Abstract 1497: PERK as a biomarker to optimize therapy of GRP78 inhibitor, BOLD-100, in gastric cancer

Abstract Background: Targeting endoplasmic reticulum (ER) stress is emerging as a potential therapeutic strategy for developing therapies against solid tumors. GRP78, also known as the master regulator of unfolded protein response (UPR), is an anti-apoptotic ER chaperone that adjusts protein folding capacity to restore ER homeostasis. Moreover, GRP78 is an inhibitory PERK-binding protein that regulates the activation of death-triggering UPR pathways to avoid cell apoptosis. In gastric cancer (GC), GRP78 expression is elevated compared to normal tissue making GRP78 an attractive therapeutic target in GC. However, there have been limited studies on targeting GRP78 in GC. This study investigates the antitumor effects of GRP78 inhibition with BOLD-100 and explore potential biomarkers for therapeutic response in GC. Methods: A total of 9 gastric cancer cell lines (KATO-III, SNU-601, NCI-N87, MKN-45, SNU-216, SNU-719, SNU-668, AGS, SNU-638) were used. BOLD-100 (GRP78 inhibitor), TUDCA (Tauroursodeoxycholic acid; ER stress inhibitor), and AZD6738 (ATR inhibitor) were used. MTT assay, RT-qPCR, western blot, cell cycle analysis, DCF-DA staining, immunofluorescence, and Annexin V assay were performed to evaluate the antitumor effects of BOLD-100. To evaluate the effect of PERK expression level on the efficacy of BOLD-100, siRNA was used to downregulate PERK and consequent BOLD-100 effects were analyzed by MTT assay, western blot, and flow cytometry. Results: The antitumor effect of BOLD-100 positively correlated with PERK expression. BOLD-100 suppressed the mRNA level of GRP78 leading to a subsequent increase in unfolded protein load and ROS level in a PERK expression-dependent manner. In PERK-high GC cells (KATO-III, SNU-601), BOLD-100 exerted pronounced activation of PERK-eIF2a-CHOP pathway, resulting in inhibited tumor cell growth and induced apoptosis. PERK knockdown reversed BOLD-100 induced ROS accumulation and UPR activation suggesting that PERK expression level regulates sensitivity to BOLD-100. Conversely, in PERK-low GC cells (AGS, SNU-638), BOLD-100 exerted minimal effects on UPR pathway. Rather, it induced R-loop accumulation and upregulated active forms of ATR and Chk1 - effects not observed in PERK-high cells - suggesting activation of DNA damage repair pathway. A combination of BOLD-100 and AZD6738 showed synergistic effects in PERK-low GC cells by suppressing BOLD-100 induced ATR phosphorylation. Conclusion: The expression of PERK determines sensitivity to GRP78 inhibition by BOLD-100 in GC. PERK-high GC cells are sensitive to BOLD-100 and show efficacy as monotherapy. In PERK-low GC cells, the combination of BOLD-100 and ATR inhibitor is an optimal therapeutic option for GC treatment. Citation Format: Sea Young Choo, Ah-Rong Nam, Kyoung-Seok Oh, Jae-Min Kim, Ju-Hee Bang, Yoojin Jeong, Hyo Jung Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh. PERK as a biomarker to optimize therapy of GRP78 inhibitor, BOLD-100, in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1497.