Signal transducer and activator of transcription 5 (STAT5) is constitutively activated by BCR/ABL, the oncogenic tyrosine kinase responsible for chronic myelogenous leukemia. The mechanism of BCR/ABLmediated STAT5 activation is unknown. We show here that the BCR/ABL SH3 and SH2 domains interact with hematopoietic cell kinase (Hck), leading to the stimulation of Hck catalytic activity. Active Hck phosphorylated STAT5B on Tyr699, which represents an essential step in STAT5B stimulation. Moreover, a kinase-dead Hck mutant and Hck inhibitor PP2 abrogated BCR/ABL-dependent activation of STAT5 and elevation of expression of STAT5 downstream effectors A1 and pim-1. These data identify a novel BCR/ ABL±Hck±STAT5 signaling pathway, which plays an important role in BCR/ABL-mediated transformation of myeloid cells. Keywords: activation/BCR/ABL/Hck/pathway/STAT5 Introduction BCR/ABL is derived from translocation of the c-ABL gene on chromosome 9 to the BCR locus on chromosome 22 [t(9;22), Philadelphia chromosome], and is present in essentially all cases of chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemia (ALL) patients (Shtivelman et al., 1986;Clark et al., 1988). BCR/ ABL hybrid genes produce p230, p210 and p185 fusion proteins with constitutive tyrosine kinase activity that transform hematopoietic cells in vitro, and cause CML-or ALL-like syndromes in mice (Daley et al., 1990;Heisterkamp et al., 1990;Gishizky and Witte, 1992).BCR/ABL activates multiple signaling pathways responsible for the protection from apoptosis, stimulation of growth factor-independent proliferation, modulation of adhesion/invasion ability and induction of resistance to genotoxic drugs and g-radiation (Raitano et al., 1997;Zou and Calame, 1999). Previous reports, including those from our laboratory, revealed that activation of signal transducer and activator of transcription 5 (STAT5) contributed to BCR/ABL-dependent changes in the phenotype of transformed cells (Ilaria et al., 1999;Nosaka et al., 1999;Kieslinger et al., 2000;Slupianek et al., 2001). STAT5A and STAT5B proteins belong to the family of STATs, which are latent transcription factors that become activated upon phosphorylation on tyrosine and also on serine (Horvath and Darnell, 1997). Phosphorylation of Tyr694 (Y694) in STAT5A and Tyr699 (Y699) in STAT5B is essential for dimerization and subsequent translocation to the nucleus (Gouilleux et al., 1994), resulting in the transactivation of several STAT5-inducible genes (Mui et al., 1996). Protein products of these genes are involved in regulation of growth factor independence, differentiation, adhesion/invasion and DNA repair/drug resistance (Ilaria et al., 1999;Nosaka et al., 1999;Kieslinger et al., 2000;Slupianek et al., 2001).The mechanism(s) of STAT5 activation by BCR/ABL remains unknown. Myeloid cells expressing the BCR/ ABL DSH3 + DSH2 mutant (BCR/ABLDD mutant, which lacks both SH3 and SH2 domains) failed to exhibit constitutive activation of STAT5, implicating the SH3 + SH2 region of BCR/ABL in recruiting...

Intercellular communication within the bone marrow niche significantly promotes leukemogenesis and provides protection of leukemic cells from therapy. Secreted factors, intercellular transfer of mitochondria and the receptor–ligand interactions have been shown as mediators of this protection. Here we report that tunneling nanotubes (TNTs)—long, thin membranous structures, which have been identified as a novel mode of intercellular cross-talk—are formed in the presence of stroma and mediate transfer of cellular vesicles from stroma to leukemic cells. Importantly, transmission of vesicles via TNTs from stromal cells increases resistance of leukemic cells to the tyrosine kinase inhibitor, imatinib. Using correlative light-electron microscopy and electron tomography we show that stromal TNTs contain vesicles, provide membrane continuity with the cell bodies and can be open-ended. Moreover, trans-SILAC studies to reveal the non-autonomous proteome showed that specific sets of proteins are transferred together with cellular vesicles from stromal to leukemic cells, with a potential role in survival and adaptation. Altogether, our findings provide evidence for the biological role of the TNT-mediated vesicle exchange between stromal and leukemic cells, implicating the direct vesicle and protein transfer in the stroma-provided protection of leukemic cells.

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Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agentdoxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short term in vitro cell re-population. It was associated with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls.Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment.