Squamous cell carcinoma in situ (SCCIS) is a prevalent precancerous lesion that can progress to cutaneous squamous cell carcinoma. Although SCCIS is common, its pathogenesis remains poorly understood. To better understand SCCIS development, we performed laser captured microdissection of human SCCIS and the adjacent epidermis to isolate genomic DNA and RNA for next-generation sequencing. Whole-exome sequencing identified UV-signature mutations in multiple genes, including NOTCH1e3 in the epidermis and SCCIS and oncogenic TP53 mutations in SCCIS. Gene families, including SLFN genes, contained UV/oxidativesignature disruptive epidermal mutations that manifested positive selection in SCCIS. The frequency and distribution of NOTCH and TP53 mutations indicate that NOTCH mutations may precede TP53 mutations. RNA sequencing identified 1,166 differentially expressed genes; the top five enriched gene ontology biological processes included (i) immune response, (ii) epidermal development, (iii) protein phosphorylation, (iv) regulation of catalytic activity, and (v) cytoskeletal regulation. The NEURL1 ubiquitin ligase, which targets Notch ligands for degradation, was upregulated in SCCIS. NEURL1 protein was found to be elevated in SCCIS suggesting that increased levels could represent a mechanism for downregulating Notch during UV-induced carcinogenesis. The data from DNA and RNA sequencing of epidermis and SCCIS provide insights regarding SCCIS formation.

Most melanomas are driven by BRAF(V600E)-activating mutations, while nevi harboring the same mutations have growth arrest. Although decreased p16 expression has been associated with melanoma formation, in recent work, p15 represented a primary effector of oncogene-induced senescence in nevomelanocytes that was diminished in melanomas. This study determined whether decreased p15 levels represent a general biomarker for the transition from nevus to melanoma. We performed p15 and p16 IHC analyses on a random series of nevi and melanomas. Staining was evaluated and graded for percentage and intensity to determine the H score. For real-time quantitative RT-PCR analysis of p15, RNA was extracted from FFPE sections from 14 nevus and melanoma samples via macrodissection. A two-sided t-test was used to evaluate between-group differences in mean H scores and qDCt values. p15 Expression was significantly increased in melanocytic nevi compared with melanomas (mean H scores, 254.8 versus 132.3; P < 0.001). On p15 staining, the H score differential was greater than that with p16 staining [122.5 (P < 0.001) and 64.8 (P Z 0.055), respectively]. Real-time quantitative RT-PCR analysis revealed a lower mean qDCt value in melanomas, consistent with lower p15 expression (P Z 0.018). Together, these data support the hypothesis that decreased p15 expression is a robust biomarker for distinguishing nevus from melanoma. (Am J Pathol 2016, 186: 3094e3099; http:// dx

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer and is associated with cumulative UV exposure. Studies have shown that prolonged voriconazole use promotes cSCC formation; however, the biological mechanisms responsible for the increased incidence remain unclear. Here, we show that voriconazole directly increases oxidative stress in human keratinocytes and promotes UV‐induced DNA damage as determined by comet assay, 8‐oxoguanine immunofluorescence and mass spectrometry. Voriconazole treatment of human keratinocytes potentiates UV‐induced apoptosis and activation of the p38 MAP kinase and 53BP1 UV stress response pathways. The p38 MAP kinase activation promoted by voriconazole exposure can be mitigated by pretreating keratinocytes with N‐acetylcysteine. Voriconazole increases oxidative stress in keratinocytes by directly inhibiting catalase leading to lower intracellular NADPH levels and the triazole moieties in voriconazole are critical for inhibiting catalase. Furthermore, voriconazole is shown to promote UV‐induced dysplasia in an in vivo model. Together, these data demonstrate that voriconazole potentiates oxidative stress in UV‐irradiated keratinocytes through catalase inhibition. Use of antioxidants may mitigate the pro‐oncogenic effects of voriconazole.

Basal cell carcinomas (BCCs) are more prevalent than any other cancer and are driven by mutations in PTCH1 or SMO causing aberrant activation of the hedgehog (HH) signaling pathway. The SMO inhibitor, Vismodegib recently received FDA approval for the treatment of advanced BCCs. Unfortunately, most BCCs become resistant and only 50% of patient resistant tumors contained additional classical pathway mutations. This suggested an alternative mechanism accounts for most resistant growth, thus, we searched for novel non-classical resistance pathways. We used multidimensional genomics in drug-resistant BCCs to identify a noncanonical hedgehog activation pathway driven by the transcription factors serum response factor (SRF) and mycardin-related transcription factor (MRTF/MKL1). Further studies indicated SRF-MRTF share chromosomal occupancy and form a novel protein complex with the HH transcriptional activator, GLI1. Cytoskeletal activation by Rho and mDia are required for MRTF-driven HH activation and resistant BCC growth. Remarkably, small molecule inhibition of MRTF suppressed tumor growth in a mouse model of resistant BCC. However, efforts to further test MRTF inhibitors in human tumors are hampered by an inability to produce viable patient-derived xenografts (PDXs) for BCCs. We overcame this obstacle by developing ex vivo culture conditions using freshly resected BCCs from Mohs patients. In addition, we found that nuclear (active) MKL1 staining serves as a biomarker to predict efficacy for MKL inhibitors. Indeed, tumors containing nuclear MRTF produced a robust response to MRTF inhibitors by displaying a significant reduction of GLI1 mRNA. Thus, our work highlights the therapeutic potential of MRTF inhibitors in BCCs and establishes a new human tumor model for preclinical testing of promising drug candidates. 211 Co-Inheritance of mutations in CDKN2A and MC1R increases melanoma predisposition independently of deregulation of cell cycle and UV response in melanocytes

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