Abemaciclib Induces G1 Arrest and Lysosomal Dysfunction in Canine Melanoma Cells: Synergistic Effects with Fenbendazole

Se-Hoon Kim¹Jun-Yeol Choi¹Yoon-Ho Suh¹Ki-Hoon Song²Min-Ok Ryu¹Kyoung-Won Seo^1*

• ¹Seoul National University, Seoul, Republic of Korea
• ²ViroCure Inc., Seoul, Republic of Korea

Abemaciclib, a CDK4/6 inhibitor, is well-established in hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+/HER2-) human breast cancer treatment by inducing G1 cell cycle arrest. However, the potential anti-tumor effects of abemaciclib in other cancers, including canine melanoma, remain underexplored. In canine melanoma, CDK4/6 copy number gains and cell cycle alterations have been reported, suggesting potential abemaciclib targets. Additionally, we previously identified mitotic catastrophe and senescence in fenbendazole-treated canine melanoma cells, suggesting cell cycle defects. This study investigated the antitumor effects of abemaciclib in canine melanoma cell lines (CMeC1, KMeC, LMeC, UCDK9M4, and UCDK9M5) and evaluated its synergistic interaction with fenbendazole in vitro and in vivo. Abemaciclib inhibited cell proliferation, as shown using Cell Counting Kit-8 assays, and reduced migration, as demonstrated using wound healing assays. Flow cytometry further confirmed that abemaciclib induces G1 cell cycle arrest and altered the expression of cell cycle regulatory genes and proteins, as shown using real-time polymerase chain reaction (RT-PCR) and western blotting. Autophagy induction, but not apoptosis, was observed using western blot. Morphological studies revealed vacuolization, with FITC-dextran studies confirming a non-extracellular origin. Co-treatment with V-ATPase inhibitors confirmed that lysosomes were the source of vacuolization. Additionally, accumulation of p62 observed in immunofluorescence analysis suggested impaired autophagy flux. Co-treatment with fenbendazole enhanced cytotoxicity and demonstrated a synergistic effect at specific dose ranges, as indicated by Highest Single Agent (HSA) synergy score. In vivo studies confirmed that abemaciclib regressed tumors, as well as in combination with fenbendazole. These findings highlight the potential of abemaciclib, and its synergy with fenbendazole, as a promising strategy for treating canine melanoma.