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Harnessing Noxa demethylation to overcome Bortezomib resistance in mantle cell lymphoma.

ABSTRACT: Bortezomib (BZM) is the first proteasome inhibitor approved for relapsed Mantle Cell Lymphoma (MCL) with durable responses seen in 30%-50% of patients. Given that a large proportion of patients will not respond, BZM resistance is a significant barrier to use this agent in MCL. We hypothesized that a subset of aberrantly methylated genes may be modulating BZM response in MCL patients. Genome-wide DNA methylation analysis using a NimbleGen array platform revealed a striking promoter hypomethylation in MCL patient samples following BZM treatment. Pathway analysis of differentially methylated genes identified molecular mechanisms of cancer as a top canonical pathway enriched among hypomethylated genes in BZM treated samples. Noxa, a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of BZM, was significantly hypomethylated and induced following BZM treatment. Therapeutically, we could demethylate Noxa and induce anti-lymphoma activity using BZM and the DNA methytransferase inhibitor Decitabine (DAC) and their combination in vitro and in vivo in BZM resistant MCL cells. These findings suggest a role for dynamic Noxa methylation for the therapeutic benefit of BZM. Potent and synergistic cytotoxicity between BZM and DAC in vitro and in vivo supports a strategy for using epigenetic priming to overcome BZM resistance in relapsed MCL patients.

SUBMITTER: Leshchenko VV

PROVIDER: S-EPMC4694993 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Harnessing Noxa demethylation to overcome Bortezomib resistance in mantle cell lymphoma.

Leshchenko Violetta V VV Kuo Pei-Yu PY Jiang Zewei Z Weniger Marc A MA Overbey Jessica J Dunleavy Kieron K Wilson Wyndham H WH Wiestner Adrian A Parekh Samir S

Oncotarget 20150901 29

Bortezomib (BZM) is the first proteasome inhibitor approved for relapsed Mantle Cell Lymphoma (MCL) with durable responses seen in 30%-50% of patients. Given that a large proportion of patients will not respond, BZM resistance is a significant barrier to use this agent in MCL. We hypothesized that a subset of aberrantly methylated genes may be modulating BZM response in MCL patients. Genome-wide DNA methylation analysis using a NimbleGen array platform revealed a striking promoter hypomethylatio ...[more]

PMID: 25714012

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Project description:BackgroundBortezomib (BTZ) is an inhibitor of the proteasome that has been used to treat patients with mantle cell lymphoma (MCL), but the resistance to BTZ in clinical cases remains a major drawback. BACH2 is a lymphoid-specific transcription repressor recognized as a tumor suppressor in MCL. Reduced BACH2 levels contribute to BTZ resistance; however, the molecular events underlying BACH2-mediated BTZ resistance are largely unclear.MethodsWe silenced BACH2 in MCL cells using a lentiviral shRNA-mediated knockdown system. Bioinformatic, real-time RT-PCR, immunoblotting and a series of functional assays were performed to describe the molecular mechanisms underlying BTZ resistance in MCL. The therapeutic effects of chemicals were evaluated on numerous cellular and molecular processes in resistant MCL cell lines and xenografts.ResultsIn resistant cells, BTZ-triggered mild oxidative stress induced a strong activation of PI3K-AKT signaling, which further blocked nuclear translocation of BACH2. Defective nuclear translocation of BACH2 or silencing BACH2 removed its transcriptional repression on HMOX1, leading to upregulation of heme oxygenase-1 (HO-1). Increased HO-1 further maintained reactive oxygen species (ROS) within a minimal tumor-promoting level and enhanced cytoprotective autophagy. Interestingly, although mild increase in ROS exhibited a pro-tumorigenic effect on resistant cells, simply blocking ROS by antioxidants did not lead to cell death but aggravated BTZ resistance via stabilizing BACH1, the other member of BACH family. Instead, 3-methyladenine (3-MA), a dual inhibitor to suppress PI3K signaling and autophagosome formation, sensitized resistant MCL cells to BTZ, both in vitro and in vivo.ConclusionOur results dissected the interconnected molecular network in resistant MCL cells in which 3-MA represents an effective therapeutic strategy to overcome BTZ resistance. Notably, BACH1 and BACH2, albeit from the same family, are likely to play opposite roles in pathogenesis and progression of MCL.

Project description:BackgroundMantle cell lymphoma (MCL) is an aggressive B-non-Hodgkin lymphoma with generally poor outcome. MCL is characterized by an aberrantly high cyclin D1-driven CDK4 activity. New molecular targeted therapies such as inhibitors of the ubiquitin-proteasome system (UPS) have shown promising results in preclinical studies and MCL patients. Our previous research revealed stabilization of the short-lived pro-apoptotic NOXA as a critical determinant for sensitivity to these inhibitors. It is currently unclear how cyclin D1 overexpression and aberrant CDK4 activity affect NOXA stabilization and treatment efficacy of UPS inhibitors in MCL.MethodsThe effect of cyclin D1-driven CDK4 activity on response of MCL cell lines and primary cells to proteasome inhibitor treatment was investigated using survival assays (Flow cytometry, AnnexinV/PI) and Western blot analysis of NOXA protein. Half-life of NOXA protein was determined by cycloheximide treatment and subsequent Western blot analysis. The role of autophagy was analyzed by LC3-II protein expression and autophagolysosome detection. Furthermore, silencing of autophagy-related genes was performed using siRNA and MCL cells were treated with autophagy inhibitors in combination with proteasome and CDK4 inhibition.ResultsIn this study, we show that proteasome inhibitor-mediated cell death in MCL depends on cyclin D1-driven CDK4 activity. Inhibition of cyclin D1/CDK4 activity significantly reduced proteasome inhibitor-mediated stabilization of NOXA protein, mainly driven by an autophagy-mediated proteolysis. Bortezomib-induced cell death was significantly potentiated by compounds that interfere with autophagosomal function. Combined treatment with bortezomib and autophagy inhibitors enhanced NOXA stability leading to super-induction of NOXA protein. In addition to established autophagy modulators, we identified the fatty acid synthase inhibitor orlistat to be an efficient autophagy inhibitor when used in combination with bortezomib. Accordingly, this combination synergistically induced apoptosis both in MCL cell lines and in patient samples.ConclusionOur data demonstrate that CDK4 activity in MCL is critical for NOXA stabilization upon treatment with UPS inhibitors allowing preferential induction of cell death in cyclin D transformed cells. Under UPS blocked conditions, autophagy appears as the critical regulator of NOXA induction. Therefore, inhibitors of autophagy are promising candidates to increase the activity of proteasome inhibitors in MCL.

Dataset Information

Harnessing Noxa demethylation to overcome Bortezomib resistance in mantle cell lymphoma.

Publications

Harnessing Noxa demethylation to overcome Bortezomib resistance in mantle cell lymphoma.

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