The Nedd8‐activating enzyme inhibitor MLN4924 (TAK‐924/Pevonedistat) induces apoptosis via c‐Myc‐Noxa axis in head and neck squamous cell carcinoma

Abstract Objectives The present study aimed to reveal expression status of the neddylation enzymes in HNSCC and to elucidate the anticancer efficacy and the underlying mechanisms of inhibiting neddylation pathway. Materials and methods The expression levels of neddylation enzymes were estimated by Western blotting in human HNSCC specimens and bioinformatics analysis of the cancer genome atlas (TCGA) database. Cell apoptosis was evaluated by Annexin V fluorescein isothiocyanate/propidium iodide (Annexin V‐FITC/PI) stain and fluorescence‐activated cell sorting (FACS). Small interfering RNA (siRNA) and the CRISPR‐Cas9 system were used to elucidate the underlying molecular mechanism of MLN4924‐induced HNSCC apoptosis. Results Expression levels of NAE1 and UBC12 were prominently higher in HNSCC tissues than that in normal tissues. Inactivation of the neddylation pathway significantly inhibited malignant phenotypes of HNSCC cells. Mechanistic studies revealed that MLN4924 induced the accumulation of CRL ligase substrate c‐Myc that transcriptionally activated pro‐apoptotic protein Noxa, which triggered apoptosis in HNSCC. Conclusions These findings determined the over‐expression levels of neddylation enzymes in HNSCC and revealed novel mechanisms underlying neddylation inhibition induced growth suppression in HNSCC cells, which provided preclinical evidence for further clinical evaluation of neddylation inhibitors (eg, MLN4924) for the treatment of HNSCC.


| INTRODUC TI ON
Protein neddylation is a type of posttranslational modification, which conjugates neural precursor cell expressed, developmentally down-regulated 8 (NEDD8), a ubiquitin-like molecule, to targeted proteins and then affects subcellular localization, stability, conformation and function of targeted proteins. [1][2][3][4][5] This process is a three-step enzymatic cascade involving NEDD8-activating enzyme E1 (NAE, a heterodimer comprising subunits NAE1 and UBA3), NEDD8-conjugating enzyme E2 M (UBC12) and substrate-specific E3s. [1][2][3][4][5] Cullin family proteins, which serve as essential components of cullin-RING E3 ubiquitin ligases (CRLs), are the best-known substrates among NEDD8-targeted proteins. 6,7 Modification of cullin by NEDD8 leads to the activation of CRL, which further triggers the ubiquitination and degradation of its substrates to regulate diverse biological processes, such as transcription, signal transduction, cellcycle progression and stress responses. The dysfunction of CRL, such as the elevated CRL neddylation modification, contributes to carcinogenesis and cancer progression. 8 Recently, the neddylation pathway, including NAE, UBC12 and NEDD8 itself, has been frequently reported to be hyperactivated in several human cancers and indicates an unfavourable prognosis, highlighting the neddylation-CRL pathway as an attractive anticancer target. [9][10][11][12][13] MLN4924 (Pevonedistat /TAK-924), an investigational smallmolecule inhibitor of NAE, has shown antitumor activity in various cancer xenograft models. 9,11,12,14,15 Mechanistically, MLN4924 abrogates cullin neddylation, and therefore inactivates CRL, leading to the accumulation of tumour-suppressive CRL substrates to suppress the growth of cancer cells by triggering cell-cycle defects, apoptosis or senescence. 3,9,[16][17][18][19] Preclinical studies have demonstrated the therapeutic efficacy of MLN4924 as a single anticancer agent 9,11,14,15 or in combination with chemo/radiotherapy. 20,21 Due to its potent anticancer efficacy and well-tolerated toxicity in preclinical studies, MLN4924 is currently tested in several Phase I/II clinical trials for relapsed/refractory lymphoma, multiple myeloma and advanced nonhematologic malignancies (http://www.clinicaltrials.gov). [22][23][24] Encouragingly, MLN4924 demonstrates anticipated pharmacodynamics effects in myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML), lymphoma and solid tumours with a tolerable safety profile in recently published clinical trials. [22][23][24] Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with an incidence of ~600 000 cases per year and mortality of ~50%. 25,26 Despite advances in therapeutic approaches over the past decades, little improvement has been achieved in the survival rate for HNSCC due to relatively low anticancer efficacy, severe treatment-associated adverse effect and acquired drug resistance, leading to high risk of local recurrences and the development of distant metastases. 27,28 This plight makes an urgent necessity to identify novel anticancer targets and develop new therapeutic agents with efficient and selective anticancer efficacy to improve the treatment of HNSCC.
A previous study has reported that highly proliferative HNSCC cells possessed up-regulated NEDD8 conjugation and MLN4924 cooperating with TRAIL-augmented apoptosis possibly through facilitating c-FLIP degradation in HNSCC cells. 29

| Cell viability assay and clonogenic survival assays
Cells were seeded in 96-well plates (3 × 10 3 cells/well) and treated with DMSO or MLN4924. Cell proliferation was determined using Conclusions: These findings determined the over-expression levels of neddylation enzymes in HNSCC and revealed novel mechanisms underlying neddylation inhibition induced growth suppression in HNSCC cells, which provided preclinical evidence for further clinical evaluation of neddylation inhibitors (eg, MLN4924) for the treatment of HNSCC.
the ATPlite Luminescence Assay Kit (PerkinElmer, Waltham, MA, USA) according to manufacturer's protocol. 9 For the clonogenic assay, 200 cells were seeded in 6-well plates and then were treated with DMSO or MLN4924 and incubated for 10 days in 6-well plates. Colonies comprising 50 cells or more were counted under an inverted microscope. 32 Three independent experiments were performed.

| Transwell cell migration and invasion assays
A standard transwell cell migration assay using a transwell polycarbonate filter (8 μm pore size; Corning Inc., Lowell, MA, USA) was performed to analyse the efficacy of MLN4924 on cell migration. 9 Briefly, cells suspended with serum-free DMEM (Gibco, Thermo were applied, and the culture time was prolonged to 36 h.

| Immunoblotting and cycloheximide (CHX)chase assay
Cell lysates were prepared and analysed by immunoblotting.

| Cell-cycle profile analysis
Cell-cycle profile was evaluated by propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS) analysis as described previously. 33

| Collection of HNSCC tissues and clinicopathological characteristics of patients
All patients underwent surgery, followed by treatment in accordance Center. Written informed consents regarding tissue and data used for scientific purposes were obtained from all participating patients.

The study was approved by the Research Ethics Committee of Sun
Yat-sen University Cancer Center.

| Generation of stable cell lines
Knockout was performed by SgRNA oligos (Table S1)   The statistical significance of differences between groups was assessed using GraphPad Prism5 software (GraphPad Software, San Diego, CA, USA). The t test was used for the comparison of parameters between groups. Data are presented as mean ± standard deviation. For all tests, three levels of significance (*P < 0.05, **P < 0.01, ***P < 0.001) were applied 9

| Inhibiting neddylation pathway impeded the maintenance of HNSCC malignant phenotypes
To determine the expression status of the neddylation enzymes in HNSCC, bioinformatics analysis of TCGA RNA-Seq database was performed to determine the expression levels of NEDD8-activating enzyme E1 (NAE1) and NEDD8-conjugating enzyme E2 (UBC12). As shown in Figure 1A, the mRNA levels of NAE1 and UBC12 in TCGA RNA-Seq database detected HNSCC tissues (n = 514) were significantly higher than those in detected adjacent normal tissues (n = 74) (both P < 0.001). Moreover, Kaplan-Meier analysis showed that the overall survival probability was lower in HNSCC patients with high expression of UBC12 than in patients with low expression of UBC12 and to immunoblotting using antibodies against cleaved-Caspase-3 (c-Caspase-3) and cleaved-Parp (c-Parp) (B) (*P < 0.05,***P < 0.001, n = 3). (C) Intrinsic apoptosis pathway was mainly activated upon neddylation inhibition. HN13 and CAL27 cells were treated by the way mentioned above and were subjected to immunoblotting using antibodies against Caspase-8, cleaved-Caspase-9 (c-Caspase-9), with actin as a loading control. These data were representative of three independent experiments. Data represented means, and error bars were standard deviation. Two-sided t test | 9 of 14 ZHANG et Al.

| MLN4924 triggered G 2 phase cell-cycle arrest by blocking the degradation of P27, P21 and WEE1
To elucidate the underlying mechanisms of MLN4924-induced inhibitory effects on HNSCC cells, cell-cycle profiles of MLN4924-treated cells were analysed by using PI staining and FACS analysis after 24-h treatment. As shown in Figure 2A To address the potential mechanisms of MLN4924-induced G 2 phase arrest, the expression levels of cell cycle-inhibitory CRL substrates, P21 and P27, were determined upon MLN4924 treatment. 43,44 As shown in Figure 2B, MLN4924 led to the accumulation of both P27 and P21. Considering that P27, P21 and WEE1 serve as the substrates of CRL E3 ligase, we hypothesized that MLN4924 induced the accumulation of these cell cycle-related proteins by blocking their degradation due to CRL inactivation.
To test this hypothesis, the degradation of P27, P21 and WEE1 in MLN4924-treated cells was determined under the condition of protein translation inhibition by CHX. As shown in Figure 2C, neddylation inhibition significantly stabilized the expression of these cell cycle-related proteins and extended their half-lives in MLN4924-treated cells.

| MLN4924-activated apoptotic pathway in HNSCC cells
To further define the cellular response to neddylation inhibition over time, HNSCC cells were exposed to MLN4924 with prolonged time  Figure 3B).
To define MLN4924-activated apoptotic pathways, the cleavage of Caspase-8 and Caspase-9, two representative hallmarks of extrinsic and intrinsic apoptotic pathways, respectively, was determined after treatment. As shown in Figure 3C, the cleavage of Caspase-9 was significantly induced by MLN4924 in both cell lines, whereas the cleaved-Caspase-8 was only detected in HN13 cell line, indicating that the intrinsic apoptotic pathway was mainly activated upon neddylation inhibition.

| MLN4924 triggered apoptosis via the up-regulation of BH3-only protein Noxa
The induction of apoptosis by MLN4924 prompted us to investigate the underlying mechanism. We measured the expression levels of

| CRL substrate c-Myc was responsible for the transcriptional activation of Noxa
Based on our above research, Noxa was transactivated upon neddylation inhibition and played a critical role in MLN4924-induced F I G U R E 4 MLN4924 triggered apoptosis via the up-regulation of BH3-only proteins. (A) Effects of MLN4924 on the expression levels of pro-apoptotic proteins. HN13 and CAL27 cells were treated with MLN4924 at increasing concentrations (0.1, 0.3 and 1.0 μmol/L) vs DMSO for 48 h, followed by immunoblotting using indicated antibodies against pro-apoptotic proteins with Actin as a loading control. (B) Effects of MLN4924 on transcriptional activation of Noxa, Bim and Bik. HN13 cells were treated by the way mentioned above and were subjected to the real-time PCR analysis (**P < 0.01, ***P < 0.001, n = 3). (C) Knockdown of Noxa attenuated the apoptosis induced by MLN4924. After HN13 cells transfected Noxa vs control siRNA for 48 h, cultures were treated with DMSO or 0.6 μmol/L MLN4924 for another 48 h.

| D ISCUSS I ON
Inhibition of neddylation pathway with NAE inhibitor MLN4924, a first-in-class anticancer agent, has been developed as a potential anticancer strategy for several human cancers. 9,11,13,47 In the pre-

Induction of cell cycle arrest in cancer cells upon neddylation
inhibition is an initial response, frequently proceeding apoptosis. 48,49 Previous studies showed that MLN4924 could induce S phase and G 1 phase cell cycle arrest in some cancer cells. 14,50,51 The later studies from our and other's groups demonstrated that MLN4924 mainly trigger cell cycle arrest at the G 2 phase in many types of cancer cells. [9][10][11]33,[52][53][54][55][56][57][58][59][60][61] Consistently, genetic inhibition of CRLs by RBX1/ROC1 knockdown induces G 2 phase arrest in cancer cells as well. 32,[62][63][64] In this study, we demonstrated that neddylation inhibition by MLN4924 triggered G 2 phase arrest in HNSCC cells by inducing the accumulation of CRL substrates P21, P27 and WEE1. These findings demonstrate that neddylation F I G U R E 5 CRL substrate c-Myc was responsible for the transcriptional activation of Noxa. (A) MLN4924 treatment led to the accumulation of c-Myc and ATF4. Immunoblotting using antibodies against c-Myc and ATF4 in HN13 and CAL27 cell lines was performed after 24 h of MLN4924 treatment at increasing concentrations (0.1, 0.3 and 1.0 μmol/L) vs DMSO. (B) MLN4924 prolonged the half-lives of c-Myc and ATF4. HN13 cells were pretreated with 0.3 μmol/L MLN4924 for 24 h and then treated with 1.0 μmol/L MLN4924 vs DMSO in combination with 50 μg/mL CHX for 0, 20, 40, 60 and 120 min and subjected to immunoblotting using antibodies against c-Myc and ATF4 with actin as a loading control. The protein level was quantified by densitometric analysis. (C) C-Myc accumulation led to Noxa transactivation. After HN13 cells transfected with control vs c-Myc siRNA for 48 h, cultures were treated with DMSO or 1.0 μmol/L MLN4924 for another 48 h and collected for real-time PCR for Noxa mRNA or subjected to immunoblotting using antibody against cleaved-Parp (c-Parp) and Noxa with actin as a loading control (**P < 0.01, n = 3). (D-G) C-Myc knockout blocked Noxa transcriptional activation.
(D) C-Myc knockout efficiency was determined by Western blotting with actin as a loading control. Noxa protein and mRNA level in c-Myc knockout cells were determined by Western blotting (D) and real-time PCR (E, ***P < 0.001, n = 3). C-Myc knockout cell lines were treated with DMSO or 1.0 μmol/L MLN4924 for 48 h and subjected to immunoblotting using antibody against Noxa with actin as a loading control (F) or collected for real-time PCR for Noxa Mrna (G, ***P < 0.001, n = 3). All data were representative of three independent experiments. Data represented means, and error bars were standard deviation. Two-sided t test F I G U R E 6 A working model of targeting neddylation pathway in HNSCC. N8 = NEDD8 inhibition induces cell cycle arrest at different phases in a cell type-dependent manner.
During addressing the mechanisms for MLN4924-induced intrinsic apoptosis in HNSCC cells, we defined that BH3-only protein Noxa plays a crucial role in this process. Chen et al 12