Effect of MLN4924 on Bim expression in CLL. CLL cells were co-cultured with CD40L-expressing stroma for 24 hours, followed by incubation with 1 μM MLN4924 or vehicle control. Cell were lysed at 24 hours and subjected to immunoblotting. Raji cells (American Type Culture Collection) were cultured in RPMI 1640 with 10% FBS without (first lane) or with (second lane) 50 μg/L phorbol 12-myristate 13-acetate for 30 min to serve as control for expression of phospho-Bim.
ARTICLE ABSTRACT
Background: Stromal-mediated signaling enhances NF-κB pathway activity in chronic lymphocytic leukemia (CLL) B cells, leading to cell survival and chemoresistance. Ubiquitination of IκBα may partially account for constitutive activation of NF-κB. MLN4924 is an investigational agent that inhibits the Nedd8-activating enzyme, thereby neutralizing Cullin-RING ubiquitin ligases and preventing degradation of their substrates.Experimental Design: We conducted a preclinical assessment of MLN4924 in CLL. Primary CLL cells were cocultured in vitro with CD40L-expressing stroma to mimic the prosurvival conditions present in lymphoid tissue. The effect of MLN4924 on CLL cell apoptosis, NF-κB pathway activity, Bcl-2 family members, and cell cycle was assessed by flow cytometry, Western blotting, PCR, and immunocytochemistry.Results: CD40L-expressing stroma protected CLL cells from spontaneous apoptosis and induced resistance to multiple drugs, accompanied by NF-κB activation and Bim repression. Treatment with MLN4924 induced CLL cell apoptosis and circumvented stroma-mediated resistance. This was accompanied by accumulation of phospho-IκBα, decreased nuclear translocation of p65 and p52 leading to inhibition of both the canonical and noncanonical NF-κB pathways, and reduced transcription of their target genes, notably chemokines. MLN4924 promoted induction of Bim and Noxa in the CLL cells leading to rebalancing of Bcl-2 family members toward the proapoptotic BH3-only proteins. siRNA-mediated knockdown of Bim or Noxa decreased sensitivity to MLN4924. MLN4924 enhanced the antitumor activity of the inhibitors of B-cell receptor (BCR)–associated kinases.Conclusions: MLN4924 disrupts NF-κB activation and induces Bim expression in CLL cells, thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL. Clin Cancer Res; 20(6); 1576–89. ©2014 AACR.
