Abstract 3706: Double impact of ABT-199 by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivation of NOXA
Authors: Weller Sandra , Schaefer Benjamin , Beigl Tobias B. , Böpple Kathrin , Hofmann Ute , Aulitzky Walter E. , Kopp Hans-Georg , Essmann Frank 
Published: June 2022

Abstract
Soft-Tissue Sarcomas (STS) are mesenchymal malignancies with high heterogeneity and poor prognosis. As recently shown, ABT-199 and Bortezomib (BTZ) synergistically induce apoptosis in STS derived cells and cell lines (Muenchow et al. 2020). This is of great importance due to limited clinical therapeutic options and lower dependence on BCL-2 expression in solid cancers. To delineate the molecular basis for the synergism of ABT-199 and BTZ, we thought to elucidate the mechanism leading to enhanced expression of the BH3-only protein NOXA. We investigated whether proteasome inhibition augmented the expression of NOXA in a TP53-regulated manner. To clarify a possible role of TP53 in increased NOXA expression, we performed knock-down of TP53 and analyzed transcription of PMAIP1 by qRT-PCR and expression of NOXA protein by Western Blot in presence of ABT-199 alone and in combination with BTZ. Previous reports also show that ABT-199 impairs complex I and II activity of the electron transport chain (ETC), resulting in induction of metabolic reprogramming confirmed by enhanced reductive carboxylation (Roca-Portoles et al. 2020). The ratio of α-ketoglutarate : citrate, indicating disbalance of the ETC, was assessed by mass spectrometric analysis in SW982/WT cells after incubation with ABT-199 alone and in combination with BTZ. Moreover, we gained further insight into the molecular mechanism of action of enhanced NOXA expression by analyzing ABT-199 and BTZ induced cell death induction in time kinetics of cell death induction in knock-down experiments using CellTOX Green as indicator. We found that ABT-199 induced the expression of NOXA by a mainly TP53-independent mechanism at the transcriptional level. Indeed, our data suggest that ABT-199 induces metabolic reprogramming leading to enhanced reductive carboxylation, evidenced by an increased ratio of α-ketoglutarate : citrate. Subsequent activation of the integrated stress response (ISR) induces expression of the known ISR markers ATF3 and ATF4 at both the transcriptional and protein levels. Silencing of ATF3 and ATF4 significantly reduced NOXA expression and NOXA mediated cell death induction. Therefore, we propose that transactivation of NOXA by ABT-199 and subsequent sensitization to proteasome inhibitors effectively induces cell death in solid cancers. The double impact of ABT-199 by i) directly blocking anti-apoptotic BCL-2 and ii) inhibiting MCL-1 via transactivated NOXA is of high clinical relevance, because combined treatment with proteasome inhibitors might overcome intrinsic or acquired resistance to BH3-mimetics. Thus, the here described novel effect of ABT-199 to transactivate NOXA expression, should prompt future efforts in the clinical evaluation of combinatorial regimens in additional malignancies. We hypothesize that these effects represent generally active principles of a tumor-agnostic mechanism of action.
Citation Format: Sandra Weller, Benjamin Schaefer, Tobias B. Beigl, Kathrin Böpple, Ute Hofmann, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. Double impact of ABT-199 by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivation of NOXA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3706.