Cohort overview and subtype-specific SV patterns. We assembled and uniformly analyzed SVs in 355 patients with melanoma WGS (116 acral, 175 cutaneous, and 64 mucosal melanoma) (1, 2, 8, 9). Of the cutaneous melanoma samples, 81, 55, 19, and 20 samples were BRAF-, (N)RAS-, or NF1-mutant or TWT, respectively. The median sequencing coverage was 57× and 37× in tumor and matched normal samples, respectively, with no statistically significant difference in tumor sample coverage between the histologies (Wilcoxon-Mann-Whitney, P = 0.08; Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI177270DS1). Additionally, there was no statistically significant difference in the median tumor purity between the histologies, ranging from 61% in mucosal melanomas to 66% in acral melanomas (Wilcoxon-Mann-Whitney, P = 0.37), while background ploidy in acral (median 3.3) and mucosal (median 2.9) melanomas was significantly higher than in cutaneous (median 2.1) melanomas (Wilcoxon-Mann-Whitney, P < 3.8 × 10–5). In total, our framework identified 106,032 somatic genomic rearrangements (>30 bp; median events per tumor: acral, 81; mucosal, 64; cutaneous, 23; Figure 1A), consisting of 46,399 translocations (TRAs), 25,401 deletions (DELs), 17,935 inversions (INVs), and 16,297 duplications (DUPs). Of the 46,399 TRA events, 13,075 (28%) were intrachromosomal while 33,324 (72%) were interchromosomal. Across acral, mucosal, and cutaneous melanomas, approximately 72.4%, 71.4%, and 70.7% of TRA events were interchromosomal, respectively.

Characteristics of histologic and cutaneous genomic subtypes in melanoma. (A) The total number of TRA, DEL, INV, and DUP events in acral, cutaneous, and mucosal melanomas. (B) The distribution of the number of TRA, DEL, INV, and DUP events across acral, cutaneous, and mucosal melanoma histologic subtypes. (C) The distribution of the sizes of TRA, DEL, INV, and DUP events across acral, cutaneous, and mucosal melanoma histologic subtypes. (D) The distribution of the number of TRA, DEL, INV, and DUP events across the cutaneous melanoma genomic subtypes. (E) The distribution of the sizes of TRA, DEL, INV, and DUP events across the cutaneous melanoma genomic subtypes. Number of events for the boxplots in B and D and SV length for the density plots in C and E are plotted on a log2 scale. (B and D) *False discovery rate (FDR) adjusted P value < 0.05 via Mann-Whitney test.

The number and features of SVs varied widely across the melanoma histologies. Both acral and mucosal melanomas had significantly more events per tumor across all SV categories compared with cutaneous melanomas (Wilcoxon-Mann-Whitney, FDR P < 4.33 × 10–9; Figure 1B). However, when compared with mucosal melanomas, acral melanomas had significantly higher numbers of TRA (Wilcoxon-Mann-Whitney, FDR P = 3.85 × 10–4) and INV (FDR P = 0.013) events per tumor, but not DEL or DUP events. Acral melanomas were also significantly associated with larger (measured by distance between breakpoints) SV events across all SV categories compared with cutaneous melanomas (Wilcoxon-Mann-Whitney, P < 0.026), but not mucosal melanomas. Furthermore, the distributions of DEL and INV sizes in cutaneous melanomas possessed distinctive peaks surrounding smaller SV events (<10 kb; Kolmogorov-Smirnov, P < 2.2 × 10–16; Figure 1C), which may suggest a distinct mechanism of generation. Indeed, pan-cancer analysis of SVs identified small deletions as being enriched in early-replicating regions near TAD boundaries, and small inversions as being enriched in late-replicating regions (7).

Within cutaneous melanomas, there was no difference in the number of TRA, INV, and DUP events per tumor between the genomic subtypes (Wilcoxon-Mann-Whitney, P > 0.05). However, NF1-mutant melanomas had significantly higher numbers of DEL events per tumor compared with the other genomic subtypes (Wilcoxon-Mann-Whitney, FDR P < 0.033; Figure 1D). Examining the distribution of DEL and INV sizes within cutaneous melanomas revealed that the majority of smaller SV events in this histology were in NF1- and (N)RAS-mutant tumors (Figure 1E). Thus, the quantity and characteristics of SVs vary widely between melanoma histologic and molecular subtypes.

Chromothripsis and chromoplexy patterns in subtypes. While chromothripsis has been identified in each of the melanoma histologic subtypes, prior studies either were unable to differentiate chromothripsis from other complex events (8, 9) or were calling SVs with low sensitivity (11, 20, 21). Additionally, the comparative importance of chromothripsis with respect to the melanoma histologic subtypes or the genomic subtypes within cutaneous melanomas is uncertain (1, 2, 10). In this cohort, acral melanomas were significantly enriched for chromothripsis events (11) compared with both mucosal (70% vs. 31%; Fisher’s exact, OR = 5.04, 95% CI = 2.51–10.44, P = 8.23 × 10–7) and cutaneous (70% vs. 25%; Fisher’s exact, OR = 6.84, 95% CI = 3.96–12.04, P = 5.01 × 10–14; Figure 2A) melanomas, with no significant difference in the rate of chromothripsis between cutaneous and mucosal melanomas (Fisher’s exact, P = 0.41). Acral melanomas uniquely exhibited more SVs than expected by chance in chromosomes 3–4, 8–10, 12–14, 16–18, 20–22, X, and Y (P < 0.05). Mucosal melanomas exhibited more SVs than expected by chance in chromosome 1 (P < 0.05). Cutaneous melanomas exhibited more SVs than expected by chance in chromosome 5 (P < 0.05).

The rate and characteristics of chromothripsis events vary by melanoma histologic and cutaneous genomic subtypes. (A) The frequency of chromothripsis across acral, cutaneous, and mucosal melanoma histologic subtypes. (B) The most extreme chromothripsis event observed in an acral melanoma tumor, which consisted of SVs spanning a total of 18 chromosomes. (C) The most extreme chromothripsis event observed in a mucosal melanoma tumor, which consisted of SVs spanning a total of 8 chromosomes. (D) The frequency of chromothripsis across the cutaneous melanoma genomic subtypes. (E) An example of an intrachromosomal chromothripsis event spanning the BRAF locus in a BRAF-mutant cutaneous melanoma. (F) An example of an intrachromosomal chromothripsis event spanning the KRAS locus in an (N)RAS-mutant cutaneous melanoma. (E and F) SV events colored blue, orange, black, and green correspond to duplication-like, deletion-like, head-to-head inversions, and tail-to-tail inversions, respectively. Intrachromosomal events are connected by arches, while breakpoints of interchromosomal events are represented by points. (A and D) *P < 0.05 via Fisher’s exact test.

Approximately 85% of chromothripsis events in acral melanomas involved interchromosomal SVs, compared with 65% of mucosal (Fisher’s exact, OR = 3.05, 95% CI = 0.85–10.49, P = 0.055) and 52% of cutaneous (Fisher’s exact, OR = 5.17, 95% CI = 2.07–13.53, P = 1.1 × 10–4) melanomas. Of the interchromosomal chromothripsis events, the majority involved more than 1 additional chromosome (>2 in total; 67% acral, 62% mucosal, and 57% cutaneous). In one extreme case, an acral melanoma tumor had a single chromothripsis event affecting 18 chromosomes (Figure 2B), whereas the greatest number of chromosomes involved in a single chromothripsis event in mucosal and cutaneous melanomas was 8 (Figure 2C) and 6, respectively. Additionally, specific chromosomes were enriched for chromothripsis in melanoma histologic subtypes. Acral melanomas uniquely exhibited more SVs than expected by chance in chromosomes 3–4, 8–10, 12–14, 16–18, 20–22, X, and Y (P < 0.05), mucosal melanomas exhibited more SVs than expected by chance in chromosome 1 (P < 0.05), and cutaneous melanomas exhibited more SVs than expected by chance in chromosome 5 (P < 0.05). Thus, chromothripsis is associated with genomic instability in the majority of acral melanomas, while cutaneous and mucosal melanomas exhibit chromothripsis at less than half the rate of acral melanomas, and have similar chromothripsis landscapes despite significantly different global SV properties.

In addition to chromothripsis events, we also quantified the number of chromoplexy events. Both acral (Mann-Whitney U, P = 3.2 × 10–4, 3.4 vs. 2.3 events per tumor) and mucosal (Mann-Whitney U, P = 4.3 × 10–4, 4.2 vs. 2.3 events per tumor) melanomas were enriched for chromoplexy events compared with cutaneous melanomas. There was no difference in the number of chromoplexy events per tumor between acral and mucosal melanomas (Mann-Whitney U, P = 0.38). Within cutaneous melanomas, 42% (8/19) of NF1-mutant melanomas harbored chromothripsis events compared with 20%–25% in the other genomic subtypes, although this did not reach statistical significance (Fisher’s exact, P = 0.09; Figure 2D). All but one (88%) NF1-mutant melanomas that harbored chromothripsis involved interchromosomal SVs, compared with just 38% of BRAF-mutant melanomas with chromothripsis. Roughly 55% and 50% of (N)RAS-mutant and TWT tumors with chromothripsis involved interchromosomal SVs, respectively. Two of three (67%) NF1-mutant melanomas with missense (putatively activating) mutations in NF1 harbored chromothripsis, compared with 6 of 16 (37.5%) NF1-mutant melanomas with putatively inactivating mutations in NF1, although this difference was not statistically significant (Fisher’s exact test, P > 0.05). There was no statistically significant difference in the proportion of V600E and V600K tumors harboring chromothripsis within BRAF-mutant melanomas.

A subset of samples in each genomic subtype had chromothripsis events that spanned the driver genes that define the subtypes. For example, one BRAF-mutant melanoma harbored an intrachromosomal chromothripsis event that affected the BRAF locus (Figure 2E), while 4 other BRAF-mutant melanomas harbored chromothripsis events that spanned (i.e., the gene is at least partially between the breakpoints of at least 1 chromothripsis-generated SV) NRAS. One tumor with an NRAS G12R mutation had an intrachromosomal chromothripsis event spanning KRAS (Figure 2F), while BRAF and NF1 were involved in chromothripsis events in one NRAS melanoma each. Additionally, 2, 4, and 1 NF1-mutant melanomas harbored chromothripsis events spanning BRAF, NRAS, and NF1, respectively. In TWT tumors, BRAF and NRAS were affected by chromothripsis events in 1 sample each. Previous studies identified extrachromosomal DNA (ecDNA) events in melanoma that affect the BRAF and NRAS locus (22), and therefore we determined whether any intrachromosomal chromothripsis events spanning these loci were actually ecDNA amplifications. Only 1 sample with an intrachromosomal event spanning the BRAF locus had an ecDNA amplification event also affecting the BRAF locus. Thus, SVs generated via chromothripsis may provide secondary mechanisms of MAPK pathway dysregulation through genes that define the genomic subtypes. Furthermore, in the case of BRAF melanomas, these events may result in resistance mechanisms to targeted therapy (22). Thus, chromothripsis events in cutaneous melanoma may be capable of generating alterations that drive tumor initiation and development.

We lastly examined whether the distribution of short (<10 kb) INVs and DELs observed in NF1- and (N)RAS-mutant melanomas was the result of chromothripsis. The distribution of small INVs observed in NF1-mutant melanomas was largely driven by 2 samples, both of which had chromothripsis. However, only 34.6% and 7.3% of small INVs in these samples were located in chromothripsis regions. Similarly, the distribution of small INVs observed in (N)RAS-mutant melanomas was largely driven by a single sample that harbored chromothripsis; only 8.5% of these small INVs were located in chromothripsis regions. While the distribution of short DELs observed in NF1- and (N)RAS-mutant melanomas was not driven by a few outlier samples, there again was no association with the numbers of these events and chromothripsis (Wilcoxon-Mann-Whitney, P > 0.05). These results suggest that despite the increased frequency of small SV events in NF1- and (N)RAS-mutant tumors, these events are not the result of chromothripsis, and the differences in the sizes of these SV events are driven by outlier samples.

Effect of SVs on topologically associated domains. Disruption of topologically associated domain (TAD) boundaries through chromothripsis or other SV events can lead to the formation of neo-TADs and dysregulation of gene expression, whereby transcription factors, enhancers (14, 23), and silencers (24) that are typically absent from a gene’s native TAD may act on the gene as a result of SVs (25). To investigate the effect of SVs on TADs in melanoma, we focused on SVs unlikely to span multiple TAD boundaries using an established cutoff defined by the PCAWG consortium (<2 Mb) (13). To infer the putative impact of boundary-affecting SVs (BA-SVs), we leveraged the 5 TAD type annotations from that same study (13), which were determined using the 15-chromatin-state model from the Roadmap Epigenomics Project (26). These 5 TAD types are heterochromatin, low, repressed, low-active, and active, which are associated with increased expression (in the order specified) for genes contained within the TADs. We observed that 17.2%, 13.6%, and 7.2% of acral, mucosal, and cutaneous melanoma SVs (<2 Mb) completely spanned the full length of a TAD boundary, respectively. The frequency of these events was enriched compared with the expected number of BA-SVs based on randomly shuffled SVs, while maintaining SV size (P < 3.9 × 10–3). All acral melanoma tumors harbored at least one SV that spanned a TAD boundary, compared with 97% and 86.3% of mucosal and cutaneous melanomas, respectively (Figure 3A). Further, when assessing the putative functional impact of BA-SVs across histologic subtypes, 97.4% of acral melanomas harbored a TAD boundary–spanning SV adjacent to an active TAD, compared with 83% of mucosal and less than 50% of cutaneous melanomas (Figure 3, B and C). While there was no significant association between chromothripsis and the presence of BA-SVs in a tumor in any histologic subtype (Fisher’s, P > 0.05), tumors with chromothripsis events were associated with higher numbers of BA-SVs per tumor in acral (Wilcoxon-Mann-Whitney, P = 2.7 × 10–5) and cutaneous (Wilcoxon-Mann-Whitney, P = 0.026) melanomas, but not mucosal melanomas (Wilcoxon-Mann-Whitney, P = 0.09). Further, the correlation between global SV frequency and boundary-altering SV frequency differed by histologic subtype. The association was relatively weak in cutaneous melanomas (Pearson’s r = 0.26, P = 6.2 × 10–4), moderate in mucosal melanomas (Pearson’s r = 0.57, P = 8.8 × 10–7), and strongest in acral melanomas (Pearson’s r = 0.75, P = 2.2 × 10–16; Supplemental Figure 2).

Melanomas frequently harbor SVs affecting boundaries adjacent to active TADs and TADs containing oncogenes or tumor suppressors. (A) The number of BA-SV spanning events per tumor across acral, mucosal, and cutaneous melanomas categorized by the type of SV event. Complex SV events are defined as overlapping concomitant DEL, DUP, INV, or TRA events. (B) The number of affected TADs per tumor across acral, mucosal, and cutaneous melanomas categorized by functional TAD type. (C) The proportion of acral, mucosal, and cutaneous melanomas with BA-SVs adjacent to active TADs. (D) Known oncogenes and tumor suppressors that are putatively affected by BA-SVs in at least 5 tumors per histologic subtype, characterized by the type of SV event. (E) The proportion of event types resulting in BA-SVs that putatively affect tumor suppressors and oncogenes. (C and E) *P < 0.05 via Fisher’s exact test.

Of the total 2,477 TAD boundaries, 399 (16.1%), 159 (6.4%), and 105 (4.2%) boundaries were affected by SVs in more than one tumor in the acral, mucosal, and cutaneous cohorts, respectively. Further, SVs affecting the recurrently altered boundaries (observed in at least 5 tumors) comprised 56.6%, 35.7%, and 28.4% of all boundary-spanning SVs in acral, mucosal, and cutaneous melanomas, respectively, and frequently affected TADs containing known cancer-associated genes (Figure 3D). Although we did not possess matched expression data for human samples with SVs affecting these genes (Figure 3D), orthogonal analysis in melanoma cell lines from the Cancer Cell Line Encyclopedia (CCLE) demonstrated that SVs affecting these genes have functional consequences (Supplemental Figure 3). There was no enrichment in the types of TADs adjacent to recurrently altered boundaries (altered in >1 sample) compared with boundaries only altered in a single tumor across the histologic subtypes (Fisher’s exact, P > 0.05). In general, BA-SVs adjacent to TADs containing tumor suppressors (Supplemental Table 1) were enriched for deletion events (Fisher’s exact, OR = 2.34, 95% CI = 1.63–3.35, P = 2.21 × 10–6; Figure 3E), whereas BA-SVs adjacent to TADs containing oncogenes (Supplemental Table 1) were enriched for complex events (chromothripsis or overlapping concomitant SVs; Fisher’s exact, OR = 2.62, 95% CI = 1.69–4.18, P = 2.71 × 10–6; Figure 3E).

The most recurrently affected TAD boundary in both acral (n = 27; 23%) and mucosal (n = 7; 11%) melanomas was chr11:77750000–77825000, which is adjacent to TADs containing the cancer genes GAB2 and PAK1 (Figure 4, A and B). PAK1 is an oncogene that is involved in activation of the MAPK pathway (27), and has been suggested as a potential target in BRAF–wild type melanomas (28). Further, PAK1 has been identified as the most recurrently altered kinase gene via fusion events in a smaller cohort of acral melanomas (2), suggesting that PAK1 may also frequently activate the MAPK pathway outside of BA-SV events. Similarly, GAB2 is involved in the activation of the MAPK and PI3K/AKT pathways, and has been proposed to play a role in angiogenesis in melanomas (29). This TAD boundary was altered in 4 cutaneous melanomas and was 650 kb away from a fragile site (FRA11H) (30). The most recurrently altered boundary in cutaneous melanomas (all DEL events; n = 7; 4%) was chr9:21700000–21775000, which is flanked by a repressed TAD and a low-active TAD (Figure 4C). This boundary is adjacent to the TADs containing the cancer genes CDKN2A, CDKN2B, and MTAP, all of which are tumor suppressors, and this boundary is located within a fragile site region (FRA9C) (31). One potential mechanism of these BA-SVs is a long-range silencer interaction between regulatory elements of the adjacent repressed TAD and these tumor suppressors (32).

Recurrently affected boundaries adjacent to cancer gene–containing TADs. (A) Contact frequency map and annotations of SV events for the most recurrently altered TAD boundary in acral melanomas. DHS, DNase I hypersensitive sites. (B) Contact frequency map and annotations of SV events for the most recurrently altered TAD boundary in mucosal melanomas. Cancer genes of interest in the adjacent TADs for both acral and mucosal melanomas include PAK1 and GAB2. Both of the adjacent TADs for this boundary were low-active TADs. (C) Contact frequency map and annotations of SV events for the most recurrently altered TAD boundary in cutaneous melanomas. Cancer genes of interest in the adjacent TADs include CDKN2A, CDKN2B, and MTAP. The TAD containing these genes is a low-active TAD, and the other adjacent TAD is a repressed TAD. (D) Contact frequency map and annotations of SV events for the second most recurrently altered TAD boundary in cutaneous melanomas. Cancer genes of interest in the adjacent TADs include HIRA, SEPTIN5, and DGCR8. HIRA is present in an active TAD, and SEPTIN5 and DGCR8 are present in a low-active TAD. The contact frequencies shown here are from the IMR90 cell line, one of the 5 cell lines used to determine the functional TAD classifications by the PCAWG consortium.

The second most recurrently altered TAD boundary (chr22:19600000–19675000) was flanked by active and low-active TADs (Figure 4D), and is adjacent to TADs containing the cancer genes SEPTIN5, DGCR8, and HIRA (33). Unlike the other highly recurrently altered TAD boundaries, this TAD boundary was located several megabases away from the nearest fragile site (8 Mb; FRA22B). Both DGCR8 and HIRA are involved in UV-induced DNA damage repair, where DGCR8 is required for transcription-coupled nucleotide excision repair (NER) at UV-induced lesions (34), and HIRA is a histone regulator required for efficiently priming chromatin for transcriptional reactivation following DNA repair at UV-induced lesions (35, 36).

These results suggest an unappreciated role of BA-SVs in tumor development and progression across melanoma histologic subtypes (13), and that BA-SVs may generate histology-enriched driver events in melanoma. Further, a subset of cutaneous melanomas exhibit BA-SVs affecting NER genes that may exacerbate the effect of UV mutagenesis on the mutational spectrum of tumors.

Relationship between mutational signatures and SVs in cutaneous melanoma. To further assess the potential functional impact of SVs in melanoma, we next assessed SV pattern relationships with mutational signatures. The predominant mutational signatures in cutaneous melanoma are signature 1 (aging), signature 7 (UV mutagenesis), signature 11 (alkylating), and signature 3 (DSB repair), the lattermost of which is enriched in TWT melanomas (10). We previously reported an association between signature 3 and indel signature 8 (ID8; non-homologous end joining [NHEJ]), as well as between signature 3 and homologous recombination deficiency–associated copy number events, in cutaneous melanoma; however, the relationship between mutational signatures and SVs in cutaneous melanoma has remained unexplored (10). Consistent with prior analyses, mutational signature 3 was enriched in TWT cutaneous tumors in our cohort (Fisher’s exact, 5/20 vs. 5/155, OR = 9.75, 95% CI = 2.00–47.89, P = 1.1 × 10–3; Figure 5A), and it was the only SNV signature that was associated with increased numbers of SVs per tumor, after correction for disease stage, genomic subtype, coverage, and tumor purity (multivariate regression, P = 3.2 × 10–3). Specifically, this association was due to increased numbers of DUP and TRA SV events (multivariate regression, P = 3.2 × 10–4; Figure 5B and Supplemental Figure 4), but not DEL or INV SV events (multivariate regression, P > 0.17). Further, when SVs were characterized as being generated by either NHEJ, microhomology-mediated end joining (MMEJ), or single-strand annealing (SSA), which are DSB repair mechanisms frequently involved in the repair of SV events and associated with distinct microhomology patterns at SV breakpoint junctions, signature 3 tumors were significantly associated with increased numbers of SVs arising from NHEJ (multivariate regression, P = 6.7 × 10–3) and decreased numbers of SVs arising from SSA (multivariate regression, P = 2.8 × 10–4). The ratio of NHEJ-associated SVs to SSA-associated SVs was also significantly higher in signature 3 tumors (Wilcoxon-Mann-Whitney, P = 1.95 × 10–3; Figure 5C). Although the effect size was smaller, higher relative contribution of UV mutagenesis to the mutational spectrum of cutaneous melanomas was associated with lower numbers of SVs (multivariate regression, P < 2.7 × 10–3), particularly TRA and DUP events (multivariate regression, P < 4.09 × 10–5). There was no association between SNV mutational signatures and chromothripsis (multivariate regression, P > 0.07).

Cutaneous signature 3 melanomas are enriched for SVs frequently caused by NHEJ and are associated with SVs affecting the MRN complex. (A) The frequency of mutational signature 3 in TWT and non-TWT cutaneous melanomas. *P < 0.05 via Fisher’s exact test. (B) The distribution of the number of events per tumor between signature 3 and non–signature 3 cutaneous melanomas, characterized by SV type. (C) The distribution of the ratio of putative NHEJ- to SSA-generated SV events per tumor by signature 3 status in cutaneous melanomas. (B and C) *P < 0.05 via Mann-Whitney test. (D) The odds ratio (yellow square) and 95% confidence interval of the odds ratio (purple line) via Fisher’s exact test for SVs overlapping MRN complex genes in signature 3 cutaneous tumors compared with non–signature 3 cutaneous tumors. (E) Olaparib sensitivity curves in one TWT melanoma cell line (MeWo) and one BRAF-mutant melanoma cell line (A375) with knockouts of ATM, NBN, and MRE11. FDR adjusted P values: P > 0.05, NS; **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

SVs affecting canonical cancer genes and mutational processes in cutaneous melanoma. We then evaluated whether specific SVs affected canonical cancer genes and may directly relate to the mutational processes observed in cutaneous melanoma. Similar to our finding that cutaneous melanomas possessed somatic mutations in distinct secondary driver genes (10), several canonical cancer genes were also enriched for SVs within each genomic subtype. The most significantly enriched alterations in BRAF-mutant compared with non-BRAF-mutant melanomas were non-duplication SV events in CDKN2A (39/81, 48%; Fisher’s exact, OR = 2.41, 95% CI = 1.24–4.78, P = 7.8 × 10–3). Only 1 BRAF-mutant and 1 BRAF–wild type melanoma had duplication events overlapping CDKN2A. NF1-mutant melanomas were significantly associated with non-duplication SV events in two RASopathy genes, RAF1 and SPRED1 (Fisher’s exact, OR = 5.03, 95% CI = 1.46–16.42, P = 4.8 × 10–3), the latter of which has also been identified as a significantly mutated gene exclusive to NF1-mutant melanomas (10, 37). The most statistically significant canonical cancer gene affected by SVs in TWT melanomas was CBFA2T3, which was not altered in any of the other genomic subtypes (Fisher’s exact, OR = infinity, 95% CI = 5.69–infinity, P = 1.3 × 10–4) and is a putative tumor suppressor in breast cancer (38, 39). CBFA2T3 exclusively harbored TRA and INV events in TWT tumors (n = 4).

MRE11 was also among the cancer genes significantly enriched for SVs in TWT tumors compared with other subtypes (Fisher’s exact, OR = 5.36, 95% CI = 1.01–25.18, P = 0.024); it is one of the core genes of the MRN complex, along with NBN and RAD50, is involved in the initial processes of DSB repair prior to homologous recombination and NHEJ, and is responsible for activating ATM (40, 41). We previously found that signature 3 in TWT tumors was associated with downregulation of ATM, although we were unable to identify recurrent alterations in somatic coding regions that might explain the downregulation of ATM in a subset of samples (10). Three of the five TWT tumors with SVs affecting MRE11 had signature 3. Expanding the analysis to all signature 3 versus non–signature 3 tumors also revealed the enrichment of NBN in signature 3 tumors (Fisher’s exact, OR = 7.26, 95% CI = 1.04–39.44, P = 0.023), another core gene of the MRN complex. All SVs affecting MRE11 and NBN in signature 3 tumors were complex events, compared with less than half (43%) of non–signature 3 tumors (Fisher’s exact, OR = 6.74, 95% CI = 1.42–32.04, P = 7.4 × 10–3; Figure 5D). Pathway overrepresentation analysis on the set of cancer genes significantly enriched for SVs in signature 3 tumors compared with others identified the MRN complex as the top enriched protein complex (q = 1.79 × 10–3).

Although SVs affecting RAD50 were not associated with signature 3 tumors, there was no difference in the association of MRE11 (r = 0.73) or NBN (r = 0.72) expression with ATM expression compared with the association of RAD50 expression (r = 0.73) with ATM expression in TWT tumors (Supplemental Figure 5A). However, the correlation between MRN complex expression and ATM expression was significantly stronger in TWT tumors than in non-TWT tumors (r = 0.82 vs. r = 0.69; Fisher’s z-transformation, P = 0.03; Supplemental Figure 5B). To assess whether the correlation observed in TWT tumors was spurious as a result of having 7-fold fewer samples, we performed downsampling analysis for 10,000 simulations. Only 2.57% of these downsampled simulations yielded a correlation coefficient higher than that initially observed for TWT tumors (P = 0.0257; Supplemental Figure 5C). These results suggest that MRN-dependent ATM activation may be more frequent in TWT tumors or that ATM activation is more tightly regulated by the MRN complex in TWT tumors, potentially explaining why the association between signature 3 and ATM downregulation was restricted to TWT tumors. Additionally, these results are consistent with our previous finding that signature 3 in TWT cutaneous melanomas is associated with dysregulation of ATM and affects genes that function early during the initiation process of DSB repair.

To determine whether melanomas that have dysregulation of the MRN complex or ATM may be sensitive to PARP inhibitors, we performed independent knockouts of ATM, NBN, and MRE11 in 2 melanoma cell lines, MeWo (TWT) and A375 (BRAF-mutant), followed by olaparib cell viability assays (Supplemental Figure 6). MeWo cells lacking ATM (FDR P < 0.01, FDR P < 0.001) and NBN (FDR P < 2.32 × 10–5), but not MRE11 (FDR P > 0.15), showed increased sensitivity to olaparib (Figure 5E), and A375 cell lines lacking ATM, NBN, and MRE11 (FDR P < 7 × 10–15) all showed increased sensitivity to olaparib (Figure 5E). These results suggest that while dysregulation of ATM and the MRN complex is specifically enriched in TWT melanomas, this dysregulation may be sufficient to cause DSB repair deficiency in both TWT and non-TWT tumors, possibly rendering them sensitive to PARP inhibitors.