Abstract 3981: Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer

Abstract

Abstract PURPOSE: Lung cancer is a major cause of morbidity and mortality world wide. Signaling receptors, adhesion molecules, and surface antigens that drive lung cancer progression are regulated by N-glycosylation, a post-translational protein modification initiated in the ER. Strategies for inhibition of N-glycosylation have recently been refined through the discovery of small molecules that partially disrupt glycan transfer. Here we screened for the target of a novel small molecule that reduces protein glycosylation with the goal of identifying new therapeutic targets for lung cancer. METHODS: Pooled results from chemical library HTS efforts identified hit compounds with activity. Counter-screening was performed in A549 cells using a fluorescence-based reporter (Halo1N) that detects aberrant N-glycosylation. The cellular targets for one class of inhibitors were evaluated by combining a whole-genome CRISPR-Cas9 screen with FACS sorting for loss of pharmacologic function. Significant changes in gRNA between populations was determined by MAGeCK analysis. The interaction between a compound and its target was determined by CETSA, and mechanism of action was verified by gene knockout and rescue experiments. Glycosylation status of glycoproteins were measured by western blot and cell compartment fluorescence. Predicted compound binding sites on the protein were mutated using site-directed mutagenesis. RESULTS: The lead tool compound (CP-9) has an IC50 in uM range and was used as a probe for target identification. CP-9 activates Halo-1N fluorescence, and whole genome CRISPR-Cas9 screening was used to identify gRNAs associated with loss of CP-9 activity. MAGeCK analysis demonstrated significant enrichment of genes associated with the translocon and ribosome (FDR P < 0.01) in the non-fluorescent population. We tested components of the signal recognition particle-receptor complex for direct interactions using the CETSA and demonstrate CP-9 binding to SR-β but not SR-α. Knockout experiments confirm that inhibition of glycosylation by CP-9 is dependent on SR-β expression, and CP-9’s effect on glycosylation and was rescued by SR-β re-expression. Mutated SR-β diminished CP-9’s effects on glycosylation and reduced SR-β thermal stabilization. CP-9 disrupted an interaction between SR-β/SR-α and the ribosome-translocon-OST complex suggesting a mechanism of action. Interestingly, a transmembrane domain mutant (ΔTM) of SR-β reduced the CP-9 effect and glycoprotein expression. ΔTM-SR-β also significantly reduced cell proliferation in A459 lung adenocarcinoma cells. CONCLUSION: CP-9 partially inhibits N-glycosylation by interfering with SR-β/SR-α and the translocon complex, leading to the disruption of protein import and glycosylation in the ER. Transmembrane domain mutants of SR-β reduce lung cancer cell growth and suggest this protein as a potential target for lung cancer drug development in the future. Citation Format: Chatchai Phoomak, Joseph N. Contessa. Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3981.