Abstract 6227: ABT-199 and Bortezomib synergistically induce apoptosis in soft-tissue sarcomas

Abstract

Abstract Soft-Tissue Sarcomas (STS) are mesenchymal malignancies with high heterogeneity that predominantly affect children and young adults. Despite routinely applied therapy strategies including radiotherapy, surgery and chemotherapy, the five-year survival rate of metastatic STS is only 50 %. Therefore, it is of high importance to focus on possible combinational therapies for the effective treatment of all kinds of STS. For such a therapy we combined the clinically approved BH3-mimetic drug ABT-199 (Venetoclax) with the proteasome inhibitor Bortezomib (Velcade). ABT-199 selectively inhibits the anti-apoptotic protein Bcl-2 whereas the proteasome inhibitor Bortezomib is effective, e.g., in multiple myeloma. Sarcoma cell lines were incubated with ABT-199 and Bortezomib alone or in combination and apoptotic cell death was flow cytometric detected by analysis of mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin V). To elucidate an underlying mechanism for apoptosis induction, we analyzed expression of members of the Bcl-2 family involved in the apoptosis pathway by Western Blotting and performed analogue experiments in knock-out cell lines. Combined treatment with ABT-199 and Bortezomib showed synergistic cell death induction in several sarcoma cell lines including Rhabdomyosarcoma, Leiomyosarcoma and Synovial sarcoma. Loss of mitochondrial membrane potential and Annexin V staining revealed apoptosis as the underlying cell death mechanism. Interestingly, expression of Bok, a homologue of the pore-forming effector proteins Bax and Bak, was increased in response to ABT-199 and Bortezomib. Also, expression was increased for the BH3-only protein Noxa and its anti-apoptotic interaction partner Mcl-1. Knock-out (KO) of effector proteins Bax, Bak or Bok in the sarcoma cell line SW982 reduced apoptosis induction by ABT-199/Bortezomib with the most pronounced reduction in SW982/Bax-KO. Additional shRNA mediated knock-down of Noxa in Bax-KO, Bak-KO or Bok-KO SW982 cells further reduced apoptosis compared to Noxa knock-down alone. ABT-199 and Bortezomib synergistically induce apoptotic cell death in various sarcoma cell lines concomitant with enhanced expression of the Bcl-2 proteins Bok, Noxa and Mcl-1. Hence, we suggest a mechanism in which the simultaneous inhibition of anti-apoptotic Bcl-2 by ABT-199 and the stabilization of pro-apoptotic proteins Bok and/or Noxa shift the equilibrium of BCL-2-proteins towards apoptosis. Experiments in Bax, Bak, Bok, and Noxa deficient SW982 cells indicate that Bax and Noxa are crucial for the observed synergistic effect. based on these results we propose the combined treatment with ABT-199 and Bortezomib as a new and highly promising therapy option for advanced STS. Future efforts, e.g. simultaneous knock-out of relevant Bcl-2 proteins, will unravel the detailed underlying mechanism of the observed synergistic cell death induction by ABT-199 and Bortezomib. Citation Format: Sandra Weller, Alina Muenchow, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. ABT-199 and Bortezomib synergistically induce apoptosis in soft-tissue sarcomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6227.