Y. Xu1, C. G. Medina1, D. Surman1, M. Helal1, R. T. Ripley1 1Baylor College Of Medicine, Department Of Surgery, Houston, TX, USA
Introduction: Malignant pleural mesothelioma (MPM) is a rare, aggressive cancer. Despite some advances, the 5-year survival rate for all stages is less than 20%. Resistance to apoptosis is a well-established hallmark of cancer that is mediated through the mitochondria. Apoptosis of damaged cells occurs through an intricate process known as Mitochondrial Outer Membrane Permeabilization (MOMP). MOMP is a switch-like event regulated by the balance of anti-apoptotic and pro-apoptotic B-cell lymphoma 2 (Bcl-2) family members. Studies have highlighted that the anti-apoptotic proteins, B-Cell Lymphoma (Bcl)-xL and Myeloid Leukemia Cell-1 (Mcl-1), are crucial for mesothelioma cell survival. We hypothesize that targeting the mitochondria through Mcl-1 or Bcl-xL increases the rate of apoptosis to overcome treatment resistance in MPM.
Methods: Patient derived cells (PDC) were developed from ex vivo tumors. Immunofluorescence performed on PDC confirmed concordance with the resected specimens. To test the threshold for apoptosis as well as changes in mitochondria induced by drugs, a live-cell bioassay called Dynamic BH3 Profiling (DBP) was performed on the mesothelioma cell line (H28) and PDCs with drugs targeting Mcl-1 and/or Bcl-xL named AZD-5991 and A1155463, respectively. Apoptosis measured by Annexin V assay and IC50 calculations were performed oncell lines and PDCs treated with AZD5991 and A1155463 compared to controls. SynergyFinder tested the synergistic effects in combination treatments. Immunoblot and immunofluorescence evaluated Mcl-1 and BcL-xL expression and localization. The mitochondrial function was determined by the Seahorse XFe96 analyzer.
Results: In H28 and PDCs cells, DBP predicted that targeting either Mcl-1 or Bcl-xL decreased threshold of apoptosis. Additionally the combination of A1155463 and AZD5991 reduced the cell viability in a concentration-dependent manner with synergetic effects. Knockdown of Mcl-1 or Bcl-xL recapitulated the results of targeting these proteins with drugs. Double knockdowns of the mitochondrial proteins that induce MOMP (BAX and BAK), abrogated the effects of co-targeting of Mcl-1 and Bcl-xL, which suggests that the synergistic mechanism is specific to mitochondrially-induced apoptosis. Furthermore, Bcl-xL inhibition induced Mcl-1 mitochondrial translocation from nuclear to mitochondrial suggesting a redundant function of these proteins. Lastly, mitochondrial respiration decreased after Bcl-xl knockdown, which suggested that this protein is critical for normal function of tumor’s mitochondria.
Conclusion: Co-targeting Mcl-1 and Bcl-xL alter the mitochondria by decreasing the threshold for apoptosis. These effects can be measured via the functional precision oncology bioassay, DBP, which measures whether mitochondrially-targeted drugs undermine tumor cells respiration. Collectively, these findings suggested that inhibition of the mitochondria will provide a novel approach to treatment of patients with MPM.