S1: Results from AK306 testing on the NCI60 cancer cell line panel. Testing was done by the National Institutes of Health Developmental Therapeutics Program. S2: AK3-BODIPY arrests YAMC cells in mitosis at 600 and 800 nM. YAMCs were treated with compound overnight and then assayed for mitotic arrest by staining for phospho-histone H3. S3: Tumor-bearing Apc�14/+ mice were treated with five injections of AK306 (30 mg/kg) over the course of three days. Animals were sacrificed and tissue analyzed for PCNA staining (A) and phospho-histone H3 staining (B). Representative staining images are shown in the right panels, with quantified staining shown in the left panels.
ARTICLE ABSTRACT
AK3 compounds are mitotic arrest agents that induce high levels of γH2AX during mitosis and apoptosis following release from arrest. We synthesized a potent AK3 derivative, AK306, that induced arrest and apoptosis of the HCT116 colon cancer cell line with an EC50 of approximately 50 nmol/L. AK306 was active on a broad spectrum of cancer cell lines with total growth inhibition values ranging from approximately 25 nmol/L to 25 μmol/L. Using biotin and BODIPY-linked derivatives of AK306, binding to clathrin heavy chain (CLTC/CHC) was observed, a protein with roles in endocytosis and mitosis. AK306 inhibited mitosis and endocytosis, while disrupting CHC cellular localization. Cells arrested in mitosis by AK306 showed the formation of multiple microtubule-organizing centers consisting of pericentrin, γ-tubulin, and Aurora A foci, without apparent centrosome amplification. Cells released from AK306 arrest were unable to form bipolar spindles, unlike nocodazole-released cells that reformed spindles and completed division. Like AK306, CHC siRNA knockdown disrupted spindle formation and activated p53. A short-term (3-day) treatment of tumor-bearing APC-mutant mice with AK306 increased apoptosis in tumors, but not normal mucosa. These findings indicate that targeting the mitotic CHC complex can selectively induce apoptosis and may have therapeutic value.Implication: Disruption of clathrin with a small-molecule inhibitor, AK306, selectively induces apoptosis in cancer cells by disrupting bipolar spindle formation. Mol Cancer Res; 16(9); 1361–72. ©2018 AACR.
