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Activation of the fibromyalgia syndrome-like tyrosine kinase 3 ( FLT3) by its ligand, FLT3 ligand ( FL), strongly augments the development of natural killer ( NK) cells from human CD34+ hematopoietic progenitor cells ( HPCs) in the presence of IL-15, compared with NK-cell development in the presence of IL-15 alone. In this study, we observed that blocking the receptor tyrosine kinase Axl/ Gas6 pathway with a soluble Axl-IgG1 Fc fusion protein ( Axl- Fc) in the presence of FL significantly diminished the absolute number of CD3− CD56+ NK cells derived from human CD34+ HPCs. Axl- Fc reduced the expression levels of the IL-2/15 receptor β chain ( CD122) and γ chain ( CD132) induced by activation of FLT3 and consequently reduced the frequency of NK precursor cells responding to IL-15. Furthermore, Axl- Fc diminished FL-induced FLT3 phosphorylation and impeded the physical interaction between Axl and FLT3 in CD34+ HPCs. Collectively, our data suggest that the Axl/ Gas6 pathway contributes to normal human NK-cell development at least in part via its positive regulatory effect on FLT3 signaling in CD34+ HPCs. [ABSTRACT FROM AUTHOR]

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA- binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCβII-dependent FUS phosphorylation. Moreover, suppression of PKCβII activity in BCR/ABL-expressing cells by treatment with the PKCβII inhibitor CGP53353, or by expression of a dominant-negative PKCβII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) β-chain expression, and accelerated G-CSF-stimulated differentiation. Down-regulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression. [ABSTRACT FROM AUTHOR]

The BCR/ABL oncogenic tyrosine kinase activates phosphatidylinositol 3-kinase (PI-3k) by a mechanism that requires binding of BCR/ABL to p85, the regulatory subunit of PI-3k, and an intact BCR/ABL SH2 domain. SH2 domain BCR/ABL mutants deficient in PI-3k activation failed to stimulate Akt kinase, a recently identified PI-3k downstream effector with oncogenic potential, but did activate p21 RAS and p70 S6 kinase. The PI-3k/Akt pathway is essential for BCR/ABL leukemogenesis as indicated by experiments demonstrating that wortmannin, a PI-3k specific inhibitor at low concentrations, suppressed BCR/ABL-dependent colony formation of murine marrow cells, and that a kinase-deficient Akt mutant with dominant-negative activity inhibited BCR/ABL-dependent transformation of murine bone marrow cells in vitro and suppressed leukemia development in SCID mice. In complementation assays using mouse marrow progenitor cells, the ability of transformation-defective SH2 domain BCR/ABL mutants to induce growth factor-independent colony formation and leukemia in SCID mice was markedly enhanced by expression of constitutively active Akt. In retrovirally infected mouse marrow cells, the BCR/ABL mutant lacking the SH2 domain was unable to upregulate the expression of c-Myc and Bcl-2; in contrast, expression of a constitutively active Akt mutant induced Bcl-2 and c-Myc expression, and stimulated the transcription activation function of c-Myc. Together, these data demonstrate the requirement for the BCR/ABL SH2 domain in PI-3k activation and document the essential role of the PI-3k/Akt pathway in BCR/ABL leukemogenesis. [ABSTRACT FROM AUTHOR]