Abstract

Mantle cell lymphoma (MCL) cells are characterized by a discrepancy between high mRNA and low protein levels of the pro-apoptotic BH3-only protein NOXA. Modulation of NOXA protein expression has been described as a major mechanism of cell death induction in MCL cells. However, the efficiency of induction of apoptosis at lower concentrations is limited despite effective stabilization of NOXA.

We therefore investigated whether the main binding partner of NOXA, the anti-apoptotic protein MCL-1 is co-regulated by these agents and whether dual targeting of NOXA and MCL-1 could be a promising strategy to enhance effectiveness of cell death induction in MCL cell lines.

Screening a panel of compounds supposed to lead to NOXA stabilization in MCL, we identified the proteasome inhibitor Bortezomib, the fatty acid synthase inhibitor Orlistat and the ROS inducing agents Helenalin, a sesquesterpenone lactone from Arnica and the naphtoquinone derivative Menadione to kill MCL cells very effectively in a NOXA -dependent manner.

Investigating the NOXA-/MCL-1-protein expression upon treatment with these agents, we could observe that at lower, sublethal concentrations of Bortezomib and Orlistat, NOXA protein was increased to a certain extent but also the anti-apoptotic MCL-1 was highly induced, counteracting induction of apoptosis.

From this observation it has to be concluded that MCL-1 limits the apoptotic activity of NOXA stabilizing agents and dual targeting of MCL-1 and NOXA is required for optimal killing of MCL cells.

In search for MCL-1 regulating agents we could identify the cdk-inhibitor Dinaciclib to be the most effective one. This compound rapidly downregulates Mcl-1 protein in a dose- and time-dependent manner presumably due to cdk 9-mediated inhibition of phosphorylation of the RNA-Polymerase II-subunit RPB1.

To study the efficiency of dual targeting of NOXA/MCL-1 in killing of MCL cells we combined sublethal doses of Dinaciclib with NOXA stabilizing agents and observed a synergistic induction of apoptosis in MCL cells. Western Blot analysis showed that combination treatment decreased Mcl-1 and increased NOXA protein expression compared to single-agents. It could be shown that induction of cell death by treatment with the combined agents depends on NOXA as transfection of cells with NOXA-siRNA rescued cells from induction of apoptosis. Cell death upon treatment with Dinaciclib and ROS-inducing agents Helenalin and Menadione could furthermore be rescued by preincubation with the antioxidant GSH.

Interestingly, combined treatment of cells with Orlistat and Dinaciclib killed most effectively when Dinaciclib was added for 8 hours after 16 hours of preincubation with the NOXA stabilizing agent. This observation contributes to the hypothesis that stabilization of NOXA protein leads to priming of MCL cells to induction of apoptosis by pharmacological downregulation of Mcl-1 with Dinaciclib.

In summary, the NOXA-MCL-1 balance is critical for survival of MCL cells. Dual targeting of MCL-1 and NOXA efficiently kills MCL cells and appears to be of particular importance especially at lower concentrations of these compounds. These culture conditions most likely resemble the limited exposure after in vivo treatment. Therefore the combination of NOXA stabilizing agents with Dinaciclib appears to be a promising strategy to be tested in clinical trials.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.