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. 2023 May 29;40(2):101–117. doi: 10.4274/tjh.galenos.2023.2022-0339

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

ENOX2 NADH Oksidaz: Kronik Myeloid Lösemi’de Hücre Dışına Salgılanan ve BCR-ABL1-Bağımlı Hücre Yüzeyi Redoks Proteini

Seda Baykal 1,2, Maud Voldoire 3, Christophe Desterke 4, Nathalie Sorel 5, Emilie Cayssials 6, Hyacinthe Johnson-Ansah 7, Agnes Guerci-Bresler 8, Annelise Bennaceur-Griscelli 9, Jean-Claude Chomel 10, Ali G Turhan 11,*
PMCID: PMC10240159  PMID: 37026766

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

Introduction

Chronic myeloid leukemia (CML) therapy has been radically modified by tyrosine kinase inhibitors (TKIs). The life expectancy of patients diagnosed with chronic-phase CML (CP-CML) and responding to TKI therapy appears to be similar to that of the general population [1]. The BCR-ABL1 tyrosine kinase responsible for CML initiates uncontrolled granulocyte proliferation, decreased adhesion to the medullar niche, apoptosis inhibition, and genetic instability. Most aberrant signaling pathways downstream of BCR-ABL1 have been known for a long time [2]. Using the human granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent erythro/megakaryoblastic UT-7 cell line, our group previously uncovered novel nonconventional actors downstream of the BCR-ABL1 signaling [3,4].

In the present study, we highlight the upregulation of ENOX2 (ecto-nicotinamide adenine dinucleotide oxidase disulfide thiol exchanger 2) in BCR-ABL1-positive cell lines. ENOX2 is a growth-related cell surface protein that combines two oscillatory enzymatic activities, those of hydroquinone NADH oxidase and protein disulfide-thiol oxidoreductase, that alternate within a period of 22 minutes, generating an ultradian cellular biological clock of 22 hours (Supplementary Figure S1) [5,6]. The ENOX2 protein is present during the embryonic period and almost entirely absent in normal adult cells, and it reappears in most cancer cells [7]. ENOX2 proteins can be released from tumor cells into extracellular fluids, in which they can be detected [8].

Supplementary Figure S1.

Supplementary Figure S1

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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.

In our experiments, increased ENOX2 mRNA expression was also found in primary cells from patients with CP-CML at diagnosis. Using the UT-7 and inducible Ba/F3 cell lines, we confirmed ENOX2 upregulation at the protein level and demonstrated that this phenomenon was linked to BCR-ABL1 tyrosine kinase activity. In addition, a significant increase in ENOX2 protein levels was observed in the plasma of patients at the time of diagnosis. Reanalyzing a publicly available database, we found that ENOX2 mRNA expression was characteristic of CP-CML.

Materials and Methods

UT-7 and TET-Inducible Ba/F3 Cell Lines

The human hematopoietic UT-7 parental cell line (UT-7/p) was kindly provided by Dr.  Komatsu et al. [9]. Its counterparts transduced with either native BCR-ABL1-p210 (UT-7/11 cells) or T315I-mutated-BCR-ABL1 (UT-7/T315I) resistant to first- and second-generation TKIs were previously characterized by our group [10,11]. The doxycycline-inducible BaF/p210 sin1.55 cell line was also previously described [12]. In this TET-OFF model, the addition of doxycycline in the cell culture medium turns off BCR-ABL1 expression.

Patients and Healthy Donors

For ENOX2 mRNA expression analysis, 36 CP-CML patients were tested at diagnosis along with a cohort of 27 healthy control donors (Supplementary Table S1). Concerning the quantification of ENOX2 protein in plasma samples, independent cohorts of 41 CP-CML patients at diagnosis and 28 healthy controls were also analyzed. This nonclinical study was approved by the INSERM UA9 Ethics Committee on February 11, 2014. All patients and healthy donors gave informed consent in accordance with the Declaration of Helsinki.

Supplementary Table S1. Patients’ characteristics in the cohort for ENOX2 mRNA expression.

graphic file with name TJH-40-101-g9.jpg

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Transcriptome Experiments

Analyses were performed using UT-7/11 cells expressing BCR-ABL1 as compared to parental UT-7 cells. Total RNA was extracted from UT-7 cells. RNA quantification was performed with a NanoDrop device (Thermo Fisher Scientific, Santa Clara, CA, USA) and sample quality was evaluated with the Bioanalyzer-2100 instrument (Agilent Technologies, Santa Clara, CA, USA). Transcriptome analysis was performed on the Human Genome U133A Array version of the Affymetrix platform (Affymetrix, Santa Clara, CA, USA). The results of each experimental group were normalized with the RMA algorithm (Affymetrix).

Quantitative RT-PCR Assays

Total RNA from whole blood was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Foster City, CA, USA) and qRT-PCR experiments were performed using the 7900 Sequence Detection System (Life Technologies). TaqMan pre-developed assay reagents were used to quantify ENOX2 (Hs00197268_m1) and B2M as an internal reference (beta-2 microglobulin, Hs00187842_m1). The Hs00197268_m1 TaqMan expression assay allows the detection of all ENOX2 mRNA splicing variants (Supplementary Figure S2). PCR reactions were prepared in duplicate using TaqMan Universal PCR Master Mix (Life Technologies) and ENOX2/B2M ratios were determined using the ∆Ct method.

Supplementary Figure S2.

Supplementary Figure S2

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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.

Western Blotting

The proteins were quantified by BCA assay, separated by polyacrylamide gel electrophoresis, and wet-transferred to PVDF membranes. Membranes were probed with specific primary and HRP-conjugated secondary antibodies and revealed by chemiluminescence with SuperSignal West Dura or Femto reagents. Data were acquired using the G-BOX-iChemi Image Capture system (Syngene, Frederick, MD, USA). The antibodies used were as follows: anti-ENOX2 (LS-C346209, LifeSpan BioSciences, Seattle, WA, USA) and anti-ABL1 (Santa Cruz Biotechnology) for the detection of translocated and non-translocated ABL1 protein, and anti-beta-actin (Sigma-Aldrich) with dilution as recommended by the manufacturer.

ENOX2 Protein Concentration in Blood Plasma

ENOX2 protein in blood plasma was quantified using the human Ecto-NOX Disulfide-Thiol Exchanger 2 ELISA Kit (MBS 943476, MyBioSource Inc., San Diego, CA, USA) according to the manufacturer’s guidelines.

Transcriptome Dataset GSE4170

Using a DNA microarray, Radich et al. al. [13] aimed to compare gene expression between the chronic (<10% blasts), accelerated (10%-30% blasts), and blast phase (>30% blasts) by analyzing 91 cases of CML using normal immature CD34+ cells as a reference. The Rosetta/Merck Human 25k v2.2.1 data matrix of normalized log-ratios from GSE4170 was downloaded from the Gene Expression Omnibus (GEO) website and annotated with the GPL2029 annotation platform to be reanalyzed, focusing on ENOX2 mRNA expression.

Bioinformatics Microarray Data Analysis

Genes whose expressions were significantly correlated with the expression of ENOX2 during CML progression were determined from the transcriptomes of hematopoietic cells from the GEO GSE4170 dataset with the Pavlidis template matching algorithm. During this analysis, taking ENOX2 expression as the outcome, the thresholds of a positive Pearson correlation coefficient greater than 0.80 and a p-value less than 1.E-6 were used to define significant genes with positive correlations to ENOX2 expression during the progression of CML [14]. These significant genes were retained to draw an expression heatmap associated with a parallel coordinate plot obtained with the MADE4 [15] and GGally R packages, respectively. Gene clustering on the expression heatmap was done with Pearson correlation distances. The expression profile correlating with ENOX2 in CML cells was used to generate functional enrichment with Go-Elite Standalone software version 1.2 in the Gene Ontology Biological Process database including the Homo sapiens EnsMart77Plus (Ensembl - Biomart) update [16]. Unsupervised principal component analysis was performed on the correlated gene expression profile with the FactoMiner R package. The p-values were calculated by group discrimination on the first principal component axis. The functional interaction network was built with functional relations identified during enrichment analysis with Cytoscape software version 3.2.1 [17].

Scatter Dot Plots, Boxplots, and Statistical Analysis

Data on ENOX2 gene expression or protein concentrations in plasma were expressed as scatter dot plots or boxplots with medians (Prism version 8.0, GraphPad Software, San Diego, CA, USA). The two-sided Welch t-test was used to determine statistical significance between data groups. Differences were considered significant at p<0.05.

Results

ENOX2 Is Overexpressed in BCR-ABL1-Expressing UT-7 Cell Lines

We performed a transcriptome assay to identify genes up- or downregulated in BCR-ABL1-expressing UT-7 cells. To this end, we compared the UT-7/11 cells (n=3), which expressed high levels of BCR-ABL1 protein, to parental UT-7 cells (UT7/p, n=3). We focused on ENOX2 mRNA, which was not previously known to be overexpressed in BCR-ABL1-positive leukemia. As shown in Figure 1A, ENOX2 was significantly upregulated (x2.4, p=0.0012) in the UT-7/11 cells.

Figure 1.

Figure 1

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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.

BCR-ABL1 Induces the Production of ENOX2 Protein in BCR-ABL1-Expressing Cell Lines

Parental UT-7, UT-7/11, and UT-7/T315I cells were used to analyze the presence of ENOX2 protein by western blot. Figure 1B confirms the presence of BCR-ABL1 protein in UT-7/11 and UT-7/T315I cells in contrast to parental UT-7 cells. Concerning ENOX2, high protein levels were produced in the BCR-ABL1-expressing UT-7 cell line (native UT-7/11 and mutated-UT-7/T315I) compared to UT-7/p control cells (Figure 1C).

To ensure that ENOX2 overexpression would be related to the presence of BCR-ABL1, we used a Ba/F3 cell line transduced with BCR-ABL1 under the control of the TET promoter (BaF/p210 sin1.55). This inducible model was appropriate insofar as the BCR-ABL1 protein was inhibited by doxycycline (Figure 1D). Decreased ENOX2 protein expression was observed in response to doxycycline added to the culture medium (Figure 1E). BCR-ABL1 and ENOX2 were re-expressed on day 12 upon washing out the doxycycline from the cell medium (Figures 1D and 1E). Consequently, ENOX2 overexpression appeared to be related to the presence of BCR-ABL1 insofar as inhibition of BCR-ABL1 expression in the TET-inducible Ba/F3 model led to a reduction in ENOX2 protein synthesis.

We then asked whether the expression of ENOX2 in CML cells was a tyrosine kinase-dependent event. Western blot experiments showed that ENOX2 protein expression was reduced in the UT7/11 cells treated with imatinib at 1 µM for 6, 18, and 24 hours (Figure 2A). The same experiments performed on the UT-7/p cell line showed no modification of basal ENOX2 expression (Figure 2B).

Figure 2.

Figure 2

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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.

ENOX2 mRNA Expression Is Increased in Primary Cells from Patients at Diagnosis

To validate the results obtained for BCR-ABL1-expressing cells, we examined ENOX2 mRNA expression by qRT-PCR in blood samples obtained from a cohort of CP-CML patients at diagnosis.  ENOX2 mRNA levels were significantly increased in samples obtained from patients with CP-CML at diagnosis (p<0.0001) compared to healthy donors with a fold change of 4.75 (Figure 3A). We did not find any correlations between ENOX2 mRNA expression and Sokal scores or patient outcomes in this cohort.

Figure 3.

Figure 3

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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.

ENOX2 Protein Levels Are Significantly Increased in the Plasma of CML Patients

We determined ENOX2 protein concentrations in the plasma of patients with CML at diagnosis compared to healthy controls using an ELISA method. A significant increase in plasma ENOX2 protein levels (p<0.0001) was shown in CP-CML patients at diagnosis before TKI therapy (Figure 3B). The extended frequency distribution of ENOX2 protein levels was undoubtedly due to heterogeneity between patients. Furthermore, no correlation was found between ENOX2 protein levels in the plasma and white blood cell count at diagnosis (Supplementary Figure S3).

Supplementary Figure S3.

Supplementary Figure S3

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

ENOX2 mRNA Overexpression Is Characteristic of the Chronic Phase of CML

We reanalyzed a publicly available transcriptome dataset from CML patients during different phases of the disease (GSE4170). ENOX2 mRNA expression data were retrieved from the GE0 database and analyzed independently. ENOX2 mRNA upregulation (using normal immature CD34+ cells as the reference) was observed in this independent study for all CP-CML patients (Figure 3C). In addition, ENOX2 expression in the accelerated and blast phases was comparable to that of normal CD34+ cells and appeared to be heterogeneous.

mRNA Expressions of ENOX2 and Related Genes Distinguish the Chronic Phase of CML from Advanced Phases

Exploiting the GSE4170 transcriptome dataset, we next tried to discover genes positively correlated with ENOX2 mRNA expression. The Pavlidis template matching algorithm used with ENOX2 as a predictor highlighted 301 related genes with a positive r correlation coefficient greater than 0.80 and a p-value threshold less than p<1E-6 (Supplementary Table S2). The high correlation with ENOX2 mRNA expression is illustrated for eight potentially relevant protein-coding genes in Supplementary Figure S4. The heatmap and parallel coordinate plot revealed that the high mRNA expression levels of these 301 genes were characteristic of the CP-CML (Figure 4A). Unsupervised principal component analysis performed with the ENOX2 pattern matching gene expression profile allowed highly significant discrimination of the chronic phase from the accelerated and blastic phases (Figure 4B, p<0.0001).

Supplementary Table S2. Genes correlated with ENOX2 mRNA expression. Genes known to be involved in essential cell processes are given in red.

graphic file with name TJH-40-101-g10.jpg

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Supplementary Figure S4.

Supplementary Figure S4

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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.

Figure 4.

Figure 4

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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.

Several Proteins of a Potential ENOX2 Network May Be Involved in Crucial Biological Processes

Functional enrichment performed with the ENOX2 pattern matching in the Gene Ontology Biological Process database emphasized 49 genes out of 301 involved in essential cell processes (Figure 4C). In the context of ENOX2 mRNA overexpression in CP-CML, several major biological functions appear to be activated: angiogenesis, NOTCH signaling, cell morphogenesis differentiation, circadian rhythm, RAS signaling, cell proliferation, G-protein receptor pathways, integrin-mediated signaling, carbohydrate homeostasis, stress-activated protein kinase, and RHO GTPase activities (Figure 4D, Supplementary Table S3).

Supplementary Table S3. Proteins of ENOX2 potential network involved in critical biological processes.

graphic file with name TJH-40-101-g11.jpg

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Discussion

The involvement of ENOX2 in BCR-ABL1-positive leukemias has yet to be shown. Here, we have established that ENOX2 mRNA was overexpressed in both experimental models of BCR-ABL1-induced cell transformation and primary leukemic cells from newly diagnosed CP-CML patients. Western blots analyses confirmed the presence of higher levels of ENOX2 protein in BCR-ABL1-expressing murine and human cell lines. This overexpression is directly influenced by the presence of the BCR-ABL1 oncoprotein and related to its constitutive tyrosine kinase activity. We also observed a significant increase in plasma ENOX2 levels in CML patients at diagnosis. Reanalyzing a transcriptomic dataset from a previously reported gene profiling study [13], we observed that ENOX2 mRNA expression is restricted to the chronic phase of the disease. Nevertheless, we could not determine here whether ENOX2 is a direct or an indirect target of BCR-ABL1.

The ENOX2 gene is only translated during early embryogenesis and cancer development. Therefore, the physiological function of ENOX2 appears to be restricted to the embryonic period [7], in which this oncofetal protein located at the plasma membrane has oscillating enzymatic activity. Little is known about the reappearance and oncogenic function of ENOX2 in adult cells. However, it has been shown that ENOX2 proteins are constitutively activated in cancers and could promote cell proliferation [18,19]. As they are not firmly anchored into the cell membrane, ENOX2 proteins can be shed into extracellular fluids. This circulating form has been detected in the sera of patients suffering from various tumors [20], whereas it is present only at very low levels in healthy subjects [21]. In the present study, significant levels of circulating ENOX2 protein detected in plasma from CP-CML patients at diagnosis are consistent with the prior data.

In silico reanalysis suggested that the mRNA expressions of several genes are positively correlated with ENOX2 expression in the context of BCR-ABL1, thereby highlighting critical biological functions. These results for a substantial number of individual genes are highly significant, and it is quite unlikely that all of these genes are not translated into functional proteins. Consequently, high ENOX2 mRNA expression probably goes along with the activation of cell proliferation and differentiation through genes encoding proteins from different pathways. ENOX2 overexpression has been linked to cell proliferation, migration, and increased expression of mesenchymal markers in some cancers [22]. Interestingly, these data are in line with the characteristics of CML dysregulation [23]. Among the genes found to be positively correlated with ENOX2 mRNA expression in a CML context, some have already been confirmed to be involved in BCR-ABL1-mediated leukemogenesis. This is the case for RAC2 GTPases, ERK MAP-kinase, FAK (focal adhesion kinase), NOTCH1, and PlGF (placental growth factor) [24,25,26,27,28].

In addition to this potential biological feature, we wondered whether ENOX2 could be a surrogate marker or even a therapeutic target. Based on our experiments, we can state that ENOX2 is the first secreted biomarker described in CML. On the other hand, we did not find any relationship between ENOX2 mRNA expression or ENOX2 plasma levels and the clinical course or the most relevant biological parameters. Some antioxidant agents (capsaicin, omega-3 polyunsaturated fatty acids, or synthetic isoflavone) could exert antitumor effects by inhibiting ENOX2 enzymatic activity [29,30]. All of these agents could represent a potential ENOX2-targeted therapy for malignant diseases. In CML, the majority of patients treated with TKIs achieve a sustained molecular response. However, two circumstances may require other therapeutic options: the complete resistance to all available TKIs and the persistence of quiescent leukemic stem cells. The potential value of ENOX2 as a druggable target in these contexts warrants further exploration.

Conclusion

Overall, we propose that BCR-ABL1 upregulates ENOX2 in the chronic phase of CML. To the best of our knowledge, the association between the reactivation of ENOX2 in adult cells and deregulated tyrosine kinase activity was never previously observed. Our results suggest that ENOX2 could play a role in the pathogenesis of CML in a BCR-ABL1-dependent manner. Further studies are required to clarify the link between BCR-ABL1 and ENOX2.

Footnotes

Ethics

Ethics Committee Approval: This non-clinical study was approved by the INSERM UA9 Ethics Committee on February 11, 2014.

Informed Consent: All patients and healthy donors gave informed consent in accordance with the Declaration of Helsinki.

Authorship Contributions

Concept: S.B., M.V., C.D., N.S., E.C., H.J-A., A.G-B., A.B-G., J.C-C., A.G.T.; Design: S.B., M.V., C.D., N.S., E.C., H.J-A., A.G-B., A.B-G., J.C-C., A.G.T.; Data Collection or Processing: S.B., M.V., C.D., N.S., E.C., H.J-A., A.G-B., A.B-G., J.C-C., A.G.T.; Analysis or Interpretation: S.B., M.V., C.D., N.S., E.C., H.J-A., A.G-B., A.B-G., J.C-C., A.G.T.; Literature Search: S.B., M.V., C.D., N.S., E.C., H.J-A., A.G-B., A.B-G., J.C-C., A.G.T.; Writing: S.B., M.V., C.D., N.S., E.C., H.J-A., A.G-B., A.B-G., J.C-C., A.G.T.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.

References

  • 1.Bower H, Björkholm M, Dickman PW, Höglund M, Lambert PC, Andersson TM. Life expectancy of patients with chronic myeloid leukemia approaches the life expectancy of the general population. J Clin Oncol. 2016;34:2851–2857. doi: 10.1200/JCO.2015.66.2866. [DOI] [PubMed] [Google Scholar]
  • 2.Cilloni D, Saglio G. Molecular pathways: BCR-ABL. Clin Cancer Res. 2012;18:930–937. doi: 10.1158/1078-0432.CCR-10-1613. [DOI] [PubMed] [Google Scholar]
  • 3.Girerd S, Tosca L, Herault O, Vignon C, Biard D, Aggoune D, Dkhissi F, Bonnet ML, Sorel N, Desterke C, Bennaceur-Griscelli A, Tachdjian G, Guilhot F, Guilhot J, Chomel JC, Turhan AG. Superoxide dismutase 2 (SOD2) contributes to genetic stability of native and T315I-mutated BCR-ABL expressing leukemic cells. Biochem Biophys Res Commun. 2018;498:715–722. doi: 10.1016/j.bbrc.2018.03.023. [DOI] [PubMed] [Google Scholar]
  • 4.Desterke C, Voldoire M, Bonnet ML, Sorel N, Pagliaro S, Rahban H, Bennaceur-Griscelli A, Cayssials E, Chomel JC, Turhan AG. Experimental and integrative analyses identify an ETS1 network downstream of BCR-ABL in chronic myeloid leukemia (CML) Exp Hematol. 2018;64:71–83.e8. doi: 10.1016/j.exphem.2018.04.007. [DOI] [PubMed] [Google Scholar]
  • 5.Chueh PJ, Kim C, Cho N, Morré DM, Morré DJ. Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface. Biochemistry. 2002;41:3732–3741. doi: 10.1021/bi012041t. [DOI] [PubMed] [Google Scholar]
  • 6.Morré DJ, Chueh PJ, Pletcher J, Tang X, Wu LY, Morré DM. Biochemical basis for the biological clock. Biochemistry. 2002;41:11941–11945. doi: 10.1021/bi020392h. [DOI] [PubMed] [Google Scholar]
  • 7.Cho N, Morré DJ. Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells. Cancer Immunol Immunother. 2009;58:547–552. doi: 10.1007/s00262-008-0577-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Morré DJ, Hostetler B, Taggart DJ, Morré DM, Musk AW, Robinson BW, Creaney J. ENOX2-based early detection (ONCOblot) of asbestos-induced malignant mesothelioma 4-10 years in advance of clinical symptoms. Clin Proteomics. 2016;13:2. doi: 10.1186/s12014-016-9103-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Komatsu N, Nakauchi H, Miwa A, Ishihara T, Eguchi M, Moroi M, Okada M, Sato Y, Wada H, Yawata Y, Suda T, Miura Y. Establishment and characterization of a human leukemic cell line with megakaryocytic features: dependency on granulocyte-macrophage colony-stimulating factor, interleukin 3, or erythropoietin for growth and survival. Cancer Res. 1991;51:341–348. [PubMed] [Google Scholar]
  • 10.Issaad C, Ahmed M, Novault S, Bonnet ML, Bennardo T, Varet B, Vainchenker W, Turhan AG. Biological effects induced by variable levels of BCR-ABL protein in the pluripotent hematopoietic cell line UT-7. Leukemia. 2000;14:662–670. doi: 10.1038/sj.leu.2401730. [DOI] [PubMed] [Google Scholar]
  • 11.Aggoune D, Tosca L, Sorel N, Bonnet ML, Dkhissi F, Tachdjian G, Bennaceur-Griscelli A, Chomel JC, Turhan AG. Modeling the influence of stromal microenvironment in the selection of ENU-induced BCR-ABL1 mutants by tyrosine kinase inhibitors. Oncoscience. 2014;1:57–68. doi: 10.18632/oncoscience.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Dugray A, Geay JF, Foudi A, Bonnet ML, Vainchenker W, Wendling F, Louache F, Turhan AG. Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette. Leukemia. 2001;15:1658–1662. doi: 10.1038/sj.leu.2402225. [DOI] [PubMed] [Google Scholar]
  • 13.Radich JP, Dai H, Mao M, Oehler V, Schelter J, Druker B, Sawyers C, Shah N, Stock W, Willman CL, Friend S, Linsley PS. Gene expression changes associated with progression and response in chronic myeloid leukemia. Proc Natl Acad Sci U S A. 2006;103:2794–2799. doi: 10.1073/pnas.0510423103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Pavlidis P, Noble WS. Analysis of strain and regional variation in gene expression in mouse brain. Genome Biol. 2001;2:RESEARCH0042. doi: 10.1186/gb-2001-2-10-research0042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Culhane AC, Thioulouse J, Perrière G, Higgins DG. MADE4: An R package for multivariate analysis of gene expression data. Bioinformatics. 2005;21:2789–2790. doi: 10.1093/bioinformatics/bti394. [DOI] [PubMed] [Google Scholar]
  • 16.Zambon AC, Gaj S, Ho I, Hanspers K, Vranizan K, Evelo CT, Conklin BR, Pico AR, Salomonis N. GO-Elite: a flexible solution for pathway and ontology over-representation. Bioinformatics. 2012;28:2209–2210. doi: 10.1093/bioinformatics/bts366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, Workman C, Christmas R, Avila-Campilo I, Creech M, Gross B, Hanspers K, Isserlin R, Kelley R, Killcoyne S, Lotia S, Maere S, Morris J, Ono K, Pavlovic V, Pico AR, Vailaya A, Wang PL, Adler A, Conklin BR, Hood L, Kuiper M, Sander C, Schmulevich I, Schwikowski B, Warner GJ, Ideker T, Bader GD. Integration of biological networks and gene expression data using Cytoscape. Nat Protoc. 2007;2:2366–2382. doi: 10.1038/nprot.2007.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Bruno M, Brightman AO, Lawrence J, Werderitsh D, Morré DM, Morre DJ. Stimulation of NADH oxidase activity from rat liver plasma membranes by growth factors and hormones is decreased or absent with hepatoma plasma membranes. Biochem J. 1992;284(Pt 3):625–628. doi: 10.1042/bj2840625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Yagiz K, Snyder PW, Morré DJ, Morré DM. Cell size increased in tissues from transgenic mice overexpressing a cell surface growth-related and cancer-specific hydroquinone oxidase, tNOX, with protein disulfide-thiol interchange activity. J Cell Biochem. 2008;105:1437–1442. doi: 10.1002/jcb.21962. [DOI] [PubMed] [Google Scholar]
  • 20.Hostetler B, Weston N, Kim C, Morré DM, Morré DJ. Cancer site-specific isoforms of ENOX2 (tNOX), a cancer-specific cell surface oxidase. Clin Proteomics. 2009;5:46–51. [Google Scholar]
  • 21.Morré DJ, Hostetler B, Weston N, Kim C, Morré DM. Cancer type-specific tNOX isoforms: a putative family of redox protein splice variants with cancer diagnostic and prognostic potential. Biofactors. 2008;34:201–207. doi: 10.3233/BIO-2009-1073. [DOI] [PubMed] [Google Scholar]
  • 22.Zeng ZM, Chuang SM, Chang TC, Hong CW, Chou JC, Yang JJ, Chueh PJ. Phosphorylation of serine-504 of tNOX (ENOX2) modulates cell proliferation and migration in cancer cells. Exp Cell Res. 2012;318:1759–1766. doi: 10.1016/j.yexcr.2012.04.021. [DOI] [PubMed] [Google Scholar]
  • 23.Clarkson BD, Strife A, Wisniewski D, Lambek C, Carpino N. New understanding of the pathogenesis of CML: a prototype of early neoplasia. Leukemia. 1997;11:1404–1428. doi: 10.1038/sj.leu.2400751. [DOI] [PubMed] [Google Scholar]
  • 24.Thomas EK, Cancelas JA, Zheng Y, Williams DA. Rac GTPases as key regulators of p210-BCR-ABL-dependent leukemogenesis. Leukemia. 2008;22:898–904. doi: 10.1038/leu.2008.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Woessmann W, Mivechi NF. Role of ERK activation in growth and erythroid differentiation of K562 cells. Exp Cell Res. 2001;264:193–200. doi: 10.1006/excr.2000.5124. [DOI] [PubMed] [Google Scholar]
  • 26.Le Y, Xu L, Lu J, Fang J, Nardi V, Chai L, Silberstein LE. FAK silencing inhibits leukemogenesis in BCR/ABL-transformed hematopoietic cells. Am J Hematol. 2009;84:273–278. doi: 10.1002/ajh.21381. [DOI] [PubMed] [Google Scholar]
  • 27.Aljedai A, Buckle AM, Hiwarkar P, Syed F. Potential role of Notch signalling in CD34+ chronic myeloid leukaemia cells: cross-talk between Notch and BCR-ABL. PLoS One. 2015;10:e0123016. doi: 10.1371/journal.pone.0123016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Schmidt T, Kharabi Masouleh B, Loges S, Cauwenberghs S, Fraisl P, Maes C, Jonckx B, De Keersmaecker K, Kleppe M, Tjwa M, Schenk T, Vinckier S, Fragoso R, De Mol M, Beel K, Dias S, Verfaillie C, Clark RE, Brümmendorf TH, Vandenberghe P, Rafii S, Holyoake T, Hochhaus A, Cools J, Karin M, Carmeliet G, Dewerchin M, Carmeliet P. Loss or inhibition of stromal-derived PlGF prolongs survival of mice with imatinib-resistant Bcr-Abl1+ leukemia. Cancer Cell. 2011;19:740–753. doi: 10.1016/j.ccr.2011.05.007. [DOI] [PubMed] [Google Scholar]
  • 29.Morre J, Morré DM, Brightmore R. Omega-3 but not omega-6 unsaturated fatty acids inhibit the cancer-specific ENOX2 of the HeLa cell surface with no effect on the constitutive ENOX1. J Diet Suppl. 2010;7:154–158. doi: 10.3109/19390211003785702. [DOI] [PubMed] [Google Scholar]
  • 30.Morré DJ, Korty T, Meadows C, Ades LM, Morré DM. ENOX2 target for the anticancer isoflavone ME-143. Oncol Res. 2014;22:1–12. doi: 10.3727/096504014X14077751730270. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Turkish Journal of Hematology are provided here courtesy of Galenos Yayinevi

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