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. 2024 Nov 27:15:20406207241301948.
doi: 10.1177/20406207241301948. eCollection 2024.

High expression of malic enzyme 1 predicts adverse prognosis in patients with cytogenetically normal acute myeloid leukaemia and promotes leukaemogenesis through the IL-6/JAK2/STAT3 pathways

Chang Zhang  1 , Wenlu Li  2 , Fei Wu  2 , Zhongwei Lu  2 , Piaorong Zeng  2 , Zeyu Luo  2 , Yixiong Cao  2 , Feng Wen  2 , Junjun Li  2 , Xi Chen  3 , Fujue Wang  3   4
Affiliations

High expression of malic enzyme 1 predicts adverse prognosis in patients with cytogenetically normal acute myeloid leukaemia and promotes leukaemogenesis through the IL-6/JAK2/STAT3 pathways

Chang Zhang et al. Ther Adv Hematol. .

Abstract

Background: Progress in improving risk stratification methods for patients with cytogenetically normal acute myeloid leukaemia (CN-AML) remains limited. This study investigates the prognostic significance and potential functional mechanism of malic enzyme 1 (ME1) in CN-AML.

Methods: Gene expression and clinical data of patients with CN-AML were obtained from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, which were subjected to analysis. The prognostic significance of ME1 was assessed through Kaplan-Meier survival analysis, as well as univariate and multivariate Cox regression analyses. A novel risk model based on ME1 expression levels was developed using TCGA data for predicting CN-AML prognosis. Furthermore, the impact of ME1 silencing on AML cell lines was investigated using the Cell Counting Kit-8 assay and flow cytometry. Gene set enrichment analysis (GSEA) analysis and Western blotting were performed to explore the mechanism of ME1 in CN-AML.

Results: CN-AML patients expressing higher ME1 levels exhibited shorter event-free survival (EFS) and overall survival (OS) compared to those with lower ME1 expression in the TCGA and multiple GEO datasets (all p < 0.05). Univariate and multivariate Cox regression analyses indicated that ME1 expression served as an independent prognostic factor for the EFS (p = 0.024 in TCGA, p = 0.035 in GSE6891) and OS (p = 0.039 in TCGA, p = 0.008 in GSE6891) in patients with CN-AML. The developed risk model demonstrated that patients with CN-AML in the high-risk group had worse survival than those in the low-risk group (hazard ratio: 2.67, 95% confidence interval: 1.54-4.65, p < 0.001) and exhibited strong predictive accuracy for 1-, 3- and 5-year OS (area under the curve = 0.69, 0.75, 0.79, respectively). ME1 knockdown significantly inhibited proliferation and increased apoptosis in AML cells (all p < 0.05). GSEA and Western blotting demonstrated that ME1 regulates the IL-6/JAK2/STAT3 pathway in CN-AML.

Conclusion: Elevated ME1 expression serves as an indicator of poorer prognosis in patients with CN-AML, potentially facilitating leukaemogenesis through the IL-6/JAK2/STAT3 pathway. This suggests that ME1 could be a promising prognostic biomarker and therapeutic target for CN-AML.

Keywords: IL-6/JAK2/STAT3 pathway; cytogenetically normal acute myeloid leukaemia (CN-AML); malic enzyme 1 (ME1); prognosis.

Plain language summary

High levels of malic enzyme 1 (ME1) predict worse outcomes in normal chromosome acute myeloid leukemia and drive cancer growth via a key pathway Background: Normal chromosome acute myeloid leukaemia (CN-AML) is a type of blood cancer where current methods to predict patient outcomes are not very effective. This study looks at the role of a protein called malic enzyme 1 (ME1) in predicting patient outcomes and how it might contribute to cancer progression. Methods: We used data from large cancer databases to analyze the gene expression and clinical outcomes of CN-AML patients. We evaluated the importance of ME1 levels for predicting survival using statistical methods. Additionally, we created a new risk model based on ME1 expression to forecast patient outcomes. We also studied how reducing ME1 levels affects cancer cells in the lab, and explored the pathways ME1 might influence using gene analysis and protein tests. Results: Patients with higher ME1 levels had shorter times without cancer events and overall survival compared to those with lower levels. Statistical analysis showed that ME1 is an independent factor for poor prognosis. Our risk model revealed that patients in the high-risk group had worse survival rates. Reducing ME1 in cancer cells decreased their growth and increased cell death. Further analysis indicated that ME1 influences a key pathway involved in cancer development. Conclusion: High ME1 levels are linked to worse outcomes in CN-AML patients and may promote cancer growth through a specific pathway. ME1 could be a useful marker for prognosis and a potential target for treatment in CN-AML.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

Figure 1.
Figure 1.
High ME1 expression is associated with worse survival in patients with CN-AML. (a) EFS analysis based on ME1 expression in patients with CN-AML from TCGA database. (b) EFS analysis based on ME1 expression in patients with CN-AML from GSE6891 dataset. (c) OS analysis based on ME1 expression in patients with CN-AML from the TCGA database. (d) OS analysis based on ME1 expression in patients with CN-AML from the GSE6891 dataset. (e) OS analysis based on ME1 expression in patients with CN-AML from the GSE71014 dataset. (f) OS analysis based on ME1 expression in patients with CN-AML from the GSE12417 dataset. CN-AML, cytogenetically normal acute myeloid leukaemia; EFS, event-free survival; ME1, malic enzyme 1; TCGA, The Cancer Genome Atlas; OS, overall survival.
Figure 2.
Figure 2.
A novel risk model for the OS of patients with CN-AML based on TCGA database. (a) Nomogram that integrated clinical factors and ME1 expression predicted the probability of the 1-, 3- and 5-year OS of patients with CN-AML from TCGA database. (b) Kaplan–Meier survival analysis of the OS for patients with CN-AML belonging to the low- and high-risk groups. (c) The sensitivity and specificity of the novel risk model for patients with CN-AML were assessed using the 1-, 3- and 5-year ROC curves and AUC values. AUC, area under the curve; CN-AML, cytogenetically normal acute myeloid leukaemia; OS, overall survival; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas.
Figure 3.
Figure 3.
Knockdown of ME1 inhibits proliferation of AML cells. (a) RT-qPCR to analyse the ME1 mRNA expression after the transfection with specific siRNAs or overexpression plasmid. (b) Western blotting to detect ME1 protein expression after the transfection with specific siRNAs or overexpression plasmid. (c) CCK-8 assay was performed to investigate cells proliferation after ME1 knockdown or overexpression for 24, 48 and 72 h. **p < 0.01, ****p and ####p < 0.001. AML, acute myeloid leukaemia; CCK-8, Cell Counting Kit-8; ME1, malic enzyme 1; NC, negative control; NS, no significance; OE, overexpression; RT-qPCR, real-time quantitative polymerase chain reaction; siRNA, small interfering RNA.
Figure 4.
Figure 4.
Knockdown of ME1 induces significant apoptosis in AML cells. (a, b) Apoptosis was analysed using flow cytometry after ME1 knockdown or overexpression for 48 h in AML cells. (c) Western blotting to detect the expression of apoptosis-related proteins after ME1 knockdown or overexpression in AML cells. **p < 0.01, ****p < 0.001. AML, acute myeloid leukaemia; ME1, malic enzyme 1; NC, negative control; NS, no significance; OE, overexpression.
Figure 5.
Figure 5.
ME1 regulates the IL-6/JAK2/STAT3 pathway in AML cells. (a) GSEA for CN-AML patients with high ME1 expression in TCGA database and GSE6891 dataset. (b) IL-6/JAK/STAT3 signaling is enriched in the TCGA database and GSE6891 dataset. (c) Western blotting to investigate the effect of ME1 silencing or overexpression on the IL-6/JAK2/STAT3 pathway in AML cells. (d) THP-1 cells with or without ME1 knockdown treated with exogenous IL-6 and the IL-6/JAK2/STAT3 pathway were detected by Western blotting. AML, acute myeloid leukaemia; CN-AML, cytogenetically normal acute myeloid leukaemia; FDR, false discovery rate; GSEA, gene set enrichment analysis; ME1, malic enzyme 1; NC, negative control; NES, normalised enrichment score; OE, overexpression.
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