in HEK293FT cells and immortalized melanocytes).
• PPP6C mutations (9% of discovery set) clustered around R264, with 60% in a 12-amino-acid window; pattern suggests gain-of-function alteration of phosphatase activity.
• STK19 D89N hotspot found in 4-5% of melanomas.
• ARID2 harbored significant loss-of-function burden (7%); together with ARID1A, ARID1B, and SMARCA4, 13% of samples had LoF mutations in SWI/SNF complex components.
• NF1 LoF mutations were enriched in BRAF/NRAS wild-type melanomas (25% vs 2%, p=5.8e-3).
• 83% of samples harbored hotspot or COSMIC-recurrent mutations in BRAF (n=73) or NRAS (n=27), mutually exclusive (p=3e-14).
• PTEN mutation/deletion co-occurred with BRAF (44%) but rarely with NRAS (4%, p=4.9e-5).
• 46% of all driver mutations were attributable to UV-induced damage (C>T or G>T); 67% when excluding BRAF/NRAS.

Genes & alterations

Gene	Alteration	Finding
BRAF	V600E/K/R hotspot	60% of samples; mutually exclusive with NRAS
NRAS	Q61L/R/K hotspot	22% of samples; mutually exclusive with BRAF
RAC1	P29S hotspot (C>T)	3.9% prevalence; gain-of-function, UVB-induced
PPP6C	R264C, S270L hotspots	9%; clustered near catalytic-regulatory interface
STK19	D89N hotspot	4-5%; predicted kinase, unknown function
SNX31	Distributed missense in FERM-like domain	Sorting nexin; potential Ras effector
TACC1	Distributed near C-terminus/TACC domain	7%; interacts with Aurora A
ARID2	Nonsense/LoF truncations	7%; SWI/SNF chromatin remodeling
NF1	LoF mutations	25% of BRAF/NRAS-WT; RAS pathway negative regulator
PTEN	Mutation/focal deletion	Co-occurs with BRAF (44%); rare in NRAS-mutant
TP53	Missense/LoF	19%; enriched in cases without CDKN2A/p14ARF loss
CDKN2A	p16INK4a LoF, p14ARF mutations	>20% (LoF in 12%); RB and p53 pathway deregulation
EZH2	COSMIC-recurrent	Epigenetic regulator; 17% with chromatin modifier mutations
IDH1	COSMIC-recurrent	Epigenetic regulator
KIT	V559A, focal amplification	In BRAF/NRAS-WT subset
CDK4	R24 activating, focal amplification	Cell cycle checkpoint

Clinical implications

• BRAF V600 mutations (50% of melanomas) predict response to RAF inhibitors such as vemurafenib.
• Activating KIT aberrations may predict response to tyrosine kinase inhibitors such as imatinib.
• RAC1 P29S, PPP6C R264C, and STK19 D89N are described as “potentially targetable” recurrent mutations.
• NF1 inactivation in the BRAF/NRAS-WT subtype suggests aberrant MAPK activation that could be therapeutically exploitable (e.g., MEK inhibition).
• The RB pathway is deregulated through somatic mutations in at least 24% of samples (via CDKN2A, RB1, CDK4), informing CDK4/6 inhibitor rationale.

Limitations & open questions

• Extension/validation sets were limited to mass spectrometric genotyping of specific hotspots rather than comprehensive sequencing.
• 76/121 samples were short-term cultures rather than primary tissue, which may introduce culture-derived artifacts.
• Functional validation was performed only for RAC1 P29S; PPP6C, STK19, SNX31, and TACC1 mutations await experimental confirmation of oncogenic activity.
• The statistical framework (InVEx) depends on sufficient intronic coverage from exome capture baits, limiting its applicability to WGS data without modification.
• STK19 has unknown protein function, making therapeutic targeting speculative.
• Acral, mucosal, and uveal subtypes are underrepresented (n=5, 2, 1 respectively).

Citations from this paper used in the wiki

• “Six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19 and ARID2), three of which - RAC1, PPP6C and STK19 - harbored recurrent and potentially targetable mutations.” (Abstract)
• “RAC1 P29S hot spot mutation in melanoma was validated to be 3.9% (14/355 patients)” (Results, RAC1 section)
• “PAK1 PBD pull-down revealed a significantly higher fraction of RAC1(P29S) in the GTP-loaded active state when compared to wild-type” (Results, RAC1 functional section)
• “13% (16/121) of the discovery samples harbored a LoF mutation in a component of the SWI/SNF complex” (Results, ARID2 section)
• “putative loss-of-function NF1 mutations occurred in 5 of 21 of these tumors (25%) compared to 2 of the remaining 100 samples (2%) (p = 5.8 x 10-3)” (Results, landscape section)
• “46% were caused by C>T (37%) or G>T (9%) mutations characteristic of UVB/UVA-induced mutations” (Results, UV mutagenesis section)

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