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

Abstract

Purpose: 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 hematological 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 mechanism of MCL-1i-induced Mcl-1 protein stability. Experimental Design: Using several B-cell leukemia and lymphoma cell lines and primary 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, deubiquitination, followed by molecular simulation and modeling. Results: 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 amongst these inhibitors. Conclusions: We conclude that MCL-1i blocked Mcl-1 ubiquitination via enhanced deubiquitination 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.

Effective Targeting of Melanoma Cells by Combination of Mcl-1 and Bcl-2/Bcl-xL/Bcl-w Inhibitors

“…Mcl-1 degradation is frequently mediated by the proteasomal pathway, which may be induced by phosphorylation of the PEST domain or through binding by proapoptotic Bcl-2 proteins and BH3 mimetics. Finally, Mcl-1 is a well-described target of caspase activity and is degraded in course of induced apoptosis [ 45 ]. In our experiments, Mcl-1 downregulation appeared to be related to induced caspase activity.…”

Mechanisms of Resistance to Small Molecules in Acute Myeloid Leukemia

“…Inhibition of MCL1 or BCLXL was similarly toxic to venetoclax-resistant AML cell lines when given in combination with venetoclax [ 107 ]. Clinical research on MCL1 inhibitors is in the early stage, and potential cardiac interactions are currently being investigated [ 108 ].…”

Decoding the mechanism behind MCL-1 inhibitors: A pathway to understanding MCL-1 protein stability

“…However, the mechanism of this protein accumulation is largely unknown. To address this knowledge gap, Tantawy et al explored underlying molecular mechanisms that contribute to MCL1i-induced MCL-1 protein accumulation and its implications [ 14 ].…”

“…However, this effect was reversible following withdrawal of MCL-1i, indicating a direct effect of the drug on MCL-1 protein stability. Using protein-protein interaction studies, immunoprecipitation (IP) and co-IP experiments, Tantawy et al [ 14 ] showed that MCL-1i-induced defective ubiquitination of MCL-1 protein. This indicates that the upregulation of MCL-1 protein is not due to direct proteasomal inhibition, but rather due to interference with the ubiquitination and/or enhancing de-ubiquitination of MCL-1.…”

“…Tantawy et al [ 14 ] further addressed the mechanisms underlying the decreased ubiquitination of MCL-1 protein and showed that MCL-1i increased MEK/ERK-mediated Thr 163 phosphorylation, leading to conformation change of MCL-1 protein. This in turn increases the surface accessible area to Thr 163 residue, thereby contributing to MCL-1 stability and accumulation.…”