Casein Kinase-2 Induces Constitutive Phosphorylation of STAT3 in CLL Cells
Authors: Rozovski Uri , Harris David M. , LI Ping , Liu Zhiming , Jain Preetesh , Ferrajoli Alessandra , Burger Jan A. , O'Brien Susan M. , Bose Prithviraj , Thompson Philip A. , Jain Nitin , Wierda William G. , Keating Michael , Estrov Zeev 
Published: October 2019

Abstract
In CLL the signal transducer and activator of transcription (STAT)-3 is constitutively phosphorylated on serine 727 residues. This phosphorylated form of STAT3 is biologically active and plays a pivotal role in activating several pathways that stimulate proliferation and provide CLL cells with survival advantage. However, what induces serine phosphorylation of STAT3 in CLL cells is currently unknown. Looking for potential kinases that phosphorylate STAT3 on serine residues, we performed mass spectrometry analysis of CLL cell proteins co-immunoprecipitated with anti-serine pSTAT3 antibodies. Of the 365 pulled-down proteins we identified the beta subunit of the serine/threonine kinase casein kinase (CK)-2. Using Western immunoblotting we confirmed that phosphorylated CK2 was readily detected in CLL cells from 7 randomly selected patients. Furthermore, immnoprecipitation studies showed that STAT3 and serine pSTAT3 co-immunoprecipitated with CK2 and that CK2 co-immunoprecipitated with STAT3, suggesting that CK2 binds STAT3. Therefore to determine whether CK2 phosphorylates STAT3 we incubated active CK2 with recombinant human (rh) STAT3 and found that CK2 phosphorylated rhSTAT3 on serine 727 residues in an ATP-enriched cell-free medium. Because unlike normal B cells CLL cells express CD5 and CD5 activates CK2, we wondered whether CD5 plays a role in STAT3 phosphorylation. Immunoprecipitation studies determined that CD5 co-immunoprecipitated with CK2 and pSTAT3, and transfection of CLL cells with CD5-siRNA significantly reduced the levels of serine pSTAT3. Because CD5 activates CD5, we incubated CLL cells with CD5 neutralizing antibodies and found that CD5 neutralizing antibodies significantly reduced the levels of serine pSTAT3. Because STAT3 is not constitutively in T lymphocytes although STAT3, CK2 and CD5 are ubiquitously expressed in these cells, we wondered whether a protein, not present in T lymphocytes, is required for the induction of STAT3 phosphorylation in CLL cells. The B cell linker protein (BLNK) is an adaptor protein that is expressed in B-lymphocytes and is required for LPS-induced STAT3 phosphorylation. To determine whether BLNK is required for STAT3 phosphorylation in CLL cells we transfected the cells with BLNK-siRNA and found that the levels of serine pSTAT3 were significantly reduced. Furthermore, we found that BLNK co-immunoprecipitated with CK2, CD5, STAT3, and phosphorylated STAT3. Taken together these findings suggested that both CD5 and BLNK are required for the induction of STAT3 phosphorylation by CK2 in CLL cells. Because CD5 is a cell membrane protein we wondered whether its role in the assebly of the STAT3-phosphorylation complex would alter its localization. Therefore, we performed confocal microscopy studies that revealed that CD5 is cell bound. Then we prepared CLL cell cytoplasmic and nuclear extracts and using Western immunoblotting found that the STAT3-phosphorylation complex proteins including CD5, CK2, BLNK, STAT3, and serine pSTAT3 were detected in the cytoplasmic extracts whereas only STAT3 and phosphoserine STAT3 were detected in the nuclear extract, suggesting that following its phosphorylation, pSTAT3 detaches from the complex and shuttles to the nucleus. In conclusion, our data unraveled the role of intracellular proteins that participate in the induction of constitutive phosphorylation of STAT3 on serine 727 residues in CLL cells. Whether disruption of this protein complex might benefit patients with CLL remains to be determined.

Disclosures
O'Brien: Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Jain:Abbvie: Research Funding; Infinity: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; BMS: Research Funding; Novimmune: Consultancy, Honoraria; Celgene: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Seattle Genetics: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding. Wierda:Novartis: Research Funding; Abbvie: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding.