Integrated Phenotypic and Transcriptomic Profiling Positions ONC212 as a Lead Imipridone in Androgen-Independent Prostate Cancer Models

Prostate cancer (PCa) remains lethal at advanced stages, partly due to stem-like subpopulations known as prostate cancer stem cells (PCSCs) that sustain tumor growth and therapeutic resistance. Imipridones are small-molecule anticancer agents, with next-generation derivatives ONC206 and ONC212 designed for enhanced potency and broader activity. This study compared their antitumor efficacy and mechanisms in advanced androgen-independent PCa (AIPC) models, namely DU145 and PC3 cells, using two- and three-dimensional systems encompassing bulk cancer cells and PCSCs. DU145 and PC3 AIPC cells were treated with ONC201 (parent compound), ONC206, or ONC212. Functional assays assessed proliferation, viability, migration, invasion, PCa spheroids formation, cell cycle distribution, and mitochondrial membrane potential and mass, while RNA sequencing defined transcriptional responses. ONC212 was the most potent derivative, inhibiting proliferation and migration and abolishing PCa spheroids at nanomolar doses, whereas ONC201 and ONC206 required higher concentrations. Transcriptomic analyses revealed shared repression of DNA replication and cell-cycle transition programs, with activation of integrated stress and unfolded protein responses (ISR/UPR) and FOXO signaling. ONC206 favored PERK–ATF4-mediated apoptosis with reduced DNA repair, while ONC212 more strongly impacted oxidative phosphorylation-related pathways and mitochondrial RNA processing. Imipridones induced a time-dependent cell-cycle redistribution with increased sub-G1 accumulation and modulated mitochondrial membrane potential and mass in a context-dependent manner. Collectively, these findings position ONC212 as a leading imipridone candidate in AIPC models, combining potent inhibition of tumor and stem-like cell functions with a coherent stress-response signature that supports further translational evaluation.