Abstract 4998: Novel N-glycosilation inhibitor blocks proliferation of EGFR-dependent NSCLC
Authors: Lopez Sambrooks Cecilia , Contessa Joseph N. 
Published: August 2015

Purpose: Receptor Tyrosine Kinase (RTK) are transmembrane glycoproteins that are known to drive cellular proliferation in non-small cell lung cancer (NSCLC). Asparagine (N) linked glycosylation (NLG) is an endoplasmic reticulum (ER) post-translational modification common to RTKs that is crucial for receptor stability, localization, and activation. We hypothesized that blockade of NLG would be a novel strategy for reducing RTK signaling in NSCLC and therefore initiated a small molecule screening program to identify novel inhibitors of this biosynthetic process that have the potential for clinical translation.

Experimental Procedures: The ER-LucT NLG reporter was used to perform a cell-based high throughput screen of 358,301 compounds and identified a novel small molecule with NLG inhibitory activity (NGI-1). Effects on protein localization were determined using confocal microscopy and by surface biotin labeling. Cell proliferation was assessed with MTT assays over 5 days of drug treatment and cell cycle analysis was performed by flow cytometry. Senescence was determined by autofluorescence and p21 and p53 protein levels measured by western blot.

Results: NGI-1 is a first in class inhibitor of the oligosaccharyltransferase that is cell permeable and has a submicromolar IC50. This small molecule partially inhibits NLG and blocks epidermal growth factor receptor (EGFR) trafficking to the cell surface. Partially glycosylated receptors are retained in the secretory pathway as demonstrated by colocalization with the ER marker calreticulin. The loss of EGFR cell surface expression correlated with a significant reduction of EGFR phosphorylation and downstream signaling through MAPK and AKT. After 48hrs, NGI-1 treatment reduced proliferation of EGFR kinase domain mutant cell lines (PC9, HCC827, H3255) by &gt;90% (p&lt;0.001) but did not significantly block proliferation of KRAS mutant A549 cells or normal fibroblasts, suggesting a preferential effect on RTK addicted NSCLC. Cell cycle analysis also showed a differential effect on G1 arrest for EGFR addicted cell lines relative to controls. This proliferative block was shown to correlate with increased levels of p53 phosphorylation and increased expression of p21, as well as accumulation of lipofuscin and cell autofluorescence. Together these data suggest that NGI-1 induces a senescence-like phenotype in EGFR addicted NSCLC. Similar results were obtained in PC9 cells with T790M mutation, demonstrating the potential advantage of this approach for overcoming TKI acquired resistance in NSCLC.

Conclusion: This study suggests that partial inhibition of NLG with NGI-1 is a novel approach for blocking proliferation of RTK dependent NSCLC.

Citation Format: Cecilia Lopez Sambrooks, Joseph N. Contessa. Novel N-glycosilation inhibitor blocks proliferation of EGFR-dependent NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4998. doi:10.1158/1538-7445.AM2015-4998