Abstract 3994: ER stress is a major pathway responsible for the osteosarcoma sensitivity to OST inhibition

Abstract

Abstract Purpose: Osteosarcoma is a rare but highly aggressive tumor that primarily arises from the long bones most often in teenagers and young adults. Despite decades of research, systemic therapy options have remained essentially unchanged. Therefore, new therapeutic approaches to treat osteosarcoma are of great need for this patient population. We have performed high throughput screening to identify NGI-1, a first in class small molecule inhibitor that targets oligosaccharyltransferase (OST), disrupts protein glycosylation, and has significant activity in osteosarcoma cell lines. We have examined the mechanism of osteosarcoma sensitivity with the goal of developing a new therapeutic approach. Methods: A drug screen of 519 solid tumor cell lines was performed to identify sensitivity to NGI-1 treatment. The screening results were validated through measurement of relative changes in proliferation of nine different osteosarcoma cell lines using MTT assay. To identify the pathways involved in osteosarcoma sensitivity to OST inhibition, a CRISPR-Cas9 kinome knockout library screen targeting 750 human kinases and related genes was performed in the HUO-3N1 cell line. Drug combination (OST+PERK/IRE1 inhibitor) studies were performed using MTT assay to validate the CRISPR screen targets, where the drug combination synergy quantification was determined using COMPUSYN software. Outcomes of inhibitor treatment were analyzed using western blot and PCR assay. Results: A drug sensitivity screen of solid tumor cell lines was performed with a nine-point NGI-1 serial dilution and analyzed for cell viability. The results show that tumor cell lines derived from bone tissue origins, comprised of osteosarcomas and Ewing’s sarcomas, have high sensitivity to NGI-1 treatment. MTT assays performed with nine different osteosarcoma cell lines validated the results from the screen showing different sensitivity of the NGI-1treatment. To identify mechanisms of sensitivity, CRISPR-Cas9 kinome gRNA library screen was used in the HUO-3N1 cell line treated with NGI-1 vs control for 21 days. The results from the screen analyses showed the negative enrichment of gRNAs targeting EIF2AK3 (PERK; p=0.0000004) and ERN1(IRE1; p=0.0006) revealing roles in mediating NGI-1 sensitivity. The effect of pharmacologic inhibitors for PERK(GSK26006414) and IRE1 (4μ8C) on the cell growth (HUO-3N1 and G-292) validated the screening results. The combination of NGI-1with either the PERK or IRE1 inhibitor showed synergistic effects on the inhibition of osteosarcoma cell growth concurrent with enhanced induction of BiP and XBP1s, major effectors of the ER stress pathway. Conclusion: This study shows ER stress as one of the major pathways regulating the osteosarcoma sensitivity to OST inhibition. Ongoing studies are testing OST inhibition and other mechanisms of ER stress activation as a new therapeutic strategy for the treatment of osteosarcomas. Citation Format: Aanchal Katoch, Joseph Contessa. ER stress is a major pathway responsible for the osteosarcoma sensitivity to OST inhibition [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 3994.