Zinc Finger Protein X-Linked Exhibits Pro-Leukemia Activity Via WNT3 in Chronic Myeloid Leukemia Stem/Progenitor Cells

Zinc Finger protein, X-linked (ZFX) is a transcriptional regulator, which controls the self-renewal of both embryonic and hematopoietic stem cells and participates in pathogenesis of various cancers. Zfx deficiency impairs Notch intracellular domain (NotchIC) induced acute T-cell leukemia (T-ALL) or MLL-AF9 induced acute myeloid leukemia (AML) in mice models. However, the function of ZFX in chronic myeloid leukemia (CML) stem/progenitor cells has not been elucidated yet.

In the present study, qRT-PCR analysis showed that ZFX expression was significantly higher in CD34⁺ cells from CML patients in chronic phase (CP) (n=8, 3.1-fold, P=0.0052) and patients in blast crisis (BC) (n=8, 18.6-fold, P=0.0050) compared with that in healthy donors (n=4). Two independent shRNA sequences against ZFX were delivered in CD34⁺ cells with lentiviral vector. The silence of ZFX had a stronger inhibitory effect on colony-forming cell (CFC) ability of CML CD34⁺ cells (75±5%) than that of healthy donor CD34⁺ cells (44±5%). Furthermore, ZFX silencing augmented Imatinib Mesylate (IM) sensitivity of CML CD34⁺ cells, especially in IM-resistant samples; due to the fact that ZFX silencing increased apoptosis induced by IM. To obtain the molecular insights of how ZFX acts, we generated transcriptome data comparing ZFX silenced CML CD34⁺ cells with control cells. qRT-PCR data validated that ZFX silencing caused a significantly declined expression of WNT3 in K562, MEG-01 and CML CD34⁺ cells (n=5). In addition, ZFX silencing decreased WNT3 protein expression as well. Interestingly, WNT3 had significantly higher expression in CD34⁺ cells from patients in CP (n=10, 5-fold, P=0.0006) compared with that in healthy donors (n=8). Silence of WNT3 inhibited the growth of K562 cells and enhanced IM sensitivity of these cells as well. Overexpression of WNT3 restored the growth inhibition and IM hypersensitivity upon ZFX silencing. Chromatin immunoprecipitation (ChIP) analysis revealed that ZFX was able to bind with WNT3 promoter, and luciferase assay showed that ZFX silencing significantly decreased the activity of WNT3 promoter. Finally, we also found that the expressions of c-Myc and cyclin D1 were reduced by ZFX silencing or WNT3 silencing, suggesting decreased WNT/Catenin signaling.

Taken together, we have demonstrated that ZFX is aberrantly expressed in CML stem/progenitor cells, and it modulates the growth and IM response of CML stem/progenitor cells via wnt/Catenin pathway indicating ZFX is a new regulator of CML stem/progenitor cells, which deepens the understanding of CML pathology and potentially provides clues for novel therapies against this disease.