ALBERT

Description: 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. Disclosures No relevant conflicts of interest to declare.

Description: Growth arrest-specific 2 (GAS2) has multiple functions including the regulation of cell morphology, cell cycle, apoptosis and calpain activity.GAS2 has a dual function in cancer cells, however its expression and underlying mechanism in human T-cell acute lymphoblastic leukemia (T-ALL) remain unclear. In the present study, qRT-PCR analysis showed that GAS2 has significantly higher expression (155.5-fold, P=0.0048) in CD3+ cells from T-ALL patients (n=25) than healthy donors (n=13). GAS2 was present in Jurkat cells, while absent in MOLT-4 or HPB-ALL cells. A tiny CpG island of GAS2 was almost fully methylated in both MOLT-4 (100%) and HPB-ALL cells (80%), while 40% methylation in Jurkat cells; suggesting that DNA methylation played a subtle role in regulating GAS2 expression. Two independent shRNA sequences were delivered into Jurkat cells with lentiviral vector. GAS2 silencing inhibited the growth and colony-forming cell (CFC) production significantly. Conversely, GAS2 overexpression enhanced the growth and CFC production of both MOLT-4 and HPB-ALL cells. In addition, GAS2 overexpression promoted HPB-ALL cell induced leukemia in a xenoengraftment model (5 mice in each control group). In GAS2 expressed group, the disease latency was shortened, the splenomegaly was more severe than control group (0.35±0.04g vs. 0.27±0.05g), and more leukemic cells were present in bone marrow (85±3% vs. 45±7%). To obtain the molecular insights of how GAS2 acts, RNA-seq data comparing GAS2 silenced Jurkat cells with control cells were generated. Several Notch signaling molecules were inhibited, including NOTCH1, HES1 and HES4. Despite the differential expression of these transcripts was validated in Jurkat cells, GAS2 overexpression did not elevated the expression of these transcripts in MOLT-4 or HPB-ALL cells, suggesting GAS2 did not have a consistent impact on Notch signaling. However, we found that GAS2 silencing reduced CXCR4 protein expression in Jurkat cells and GAS2 overexpression enhanced CXCR4 protein expression in MOLT-4 cells, while CXCR4 transcript was not altered upon GAS2 manipulation. Consequently, GAS2 silencing significantly reduced migration ability of Jurkat cells and GAS2 overexpression enhanced migration ability of MOLT-4 cells. Overexpression of CXCR4 "rescued" the inhibited CFC production and migration upon GAS2 silencing. A truncated GAS2 (Δ171-313) coined as GAS2DN (dominant negative form of GAS2) has been known to inhibit normal function of GAS2. Herein, we showed that GAS2DN inhibited the growth of Jurkat cells and the expression of CXCR4. To delineate the role of calpain1 and calpain2 in GAS2 function, shRNA sequences against calpain1 and calpain2 was delivered into GAS2DN expressed Jurkat cells respectively, the results showed that calpain2 but not calpain1 silencing was able to enhance the cell growth and CXCR4 expression. Taken together, the present study has demonstrated that GAS2 is aberrantly expressed in human T-ALL cells, which promotes the growth of T-ALL cells partially via its post-transcriptional regulation of CXCR4 depending on calpain2 activity. These data provide new insights of the pathogenesis of T-ALL and possibly new clues to improve the management of the disease. Disclosures No relevant conflicts of interest to declare.