Peggy Jarrier

BCR–ABL activates STAT3 via JAK and MEK pathways in human cells

Chronic myeloid leukaemia (CML) is characterised by a progression from a chronic towards an acute phase. We previously reported that signal transducer and activator of transcription 3 (STAT3), a major oncogenic signalling protein, is the target of p210–BCR–ABL in a murine embryonic stem (ES) cell model and in primary CD34+ CML cells. This activation was associated with inhibition of differentiation in ES cells. The present study found that BCR–ABL greatly phosphorylated STAT3 Ser727 residue and, to a lesser extent, Tyr705 residue in BCR–ABL‐expressing cell lines (UT7‐p210, MO7E‐p210, and K562) and in primary CD34+ CML cells. Using BCR–ABL mutants, it was shown that BCR–ABL tyrosine kinase activity and its Tyr177 residue were necessary for STAT3 Ser727 phosphorylation. Constitutive STAT3 Tyr705 phosphorylation was associated with constitutive phosphorylation of Janus kinase (JAK)1 and JAK2, and was inhibited by the JAK inhibitor AG490, suggesting the involvement of JAK proteins in this process. Specific MEK [mitogen‐activated protein (MAP) kinase/extracellular signal‐regulated kinase (ERK) kinase] inhibitors PD98056 and UO126, as well as the use of a dominant‐negative form of MEK1 abrogated STAT3 Ser727 phosphorylation, suggesting involvement of MAP‐Kinase/Erk pathway. Inhibition of BCR–ABL with imatinib mesylate led to a dose‐dependent downregulation of total STAT3 protein and mRNA, suggesting that BCR–ABL is involved in the transcriptional regulation of STAT3. Targeting JAK, MEK and STAT3 pathways could therefore be of therapeutic value, especially in advanced stage CML.

p210BCR-ABL inhibits SDF-1 Chemotactic Response via Alteration of CXCR4 Signaling and Down-regulation of CXCR4 Expression

It has been shown that p210(BCR-ABL) significantly impairs CXCR4 signaling. We report here that the migratory response to SDF-1 was profoundly altered in blast crisis, whereas chronic-phase CD34(+) cells migrated normally to this chemokine. This migratory defect was associated with a low CXCR4 membrane expression. In vitro STI-571 treatment of CD34(+) cells from patients in blast crisis markedly increased the CXCR4 transcript and CXCR4 membrane expression. Because p210(BCR-ABL) frequently increases with disease progression, we determined the effects of high and low p210(BCR-ABL) expression on CXCR4 protein in the granulocyte macrophage colony-stimulating factor-dependent human cell line MO7e. p210(BCR-ABL) expression distinctly alters CXCR4 protein through two different mechanisms depending on its expression level. At low expression, a signaling defect was detected with no modification of CXCR4 expression. However, higher p210(BCR-ABL) expression induced a marked down-regulation of CXCR4 that is related to its decreased transcription. The effect of p210(BCR-ABL) required its tyrosine kinase activity. Collectively, these data indicate that p210(BCR-ABL) could affect CXCR4 by more than one mechanism and suggest that down-regulation of CXCR4 may have important implications in chronic myelogenous leukemia pathogenesis.

Identification of CXCR4 as a new nitric oxide-regulated gene in human CD34+ cells.

As an intracellular second messenger, nitric oxide (NO) is increasingly implicated in the control of transcriptional machinery and gene expression. Here, we show that cell surface expression of CXCR4 on CD34+ cells was increased in a dose‐ and time‐dependent manner in response to NO donors. Augmented surface expression was correlated with an increase in CXCR4 mRNA level. A specific NO scavenger prevented the elevation in CXCR4 mRNA caused by NO donors, suggesting a direct signaling action mediated by NO on CXCR4 transcription. NO treatment had no significant effect on CXCR4 mRNA stability. However, induction of CXCR4 mRNA by NO was still observed in conditions in which initiation of translation was inhibited, suggesting that the NO effect must be mediated by a pre‐existing protein. CXCR4 mRNA induction did not involve cGMP (guanosine 3′, 5′‐cyclic monophosphate) generation but was most likely mediated via oxidation of intracellular protein thiols. Finally, CD34+ cells pretreated with NO donors exhibited an increased chemotactic response. This study demonstrates that the NO pathway can modulate CXCR4 expression in human CD34+ cells and suggests that NO may play a critical role in the trafficking of hematopoietic progenitors.