Intrinsic and Estrinsic Mechanism Contributes to Maintain the JAK/STAT Pathway Aberrantly Activated in T-Type Large Granular Lymphocyte Leukemia

Abstract 1375

T-cell large granular lymphocyte (T-LGL) leukemia is a heterogeneous disorder characterized by the chronic expansion of a terminally differentiated cytotoxic T lymphocytes (CTLs) with a CD3+/CD8+/CD57+ phenotype. Although the pathogenesis of T-LGL leukemia is still unknown, the hallmark of the disease is the abnormal clonal expansion of antigen-primed mature CTLs that successfully escape activation-induced cell death (AICD) and remain long-term competent. Similar to normal activated CTLs, leukemic T-LGLs exhibit activation of multiple survival signaling pathways. Among the intracellular signaling pathways altered in T-LGL leukemia, JAK/STAT (Janus Kinase/signal transducer and transcription factor) signaling has been associated with LGL transformation. In particular, it has been demonstrated that the STAT3, a pivotal element of this pathway, is over-expressed and constitutively activated in leukemic LGLs as compared to normal peripheral blood mononuclear cells (PBMCs). STAT3 is a transcription factor inducing a number of genes promoting cell survival and the involvement of an aberrant STAT3 expression has been suggested to play a role in the pathogenesis of this disease.

In order to investigate the mechanism through which STAT3 is maintained in a state of activation in T-LGL leukemia, in patients in 27 patients we studied the expression of “suppressor of cytokine signaling 3 protein” (SOCS3) which is the specific negative regulator of STAT3 signaling, and the interleukin 6 (IL-6), which strongly induces STAT3 activation. Expression analysis was performed by Real Time-PCR and Western Blot assay in highly purified LGLs from PBMCs of patients by magnetic microbeads system.

By Real Time-PCR, the expression level of IL-6 was shown to be increased in patients' PBMCs (56.84 ± 17.01) as compared to normal PBMCs (0.53 ± 0.14, p<0.05). However, we found that SOCS3 was down-expressed, both at mRNA and protein level, in neoplastic LGLs as compared to control LGL (mRNA: 0.26 ± 0.04 vs 0.63 ± 0.17, p<0.01; protein: 0.59 ± 0.05 vs 0.85 ± 0.10, p<0.01). We also observed that in vitro SOCS3 was unresponsive to IL-6 in patients' LGL, whereas in LGLs of healthy individuals this cytokine was definitely able to increase SOCS3 expression level.

To better understand the mechanism responsible for SOCS3 low expression and unresponsiveness to IL-6 and since we found that this epigenetic mechanism is implicated in the NK type of this disease (Haematologica 2010. 95:1722), we evaluated whether an aberrant methylation of SOCS3 promoter takes place in T-LGL patients. Neoplastic LGLs were treated with the demethylating agent 5-aza-2'-deoxycytidine (DAC) and we observed that DAC re-established SOCS3 expression after IL-6 stimulation. Interestingly, as consequence of IL-6-induced SOCS3 expression we observed the inhibition of STAT3 phosphorylation and a reduction of Mcl1 protein level. These results support that in LGLs, SOCS3 tightly controls STAT3 and is able to modulate STAT3-induced gene Mcl1, that could be involved in LGL survival. Studies are now in progress to confirm the hypothesis that SOCS3 silencing is caused by the SOCS3 promoter methylation.

In conclusion, our results suggest that the downregulation of SOCS3, likely related to aberrant methylation, cooperates with IL-6 in the activation of JAK/STAT pathway, leading to the constitutive phosphorylation of STAT3, and thus ultimately resulting into increased survival of leukemic LGLs.