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Purpose:G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether LC3B, a G9a target gene, can predict treatment response.Experimental Design:Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model.Results:Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of MAP1LC3B or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death.Conclusions:Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.

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Purpose:G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether LC3B, a G9a target gene, can predict treatment response.Experimental Design:Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model.Results:Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of MAP1LC3B or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death.Conclusions:Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.

The long-standing perception of Protein Kinase C (PKC) as a family of oncoproteins has increasingly been challenged by evidence that some PKC isoforms may act as tumor suppressors. To explore the hypothesis that activation, rather than inhibition, of these isoforms is critical for anticancer activity, we isolated and characterized a family of 16 novel phorboids closely-related to tigilanol tiglate (EBC-46), a PKC-activating epoxytigliane showing promising clinical safety and efficacy for intratumoral treatment of cancers. While alkyl branching features of the C12-ester influenced potency, the 6,7-epoxide structural motif and position was critical to PKC activation in vitro. A subset of the 6,7-epoxytiglianes were efficacious against established tumors in mice; which generally correlated with in vitro activation of PKC. Importantly, epoxytiglianes without evidence of PKC activation showed limited antitumor efficacy. Taken together, these findings provide a strong rationale to reassess the role of PKC isoforms in cancer, and suggest in some situations their activation can be a promising strategy for anticancer drug discovery.

Melanocytes can group together in nevi, commonly thought to form because of intrinsic somatic mutations involving MAPK pathway activation. However, the role of the microenvironment, in particular keratinocytes, in nevogenesis is rarely studied. Melanocytes proliferate during the hair follicle growth phase and in some basal cell carcinomas, allowing us to construct keratinocyte gene expression clusters correlated with melanocyte activation. We asked whether such correlations are evident in the more subtle context of regulation of melanocyte behavior in normal skin. We considered genes which, when mutated in keratinocytes in mice, lead to nevogenesis. Across the human GTEx normal skin database, their expression was correlated with that of keratinocyte cytokines KITLG, HGF, FGF2, EDN1, and melanocyte markers. These cytokines have pleiotropic effects on melanocyte-specific and pigmentation genes and also influence mast cell gene expression. We show five classes of keratinocyte genes that, via germline genetic variation, influence melanocyte activity. These include genes involved in SHH signaling, structural keratins, ribosomal biogenesis, and stem cell governance. In agreement with the finding of KITLG linked to nevogenesis in human genome-wide association studies, we provide evidence that specific keratinocyte cytokines are components of networks that may drive or exacerbate nevus development.

Genetic variation conferring resistance and susceptibility to carcinogen-induced tumorigenesis is frequently studied in mice. We have now turned this idea to melanoma using the collaborative cross (CC), a resource of mouse strains designed to discover genes for complex diseases. We studied melanoma-prone transgenic progeny across seventy CC genetic backgrounds. We mapped a strong quantitative trait locus for rapid onset spontaneous melanoma onset to Prkdc, a gene involved in detection and repair of DNA damage. In contrast, rapid onset UVR-induced melanoma was linked to the ribosomal subunit gene Rrp15. Ribosome biogenesis was upregulated in skin shortly after UVR exposure. Mechanistically, variation in the ‘usual suspects’ by which UVR may exacerbate melanoma, defective DNA repair, melanocyte proliferation, or inflammatory cell infiltration, did not explain melanoma susceptibility or resistance across the CC. Instead, events occurring soon after exposure, such as dysregulation of ribosome function, which alters many aspects of cellular metabolism, may be important., eLife digest Melanoma is a type of skin cancer. Melanoma tumors form in the skin’s pigment-producing cells or melanocytes. Growing evidence points to complex interactions between genetics and environmental exposures that contribute to the risk of developing melanoma. Ultraviolet (UV) radiation from the sun causes genetic mutations in melanocytes. This sun exposure interacts with genetic variations that may make people more or less vulnerable to such DNA damage. For example, genetic variations that control skin color or the cell’s ability to repair DNA, and that influence how easily people develop sunburn, all affect whether UV damage leads to melanoma. However, some forms of melanoma are not caused by sun exposure at all. Most of the genetic variations linked to melanoma have a small effect on the risk of developing the disease. So, it is unlikely that these genes alone cause melanoma. Few studies have been able to map the complex interactions between genes and the environment that lead to melanoma. So far, it has been unclear if there are different genetic mechanisms that lead to an increased risk for sun-exposure linked melanoma and non-sun linked melanoma. Now, Ferguson et al. show that variations in the genes involved in DNA repair during normal cell growth are linked to non-sun linked melanoma. Sun-linked melanoma, on the other hand, was associated with genes involved in the production of proteins in part of the cell called ribosomes. In the experiments, the effects of both UV light and various genetic variations were assessed across many different strains of mice. Mutations that impair the cell’s ability to repair UV-induced DNA damage or that contribute to excessive inflammation in response to sunburn did not increase melanoma susceptibility in these experiments. Ferguson et al. show that the amount of UV-induced DNA damage alone does not explain melanoma risk, which may not always depend on skin pigmentation. The experiments also suggest that non-UV linked melanoma is caused by a different mechanism than sun exposure-linked melanoma. Learning more about different genetic factors that affect the risk of developing different types of melanoma may help scientists develop more specific treatments.

Ultraviolet radiation (UVR) exposure increases malignant melanoma (MM) risk, but in the context of acute, not cumulative exposure. CT and CCTT 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. CT/GA 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 CT 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 CCTT changes. In addition, we also found UVR 'footprints' at TA/ATs (at NpTpT) and TC/AG (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 CT/GA, 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.

Giant congenital nevi are associated with clinical complications such as neurocutaneous melanosis and melanoma. Virtually nothing is known about why some individuals develop these lesions. We previously identified the sonic hedgehog (Shh) pathway regulator Cdon as a candidate nevus modifier gene. Here we validate this by studying Cdon knockout mice, and go on to establishing the mechanism by which Shh exacerbates nevogenesis. Cdon knockout mice develop blue nevi without the need for somatic melanocyte oncogenic mutation. In a mouse model carrying melanocyte NRASQ61K, we found that strain backgrounds that carry genetic variants that cause increased keratinocyte Shh pathway activity, as measured by Gli1 and Gli2 expression, develop giant congenital nevi. Shh components are also active adjacent to human congenital nevi. Mechanistically, this exacerbation of nevogenesis is driven via the release of the melanocyte mitogen endothelin-1 from keratinocytes. We then suppressed nevus development in mice using Shh and endothelin antagonists. Our work suggests an aspect of nevus development whereby keratinocyte cytokines such as endothelin-1 can exacerbate nevogenesis, and provides potential therapeutic approaches for giant congenital nevi. Furthermore, it highlights the notion that germline genetic variation, in addition to somatic melanocyte mutation, can strongly influence the histopathological features of melanocytic nevi.

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, CDK4(R24C) and NRAS(Q61K). 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.

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.

We previously noted that melanomas developing in Cdk4 R24C/R24C ::Tyr-NRAS , Arf −/− ::Tyr-NRAS and Trp53 F/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 Trp53 F/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.

Terahertz (THz) quantum cascade lasers (QCLs) are compact sources of radiation in the 1–5 THz range with significant potential for applications in sensing and imaging. Laser feedback interferometry (LFI) with THz QCLs is a technique utilizing the sensitivity of the QCL to the radiation reflected back into the laser cavity from an external target. We will discuss modelling techniques and explore the applications of LFI in biological tissue imaging and will show that the confocal nature of the QCL in LFI systems, with their innate capacity for depth sectioning, makes them suitable for skin diagnostics with the well-known advantages of more conventional confocal microscopes. A demonstration of discrimination of neoplasia from healthy tissue using a THz, LFI-based system in the context of melanoma is presented using a transgenic mouse model. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.

Deregulation of p16INK4A is a critical event in melanoma susceptibility and progression. It is generally assumed that the major effect of loss of p16 function is mediated through the CDK-cyclin pathway via its influence on the pocket protein (PP) pRb. However, there are also two other PPs, p107 and p130, which, when phosphorylated by CDK-cyclin complexes, play a role in permitting cell progression. Cohorts of mice carrying melanocyte-specific knockouts (KOs) of various combinations of the three PPs were generated. Mice null for pRb, p107, p130 or any combination of double mutants did not develop melanoma. Surprisingly, melanocyte-specific loss of all three PPs facilitated melanoma development (median age of onset 308 days, penetrance 40% at 1 year). Tumorigenesis was exacerbated by Trp53 co-deletion (median age of onset 275 days, penetrance 82% at 1 year), with cell culture studies indicating that this difference may result from the apoptotic role of Trp53. Melanomas in PP;Trp53-deficient mice lacked either Ras or Braf mutations, and hence developed in the absence of constitutive MAPK pathway activation. The lag period between induction of total PP or PP/Trp53 KO and melanoma development indicates that additional genetic or epigenetic alterations may account for neoplastic progression. However, exome sequencing of PP;Trp53 KO melanomas failed to reveal any additional recurrent driver mutations. Analysis of the putative mutation signature of the PP;Trp53 KO melanomas suggests that melanocytes are primed for transformation via a mutagenic mechanism involving an excess of T>G substitutions, but not involving a preponderance of C>T substitutions at CpG sites, which is the case for most spontaneous cancers not driven by a specific carcinogen. In sum, deregulation of all three PPs appears central to neoplastic progression for melanoma, and the customary reference to the p16INKA/CDK4/pRB pathway may no longer be accurate; all PPs are potentially critical targets of CDK-cyclins in melanoma.

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 Cdk4(R24C) ::Tyr-NRAS(Q) (61K) 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.

We report on a systematic analysis of genotype-specific melanocyte (MC) UVR responses in transgenic mouse melanoma models along with tumour penetrance and comparative histopathology. pRb or p53 pathway mutations cooperated with Nras(Q61K) to transform MCs. We previously reported that MCs migrate from the follicular outer root sheath into the epidermis after neonatal UVR. Here, we found that Arf or p53 loss markedly diminished this response. Despite this, mice carrying these mutations developed melanoma with very early age of onset after neonatal UVR. Cdk4(R24C) did not affect the MC migration. Instead, independent of UVR exposure, interfollicular dermal MCs were more prevalent in Cdk4(R24C) mice. Subsequently, in adulthood, these mutants developed dermal MC proliferations reminiscent of superficial congenital naevi. Two types of melanoma were observed in this model. The location and growth pattern of the first was consistent with derivation from the naevi, while the second appeared to be of deep dermal origin. In animals carrying the Arf or p53 defects, no naevi were detected, with all tumours ostensibly skipping the benign precursor stage in progression.

Melanocyte stem cells (MCSCs) in the upper portion of the hair follicle periodically supply melanocytes (MCs) that migrate downward into the hair bulb during anagen, the growth phase of the hair cycle. However MCs can also migrate upwards. We previously observed an increase in epidermal MC density in the mouse epidermis after a single ultraviolet radiation (UVR) exposure in neonatal, but not adult mice. To better understand MCSC activation by UVR we methodically studied the response of MCs to narrow band UVB (since UVA does not invoke this response) exposure in neonatal mice, and in adults at different stages of the hair cycle. We found that a single exposure of adult mice did not induce activation of MCSCs, in any stage of the hair cycle. When adult mice MCSCs were isolated in telogen, multiple UVB exposures resulted in their activation and production of daughter cells, which migrated upwards to the epidermis. Importantly, the MCSCs produced new progeny without themselves having incurred DNA damage after UVB exposure. This, together with examination of MC localisation in the skin of mice overexpressing stem cell factor in their keratinocytes, leads us to conclude that MCSC activation by UVB is driven via paracrine production of either SCF and/or other keratinocyte cytokines. We re-examined the increase in epidermal MC density in neonatal mouse skin. This effect was much more profound after only a single exposure than that of even multiple exposures to adult skin, and we show that in this setting also, the epidermal MCs mostly derive from activation of MC precursors in the upper hair follicle, and most likely via a cell extrinsic mechanism. Hence, although adaptive changes in the skin induced by repetitive UVB exposures are necessary in adult mice, in both the adult and neonatal context the division and migration upwards of follicular MCSCs is the major mode by which epidermal MC numbers increase after UVR exposure.

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.

This manuscript focuses on the use of mice to study the genetics and biology of cutaneous squamous cell carcinoma (SCC). Mice develop actinic keratosis-like lesions and SCC resembling those seen in humans. As an animal model, the mouse provides great experimental flexibility and has been useful in investigating aspects of the genetics and biology of SCC that are difficult to study in humans. We discuss the pros and cons of the various murine models available. How well mouse pathology in general mimics human disease remains an open question due to the vast differences in animal strain backgrounds and the fact that only one strain is typically tested in any particular experiment. Nonetheless, the murine epidermis is thinner than the human epidermis, and this must be kept in mind when making inferences from mechanistic data obtained with mice. We outline new strategies for non-biased screens to discover genes driving SCC progression. Such work has revealed a very complex interactive molecular network, and as with other complex diseases, the picture is being pieced together using systems biology strategies to which mouse tumour models are amenable. Such approaches do not focus on single genes or proteins but try to integrate the complex interactions of many types of genetic and biological information.

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. Here we concentrate on the clinicopathological analysis of melanocytic tumors.

This manuscript focuses on the use of mice to study the genetics and biology of cutaneous squamous cell carcinoma (SCC). Mice develop actinic keratosis-like lesions and SCC resembling those seen in humans. As an animal model, the mouse provides great experimental flexibility and has been useful in investigating aspects of the genetics and biology of SCC that are difficult to study in humans. We discuss the pros and cons of the various murine models available. How well mouse pathology in general mimics human disease remains an open question due to the vast differences in animal strain backgrounds and the fact that only one strain is typically tested in any particular experiment. Nonetheless, the murine epidermis is thinner than the human epidermis, and this must be kept in mind when making inferences from mechanistic data obtained with mice. We outline new strategies for non-biased screens to discover genes driving SCC progression. Such work has revealed a very complex interactive molecular network, and as with other complex diseases, the picture is being pieced together using systems biology strategies to which mouse tumour models are amenable. Such approaches do not focus on single genes or proteins but try to integrate the complex interactions of many types of genetic and biological information.

In this paper, we introduce the self-mixing phenomenon in terahertz quantum cascade lasers (THz QCLs) and present recent advancements in the development of coherent THz imaging and sensing systems that exploit the self-mixing effect. We describe an imaging method which utilises the interferometric nature of optical feedback in a THz QCL to employ it as a homodyning transceiver. This results in a highly sensitive and compact scheme. Due to the inherently low penetration depth of THz radiation in hydrated biological tissue, imaging of superficial skin is an ideal application for this technique. We present results for imaging of excised skin tissue, showing high-contrast between different tissue types and pathologies.

Refereed/Peer-reviewed

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.

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.