Abstract C054: PARP16 modulates MYC expression and susceptibility of Ewing’s Sarcoma cells to PARP1 inhibition

Abstract

Abstract BACKGROUND: Ewing’s sarcoma (EWS) is a highly aggressive bone and soft tissue tumor with poor prognosis and an urgent need for novel therapies. We have identified that silencing of mono-ADP-ribosyltransferase PARP16 enhanced sensitivity of EWS cell lines to PARP1 inhibition. In this study, we explored the molecular mechanism of EWS vulnerability towards PARP16 targeting, alone and in conjunction with PARP1 inhibition, which may lead to development of PARP16-selective or dual targeting PARP1/16 inhibitors as new drugs for EWS. METHODS: The effect of PARP16 on cell viability was analyzed using RNA interference with various siRNAs and doxycycline-inducible PARP16 shRNAs, as well as CRISPR/Cas9-mediated genomic deletion. The changes on total protein expression upon silencing of PARP16 with or without PARP1 inhibition was determined by mass spectrometry (MS). Protein interaction partners and downstream effectors of PARP16 were determined by Co-IP/MS and ADP-ribosylation proteomics, respectively. RESULTS: Transient knockdown of PARP16 significantly reduced cell survival in EWS cell lines, particularly in combination with olaparib. Quantitative protein expression analysis by MS suggested doxycycline-induced shRNA-mediated PARP16 knockdown significantly reduced MYC protein expression in addition to PARP16 itself. Depletion of PARP16 in Cas9-expressing SK-N-MC EWS cells using different lentiviral sgRNAs further confirmed that targeting of PARP16 reduces cell survival and MYC expression, particularly in combination with PARP1 inhibition. Co-IP/MS using GFP-Trap agarose beads in mGFP/mGFP-PARP16 expressing cells suggested interaction of PARP16 with proteins that regulate post-translational modification of MYC in EWS cells. Further validation is ongoing. CONCLUSION: Our data suggest that targeting PARP16 confers vulnerability to EWS cells either alone or in conjunction with PARP1 inhibition and that it is involved in regulation of MYC. Identification of novel PARP16 substrates and interaction partners is expected to produce molecular markers that enable the development of novel PARP16 inhibitors. Citation Format: Ou Deng, Xueli Li, Vinayak C Palve, Bin Fang, Damon R Reed, Uwe Rix. PARP16 modulates MYC expression and susceptibility of Ewing’s Sarcoma cells to PARP1 inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C054.