Melanoma genome sequencing reveals frequent PREX2 mutations

Abstract

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5-55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)--a PTEN-interacting protein and negative regulator of PTEN in breast cancer--as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.

Figure 1

Elevated mutation rates and spectra indicative of UV radiation damage. Top bar plot shows somatic mutation rate of 25 sequenced melanoma genomes, in decreasing order. Middle matrix indicates BRAF/NRAS somatic mutation status, with left-adjacent bar plot indicating total number of mutations in each oncogene as well as percent frequency. Bottom plot displays each tumor’s somatic mutation spectrum. Tumor sample names are indicated at the bottom of the figure, with acral melanomas in red.

Figure 2

Hubs of rearrangement breakpoints affect known and putative oncogenes. (a) Circos plots representing 4 melanoma genomes with notable structural alterations. Interchromosomal and intrachromosomal rearrangements are shown in purple and green, respectively. (b) Location of breakpoints associated with ETV1 in melanoma ME032. (c) Location of breakpoints associated with PREX2 in melanoma ME032. The red arrow indicates a premature stop codon (E824*). All rearrangements in ETV1 and PREX2 were validated by multiplexed PCR and 454 sequencing. (d) Confirmation of high-level amplification and rearrangement in PREX2 by fluorescence in situ hybridization (FISH).

Figure 3

Mutant PREX2 expression promotes melanoma genesis. (a) Non-synonymous sequence mutations detected from Illumina sequencing of 25 melanomas (green) or from capillary sequencing of a validation cohort of 107 additional melanomas (purple). Mutations are dispersed throughout all annotated structural domains of PREX2. (DH = DBL Homology domain; PH = Plekstrin Homology domain). The C-terminal half of PREX2 exhibits sequence homology to an inositol phosphatase domain. Engineered PREX2 mutants are labeled with red arrowheads. (b) Kaplan-Meier curve showing tumor free survival of NUDE mice (n=10) injected with pMEL-NRAS* cells expressing GFP, WT, truncated, and mutated PREX2 subcutaneously.