Blake Ferguson

Ingenol Mebutate Field-Directed Treatment of UVB-Damaged Skin Reduces Lesion Formation and Removes Mutant p53 Patches

Skin cancer is the most prevalent cancer worldwide and is primarily caused by chronic UV exposure. Here, we describe the topical field-directed treatment of SKH1/hr mice with UVB-damaged skin with ingenol mebutate, a new topical drug shown to be effective for the treatment of actinic keratosis (AK). Application of 0.05% ingenol mebutate gel to photo-damaged skin resulted in a ≈70% reduction in the number of skin lesions that subsequently emerged compared with placebo treatment. Ingenol mebutate treatment also reduced the number of mutant p53 keratinocyte patches by ≈70%. The treatment resulted in epidermal cell death, acute inflammation, recruitment of neutrophils, hemorrhage, and eschar formation, all of which resolved over several weeks. Ingenol mebutate field-directed treatment might thus find utility in the removal of subclinical precancerous cells from UV-damaged skin. Field-directed treatment may be particularly suitable for patients who have AKs surrounded by UV-damaged skin.

UVB-Induced Melanocyte Proliferation in Neonatal Mice Driven by CCR2-Independent Recruitment of Ly6clowMHCIIhi Macrophages

Intermittent sunburns, particularly in childhood, are the strongest environmental risk factor for malignant melanoma (MM). In mice, a single neonatal UVR exposure induces MM, whereas chronic doses to adult mice do not. Neonatal UVR alters melanocyte migration dynamics by inducing their movement upward out of hair follicles into the epidermis. UVR is known to induce inflammation and recruitment of macrophages into the skin. In this study, we have used a liposomal clodronate strategy to deplete macrophages at the time of neonatal UVR, and have shown functionally that this reduces the melanocyte proliferative response. This effect was not reproduced by depletion of CD11c-expressing populations of dendritic cells. On the basis of epidermal expression array data at various time points after UVR, we selected mouse strains defective in various aspects of macrophage recruitment, activation, and effector functions, and measured their melanocyte UVR response. We identified Ly6c(low)MHCII(hi) macrophages as the major population promoting the melanocyte response across multiple strains. The activity of this subpopulation was CCR2 (C-C chemokine receptor type 2) independent and partly IL-17 dependent. By helping induce this effect, the infiltration of specific macrophage subpopulations after sunburn may be a factor in increasing the risk of subsequent neoplastic transformation of melanocytes.

Melanoma susceptibility as a complex trait: genetic variation controls all stages of tumor progression

Susceptibility to most common cancers is likely to involve interaction between multiple low risk genetic variants. Although there has been great progress in identifying such variants, their effect on phenotype and the mechanisms by which they contribute to disease remain largely unknown. We have developed a mouse melanoma model harboring two mutant oncogenes implicated in human melanoma, CDK4R24C and NRASQ61K. In these mice, tumors arise from benign precursor lesions that are a recognized strong risk factor for this neoplasm in humans. To define molecular events involved in the pathway to melanoma, we have for the first time applied the Collaborative Cross (CC) to cancer research. The CC is a powerful resource designed to expedite discovery of genes for complex traits. We characterized melanoma genesis in more than 50 CC strains and observed tremendous variation in all traits, including nevus and melanoma age of onset and multiplicity, anatomical site predilection, time for conversion of nevi to melanoma and metastases. Intriguingly, neonatal ultraviolet radiation exposure exacerbated nevus and melanoma formation in most, but not all CC strain backgrounds, suggesting that genetic variation within the CC will help explain individual sensitivity to sun exposure, the major environmental skin carcinogen. As genetic variation brings about dramatic phenotypic diversity in a single mouse model, melanoma-related endophenotype comparisons provide us with information about mechanisms of carcinogenesis, such as whether melanoma incidence is dependent upon the density of pre-existing nevus cells. Mouse models have been used to examine the functional role of gene mutations in tumorigenesis. This work represents their next phase of development to study how biological variation greatly influences lesion onset and aggressiveness even in the setting of known somatic driver mutations.

A blueprint for staging of murine melanocytic lesions based on the Cdk4 R24C/R24C ::Tyr- NRAS Q 61K model

It has been shown that gene mutations which drive the development of malignant melanoma (MM) in humans also lead to emergence of MM when engineered mice. However, little attention has been paid to the clinical and histopathological features of melanocytic lesions and their natural history in a given mouse model. This knowledge is crucial to enable us to understand how engineered mutations influence the initiation and evolution of melanocytic lesions, and/or for the use of mice as a preclinical model to test specific treatments. We recently reported the development of melanocytic proliferations along the spectrum of naevi to MM in a Cdk4 R24C/R24C ::Tyr‐ NRAS Q 61K mouse model. In this study, we followed the development of lesions over time using digital photography and dermoscopy with the aim to correlate the clinical and histopathological features of lesions developing in this model. We identified two types of lesions. The first are slow‐growing dermal MMs that emanate from dermal naevi. The second did not emanate from naevi, grew rapidly, and appeared to be solely confined to the subcutaneous fat. We present a simple staging system for the MMs that progress from naevi, based on depth of extension into the dermis and subcutis. This represents a blueprint for documentation and follow‐up of MMs in the live animal, which is critical for the proper use of murine melanoma models.

Plasticity of melanoma in vivo: murine lesions resulting from Trp53, but not Cdk4 or Arf deregulation, display neural transdifferentiation

We previously noted that melanomas developing in Cdk4R24C/R24C::Tyr‐NRAS, Arf−/−::Tyr‐NRAS and Trp53F/F::Tyr‐Cre(ER)::Tyr‐NRAS mice exhibited differences in behaviour in vivo. We investigated this phenomenon using global gene expression profiling of lesions from the respective genotypes. While those from the Cdk4‐ and Arf‐mutant mice exhibited similar profiles, the Trp53F/F::Tyr‐Cre(ER)::Tyr‐NRAS melanomas were strikingly different, showing relative down‐regulation of melanocyte‐related genes, and up‐regulation of genes related to neural differentiation. Specifically, they highly expressed genes representative of the myelin‐producing peripheral oligodendrite (Schwann cell) lineage, although histopathologically the lesions did not exhibit the classical features of schwannoma. As Schwann cell precursors can be a cellular origin of melanocytes, it is unsurprising that plasticity with respect to melanocyte‐neural differentiation can occur in melanoma. What is surprising is the genotype proclivity. Comparison of gene expression signatures revealed that melanomas from the Trp53‐mutant mice show significant similarities with a subset of aggressive human melanomas with relatively low levels of MITF.

Murine melanomas accelerated by a single UVR exposure carry photoproduct footprints but lack UV signature C>T mutations in critical genes.

Ultraviolet radiation (UVR) exposure increases malignant melanoma (MM) risk, but in the context of acute, not cumulative exposure. C>T and CC>TT changes make up the overwhelming majority of single base substitutions (SBS) in MM DNA, as both precursor melanocytes and melanocytic lesions have incurred incidental exposures to sunlight. To study the mutagenic mechanisms by which acute sunburn accelerates MM, we sequenced the exomes of spontaneous and neonatal UVB-induced Cdk4-R24C::Tyr-NRASQ61K mouse MMs. UVR-induced MMs carried more SBSs than spontaneous MMs, but the levels of genomic instability, reflected by translocations and copy number changes, were not different. C>T/G>A was the most common SBS in spontaneous and UVR-induced MMs, only modestly increased in the latter. However, they tended to occur at the motif A/GpCpG (reflecting C>T transition due to spontaneous deamination of cytosine at CpG) in spontaneous MMs, and T/CpCpC/T (reflecting the effects of pyrimidine dimers on either side of the mutated C) in UVR-induced MMs. Unlike MMs associated with repetitive exposures, we observed no CC>TT changes. In addition, we also found UVR ‘footprints’ at T>A/A>Ts (at NpTpT) and T>C/A>G (at CpTpC). These footprints are also present in MMs from a chronic UVR mouse model, and in some human MMs, suggesting that they may be minor UVR signature changes. We found few significantly somatically mutated genes (~6 per spontaneous and 15 per UVR-induced melanoma) in addition to the Cdk4 and NRAS mutations already present. Trp53 was the most convincing recurrently mutated gene; however, in the UVR-induced MMs no Trp53 mutations were at C>T/G>A, suggesting that it was probably mutated during tumour progression, not directly induced by UVR photoproducts. The very low load of recurrent mutations convincingly induced by classical UVB-induced dimer photoproducts may support a role for cell extrinsic mechanisms, such as photoimmunosuppression and inflammation in driving MM after acute UVB exposure.

Superficial Spreading-Like Melanoma in Arf−/−::Tyr-NrasQ61K::K14-Kitl Mice: Keratinocyte Kit Ligand Expression Sufficient to “Translocate” Melanomas from Dermis to Epidermis

TO THE EDITOR Being amenable to genetic modification, mice are critical in vivo tools to test the role of gene mutations in malignant melanoma (MM) development. They are the animal model of choice for studying MM growth and treatments either as autochthonous tumors or as xenografts. However, adult mice do not have epidermal melanocytes (MCs), and primary murine MMs are usually dermal, reminiscent of human malignant blue nevi (see, e.g., Dhomen et al., 2009), animal type, or nodular MM. In contrast, most human MMs appear to originate in the epidermis, although they can progress to a vertical growth phase and invade the dermis. Although most murine MMs are dermal, lesions from the hepatocyte growth factor model often exhibit epidermal ‘‘pagetoid’’ spread on the albino (FVB) but not C57BL6 background (Noonan et al., 2001; Florell et al., 2007). Focal pagetoid spread is also seen in Pten::Tyr-CreERT2::LSL-Braf mice (Dankort et al., 2009). One factor that differentiates mouse from human skin is the expression of KIT receptor ligand (KITL), highly expressed in the human but not murine epidermis (Kunisada et al., 1998; Longley and Carter, 1999). It is expressed in 76% of human MMs, not only in adjacent keratinocytes, but also sometimes in the tumor cells (Giehl et al., 2007). Mice with engineered keratinocyte Kitl expression (K14-Kitl) have epidermal MCs throughout life (Kunisada et al., 1998). Remarkably, they do not develop MMs even after chronic UVR exposures (Yamazaki et al., 2004). When crossed onto a DNA repair defective (Xpa-null) background, they developed lentigo-like lesions (Yamazaki et al., 2005) after a harsh chronic UVR regimen. Although the physiological relevance of this study is questionable, as many animals died from severe sunburn, epidermal MMs did develop. Recently, K14-Kitl mice were crossed with animals carrying activation of the metabotropic glutamate receptor-1 (Abdel-Daim et al., 2010), but only dermal MMs were reported. We previously reported the development of dermal MMs in Arf / ::TyrAbbreviations: MC, melanocyte; MM, malignant melanoma

A mutation in the Cdon gene potentiates congenital nevus development mediated by NRASQ61K

Congenital nevi develop before birth and sometimes cover large areas of the body. They are presumed to arise from the acquisition of a gene mutation in an embryonic melanocyte that becomes trapped in the dermis during development. Mice bearing the Cdk4R24C::Tyr‐NRASQ61K transgenes develop congenital nevus‐like lesions by post‐natal day 10, from melanocytes escaping the confines of hair follicles. We interbred these mice with the collaborative cross (CC), a resource that enables identification of modifier genes for complex diseases (those where multiple genes are involved). We examined variation in nevus cell density in 66 CC strains and mapped a large‐effect quantitative trait locus (QTL) controlling nevus cell density to murine chromosome 9. The best candidate for a gene that exacerbates congenital nevus development in the context of an NRAS mutation is Cdon, a positive regulator of sonic hedgehog (Shh) that is expressed mainly in keratinocytes.

Clinicopathological Characterization of Mouse Models of Melanoma

Mouse models of melanoma have proven invaluable in the delineation of key molecular events involved in disease progression in humans and provide potential preclinical models for therapeutic testing (Damsky and Bosenberg, Pigment Cell Melanoma Res 25(4):404-405, 2012; Walker et al., Pigment Cell Melanoma Res 24(6):1158-1176, 2011). Here we concentrate on the clinicopathological analysis of melanocytic tumors.

Superficial spreading-like melanoma in Arf -/- ::Tyr-Nras Q61K ::K14-Kitl mice: Keratinocyte kit ligand expression sufficient to 'translocate' melanomas from dermis to epidermis

TO THE EDITOR Being amenable to genetic modification, mice are critical in vivo tools to test the role of gene mutations in malignant melanoma (MM) development. They are the animal model of choice for studying MM growth and treatments either as autochthonous tumors or as xenografts. However, adult mice do not have epidermal melanocytes (MCs), and primary murine MMs are usually dermal, reminiscent of human malignant blue nevi (see, e.g., Dhomen et al., 2009), animal type, or nodular MM. In contrast, most human MMs appear to originate in the epidermis, although they can progress to a vertical growth phase and invade the dermis. Although most murine MMs are dermal, lesions from the hepatocyte growth factor model often exhibit epidermal ‘‘pagetoid’’ spread on the albino (FVB) but not C57BL6 background (Noonan et al., 2001; Florell et al., 2007). Focal pagetoid spread is also seen in Pten::Tyr-CreERT2::LSL-Braf mice (Dankort et al., 2009). One factor that differentiates mouse from human skin is the expression of KIT receptor ligand (KITL), highly expressed in the human but not murine epidermis (Kunisada et al., 1998; Longley and Carter, 1999). It is expressed in 76% of human MMs, not only in adjacent keratinocytes, but also sometimes in the tumor cells (Giehl et al., 2007). Mice with engineered keratinocyte Kitl expression (K14-Kitl) have epidermal MCs throughout life (Kunisada et al., 1998). Remarkably, they do not develop MMs even after chronic UVR exposures (Yamazaki et al., 2004). When crossed onto a DNA repair defective (Xpa-null) background, they developed lentigo-like lesions (Yamazaki et al., 2005) after a harsh chronic UVR regimen. Although the physiological relevance of this study is questionable, as many animals died from severe sunburn, epidermal MMs did develop. Recently, K14-Kitl mice were crossed with animals carrying activation of the metabotropic glutamate receptor-1 (Abdel-Daim et al., 2010), but only dermal MMs were reported. We previously reported the development of dermal MMs in Arf / ::TyrAbbreviations: MC, melanocyte; MM, malignant melanoma