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Proteasome inhibition promotes Foxn1 expression in thymic epithelial cells and induces thymic regeneration in mice

Cell Death & Differentiation (2026) Cite this article

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Abstract

The thymus plays a critical role in sustaining T-cell immunity, although its function is highly vulnerable to acute injury and physiologically declines with age, resulting in compromised immune responses. Impaired thymic function represents a major clinical challenge, particularly in settings of immunosuppression associated with cancer therapy and aging. Yet, effective strategies to rejuvenate the thymus remain limited. To explore novel regenerative approaches, we focused on FOXN1, a master regulator of thymic epithelial cell (TEC) development and function. By developing a custom screening platform, we tested a library of FDA-approved compounds for their ability to induce FOXN1 in TECs. Proteasome inhibition emerged as a potent and previously unrecognized mechanism for upregulating FOXN1 in both murine and human primary TECs. Among the hits identified in the screening, the antiparasitic drug nitazoxanide (NTZ) stood out for its proteasome inhibitory activity and for inducing Foxn1 expression while preserving cell viability, unlike other proteasome inhibitors. Mechanistically, NTZ-induced proteasome inhibition triggered endoplasmic reticulum stress (ER) and the adaptive unfolded protein response (UPR), ultimately engaging autophagy in TECs. In this context, the induction of autophagy acted as a compensatory mechanism to support cell survival in response to proteasome inhibition. Notably, when administered in mice, NTZ significantly accelerated functional thymic recovery after radiation-induced damage, promoting restoration of thymic architecture and cellularity of both stromal and hematopoietic compartments without disrupting physiological T-cell selection or tolerance mechanisms. Consistent with our in vitro findings, NTZ treatment induced Foxn1 and its downstream targets in TECs in vivo and conferred protection to TECs following irradiation. These findings uncover proteasome inhibition and, more broadly, modulation of ER stress and UPR pathways as a previously unrecognized mechanism regulating Foxn1 expression and position NTZ as a promising pharmacological strategy to enhance immunity in patients experiencing T-cell deficiencies due to cancer-related immunosuppression, infections, and age-related thymic atrophy.

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Fig. 1: Drug screening identifies proteasome inhibitors as FOXN1 inducers.
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Fig. 2: Nitazoxanide inhibits proteasome activity and promotes Foxn1 expression in TECs.
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Fig. 3: Nitazoxanide leads to ER stress followed by UPR activation and autophagy.
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Fig. 4: Nitazoxanide promotes thymic regeneration after radiation in mice.
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Fig. 5: Thymocyte selection stages and regulatory T-cell output after nitazoxanide treatment.
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Fig. 6: Effects of nitazoxanide on thymic epithelial cell recovery after irradiation.
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Fig. 7: Nitazoxanide induces Foxn1 and UPR-related gene expression in thymic epithelial cells after irradiation.
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Fig. 8: Proposed hypothetical mechanism.
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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. However, the full drug screening dataset is not publicly available due to the sensitive and proprietary nature of the information. These data may form the basis of future patent applications, and, as such, access is restricted to protect potential intellectual property. Requests for access will be evaluated on a case-by-case basis, and may require a confidentiality agreement and institutional approval.

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Acknowledgements

We thank Dr.Angelo Condorelli for his valuable support in establishing and monitoring the human primary TEC cultures. We also thank Dr. Cristiano De Stefanis from the histology facility of our institute for his help and support with tissue sectioning and staining.

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Authors and Affiliations

  1. Pediatric Hematology and Oncology Research Area, Bambino Gesù Children’s Hospital IRCCS, Rome, Italy

    Shirley Genah, Marsha Pellegrino, Angelica Mancusi, Alessio Taviani, Antonella Cardinale, Marco Rosichini, Sara Flamini, Maria Luigia Catanoso, Franco Locatelli & Enrico Velardi

  2. Immunology Research Area, Innate Lymphoid Cells, Bambino Gesù Children’s Hospital IRCCS, Rome, Italy

    Manuela Giansanti & Francesca Nazio

  3. Immunotherapy Integrated Research Center, Division of Translational Science and Therapeutics, Fred Hutchinson Cancer Center, Seattle, WA, USA

    Marco Rosichini & Jarrod A. Dudakov

  4. Research Laboratories, Bambino Gesù Children’s Hospital IRCCS, Rome, Italy

    Ezio Giorda, Gabriele Volpe & Michela Piccione

  5. Department of Biology, University of Rome Tor Vergata, Rome, Italy

    Francesca Nazio

  6. City of Hope Los Angeles and National Medical Center, Duarte, CA, USA

    Marcel R. M. van den Brink

  7. Catholic University of the Sacred Heart, Department of Basic Biotechnological Sciences, Intensive Care and Perioperative Clinics, Rome, Italy

    Emmanuel de Billy

  8. Department of Maternal and Child Health, Catholic University of the Sacred Heart, Rome, Italy

    Franco Locatelli

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  1. Shirley Genah
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Contributions

SG designed and performed experiments, data curation and analysis and wrote the manuscript. MP, MG, AC, MR, AM, AT, SF, and MLC conducted experiments and contributed to data curation and analysis. GV and EG provided support for flow cytometry experiments and cell sorting, and MP contributed to confocal imaging acquisition. FN contributed expertise in autophagy and reviewed and edited the manuscript. EDB contributed expertise in drug screening and reviewed and edited the manuscript. JAD, MRMvdB, and FL provided conceptual know-how, reviewed and edited the manuscript. EV conceived and supervised the project, designed experiments, and wrote the manuscript.

Corresponding author

Correspondence to Enrico Velardi.

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Competing interests

GS is supported by a FIRC-AIRC fellowship for Italy. RM was supported by an AIRC Fellowship for Abroad. VE was supported by grants from the Rally Foundation Investigator Award, Amy Stelzer Manasevit Research Program, the Italian Association for Cancer Research, and by the Italian Ministry of Health with “Current Research funds. LF was supported by grants from AIRC (Special Program Metastatic disease: the key unmet need in oncology 5 per mille 2018 Project Code 21147 and Accelerator Award 2017 INCAR); Ministero dell’Istruzione, dell’Università e della Ricerca, PRIN ID 2017 WC8499_004; Ministero della Salute, RF-2016-02364388. EdB received funding from Research (PRIN PNRR P2022JLHZZ), Ministero della Salute (GR-2019-12369231), by MIA Neri Foundation and by Heal Foundation. FN lab is supported by the “Associazione Italiana Ricerca sul Cancro” (My First Airc Grant -Ref. 27019), by “CureSearch Young Investigator Awards in Pediatric Oncology Drug Development”, by “Fondazione Roche per la Ricerca Indipendente”, by the Italian Ministry of University and Research (PRIN PNRR P2022JLHZZ), Ministero della Salute (GR-2019-12369231) and by MIA Neri Foundation. EV has received research funding from ToleranceBio for projects outside the submitted work. FL reports personal fees from Amgen, personal fees from Novartis, other from Bellicum Pharmaceutical, other from Neovii, personal fees from Miltenyi, personal fees from Medac, personal fees from Jazz Pharmaceutical, personal fees from Takeda, outside the submitted work. A patent application entitled “FOXN1-activating compounds and their use in thymic regeneration” has been filed (PCT/IT2025/050304).

Ethics approval

All animals were purchased from Charles River and maintained in the Plaisant Castel Romano animal facility in Rome. Mice experiments were conducted in compliance with the EU and national ethical requirements and were approved by the Italian Health Ministry. Human thymic samples for hTEC culture were obtained from pediatric patients undergoing corrective cardiac surgery. All samples were anonymously collected in accordance with local ethical guidelines, written informed consent was obtained, and the protocol was approved by the Ethics Committee of OPBG Hospital.

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Genah, S., Pellegrino, M., Giansanti, M. et al. Proteasome inhibition promotes Foxn1 expression in thymic epithelial cells and induces thymic regeneration in mice. Cell Death Differ (2026). https://doi.org/10.1038/s41418-026-01724-7

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