A bi-functional PARP-HDAC inhibitor with activity in Ewing sarcoma

Histone deacetylase (HDAC) inhibition has been shown to induce pharmacological "BRCAness" in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and poly-(ADP-ribose)-polymerase (PARP) inhibition in cancer types that are insensitive to single-agent PARP inhibitors. Here, we report the concept and characterization of a novel bi-functional PARP inhibitor (kt-3283) with dual activity towards PARP1/2 and HDAC enzymes in Ewing sarcoma cells. Inhibition of PARP1/2 and HDACs was measured using PARP1/2, HDAC activity, and PAR formation assays. Cytotoxicity was assessed by IncuCyte live cell imaging, CellTiter-Glo®, and spheroid assays. Cell cycle profiles were determined using propidium iodide staining and flow cytometry. DNA damage was examined by γH2AX expression and comet assay. Inhibition of metastatic potential by kt-3283 was evaluated via ex vivo pulmonary metastasis assay (PuMA). Compared to FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, kt-3283 displayed enhanced cytotoxicity in Ewing sarcoma models. The kt-3283-induced cytotoxicity was associated with strong S and G2/M cell cycle arrest in nanomolar concentration range and elevated DNA damage as assessed by γH2AX tracking and comet assays. In three-dimensional spheroid models of Ewing sarcoma, kt-3283 showed efficacy in lower concentrations than olaparib and vorinostat and kt-3283 inhibited colonization of Ewing sarcoma cells in the ex vivo PuMA model. Our data demonstrates the preclinical justification for studying the benefit of dual PARP and HDAC inhibition in the treatment of Ewing sarcoma in a clinical trial and provides proof-of-concept for a bi-functional single-molecule therapeutic strategy.