The Nedd8-Activating Enzyme Inhibitor MLN4924 Thwarts Microenvironment-Driven NF-κB Activation and Induces Apoptosis in Chronic Lymphocytic Leukemia B Cells

Abstract

Background Stromal-mediated signaling enhances NFκB pathway activity in chronic lymphocytic leukemia B-cells (CLL), leading to cell survival and chemoresistance. Ubiquitination of IκBα may partially account for constitutive activation of NFκB. 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 pre-clinical assessment of MLN4924 in CLL. Primary CLL cells 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. Results CD40L-expressing stroma protected CLL cells from spontaneous apoptosis and induced resistance to multiple drugs, accompanied by NFκB 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 canonical and non-canonical NFκB 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 towards the pro-apoptotic BH3-only proteins. siRNA-mediated knockdown of Bim or Noxa decreased sensitivity to MLN4924. MLN4924 enhanced the antitumor activity of the inhibitors of BCR-associated kinases. Conclusions MLN4924 disrupts NFκB activation and induces Bim expression in CLL cells thereby preventing stroma-mediated resistance. Our data provide rationale for further evaluation of MLN4924 in CLL.

MLN4924 induces Noxa upregulation in acute myelogenous leukemia and synergizes with Bcl-2 inhibitors

“…9,36,37 As indicated above, p53 is not critical for MLN4924-induced Noxa upregulation, as this upregulation was seen in p53 null (HL-60) as well as p53 wild-type cells (ML-1; Figures 3a and c). NF-κB does not appear to have a critical role because, consistent with previous reports, 10,38 NF-κB levels decreased upon MLN4924 treatment and levels of the NF-κB inhibitor (I-κB) increased (Supplementary Figure S2c). Levels of ATF4, another transcription factor implicated in Noxa upregulation, 39 also did not increase (Supplementary Figure S1c), consistent with the observation that MLN4924-induced Noxa upregulation and apoptosis in these AML lines occurs without evidence of elevated ROS or ER stress (Supplementary Figures S1c, d and S2a, b).…”

“…These results complement and extend prior work showing that c-Myc, which is generally considered a contributor to proliferation, can contribute to apoptosis, 44,45 particularly under unfavorable growth conditions such as those resulting from environmental alterations or pharmacological manipulation. [18][19][20]36 Collectively, these studies are consistent with the pathway shown in Figure 6, which is different from the cytotoxic mechanism reported in solid tumor cells, 1,33 chronic lymphocytic leukemia, 38 lymphoma, 10 or myeloma, 12 and support the notion that the mechanism of MLN4924-induced cell death downstream of NAE inhibition will vary from cell type to cell type. 1 Importantly, the increases in c-Myc protein, Noxa mRNA and Noxa protein observed in AML cell lines were also observed in AML samples exposed to MLN4924 ex vivo, pointing to a pharmacodynamic marker of NAE inhibition (Noxa upregulation) and possible mechanisms of MLN4924 resistance (diminished c-Myc or Noxa upregulation) that could be investigated in future trials of MLN4924 in AML.…”

The Neddylation Inhibitor Pevonedistat (MLN4924) Suppresses and Radiosensitizes Head and Neck Squamous Carcinoma Cells and Tumors

“…This significant apoptotic response was attributed to the induction of c-FLIP degradation, which the authors showed to be independent of NEDD8 inhibition (67). Furthermore, it is now abundantly clear that hundereds of cellular proteins are regulated through neddylation, and consistently, pevonedistat inhibits several signal transduction pathways, including the NFkB, AKT, and the mTOR signaling pathways, in addition to cullin-signaling (28,29,(63)(64)(65)(66). Thus, pevonedistat may exhibit differential toxicities in HNSCC cells and tumors depending on the genetic backgrounds of the cells or tumors as well as on the concentrations employed.…”

“…Pevonedistat inhibits all cullin-based E3 ligases and additionally exhibits cullin-independent activity (28,29,(63)(64)(65)(66)(67). However, the results described above suggest that the radiosensitizing activity of pevonedistat is due to its ability to induce rereplication, which can be enhanced by IR.…”

SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation

“…MLN4924 targets the NEDD8-activating enzyme, thereby preventing the neddylation (activation) of cullin-RING proteins. This prevents ubiquitination by SCF complexes, promoting apoptosis and autophagy in targeted cells (56,57). We tested MLN4924 for efficacy in rescuing ⌬F508-CFTR-mediated Cl Ϫ transport in CFBE cells at six different doses ranging from 1 nM to 100 M. Unfortunately, MLN4924 was toxic to epithelial cells and was only tolerated at doses that did not inhibit neddylation.…”