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A Bifunctional PARP-HDAC Inhibitor with Activity in Ewing Sarcoma
Louise Ramos1,2,3, Sarah Truong2,3, Beibei Zhai1,2,3
1Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
Purpose:
Histone deacetylase (HDAC) inhibition has been shown to induce pharmacologic "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 (PARPi). Here, we report the concept and characterization of a novel bifunctional PARPi (kt-3283) with dual activity toward PARP1/2 and HDAC enzymes in Ewing sarcoma cells.
Experimental Design:
Inhibition of PARP1/2 and HDAC 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).
Results:
Compared with 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.
Conclusions:
Our data demonstrate 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 bifunctional single-molecule therapeutic strategy.
Insights
A novel bifunctional inhibitor, kt-3283, targets both PARP and HDAC enzymes, showing enhanced efficacy against Ewing sarcoma. This dual-action molecule offers a promising single-drug strategy for treating this rare cancer.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Histone deacetylase (HDAC) inhibition can induce "BRCAness," a state of synthetic lethality in cancers with proficient DNA repair.
- Combining HDAC and poly (ADP-ribose) polymerase (PARP) inhibitors is a strategy for cancers resistant to single-agent PARP inhibitors (PARPi).
Purpose of the Study:
- To report the development and characterization of a novel bifunctional inhibitor, kt-3283, with dual PARP1/2 and HDAC activity.
- To evaluate the efficacy of kt-3283 in Ewing sarcoma models.
Main Methods:
- Assays for PARP1/2, HDAC activity, and PAR formation.
- Cytotoxicity, cell-cycle, and DNA damage assessments (γH2AX, comet assay).
- Evaluation of metastatic potential using an ex vivo pulmonary metastasis assay (PuMA).
Main Results:
- Kt-3283 demonstrated superior cytotoxicity compared to FDA-approved PARPi (olaparib) and HDAC inhibitors (vorinostat) in Ewing sarcoma models.
- Kt-3283 induced significant S and G2-M cell-cycle arrest and elevated DNA damage at nanomolar concentrations.
- Kt-3283 inhibited Ewing sarcoma cell colonization in the ex vivo PuMA model.
Conclusions:
- Kt-3283 provides preclinical justification for a clinical trial of dual PARP and HDAC inhibition in Ewing sarcoma.
- This study presents proof-of-concept for a single-molecule bifunctional therapeutic strategy targeting Ewing sarcoma.
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