Characterization of the Osimertinib resistant H1975 cells.

List of antibodies used in the study

Dose response of NGI-1, temozolomide and etoposide in glioma cell lines

Apoptosis susceptibility of KRAS mutant A549 cell line following treatment with NGI-1, Erlotinib or both.

Time course resolution of γH2AX foci formation in D54 cells

Legends for supplementary figures 1 and 2

Deep sequencing of lung cancer cell lines to determine the proportion of EGFR mutations.

Results of the Lung Cancer Cell Line Screen for OST Inhibitor Sensitivity

Supplementary Figure S1, Supplementary Figure S2 and Supplementary Table 1

Abstract EGFR signaling confers resistance to radiotherapy and is a validated target in head and neck squamous cell carcinoma (HNSCC). The inhibition of EGFR in combination with radiotherapy improves local control and overall survival in these patients; however, therapeutic resistance limits the efficacy of this approach. We therefore sought to identify cellular mechanisms that cause resistance to EGFR inhibition and radiotherapy in HNSCC. Though clonal isolation of carcinoma cells exposed to increasing concentrations of cetuximab, we found that resistant cells upregulate prosurvival ErbB3 and AKT signaling. Using EFM-19 cells and confirmatory analysis of protein levels, we demonstrate that cetuximab resistance is characterized by enhanced neuregulin expression identifying a novel adaptive mechanism of therapeutic resistance. Inhibition of this autocrine loop with CDX-3379 (an ErbB3 specific antibody) was sufficient to block ErbB3/AKT signaling in cetuximab resistant cells. The combination of CDX-3379 and cetuximab reduced proliferation and survival after radiotherapy in several HNSCC cell lines. These in vitro findings were confirmed in xenograft tumor growth experiments including an approach using growth factor–supplemented Matrigel. In vivo, the delivery of EGFR and ErbB3 antibodies significantly reduced tumor growth in cetuximab-resistant FaDu and CAL27 xenografts. In summary, this work demonstrates that autocrine NRG ligand secretion is a mechanism for therapeutic resistance to cetuximab and radiotherapy. This cross-resistance to both therapeutic modalities identifies NRG as an actionable therapeutic target for improving treatment regimens in HNSCC.

Abstract Purpose: Parallel signaling reduces the effects of receptor tyrosine kinase (RTK)–targeted therapies in glioma. We hypothesized that inhibition of protein N-linked glycosylation, an endoplasmic reticulum co- and posttranslational modification crucial for RTK maturation and activation, could provide a new therapeutic approach for glioma radiosensitization. Experimental Design: We investigated the effects of a small-molecule inhibitor of the oligosaccharyltransferase (NGI-1) on EGFR family receptors, MET, PDGFR, and FGFR1. The influence of glycosylation state on tumor cell radiosensitivity, chemotherapy-induced cell toxicity, DNA damage, and cell-cycle arrest were determined and correlated with glioma cell receptor expression profiles. The effects of NGI-1 on xenograft tumor growth were tested using a nanoparticle formulation validated by in vivo molecular imaging. A mechanistic role for RTK signaling was evaluated through the expression of a glycosylation-independent CD8-EGFR chimera. Results: NGI-1 reduced glycosylation, protein levels, and activation of most RTKs. NGI-1 also enhanced the radiosensitivity and cytotoxic effects of chemotherapy in those glioma cells with elevated ErbB family activation, but not in cells without high levels of RTK activation. NGI-1 radiosensitization was associated with increases in both DNA damage and G1 cell-cycle arrest. Combined treatment of glioma xenografts with fractionated radiotherapy and NGI-1 significantly reduced tumor growth compared with controls. Expression of the CD8-EGFR eliminated the effects of NGI-1 on G1 arrest, DNA damage, and cellular radiosensitivity, identifying RTK inhibition as the principal mechanism for the NGI-1 effect. Conclusions: This study suggests that oligosaccharyltransferase inhibition with NGI-1 is a novel approach to radiosensitize malignant gliomas with enhanced RTK signaling. See related commentary by Wahl and Lawrence, p. 455

Abstract Asparagine (N)-linked glycosylation is a posttranslational modification essential for the function of complex transmembrane proteins. However, targeting glycosylation for cancer therapy has not been feasible due to generalized effects on all glycoproteins. Here, we perform sensitivity screening of 94 lung cancer cell lines using NGI-1, a small-molecule inhibitor of the oligosaccharyltransferase (OST) that partially disrupts N-linked glycosylation, and demonstrate a selective loss of tumor cell viability. This screen revealed NGI-1 sensitivity in just 11 of 94 (12%) cell lines, with a significant correlation between OST and EGFR inhibitors. In EGFR-mutant non-small cell lung cancer with EGFR tyrosine kinase inhibitor (TKI) resistance (PC9-GR, HCC827-GR, and H1975-OR), OST inhibition maintained its ability to induce cell-cycle arrest and a proliferative block. Addition of NGI-1 to EGFR TKI treatment was synthetic lethal in cells resistant to gefitinib, erlotinib, or osimertinib. OST inhibition invariably disrupted EGFR N-linked glycosylation and reduced activation of receptors either with or without the T790M TKI resistance mutation. OST inhibition also dissociated EGFR signaling from other coexpressed receptors like MET via altered receptor compartmentalization. Translation of this approach to preclinical models was accomplished through synthesis and delivery of NGI-1 nanoparticles, confirmation of in vivo activity through molecular imaging, and demonstration of significant tumor growth delay in TKI-resistant HCC827 and H1975 xenografts. This therapeutic strategy breaks from kinase-targeted approaches and validates N-linked glycosylation as an effective target in tumors driven by glycoprotein signaling. Significance: EGFR-mutant NSCLC is incurable despite the marked sensitivity of these tumors to EGFR TKIs. These findings identify N-linked glycosylation, a posttranslational modification common to EGFR and other oncogenic signaling proteins, as an effective therapeutic target that enhances tumor responses for EGFR-mutant NSCLC. Cancer Res; 78(17); 5094–106. ©2018 AACR.

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

Abstract 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 >90% (p<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

Asparagine-linked glycosylation is an endoplasmic reticulum co- and post-translational modification that enables the transit and function of receptor tyrosine kinase (RTK) glycoproteins. To gain insight into the regulatory role of glycosylation enzymes on RTK function, we investigated shRNA and siRNA knockdown of mannose phosphate isomerase (MPI), an enzyme required for mature glycan precursor biosynthesis. Loss of MPI activity reduced phosphorylation of FGFR family receptors in U-251 and SKMG-3 malignant glioma cell lines and also resulted in significant decreases in FRS2, Akt, and MAPK signaling. However, MPI knockdown did not affect ligand-induced activation or signaling of EGFR or MET RTKs, suggesting that FGFRs are more susceptible to MPI inhibition. The reductions in FGFR signaling were not caused by loss of FGF ligands or receptors, but instead were caused by interference with receptor dimerization. Investigations into the cellular consequences of MPI knockdown showed that cellular programs driven by FGFR signaling, and integral to the clinical progression of malignant glioma, were impaired. In addition to a blockade of cellular migration, MPI knockdown also significantly reduced glioma cell clonogenic survival following ionizing radiation. Therefore our results suggest that targeted inhibition of enzymes required for cell surface receptor glycosylation can be manipulated to produce discrete and limited consequences for critical client glycoproteins expressed by tumor cells. Furthermore, this work identifies MPI as a potential enzymatic target for disrupting cell surface receptor-dependent survival signaling and as a novel approach for therapeutic radiosensitization.