350 Poly (ADP-ribose) Polymerase-1 (PARP-1) Inhibitors Potentiate Trabectedin Activity in Preclinical Models of Bone and Soft Tissue Sarcomas

Abstract

Background: Trabectedin (TR) is a DNA-binding alkaloid approved in Europe for the treatment of soft tissue sarcoma as second/third line therapy. TR binds to the minor groove of DNA and interferes with gene transcription and nucleotide excision repair, inducing DNA double strand breaks (DSBs), and S/G2 cell cycle arrest. There is a strong clinical interest to increase TR activity by combination with other anti-cancer drugs. PARP-1 inhibitors (PARP1-i) which disable DNA base-excision repair mechanism causing the accumulation of DSBs, look like a worth to explore TR therapeutic partners. We focused our in vitro studies on the effects of the combination of TR with the PARP1-i olaparib (OL) and veliparib (VEL). Methods:We explored DNA damage by comet assay and phospho-histone H2AX determination and PARP-1 activity by polyADPribosylation assay in a panel of sarcoma cell lines treated with TR, OL,VEL and combinations. We evaluated cell viability after 72h treatment with escalating doses of TR (0−2nM), PARP1-i (VEL:0−80mcM; OL:0−20mcM), and their constant combinations. Following colony formation, cell cycle, apoptosis and DNA damage response were checked. Results: We demonstrated that TR-induced DNA damage and PARP1 activation in sarcoma sensitive cell line. The addition of PARP1i completely blocked basal and TR-induced PARP-1 activation. DNA damage response and checkpoints (ATM, ATR, BRCA1, CHK1, CHK2, p53) are activated after treatments with single agents and combinations inducing cell cycle arrest in S/G2 phase. This block was release after 48h in single agents-treated cells but not in combinations-treated cells leading to apoptotic cell death. Colony growth assay and viability tests revealed a strong synergism of the two drugs (Combination Index leyomiosarcoma > osteosarcoma undifferentiated pleomorphic > fibrosarcoma the most resistant). Determination of molecular markers of sensitivity and in vivo antitumoral activity studies are ongoing. Conclusions: Our results validate the biological rationale to combine TR and PARP1-i in sarcomas and suggest exploring this pharmacological approach in the clinical setting.