Abstract PS5-03-27: Multifaceted Therapeutic Approaches Targeting Oncogenic Pathways to Combat Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype, characterized by the lack of targetable bioreceptors, high chemoresistance, metastasis and limited treatment options. Therefore, identifying novel biological targets and therapeutic avenues is crucial to improve TNBC outcomes. It is known that the plasticity of key signaling pathways like Wnt and MAPK, contributes to TNBC progression and metastasis. In the efforts to identify actionable targets, we performed a high-throughput analysis of TNBC microarray datasets and identified Maternal embryonic leucine zipper kinase (MELK), a component of MAPK pathway, to be significantly upregulated, with high MELK expression correlated with poor overall survival of TNBC patients. Additionally, our bioinformatic and RT-PCR analyses identified Pigment epithelium-derived factor (PEDF), a suppressor of the Wnt pathway through the inhibition of its co-receptor LRP6, to be significantly downregulated in TNBC cells. Building on these findings, our current study aims to target these dysregulated pathways by leveraging drug repurposing approach to target MELK in the MAPK pathway and PEDF recombinant protein therapy to restore Wnt suppression in TNBC. Methods: To inhibit MELK, an FDA-approved drug library was screened utilizing molecular docking, molecular dynamics simulations (MDS), and MMPBSA energy calculations. The top drug candidates were validated for their cellular activity using functional assays, flow cytometry, RT-PCR, and western blot analyses in TNBC cell lines (MDA-MB-231 and MDA-MB-468). The recombinant PEDF (rPEDF) protein was produced in E.coli with His-tag for purification using affinity chromatography and characterized by circular dichroism (CD) and MALDI-TOF biophysical analyses. The effect of rPEDF in EMT-induced TNBC cell lines and free-floating generated 3D spheroid cultures was evaluated by MTT assay, scratch assay, matrigel invasion assay and cellular signaling by RT-PCR, western blot and confocal microscopy. Results: Virtual Screening of a library of 1293 FDA-approved drugs and MDS parameters like RMSD, RMSF and pair distance revealed that the top 10 drugs (binding energy -67.75 kJ/mol) were strongly stabilized in the kinase domain of MELK. The top two candidates: Netarsudil and Dutasteride, led to a dose-dependent reduction in cell proliferation, migration, G1-phase cell cycle arrest, significant reduction of MELK expression and its target genes, including cyclin B1, cyclin D1, p21 and p27 in TNBC cell lines. In protein therapy, MALDI-TOF confirmed the purified rPEDF had the legitimate 54 kDa molecular weight, and CD demonstrated the protein retained its secondary structure integrity. rPEDF treatment demonstrated anti-proliferative effects in EMT-induced TNBC cells and 3D tumor spheroids along with increased intracellular ROS and mitochondrial membrane depolarization. A significant decrease in colony and sphere formation, invasion and migration ability of TNBC cells was also observed following rPEDF treatment. The reduction in migration ability was corroborated by significant reduction in EMT markers and Wnt signaling proteins: N-cadherin, Vimentin, and β-catenin, underscoring the role of rPEDF in suppressing Wnt pathway-mediated TNBC survival and metastasis. Conclusion: Our findings accentuate the translational potential of repurposed drugs Netarsudil and Dutasteride and rPDEF protein as novel therapeutic agents, effectively targeting the MAPK and Wnt pathways, leading to reduced proliferation and metastasis in TNBC cell lines. By leveraging these approaches, including ongoing in vivo validation, our research aims to bridge the gap between identification of therapeutic targets and development of tailored treatment options, facilitating future clinical investigations in TNBC. Citation Format: A. Arora, S. S. Ghosh. Multifaceted Therapeutic Approaches Targeting Oncogenic Pathways to Combat Triple-Negative Breast Cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS5-03-27.