ALBERT

Description: 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.

Description: Key Points MALT1 protease activity stabilizes MYC. The MALT1-MYC network might represent a therapeutic target for MCL patients.

Description: Mantle cell lymphoma (MCL) is a rare B-cell-neoplasm with an aggressive clinical course characterized by the hallmark translocation t(11;14)(q13;q32) resulting in overexpression of cyclin D1. Furthermore, genomic profiling revealed numerous secondary genetic alterations and recurrent mutations contributing to MCL pathogenesis. Among them, mutations in the NOTCH1 gene have been described with a frequency of ~10% and are associated with significantly shorter survival rates. Therefore, further investigation of the biological impact of this mutation in MCL and its potential as a target for a specific inhibitory antibody therapy is of great interest. Here we investigated the role of the Notch receptor ligands Jagged1 (JAG1), Jagged2 (JAG2), Delta-like canonical Notch ligand 1 (DLL1) and Delta-like canonical Notch ligand 4 (DLL4) in activating the Notch1-signaling pathway in NOTCH1-mutated Mino and NOTCH1-wt Jeko1 MCL cell lines and evaluated the effects of the novel and specific monoclonal Notch1-antibody OMP-52M51. Mino cells are characterized by a single nucleotide substitution in exon 34 of the NOTCH1 gene, leading to truncation of the C-terminal PEST-domain. These mutations are known to remove the recognition site from the ubiquitin ligase degradation complex, resulting in increased stability and transcriptional activity of Notch1 upon ligand-stimulation. We confirmed the predicted truncation of Notch1 in Mino cell lysates and demonstrated that the expression of cleaved Notch1 was potently stimulated by recombinant DLL4 protein. In contrast, in the NOTCH1-wt cell line Jeko1, no stable overexpression of cleaved Notch1 could be observed upon any ligand-stimulation. Treatment of the NOTCH1-mutated cells with OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed transcriptional induction of well-known Notch1-target genes HES1 (hairy and enhancer of-split 1), DTX1 (deltex E3 ubiquitin ligase 1) and c-MYC as observed by real-time quantitative PCR. Gene expression profiling furthermore revealed that OMP-52M51 significantly impeded DLL4-induced upregulation of numerous genes involved in lymphoid biology and lymphomagenesis, as well as genes related to the cell cycle and the MAP-Kinase signaling pathway. Western blot analysis confirmed Notch1-antibody-mediated abrogation of enhanced expression of p-ERK and p-MEK upon DLL4-ligand-stimulation. Further investigations of functional effects revealed inhibitory effects of OMP-52M51 on enhanced tumor cell migration of DLL4-stimulated cells. As the DLL4-Notch-axis is known to be involved in regulating angiogenesis, we investigated the impact of activating NOTCH1-mutations in MCL on HUVEC tube formation and observed increased vessel sprouting upon DLL4-stimulation that could be abolished by treatment with OMP-52M51. Importantly, all these effects were specific for NOTCH1-mutated cells and did not occur in the NOTCH1-wt cell line Jeko1. In conclusion, we demonstrate for the first time that DLL4 is the most important ligand to activate Notch1 in MCL harbouring NOTCH1-mutations in the PEST-domain. Our findings indicate that enhanced Notch1 activity promotes a more aggressive behavior of the disease and specific antibody-inhibition of the Notch1-signaling pathway might provide an interesting therapeutic alternative for a subset of MCL patients, warranting further investigation. Disclosures No relevant conflicts of interest to declare.

Description: Mantle cell lymphoma (MCL) is a mature B-cell lymphoma characterized by poor prognosis. Recent studies revealed the importance of constitutive B-cell receptor (BCR) signaling in maintaining survival of a subset of MCLs. MALT1 is an essential component of the CARD11-BCL10-MALT1 (CBM) complex that links BCR signaling to the nuclear factor kappa-B (NF-κB) pathway. Additionally, MALT1 functions as a protease that cleaves various substrates to promote proliferation and survival. However, its role in the molecular pathogenesis of MCL is unknown. To elucidate the functional role of MALT1 in the biology of MCL, we determined its proteolytic activity in primary MCL cells and in MCL derived cell lines. A large fraction of MCLs displayed constitutive activity of MALT1. This MALT1 activity is driven by constitutive BCR signaling, as we were able to show that RNA interference-mediated knockdown of central components of the BCR cascade abolished MALT1 activity. To gain insights into the functional significance of MALT1 in MCL, we knocked down its expression by different MALT1 shRNAs. Transduction of these shRNAs induced cytotoxicity in models that are characterized by constitutive MALT1 activity, whereas no effect on survival was observable in MCLs without MALT1 activation. To determine if this MALT1 addiction translates into an in vivosetting, we knocked down MALT1 in mouse MCL models and detected a significant inhibition of tumor growth. This indicates that MALT1-activated MCLs are dependent on the function of MALT1. These results were confirmed as pharmacologic inhibition of MALT1 significantly reduced cell viability in MALT1-activated MCLs, implying that MALT1 inhibition might represent a promising therapeutic strategy for MCL patients. To understand which biologic processes are regulated by MALT1 in MCL, we profiled gene expression changes at different time points following MALT1 inhibition. An unbiased gene set enrichment analysis identified various previously described MYC gene expression signatures to be among the top downregulated signatures, suggesting that MALT1 regulates MYC expression. MYC downregulation following MALT1 inhibition or MALT1 knockdown was confirmed at the protein level and various analyses revealed that MALT1 regulates MYC expression posttranslationally by preventing its proteasomal degradation. These results were further confirmed in primary mouse splenocytes, indicating that this novel molecular mechanism of regulating MYC expression is not restricted to MCL. To confirm that MYC is indeed expressed in primary MCLs, we determined MYC expression in 234 primary MCL samples by immunohistochemistry. These analyses revealed that 75 samples (32.1%) displayed an intermediate and 55 samples (23.5%) a high MYC positivity, suggesting that MYC is expressed in a substantial number of primary MCLs. As common alterations such as MYC high-levelamplifications and translocations determined by FISH occurred extremely rarely in our primary MCL samples (1% of samples), it is conceivable that BCR-driven MALT1 signaling is the predominant mechanism of MYC upregulation in MCL. In summary, we report for the first time that a substantial fraction of MCLs is addicted to constitutive MALT1 signaling. Thus, MCLs can be divided based on their MALT1 activation status into two distinct subgroups. We further identified a novel regulatory mechanism of MYC expression by MALT1. Thus, our study provides a strong mechanistic rationale to investigate the therapeutic efficacy in targeting the MALT1-MYC axis in MCL patients. Disclosures Trneny: Janssen Research & Development: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreyling:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau. Tzankov:Novartis: Speakers Bureau; Abbott: Speakers Bureau.