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. 2022 Aug 31;42(8):BSR20220994.
doi: 10.1042/BSR20220994.

Inhibition of NEDD8 NEDDylation induced apoptosis in acute myeloid leukemia cells via p53 signaling pathway

Yanli Chen  1 , Ling Sun  1
Affiliations

Inhibition of NEDD8 NEDDylation induced apoptosis in acute myeloid leukemia cells via p53 signaling pathway

Yanli Chen et al. Biosci Rep. .

Abstract

MLN4924 is a potent and selective small-molecule inhibitor of NEDD8-activating enzyme, which showed antitumor effect in several types of malignant tumor types. However, the mechanism of action of MLN4924 in acute myeloid leukemia (AML) requires further investigation. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was conducted to detect the mRNA levels of genes. Gene expression was knocked down by short hairpin RNA (shRNA). Moreover, the protein expression was detected by Western blotting (WB) assay. The proliferation and apoptosis of AML cells were measured by Cell Counting Kit-8 (CCK8) assay and flow cytometry (FCM). In the present study, we observed that the mRNA expression levels of NEDD8, UBA3, UBE2M and RBX1 in AML patients were up-regulated compared with healthy controls, which were correlated with worse overall survival (OS) of patients. Besides, knockdown of UBA3, UBE2M and RBX1 inhibited the NEDDylation of CULs and increased the protein expression of p53 and p21 in MOLM-13 cell line. In AML cells, MLN4924 inhibited cell proliferation, promoted cell apoptosis, and induced cell cycle arrest at the G2/M phase. As revealed by experiments in vivo and in vitro, the NEDDylation of CULs was significantly inhibited and the p53 signaling pathway was activated after MLN4924 treatment. So, we concluded that NEDD8, UBA3, UBE2M and RBX1 may serve as the prognostic biomarkers and novel therapeutic targets for AML. Inhibition of the NEDDylation pathway resulted in an anti-leukemia effect by activating the p53 signaling pathway.

Keywords: AML; MLN4924; NEDD8; p21; p53.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. NEDD8, UBA3, UBE2M and RBX1 were over-expressed in AML patients and were correlated with worse OS
(A–D) Compared with normal control, the mRNA levels of NEDD8, UBA3, UBE2M and RBX1 were significantly higher in AML patients. (E–G) As revealed by FAB subtype analysis, the mRNA expression levels of NEDD8, UBE2M and RBX1 were higher in patients with AML-M2, M4 and M5 than those in healthy controls. In patients with AML-M5, the mRNA expression of UBA3 was higher than that in the control group. (H–K) Survival analysis revealed that the OS rate of patients with overexpression of NEDD8, UBA3, UBE2M and RBX1 was lower than that of patients with low-expression. ROC curves were plotted based on the mRNA expression levels of NEDD8, UBA3, UBE2M and RBX1, respectively, and Jorden index was calculated to determine the cut-off value (NEDD8, 1.4257; UBA3, 1.6511; UBE2M, 1.6146; RBX1, 1.4026) to divide the samples into high and low expression groups. An independent sample t-test was conducted to compare the means between the two groups. Triplicates were set for each gene in each sample for RT-qPCR. Chi-square test was performed to compare the qualitative data. The KM method was employed to draw the survival curves of patients, and log-rank tests were utilized to compare the survival rates. Univariate and multivariate Cox analyses were adopted to determine whether the expression of the interested genes was the independent prognostic factor for OS; ***P<0.001, **P<0.01, *P<0.05.
Figure 2
Figure 2. UBA3, UBE2M and RBX1 knockdown inhibited the NEDD8 NEDDylation of CULs and activated the p53 signaling pathway in MOLM-13 cells
(A) The knockdown efficiency of shRNA was >70% by RT-qPCR. (B and E) After transfection of shRNA in MOLM-13 cells, the expression of UBA3, UBE2M and RBX1 proteins was reduced. (C) and (F–H) NEDD8 NEDDylation of CUL1-4 was significantly inhibited following the knockdown of UBA3, UBE2M and RBX1. The NEDDylation of CUL5 was suppressed by the knockdown of UBA3 but up-regulated by the knockdown of UBE2M and RBX1. (D) and (I–K) The expression of p21 and p53 significantly increased following UBA3, UBE2M and RBX1; ***P<0.001, **P<0.01. An independent sample t-test was adopted to compare the means between two groups. Each experiment was carried out in triplicate.
Figure 3
Figure 3. MLN4924 inhibited the proliferation and induced the apoptosis of AML cells
(A) MLN4924 significantly inhibited the proliferation of THP-1 cells, MOLM-13 cells and the primary cells collected from two AML patients in a concentration- and time-dependent manner. (B–E) The numbers of apoptotic cells of THP-1 and MOLM-13 cells significantly increased following treatment with MLN4924 (except for the 0.1 μM dose group of MLN4924 in MOLM-13 cells, P>0.05). (F–H) MLN4924 induced cell cycle arrest in both THP-1 and MOLM-13 cells at the G2/M phase, and significantly increased the cells at G2/M phase; ***P<0.001. The means among multiple groups were compared by one-way ANONA and LSD-t test. Each experiment was carried out in triplicate.
Figure 4
Figure 4. MLN4924 inhibited the NEDDylation of CULs in AML cells
(A) MLN4924 significantly inhibited the NEDD8 NEDDylation of CUL1-5 in THP-1 cells. (B) In MOLM-13 cells, the NEDDylation of CUL1-5 was also inhibited. (C) MLN4924 inhibited the NEDD8 NEDDylation of CUL1-5 in primary AML cells collected from two AML patients. (D–G) Statistical analysis revealed that MLN4924 inhibited the NEDD8 NEDDylation of CULs in THP-1 cells; ***P<0.001, **P<0.01. An independent sample t-test was adopted to compare the means between two groups. Each experiment was conducted in trplicate.
Figure 5
Figure 5. MLN4924 activated the p53 signaling pathway in AML cells
(A,B) In THP-1 and MOLM-13 cells, the expression levels of p21 and p53 proteins were significantly up-regulated after MLN4924 treatment. (C) In primary AML cells harvested from two AML patients, MLN4924 increased the expression of both p21 and p53 proteins. (D,E) Statistical analysis indicated that the expression of p21 and p53 was up-regulated; ***P<0.001, **P<0.01. An independent sample t-test was conducted to compare the means between two groups. Each experiment was carried out in triplicate.
Figure 6
Figure 6. MLN4924 inhibited the growth of xenograft tumors in mice
(A) The xenograft tumors were detected by in vivo bioluminescence imaging. (B) Compared with the control group, the growth of the xenograft tumors in the experimental group was significantly inhibited. (C,D) The NEDDylation of CULs was inhibited and the protein expression levels of p21 and p53 significantly increased in xenograft tumors of mice treated with MLN4924 (n=5). ***P<0.001. An independent sample t-test was conducted to compare the means between two groups. Each experiment was carried out in triplicate.
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