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.

Keywords: CD133; CD34; JAK2V617F; c-MYC; Myeloproliferative neoplasms; Hematopoieticstem cells; Gold-nanoflare; Endothelialprogenitor cell.

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.

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.