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<p>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 <i>NAE1</i> and <i>UBA3</i> 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 <i>NAE1</i> and <i>UBA3</i> 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. <i>NAE1</i> and <i>UBA3</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月3日

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  • Francesca Antonella Aiello,
  • Lucrezia D’Archivio,
  • Marika Attili,
  • Erika Ferraro,
  • Elisa Macrì,
  • Riccardo Mazzocchi,
  • Matteo Cassandri,
  • Silvia Pomella,
  • Valeria Tocco,
  • Marco Pezzullo,
  • Cristiano De Stefanis,
  • Silvia Codenotti,
  • Giovanni Barillari,
  • Cinzia Marchese,
  • Alessandro Fanzani,
  • Francesca Megiorni,
  • Janet Shipley,
  • Marielle Yohe,
  • Susanne A. Gatz,
  • Peter J. Houghton,
  • Giovanni Cenci,
  • Concetta Quintarelli,
  • Franco Locatelli,
  • Francesco Marampon,
  • Biagio De Angelis,
  • Rossella Rota

摘要

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.