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.

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<p>Supplemental Figure S1. Changes in protein expression levels induced by MCL-1 inhibitor. Supplemental Figure S2. Impact of MCL-1 inhibitors on total Mcl-1, T163 and S159 phosphorylation.Supplemental Figure S3. MCL-1 inhibitors induce a transient decrease in Mule.Supplemental Figure S4. In vitro De-ubiquitinating activity assay of USP9x with MCL-1 inhibitors.</p>