High throughput screening identifies novel drugs for calreticulin surface translocation in pancreatic ductal adenocarcinoma

Background: Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related deaths in the United States and is projected to increase within the next decade due to its poor prognosis and increasing incidence. There is an urgent need to develop more effective therapeutic options and detection strategies to improve survival outcomes for PDAC patients. Calreticulin (CALR), an endoplasmic reticulum protein, is known to translocate to the cell surface following cellular stress. Surface-expressed CALR holds promise for cancer detection or therapeutic delivery. To explore this potential, we performed high-throughput screening to identify compounds which induce surface CALR expression. Methods: Two human PDAC cell lines (Panc1, MIA Pa Ca-2) were engineered using the Nano-Glo HiBiT Extracellular Detection System to stably integrate HiBiT into the CALR gene. A high throughput screen for surface CALR-inducing compounds was performed at the National Center for Advancing Translational Sciences (NCATS). Cell lines expressing HiBiT-CALR were treated with approximately 3,000 drugs from the Mechanism interrogation PlatEs (MIPE) library for 24 or 48 hours and then assessed for cell viability using CellTiter-Glo and surface CALR levels by luminescence. Surface HiBiT-CALR and cell viability signals were quantified using a ViewLux microplate imager and automated processes utilizing the XTS Staubli UniVAL robotic system. The top ten drugs demonstrating high CALR signal (AUC) with minimal toxicity were validated for surface CALR induction by flowcytometry. Cells were also analyzed for viability using MTT assay, with P < 0.05 considered significant. Results: Out of the compounds evaluated, 102 compounds showed increased CALR surface expression and were selected for secondary screening, from which ten were further chosen for in-lab validation. Among these, bisindolylmaleimide IV and AEM1 were identified to reliably increase CALR. Flow cytometry analysis confirmed significant increases in live surface CALR+ cells, with bisindolylmaleimide IV (1.1µM) increasing CALR by 1.7-fold in MIA Pa Ca-2 and 1.32-fold in Panc1, while AEM1 (10µM) increased CALR by 7-fold in MIA PaCa-2 and 3.7-fold in PANC-1. Viability assays confirmed low cytotoxicity for bisindolylmaleimide IV (1.1 µM), with 77% and 84% cell viability observed in MIA PaCa-2 and PANC-1 cells, respectively. Similarly, AEM1 (10 µM) showed 81% viability in MIA PaCa-2 and 76% in PANC-1 cells. Conclusion: Our results identified the potential of two novel drugs, bisindolylmaleimide IV and AEM1, as promising candidates for enhancing surface CALR expression, offering new avenues for PDAC detection and tumor specific delivery of therapies. Keywords: Calreticulin, pancreatic ductal adenocarcinoma, drug discovery, therapeutic delivery, HiBiT-CALR.