STAT5 Activation Is Mandatory for IL-7-Mediated Viability and Growth of T-Cell Acute Lymphoblastic Leukemia Cells

Abstract 3530

T-cell acute lymphoblastic leukemia (T-ALL) constitutes, in general, an aggressive subset of ALL, the most frequent childhood malignancy. Although risk-adjusted chemotherapeutic regimens are currently extremely effective, their efficacy is associated with significant long-term side effects and those cases that relapse have dismal prognosis. New, more specific, therapies are therefore required. To achieve this goal it is essential to have a better understanding of T-ALL biology, including the contribution of tumor microenvironmental factors for leukemia progression. Interleukin 7 (IL-7) is produced by stromal cells in the bone marrow and thymus. While IL-7 is essential for normal T-cell development, there is also considerable evidence that it can partake in leukemia expansion. Previously, we have shown that IL-7 promotes T-ALL expansion in vivo (Silva et al, Cancer Res. 2011) and leukemia cell survival and proliferation in vitro by activating PI3K/Akt/mTOR signaling pathway (Barata et al, J Exp Med. 2004), consequently leading to p27^kip1 downregulation and Bcl-2 upregulation. However, it is also known that T-cell lymphomas arising spontaneously in IL-7 transgenic mice depend on STAT5 activity. Thus, we investigated whether STAT5 could also be involved in the IL-7 pro-leukemia effects in human T-ALL cells. Using an IL-7-dependent leukemia T-cell line (TAIL7), we show that IL-7 activates JAK-STAT5 pathway in a dose- and time-dependent manner, as measured by phosphorylation of JAK1, JAK3 and STAT5. We further demonstrate that IL-7 induces STAT5 DNA binding and transcriptional activity. To establish the role of STAT5, we stably transduced HPB-ALL cells with lentiviral vectors driving the expression of STAT5a shRNA or scramble control, and confirmed the efficiency of STAT5a knock down at the protein level. Flow cytometry analysis of cell size and annexin V binding, indicate that STAT5 is indispensable for IL-7-mediated T-ALL cell growth and viability. To test the potential clinical applicability of these observations, we treated TAIL7 and primary leukemia cells collected from pediatric T-ALL patients at diagnosis with pharmacological inhibitors of JAK3 (WHI-P131), STATs in general (parthenolide) and STAT5 in particular (N-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide). All three inhibitors abrogate IL-7-mediated T-ALL cell viability, growth and proliferation. At the molecular level, these observations correlate with complete inhibition of IL-7-induced modulation of p27^kip1, cyclin A and cyclin D2 expression and with prevention of transferrin receptor (CD71) upregulation. Interestingly, Bcl-2 expression does not appear to be significantly affected by inhibition of STAT5 activity. This contrasts with PI3K/Akt pathway and suggests that STAT5 regulates leukemia T-cell survival, at least in part, by an alternative, Bcl-2-independent mechanism. Overall, these results extend our observations that JAK/STAT5 pathway inhibitors can eliminate cells displaying IL-7Rα gain-of-function mutations that we recently found in T-ALL (Zenatti et al, Nat Genet. 2011), and indicate that STAT5 plays a major role in mediating IL-7/IL-7R signaling effects in T-ALL cells, therefore constituting a promising target for therapeutic intervention in this malignancy.