The Phenotype High Noxa mRNA/Low NOXA Protein Levels Constitutes a Critical Achilles Heel Of Mantle Cell Lymphoma (MCL) Cells

Mantle cell lymphoma (MCL) is an aggressive type of non-Hodgkin lymphoma with comparatively short response to chemotherapy and frequent relapses. New treatment strategies for this malignancy are urgently needed. The genetic hallmark of MCL is the t(11;14)(q13;q32) translocation. This alteration leads to deregulated expression of the oncogene Cyclin D1 and is considered the primary event in the pathogenesis of MCL. Additionally, deregulations of different oncogenic signaling and cell death pathways have been described in MCL.

In this study, we investigated the role of the BH3-only protein NOXA for life/death decision in MCL. We found a stunning discrepancy between constitutive Noxa (PMAIP1) mRNA and NOXA protein levels in MCL cell lines and primary cells. Noxa mRNA was found to be highly expressed whereas NOXA protein levels were low. Remarkably, constitutive high gene expression of this pro-apoptotic Bcl2 family member was dependent on Cyclin D1 overexpression and chronic active B cell receptor (BCR) signaling, two of the major oncogenic alterations in MCL. We identified the PI3K/AKT/mTOR pathway to be crucial in the maintenance of Noxa transcript levels downstream of BCR. Cyclin D1 overexpression contributed to the high Noxa mRNA levels by exerting a positive feedback loop on PI3K/AKT/mTOR signaling. Intriguingly, the high constitutive Noxa transcript levels do not impair cell viability. MCL cells adapt to this constitutive pro-apoptotic signal by keeping NOXA protein low due to extensive ubiquitination and rapid proteasomal degradation of NOXA protein (T_½ ∼ 15-30 min). As expected, treatment of the cells with the proteasome inhibitor Bortezomib accumulated NOXA protein and efficiently induced cell death. Additionally, we identified the neddylation inhibitor MNL4924 and the fatty acid synthase (FASN) inhibitor Orlistat as potent inducers of NOXA protein, and thereby apoptosis, in MCL. Cell death upon Bortezomib as well as MLN4924 and Orlistat treatment was dependent on NOXA since RNAi mediated silencing of the pro-apoptotic protein significantly reduced induction of apoptosis. We found that all three inhibitors targeted the rapid NOXA protein turnover by stabilizing the preexisting pool of NOXA. In contrast to Bortezomib, however, MLN4924 and Orlistat inhibited the ubiqutination process of NOXA protein and stabilized the pro-apoptotic protein by a proteasome independent manner. These findings could be of great clinical relevance as Bortezomib resistance is a frequently observed phenomenon. Indeed, both inhibitors were still able to induce NOXA and cell death in Bortezomib resistant clones through targeting ubiquitine-proteasome system-mediated NOXA turnover upstream of the proteasome. Interestingly, active PI3K/AKT/mTOR signaling was needed for effective accumulation of NOXA protein and induction of cell death by all these compounds indicating that the high constitutive Noxa mRNA levels are essential for sensitivity of MCL cells.

Together, our data highlight that NOXA regulation may represent an important Achilles heel of MCL cells. Stabilization of NOXA protein by inhibition of the ubiquitin-proteasome system on different levels might be an effective strategy to kill MCL cells and offer novel treatment options.