Department of Urologic Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
Vancouver Prostate Centre, Vancouver, British Columbia, Canada.
Clin Cancer Res. 2023 Sep 1;29(17):3541-3553. doi: 10.1158/1078-0432.CCR-22-3897.
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.
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).
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.
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.
已有研究表明，组蛋白去乙酰化酶（HDAC）抑制可诱导具有健全 DNA 修复活性的癌细胞产生药理学上的“BRCAness”。这为探索在对单药 PARP 抑制剂（PARPi）不敏感的癌症类型中联合使用 HDAC 和 PARP 抑制剂的治疗方法提供了理论依据。在这里，我们报告了一种新型双功能 PARPi（kt-3283）的概念和特征，该化合物对 PARP1/2 和 Ewing 肉瘤细胞中的 HDAC 酶具有双重活性。
使用 PARP1/2、HDAC 活性和 PAR 形成测定法来测定 PARP1/2 和 HDAC 的抑制作用。通过 IncuCyte 活细胞成像、CellTiter-Glo 和球体测定法评估细胞毒性。通过碘化丙啶染色和流式细胞术确定细胞周期谱。通过 γH2AX 表达和彗星试验检查 DNA 损伤。通过体外肺转移测定（PuMA）评估 kt-3283 对转移潜力的抑制作用。
与 FDA 批准的 PARP（奥拉帕利）和 HDAC（伏立诺他）抑制剂相比，kt-3283 在 Ewing 肉瘤模型中显示出更强的细胞毒性。kt-3283 诱导的细胞毒性与在纳摩尔浓度范围内强烈的 S 和 G2-M 细胞周期阻滞以及通过 γH2AX 跟踪和彗星试验评估的升高的 DNA 损伤相关。在 Ewing 肉瘤的三维球体模型中，kt-3283 在比奥拉帕利和伏立诺他更低的浓度下显示出疗效，并且 kt-3283 抑制了 Ewing 肉瘤细胞在体外 PuMA 模型中的定植。
我们的数据证明了在临床试验中研究双重 PARP 和 HDAC 抑制在治疗 Ewing 肉瘤中的益处的临床前合理性，并为一种双功能单分子治疗策略提供了概念验证。
