High NRF2 expression controls endoplasmic reticulum stress induced apoptosis in multiple myeloma

Highlights

• We report for the first time that NRF2 is constitutively activated in circa 50% of MM primary samples.

• Proteasome inhibition induces further activation of NRF2 in primary MM.

• Inhibition of NRF2 in combination with PI treatment significantly increased apoptosis in primary MM.

Abstract

Multiple myeloma (MM) is an incurable disease characterized by clonal plasma cell proliferation. The stress response transcription factor Nuclear factor erythroid 2 [NF-E2]-related factor 2 (NRF2) is known to be activated in MM in response to proteasome inhibitors (PI). Here, we hypothesize that the transcription factor NRF2 whose physiological role is to protect cells from reactive oxygen species via the regulation of drug metabolism and antioxidant gene plays an important role in MM cells survival and proliferation. We report for the first time that NRF2 is constitutively activated in circa 50% of MM primary samples and all MM cell lines. Moreover, genetic inhibition of constitutively expressed NRF2 reduced MM cell viability. We confirm that PI induced further expression of NRF2 in MM cell lines and primary MM. Furthermore, genetic inhibition of NRF2 of PI treated MM cells increased ER-stress through the regulation of CCAAT-enhancer-binding protein homologous protein (CHOP). Finally, inhibition of NRF2 in combination with PI treatment significantly increased apoptosis in MM cells. Here we identify NRF2 as a key regulator of MM survival in treatment naive and PI treated cells.

Introduction

Multiple myeloma (MM) is an incurable disease characterized by clonal plasma cell proliferation [1], [2], [3]. Genetic studies demonstrate that MM is a highly complex and heterogenous disease that undergoes clonal evolution towards a multi-drug resistant disease over time [4], [5], [6], [7]. Thus, treatment relapse from the development of drug resistance clones is inevitable and presently MM remains incurable [8]. Therefore, better patient outcomes are expected to come from an improved understanding of the mechanisms of drug resistance which results in the development of novel treatment strategies that ‘re-sensitise’ MM cells to chemotherapy.

MM cells are dependent on the unfolded protein response to alleviate the endoplasmic reticulum (ER) stress caused by the excessive amounts of paraprotein being produced [9]. The proteasome inhibitors bortezomib and carfilzomib increase the accumulation of proteins, which elevate ER-stress and increase intracellular oxidative stress. This, in part accounts for proteasome inhibitor induced apoptosis in MM cells [10]. The transcription factor (nuclear factor erythroid 2 [NF-E2]-related factor 2 (NRF2)) is a key mediator of oxidative stress through the direct regulation of over 200 genes, as well as through mechanisms of post transcriptional modification [11], [12], [13]. These genes are involved in various cellular processes including the regulation of glutathione (GSH) synthesis, detoxification and the regulation of inflammatory processes [14], [15], [16], [17]. The transcription factor NRF2 has been shown to contribute to the malignant phenotypes of several cancers through effects on proliferation and drug sensitivity [18]. Moreover, in MM we identified the pro-tumoural function of heme oxygenase-1 (HO-1), an NRF2 regulated gene, through chemotherapy resistance [19].

NRF2 is regulated by Kelch-like ECH-associated protein 1 (KEAP1), which facilitates the ubiquitination and subsequent degradation of NRF2 by the proteosome [18]. Therefore, because proteosome inhibitors prevent the degradation of NRF2 by KEAP1, an increased transcriptional activity is induced in most cell types including malignant plasma cells [15], [20]. Recently, NRF2 has also been shown to be involved in regulating ER-stress through the negative regulation of CCAAT-enhancer-binding protein homologous protein (CHOP) [21]. CHOP is induced by the transcription factor, Activating Transcription Factor 4 (ATF4), as part of the ER-stress response which then mediates apoptosis. Studies have shown that high NRF2 levels inhibit the expression of CHOP and therefore prevent ER-stress induced apoptosis [22]. Others have shown that modulating redox homeostasis in MM could increase sensitivity of MM to bortezomib [20]. Finally, a recent study has shown that elevated glutathione levels can block bortezomib induced stress responses [23]. Therefore, since NRF2 activation positively regulates glutathione levels and negatively regulates CHOP we wanted to determine in MM if NRF2 is highly expressed and if silencing the expression of NRF2 reduced cell viability. In addition, we aimed to determine the relationship between NRF2 activation, increased glutathione levels and CHOP deregulation in response to proteasome inhibitors.