Synergistic induction of apoptosis by a polo-like kinase 1 inhibitor and microtubule-interfering drugs in Ewing sarcoma cells.

Since polo-like kinase 1 (PLK1) is highly expressed in Ewing sarcoma (ES), we evaluated the therapeutic potential of the PLK1 inhibitor BI 6727. Here, we identify a synergistic induction of apoptosis by BI 6727 and several microtubule-interfering drugs in ES cells, including vincristine (VCR), vinblastine (VBL), vinorelbine (VNR) and eribulin. Synergistic drug interaction is confirmed by calculation of combination index (CI). Also, BI 6727 and VCR act in concert to reduce long-term clonogenic survival. Mechanistically, BI 6727/VCR co-treatment cooperates to trigger mitotic arrest, phosphorylation of BCL-2 and BCL-XL and downregulation of MCL-1. This inactivation of anti-apoptotic BCL-2 family proteins in turn promotes activation of BAX and BAK, activation of caspase-9 and -3 and caspase-dependent apoptosis. Overexpression of BCL-2 or simultaneous knockdown of BAX and BAK significantly rescue BI 6727/VCR-induced apoptosis, indicating that engagement of the mitochondrial pathway is critical for BI 6727/VCR-mediated apoptosis. The clinical relevance of PLK1 inhibitor-based combination therapies is underscored by the fact that BI 6727 is currently evaluated in phase I clinical trials in childhood cancer. In conclusion, PLK1 inhibitors such as BI 6727 may provide a new strategy to chemosensitize ES.

PARP Inhibitors Sensitize Ewing Sarcoma Cells to Temozolomide-Induced Apoptosis via the Mitochondrial Pathway.

Ewing sarcoma has recently been reported to be sensitive to poly(ADP)-ribose polymerase (PARP) inhibitors. Searching for synergistic drug combinations, we tested several PARP inhibitors (talazoparib, niraparib, olaparib, veliparib) together with chemotherapeutics. Here, we report that PARP inhibitors synergize with temozolomide (TMZ) or SN-38 to induce apoptosis and also somewhat enhance the cytotoxicity of doxorubicin, etoposide, or ifosfamide, whereas actinomycin D and vincristine show little synergism. Furthermore, triple therapy of olaparib, TMZ, and SN-38 is significantly more effective compared with double or monotherapy. Mechanistic studies revealed that the mitochondrial pathway of apoptosis plays a critical role in mediating the synergy of PARP inhibition and TMZ. We show that subsequent to DNA damage-imposed checkpoint activation and G2 cell-cycle arrest, olaparib/TMZ cotreatment causes downregulation of the antiapoptotic protein MCL-1, followed by activation of the proapoptotic proteins BAX and BAK, mitochondrial outer membrane permeabilization (MOMP), activation of caspases, and caspase-dependent cell death. Overexpression of a nondegradable MCL-1 mutant or BCL-2, knockdown of NOXA or BAX and BAK, or the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) all significantly reduce olaparib/TMZ-mediated apoptosis. These findings emphasize the role of PARP inhibitors for chemosensitization of Ewing sarcoma with important implications for further (pre)clinical studies.