CDKN2B Loss Promotes Progression from Benign Melanocytic Nevus to Melanoma

Published OnlineFirst July 16, 2015; DOI: 10.1158/2159-8290.CD-15-0196

Research Article

Andrew S. McNeal1, Kevin Liu1, Vihang Nakhate1, Christopher A. Natale1, Elizabeth K. Duperret1, Brian C. Capell1,2, Tzvete Dentchev1, Shelley L. Berger2, Meenhard Herlyn3, John T. Seykora1, and Todd W. Ridky1

Abstract Deletion of the entire CDKN2B–CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here, we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAFV600E-activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAFV600E, we show that BRAF activation results in reversible, TGFβ-dependent, p15 induction that halts proliferation. Furthermore, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma.

SIGNIFICANCE: Although BRAFV600E mutations cause melanocytes to initially proliferate into benign moles, mechanisms responsible for their eventual growth arrest are unknown. Using melanocytes from human moles, we show that BRAF activation leads to a CDKN2B induction that is critical for restrain- ing BRAF oncogenic effects, and when lost, contributes to melanoma. Cancer Discov; 5(10); 1072–85. ©2015 AACR.

1Department of Dermatology, Perelman School of Medicine, University of Corresponding Author: Todd Ridky, Department of Dermatology, Perel- Pennsylvania, Philadelphia, Pennsylvania. 2Department of Cell and Develop- man School of Medicine, University of Pennsylvania, BRB1010, 421 Curie mental Biology, Perelman School of Medicine, University of Pennsylvania, Boulevard, Philadelphia, PA 19104. Phone: 215-573-5709; Fax: 215-573- Philadelphia, Pennsylvania. 3Wistar Institute, Philadelphia, Pennsylvania. 2033; E-mail: [email protected] Note: Supplementary data for this article are available at Cancer Discovery doi: 10.1158/2159-8290.CD-15-0196 Online (http://cancerdiscovery.aacrjournals.org/). ©2015 American Association for Cancer Research.

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INTRODUCTION cytic nevi (moles; refs. 4, 5). Nevi typically develop in the first 25 years of life, when constitutive BRAFV600E signaling causes The CDKN2A locus on chromosome 9p21 encodes two melanocytes to transiently proliferate, forming the common tumor suppressors: the cyclin-dependent kinase (CDK) inhib- brown skin papule present on most adults. Nevus growth itor p16INK4A and the MDM2 regulator p14ARF, which typically halts after reaching 3 to 5 mm in diameter, where together help maintain functional RB and p53. Homozygous melanocytes then remain quiescent for the remainder of a deletion of this locus is among the most common genetic person’s life, despite continued expression of the BRAF V600E events in human cancer across tissues, with the resultant oncogene (6). Mechanisms responsible for proliferation selective advantage to cancer cells generally attributed to arrest at this stage have not been previously established, but the loss of CDKN2A proteins. However, in greater than 90% are likely to include induction of CDK inhibitors, as bypass of of cancer tissues harboring CDKN2A deletion, the adjacent RB-mediated cell-cycle restraints via CDK4 activation occurs CDKN2B gene encoding the p15INK4B CDK inhibitor is in human melanoma, and was shown previously to promote also deleted (1, 2). The role of p15 in normal or neoplastic melanoma formation in a human model driven by virally human tissues has not been clearly established. Here, we overexpressed active RAS or BRAF (7). Although most nevi use primary cells isolated directly from normal human skin remain innocuous, up to half of malignant melanomas arise and benign melanocytic nevi, along with genetically defined from preexisting nevi, suggesting that the growth arrest human skin tissue xenografts engineered in vivo, to establish is likely reversible (8–10). However, no previous work has p15 induction as a key element arresting common nevi in established the reversibility of the growth arrest observed in their premalignant state. p15 depletion in benign nevi pro- human nevus melanocytes, nor demonstrated that BRAFV600E motes progression to melanoma, establishing the functional nevus melanocytes within common benign human moles importance of CDKN2B loss in human cancer. are capable of progressing to malignancy. Determining the Melanoma is a potentially fatal skin cancer arising from mechanisms through which benign nevus cells ultimately the pigment-producing melanocyte cells within the basal restrain BRAF proliferative signaling, and how this capacity layer of the human epidermis. Most melanomas are driven by is lost in the progression to malignant melanoma, may be an acquired, activating V600E mutation in the BRAF proto- crucial for the development of new melanoma therapeutics. oncogene, and are attributed to nucleotide sequence errors To delineate the signaling events responsible for the gen- incorporated during imperfect repair of ultraviolet DNA esis of nevi and their subsequent growth arrest, we first damage (3). This same BRAFV600E mutation is also responsible developed methods to isolate primary BRAF V600E-expressing for the formation of the majority of common benign melano- melanocytes directly from freshly excised benign human nevi,

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RESEARCH ARTICLE McNeal et al.

A B Normal MC Nevus-MC Figure 1. BRAFV600E-driven melanocyte growth arrest. A, clinical image of a benign nevus used in this study. B, ×10 brightfield images of representative normal melanocytes (MC; left) and nevus-MCs (right). Scale bars, 20 μm. C, Sanger sequencing of nevus- associated keratinocytes expressing wild- type (WT) BRAF (left), and nevus-MCs expressing BRAFV600E (right). D, Ki67 proliferation index of normal MCs and nevus- MCs. E, growth of diBRAF melanoctyes ± 0.25 μg/mL doxycycline showing prolif- C D 50 * V600E BRAF WT BRAF V600E eration arrest in BRAF -expressing 45 melanocytes. F, relative mRNA expression G A TTT CCA T GGT A G A TTT CCW T GGT A 40 of cell-cycle regulators in normal versus 35 nevus-derived melanocytes. *, P < 0.05; 30 n = 3 biologic replicates of distinct normal cells + 25 and nevus melanocyte populations used in 20 each experiment.

% Ki67 15 10 5 0 E F Normal MC Nevus-MC 160 Normal MC * 100 140 Nevus-MC 90 diBRAF –dox 80

6 70 diBRAF +dox 120 60 50 40 100 30 Cells, × 10 Cells, 20 80 10 0 0 5 10 15 20 60 Time (days) 40

Relative mRNA expression Relative 20

p14 p15 p16 p18 p21 CDK2 CDK4 CDK6

a significant technical advance that has not been demon- from clinically benign nevi excised from 27 human donors strated previously. Nevi are naturally occurring “knockin” (Fig. 1A). Only long-standing nevi with benign appearance experiments where melanocytes harbor a single copy of the and no history of recent morphologic changes were col- BRAF V600E gene regulated by the endogenous promoter, in lected. Nevi were microdissected from surrounding tissue, the context of native human stroma, and in most adult cases and melanocytes were separated from stroma and other skin have been growth arrested in vivo for decades. By examining cell types using physical and enzymatic dissociation followed these nevus melanocytes, as well as normal primary melano- by culture in selective media. Cultured nevus-MCs were mor- cytes engineered to inducibly express BRAFV600E, we were able phologically indistinguishable from melanocytes isolated to link BRAF activation to a TGFβ-dependent p15 induction from unremarkable skin (Fig. 1B). Sequencing BRAF cDNA that arrests the cell cycle and antagonizes oncogenic BRAF from cultured nevus-MC revealed overlapping, equal-inten- signaling. We demonstrate that the growth arrest is revers- sity peaks at nucleotide 1799, corresponding to expression of ible and use primary nevus cells to establish a new geneti- both wild-type (1799T) and mutant V600E (1799T>A) BRAF cally defined human melanoma xenograft model in vivo, alleles (Fig. 1C). Consistent with the high frequency of BRAF which maximizes medical relevance by supporting tissues in mutation in benign nevi, every nevus-MC culture we success- their native three-dimensional environment (11). With this fully initiated harbored an identical BRAFV600E mutation on a approach, we show that p15 loss is a major factor promoting single allele. Keratinocytes isolated from epidermis associated the transition from benign nevus to melanoma. with the nevi expressed only wild-type BRAF (Fig. 1C). Although nevus-MCs remained viable in vitro, they failed RESULTS to proliferate, independent of cell plating density, reflecting their in vivo growth arrest. Consistent with this, the Ki67+ V600E BRAF Nevus Melanocytes proliferative index of nevus-MCs was only 4%, compared with Are Growth Arrested 38% for normal melanocytes (Fig. 1D and Supplementary To establish mechanisms promoting growth arrest in Fig. S1). The significance of the few, weakly Ki67-positive BRAF V600E nevi, we isolated primary melanocytes (nevus-MC) nevus-MCs is unclear, but as Ki67 is expressed in all proliferative

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CDKN2B Loss Drives Melanoma RESEARCH ARTICLE

Figure 2. p15 expressed in nevi is a key A p15 B Ab ctrl factor mediating BRAF-induced growth arrest. A, p15 expression in a benign intradermal nevus and B, antibody control C, coimmunofluorescence of p15 and Melan-A in a representative intradermal benign nevus. Scale bars, 100 μm. D, nevus melanocytes express p15, whereas non- nevus melanocytes (white arrows) are p15-negative. Scale bar, 25 μm.

C p15 D p15 MeIan-A MeIan-A

phases of the cell cycle, cells arrested beyond G0 may retain To test the hypothesis that p15 and/or p16 induction is a some Ki67 expression, without actually completing cell divi- necessary component for BRAFV600E-induced growth arrest, sion (12). and to determine whether the growth arrest is reversible, To determine whether BRAFV600E expression was suffi- we first sought to circumvent p15/p16 function through cient to drive melanocytes’ proliferative arrest in the absence expression of CDK4R24C in arrested nevus and diBRAFV600E of additional in vivo cues, we engineered normal primary melanocytes. The activating R24C mutation, which occurs in melanocytes to express doxycycline-inducible BRAFV600E human melanoma (15, 16), rendered CDK4 resistant to p15/ (diBRAFV600E). Consistent with a key role for BRAFV600E in p16 and restored proliferation in both nevus and engineered nevus-MC growth arrest, doxycycline-treated diBRAF-MCs diBRAFV600E melanocytes (Supplementary Fig. S2A–S2C). ceased to proliferate, whereas untreated diBRAF cells proliferated normally (Fig. 1E). p15 Induction by BRAF Is Both Sufficient and Necessary for Melanocyte Growth Arrest The CDK Inhibitor p15 Is Induced in Nevi To test whether expression of BRAFV600E is sufficient on its To elucidate the mechanisms underlying BRAFV600E- own to upregulate p15 and p16, we compared their expres- induced growth arrest, we compared expression of cell-cycle sion in doxycycline-treated diBRAFV600E-MCs, nevus-MCs, and regulatory genes in nevus-MCs versus normal MCs by quanti- normal melanocytes. Whereas normal melanocytes expressed tative reverse transcriptase (qRT) PCR analysis. This revealed very little p15 or p16, doxycycline-treated diBRAF-MCs and altered expression of multiple cell-cycle regulators, the most nevus-MCs both expressed p15 and p16 at high levels in pri- prominent being CDKN2B (p15), which was induced 138- mary culture and in vivo (Fig. 3A and Supplementary Fig. S3). fold (Fig. 1F). Consistent with this finding, benign human Extended culture of primary nevus-MCs in vitro showed that nevi in vivo express high levels of p15 (Fig. 2A and B and elevated levels of p15 and p16 were maintained along with Supplementary Fig. S1). Epidermal melanocytes in adjacent the growth-arrested phenotype. Importantly, p15 induction tissue, not associated with the nevus, appeared p15 negative and the associated growth arrest are both dependent on the (Fig. 2C and D). As p15 inhibits cyclin D–CDK4/6 complexes level of BRAFV600E expression. Levels of p15 initially increase V600E and promotes RB-dependent maintenance of the G1–S cell- proportionally with increasing BRAF protein. However, at cycle checkpoint (13), we hypothesized that p15 plays a role the highest doxycycline-induced BRAF levels, or with high-level in arresting nevus growth, and may cooperate with other BRAF expression driven by a conventional lentivirus using a upregulated cell-cycle regulators, including p14, p16, and constitutive PGK promoter, p15 protein induction is attenu- p21, to maintain proliferative arrest in vivo. In line with this ated, and melanocytes continue to proliferate (Fig. 3A and data hypothesis, previous efforts to elucidate the basis for BRAF- not shown). This highlights the need to consider the BRAF mediated growth arrest in human cells using viral-driven protein level when using model systems, and led us to rely on transgene expression of BRAF V600E in fibroblasts, orNRAS Q61K the native nevus cells for functional in vivo experiments. in melanocytes, noted p16 induction, but concluded that To determine whether p15 is sufficient to cause melano- other factors must contribute, as p16 antagonism was insuf- cyte growth arrest without simultaneous induction of other ficient to rescue proliferation (6, 14). growth regulators, including p16, we transduced primary

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A B

1,400,000 Luciferase 1,200,000 p16 no doxycycline 0.015 µg/mL 0.031 µg/mL 0.0625 µg/mL 0.125 µg/mL 0.25 µg/mL 0.5 µg/mL 2.0 µg/mL p15 1,000,000 BRAF 800,000 p16 600,000

p15 number Cell 400,000

β-Actin 200,000 0 diBRAFV600E MC + doxycycline 0 4 7 10 Time (days) C D 2,500,000 3,000,000 p15i + dox NSi dox NSi p15i p16i NSi p15i p16i 2,000,000 − 2,500,000 p16i + dox p15i − dox NSi + dox BRAF p16i − dox 2,000,000 1,500,000 BRAFV600E 1,500,000 1,000,000 p16

Cell number Cell 1,000,000 p15 500,000 500,000 β-Actin 0 0 0 4 8 0 4 8 –dox +dox Time (days) Time (days) –dox +dox

Figure 3. p15 is necessary and sufficient to arrest melanocyte proliferation. A, Western blot analysis of BRAF, p15, and p16 expression in diBRAFV600E melanocytes in response to increasing levels of doxycycline. β-Actin was used as a loading control. B, proliferation of normal human melanocytes engineered to express p15 or p16. C, proliferation of diBRAFV600E melanocytes with nonsilencing control (NSi), p15 (p15i) or p16 (p16i) shRNA knockdown, without (left) or with (right) 0.25 μg/mL doxycycline. Data, average ± SEM for B and C; n = 3 biologic replicates for each group. D, Western blot analysis of BRAF, BRAFV600E, p15, and p16 in diBRAFV600E cells with nonsilencing control (NSi), p15 (p15i), and p16 (p16i) shRNA knockdowns. β-Actin was used as a loading control. Western blot analysis results and growth curves were representative of three biologic replicates composed of distinct melanocyte populations.

melanocytes with p15 or p16. Melanocytes expressing p15 ing proteins and pathways mediating this additional p15 halted proliferation, whereas melanocytes expressing p16 activity are not yet established, they are likely functionally continued to divide, albeit more slowly than controls (Fig. 3B important in nevus melanocytes. and Supplementary Fig. S4A). Through virus titering and To test whether BRAFV600E-induced growth arrest requires quantitative PCR, we demonstrated that these proliferation p15 or p16, we transduced diBRAFV600E cells with shRNAs target- differences did not result from relatively lower levels of p16 ing p15 (p15i) or p16 (p16i; Fig. 3C and D). Without doxycycline, gene expression. The p16 virus titer was actually higher than diBRAFV600E cells harboring p15i or p16i proliferated normally that of the p15 virus used, and this corresponded to higher as expected. With doxycycline, proliferation of diBRAFV600E cells levels of p16 transcript (Supplementary Fig. S4B and S4C). expressing control or p16i slowed dramatically, whereas p15i The relative proliferation effects correlated well with pRB cells continued to proliferate. Together, these data indicate that protein, with pRB levels lowest in the p15-expressing melano- p15 is both necessary and sufficient for BRAFV600E-mediated cytes (Supplementary Fig. S4D and S4E). Although p15 and growth arrest. To ensure that the observed proliferation phe- p16 share structural homology through their ankyrin repeat notype of the p15 knockdown was due to loss of p15 and not domains, there are significant regions of divergent sequence, an off-target effect of the shRNA, we restored p15 expression in particularly in the N-terminus, that may serve to make p15 diBRAFV600E-MCs expressing the p15 shRNA, and demonstrated a more efficient CDK inhibitor (reflected by lower pRB). that cells again growth arrested as expected (Supplementary In addition, previous work using a series of cell lines and Fig. S5A and S5B). As an additional control, we also validated chimeric p15/p16 domain swap constructs established that the phenotype using a CRISPR-Cas9 approach targeting the sequences unique to the p15 N-terminus confer p15 with the CDKN2B locus. As with shRNAs targeting p15 mRNA, CDKN2B ability to influence cell cycle beyond RB, and that p16 does ablation by CRISPR-Cas9 prevented BRAFV600E-induced growth not possess this activity (17). Although the specific interact- arrest (Supplementary Fig. S5C and S5D).

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CDKN2B Loss Drives Melanoma RESEARCH ARTICLE

As CDKN2A and CDKN2B are frequently simultaneously A deleted in melanoma, we next questioned whether simulta- 4 5 6 7 neous loss of p15 and p14/16 activity had an additive effect on overcoming BRAFV600E-mediated growth arrest. We used the melanoma-associated dominant-negative p53 (dnp53) mutant (R248W) to recapitulate the functional effects of p14 loss. Accordingly, we compared the relative proliferation of diBRAFV600E-MCs expressing shRNAs targeting p15 to matched cells with the p16 shRNA and dnp53, or the com- Melanoma bination of p15 and p16 shRNAs and dnp53. In the absence Nevus of BRAFV600E expression, all cell populations proliferated normally, indicating that no off-target effects compromised B C general cell-cycle regulation. As seen previously, melanocytes expressing BRAFV600E growth arrested, whereas BRAFV600E cells with p15 knockdown proliferated, albeit more slowly than normal controls lacking BRAFV600E. BRAFV600E-expressing cells with p16 knockdown and dnp53 expression failed to proliferate. However, cells with the combination of BRAFV600E, p15/ p16 knockdown, and dnp53 proliferated faster than the cells with p15 knockdown alone (Supplementary Fig. S6). There- fore, the combinatorial loss of p15, p14 (through dnp53), and p15 p16 activity promoted proliferation in the face of BRAFV600E to a greater extent than the loss of the individual elements. These results are consistent with our data showing that although Figure 4. BRAF-induced p15 is decreased in melanoma. A, representa- each of the proteins encoded by the CDKN2A–CDKN2B locus tive clinical image from n = 5 cases in which melanoma developed in are important in melanomagenesis, p15 is an especially impor- continuity with a preexisting nevus. B, p15 expression is markedly dimin- tant mediator of BRAF-induced growth arrest. ished in the malignant proliferative nests of the melanoma portion of A compared with C, the benign nevus portion. Scale bars, 100 μm. p15 Is Diminished in Melanoma To discover whether p15 helps restrain the oncogenic malignant melanocytic neoplasias that are otherwise histo- activity of BRAFV600E in vivo, we examined informative clinical logically ambiguous. cases in which invasive melanoma arose in clear continuity V600E with a preexisting benign nevus (Fig. 4A). p15 protein was BRAF Activates p15 Expression diminished in the malignant melanomas compared with through TGFb Signaling adjacent nevi (Fig. 4B and C). To determine the general In other cell contexts, p15 is induced via TGFβ/SMAD relevance of this finding, we examined a series of 12 ran- activity (13, 20). Increased TGFβ secretion in response to domly selected malignant melanomas and 15 benign nevi, BRAF has not been previously demonstrated in melanocytes, along with five nevus-melanoma cases. The majority of nevi but has been observed in immortalized rat thyroid epithelial strongly expressed both p15 and p16, whereas the major- cells (21). We questioned whether BRAFV600E induced melano- ity of melanomas showed very low to undetectable p15 and cyte TGFβ expression. BRAFV600E increased both TGFβ mRNA p16 (Supplementary Fig. S7A). This pattern of coordinate and protein secretion in nevus-MCs and diBRAFV600E-MCs regulation of p15 and p16 is consistent with the latest Cancer (Fig. 5A and B). To determine whether TGFβ was sufficient Genome Atlas (TCGA) analysis of 278 melanomas in which to recapitulate the antiproliferative effects of BRAFV600E, we 93% of melanomas with homozygous deletion of CDKN2A added TGFβ to primary melanocyte cultures. As expected, (p16) also harbor homozygous deletions of CDKN2B (p15; this induced p15 and arrested growth (Fig. 5C). p15 induc- Supplementary Fig. S7B and S7C; refs. 1, 2). Simultaneous tion resulting from either exogenous TGFβ or BRAFV600E loss of these two cell-cycle inhibitors likely reflects the close is dependent on the TGFβ receptor, as the TGFβ receptor proximity of the adjacent CDKN2A–CDKN2B loci on chromo- inhibitor SB-431542 blocked p15 induction in both cases some 9p21.3. Importantly, the TCGA series includes a tumor (Fig. 5D). These data are consistent with human nevus tissue, in which CDKN2B is lost but p16 remains intact, a genetic which displays robust SMAD activation (22). event that has also been described in other series (18, 19). The specific transcription complexes downstream of Consistent with these previous reports, we also identified BRAF responsible for directly activating TGFB transcrip- a clinical case in our sample group in which a p16-positive tion are not definitively established, but are likely to include melanoma developed in the epidermis overlying a benign EGR1. In other human cell contexts, EGR1 binds to the nevus (Supplementary Fig. S8). Although the nevus-MCs regulatory regions of the TGFB promoter to drive transcrip- robustly expressed both p15 and p16, the melanoma retained tion (23, 24), and EGR1 is a firmly established ERK1/2 only p16 and lacked p15 expression. Together, these findings target (25). Consistent with these prior studies, EGR1 is highlight the functional importance of p15 loss in human upregulated in BRAFV600E nevus melanocytes (Supplemen- melanoma and suggest that p15 expression may serve as a tary Fig. S9A). Also consistent with this ERK-dependent clinically useful tool to help distinguish between benign and p15 induction, inhibition of ERK signaling by the MEK

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A 16 B Figure 5. TGFβ secretion 14 * induces p15 expression and drives melanocyte growth 12 arrest. A, relative transcription * 25 Normal MC of TGFB1, TGFB2, and TGFB3 in 10 nevus-MCs and normal melano- Nevus-MC 20 cytes determined by TaqMan 8 qPCR. n = 3 biologic replicates 15 6 for normal melanocyte and n = 4 biologic replicates for nevus- 10 4 MCs. *, P < 0.05. B, TGFβ levels in culture media from diBRAFV600E Relative mRNA expression Relative 5 2 or normal melanocytes. Data, average ± SEM; n = 3 biologic Relative TGF β 1 expression Relative 0 0 replicates for diBRAFV600E TGFB1 TGFB2 TGFB3 BRAFV600E MC media Control MC media melanocytes and normal melanocytes. C, proliferation of C 2,000,000 D luciferase-transduced melano- 1,800,000 cytes with or without 100 pmol/L 1,600,000 exogenous TGFβ. Data, average ± SEM; n = 3 biologic replicates 1,400,000 + SB4 for each group. D, 100 pmol/L TGFβ or BRAFV600E-driven p15 1,200,000 Ctrl vehicle V600E V600E induction is inhibited by 50 1,000,000 μmol/L SB-431542 (SB4) TGFβ 100 pmol/L Control TGFβ1 SB4 TGFβ1 + SB4BRAF BRAF 800,000 TGFβ1 receptor blockade in human Cell number 600,000 BRAF melanocytes. 400,000 p15 200,000 β-Actin 0 0 853 Time (days)

inhibitor U0126 blocked BRAF-driven p15 induction (Sup- after 3 days of doxycycline administration were subsequently plementary Fig. S9B). cultured in media lacking doxycycline. Those cells remained We next sought to determine whether inhibiting BRAFV600E growth arrested (Supplementary Fig. S10C and S10D), rein- itself would restore proliferation in melanocytes harboring forcing the idea that melanocytes may use additional BRAF- constitutively active BRAF. We treated both our engineered independent mechanisms that maintain the growth-arrested doxycycline-inducible diBRAFV600E melanocytes and primary state, once established. nevus-derived V600E melanocytes with the selective BRAFV600E V600E small-molecule inhibitor PLX4720. As expected, PLX4720 BRAF Alters the Epigenetic Chromatin treatment, when delivered with doxycycline to diBRAFV600E Landscape at the CDKN2A and CDKN2B Loci cells, prevented both p15 induction and the corresponding CDKN2A and CDKN2B are normally suppressed in pro- growth arrest. In contrast, when the inhibitor was delivered liferating melanocytes, suggesting that the loci are epige- to diBRAF cells in which growth arrest had been previously netically repressed. Protein induction in response to BRAF established by antecedent doxycycline administration, cells activation would therefore require not only engagement of remained growth arrested, and p15 levels, although lower TGFβ-activated transcription complexes, but also removal of than those observed without PLX4720, remained higher than repressive epigenetic chromatin marks. Chromatin immuno- in normal cells (Supplementary Fig. S10A). This suggests that precipitation targeting the H3K27me3 and H3K9me3 repres- under in vitro culture conditions, BRAF activity and the cor- sive histone modifications showed that BRAFV600E expression responding p15 induction are necessary for establishing the results in reduction of both marks at the p15 (Supplementary growth arrest, but that additional events may also serve to Fig. S11A) and p16 (Supplementary Fig. S11B) transcription reinforce and maintain it. These other events are unclear, but start sites. are likely to involve epigenetic transcriptional memory influ- encing core cell-cycle regulators. This finding of persistent Nevus Melanocyte Growth Arrest Is Reversible and growth arrest despite BRAFV600E inhibition is consistent with Associated with Progression to Melanoma In Vivo clinical observations, in which vemurafenib-treated patients We next sought to use BRAFV600E nevus cells to develop new do not typically display obvious morphologic changes in pre- in vivo human melanoma models to determine the functional existing benign nevi. Also consistent with this is our observa- role of p15 in melanocyte neoplasia. To determine whether the tion that PLX4720 does not reverse growth arrest in primary findings from our in vitro studies are relevant in vivo, we first melanocytes isolated directly from nevus specimens (Supple- used nevus-MCs transduced with CDK4R24C together with mentary Fig. S10B). To examine this issue further, we used epidermal keratinocytes from unremarkable skin, and native an alternative approach to “turn off” BRAFV600E. Melanocytes human dermis, to regenerate three-dimensional human skin tissue expressing inducible BRAFV600E that underwent growth arrest xenografts, using modifications to previous methodologies

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CDKN2B Loss Drives Melanoma RESEARCH ARTICLE

A B

Nevus-MC CDK4R24C

Nevus-MC CDK4R24C hTERT

Nevus-MC CDK4R24C hTERT dnp53R248W

H&E Melan-A MITF Ki67

Figure 6. In vivo melanoma model using nevus-derived BRAFV600E melanocytes. A, in vitro organotypic skin tissue prior to skin grafting. B, human skin xenograft harboring BRAFV600E-expressing melanocytes. C, hemotoxylin and eosin (H&E) staining, with Melan-A, microphthalmia-associated transcription factor (MITF), and Ki67 immunohistochemistry (columns, left to right) from xenografts incorporating nevus-MC expressing CDK4R24C (row 1), CDK4R24C and hTERT (row 2), or CDK4R24C, hTERT, and dnp53R248W (row 3). Images are representative of 3 biologic replicates. hTERT, human telomerase reverse transcriptase. Scale bars, 100 μm.

(Fig. 6A and B; refs. 7, 11, 26). Although CDK4 activation (hTERT) and CDK4R24C prior to xenografting. These cells overcame nevus-MC growth arrest in culture, it was insuf- displayed a 4-fold increase in telomerase activity (Supple- ficient to sustain proliferation in vivo. Melanocytes survived mentary Fig. S12A), consistent with the magnitude of the in the orthotopic location, but demonstrated a low Ki67+ increase seen in spontaneous melanoma. Tissue grafts with index (Fig. 6C). As a cell’s ability to progress through the cell nevus-MC expressing hTERT and CDK4 contained nests cycle depends on the relative balance of proliferative versus of pigmented melanocytes similar in architecture to those antiproliferative signals, this difference between in vitro and seen in benign human nevi (Fig. 6C), indicating that after in vivo proliferation likely results from a relative lack in vivo of a brief proliferative period, the nevus-MCs ceased to divide. the mitogenic growth factors incorporated into melanocyte The initial proliferation increase stimulated by the intro- tissue culture media that cooperate with activated BRAF duction of hTERT prompted us to ask whether TERT may and CDK4 to overcome the antiproliferative effects of TGFβ. play a role in downregulating p15 expression in melano- These data are consistent with the observation that although cytes; however, hTERT expression did not inhibit p15 induc- normal interfollicular melanocytes proliferate under culture tion in BRAFV600E melanocytes (Supplementary Fig. S12B). conditions optimized to support normal melanocyte cell Therefore, nevus-MCs expressing the combination of hTERT, division, the mitotic rate of normal melanocytes in vivo is CDK4, and BRAFV600E still lack elements required to fully extremely low (27), and underscore the importance of assays overcome the senescence-like program in vivo and progress that mimic the in vivo setting. These data also suggest that to melanoma. To determine whether these growth-arrested additional genetic events promote melanoma cell-cycle pro- melanocytes (shown in Fig. 6C) also expressed other tradi- gression in vivo. As most melanomas express increased levels tional “senescence” markers in addition to absence of Ki67, of telomerase (28), and telomerase upregulation has been we next examined the tissues for melanocyte expression of shown to decrease the requirements for exogenous mitogens p16, which was highly expressed in all melanocytic nests, to maintain human cell growth in culture (29) in addition but largely absent in single nevus-derived melanocytes that to maintaining telomere length, we next transduced nevus- failed to proliferate, even transiently, in xenografts (Supple- MCs with both human telomerase reverse transcriptase mentary Fig. S13A). As p16 is generally expressed in response

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Nevus-MC NSi dnp53R248W hTERT

Nevus-MC p15i dnp53R248W hTERT

H&E Melan-A H&E Melan-A 60 days 100 days

Figure 7. p15 loss reverses nevus-MC growth arrest and promotes progression to melanoma in vivo. Hemotoxylin and eosin (H&E) staining with Melan- A immunofluorescence of nevus-MC expressing control shRNA (NSi), dnp53R248W, and hTERT (row 1), and nevus-MC expressing shRNA against p15 (p15i), dnp53R248W, and hTERT (row 2). Tissue from human skin xenografts was harvested at either 60 days (left) or 100 days (right) and is representative of 3 biologic replicates. Arrows indicate upward epidermal pagetoid scattering of neoplastic melanocytes. Scale bars, 100 μm.

to proliferation and mitogenic stimulation, the growth- p15 Loss Reverses Nevus-MC Growth Arrest arrested nevus cells that remain as single cells likely do not In Vivo and Promotes Progression to Melanoma reach the mitogenic threshold in vivo required to trigger To determine whether p15 loss functionally substitutes for p16 expression. The functional significance of these variably CDK4 activation in vivo to promote progression from benign positive senescence markers is somewhat unclear, as previous nevus to melanoma, nevus-MCs transduced with shRNA efforts have determined that there are currently no avail- targeting p15 (p15i), or control shRNA (NSi), dnp53, and able senescence markers that reliably distinguish between hTERT were seeded along with unremarkable keratinocytes normal melanocytes, nevi, and melanoma (30). To determine on native human dermis. Xenografts were harvested at 60 whether cell death was increased in the growth-arrested nests, and 100 days. At 60 days after grafting, nevus-MCs harboring we next analyzed the sections by terminal deoxynucleotidyl anti-p15 shRNAs had clearly overcome their growth-arrested transferase–mediated dUTP nick end labeling (TUNEL). As a state and proliferated to form nests of melanocytes at the positive control, we used skin that had been acutely exposed dermal–epidermal junction. Conversely, nevus-MCs harbor- to UVB radiation, which triggers an apoptotic response in the ing the control hairpin survived the grafting process, but epidermis. Although many apoptotic cells were seen in con- were present only as single cells at regular intervals along trols, there were very few, if any, apoptotic cells noted in the the basement membrane of the tissue and did not prolifer- xenografts (Supplementary Fig. S13B). ate into melanocytic nests (Fig. 7). At 100 days after graft- In addition to p15 induction, nevus-MCs displayed ing, nevus-MCs harboring control shRNAs maintained their increased levels of the p53-regulated CDK inhibitor p21 (Sup- growth-arrested state, whereas those with p15 knockdown plementary Fig. S14). Most melanomas lack functional p53 proliferated into large nests and displayed hallmark features as a result of either p14ARF loss or dominant-negative p53 of melanoma in situ, including large histologically atypi- mutation (31). To recapitulate this functional loss, we engi- cal melanocytes with high mitotic index and pagetoid cells neered xenografts with nevus-MCs expressing dominant-neg- migrating upward through epidermis (Fig. 7). R248W R24C ative p53 together with CDK4 and hTERT. Resulting To determine whether loss of p16 was also sufficient to tissues displayed many features consistent with melanoma reverse the BRAFV600E-induced growth arrest of nevus-MCs in including cytologically atypical, asymmetric melanocytic nests vivo, we repeated the experiment with nevus-MCs harboring associated with disorganized growth, high mitotic index, and shRNAs targeting p16. Consistent with our in vitro data, we pagetoid melanocyte migration upward through the epider- found that p16 knockdown nevus-MCs proliferated in the mis. The requirement for p53 antagonism was previously xenograft setting, but did so less robustly than melanocytes seen in a melanoma model driven by high-level expression of lacking p15 (Supplementary Fig. S15A). Loss of p16 led to HRAS (7) and may reflect an in vivo requirement for overcom- modest increases in melanocyte density that were intermedi- ing the antiproliferative effect of WAF/KIP CDK inhibitors, ate between the controls and that of the p15 knockdown tis- including p21, which remain active against the p15-resistant sues (Supplementary Fig. S15B). CDK4R24C mutant (15). Melanoma therefore can arise from precursor BRAFV600E nevi through acquisition of a minimum of three additional genetic events. This contrasts with skin DISCUSSION squamous cell carcinoma (SCC), which appears to require The initial identification of activatingBRAF V600E mutations only two mutations for malignant invasive growth (26, 32), in common acquired melanocytic nevi provided an expla- and supports the observation that the U.S. incidence of SCC nation for how nevi originate, as BRAF activation engages is approximately five times higher than that of melanoma (33). MAPK signaling to drive cell-cycle progression. However,

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CDKN2B Loss Drives Melanoma RESEARCH ARTICLE

identifying the mechanistic basis for the subsequent growth Although an exact mechanism underlying the reduction of arrest has remained elusive. A striking clinical feature is that p15 expression at the highest levels of BRAFV600E signaling most acquired nevi, independent of body site, sex, or age of remains elusive, increased MAPK flux and potential cross-talk onset, are similarly sized, suggesting that nevi engage a com- with the PI3K–AKT pathway may influence core cell-cycle mon feedback mechanism that ultimately restrains BRAF sig- machinery and lead to negative feedback on p15. This find- naling, and that this process is related to lesion size. Telomere ing provides direct evidence that the effects of oncogenes on erosion with replicative senescence could potentially limit human cells are highly dependent on their absolute expres- nevus size; however, telomere length in nevi is not shortened sion level, and underscores the importance of controlling (6). Our results identify a novel mechanism for nevus growth oncogene expression level. A similar observation was recently arrest in which BRAFV600E activation induces expression of made in mice, where relatively modest changes in KRAS secreted TGFβ via signaling through the canonical MEK/ translation, driven by alternative codon usage, altered its ERK MAPK cascade. TGFβ signaling, in turn, upregulates oncogenic potential (35). p15 and halts proliferation. The involvement of a secreted To our knowledge, this is the first research that formally molecule, TGFβ, may provide a mechanistic explanation for demonstrates a proliferation arrest of human melanocytes in why most human nevi fall within the narrow size range. response to BRAFV600E. Previous studies using viral-mediated Because TGFβ is secreted as an inactive precursor molecule BRAF expression in primary cells did not include melano- that requires proteolytic processing in the extracellular space cyte growth curves, and instead relied on senescence-asso- in order to activate the TGFβ receptor, the local tissue con- ciated β-galactosidase, Ki67 positivity, or decreased growth centration of TGFβ would need to be high enough to signal in another cell type (fibroblasts) as proliferation surrogates back through the TGFβ receptor to halt proliferation. Small (6). Indeed, our initial experiments with constitutive BRAF- lesions may not produce sufficient TGFβ to reach the thresh- expressing vectors failed to arrest growth, and it was for that old level required to counteract BRAF mitogenic activity. In reason that we developed the tunable doxycycline-inducible this model, TGFβ’s rate of secretion, diffusion, and half-life approach. in skin would together dictate the number and density of Our data from in vivo xenograft experiments demon- melanocytes necessary for growth arrest. It seems likely that strate that human nevus melanocytes are not irreversibly for human nevi, this critical mass often corresponds to a 3 growth arrested and can become malignant with the acqui- to 5 mm sphere, although the geometry of the nascent nevus sition of a minimal set of melanoma-associated secondary nest and the local ratio of receptor to ligand may contribute mutations, including CDKN2A/B loss (or CDK4 activation), to the variations in three-dimensional nevus architecture dominant-negative p53, and telomerase upregulation. This observed in human skin. malignant combination of genetic insults counteracts the Previous studies exploring the genesis of melanocytic nevi cell-cycle inhibition, resulting in conversion from benign and subsequent progression to melanoma have relied on nevus to melanoma. The melanoma-promoting effects of either murine models or viral transduction of human cells hTERT, CDK4, and dominant-negative p53 were shown in with activated BRAF. Although these efforts have yielded use- a prior xenograft model (7), but that study did not use ful insights, their medical relevance is questionable, as mice nevus melanocytes, used high-level oncodriver expression do not naturally form nevi, and most mouse skin contains using constitutive retroviral promoters, and did not examine melanocytes only within hair follicles. In contrast, human the role of p15. The functional consequence of CDKN2B loss skin contains melanocytes both within hair follicles and also in human tissue has not previously been examined directly, along the basal layer of the intervening epidermis, where they but our observation that CDKN2B was induced 138-fold in are exposed to higher levels of ultraviolet radiation within nevus versus normal melanocytes suggested that p15 is likely a three-dimensional environment architecturally distinct functionally important. Indeed, all of the benign nevi tested from the hair follicle. Using the naturally occurring human displayed high p15 expression, whereas malignant tissues were BRAF V600E nevus cells in the context of native human tissue frequently p15-negative, indicating that p15 expression may also avoids the potential of being misled by murine-specific be a useful marker to help distinguish benign versus malig- oncogene effects. This is an important point to consider nant melanocytic tumors. These clinical data are consist- when developing models for testing potential therapeutics, ent with our functional studies in vitro, as well as with our as murine and human cells can differ in their response to genetically defined human skin tissue in vivo, demonstrating oncogenes activating the MAPK pathway (34). clearly that p15 loss promotes progression from benign nevus In characterizing nevus-MCs and primary melanocytes melanocyte to melanoma. engineered to express inducible or constitutive BRAF V600E, In human cancer, nonsense and missense mutations are we noted that the proliferative capacity depends on the level more commonly observed in CDKN2A than in CDKN2B. This of BRAFV600E protein. Increasing BRAFV600E expression ini- likely reflects the fact that CDKN2A mutations frequently tially correlates with proportionally increased p15 expression. compromise function of the overlapping p14ARF gene (36), However, at the highest levels of BRAF expression (achieved decreasing p53 activity and conferring added proliferative with either diBRAF cells treated with high doxycycline doses, advantage to melanocytes harboring these mutations. Con- or cells harboring a constitutive BRAFV600E gene driven by a versely, CDKN2B mutations do not directly affect ARF. The standard PGK promoter), p15 protein levels were attenuated most recent TCGA melanoma data include 36 of 278 cases and melanocytes continued to proliferate. In this case, it (13%) in which CDKN2A contains nonsense, missense, or appears that supraphysiologic BRAF activity overcomes the frameshift mutations, most of which likely negatively affect TGFβ-mediated growth arrest observed at lower BRAF levels. p16 activity (1, 2). The vast majority of these cases (31 of 36; 86%)

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are in exons shared with ARF. Exon 1 from p16, which does as melanoma-initiating cells. However, mitotically active not have overlapping sequence with ARF, is mutated in melanocytes are seen very rarely in benign nevi in adults. only 5 of 278 cases. Remarkably, 3 (60%) of those cases with Furthermore, we have maintained primary nevus-derived CDKN2A exon 1 mutations also harbor inactivating TP53 melanocytes in culture for up to 2 months and have not mutations, which are otherwise rare in melanoma. (The other observed emergence of proliferative clones. two cases are tumors driven by RAS, not BRAF.) This indicates As melanocytic nevi are present on nearly all adults, future that p16 loss on its own is likely not sufficient to provide a melanoma studies will likely benefit from this new technical significant fitness advantage to human melanocytes in vivo, capacity to directly examine primary human nevus melano- and suggests that CDKN2A mutations may be more com- cytes, in order to gain insight into mechanisms promoting mon than CDKN2B mutations, at least in part because of the their malignant progression. The in vivo melanoma tissue associated ARF effects. The idea that p53/ARF effects may grafts developed here recapitulate progression from benign play a relatively larger role than p16 effects in melanoma is nevus to melanoma in an architecturally faithful tissue envi- consistent with a mouse melanoma model driven by HRAS ronment that provides a human alternative to murine or where tumors form more quickly on a germline ARF-null melanoma cell-line surrogates for mechanistic studies and background than on a p16-null background (37), and in the testing of potential therapeutics. murine melanoma models driven by BRAFV600E with intact p16, where ARF loss promotes melanoma formation without altering the initial growth-arrested senescent phenotype in METHODS vivo (38). Additional experimental details are included in the Supplementary Together, our in vitro and in vivo data indicate that although Methods. each of the proteins encoded by the CDKN2A–CDKN2B locus are important in melanomagenesis, p15 is an espe- Isolation of Melanocytes from Human Nevi cially important element and a critical mediator of BRAF Benign human melanocytic nevi were excised with informed con- growth arrest. Combined antagonism of p15, p16, and p14 sent from patient donors seen in the Dermatology clinic at the Hos- (via dnp53) promoted proliferation of diBRAFV600E melano- pital of the University of Pennsylvania (Philadelphia, PA) according cytes beyond that seen with the individual elements. In to an Institutional Review Board–approved protocol. Patient studies contrast, we have been unable to restore proliferation in pri- were conducted in accordance with the Declaration of Helsinki. mary BRAFV600E nevus melanocytes in vitro using individual A portion of each specimen was processed for routine histologic examination to confirm the clinical diagnosis of benign nevus. The or combinatorial loss of p15, p16, and dnp53. This likely tissue samples were microdissected to isolate nevus from surround- reflects technical differences encountered when moving ing normal tissue. Nevus tissue was then mechanically separated into nevus cells from the three-dimensional in vivo to the two- fine pieces, and enzymatically dissociated in a mixture of dispase and dimensional in vitro environment, and is consistent with collagenase for 2 hours. Melanocytes were further isolated from con- the results from the PLX4720 and doxycycline withdrawal taminating fibroblasts by selective trypsinization and 4 day exposure experiments where additional factors beyond BRAF and to 100 μg/mL G418. CDKN2A/B seem to maintain the previously established growth arrest in vitro. Quantification of Immunohistochemistry Staining Our data establishing that p15 is important for restrain- Tissue sections from human nevi, melanomas, and melanomas ing human BRAF oncogenic activity is consistent with the arising in continuity with existing nevi were stained for p15 or p16 existence of benign nevi in p16-null humans who retain p15 expression using methods described in the immunohistochemistry (39), indicating that melanocytes are capable of using addi- section. Photomicrograph images (×10) of representative tissue sec- tional mechanisms beyond p16 to restrain BRAF proliferative tions were taken using the Zeiss microscope. TIF files of the images were saved and transferred to Adobe Photoshop, where pixels cor- signals. Also suggestive of an important role for p15 is the responding to p15 or p16 IHC staining were selected using the color previous identification of inactivatingp15 mutations in spon- selection tool. Images corresponding to the single specific color were taneous melanomas in which p16 is preserved (40). then analyzed using FIJI (ImageJ) to determine the number of pixels V600E Although it is likely that most BRAF nevi undergo in each sample. The total number of pixels comprising the IHC stain proliferative arrest through a common pathway involving and the DAPI counterstain was also obtained so that the levels of p15 induction, there are likely multiple mechanisms through p15 and p16 expression could be normalized to the total amount which the growth arrest can be subsequently bypassed in of label in each section. Final ratios of p15 and p16 expression were conversion to melanoma. In addition to the p15 loss dem- calculated by dividing the number of p15 or p16 pixels by the total onstrated here in human tissue, recent murine models have pixels in each section. suggested that mTOR activation may play a role in some cases, as STK11 loss (which occurs in about 5% of human Generation of Lentiviral Vectors melanoma and leads to increased mTOR activity) promotes The following lentiviral plasmids were used to express the cor- R24C BRAF-driven mouse melanoma (41). PTEN loss or AKT3 responding human genes: diBRAF, p15, p16, CDK4 , dominant- R248W V600E activation, which both increase mTOR signaling, are also negative p53 , hTERT, and luciferase. The human BRAF gene was inserted immediately after the TetO operator in a modi- frequent events in nevus-associated human melanoma (42). fied version of the doxycycline-inducible lentiviral pTRIPZ vector Although our studies suggest that melanoma can develop (Thermo Scientific), in which the shRNA hairpin sequences were from growth-arrested precursor cells, we cannot formally deleted. Human p15 (Addgene 16454), CDK4R24C, p53R248W, and exclude the possibility that there are rare nevus-associated hTERT (7) were cloned into the pRRL lentivector (43). Human melanocytes that are not growth arrested and that serve p16 was expressed using Addgene plasmid #22263 (Eric Campeau).

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CDKN2B Loss Drives Melanoma RESEARCH ARTICLE

shRNAs were expressed from pLKO.1 and are available through supplement-free DMEM. The resulting mixture was incubated at OpenBiosystems. room temperature for 15 minutes and then added into the HEK293T culture media. At 16 hours after transfection, 10 mmol/L sodium qRT-PCR butyrate was added onto the cells. At 24 hours, the culture media mRNA was extracted from melanocytes according to the RNeasy was replaced and the cells were incubated at 32°C. At 45 hours, viral Mini Kit protocol (Qiagen), and reverse transcribed to cDNA using supernatant was collected by filtering the media through a 0.45-μm 6 the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). Quanti- syringe filter (Argos). Melanocytes seeded at 2.5 × 10 cells per well tative PCR of the resulting cDNA was carried out using Power SYBR were incubated in viral supernatant in the presence of 5 μg/mL poly- Green Master Mix (Applied Biosystems) and gene-specific primers, brene and centrifuged at 300 × g for 60 minutes at room temperature. in triplicate, on a ViiA 7 Real-Time PCR System (Life Technolo- After incubating the transduced melanocytes for 15 minutes at 37°C, gies). Relative expression was determined using the 2-[delta][delta] viral supernatant was removed and replaced with growth media.

Ct method followed by normalization to the wild-type melanocyte transcript levels. CRISPR-Cas9 Cloning and Transduction Guide RNAs were designed using software tools developed by the Preparation of 3-D Organotypic Skin Culture Zhang laboratory (45). Guide RNAs were subsequently cloned into Organotypic skin grafts containing melanocytes were established lentiCRISPRv2 (Addgene #52961). Guide RNA sequences are as follows: using modifications to previously detailed methods (7, 11). The lentiCRISPR GFP 5′-GAA GTT CGA GGG CGA CAC CC-3′; lenti Keratinocyte Growth Media (KGM) used for keratinocyte-only CRISPR p15.2 5′-CCC GAA ACG GTT GAC TCC GT-3′. LentiCRISPR skin grafts was replaced with Melanocyte Xenograft Seeding Media transductions in human melanocytes were conducted as previously (MXSM). MXSM is a 1:1 mixture of KGM and Keratinocyte Media described for the other lentiviral constructs used in this study. 50/50 (Gibco) containing 2% FBS, 1.2 mmol/L calcium chloride, 100 nmol/L Et-3 (endothelin 3), 10 ng/mL rhSCF (recombinant human Statistical Analysis stem cell factor), and 4.5 ng/mL r-basic FGF (recombinant basic Mean values were compared using an unpaired Student two-tailed fibroblast growth factor). Melanocytes (1.5 × 105) and keratinocytes t test. (5.0 × 105) were suspended in 80 μL MXSM, seeded onto the dermis, and incubated at 37°C for 4 days at the air–liquid interface. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Mouse Xenografting Organotypic skin tisssues were grafted onto 5- to 7-week-old female Authors’ Contributions ICR SCID mice (Taconic) according to an International Animal Care Conception and design: A.S. McNeal, M. Herlyn, J.T. Seykora, and Use Committee–approved protocol at the University of Pennsyl- T.W. Ridky vania. Mice were anesthetized in an isoflurane chamber. Murine skin Development of methodology: A.S. McNeal, M. Herlyn, T.W. Ridky was removed from the upper dorsal region of the mouse. Reconstituted Acquisition of data (provided animals, acquired and managed human skin was reduced to a uniform 11 mm × 11 mm square and patients, provided facilities, etc.): A.S. McNeal, K. Liu, V. Nakhate, grafted onto the back of the mouse with individual interrupted 6-0 nylon C.A. Natale, E.K. Duperret, B.C. Capell, S.L. Berger, J.T. Seykora, sutures. Mice were dressed with Bactroban ointment, Adaptic, Telfa pad, T.W. Ridky and Coban wrap. Dressings were removed 2 weeks after grafting. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A.S. McNeal, K. Liu, Chromatin Immunoprecipitation V. Nakhate, C.A. Natale, E.K. Duperret, B.C. Capell, J.T. Seykora, Followed by Quantitative PCR (ChIP-qPCR) T.W. Ridky Cells in 10-cm2 dishes were fixed in 1% formaldehyde for 5 minutes Writing, review, and/or revision of the manuscript: A.S. McNeal, and fixation was quenched with the addition of glycine to 125 mmol/L K. Liu, V. Nakhate, C.A. Natale, E.K. Duperret, B.C. Capell, T. Dentchev, for an additional 5 minutes. Cells were harvested by scraping from J.T. Seykora, T.W. Ridky plates, and washed twice in ×1 PBS before storage at −80°C. ChIP Administrative, technical, or material support (i.e., reporting was performed as previously described (44) except that extracts were or organizing data, constructing databases): A.S. McNeal, K. Liu, sonicated six times for 7.5 minutes each round (30 seconds sonica- T.W. Ridky tion with intermediate incubation of 30 seconds per round) using Study supervision: S.L. Berger, T.W. Ridky a Bioruptor (Diagenode). All ChIPs were performed using 150 μg of extract and 2 μg of antibody per sample. Protein G Dynabeads (30 μL; Acknowledgments Invitrogen 100.02D) were used per ChIP. Antibodies included anti- The authors thank the many patients who graciously donated histone H3 (Abcam ab1791), anti-histone H3 (trimethyl K9; Abcam tissue for this study; the University of Pennsylvania Skin Disease ab8898), anti-histone H3 (trimethyl K4; Abcam ab8580), and anti- Research Center for primary keratinocytes and immunohistochemis- histone H3 (trimethyl K27; Abcam ab6002). Following elution, ChIP try; and John Stanley, Sarah Millar, George Cotsarelis, and Alan Diehl DNA was analyzed by standard qPCR methods on a 7900HT Fast- for critical presubmission manuscript review. Real-Time PCR (ABI). Primer sequences are available upon request. Grant Support Viral Transfection and Transduction This work was supported by the NIH (R01CA163566 to T.W. Viral transductions were performed as described previously (7, Ridky; RO1CA165836 to J.T. Seykora and T.W. Ridky; F31CA186446 11, 26). HEK293T cells were cultured to 40% confluency and incu- to E.K. Duperret; CA076674 and CA182890 to M. Herlyn; and bated in DMEM supplemented with 5% FBS and 1% antibiotic and P01AG031862 to S.L. Berger), Melanoma Research Alliance (T.W. antimycotic in 6-well plates. For each well, 1.22 μg lentiviral vector Ridky and M. Herlyn), Dermatology Foundation (B.C. Capell), Amer- was mixed with viral packaging plasmids pCMVΔR8.91 (0.915 μg) ican Skin Association (B.C. Capell), Melanoma Research Foundation and pUC-MDG (0.305 μg). This plasmid solution and 7.2 μL of (B.C. Capell), and the University of Pennsylvania Undergraduate Fugene 6 Transfection Reagent (Promega) were added to 96 μL of Research Mentoring Program (K. Liu and V. Nakhate).

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The costs of publication of this article were defrayed in part by 19. Robinson WA, Elefanty AG, Hersey P. Expression of the tumour sup- the payment of page charges. This article must therefore be hereby pressor genes p15 and p16 in malignant melanoma. Melanoma Res marked advertisement in accordance with 18 U.S.C. Section 1734 1996;6:285–9. solely to indicate this fact. 20. Seoane J, Pouponnot C, Staller P, Schader M, Eilers M, Massague J. TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibi- Received February 12, 2015; revised July 8, 2015; accepted July 9, tor p15INK4b. Nat Cell Biol 2001;3:400–8. 21. Riesco-Eizaguirre G, Rodriguez I, De la Vieja A, Costamagna E, 2015; published OnlineFirst July 16, 2015. Carrasco N, Nistal M, et al. The BRAFV600E oncogene induces transforming growth factor beta secretion leading to sodium iodide symporter repression and increased malignancy in thyroid cancer. 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CDKN2B Loss Drives Melanoma RESEARCH ARTICLE

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CDKN2B Loss Promotes Progression from Benign Melanocytic Nevus to Melanoma

Andrew S. McNeal, Kevin Liu, Vihang Nakhate, et al.

Cancer Discovery 2015;5:1072-1085. Published OnlineFirst July 16, 2015.

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