Circulating CD133+/–CD34– Have Increased c-MYC Expression in Myeloproliferative Neoplasms

Abstract

Objective:

Myeloproliferative neoplasms (MPNs) are hematopoietic stem cell (HSC)-originated diseases with clonal myeloproliferation. The constitutive activation of the JAK/STAT pathway is frequently detected in patients with Philadelphia chromosome-negative (Ph^–) MPNs with an acquired JAK2V617F mutation. The c-MYC proto-oncogene is associated with malignant growth and cellular transformation, and JAK2V617F was previously shown to induce constitutive expression of c-MYC. This study examines the expressional profile of c-MYC in Ph^– MPNs with JAK2V617F and highlights its hierarchical level of activation in circulating hematopoietic stem/progenitor cell (HSPC) subgroups.

Materials and Methods:

Mononuclear cells (MNCs) of Ph^– MPNs were fluorochrome-labeled in situ with wild-type (wt) JAK2 or JAK2V617F mRNA gold nanoparticle technology and sorted simultaneously. Isolated populations of JAK2wt or JAK2V617F were evaluated for their c-MYC expressions. The MNCs of 14 Ph^– MPNs were further isolated for the study of HSPC subgroups regarding their CD34 and CD133 expressions, evaluated for the presence of JAK2V617F, and compared to cord blood (CB) counterparts for the expression of c-MYC.

Results:

The mRNA-labeled gold nanoparticle-treated MNCs were determined to have the highest ratio of c-MYC relative fold-change expression in the biallelic JAK2V617F compartment compared to JAK2wt. The relative c-MYC expression in MNCs of MPNs was significantly increased compared to CB (p=0.01). The circulating HSPCs of CD133^+/–CD34⁻ MPNs had statistically significantly elevated c-MYC expression compared to CB.

Conclusion:

This is the first study of circulating CD133^+/–CD34⁻ cells in Ph^– MPNs and it has revealed elevated c-MYC expression levels in HSCs/endothelial progenitor cells (HSCs/EPCs) and EPCs. Furthermore, the steady increase in the expression of c-MYC within MNCs carrying no mutations and monoallelic or biallelic JAK2V617F transcripts was notable. The presence of JAK2V617F with respect to c-MYC expression in the circulating HSCs/EPCs and EPCs of MPNs might provide some evidence for the initiation of JAK2V617F and propagation of disease. Further studies are needed to clarify the implications of increased c-MYC expression in such populations.

Introduction

Philadelphia chromosome-negative (Ph^–) myeloproliferative neoplasms (MPNs) are clonal disorders originating from hematopoietic stem cells (HSCs) [1,2,3]. Patients with MPNs are characterized by increases in the myeloid, erythroid, and megakaryoid cells in peripheral blood, leading to varying phenotypic diseases, including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF) [4,5]. A cytosolic tyrosine kinase, Janus kinase 2 (JAK2), plays a significant role in hematopoiesis and immune responses in cytokine-dependent cancers [6]. An acquired mutation (V617F) of JAK2 is frequently detected in patients with MPNs, causing constitutive activation of the intracellular JAK/STAT pathway and leading to uncontrolled myeloproliferation and cytokine-independent cell survival [7,8].

MYC is a family of basic helix-loop-helix leucine zipper transcription factors, including c-MYC, N-MYC, and L-MYC, that regulate 11%-15% of the human genome and play diverse roles in cell cycle progression, apoptosis, and cellular transformation [9,10,11]. MYC controls HSCs by fine-tuning the balance between self-renewal, differentiation, proliferation, survival, and hematopoiesis [12,13,14,15,16]. In nearly half of all human tumors, overexpression of MYC is associated with malignant growth [17], with which enhanced protein synthesis, energy metabolism, and genomic instability have been associated [9,18,19]. This most common oncogene is also frequently associated with hematopoietic tumors, such as lymphoma and leukemia [14,20,21], and its driving role in myeloid malignancies is increasingly being indicated [22,23,24]. Recent studies have demonstrated JAK2V617F-induced constitutive expression of c-MYC, which is mediated by STAT5 activation [25], and this activation is FERM domain-dependent [26]. Upregulated expression of c-MYC mRNA is detected in bone marrow cells in ET but not in PV [27]. In addition, knockdown of c-MYC significantly inhibits the proliferation of JAK2V617F-positive cells [25]. Since myeloproliferation is remarkable in MPNs, it might be interesting to estimate the hierarchical level of c-MYC activation in JAK2V617F-positive MPNs.

CD133 is a commonly expressed antigen at the cell surface of several somatic stem cells, particularly human hematopoietic stem/progenitor cells (HSPCs) [28]. Studies have shown that CD133 might further characterize HSPCs [29,30,31], with CD133⁺CD34⁺CD45RA^- cell fractions being enriched for HSCs and multipotent progenitor cells (MPPs), giving rise to CD133^lowCD34⁺CD45RA^- erythro-myeloid restricted progenitors (EMPs) [32]. This suggests that adult/postnatal multipotent HSCs/MPPs may be easily identified as CD133⁺CD34⁺ cells with erythroid potential [33,34]. Surprisingly, some other studies have revealed that CD133^-CD34^-CD45RA^- cells show a stem cell ability making them compatible with both hematopoietic and endothelial potential by reconstituting hematopoietic tissue and generating functional HSCs and endothelial progenitor cells (EPCs) in vivo [35], and that CD34^-CD133⁺ progenitors may further differentiate into EPCs [36].

In the present study, we establish various approaches for investigating the c-MYC expression of MPNs with JAK2V617F and its hierarchical activation level. Circulating CD133^+/-CD34^- progenitors in MPNs have elevated c-MYC expression levels in such compartments. Additionally, genes known to be involved in the regulatory mechanisms of MPN pathogenesis related to c-MYC, including STAT5A and STAT5B, are investigated in this study at the mRNA level.

The results of this study may explain the development of clonal myelopoiesis, reveal the high frequency of EPCs in MPNs, and further illuminate the phenotypic characteristics of MPNs with JAK2V617F, which remain elusive.

Materials and Methods

Patients and Samples

Fourteen patients who met the revised 2008 World Health Organization diagnostic criteria for MPN [37] and five cord blood (CB) samples used as controls were included in this study. All patients were diagnosed in the Hematology Clinic of the İstanbul Medical Faculty, İstanbul University. Informed consent was obtained according to the guidelines outlined by the Ethical Review Board of the İstanbul Medical Faculty of İstanbul University.

Mononuclear Cell Isolation and FACS Sorting of the Cells

Mononuclear cells (MNCs) were isolated using the Ficoll gradient centrifugation technique (Ficoll-Paque Premium, GE Healthcare, Uppsala, Sweden) from the peripheral blood of patients with MPNs after phlebotomy and from CB samples in blood bags containing 10% sodium citrate.

Following centrifugation at 400x g and 20 °C for 30 min, the collected MNCs were counted and resuspended in phosphate-buffered saline (PBS)-based buffer containing 500 mL PBS with 7.5% bovine serum albumin +0.5 mM EDTA, referred to hereafter as “buffer.”

The MNCs were labeled with CD45-FITC (BD Pharmingen, San Diego, CA, USA), CD34-PE (BD Pharmingen), and CD133-APC (BD Pharmingen). Each antibody was added to 1 µL (1x10⁶) of cells. After 30 min of incubation with the antibodies on ice, the MNCs were washed twice with buffer and resuspended in 500 µL of buffer containing 0.1% propidium iodide (PI) (Sigma Aldrich, St. Louis, MO, USA). Each sample was acquired on a FACSAria II instrument (BD Bioscience, Franklin Lakes, NJ, USA). The acquisition was performed with a CD45-negative gate using the following steps: 1) dead cells were excluded using PI; 2) doublets were excluded by gating outliers on SSC-A vs. SSC-H and FSC-A vs. FSC-H plots; 3) negative CD45 populations were segregated into four different quadrants (CD133^–CD34⁺, CD133⁺CD34⁺, CD133^–CD34^–, and CD133⁺CD34^–) for subpopulation analysis. Each gate was then sorted with ~98% purity.

JAK2 Wild-Type and JAK2V617F mRNA-Labeled Gold Nanoflare Infection of MPN Cells and FACS Sorting

Seven different probes were applied for the detection of JAK2 wild-type (wt) and JAK2V617F transcripts in live cells as follows: two target-specific probes (JAK2wt-Cy5 and JAK2V617F-Cy3) with a housekeeping control; beta-actin human-Cy3 and beta-actin human-Cy5; and three control probes (JAK2 Scramble-Cy3, Uptake-Cy3, and Scramble-Cy3+Uptake-Cy5) (EMD Millipore Company, Merck KGaA, Darmstadt, Germany), as explained elsewhere [38]. In brief, the MNCs were incubated with either the specific or control probes using 30000 cells at 200 µL/well in 96-well plates. After 16 h of incubation at 37°C in 5% CO₂ with ≥95% humidity, the cells were collected and resuspended in 500 µL of buffer using a FACSAria II instrument (BD Bioscience) with ~90% purity.

Allele-Specific Nested PCR

Genomic DNA was extracted using the Quick gDNA Micro Prep Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. The JAK2V617F mutation was detected by nested allele-specific PCR for the 14 MPN patients and the sorted compartments of 12 MPN patients as described previously [39]. Agarose gel electrophoresis revealed a band for a mutant allele of JAK2V617F with 279 bp and a wt allele of JAK2 with 229 bp with a 50-bp ladder (Bio-Rad, Hercules, CA, USA).

RNA Isolation and Quantitative Real-Time Reverse Transcription PCR

Total RNA was extracted using a Pico Pure RNA Extraction Kit (Applied Biosystems, Bedford, MA, USA). According to the manufacturer’s instructions, first-strand complementary DNA (cDNA) was synthesized from 60 ng of total RNA using a SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany). Differences in each gene expression were detected by applying Universal Probe Library (UPL) probes (Roche, Basel, Switzerland). Expression analysis was performed using a primer designed for the c-MYC gene of the NM_002467.4 transcript variant with forward primer 5’-GCT GCT TAG ACG CTG GAT TT-3’ and reverse primer 5’-TAA CGT TGA GGG GCA TCG-3’ combined with UPL probe #66 (Roche). The beta-actin gene (ACTB) was applied as a reference housekeeping gene for quantitative real-time PCR with the NM_001101.3 transcript variant using forward primer 5’-CCA ACC GCG AGA AGA TGA-3’ and reverse primer 5’-CCA GAG GCG TAC AGG GAT AG-3’ with UPL probe #64 (Roche). A total volume of 20 µL, composed of 1X ORA qPCR Probe Mix (HighQu, Kraichtal, Germany), forward primer (200 nM), reverse primer (200 nM), UPL probe (0.04 U), and dH₂O, was prepared and run with an initial denaturation of 2 min at 95 °C, then 40 cycles of 5 s at 95°C and 40 cycles of 20 s at 60°C, ending with 30 s at 40°C using a real-time quantitative RT-PCR instrument (LightCycler II 480, Roche).

In order to detect expressional gene changes for JAK2, STAT5A, and STAT5B, the commercially available PCR Array for the Human JAK/STAT Signaling Pathway (QIAGEN, Dusseldorf, Germany) was used. Comparative measurements with real-time RT-PCR were performed using SYBR Green (RT² SYBR Green qPCR Mastermix, QIAGEN). Five different housekeeping genes were applied as the reference genes included in the array and run on the instrument.

Statistical Analysis

Gene expression fold changes were examined for MNCs and sorted stem cell compartments of patients and CB. The specified primers were applied for amplification. The fluorescence emitted by dye above the baseline signal was detected using the software in real time, recorded, and presented as the cycle threshold (C_T). The arithmetic mean values of C_Ts, obtained twice, were calculated. All samples were studied in duplicate. The relative c-MYC gene expressions were calculated using the formula of 2^-∆Ct= 2^{-(CTExample - CTReferance)} and applying the ACTB reference gene for samples [40]. GraphPad Prism 8.0 for Windows (GraphPad Software, San Diego, CA, USA) was used for the statistical analysis of gene expression data.

Significant differences in HSPC subgroups were determined using the Mann-Whitney U test as a nonparametric statistical test to compare the relative mRNA levels of patients and CB samples. The variables used included mean values, standard deviation of the mean (SD), 95% confidence intervals, and percentage and frequency values. Statistical significance was set at p<0.05 (**) and p<0.01 (***).

Results

The clinical characteristics of the patients with Ph^- MPNs enrolled in this study are summarized in Table 1.

Table 1. Clinical characteristics of the patients with Philadelphia chromosome-negative myeloproliferative neoplasms included in this study.

JAK2V617F-Positive Cells Have High Relative c-MYC Gene Expression

In order to ascertain whether c-MYC expression has an association with JAK2V617F mutation, a previously established method for live cell analysis of JAK2V617F mRNA and JAK2wt transcripts with fluorochrome-conjugated gold nanoparticles [38,41] was applied to the MNCs of two Ph^- MPN samples that had monoallelic JAK2V617F. The JAK2V617F mRNA- and JAK2wt mRNA-labeled gold nanoparticle-treated MNCs of MPN patients were sorted with a fluorescence-activated cell sorter for the JAK2V617F and JAK2wt transcripts, followed by analysis for relative c-MYC gene expression. Representative images of the flow cytometry analysis of distinct populations of JAK2wt⁺ and JAK2V617F⁺ were obtained (Figure 1). The MNCs of MPN patients were initially determined as monoallelic JAK2V617F by nested PCR analysis (Table 1).

Figure 1.

Fluorescence‐activated cell sorting (FACS) plot of peripheral blood mononuclear cells of a patient (P29) with Philadelphia chromosome-negative Ph^– myeloproliferative neoplasm with polycythemia vera using gold nanoparticle probes. For all analyses, the main population of cells was gated excluding dead and fractured cells. After settings were completed for the background and control probes, including uptake fluorescence, analysis and sorting of the gold nanoparticle‐treated cells were performed according to their signals in channels of FL‐2 for JAK2V617F‐CY3 and FL‐4 for JAK2 wild‐type (wt)‐CY5 transcripts. The FACS of the gold nanoparticle-treated cells was performed in four quadrants: JAK2V617F–JAK2wt‐ for the negative control, JAK2V617F+JAK2wt– for JAK2V617F mutant mRNA, JAK2V617F+JAK2wt+ for both JAK2wt and JAK2V617F mRNA-positive cells, and JAK2V617F−JAK2wt+ for JAK2wt mRNA.

Surprisingly, there were heterogeneous subgroups of transcripts within the MNCs, including solely monoallelic and biallelic JAK2V617F and JAK2wt transcripts. In addition, populations of JAK2wt⁺JAK2V617F⁺, JAK2wt⁺JAK2V617F^-, and JAK2wt^-JAK2V617F⁺ were detected at rates of 26.3±4.1%, 3.5±2.3%, and 38.5±5.7%, respectively. Each of the sorted cell populations was analyzed for relative changes in expressions of c-MYC, JAK2, STAT5A, and STAT5B. The ratio of the relative fold change of c-MYC was found to be highest (5.9-fold) in the JAK2V617F⁺ compartment compared to the JAK2wt⁺ compartment (biallelic/wt). The relative fold change of JAK2 expression in the same compartment was determined to be 3.85-fold, while the STAT5A and STAT5B expressions were 2.78-fold and 4.87-fold, respectively (Table 2). Surprisingly, the ratio of gene expression fold changes in the compartments of biallelic/monoallelic JAK2V617F was below 2-fold. However, the relative gene expression fold changes for c-MYC, JAK2, and STAT5B in JAK2V617F monoallelic/wt compartments were 3.03-, 2.53-, and 3.89-fold, respectively.

Table 2. Relative gene expression fold changes of JAK2 wild-type and JAK2V617F mRNA sorted Philadelphia chromosome-negative myeloproliferative neoplasms.

JAK2V617F Is Detected in MPP, EMP, HSC/EPC, and EPC Populations

To evaluate the circulating HSPCs (MPP, EMP, HSC/EPC, and EPC populations) of Ph^- MPN samples and the status of JAK2V617F in each population, MNCs labeled with CD45, CD34, and CD133 antibodies were sorted accordingly and further analyzed for the presence of JAK2V617F.

In the cohort of 14 Ph^- MPN samples, MPPs (CD133⁺CD34⁺) accounted for 0.22±0.18% of the total cells, EMPs (CD133^-CD34⁺) accounted for 4.58±3.61%, and EPCs (CD133⁺CD34^-) accounted for 0.79±0.42% (Table 3). HSCs/EPCs (CD133^-CD34^-) demonstrated the highest percentage in this compartmentalization.

Table 3. Percentage of sorted cell compartments of CD133 and CD34 cells of cord blood and mononuclear cells of Philadelphia chromosome-negative myeloproliferative. neoplasms.

Results from allele-specific nested PCR analysis are summarized in Tables 2 and 4. The MNCs of 8 of the 14 patients had monoallelic JAK2V617F, 2 patients had biallelic JAK2V617F, and 4 patients had the JAK2wt allele.

Table 4. JAK2V617F allele status of mononuclear cells and sorted populations of Philadelphia chromosome-negative myeloproliferative neoplasm samples.

JAK2V617F characterization was performed for the sorted cell compartments of 12 Ph^- MPN samples. JAK2V617F was not detected in the sorted cell compartments of 4 patients who did not carry the mutation in their MNCs. These 4 patients also did not carry the JAK2 exon 12 mutation. The Ph^- MPN patients who carried monoallelic JAK2V617F in their MNCs had varying presentations of mutation, including no mutation (wt) or monoallelic JAK2V617F, in each sorted cell compartment. All patients with a mutation in the MNC samples carried the monoallelic JAK2V617F mutation in the EMP (CD133^-CD34⁺) cell population. Three of 8 MPN samples had no mutation in the MPP (CD133⁺CD34⁺) population, 2 of 8 monoallelic JAK2V617F Ph^- MPN samples had no mutation in the EPC (CD133⁺CD34^-) population, and 1 of 8 Ph^- MPN patients had no mutation in the HSC/EPC (CD133⁺CD34^-) population (Table 4).

Relative c-MYC Expression Increased in Progenitor Cell Populations and MNCs

To identify the activation level of c-MYC expressions in the HSPC subgroups, 14 patients with Ph^– MPNs and 5 CB samples were studied. The overall analysis of the relative c-MYC expression in the MNCs of the MPN samples and CB showed increases of 0.026±0.015 and 0.009±0.004, respectively (p=0.01) (Figure 2A).

Figure 2.

Relative c-MYC gene expression analysis of (A) mononuclear cells (p=0.01, Mann-Whitney U), (B) multipotent progenitor cells (MPPs, CD133⁺CD34⁺) (p>0.05, Mann-Whitney U), (C) erythro-myeloid restricted progenitors (EMPs, CD133^-CD34⁺) (p=0.02 Mann-Whitney U), (D) hematopoietic and endothelial progenitor cells (HSCs/EPCs, CD133–CD34–) (p=0.01, Mann-Whitney U), and (E) endothelial progenitor cells (EPCs, CD133⁺CD34^-) (p=0.03, Mann-Whitney U) in samples from 14 patients with myeloproliferative neoplasms and 5 control samples of cord blood. Relative c-MYC gene expressions were calculated with the formula 2^-∆Ct = 2^{-(CTExample – CTReferance)} applying ACTB as a reference gene. GraphPad Prism 8.0 software was used for the statistical analysis of gene expression data; in each figure, the standard deviation of the mean (SD) is shown with a bar; **p< 0.05.

Relative gene expression analysis was performed for Ph^- MPNs and CB in the MPP (CD133⁺CD34⁺), EMP (CD133^-CD34⁺), HSC/EPC (CD133^-CD34^-), and EPC (CD133⁺CD34^-) populations. The relative c-MYC expression levels detected in the MPPs of the MPN and CB samples were 0.083±0.060 and 0.057±0.017, respectively (p>0.05) (Figure 2B). In the HSCs/EPCs, relative c-MYC expression was determined as being significantly higher in MPNs compared to CB, at 0.026±0.021 and 0.003±0.004, respectively (p=0.01) (Figure 2D). In the EPCs of MPNs, relative c-MYC expression was statistically significant compared to the CB samples, at 0.019±0.013 and 0.004±0.004, respectively (p=0.03) (Figure 2E). Contrarily, EMPs had higher relative c-MYC expression in the CB than the MPN samples, at 0.037±0.015 and 0.018±0.013, respectively (p=0.02) (Figure 2C).

Discussion

The increased oncogenic activity of c-MYC has been previously associated with JAK2V617F but the exact level of upregulation of HSCs is lacking [42]. The determination of such a process might link the initiation and progression of Ph^- MPNs with JAK2V617F.

The transcript-labeled gold nanoparticle technology method was applied in this study for the mutant and wild-type allele-specific isolation of cells and distinct Ph^- MPN MNC populations of JAK2wt⁺ and JAK2V617F⁺ were created [38]. This showed that the c-MYC, JAK2, STAT5A, and STAT5B genes were overexpressed in JAK2V617F⁺ compared to JAK2wt⁺.

It is well known that JAK2V617F induces aberrant activation of a transcription factor, STAT5, which plays a role in the JAK/STAT pathway, is critical for antiapoptotic and oncogenic activities [25], and directly regulates the synthesis of proteins in growth and survival [20]. Activation of the JAK/STAT pathway is required to induce the MYC protein level whereby MYC protein synthesis is found to be due to JAK2 kinase activation [26]. Therefore, the increase in c-MYC, JAK2, and STAT5B expressions in the presence of JAK2V617F in MNCs shown here is in concordance with previous findings. Furthermore, the steady increase in the expression of these genes within MNCs carrying no mutation (JAK2wt) and monoallelic or biallelic JAK2V617F transcripts was notable.

The JAK2V617F allele varies in the HSC compartments of MPNs, and the role of c-MYC expression in such environments is not clear. Therefore, we further evaluated the c-MYC expression in circulating HSPCs and their subgroups. CD34⁺ cells are commonly detected in peripheral blood circulation [33] and are found to be increased among inpatients with MPNs [43]. CD133 expression among CD34⁺ cells has been reported to enrich the stem cell fraction of circulating cells [31,44] and could also be applied as a marker of neoplastic stem cell activity in the peripheral blood circulation of patients with PMF [45]. Thus, the presence of JAK2V617F with respect to c-MYC expression in the circulating HSC subpopulation including the MPPs, EMPs, HSCs/EPCs, and EPCs of MPNs might provide evidence for the initiation of JAK2V617F and the propagation of disease.

Our study revealed that a minor proportion of circulating HSCs are co-expressed with CD133. However, HSCs appear to be roughly split between circulating CD133⁺ and CD133^- cells. The majority of the cells closely related to HSCs, MPPs, and EMPs are also divided between CD133⁺ and CD133^- cells and predominantly enhanced in the CD133^- population. Although each population needs further characterization with in vivo xenograft experiments, the indication of circulating CD133⁺ cells being more enriched for a more primitive state of HSPCs might be partially correct [44]. Furthermore, the determination of the least common population being the CD133⁺CD34⁺ phenotype [31,46] is consistent with a previous study in which patients with PMF had more circulating CD133⁺CD34⁺ cells than healthy donors [45].

The JAK2V617F allele statuses of the MNCs and the circulating MPP, EMP, HSC/EPC, and EPC populations of the MPN cohort, including 9 PV, 2 ET, and 3 PMF cases, were compared in the present study. The allele-specific mutational analysis of the subpopulations of HSCs of 12 MPN samples that did not have mutations in the MNCs suggests that mutations other than JAK2V617F also affect disease development [47].

Regarding circulating EMPs with the CD133^-CD34⁺ immunophenotype, all 8 cases with monoallelic JAK2V617F in MNCs revealed the monoallelic JAK2V617F genotype. Considering malignant clones with myeloid proliferation and progression, this was anticipated. For the more primitive population of circulating MPPs with the CD133⁺CD34⁺ phenotype, the mutant monoallelic genotype was detected in 5 of 8 MPN samples.

Surprisingly, the circulating CD34^– cells with the CD133^+/- phenotype with the capacity to differentiate into endothelial cell (ECs) revealed JAK2V617F-positive cases. Since the discovery of JAK2V617F-positive ECs [48], the presence of JAK2V617F in ECs has led to many theories about the origin of malignant clones based on experiments with CD34⁺ MPN cells [39,49,50]. Although circulating HSCs/EPCs and EPCs require further conformational studies, this preliminary finding is exciting because high levels of EPCs in MPNs [51] might originate from circulating CD34^- cells.

An abundance of c-MYC protein increases as HSCs differentiate into myelo-erythroid and myeloid progenitor lineages [16] and cause cells to leave the niche [10]. An in vivo study performed by Franke et al. [52] showed that the constitutive overexpression of c-MYC in the HSPC compartment resulted in a myeloproliferative disorder. The present study showed increased c-MYC expression in MNCs and circulating MPPs, HSCs/EPCs, and EPCs of Ph^- MPNs compared to CB. Remarkably, the relative c-MYC expression in the circulating EMPs of Ph^- MPN cases was lower compared to the EMPs of CB. Previous studies have revealed that CB cells enriched with CD34 have increased c-MYC expression compared to the adult bone marrow cells of healthy donors [53]. Possible limitations of the present study might include the comparison of c-MYC expression with CB, which could be deceptive in some cases. Although there are examples of studies in the literature utilizing CB cells as controls [53], better optimization with more appropriate controls or more Ph^- MPN cases with no mutations and detailed analysis for other driver mutations might be necessary.

Nevertheless, the elevated circulating HSC/EPC and EPC populations in MPNs might still be driven by c-MYC. One study in which c-MYC mRNA was determined in formalin-fixed and paraffin-embedded bone marrow samples from patients with ET revealed c-MYC overexpression. However, a lack of significant expression in PV might also be related to the phenotype of the bone marrow cells analyzed, as bone marrow cells in cases of PV might be enriched with CD133^-CD34⁺ [27]. Furthermore, the regulation of gene expression profiles and methylation patterns vary significantly between bone marrow cells and circulating HSPCs, such that the comparative effect of JAK2V617F with c-MYC expression in varying cell types might be diverse. Since more than 95% of PV cases carry JAK2V617F, and JAK2V617F is known to induce c-MYC expression in cell lines [14,27], the present study further confirms the increased expression of the JAK2V617F population.

Conclusion

The data obtained in this work support the increased c-MYC expression in Ph^- MPNs with JAK2V617F and further reveal the overexpression of c-MYC in HSCs/EPCs and EPCs of MPNs. The effects of c-MYC on the molecular mechanisms of these compartments and its functional relevance remain elusive and require further study.