LAUSR

Background: Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States. Despite initial response to chemotherapy, most OCa patients become chemo resistant and progress to metastatic disease. Here, we tested the hypothesis that the high basal level of endoplasmic reticulum stress (ERS) in OCa represents a critical vulnerability and drugs that further aggravate this already engaged system in OCa may exhaust its protective features and contribute to apoptosis induction. The objective of this proposal is to identify a hit compound that enhances ERS in OCa and to conduct mechanistic studies. Methods: We synthesized a small library of >200 chemically distinct oligobenzamide analogs with maintenance of the chemical backbone but altered R groups of ERX-11. We performed the primary screening of this library to evaluate the induction of mRNA levels of two canonical ERS/UPR (unfolded protein response) genes- sXBP1 and CHOP. Biological activity of ERX-208 was validated using multiple OCa cells. Mechanistic studies were conducted using CRISPR/Cas9 KO, Western blotting, reporter gene assays, IHC and RNA-seq analysis. PK (pharmacokinetics) and toxicity studies were done using C57BL/6 mice. Cell line-derived xenografts (CDXs), patient-derived xenografts (PDXs), patient-derived explants (PDEs), and patient-derived organoids (PDO) were used for preclinical evaluation. Results: From a screen of a curated ERX-11 derived oligobenzamide library, we identified a hit compound, ERX-208 that potently (IC50~100nM) induces ERS/UPR and apoptosis in multiple OCa cells in vitro. CRISPR KO screen identified the lysosomal acid lipase A (LIPA) protein as the critical target of ERX-208. LIPA KO abrogates response to ERX-208, while reconstitution of LIPA restores ERX-208 response. The time course studies showed a robust and consistent induction (>15-fold CHOP, and >10-fold sXBP1) by ERX-208 treatment within 24h. We confirmed induction of classic UPR components peIF2α, CHOP and LC3B using Western blotting in multiple OCa cells. Functionally, ERX-208 causes growth inhibition of OCa cells, as noted by MTT cell viability assays using 15 OCa cells with an IC50 of ~50-100nM. The activity of ERX-208 is distinct among oligobenzamides as ERX-11 has limited/no activity against OCa cells. RNA-seq analysis confirmed that ERX-208 induces significant ERS, UPR, and apoptosis. Further, ERX-208 reduced the growth of OCa PDO’s in vitro, PDEs ex vivo and CDXs and PDXs in vivo. ERX-208 treatment did not show any signs of toxicity and body weight of mice was not affected. IHC analyses showed increased activation of ERS/UPR markers such as GRP78, p-PERK and decreased proliferation measured by Ki67. Conclusions: Collectively, our results demonstrated the utility of ERX-208 and will establish a novel therapeutic paradigm in OCa that overcomes tumor heterogeneity by targeting LIPA and enhancing ERS leading to apoptosis. Citation Format: Suryavathi Viswanadhapalli, Tae-Kyung Lee, Kara Kassees, Gaurav Sharma, Rahul Gopalam, Karla Parra, Tanner Reese, Michael Hsieh, Uday P. Pratap, Xue Yang, Behnam Ebrahimi, Chia Yuan Chen, Scott Terry Elmore, Christian Cervantes, Zhenming Xu, Edward Kost, Gangadhara Reddy Sareddy, Rajeshwar Rao Tekmal, Jung-Mo Ann, Ganesh V. Raj, Ratna K. Vadlamudi. ERX-208 as a novel therapeutic for treating ovarian cancer by enhancing endoplasmic reticulum stress. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4813.