Suppression of glycosidase NGLY1 induces multifaceted anticancer responses.

Purpose: NGLY1 is a pivotal enzyme that catalyzes the deglycosylation of denatured glycoproteins and facilitates proteasome-mediated protein degradation. However, there is limited information regarding the responses of human normal and cancer cells to NGLY1 suppression. The objective of our study is to determine the significance of NGLY1 for melanoma cell viability and how it may be exploited as a novel anticancer target. Methods: We used cellular and molecular biology tools such as Crispr-Cas9-mediated gene editing and shRNA for NGLY1 suppression. Computational modelling and a rational design approach was used to design and synthesize novel small molecules that can covalently modify NGLY1 to irreversibly inhibit its activity. We also used systems biology approaches including global gene expression profiling and proteomics analysis to uncover mechanisms through which inhibition of NGLY1 preferentially leads to cancer suppression. Results: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumor samples. NGLY1 knockdown caused melanoma cell death in vitro and tumor growth retardation in vivo. Mechanistically, NGLY1 suppression induced pleiotropic responses which can synergize with the anti-melanoma activity of chemotherapy and targeted therapy agents. We have discovered a series of novel small-molecule inhibitors of human NGLY1. Both pharmacological and molecular biology tools that inhibit NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. Conclusion: Our work represents the first comprehensive characterization of multifaceted anti-melanoma responses by targeting NGLY1. This study revealed the biological significance of NGLY1 in melanoma cells and provided mechanistic insights regarding how NGLY1 inactivation preferentially leads to eradication of melanoma with limited impact on normal cells. Collectively, our findings attest that the inactivation of NGLY1 represents a novel and promising anti-melanoma strategy.