MLN4924, An Investigational Inhibitor Of NEDD8 Activating Enzyme (NAE), Is Active In Mantle Cell Lymphoma (MCL) Pre-Clinical Models and Enhances Rituximab Activity In Vivo

MCL is characterized by an aggressive clinical course and inevitable development of refractory disease despite early interventions that include immunotherapy (i.e. rituximab), multi-agent induction chemotherapy (that contains high-dose cytarabine or bortezomib) and often consolidation with high- dose chemotherapy and autologous stem cell support (HDC-ASCS) in first remission. The acquisition of resistant disease resulting in poor clinical outcomes stresses the need to develop alternative therapeutic strategies for MCL patients. To this end, we evaluated MLN4924, an NAE inhibitor which prevents the neddylation and activation of cullin-RING ubiquitin ligases, in MCL pre-clinical models. A panel of MCL cell lines and primary tumor cells isolated from MCL patients were exposed to escalating doses of MLN4924 alone or in the presence of caspase inhibitors. Changes in: apoptosis, cell cycle distribution, NFkB activity, protein or mRNA expression levels of Bcl-2 family members/cell cycle regulators were determined using the cell titer glo assay, flow cytometry, imagestream technology, and Western blotting, respectively. Subsequently, MCL cells were exposed to MLN4924 alone or in combination with various chemotherapy agents, and differences in cell viability were determined by measuring changes in ATP content. Standard ⁵¹Cr release assays were conducted to study the effects of MLN4924 on the anti-tumor activity of rituximab against MCL cell lines. For in vivo studies, severe combined immunodeficiency (SCID) mice were inoculated via tail vein injection (iv) with Granta cells (day 0) and assigned to observation, rituximab (at 10mg/kg/dose on days +2,+9,+16 and +23), cytarabine (at 20mg/kg/dose on days +3,+10,+17 and +24) or MLN4924 (at 180mg/kg/dose on days +1,+4,+8,+11,+15 and +18) with or without rituximab (same dose-schedule) or cytarabine (same dose-schedule). Differences in survival (measured as the time to development of limb paralysis) were evaluated by log-rank test across treatment arms. MLN4924 exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested (IC₅₀=0.1-8.4µM). Of interest in MCL cell lines with lower IC₅₀ (0.1-0.5µM), MLN4924 induced G1 phase cell cycle arrest, down-regulation of both Bcl-XL mRNA and protein levels, down-regulation of NFkB activity, and apoptosis (PARP cleavage). In addition, MLN4924 exhibited additive/synergistic effects when combined with chemotherapy agents (i.e. cytarabine, bortezomib or bendamustine). As a single agent, MLN4924 prolonged the survival of MCL-bearing SCID mice when compared to controls (median=56 vs 21 days; p<0.05). More importantly, MLN4924 in combination with rituximab (median survival not reached at 90 days) led to an improved survival compared to rituximab (median survival 40 days) or MLN4924 alone (56 days) (P=0.045). Our data suggest that MLN4924 has significant activity in MCL pre-clinical models, possibly related to effects on NF-κB activity and Bcl-XL down-regulation, and can potentiate rituximab activity in vivo. These findings support further investigation of MLN4924 +/- rituximab in the treatment of MCL. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund)