Targeting protein neddylation with an NEDD8-activating enzyme inhibitor MLN4924 induced apoptosis or senescence in human lymphoma cells

Abstract

Recent studies indicate that post-translational protein neddylation is required for the maintenance of cell viability in several lymphoma cell lines, while inhibition of the neddylation pathway with an NEDD8-activating enzyme (NAE) inhibitor MLN4924 induces apoptosis in lymphoma cells. However, the mechanism by which neddylation inhibition induces apoptosis in lymphoma cells has not been fully elucidated. Moreover, it is unknown whether neddylation inhibition triggers non-apoptotic cell-killing responses, such as cell senescence, in lymphoma cells. Here, we report that MLN4924 specifically inhibited protein neddylation, inactivated cullin-RING E3 ligase (CRL), the best-known neddylation substrate, and induced the accumulation of tumor-suppressive CRL substrates in lymphoma cells. Moreover, MLN4924 potently suppressed the growth of lymphoma cells by inducing G2 cell-cycle arrest, followed by apoptosis or senescence in a cell line-dependent manner. MLN4924-induced apoptosis was mediated by intrinsic apoptotic signaling with substantial up-regulation of pro-apoptotic Bik and Noxa as well as down-regulation of anti-apoptotic XIAP, c-IAP1 and c-IAP2, while senescence induction upon neddylation inhibition seemed dependent on the expression of tumor suppressor p21/p27. Together, these findings expand our understanding on how lymphoma cells respond to neddylation inhibition and support the development of neddylation inhibitors (e.g. MLN4924) for the treatment of lymphoma.

Keywords: CRL, cullin-RING E3 ligase; GCB-DLBCL, germinal-center B cell-like diffuse large B-cell lymphoma; IAP, inhibitor of apoptosis; MLN4924; NAE, NEDD8-activating enzyme; NHL, non-Hodgkin lymphoma; SA-β-gal, senescence-associated β-galactosidase; apoptosis; lymphoma; neddylation; senescence.

Figure 1.

MLN4924 specifically inhibits protein neddylation and inactivates CRL E3 ligases. (A) MLN4924 specifically inhibited protein neddylation. Raji, U937, SU-DHL-4 and Toledo cells were seeded into 6-well plates, cultured overnight, treated with 0.3 μM MLN4924, 10 μM proteasome inhibitor MG132 or DMSO for 1 h, and subjected to immunoblotting using antibodies against NEDD8 with GAPDH as a loading control. (B) Effects of MLN4924 on neddylated Cullin 1 and downstream effectors. The four lymphoma cells were treated with MLN4924 at 0.1 and 0.3 μM or DMSO for 48 h, and subjected to immunoblotting using indicated antibodies with GAPDH as a loading control.

Figure 2.

Neddylation inhibition with MLN4924 triggers G2 cell-cycle arrest and suppresses the growth of lymphoma cells. (A) MLN4924 induced G2-M cell-cycle arrest. Raji, U937, SU-DHL-4 and Toledo cells seeded into 6-well plates were treated with MLN4924 at 0.1 and 0.3 μM or DMSO for 48 h, followed by PI staining and FACS analysis for cell-cycle profile. (B) MLN4924 induced accumulation of Wee1 and decrease of p-H3. The four lymphoma cells were treated with MLN4924 at 0.1 and 0.3 μM or DMSO for 48 h, and subjected to immunoblotting using antibodies against Wee1 and p-H3 (S10) with GAPDH as a loading control. (C) MLN4924 inhibited the proliferation of lymphoma cells. The four cells seeded into 24-well plates were treated with 0.3 μM MLN4924 or DMSO for indicated time points, followed by cell counting (n = 3). (D) MLN4924 impaired cell viability in lymphoma cells. The four cells seeded into 96-well black plates were treated with MLN4924 at 0.1 and 0.3 μM or DMSO for 96 h, followed by cell viability analysis using the ATPlite assay (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Neddylation inhibition with MLN4924 triggers cell line-dependent induction of apoptosis or senescence in lymphoma cells. (A) Changes in cellular morphology upon MLN4924 treatment. Raji, U937, SU-DHL-4 and Toledo cells were treated with 0.1 μM MLN4924 or DMSO for 96 h, followed by morphological observation. Scale bar, 100 μm. (B) MLN4924 significantly induced apoptosis in SU-DHL-4 and Toledo cells, but not Raji and U937 cells. The four lymphoma cells were treated with MLN4924 at 0.1 and 0.3 μM or DMSO for 48 h, and subjected to immunoblotting using antibodies against cleaved caspase-3 and cleaved PARP with GAPDH as a loading control. (C) MLN4924 induced senescence in Raji and U937 cells. Raji and U937 cells, treated with 0.3 μM MLN4924 or DMSO for 96 h, were subjected to senescence-associated β-galactosidase (SA-β-gal) staining assay. Representative pictures were shown (left panel), and positively stained cells were counted and plotted as percentage of total cell numbers (right panel) (n = 3). Scale bar, 100 μm. (D) MLN4924 extended the half-life of p21/p27. Raji and U937 cells were treated with 0.3 μM MLN4924 or DMSO in combination with 50 μg/mL cycloheximide (CHX) for indicated time points, and subjected to immunoblotting using antibodies against p21 and p27 with GAPDH as a loading control. (E) MLN4924 had little effect on the transactivation of p21/p27. Raji and U937 cells were treated with 0.3 μM MLN4924 or DMSO for 6 h, and subjected to real-time PCR for p21 and p27 with GAPDH as a normalizer (n = 3). **P < 0.01, ***P < 0.001.

Figure 4

(See previous page). MLN4924-induced apoptosis is mediated through the intrinsic apoptotic signaling and associated with a coordinated dysregulation of pro-apoptotic and anti-apoptotic proteins. (A) MLN4924 induced apoptosis at early time post treatment. SU-DHL-4 and Toledo cells were treated with MLN4924 at 0.1 and 0.3 μM or DMSO for indicated time points, and subjected to immunoblotting using the antibody against cleaved PARP with GAPDH as a loading control. (B) MLN4924 induced cell apoptosis mainly via the intrinsic apoptotic signaling pathway. SU-DHL-4 and Toledo cells, treated with MLN4924 at 0.1 and 0.3 μM or DMSO for 48 h, were subjected to immunoblotting using antibodies against caspase-8 and cleaved caspase-9 with GAPDH as a loading control. (C) Effects of MLN4924 on expression of pro-apoptotic and anti-apoptotic proteins. SU-DHL-4 and Toledo cells were treated with MLN4924 at 0.1 and 0.3 μM or DMSO for 48 h, followed by immunoblotting using indicated antibodies against pro-apoptotic (left panel) and anti-apoptotic (right panel) proteins with GAPDH as a loading control. (D) Effects of MLN4924 on transcriptional activation of apoptosis-regulatory proteins. Toledo cells, treated with 0.3 μM MLN4924 or DMSO for 36 h, were subjected to the real-time PCR analysis using human apoptosis PCR array as described in Materials and Methods (n = 3).

Figure 5.

A working model. Ub, ubiquitin; N8, NEDD8; SRS, substrate recognition subunit.