Ingenol Mebutate: Potential for Further Development of Cancer Immunotherapy

Abstract

Ingenol mebutate is a diterpene ester derived from the plant Euphorbia peplus and is FDA approved for the topical treatment of actinic keratoses (AK). Shown to be efficacious with as little as a 3-day trial, this compound is being further tested for the topical treatment of other nonmelanoma skin cancers with promising preclinical data. In an effort to elucidate the molecular mechanism of this novel drug, Stahlhut et al.,(2012) suggest a role for calcium and apoptosis. Further studies are needed to evaluate the intracellular mechanisms of ingenol mebutate-mediated cytotoxicity. Additionally, studies such as this not only shed light on the mechanism of ingenol mebutate and its derivatives, but also pave the way for evaluating the involvement of the immune system in elim1nating drug-treated cells and tissues. This has important implications for the development of novel topical immune modulatory products and the field of topical immunotherapy.

INTRODUCTION

Ingenol mebutate (Picato®) is a recently FDA approved topical agent for the treatment of actinic keratosis with as little as a 2 or 3 day treatment. ^{1, 2} This drug has also been shown to be effective in treating superficial basal-cell cancer and is potentially effective in killing melanoma cells.^3-5 How it works is still speculative, but this natural product, derived from the sap of the tree Euphorbia peplus, has been used extensively for its medicinal properties.² 1n this issue of the Journal, Stahlhut et al. reveal for the first time evidence for the role of apoptosis and mitochondrial permeability as a possible mechanism of ingenol mebutate-mediated cytotoxicity. The authors demonstrate that ingenol mebutate elicits a strong and sustained increase in intracellular calcium that involves both ER-associated and mitochondrial-associated calcium stores. Interestingly, cancer cells take up ingenol mebutate and have a more robust ca lcium release promoting cell death upon treatment as compared to the same dose of ingenol mebutate in cultured donor keratinocytes. These findings are consistent with previous reports showing that ingenol mebutate specifically targets rapidly dividing cells in the basal cell layer (ie, dysplastic keratinocytes).

The findings in this study are the first to evaluate the intracellular mechanisms involved in how ingenol mebutate may promote dysplastic and neoplastic keratinocyte cell death and elimination while sparing normal keratinocytes. This study employed an in vitro method and utilized a model of reconstituted skin to demonstrate the localization of this novel compound. Future studies are needed to evaluate how this novel compound functions in vivo, especially considering how this compound implicates apoptosis and alternative death pathways in promoting cell death.

Ingenol mebutate treatment was shown to promote death of dysplastic and neoplastic skin cel ls and subsequently promoted keratinocyte proliferation in a mouse model of UVB-induced actinic keratosis and skin cancer.⁶ The authors of this earlier study suggest that early inflammation and neutrophil infiltration were the initiating events for regenerating keratinocytes at the basal layer. Additionally, Le et al. have further shown an immunostimulatory effect of ingenol mebu-tate resulting in an increase in anti-tumor CD8+ cells.⁷ Ingenol mebutate was also shown to differentially regulate apoptosis and TNF-α related apoptosis ligand (TRAIL) induced apoptosis in melanoma cancer cells.⁴ However, the apoptosis regulators are yet to be determined. Cozzi et al., showed that ingenol mebutate specifically targeted cells with mutant regulation of p53.⁶ Indeed, p53 expression and function are altered in cancer cells and thus ingenol mebutate may be acting on regulators of apoptosis. For instance, novel regu lators such as the anti-apoptotic protein Fortilin are known to regulate p53, yet little else is known of this protein in relation to how apoptosis is modulated based on Forti lin-mediated regulation of p53.⁸ B-cell lymphoma-extra large (Bcl-xl) has further been shown to differentially regulate mitochondrial and ER-calcium stores.⁹ Other studies suggest a role for apoptotic regulators including Bel proteins and the Protein Kinase C regulated M itogen Activated Protein Kinase pathways (PKC-MAPK) in ingenol mebutate-mediated cell death.^8-10 Additionally, the authors of the current study show a differential regulation of mitochondrial and ER stores of calcium. Apoptosis regulators in the Bcl-2 family, including Bcl-xL and Bax, are known to regulate calcium-mediated cell death yet the mechanisms are still to be worked out.¹¹ Based on these published findings and the known role of the immune system in tumor surveillance, one possible mechanism of ingenol mebutate is through antigen presentation of apoptotic keratinocytes.^7-12 The findings from this work strongly suggest such a mechanism.

Several lines of evidence suggest cross-presentation as a possible mechanism specifically targeting dying neoplastic cells. Similar mechanisms have been shown in an infectious model of Mycobacterium tuberculosis where dying infected macrophages enhance cross-presentation of antigenic apoptotic bodies to CD8+ cytotoxic T-cells.^13,14 In humans, BDCA3+ dendritic cells are considered to be responsible for cross-presentation to CD8+ T-cells.¹⁵ Stahlhut's present study suggests a role of both mitochondrial and ER-associated calcium efflux as a downstream effector of cytotoxicity. Thus, evaluating which proteins and pathways in neoplastic keratinocytes are aberrantly upregulated can shed light on the molecular targets of ingenol mebutate.

While this novel drug shows effective antitumor effects in previously published studies, the greatest limitation is that it is a derivative of a natural compound. By evaluating the mechanism of action of this drug, novel drugs can be developed synthetically which can concomitantly activate the immune system. For instance, current effort is underway to evaluate the effectiveness of the novel immunomodulatory compound diphenylcyclopropenone (DPCP).¹⁶ Other novel avenues of development are through the investigat ion of micro RNAs (miRNAs), short RNA oligonucleotides t hat can post-transcriptionally regulate genes and in turn are regulated by other miRNAs. Specifically, miR221/222 have been shown to be involved in regulating several pathways important in the oncogenic transformation of melanoma cells.¹⁷ With the probable immunomodulatory mechanism of ingenol mebutate and pre-clinical models showing an effect on melanoma cells, it stands to reason t hat miRNAs are involved. With this in mind, the manufacturer of Picatd®, LEO Pharmaceuticals, Exiqon, and the University of Copenhagen are currently developing a discovery platform, miRskin (www.mirskin.dk), which allows researchers to detect and classify specific chronic skin diseases by a novel technique to evaluate miRNAs in the skin. The findings from this platform will provide very useful information to further define and develop novel drug targets specific to dermatology.

The current study as well as previous studies have shed light on possible molecular targets but were limited by in vitro outputs; to more fully elucidate the mechanisms of immune activation future studies would require more robust in vivo models. Novel in vivo models on the role of immune surveillance are necessary to investigate the role of antigen presentation and cross-presentation at the dermal surface and which cell types are involved. The current study is the first step in elucidating how this novel compound can modulate dysplastic and neoplastic skin cells. Future studies can continue to delineate how this novel compound is immunostimulatory increasing CD8+ cells. Also, it is not known which cells are presenting antigen, the involvement and extent of cross-presentation, and how the dying keratinocytes are involved in immune modulation. Such studies will reveal not only the subtle activation and complexity of the anti-tumor effects of CD8+ cytotoxicT-cells, but also allow for discovery of novel interactions and targets for further drug development.