pevonedistat and Leukemia--Lymphocytic--Chronic--B-Cell

Topics: Cells, Cultured; Cyclopentanes; Enzyme Inhibitors; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lysine; Mass Spectrometry; NEDD8 Protein; Protein Processing, Post-Translational; Pyrimidines; Tumor Suppressor Protein p53; Ubiquitins

Chronic lymphocytic leukemia (CLL) B-cells demonstrate both constitutive and stroma-mediated activation of nuclear factor-κB (NF-κB). NEDD8, a ubiquitin-like protein, regulates activity of Cullin-RING ubiquitin ligases (CRLs) and thus indirectly controls NF-κB activity. Inhibition of CRLs with MLN4924, an investigational agent that targets the NEDD8-activating enzyme, induces accumulation of CRL substrates, including inhibitor of NF-κB (IκB), a negative pathway modulator. We demonstrate that both continuous and pulse treatments with MLN4924 abrogate NF-κB activity in CLL B-cells ex vivo in a CD40L-expressing stromal co-culture system and identify pathways potentially responsible for resistance to MLN4924. To achieve long-lasting therapeutic effects in CLL, combination strategies are likely necessary.

Topics: Apoptosis; Cell Line, Tumor; Cyclopentanes; Gene Expression Regulation, Leukemic; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; NEDD8 Protein; NF-kappa B; Pyrimidines; Transcription, Genetic; Ubiquitins

Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

Topics: Alkylating Agents; Apoptosis; B-Lymphocytes; Bendamustine Hydrochloride; CD40 Antigens; CD40 Ligand; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclopentanes; DNA Damage; Gene Expression Regulation, Neoplastic; Humans; Interleukins; Leukemia, Lymphocytic, Chronic, B-Cell; NEDD8 Protein; NF-kappa B; Pyrimidines; Ubiquitin-Activating Enzymes; Ubiquitins

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.. 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.. 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.. 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.

Topics: Animals; Antineoplastic Agents; Apoptosis; B-Lymphocytes; Blotting, Western; Cell Line, Tumor; Cyclopentanes; Enzyme Inhibitors; Flow Cytometry; Humans; Immunohistochemistry; Leukemia, Lymphocytic, Chronic, B-Cell; Mice; NF-kappa B; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Pyrimidines; Real-Time Polymerase Chain Reaction; Signal Transduction; Tumor Microenvironment; Ubiquitin-Activating Enzymes