Abstract 1398: Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199

Abstract

Abstract Metastatic Soft-Tissue Sarcomas (STS) are a rare and highly heterogeneous group of mesenchymal malignancies that carry a poor prognosis. Therapy is hampered by a limited number of effective treatments, and the almost non-existing long-term survival rate illustrates the need of effective targeted treatment. As a potential approach, we investigated combination of the clinically approved BH3-mimetic drug ABT-199 with different proteasome inhibitors (PIs): Bortezomib, Carfilzomib and Ixazomib, each with proven efficacy, e.g., in multiple myeloma. ABT-199 selectively inhibits the anti-apoptotic protein Bcl-2 while Bortezomib (BOZ) and Ixazomib (IXZ) bind reversible to the β1 and β5 subunits of the 20S proteasome and Carfilzomib (CFZ) irreversibly blocks the subunits β2 and β5. We investigated whether the combination of ABT-199 with CFZ or IXZ shows synergistic activity as recently published for BOZ. SW982 sarcoma cells were cultured with PIs alone or in combination with ABT-199. Cell death was detected by flow cytometric analysis of mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin V). To elucidate potential differences due to specificity of PIs, we analyzed expression of BCL-2 family proteins by Western Blot and performed analogue experiments in knock-out (BAXKO, BAKKO, BOKKO) cell lines. In combination, ABT-199&BOZ or ABT-199&CFZ showed comparable synergistic cell death induction in SW982 while ABT-199&IXZ less efficiently induced cell death. Engineered SW982 knock-out cell lines suggests specific relevance of BOK in IXZ induced apoptosis, whereas all tested PIs crucially depend on BAX for apoptosis induction in combination with ABT-199. Also, as shown for ABT-199&BOZ, both CFZ and IXZ, alone and in combination with ABT-199, efficiently induced expression of NOXA. Strikingly, and in line with augmented cell death induction, ABT-199&CFZ resulted in highest expression of NOXA as compared to BOZ and IXZ. ABT-199&PIs synergistically induce apoptotic cell death in SW982 and corresponding knock-out cell lines with CFZ showing exacerbated expression of NOXA. ABT-199&CFZ also induced strongest stabilization of p53, a transcriptional key regulator of NOXA. We suggest that simultaneous inhibition of anti-apoptotic BCL-2 by ABT-199 and the enhanced expression of NOXA double-hit on the BCL-2 and the MCL-1 signaling axis force the cells over the threshold of apoptosis. Enhanced efficacy of CFZ is dually caused by reduced degradation of NOXA and enhanced stabilization of p53, which in turn transactivates expression of NOXA. In conclusion, PI specificity and efficacy modulate apoptosis induction by combination with ABT-199. Future efforts, e.g. knock-out of relevant BH3-only proteins, will elucidate the observed variable efficacy of these and other PIs for prospective translation in clinical applications. Citation Format: Sandra Weller, Benjamin Schaefer, Tobias Beigl, Kathrin Böpple, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1398.