SOCS5 Regulates JAK-STAT Signaling and T-ALL Development

T-lineage acute lymphoblastic leukemia (T-ALL) is a hematopoietic disease that tends to present with high-risk features in children, adolescents and young adults. While cure rates have improved in recent decades, event free survival is <10% in patients with relapsed disease. Activating mutations in IL-7, JAK1, JAK2, JAK3 have been implicated in aberrant JAK/STAT signal transduction and disease progression. However, the roles played by suppressors of the cytokine signaling (SOCS) remain incompletely understood. Because SOCS5 plays a key regulatory role in T-helper (Th) cell differentiation, we hypothesized that this gene is a critical determinant in mediating aberrant JAK/STAT signaling in T-ALL. To test regulatory effects of SOCS5 on T-ALL signal transduction and progression, we utilized lentiviral shRNA-mediated methods to knock down of SOCS5 in PF382 and KoptK1 T-ALL cell lines or, alternatively, to force over-expression of SOCS5 in ALL-SIL cells, respectively. We found that SOCS5 silencing resulted in enhanced cellular proliferation (P < 0.05), and that forced expression of SOCS5 resulted in significant decrease in cell proliferation rate (p < 0.05), suggesting that SOCS5 negatively regulates T-ALL cell growth in vitro. We utilized flow-based assays to examine the effects of SOCS5 on cell cycle control, and found that SOCS5 depletion significantly increased cell cycle progression in S/G2/M phases and decreased G1 (p < 0.05), but had no effect on apoptotic cell death. Forced expression of SOCS5 delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of cell cycle (p < 0.05). We next utilized protein analyses to examine the effects of SOCS5 on JAK-STAT signaling and found that suppressed SOCS5 expression induced phosphorylation of STAT1, STAT5, STAT6 while forced SOCS5 expression decreased STATs activation. Because STAT6 is involved in regulation of IL4 signaling we tested whether SOCS5 regulates the expression of this cytokine and its receptor, IL4R and demonstrated that SOCS5 depletion upregulated IL4 and its receptor in CCRF-CEM and KoptK1 cells. We next asked whether SOCS5 regulates IL7 signaling, which is critical for T-ALL development and resistance. Down-regulation of SOCS5 expression enhanced expression of both, IL7 and IL7R. On the contrary, SOCS5 overexpression decreased the levels of IL7 and IL7R. These findings suggest that SOCS5 is involved in negative regulation of IL4 and IL7 signaling. Similar experiments showed the SOCS5 leads to aberrant signaling and expression of c-myc oncogene. To study the effect of SOCS5 on leukemia development in a murine xenograft model, PF382 cells transduced with SOCS5 shRNA and control plasmid were transplanted intravenously into the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (10 mice per group) followed by survival analyses. We observed a significant decrease in mouse survival after SOCS5 depletion (median survival - 21 days) compared to animals engrafted with negative control cells (median survival - 30 days; p < 0.0002), indicating that SOCS5 inactivation promotes T-ALL progression. Using the same murine model, depletion of SOCS5 significantly increased the number of leukemic cells in bone marrow (92.4 ± 3.5 % vs. 13.6 ± 6 %; p < 0.0001), brain (88.4 ± 1.5 % vs. 14.7 ± 5.6 %; p < 0.0001) and liver (49.5 ± 8.2 % vs. 6.1 ± 1.5 %; p < 0.002) compared to negative control group. Our results link the aberrant SOCS5 expression to the up-regulation of the JAK-STAT and cytokine receptor-signaling cascade in T-ALL. We propose deregulation of SOCS5 expression as a novel mechanism governing aberrant cytokine signaling and JAK-STAT signal transduction in T-ALL. We conclude that SOCS5 is an important regulator of T-ALL cell proliferation and leukemic progression in sanctuary and non-sanctuary sites.