Mechanisms of MCL-1 Protein Stability Induced by MCL-1 Antagonists in B-Cell Malignancies

• Published in: Clinical cancer research Vol. 29; no. 2; pp. 446 - 457
• Main Authors: Tantawy, Shady I., Sarkar, Aloke, Hubner, Stefan, Tan, Zhi, Wierda, William G., Eldeib, Abdelraouf, Zhang, Shuxing, Kornblau, Steven, Gandhi, Varsha
• Format: Journal Article
• Language: English
• Published: United States 17.01.2023
• Subjects: Antineoplastic Agents - therapeutic use; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Humans; Leukemia, Lymphocytic, Chronic, B-Cell - drug therapy; Leukemia, Lymphocytic, Chronic, B-Cell - genetics; Myeloid Cell Leukemia Sequence 1 Protein - genetics; Myeloid Cell Leukemia Sequence 1 Protein - metabolism; Protein Stability; Proto-Oncogene Proteins c-bcl-2 - genetics; Proto-Oncogene Proteins c-bcl-2 - metabolism; Ubiquitin Thiolesterase - metabolism
• ISSN: 1078-0432; 1557-3265; 1557-3265
• DOI: 10.1158/1078-0432.CCR-22-2088

Several MCL-1 inhibitors (MCL-1i), including AMG-176 and AZD5991, have shown promise in preclinical studies and are being tested for the treatment of hematologic malignancies. A unique feature of these agents is induction and stability of Mcl-1 protein; however, the precise mechanism is unknown. We aim to study the mechanism of MCL-1i-induced Mcl-1 protein stability. Using several B-cell leukemia and lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) lymphocytes, we evaluated molecular events associated with Mcl-1 protein stability including protein half-life, reverse-phase protein array, protein-protein interaction, phosphorylation, ubiquitination, and de-ubiquitination, followed by molecular simulation and modeling. Using both in vivo and in vitro analysis, we demonstrate that MCL-1i-induced Mcl-1 protein stability is predominantly associated with defective Mcl-1 ubiquitination and concurrent apoptosis induction in both cell lines and primary CLL subjects. These MCL1i also induced ERK-mediated Mcl-1Thr163 phosphorylation, which partially contributed to Mcl-1 stability. Disruption of Mcl-1:Noxa interaction followed by Noxa degradation, enhanced Mcl-1 de-ubiquitination by USP9x, and Mule destabilization are the major effects of these inhibitors. However, unlike other BH3 proteins, Mule:Mcl-1 interaction was unaffected by MCL-1i. WP1130, a global deubiquitinase (DUB) inhibitor, abrogated Mcl-1 induction reaffirming a critical role of DUBs in the observed Mcl-1 protein stability. Further, in vitro ubiquitination studies of Mcl-1 showed distinct difference among these inhibitors. We conclude that MCL-1i blocked Mcl-1 ubiquitination via enhanced de-ubiquitination and dissociation of Mcl-1 from Noxa, Bak and Bax, and Mule de-stabilization. These are critical events associated with increased Mcl-1 protein stability with AMG-176 and AZD5991.