Single-cell spatial proteomics identifies the JAK/STAT pathway as an actionable therapeutic target in lethal cutaneous drug reactions

Toxic epidermal necrolysis (TEN) is a fatal drug-induced skin reaction and an emerging public health issue. Triggered by common medications, TEN patients undergo severe and sudden epidermal detachment caused by keratinocyte cell death. Although molecular mechanisms driving keratinocyte cell death have been proposed, the main drivers remain unknown and no effective therapy exists. To systematically map molecular changes that are associated with TEN and identify potential druggable targets, we employed the single-cell spatial proteomics technique Deep Visual Proteomics. We analyzed formalin-fixed paraffin-embedded archived skin-tissue biopsies of three types of cutaneous drug reactions with varying severity and quantified over 5,000 proteins in keratinocytes and skin-infiltrating immune cells. Most strikingly, this revealed a robust enrichment of Type-I and -II interferon signature in the immune cell and keratinocyte compartment of TEN patients, along with a drastic activation of pSTAT1. Targeted inhibition with pan-JAK inhibitor (JAKi) tofacitinib reduced keratinocyte-directed cytotoxicity in a novel live-cell imaging assay, using patient-derived keratinocytes and peripheral blood mononuclear cells (PBMCs). Furthermore, oral administration of pan-JAKi tofacitinib or baricitinib ameliorated clinical and histological disease severity in two distinct mouse models of TEN. Lastly, JAKi treatment was safe and associated with rapid cutaneous re-epithelialization and recovery in four patients with TEN. This study uncovers the JAK-STAT and interferon signaling pathways as key pathogenic drivers of TEN and demonstrates the potential of targeted JAK inhibition as a curative therapy. ### Competing Interest Statement A patent for treatment of TEN, SJS/TEN and SJS with JAKi has been filed (US patent: provisional application no. 63/531,677; European patent: provisional application no. 23 190 686.8 inventors, T.M.N., L.E.F. and M.M.). The patent is owned by Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.. The authors declare no other competing interests. ### Clinical Trial NCT06119490 ### Funding Statement T.M.N. is supported by a Swiss National Science Foundation (SNSF) Early Postdoc Mobility (P2ZHP3-199648) and Postdoc Mobility Fellowship (P500PM_210917). M.T. is supported by the European Unions Horizon 2020 research and innovation program under grant agreement No 874839 (ISLET). J.S., H.A. and N.S. are supported by NHMRC grants Investigator Grant (1195038) and through the Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS (GNT9000719). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All obtained disease biopsies were performed in the context of routine clinical workup for retrospective analysis with informed consent and ethical approval in place (Munich: 22-0342, 22-0343; Zurich: BASEC: Req-2021-00226 and 2017--00494; Fujian: MRCTA, ECFAH of FMU[2023]400. Smac mimetic mouse model experiments were approved by the local ethics committee (WEHI AEC# 2022.009). Humanized mouse model experiments were approved by the local ethics committee and the institutional review board of Niigata University, and the patient was provided written informed consent. Treatment of patients with JAK inhibitors was approved by the local ethics committee and the institutional review board of the First Affiliated Hospital of Fujian Medical University (Fujian: MRCTA, ECFAH of FMU[2023]400), and the patient was provided written informed consent. Evaluation of the Efficacy and Safety of Methylprednisolone Combined With the JAK Inhibitors in the Treatment of Toxic Epidermal Necrolysis (TEN) is registered on ClinicalTrials.gov (ID: [NCT06119490][1]) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study will be available after peer-review, when published. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT06119490&atom=%2Fmedrxiv%2Fearly%2F2023%2F11%2F12%2F2023.11.11.23295492.atom