STAT3 Is Constitutively Phosphorylated on Serine 727 Residues, Activates Transcription, and Plays a Major Role in the Pathogenesis of B-Cell Chronic Lymphocytic Leukemia.
Authors: Hazan-Halevy Inbal , Harris David , Liu Zhiming , Ferrajoli Alessandra , Keating Michael J , Estrov Zeev 
Published: April 2020

Abstract
Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in cellular physiology and organ development. Tyrosine phosphorylation of STAT3 provides cells with survival advantage and induces proliferation, whereas deletion of STAT3 results in embryonic lethality. As might be expected, STAT3 is commonly activated in neoplastic cells. Constitutive phosphorylation of STAT3 on tyrosine 705 residues has been found in a number of solid tumors and hematologic malignancies. A decade ago, Frank et al. (JCI100:3149, 1997) reported that unlike other neoplasms in B-cell chronic lymphocytic leukemia (CLL), STAT3 is constitutively phosphorylated (p) on serine 727 but not on tyrosine residues. This observation prompted us to investigate the role of STAT3 in CLL. We studied peripheral blood (PB) cells from ~100 patients with CLL using Western immunoblotting and found that in all patients, regardless of PB count, disease stage, or treatment status, STAT3 is constitutively phosphorylated on serine 727 but not on tyrosine residues. Serine pSTAT3 was detected in CD19+ but not in CD19− CLL PB-derived cells or in normal PB-derived B-lymphocytes isolated by immunomagnetic beads. Unlike constitutive serine pSTAT3, tyrosine pSTAT3 could be induced by incubating CLL cells with cytokines, such as interleukin (IL)-6, or anti-IgM antibodies. However, expression of cytokine-induced tyrosine pSTAT3 was transient and was no longer detected shortly (2 hours) after removal of the stimulating agent. In contrast, serine pSTAT3 levels remained stable in vitro for up to 72 hours, confirming that serine phosphorylation of STAT3 is constitutive in CLL cells. Because CLL is the only disease in which STAT3 is constitutively phosphorylated exclusively on serine residues and since tyrosine phosphorylation is thought to be required for STAT3 activation, we sought to determine whether serine pSTAST3 is biologically active. First, we isolated cytoplasmic and nuclear extracts and used Western immunoblotting to demonstrate that pSTAT3 is present both in the cytoplasm and the nucleus. Then, we confirmed these findings using confocal microscopy. After confirming that serine pSTAT3 translocates to the nucleus, we asked whether serine pSTAT3 binds to DNA. Using the electrophoretic mobility shift assay we demonstrated that nuclear STAT3 binds to a STAT3-specific DNA probe and that STAT3-DNA binding was eliminated by both anti-STAT3 and anti-serine pSTAT3 antibodies. Furthermore, although induction of tyrosine pSTAT3 by exogenous IL-6 slightly increased STAT3-DNA binding, complete dephosphorylation of tyrosine pSTAT3 by T-cell tyrosine phosphatase did not affect STAT3-DNA binding, suggesting that STAT3-DNA binding does not require tyrosine phosphorylation. Finally, pull-down of STAT3 with biotin-labeled DNA further confirmed that serine pSTAT3 binds to DNA in CLL cells. We then asked whether serine pSTAT3 initiates transcription. To answer this question we infected CLL cells with a lentiviral GFP-STAT3-shRNA and, as control, with GFP-empty vector and calculated changes in RNA levels by relative real-time PCR. In several CLL samples, infection with GFP-STAT3-shRNA induced a ~4-fold time-dependent reduction in the STAT3-regulated genes Bcl2, Pim1, Bcl-xL, Cyclin D1, p21 Waf1, and c-Myc, implying that serine pSTAT3 initiates transcription. Taken together, our data suggest that constitutive phosphorylation of STAT3 on serine 727 residues is a hallmark of CLL and induces activation of proliferation and survival genes. Therefore, STAT3 should be considered a therapeutic target in this disease.