Inhibition of JAK2/STAT3 Pathway Leads to Apoptosis in Chronic Lymphocytic Leukemia Cells
Authors: Severin Filippo , Frezzato Federica , Martini Veronica , Raggi Flavia , Trimarco Valentina , Martinello Leonardo , Visentin Andrea , Facco Monica , Semenzato Gianpietro , Trentin Livio 
Published: October 2019

Abstract
INTRODUCTION
Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Despite their in vivo prolonged lifespan due to intrinsic defects, CLL leukemic cells rapidly undergo spontaneous apoptosis in vitro, highlighting the need of extrinsic signals delivered by the microenvironment.
Several molecules, including those released by mesenchymal stromal cells (MSCs), signal through JAK (Janus kinases)-STAT (Signal Transducers and Activators of Transcription) pathways. We particularly focused on the JAK2/STAT3 axis since Interleukin-6 (IL-6), one of the most abundant cytokines released in the CLL microenvironment, is the key ligand of the receptor triggering this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells.
METHODS
B cells were collected from 12 controls and 46 CLL patients. Purified cells (2x106cells/ml) were cultured, and treated with AG490 (10, 50 and 100μM), AZD1480 (1, 4 and 10μM), Fedratinib (1, 5 and 10μM), and Ruxolitinib (0.313, 2.5 and 10μM) (which are JAK2 inhibitors), and the STAT3 inhibitor Stattic (5, 7.5, and 10μM) for 24, 48 and 72h. Experiments with AG490 and Stattic were performed with/without MSCs.
STAT3 expression and phosphorylation were evaluated by Western Blotting (WB) and Flow Cytometry (FC), and its localization was analyzed by confocal microscopy and subcellular fractionation. CLL and normal B cell viability was tested by FC with Annexin V/PI test.
RESULTS
We demonstrated that STAT3 was highly expressed in malignant B cells with respect to normal B lymphocytes. As far as STAT3 phosphorylation at Tyr705, that is an essential step for STAT3 activation, we demonstrated a constitutive phosphorylation in CLL cells by FC and WB analyses, although in some patients STAT3 Tyr705 phosphorylation is barely detected.
We also pointed out that the in vitroincubation of leukemic B cells with AG490 and Stattic and Fedratinib, induces a dose-dependent apoptosis of CLL B cells. However, the tested doses of Ruxolitinib and AZD1480 did not seem to CLL B cell viability but only STAT3 phosphorylation. Both AG490 and Stattic were able to bypass the microenvironmental protection when neoplastic B cells were co-cultured with MSCs.
STAT3 Tyr705 localization was analyzed in normal and leukemic B cells by a subcellular protein fractionation. We separated nuclei from cytosol, detecting STAT3 Tyr705 both in the cytosolic and in the nuclear fractions of CLL B cells.
We showed that AG490 and Fedratinib treatment on CLL cells can mediate other effects: i) SHP-1 activity is turned on by JAK2 inhibition, decreasing its phosphorylation at Ser591; ii) AG490 administration inactivates protein Lyn, reducing the phosphorylation in its active site at Tyr396.
Lyn, a Tyr-kinase, and SH2-domain containing Tyrosine Phosphatase (SHP-1), a Tyr-phosphatase, are both involved in the prolonged lifespan of neoplastic CLL cells. To confirm the link between JAK2 inhibition by AG490 and Lyn dephosphorylation, we added sodium orthovanadate (Na3VO4, 100 μM), a phosphatase inhibitor, to cell culture to restore Lyn activation (resulting from the inactivation of SHP-1 phosphatase); as a result, Lyn Tyr396 phosphorylation was restored. On the contrary, the treatment of CLL cells with Stattic did not induce any change in SHP-1 status with respect to untreated cells since Stattic is effective on STAT3, that is a downstream protein with respect to JAK2. Since Stattic did not affect SHP-1 activation, it does not impact on Lyn activation/phosphorylation.
CONCLUSIONS
The ability of AG490 and Stattic to induce apoptosis in leukemic B cells bypassing the pro-survival stimuli provided by the tumor microenvironment and the Fedratinib effectiveness at low doses, represents a starting point for the development of new therapeutic strategies in CLL.
This study also provides new insights for the investigation of the pathogenesis of CLL focusing the attention on the cross-talk between JAK/STAT and BCR/Lyn axes.

Disclosures
No relevant conflicts of interest to declare.