Targeting NAE1-mediated protein hyper-NEDDylation halts cholangiocarcinogenesis and impacts on tumor-stroma crosstalk in experimental models

Background and aims: cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Here, we investigate the role of NEDDylation in CCA development and progression. Methods: levels and function of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated in vitro, in vivo and/or in patients with CCA. Development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. Results: NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation in vitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA in vivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells in vitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, NEDDylation impairment in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. Conclusion: aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. Lay summary: Little is known about the role of PTMs in CCA development and progression. Here, we show that protein NEDDylation is upregulated and hyperactivated in CCA, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis, emerging as a promising therapeutic strategy to tackle these tumors.