Triple-negative breast cancer (TNBC) has a poor clinical outcome, due to a lack of actionable therapeutic targets. Herein we define lysosomal acid lipase A (LIPA) as a viable molecular target in TNBC and identify a stereospecific small molecule (ERX-41) that binds LIPA. ERX-41 induces endoplasmic reticulum (ER) stress resulting in cell death, and this effect is on target as evidenced by specific LIPA mutations providing resistance. Importantly, we demonstrate that ERX-41 activity is independent of LIPA lipase function but dependent on its ER localization. Mechanistically, ERX-41 binding of LIPA decreases expression of multiple ER-resident proteins involved in protein folding. This targeted vulnerability has a large therapeutic window, with no adverse effects either on normal mammary epithelial cells or in mice. Our study implicates a targeted strategy for solid tumors, including breast, brain, pancreatic and ovarian, whereby small, orally bioavailable molecules targeting LIPA block protein folding, induce ER stress and result in tumor cell death.

Cell competition, a fitness sensing process is essential for tissue homeostasis. Employing cancer metastatic latency models, we show that cell competition results in displacement of latent metastatic (Lat-M) cells from the primary tumor. Lat-M cells resist anoikis and survive as residual metastatic disease. Remodelled extracellular matrix facilitates Lat-M cell displacement and survival in circulation. Disrupting cell competition dynamics by depleting SPARC reduced displacement from orthotopic tumors and attenuated metastases. In contrast, depletion of SPARC post-extravasation in lung resident Lat-M cells increased metastatic outgrowth. Furthermore, multi-regional transcriptomic analyses of matched primary tumors and metachronous metastases from kidney cancer patients identified tumor subclones with latent metastatic traits. Kidney cancer enriched for these latent metastatic traits had rapid onset of metachronous metastases and significantly reduced disease-free survival. Thus, an unexpected consequence of cell competition is displacement of cells with latent metastatic potential, thereby shaping metastatic latency and relapse.

Bioactive molecule library screening may empirically identify effective combination therapies, but molecular mechanisms underlying favorable drug–drug interactions often remain unclear, precluding further rational design. In the absence of an accepted systems theory to interrogate synergistic responses, we introduce Omics-Based Interaction Framework (OBIF) to reveal molecular drivers of synergy through integration of statistical and biological interactions in synergistic biological responses. OBIF performs full factorial analysis of feature expression data from single versus dual exposures to identify molecular clusters that reveal synergy-mediating pathways, functions and regulators. As a practical demonstration, OBIF analyzed transcriptomic and proteomic data of a dyad of immunostimulatory molecules that induces synergistic protection against influenza A and revealed unanticipated NF-κB/AP-1 cooperation that is required for antiviral protection. To demonstrate generalizability, OBIF analyzed data from a diverse array of Omics platforms and experimental conditions, successfully identifying the molecular clusters driving their synergistic responses. Hence, unlike existing synergy quantification and prediction methods, OBIF is a phenotype-driven systems model that supports multiplatform interrogation of synergy mechanisms.

BACKGROUND: Ovarian cancer (OCa) is the deadliest of all gynecologic cancers in the United States. Currently approved therapies have improved OCa survival for clinically localized disease, however, the majority (~90%) of patients with high-grade serous OCa (HGSOC) experience relapse with incurable metastases. There is a dire need for new therapeutic approaches. We hypothesized that the high basal endoplasmic reticulum stress (ERS) in OCa represents a critical and targetable vulnerability and may overcome the tumor heterogeneity. The objective of this project is to exploit increased ERS in ovarian cancer cells by engaging the novel target LIPA using the unique compound ERX-41. METHODS: The utility of ERX-41 as a new therapy was evaluated using MTT and CellTiter-Glo Cell Viability Assays. We used multiple established and patient derived OCa cell lines. The effect of ERX-41 on the Cell viability of patient-derived organoids (PDO) was measured using CellTiter-Glo 3D Assay. Long term effects of ERX-41 on cell survival were measured using colony formation assays. Apoptosis was measured using Annexin V and Caspase-Glo® 3/7 Assays. Cell cycle analysis was analyzed by Flow Cytometry. Mechanistic studies were done using LIPA knockout (KO) cells, RT-qPCR, and western blotting. Status of LIPA in OCa was determined using TNMplot database. In vivo efficacy of ERX-41 was tested using both cell line derived (CDX) and patient derived (PDXs) xenografts. RESULTS: TNM plot results showed that LIPA is highly expressed in OCa tumors compared to normal tissues and LIPA expression correlated with clinical grade. Kaplan-Meier plotter analyses of TCGA data revealed that LIPA expression is negatively correlated with overall survival in OCa patients. MTT and CellTitre-Glo assay results showed that ERX-41 significantly reduced the cell viability of both established and primary OCa cells, and PDO’s with an IC50 of ~500nM. ERX-41 treatment also significantly reduced the cell survival, increased S-phase arrest, and promoted apoptosis of OCa cells. A time course study revealed a robust and consistent induction of ERS markers (CHOP and sXBP1) in OCa cells by ERX-41 within 4h. Western blotting analyses also confirmed increased expression of ERS markers including CHOP, elF2α, PERK, and ATF4 upon ERX-41 treatment confirming that ERX-41 induces ERS. In xenograft studies, ERX-41 treatment resulted in ~66% reduction of tumor volume measured by Xenogen-IVIS. Further, in studies using PDX tumors, treatment with ERX-41 resulted in a significant reduction (~60%) of tumor volume and tumor weight. CONCLUSION: Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of action and implicate ERX-41 binding to LIPA induces ER stress, and apoptosis of OCa cells. Further molecular characterization of how ERX-41 binding to LIPA induces ER stress in OCa cells is ongoing. Citation Format: Alexia B. Collier, Suryavathi Viswanadhapalli, Tae-Kyung Lee, Kara Kassees, Karla Parra, Gaurav Sharma, Tanner Reese, Michael Hsieh, Xihui Liu, Xue Yang, Behnam Ebrahimi, Uday P. Pratap, Rahul Gopalam, Chia Yuan Chen, Scott Terry Elmore, Gangadhara Reddy Sareddy, Edward R. Kost, Jung-Mo Ahn, Ganesh V. Raj, Ratna K. Vadlamudi. Novel LIPA targeted therapy for treating ovarian cancer. [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 3986.