ENOX2 NADH Oxidase: A BCR-ABL1-Dependent Cell Surface and Secreted Redox Protein in Chronic Myeloid Leukemia

Abstract

Objective: Chronic myeloid leukemia (CML) is a disease caused by the acquisition of BCR-ABL1 fusion in hematopoietic stem cells. In this study, we focus on the oncofetal ENOX2 protein as a potential secretable biomarker in CML.

Materials and methods: We used cell culture, western blot, quantitative RT-PCR, ELISA, transcriptome analyses, and bioinformatics techniques to investigate ENOX2 mRNA and protein expression.

Results: Western blot analyses of UT-7 and TET-inducible Ba/F3 cell lines demonstrated the upregulation of the ENOX2 protein. BCR-ABL1 was found to induce ENOX2 overexpression in a kinase-dependent manner. We confirmed increased ENOX2 mRNA expression in a cohort of CML patients at diagnosis. In a series of CML patients, ELISA assays showed a highly significant increase of ENOX2 protein levels in the plasma of patients with CML compared to controls. Reanalyzing the transcriptomic dataset confirmed ENOX2 mRNA overexpression in the chronic phase of the disease. Bioinformatic analyses identified several genes whose mRNA expressions were positively correlated with ENOX2 in the context of BCR-ABL1. Some of them encode proteins involved in cellular functions compatible with the growth deregulation observed in CML.

Conclusion: Our results highlight the upregulation of a secreted redox protein in a BCR-ABL1-dependent manner in CML. The data presented here suggest that ENOX2, through its transcriptional mechanism, plays a significant role in BCR-ABL1 leukemogenesis.

Keywords: CML; ENOX2; Biomarker; Chronic myeloid leukemia; Redox protein; Secreted protein.

Figure 1

ENOX2 is upregulated by BCR-ABL1 in UT-7/11 and TET-inducible Ba/F3 cell lines. A: mRNA expression in UT-7/11 (transfected by BCR-ABL1) compared with UT-7/Parental. B, C, D, E: Western blot analyses of BCR-ABL and ENOX-2 protein in UT-7 cell lines and TET-inducible Ba/F3 cell line.

Figure 2

ENOX2 protein upregulation depends on the BCR-ABL1 tyrosine kinase-activity. A: Western blot analysis performed with the UT-7/11 cell line under tyrosine kinase inhibition conditions (imatinib at 1 μM for 6, 18, and 24 hours) showed the reduction of ENOX2 protein expression. B: Western blot analysis performed with the UT-7/p cell line as a control.

Figure 3

ENOX2 is overexpressed in primary cells from patients with chronic-phase chronic myeloid leukemia (CP-CML). A: Scatter dot plot analyses of ENOX2 mRNA expression of CML patients at diagnosis. B: Boxplot shows high ENOX2 protein in plasma levels of CP-CML patients at diagnosis. C: GSE4170 GEO dataset reanalysis. ENOX2 mRNA relative expressions in CML cells corresponding to patients in the chronic phase (CP), acute phase (AP), and blast phase (BC) compared to normal immature CD34+ cells.

Figure 4

A potential network correlated with ENOX2 gene expression in patients with chronic-phase chronic myeloid leukemia (CP-CML) is highlighted. A: Heatmap and parallel coordinate plot revealed 301 genes positively correlated with ENOX2 mRNA expression. B: Unsupervised principal component analysis performed with ENOX2 pattern matching gene expression profile distinguished the chronic phase of CML from the accelerated and blast phases. Correlation p-values were evaluated for phase discrimination on the first principal axis; ellipses around the barycenter were estimated with 75% confidence. C: Functional enrichment performed with ENOX2 pattern matching in the Gene Ontology Biological Process database revealed 49 genes involved in essential cellular processes. Results are presented as a Cytoscape network view showing both biological functions and related genes. The color of nodes in the network is relative to the Z scores obtained during functional enrichment. The size of function nodes is relative to the number of genes enriched in the corresponding function mapped on the network. D: Bar plots represent both Z scores and gene expression fold changes for the different molecular functions defined by the Gene Ontology Biological Process database.

Supplementary Figure S1

Schematic representation of ENOX2 dimer functions at the plasma membrane of a cancer cell. ENOX2 combines two main enzymatic oscillatory activities (hydroquinone NADH oxidase and protein disulfide-thiol oxidoreductase) that alternate with a period length of approximately 22 minutes, generating an ultradian cellular biological clock of 22 hours. Figure from Morré DJ, Morré DM. ECTO-NOX Proteins, Growth, Cancer, and Aging. New York, Springer, 2013.

Supplementary Figure S2

Schematic representation of some mature transcript types and alternative splicing observed in ENOX2 gene. Data are from REFSEQ mRNAs (NM_182314 was taken as the reference for exon numbering). The blue bar represents exon 10-11 boundaries for qRT-PCR experiments using ENOX2 TaqMan pre-developed assay reagent (Hs00197268_m1). Theoretically derived molecular weights of proteins translated from the open reading frame of different mRNA isoforms are given as indications. Exons in red and orange correspond to full-length cDNA and exons potentially translated in the case of skipping of exon 7 (alternative translation initiation sites), respectively.

Supplementary Figure S3

No correlation was found between ENOX2 protein levels in the plasma and white blood cell (WBC) count at diagnosis.

Supplementary Figure S4

Several potentially relevant protein-coding genes displaying mRNA expressions significantly correlated with ENOX2 mRNA expression. Linear regression revealed high positive correlation degrees between ENOX2 and EPHB3, HEYL, ERK1, PlGF, FAK, RHOG, THY1, and TRIB1 gene mRNA expression. The r² and p parameters are shown on each graph.