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 PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors. Here, we report the concept and characterization of a novel bifunctional PARP inhibitor (kt-3283) with dual activity towards PARP1/2 and HDAC enzymes in Ewing sarcoma cells. Compared to the 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 a strong S and G2/M cell cycle arrest in the 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 an ex vivo lung metastasis model. In summary, our data indicate a potential benefit of dual PARP and HDAC inhibition in the treatment of Ewing sarcoma and provide proof-of-concept for a bi-functional single-molecule therapeutic strategy.
Introduction: Poly(ADP-ribose) polymerase (PARP) plays a major role in DNA repair and PARP inhibitors (PARPi) have shown promise in pre-clinical studies for the treatment of Ewing sarcoma (ES). While a clinical trial using olaparib as a single agent failed to show significant response against ES, combination therapies with PARPi have emerged as an area of interest. Deacetylation of histones, controlled by histone deacetylases (HDACs) is a key regulatory event in DNA repair and inhibition of HDACs has been shown to reduce ES tumor growth in vitro and in vivo. PARP inhibition combined with HDAC inhibition has demonstrated enhanced efficacy in pre-clinical studies in various tumor indications, and a clinical trial of olaparib and vorinostat combination therapy against metastatic breast cancer is currently ongoing. However, combination therapies can be limited in clinical utility due to overlapping toxicities and different pharmacokinetic profiles. Here, we report the efficacy of a novel bifunctional small-molecule compound, kt-3283, designed to have both PARP and HDAC inhibitory activities. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte® S3 system and CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by western blot, immunofluorescence, and comet assay. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module and inhibition of metastases was assessed in a PUMA ES mouse model. Results and discussion: Kt-3283 showed potent inhibition of PARP1/2 activity and PAR synthesis with IC50 values comparable to olaparib. Kt-3283 also showed inhibition of HDACs with an IC50 value in the low µM range. Cell survival EC50 values for the compound were also superior to those of olaparib and vorinostat in ES cell lines. Cell cycle and DNA damage analyses indicated S/G2/M cell cycle arrest and strong DNA damage upon treatment with kt-3283 at lower concentration range compared to olaparib and vorinostat. This compound also exhibited potent inhibition of 3D spheroid growth of ES cells with low µM EC50 values, and inhibited metastatic growth in a PUMA mouse model. Conclusion: Kt-3283 shows potent inhibition of PARP1/2 and HDAC activities. It induces S and G2/M cell cycle arrest and DNA damage, and inhibits 3D spheroid growth and metastatic potential of ES cells. Further investigation of this bifunctional single-molecule inhibitor may offer a novel treatment opportunity for ES and other solid tumors with limited responses to PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Jay Joshi, Fariba Ghaidi, Michael M. Lizardo, Taras Shyp, John Langlands, Dennis Brown, Jeffrey Bacha, Poul Sorensen, Wang Shen, Mads Daugaard. A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6194.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.