STAT3-Activated GM-CSFRα Shuttles To The Nucleus and Protects CLL Cells From Apoptosis

Granulocyte-macrophage colony stimulating factor (GM-CSF) regulates the survival, proliferation, differentiation, and activation of hematopoietic cells, dendritic cells, and T cells. Upon GM-CSF binding, the α and β subunits of the GM-CSF receptor (GM-CSFR) dimerize and activate signaling. While exploring a possible role for GM-CSF in treating chronic lymphocytic leukemia (CLL), we found that GM-CSF did not enhance the phosphorylation of STAT3, Akt, or ERK, suggesting that GM-CSF does not directly affect CLL cells. Indeed, as in normal B cells, flow cytometry analysis of CLL cells did not detect GM-CSFRβ. However, unlike in normal B cells, GM-CSFRα (CD116) was present in CLL cells. Using confocal microscopy, cell fractionation studies, and GM-CSFRα antibody epitope mapping, we detected GM-CSFRα not only on the surfaces but also in the cytosol and nuclei of CLL cells, suggesting that GM-CSFRα has functions that are unrelated to receptor-ligand interaction. Because STAT3 is constitutively activated in CLL cells and because the promoter of the GM-CSFRα gene harbors putative STAT3 binding sites, we hypothesized that STAT3 activates GM-CSFRα in CLL cells. Both chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) analyses revealed that STAT3 binds to the promoter of GM-CSFRα. We therefore cloned the GM-CSFRα promoter in multiple myeloma cell line MM1 and used luciferase assay we identified STAT3 binding activity. To investigate the function of GM-CSFRα, we induced the overexpression of GM-CSFRα in 293FT cells and analyzed the RNA and protein that co-immunoprecipitated with GM-CSFRα. We identified 7200 RNA transcripts that co-immunoprecipitated with GM-CSFRα, including SKI and MAFA oncogenes, two serine/threonine kinase genes, and DEFT1P2 and DEFP1P, which encode proteins that belong to members of the death effector domains family, known to have a role in modulating apoptosis. Overall, the transcripts that co-immunoprecipitated with GM-CSFRα belong to survival pathways, the JAK/STAT pathway, and hematopoietic lineage pathways. Mass spectrometry analysis revealed that housekeeping proteins, chaperon proteins, KAP1, and ISG-15 co-immunoprecipitated with GM-CSFRα. We found that GM-CSFRα–bound KAP1 enhanced the transcriptional activity of STAT3, whereas GM-CSFRα–bound ISG-15 inhibited the nuclear factor-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα-siRNA induced the apoptosis of CLL cells. Taken together, our data suggest that STAT3 induces the transcription of GM-CSFRα, which has distinct, ligand-independent, anti-apoptotic activities.