Greater mechanistic understanding of the cutaneous pathogenesis of Stevens-Johnson syndrome/toxic epidermal necrolysis can shed light on novel therapeutic strategies: a comprehensive review.

PURPOSE OF REVIEW: Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) are severe cutaneous adverse drug reactions (SCARs) characterized by widespread epithelial detachment and blistering, which affects the skin and mucocutaneous membranes. To date, therapeutic interventions for SJS/TEN have focused on systematic suppression of the inflammatory response using high-dose corticosteroids or intravenous immunoglobulin G (IgG), for example. No targeted therapies for SJS/TEN currently exist. RECENT FINDINGS: Though our understanding of the pathogenesis of SJS/TEN has advanced from both an immunological and dermatological perspective, this knowledge is yet to translate into the development of new targeted therapies. SUMMARY: Greater mechanistic insight into SJS/TEN would potentially unlock new opportunities for identifying or repurposing targeted therapies to limit or even prevent epidermal injury and blistering.

TNF-α induced extracellular release of keratinocyte high-mobility group box 1 in Stevens-Johnson syndrome/toxic epidermal necrolysis: Biomarker and putative mechanism of pathogenesis.

Decreased epidermal high-mobility group box 1 (HMGB1) expression is an early marker of epidermal injury in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Etanercept, an anti-tumor necrosis factor therapeutic, is effective in the treatment of SJS/TEN. The objective was to characterize antitumor necrosis factor-alpha (TNF-α)-mediated HMGB1 keratinocyte/epidermal release and etanercept modulation. HMGB1 release from TNF-α treated (± etanercept), or doxycycline-inducible RIPK3 or Bak-expressing human keratinocyte cells (HaCaTs) was determined by western blot/ELISA. Healthy skin explants were treated with TNF-α or serum (1:10 dilution) from immune checkpoint inhibitor-tolerant, lichenoid dermatitis or SJS/TEN patients ± etanercept. Histological and immunohistochemical analysis of HMGB1 was undertaken. TNF-α induced HMGB1 release in vitro via both necroptosis and apoptosis. Exposure of skin explants to TNF-α or SJS/TEN serum resulted in significant epidermal toxicity/detachment with substantial HMGB1 release which was attenuated by etanercept. Whole-slide image analysis of biopsies demonstrated significantly lower epidermal HMGB1 in pre-blistered SJS/TEN versus control (P < 0.05). Keratinocyte HMGB1 release, predominantly caused by necroptosis, can be attenuated by etanercept. Although TNF-α is a key mediator of epidermal HMGB1 release, other cytokines/cytotoxic proteins also contribute. Skin explant models represent a potential model of SJS/TEN that could be utilized for further mechanistic studies and targeted therapy screening.

The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury.

Background: Evidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here, we describe the generation of HepG2-derived transmitochondrial cybrids, to investigate the impact of mtDNA variation on mitochondrial (dys)function and susceptibility to iDILI. This study created 10 cybrid cell lines, each containing distinct mitochondrial genotypes of haplogroup H or haplogroup J backgrounds. Methods: HepG2 cells were depleted of mtDNA to make rho zero cells, before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide, and tolcapone, and their less toxic counterparts bicalutamide and entacapone utilizing ATP assays and extracellular flux analysis. Results: Whilst only slight variations in basal mitochondrial function were observed between haplogroups H and J, haplogroup-specific responses were observed to the mitotoxic drugs. Haplogroup J showed increased susceptibility to inhibition by flutamide, 2-hydroxyflutamide, and tolcapone, via effects on selected mitochondrial complexes (I and II), and an uncoupling of the respiratory chain. Conclusions: This study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest. This provides a practical and reproducible system to investigate the cellular consequences of variation in the mitochondrial genome, against a constant nuclear background. Additionally, the results show that inter-individual variation in mitochondrial haplogroup may be a factor in determining sensitivity to mitochondrial toxicants. Funding: This work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).

TNF-α‒Mediated Keratinocyte Expression and Release of Matrix Metalloproteinase 9: Putative Mechanism of Pathogenesis in Stevens‒Johnson Syndrome/Toxic Epidermal Necrolysis.

Stevens‒Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are severe cutaneous adverse drug reactions characterized by widespread keratinocyte cell death and epidermal detachment. At present, there is little understanding of how the detachment occurs or how it is abrogated by the TNF-α inhibitor etanercept, an effective SJS/TEN treatment. RNA sequencing was used to identify upregulated transcripts in formalin-fixed paraffin-embedded SJS/TEN skin biopsies. Epidermal matrix metalloproteinase 9 (MMP9) expression was assessed by immunohistochemistry in skin biopsies and cultured human skin explants exposed to serum from patients with cutaneous adverse drug reactions. TNF-α‒induced MMP9 expression and activity and its abrogation by etanercept were determined using the HaCaT immortalized keratinocyte cell line. Epidermal MMP9 expression was significantly higher in SJS/TEN skin (70.6%) than in healthy control skin (0%) (P = 0.0098) and nonbullous skin reactions (10.7%) (P = 0.0002). SJS/TEN serum induced significant MMP9 expression and collagenase activity in healthy skin explants, which was reduced by etanercept. Etanercept was also able to negate the TNF-α‒induced MMP9 expression in the HaCaT cell line. Data suggest that elevated epidermal MMP9 expression and collagenase activity are a putative pathogenic mechanism in SJS/TEN, which is limited by etanercept. Modulation of MMP9 expression and activity represents, to our knowledge, a previously unreported therapeutic target for the treatment of SJS/TEN.

Checkpoint Inhibition Reduces the Threshold for Drug-Specific T-Cell Priming and Increases the Incidence of Sulfasalazine Hypersensitivity.

An emerging clinical issue associated with immune-oncology agents is the collateral effects on the tolerability of concomitant medications. One report of this phenomenon was the increased incidence of hypersensitivity reactions observed in patients receiving concurrent immune checkpoint inhibitors (ICIs) and sulfasalazine (SLZ). Thus, the aim of this study was to characterize the T cells involved in the pathogenesis of such reactions, and recapitulate the effects of inhibitory checkpoint blockade on de-novo priming responses to compounds within in vitro platforms. A regulatory competent human dendritic cell/T-cell coculture assay was used to model the effects of ICIs on de novo nitroso sulfamethoxazole- and sulfapyridine (SP) (the sulfonamide component of SLZ) hydroxylamine-specific priming responses. The role of T cells in the pathogenesis of the observed reactions was explored in 3 patients through phenotypic characterization of SP/sulfapyridine hydroxylamine (SPHA)-responsive T-cell clones (TCC), and assessment of cross-reactivity and pathways of T-cell activation. Augmentation of the frequency of responding drug-specific T cells and intensity of the T-cell response was observed with PD-1/PD-L1 blockade. Monoclonal populations of SP- and SPHA-responsive T cells were isolated from all 3 patients. A core secretory effector molecule profile (IFN-γ, IL-13, granzyme B, and perforin) was identified for SP and SPHA-responsive TCC, which proceeded through Pi and hapten mechanisms, respectively. Data presented herein provides evidence that drug-responsive T cells are effectors of hypersensitivity reactions observed in oncology patients administered ICIs and SLZ. Perturbation of drug-specific T-cell priming is a plausible explanation for clinical observations of how an increased incidence of these adverse events is occurring.

T cell mediated hypersensitivity to previously tolerated iodinated contrast media precipitated by introduction of atezolizumab.

Many adverse reactions associated with immune checkpoint inhibitor (ICI) treatments are immunologically driven and may necessitate discontinuation of the ICI. Herein, we present a patient who had been administered the radio contrast media amidotrizoate multiple times without issue but who then developed a Stevens-Johnson syndrome reaction after coadministration of atezolizumab. Causality was confirmed by a positive re-challenge with amidotrizoate and laboratory investigations that implicated T cells. Importantly, the introduction of atezolizumab appears to have altered the immunologic response to amidotrizoate in terms of the tolerance-elicitation continuum. Proof of concept studies demonstrated enhancement of recall responses to a surrogate antigen panel following in-vitro (healthy donors) and in-vivo (ICI patients) administrations of ICIs. Our findings highlight the importance of considering all concomitant medications in patients on ICIs who develop immune-mediated adverse reactions. In the event of some immune-related adverse reactions, it may be critical to identify the culprit antigen-forming entity that the ICIs have altered the perception of rather than simply attribute causality to the ICI itself in order to optimize both patient safety and treatment of malignancies.

The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine.

During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 µM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants.

Acute Metabolic Switch Assay Using Glucose/Galactose Medium in HepaRG Cells to Detect Mitochondrial Toxicity.

Using galactose instead of glucose in the culture medium of hepatoma cell lines, such as HepG2 cells, has been utilized for a decade to unmask the mitochondrial liability of chemical compounds. A modified glucose-galactose assay on HepG2 cells, reducing the experimental period for screening of mitochondrial toxicity to 2 to 4 hr, has been previously reported. HepaRG cells are one of the few cell lines that retain some of the important characteristics of human hepatocytes, offering advantages of working with a cell line, therefore, are considered an alternative for HepG2 cells in drug toxicity screening. A method is described here using HepaRG cells in an acute metabolic switch assay utilizing specific glucose/galactose media, a combined ATP-protein-LDH assay measuring three endpoints from one 96-well plate, and a criteria to label a compound as a mitochondrial toxin. © 2019 by John Wiley & Sons, Inc.

The utility of HepaRG cells for bioenergetic investigation and detection of drug-induced mitochondrial toxicity.

The importance of mitochondrial toxicity in drug-induced liver injury is well established. The bioenergetic phenotype of the HepaRG cell line was defined in order to assess their suitability as a model of mitochondrial hepatotoxicity. Bioenergetic phenotyping categorised the HepaRG cells as less metabolically active when measured beside the more energetic HepG2 cells. However, inhibition of mitochondrial ATP synthase induced an increase in glycolytic activity of both HepaRG and HepG2 cells suggesting an active Crabtree Effect in both cell lines. The suitability of HepaRG cells for the acute metabolic modification assay as a screen for mitotoxicity was confirmed using a panel of compounds, including both positive and negative mitotoxic compounds. Seahorse respirometry studies demonstrated that a statistically significant decrease in spare respiratory capacity is the first indication of mitochondrial dysfunction. Furthermore, based upon comparing changes in respiratory parameters to those of the positive controls, rotenone and carbonyl cyanide m-chlorophenyl hydrazone, compounds were categorised into two mechanistic groups; inhibitors or uncouplers of the electron transport chain. Overall, the findings from this study have demonstrated that HepaRG cells, despite having different resting bioenergetic phenotype to HepG2 cells are a suitable model to detect drug-induced mitochondrial toxicity with similar detection rates to HepG2 cells.

Transcriptional repression of E-cadherin by human papillomavirus type 16 E6.

There is increasing evidence supporting DNA virus regulation of the cell adhesion and tumour suppressor protein, E-cadherin. We previously reported that loss of E-cadherin in human papillomavirus (HPV) type 16-infected epidermis is contributed to by the major viral proto-oncogene E6 and is associated with reduced Langerhans cells density, potentially regulating the immune response. The focus of this study is determining how the HPV16 E6 protein mediates E-cadherin repression. We found that the E-cadherin promoter is repressed in cells expressing E6, resulting in fewer E-cadherin transcripts. On exploring the mechanism for this, repression by increased histone deacetylase activity or by increased binding of trans-repressors to the E-cadherin promoter Epal element was discounted. In contrast, DNA methyltransferase (DNMT) activity was increased in E6 expressing cells. Upon inhibiting DNMT activity using 5-Aza-2'-deoxycytidine, E-cadherin transcription was restored in the presence of HPV16 E6. The E-cadherin promoter was not directly methylated, however a mutational analysis showed general promoter repression and reduced binding of the transactivators Sp1 and AML1 and the repressor Slug. Expression of E7 with E6 resulted in a further reduction in surface E-cadherin levels. This is the first report of HPV16 E6-mediated transcriptional repression of this adhesion molecule and tumour suppressor protein.

Leptomycin B induces apoptosis in cells containing the whole HPV 16 genome.

Cervical cancer is a major cause of death in women worldwide and is strongly associated with human papillomavirus (HPV) infection. Integration of HPV is thought to be a key step in malignant progression, and is associated with loss of regulation of the viral E6 and E7 oncogenes. Leptomycin B (LMB), a nuclear export inhibitor, has previously been shown to induce apoptosis in primary keratinocytes transduced with the HPV 16 E7 or E6/E7 genes, but not in normal cells. We show here that LMB can also induce apoptosis in derivatives of the W12 cell line that contain either episomal or integrated HPV 16. Cells transduced with HPV 16 E7 or E6/E7, and the episomal and integrated W12 derivatives showed distinct temporal expression patterns of the apoptotic markers activated caspase-3 and M30. The expression of both markers occurred later in the episomal derivatives than in either transduced cells or W12 derivatives containing integrated HPV. These findings suggest that, although LMB can induce apoptosis in keratinocytes containing episomal or integrated HPV 16, genome status is likely to influence the response of HPV-associated anogenital lesions to LMB treatment.

Selective induction of apoptosis by leptomycin B in keratinocytes expressing HPV oncogenes.

Human papillomavirus (HPV) infection is strongly associated with the development of anogenital neoplasia, particularly cervical cancer. It has been estimated that 99.7% of all cervical carcinomas are attributable to infection with HPV, and types 16 and 18 account for the vast majority of such cases. Both of these 'high risk' HPV types encode the oncoproteins E6 and E7, which exert multiple effects on many proteins involved in cell-cycle regulation, including p53. The nuclear export protein inhibitor leptomycin B (LMB) has been shown to cause the nuclear sequestration of p53 in cervical carcinoma cells. We demonstrate that LMB induces apoptosis selectively at nanomolar concentrations in primary human keratinocytes (PHKs) expressing HPV oncogenes. Both monolayer and organotypic raft cultures of transduced PHKs were highly susceptible to treatment with LMB. By contrast, although LMB stimulated p53 accumulation in normal PHKs, no significant induction of apoptosis was detected on Western blots or immunostained monolayer/raft cells, or following pulsed exposure to the drug. Furthermore, topical application of microM concentrations of LMB to mouse skin was non-toxic. These data suggest that the topical application of LMB to HPV-infected intra-epithelial lesions may represent a specific and effective therapeutic strategy against HPV-associated anogenital neoplasia.