Targeting Microenvironment-Mediated NFκb Activation With MLN4924, An Inhibitor Of The Nedd8-Activating Enzyme, In Chronic Lymphocytic Leukemia B Cells

Gene expression profiling identified lymph nodes as the site of cell activation and proliferation with upregulation of NFκB. B-cell receptor (BCR) and CD40 signaling enhances NFκB pathway activity in CLL cells resident in the microenvironment, leading to their survival, proliferation and chemoresistance. The NFκB pathway has been successfully targeted in CLL in vitro, yet lack of clinical advances with those agents necessitates development of novel approaches. Ubiquitination of IκBα may partially account for constitutive activation of NFκB in this setting. MLN4924 is an investigational agent that inhibits the Nedd8-activating enzyme and thereby prevents neddylation of Cullin-RING ubiquitin ligases, resulting in a decrease of their activity and stabilization of their protein substrates. This study reports that MLN4924 abrogates pro-survival microenvironment stimuli in CLL cells in vitro.

Primary B cells from 50 patients with CLL were co-cultured in vitro with CD40L-expressing stroma to mimic the pro-survival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NFκB pathway activity, Bcl-2 family members and cell cycle was assessed by flow cytometry, western blotting, PCR and immunocytochemistry. MLN4924 was provided by Millennium Pharmaceuticals.

Treatment with MLN4924 led to reduced neddylation of Cullin-RING ubiquitin ligases, accumulation of phospho-IκBa and induced modest apoptosis in CLL cells cultured off stroma (12.8±1.7% after a 24 hour incubation with 1 μM MLN4924 compared to untreated control). Incubation with the CD40L-expressing stroma resulted in activation of the canonical and non-canonical NFκB pathways in the CLL cells, induction of Mcl-1 and Bcl-xL, leading to protection from spontaneous apoptosis. This was accompanied by repression of Bim, a pro-apoptotic BH3-only protein of the Bcl-2 family. CD40L-expressing stroma rescued CLL cell from drug-induced apoptosis, including bendamustine, fludarabine, chlorambucil and CAL-101. By contrast, CD40L-expressing stroma failed to elicit resistance to MLN4924. Moreover, CLL cells cultured on stroma were more sensitive to Nedd8-activating enzyme inhibition compared to cells off stroma (1 μM MLN4924 induced apoptosis in 55.3±3.4% cells cultured on stroma). MLN4924 promoted apoptosis regardless of the common adverse prognostic factors (IGHV mutational status, cytogenetic markers, or ZAP-70 expression). In the presence of MLN4924, CLL cells demonstrated a significant decrease in nuclear translocation of the NFκB subunits p65 and p52 thus marking de-activation of both canonical and non-canonical pathways. Using gene microarray analysis we determined that receptor signaling and expression target pathways involving NFκB were most significantly associated with genes downregulated in CLL cells by MLN4924 (p<0.0001). We noted reduced transcription of several groups of NFκB target genes, including anti-apoptotic Bcl-2 family members and genes involved in cell cycle progression (p<0.01). Furthermore, treatment with MLN4924 resulted in a significant downregulation of a number of important cytokine ligands and receptors expressed by the CLL cells, including CCL5, CCL22, CXCR7, CXCR5 and CD40. Concomitantly, MLN4924 promoted induction of Bim and Noxa in the CLL cells which preceded apoptosis as evidenced by PARP cleavage. siRNA-mediated knockdown of either Bim or Noxa resulted in partial protection from MLN4924-mediated apoptosis irrespective of expression of the pro-survival Bcl-2 family members (Bcl-2, Bcl-xL or Mcl-1). This indicates that MLN4924 leads to rebalancing of Bcl-2 family members towards the pro-apoptotic BH3-only proteins in CLL. Furthermore, MLN4924 reversed stroma-mediated protection from fludarabine and the alkylators.

In summary, we were able to abrogate the protective effect of the CLL microenvironment in vitro using MLN4924, a Nedd8-activating enzyme inhibitor. MLN4924 reduced NFκB activity in CLL cells, led to re-expression of the pro-apoptotic BH3-only proteins and interfered the chemokine network thus carrying a potential to disrupt cell homing. These results suggest a rationale for clinical investigation of MLN4924 as a novel tool to target microenvironment in CLL.