Oncogenesis and tumor progression are supported by alterations in cellular signaling. We used phospho-specific antibodies in flow cytometry to analyze specific signaling profiles of leukemia cells at a single cell level in 7 B cell precursor (BCP)-ALL leukemia cell lines and 7 primary pediatric BCP-ALL xenograft samples. Peripheral blood lymphocytes gated on CD19-positive B cells were used as normal nonmalignant controls. Cells were stimulated by different stimulants and cytokines (PMA, anisomycin, IL-4, IL-6, IL-7, IL-10 and IFN-α) and activation of various phosphoepitopes (pERK, pp38, pJNK, pStat1, pStat3, pStat5, pStat6) was analyzed and compared to the basal state of unstimulated samples. Signaling profiles of normal B-lymphocytes were compared to those of the BCPALL cell lines as well as to the BCP-ALL xenograft samples. Significance of differences was assessed by the nonparametric Mann-Whitney U-test. Basal phosphorylation was significantly higher in the leukemia cell lines than in normal lymphocytes. Similarly, basal phosphorylation of all analyzed epitopes in xenografts exceeded the phosphorylation state of normal B-lymphocytes (with the exception of p38 phosphorylation, where there was no significant difference). Interestingly, the BCP-leukemia cell lines also had significantly higher basal phosphorylation levels than the primary BCP-ALL xenografts. However, when comparing the amounts of phosphorylation before and after stimulation mature normal B-cells displayed significantly higher profiles compared to the leukemia cell lines e.g. for pp38 and pJNK after stimulation with PMA (P= .001), for pStat3 after stimulation with IL-6 (P= .002) and IL-10 (P= .037) and for pStat6 (P= .001) after stimulation with IL-4. Conversely, the leukemia cell lines showed increased phosphorylation of p38 after stimulation with anisomycin (P= .021) as well as higher Stat5 phosphorylation after stimulation with IL-7 (P= .021) compared to normal lymphocytes. In normal B-cells compared to xenografts higher levels were found after stimulation with PMA for pp38 (P= .007), for pJNK after PMA stimulation (P= .001), for pStat3 after IL-6 (P=.003) and for pStat6 after IL-4 (P= .002) stimulation while the xenograft samples displayed stronger reaction to stimulation with anisomycin for pp38 (P= .037) and to stimulation with IL-7 for pStat5 (P= .028). The level of phosphorylation after treatment with different stimulants in the xenografted leukemia samples was similar to that of the leukemia cell lines although the cell lines displayed higher basal phosphorylation values. The BCP-leukemia cell lines and the BCP xenograft samples both displayed high levels of constitutive phosphorylation in general reducing their ability to react to a given stimulus compared to normal B-lymphocytes. With the most important exception of Stat5: we consistently found that Stat5 phosphorylation is increased in acute lymphoblastic leukemia cell lines and primary xenografts after stimulation with IL-7 compared to normal B-lymphocytes. Stat5 is known to enhance proliferation and protect from apoptosis and our data now strongly suggest that Stat5 and Stat5 dependent pathways are critically involved in leukemogenesis. Since we could identify significant and specific phosphorylation signatures characteristic for leukemia cells, this provides a strategy to define pathways important for continued survival, proliferation and resistance of leukemia and allows identification of therapeutic targets and novel biomarkers associated with clinical outcome.

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