Abstract A024: In vitro efficacy of a novel dual PARP-HDAC inhibitor in ewing sarcoma
Authors: Truong Sarah , Zhai Beibei , Ghaidi Fariba , Ramos Louise , Joshi Jay , Brown Dennis , Sankar Neil , Langlands John , Bacha Jeffrey , Shen Wang , Sorensen Poul , Daugaard Mads 
Published: September 2022

Abstract
Introduction: Inhibition of poly-adenosine diphosphate-ribose polymerase (PARP) is an effective therapy against cancers with DNA damage repair (DDR) deficiencies, such as BRCA1 and BRCA2 defects. In preclinical studies, PARP inhibitors demonstrated potential therapeutic value in Ewing sarcoma (ES), though clinical trials with olaparib failed to show significant clinical benefit. While single agent therapy proved inefficacious in the clinical treatment of ES, combination therapies may show anti-tumour activity. A key regulatory event in DNA damage repair is acetylation and deacetylation of histones, controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Increased expression of HDACs have been correlated to more malignant phenotypes in sarcomas and inhibition of HDAC in ES has been shown to be effective in inhibiting tumor growth. HDAC inhibition combined with PARP inhibition has been shown to sensitize cells to treatment in vitro, however clinically, combination therapies often require sequential administration due to different pharmacokinetic profiles and overlapping toxicities, severely limiting clinical utility. Here, we evaluate the activity and efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibiting activity. Methods: PARP1 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit and PARP2 activity was measured using the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit. HDAC activity was measured using HeLa nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. Accumulation of phospho-histone H2AX (pH2AX) was detected by western blot using anti-phospho histone H2AX (Ser139) antibody from Cell Signaling Technologies. Results: A representative compound from the kt-3000 series showed potent inhibition of PARP1 and PARP2 with IC50 values in the low nM range, comparable to FDA-approved PARP inhibitors. The compound also showed inhibition of HDAC enzymes with IC50 values in the low µM range, slightly lower than the FDA-approved HDAC inhibitor, vorinostat. Cell survival EC50 values were superior to olaparib in ES cell lines in vitro. Treatment with the kt-3000 compound also resulted in the increased accumulation of pH2AX by western blot and increased S and G2/M cell cycle arrest compared to olaparib. Conclusion: Our kt-3000 compound shows potent inhibition of PARP1, PARP2, and HDAC, as well as induction of DNA damage and cell cycle arrest. Further development of these bifunctional single molecule inhibitors may result in a novel treatment opportunity for Ewing sarcoma.
Citation Format: Sarah Truong, Beibei Zhai, Fariba Ghaidi, Louise Ramos, Jay Joshi, Dennis Brown, Neil Sankar, John Langlands, Jeffrey Bacha, Wang Shen, Poul Sorensen, Mads Daugaard. In vitro efficacy of a novel dual PARP-HDAC inhibitor in ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A024.