The NEDD8 Conjugation Pathway Inhibitor MLN4924 Activates AMPK and Induces The ER Stress/UPR-Mediated Cell Death Pathway In Acute Lymphoblastic Leukemia
Authors: Leclerc Gilles M. , Zheng Shuhua , Swords Ronan T. , Leclerc Guy J. , Barredo Julio C. 
Published: October 2019

Abstract
Acute lymphoblastic leukemia (ALL) is the most common cancer in children, and cure rates for patients with resistant/relapsed ALL remain poor. In addition, many childhood cancer survivors experience significant long-term therapy related toxicity, highlighting the need for novel targeted anti-leukemic agents. We recently uncovered that ALL cells are vulnerable to undergo apoptosis via endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated mechanisms (Mol Cancer Res 2012;10:969-978). We reported that the AMP activated protein kinase (AMPK) is activated in response to ER stressors (metformin, 2-DG, tunicamycin), and its activation suppressed the UPR preventing ALL cells from processing misfolded/unfolded proteins in the ER lumen, leading to apoptosis. In search for novel agents targeting AMPK, we investigated the anti-leukemic activity of a new AMP analogue, MLN4924, in Bp- and T-ALL cell line models. This agent targets the ubiquitin-like protein NEDD8 conjugation pathway leading to inhibition of cullin-based E3 ligase dependent proteasomal degradation of proteins essential for cancer cell growth and survival. We demonstrated that MLN4924 induced dose-dependent cell growth inhibition and apoptosis in both Bp-ALL (EC50 = 372 nM, p<0.0001 for MLN4924-treated NALM6 cells vs. controls) and T-ALL (EC50 = 421 nM, p<0.0001 for MLN4924-treated CCRF-CEM cells vs. controls) cell line models. To investigate the mechanisms of cell death induced by MLN4924 in ALL cells, we determined its effect on AMPK, UPR, and NF-κB signaling pathways using Western immunoblots. Our results show that MLN4924 induced phosphorylation of AMPK (Thr172) and down-regulated its downstream target p-mTOR (Ser2448), suggesting that MLN4924 as an AMP-analogue can allosterically induce activation of AMPK and affect downstream AMPK-associated signaling pathways. As expected, MLN4924 decreased significantly the expression of NEDDylated cullins confirming the inhibitory effect of MLN4924 on the NEDD8 conjugation pathway. We also demonstrated that MLN4924 induced ER stress as evidenced by increased expression of the main UPR regulator GRP78 and the UPR cell death mediator CHOP, suggesting that MLN4924 induces cell death in ALL cell models via an UPR-mediated mechanism. Additional Western blots revealed that MLN4924 inhibited the degradation of the inhibitor of NF-κB, p-IκBα (Ser32), with concomitant down-regulation of phosphorylated RelA (p65) at Ser 536, suggesting that stabilization of p-IκBα (Ser32) following inhibition of NEDD8 conjugation pathway by MLN4924 prevents NF-κB activation in ALL. To further characterize the cytotocixity of MLN4924 in ALL cells, we examined the effects of MLN4924 on ROS generation. Using the cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorogenic dye, we did not detect ROS induction in MLN4924-treated ALL cells and co-treatment of ALL cells with the antioxidant N-Acetyl-L-Cysteine (NAC) did not rescue MLN4924’s cytotoxicity in ALL cells. In contrast to data reported in AML (Blood 2010; 115:3796-3800), our findings suggest that ROS does not mediate MLN4924-induced cell death in ALL. Taken together, our data indicate that MLN4924 induces cell death in ALL cells by inhibiting the NEDD8 conjugation pathway and activating AMPK, which respectively trigger down-regulation of the NF-κB pathway and ER stress/UPR-mediated cell death. These findings provide a rationale to further investigate the mechanism of action of MLN4924 either alone or in combination with agents that target the NF-κB pathway and ER stress/UPR pathways in ALL.

Disclosures:
No relevant conflicts of interest to declare.