Abstract P209: Collaborative crosstalk between two apoptosis pathways drives synergy of dual inhibition of BCL-2/MDM2 in preclinical models of neuroblastoma

Abstract

Abstract Introduction: The prognosis of patients with neuroblastoma is poor, and novel therapeutic strategies are needed to improve clinical outcomes. Neuroblastoma often exhibits a low TP53 mutation rate and overexpresses MDM2 and BCL-2 genes. Dual targeting of the MDM2-P53 and BCL-2 apoptotic pathways is an attractive treatment strategy (Vernooij et al, Mol Cancer Ther 2021; Dalton et al, Mol Cancer Ther 2021) and is under clinical development using idasanutlin and venetoclax. However, the mechanisms of action (MOAs) of this combination in neuroblastoma are not fully understood. Alrizomadlin (APG-115) is a potent, orally bioactive MDM2 inhibitor. Lisaftoclax (APG-2575) is a novel BCL-2 selective inhibitor. Both agents have shown clinical activity in solid and hematologic malignancies (Tolcher et al, J Clin Oncol 2021:2506; Ailawadhi et al, J Clin Oncol 2021:7502). We investigated the effects of combined alrizomadlin and lisaftoclax treatment in preclinical models of neuroblastoma and examined novel MOAs associated with this combination. Materials and Methods: Antiproliferative activity was measured by CellTiter-Glo luminescent cell viability assay. Apoptosis was evaluated by annexin V/propidium iodide staining and flow cytometry. Meso-Scale Discovery ELISA and western blot analyses characterized the mechanisms of this combination’s synergistic effects. In animal studies, clinically relevant doses of lisaftoclax were employed. A pulsed high-dose regimen of alrizomadlin was used to achieve best efficacy and manage on-target toxicity. Results: In TP53WT neuroblastoma cell lines IMR-32 and SH-SY5Y, the combination of lisaftoclax and alrizomadlin synergistically inhibited proliferation and induced apoptosis, as evidenced by elevations in caspase-3/7, cleaved PARP, and annexin V+ subpopulations. Tumor growth was synergistically inhibited in 3 distinct subcutaneous mouse xenograft tumor models derived from neuroblastoma cell lines or patient tumors. In the cell lines, alrizomadlin treatment upregulated proapoptotic BCL-2 family member expression (NOXA, BAX, and PUMA) and downregulated antiapoptotic BCL-2 family member MCL-1, likely sensitizing cancer cells to lisaftoclax-induced apoptosis. Treatment of these cells with lisaftoclax and alrizomadlin reduced all 3 major BIM-related complexes, thereby increasing free BIM and triggering cellular apoptosis. Lisaftoclax and alrizomadlin downregulated NMYC and its transcription target MDM2, preventing the development of alrizomadlin resistance mediated by a feedback upregulation of MDM2, and inhibition of the MDM2-P53 apoptotic pathway by alrizomadlin primed cancer cells to BCL-2 inhibition. Conclusions: Alrizomadlin synergizes with lisaftoclax to achieve synthetic lethality through a regulated interaction at multiple molecular nodules, and inhibition of BCL-2 apoptotic pathway by lisaftoclax may prevent cancer cells from developing resistance to MDM2-P53 inhibition. These data warrant clinical development of combined alrizomadlin and lisaftoclax treatment in patients with neuroblastoma. Citation Format: Douglas D. Fang, Qiuqiong Tang, Qixin Wang, Yanhui Kong, Na Li, Feng Zhou, Ran Tao, Dajun Yang, Yifan Zhai. Collaborative crosstalk between two apoptosis pathways drives synergy of dual inhibition of BCL-2/MDM2 in preclinical models of neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P209.