Signal Transducer and Activator of Transcription (STAT)-3 Induces Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) Receptor-α Expression in Chronic Lymphocytic Leukemia (CLL) Cells,
Authors: Li Ping , Liu Zhiming , Harris David , Ferrajoli Alessandra , Wang Yongtao , Hazan-Halevy Inbal , Grgurevic Srdana , Wierda William G. , Burger Jan A. , O'Brien Susan , Faderl Stefan , Keating Michael J , Estrov Zeev 
Published: October 2019

Abstract
Abstract 3879
GM-CSF stimulates proliferation of granulocytes, macrophages, and hematopoietic progenitors. Upon binding to its cellular receptor (R), GM-CSF induces dimerization of the GM-CSFR α and β subunits, phosphorylation of Janus kinase (JAK)-2, and activation of downstream signaling pathways. Because GM-CSF improved the response to Rituximab monotherapy in patients with CLL (Ferrajoli A. Leuk Lymphoma 50:514, 2009) and was found to upregulate CD20 cell surface antigen expression (Vanugopal P. et al. Leuk Res 24:411, 2000), we investigated the effect of GM-CSF on CLL cells. Incubation of peripheral blood (PB) CLL cells with increasing concentrations of GM-CSF (0.05 to 1.0 μM) did not induce the phosphorylation of STAT3, STAT5, AKT, or ERK as assessed by western immunoblot, or phosphorylation of JAK2 as assessed by immunoprecipitation. Therefore, we investigated whether CLL cells express GM-CSFR. Western immunoblot studies revealed that, like normal B lymphocytes, CLL cells do not express GM-CSFRβ but, unlike normal B cells, CLL cells express high levels of GM-CSFRα. Flow cytometry analysis of PB cells from 8 patients with CLL showed that 13 to 59% of CLL cells (CD19+/CD5+) co-expressed CD116 (GM-CSFRα) but not CD131 (GM-CSFRβ). Thus, we wondered what induces GM-CSFRα expression in CLL cells. Because STAT3 is constitutively activated in CLL and sequence analysis revealed that the GM-CSFRα promoter harbors γ-interferon activation sequence (GAS)-like elements typically activated by STAT3, we sought to determine whether STAT3 activates GM-CSFRα. In MM1 cells, interleukin (IL)-6 induced STAT3 phosphorylation and up-regulated GM-CSFRα whereas STAT3-siRNA down-regulated both STAT3 and GM-CSFRα protein levels, suggesting that STAT3 activates transcription of GM-CSFRα. To clarify these findings, we cloned the human GM-CSFRα promoter, generated a series of truncated promoter constructs and assessed their activity using a luciferase assay. We found that IL-6 augmented luciferase activity of GM-CSFRα promoter −4012 – +23, −3018 – +23, −2517 – +23, and −496 – +23, suggesting that IL-6 enhanced GM-CSFRα expression by activating STAT3. Furthermore, we established that regions, located between bp −3581 TTGTTGAAAA −3572, −2984 TTTTCTTAA −2976 and −77 TTTCCCAA −70, harbor GAS-like elements that activated the GM-CSFRα promoter upon exposure to IL-6. Binding of STAT3 to those regions in IL-6-stimulated MM1 cells was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether STAT3 induced transcription of GM-CSFRα in CLL, we obtained fresh PB CLL cells and, by using the same GAS-like element-containing probes, performed EMSA. CLL cell nuclear protein bound these probes and anti-STAT3 and -phosphoserine STAT3 antibodies attenuated the binding. CLL cell ChIP confirmed that STAT3 binds to the promoter of GM-CSFRα as well as the promoters of the STAT3-regulated genes STAT3, c-Myc and P21, but not to that of the control gene RPL30. Finally, using qRT-PCR and western blot analysis we determined that STAT3-shRNA down-regulated GM-CSFRα, STAT3 and STAT3-regulated gene mRNAs, and STAT3 and GM-CSFRα protein levels. Taken together, these data suggest that constitutively activated STAT3 binds to the GM-CSFRα promoter, activates its transcription, and induces production of GM-CSFRα protein in CLL cells.

Disclosures:
No relevant conflicts of interest to declare.