Chromobox2 inhibition: a novel activity of alisertib, an aurora A kinase inhibitor

Abstract Chromobox 2 (CBX2), a subunit of Polycomb Repressor Complex 1 (PRC1), is expressed in high-grade serous carcinoma. CBX2 inhibitory peptide (CBX2i) has demonstrated efficacy in a syngeneic mouse model, but has limitations. We sought to identify an alternative approach to CBX2 inhibition. A computational-based molecular docking screen was performed using the SelleckChem Bioactive library to identify inhibitors of CBX2. A similarity screen of top hits against the bound conformation of CBX2i pharmacophore model was performed in parallel. A series of in vitro validation studies evaluated the effect of alisertib on proliferation, a CBX2 target gene, and stemness. CBX2 knockdown cell lines and a syngeneic murine model were utilized to evaluate alisertib response in the context of CBX2 loss. Cell target engagement assay was performed. PRC1-activity was measured by H2AK119ub levels. Immune profiling of treated tumors defined the immune microenvironment. The computational-based screen identified 10 candidate compounds. In vitro validation narrowed compounds of interest to raltitrexed, alisertib, GTX-007, LY315920, and PD0325901. Ultra-low dilution assay demonstrated dramatic decrease in spheroid formation with alisertib, an aurora A kinase (AURKA) inhibitor. Good structural overlap was observed between CBX2i and alisertib. Cell target engagement assay confirmed alisertib selectivity for both aurora A kinase and CBX2. Loss of CBX2 attenuated alisertib efficacy in vitro and in vivo. Treatment with alisertib leads to decrease in H2AK119ub and shift in the immune tumor microenvironment. Alisertib efficacy in HGSC is dependent on functional CBX2 and cell target engagement confirms selectivity for CBX2, supporting that alisertib activity involves CBX2 inhibition.