Abstract 3045: Development of a first-in-class PDIA6/IRE1 modulator for selectively sensitizing cancer cells to ER stress and regulated cell death

Abstract Background: A high proliferation rate of cancer cells requires increased protein synthesis leading to ER stress, a condition characterized by accumulation of unfolded or misfolded proteins in the ER lumen. Three interconnected Unfolded Protein Response (UPR) pathways, IRE1, PERK, and ATF6 pathways, are activated to relieve ER stress or to initiate apoptosis if prolonged strong ER stress remains unsolved. Thus, the chronically elevated ER stress levels in cancer cells constitute an Achilles’ heel and provide a window of opportunity for development of therapeutic regimens. Despite multiple ongoing preclinical and clinical studies using various agents and regimens to overactivate ER stress and UPR, agents that can specifically induce UPR in cancer cells but not in normal cells are still lacking. Methods: Metabolic activity-based phenotypic assay was employed to identify NAI003 as the most potent anticancer compound during lead optimization where over three hundred small-molecule NCEs were derived from the hit compounds. HuProt™ Human Proteome Microarray and chemoproteomics approaches were used to identify target proteins to which NAI003 can directly bind. The top ranked target proteins were each validated by specific siRNAs, followed by Biacore analysis to quantify target binding affinity. Subsequently, multiple approaches were used to further understand the MoA of NAI003 and to assess its developability. Results: Our preclinical candidate compound NAI003 directly binds with high affinity (Kd=1.8 nM) and selectivity to PDIA6 protein that is known to catalyze protein folding and thiol-disulfide interchange reactions in the ER. NAI003 blocks the PDIA6/IRE1 protein-protein interaction in ER-stressed cancer cells and thereby inducing constitutively activated IRE1 signaling that ultimately leads to regulated cell death (RCD). NAI003 series compounds have subnanomolar to low micromolar IC50s against selected cervical, lung, ovarian, liver, pancreatic, skin and brain cancer cells in vitro, and potently inhibit their in vivo propagation in multiple CDX and PDX models. Remarkably, they completely spare normal cells that usually have much lower levels of ER stress. Furthermore, in rodent models, NAI003 compounds exhibit low acute toxicity, desirable pharmacokinetics profile and reasonable oral bioavailability. Importantly, NAI003 compounds exhibit synergism with PARP inhibitors (e.g. Talazoparib), proteasome inhibitors (e.g. Ixazomib) and metabolic stressors (e.g. glucose deficiency) that are all known to induce ER stress in cancer cells. Conclusion: NAI003 is a potential first-in-class small-molecule PDIA6/IRE1 modulator that sensitizes cancer cells to ER stress and RCD by selectively overactivating IRE1 signaling. It holds great promise in treating selected cancers either as a single agent or as a combination therapy with marketed anticancer drugs. Citation Format: Wei Jia, Xiaobing Zhang, Bo Kang, Zhao Xu, Chencong Bo, Mingxing Yin, Shujian Ren, Zhengshu Chen, Amar Al-Ansi, Hongban Zhong, Ying-Jie Wang. Development of a first-in-class PDIA6/IRE1 modulator for selectively sensitizing cancer cells to ER stress and regulated cell death [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 3045.