A novel dual PARP-HDAC inhibitor for treatment of Ewing sarcoma

Background: 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), but clinical trials with olaparib failed to show significant clinical benefit. 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 correlate with more malignant phenotypes in sarcomas and inhibition of HDAC in pre-clinical models of ES inhibits tumor growth. HDAC inhibition combined with PARP inhibition has promising results in vitro, however clinically, combination therapies often require complicated sequential dosing protocols due to different pharmacokinetic profiles and overlapping toxicities, that severely limits clinical utility. Here, we report the efficacy of a novel bifunctional small-molecule compound designed to have both PARP and HDAC inhibition activity. Methods: PARP1 and PARP2 activity was measured using the Trevigen Universal Colorimetric PARP Assay Kit, the BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and by PARylation assays. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50 s were determined using live cell imaging with an Incucyte S3 system and the CellTiter Glo viability assay. DNA damage was detected by western blot, immuno-fluorescence, and comet assays. 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 increased DNA damage and S and G2/M cell cycle arrest as 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 offer a novel treatment opportunity for Ewing sarcoma and other solid tumors with limited responses to PARPi monotherapies. Conflict of interest: Board of Directors: J. Bacha and D. Brown are directors of Rakovina Therapeutics. Corporate-sponsored Research: S. Truong, B. Zhai, F. Ghaidi, L. Ramos, J. Joshi are employees of Rakovina Therapeutics. J. Langlands, M. Daugaard and W. Shen are consultants to Rakovina Therapeutics.