Targeting Bmi-1 suppresses cancer stemness and tumor growth in mucoepidermoid carcinoma

Salivary gland carcinomas display considerable histopathological diversity and mucoepidermoid carcinoma (MEC) is recognized as the most frequent malignant subtype. However, chemotherapy provides limited benefit in MEC, highlighting the need for novel therapeutic approaches. Recent studies have highlighted the impact of cancer stemness in treatment resistance and tumor progression. Bmi-1 is a polycomb group protein and master regulator of stemness in head and neck cancers and therefore is a potential therapeutic target. This study aimed to evaluate the impact of therapeutic Bmi-1 in salivary gland MEC. Three MEC cell lines (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) were treated with the Bmi-1 inhibitor PTC596 (Unesbulin), currently under clinical evaluation for solid tumors. Western blotting, flow cytometry, and sphere formation assays were performed. For Western blotting and flow cytometry, PTC596 was administered at 0-500 nM for 24-72 hours. Sphere assays in ultra-low attachment plates were performed with 0-100 nM for 10 days. Xenograft models were generated by implanting cell-seeded scaffolds of UM-HMC-3A and UM-HMC-3B into immunodeficient mice. When tumors reached 200 mm³, mice were randomized to receive vehicle, 5 mg/kg or 10 mg/kg PTC596 every three days for 30 days. Tumor volume and body weight were recorded throughout treatment. We observed dose- and time-dependent inhibition of Bmi-1 protein expression in all MEC cell lines following PTC596 exposure. Flow cytometry revealed marked reductions in the fraction of ALDH high CD44 high cells, indicating effective targeting of cancer stem cells. Sphere formation assays further confirmed a significant dose-dependent decrease in sphere-forming capacity. In vivo , PTC596 induced rapid tumor regression, with tumors shrinking and remaining stably suppressed thereafter. Both 5 mg/kg and 10 mg/kg PTC596 regimens produced comparable antitumor effects. Importantly, mice maintained or gained body weight, and no overt treatment-associated toxicity was observed. Notably, PTC596 ablated cancer stem cells (ALDH high CD44 high ) and produced antitumor responses in preclinical salivary gland MEC models. Collectively, these findings provide compelling preclinical evidence that Bmi-1 represents a promising and clinically relevant therapeutic target for salivary gland mucoepidermoid carcinoma, supporting further translational and clinical investigation.