Neddylation inhibition induces DNA double-strand breaks, hampering tumor growth in vivo, and promotes radiosensitivity in PAX3-FOXO1 rhabdomyosarcoma PMID: 41184241

ADC Targets in Oncology

2026-04-10

Neddylation inhibition induces DNA double-strand breaks, hampering tumor growth in vivo, and promotes radiosensitivity in PAX3-FOXO1 rhabdomyosarcoma

Cell Death Discov. 2025 Nov 3;11(1):496. doi: 10.1038/s41420-025-02787-0.

Francesca Antonella Aiello ^# ¹ , Lucrezia D'Archivio ^# ¹ , Marika Attili ^# ¹ , Erika Ferraro ¹ , Elisa Macrì ¹ , Riccardo Mazzocchi ¹ , Matteo Cassandri ² ³ , Silvia Pomella ⁴ ⁵ , Valeria Tocco ⁶ , Marco Pezzullo ⁶ , Cristiano De Stefanis ⁶ , Silvia Codenotti ⁷ , Giovanni Barillari ⁸ , Cinzia Marchese ⁹ , Alessandro Fanzani ⁷ , Francesca Megiorni ⁹ , Janet Shipley ¹⁰ , Marielle Yohe ¹¹ , Susanne A Gatz ¹² , Peter J Houghton ¹³ , Giovanni Cenci ¹⁴ , Concetta Quintarelli ¹ ¹⁵ , Franco Locatelli ¹ ¹⁶ , Francesco Marampon ¹⁷ , Biagio De Angelis ¹ , Rossella Rota ¹⁸

Affiliations

1. Department of Hematology/Oncology, Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
2. Department of Hematology/Oncology, Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy. [email protected].
3. Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy. [email protected].
4. Department of Hematology/Oncology, Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy. [email protected].
5. Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy. [email protected].
6. Core Facilities Research Laboratories, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy.
7. Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
8. Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy.
9. Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy.
10. Sarcoma Molecular Pathology, Divisions of Molecular Pathology, The Institute of Cancer Research, London, UK.
11. Laboratory of Cell and Developmental Signaling, National Cancer Institute, NIH, Frederick, MD, USA.
12. Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, West Midlands, UK.
13. Greehey Children's Cancer Research Institute (GCCRI), UT Health Science Center, San Antonio, TX, USA.
14. Department of Biology and Biotechnologies "C. Darwin", "Sapienza" University of Rome, Rome, Italy.
15. Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy.
16. Department of Life Sciences and Public Health, Catholic University of the Sacred Heart, Rome, Italy.
17. Department of Radiotherapy, Policlinico Umberto I, "Sapienza" University of Rome, Rome, Italy.
18. Department of Hematology/Oncology, Cell and Gene Therapy, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy. [email protected].
# Contributed equally.

Abstract

Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma with myogenic features affecting children and adolescents. The high-risk fusion-positive RMS subtype (FP-RMS), driven by the oncogenic chimeric transcription factor PAX3-FOXO1, shows 5-year overall survival not exceeding 30%. Here, we examine the impact of neddylation inhibition, a post-translational modification in which the NEDD8 peptide is conjugated to proteins, on the tumorigenic properties of FP-RMS. Here, we report that the NAE1 and UBA3 genes encoding the two subunits of the NEDD8-activating Enzyme (NAE) heterodimer are upregulated in FP-RMS patients compared to healthy skeletal muscle tissues and highly expressed in RMS among several tumor types. Furthermore, DepMap analyses showed that FP-RMS cell lines are among the most sensitive to both NAE1 and UBA3 CRISPR-mediated knockout as well as to NAE pharmacological inhibition with MLN4924 compared to other Cancer cell lines. In agreement, FP-RMS cells treated in vitro with MLN4924 (Pevonedistat) exhibited cell proliferation decrease, G2/M cell cycle arrest, senescence, and caspase- and PARP1-dependent Apoptosis. These phenotypes were associated with increased γH2AX nuclear foci and protein levels, DNA double-strand breaks (DSB), and reduced RAD51 levels. NAE1 and UBA3 individual silencing mirrors the major effects of MLN4924. In addition, MLN4924 also prevented FP-RMS tumor growth in vivo. Combining MLN4924 with irradiation enhanced Apoptosis and the inhibition of colony formation, cell cycle progression, and anchorage-independent and tumor spheroids growth compared to single treatments. Molecularly, MLN4924 amplified the irradiation-induced DNA damage by increasing γH2AX and DSBs, while reducing RAD51 expression and DNA-PKcs activation, both of which are involved in DNA repair. Collectively, our results suggest that the neddylation pathway is deregulated in FP-RMS, representing a potential therapeutic target. Therefore, MLN4924 could be considered as an anti-tumorigenic compound and a novel radiosensitizer in FP-RMS.