Abstract
CREB (cAMP Response Element Binding Protein) is a nuclear transcription factor that plays a critical role in the pathogenesis of Acute Myeloid Leukemia (AML). CREB is overexpressed in the majority of AML patients, and this is associated with a poor prognosis. CREB overexpression leads to increased AML cell proliferation and resistance to apoptosis in vitro. For CREB to be transcriptionally active, however, it must first be phosphorylated at Serine 133. Previous work has suggested that Ribosomal S6 Kinase (pp90rsk or RSK) is the primary kinase responsible for growth factor-induced phosphorylation of CREB, and that RSK is activated downstream of the Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Granulocyte-Colony Stimulating Factor (G-CSF) in AML cells. The overall role and regulation of RSK in AML cells, however, remains unknown. Thus, the goal of this study was to characterize the RSK-CREB signaling pathway in AML, with the overall hypothesis that disruption of this pathway represents a potential therapeutic strategy for the treatment of AML. We report that of the four known isoforms of RSK, RSK1 and RSK2 appear to be the predominant subtypes expressed in AML cells. To identify additional upstream pathways responsible for activation of these isoforms in AML cells, we performed cytokine stimulation experiments. Granulocyte-Colony Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), Thrombopoietin (TPO), and Interleukin 3 (IL-3) were all capable of stimulating phosphorylation and activation of RSK in KG-1 and HL-60 cells, implicating that multiple signaling pathways converge on RSK. G-CSF and GM-CSF had the greatest effects, stimulating increased phosphorylation and activation of RSK by 2.8 and 2.6 fold, respectively. Using shRNA technology, we then generated AML cell lines (HL-60 and KG-1) in which the expression of each isoform was ‘knocked-down’ to examine whether these two isoforms play unique roles in AML cells. Interestingly, RSK1 appears to be the isoform primarily responsible for phosphorylating CREB downstream of the G-CSF receptor. We demonstrate that G-CSF treatment of RSK1 knockdown cells did not induce an increase in CREB phosphorylation, and baseline CREB phosphorylation was also significantly decreased in these cells. Previous data had shown that blockade of total RSK activity using the non-selective RSK inhibitor BI-D1870 induced cell death in both AML cell lines and primary AML patient samples. RSK1 knockdown in HL-60 cells sensitized them to this agent (IC50 1.2 microM vs 3.3 microM), while the sensitivity of RSK2 knockdown cells was unchanged. Finally, since targets of RSK also include regulators of apoptosis (BAD) and cellular stress signaling pathways (IkB), we examined the effects of inhibiting RSK on the phosphorylation of these proteins. Levels of phosphorylated CREB and BAD decreased by 50% in HL-60 cells after 2 hours of treatment with the RSK inhibitor, suggesting that this treatment induces apoptosis. In summary, targeting the RSK-CREB signaling axis may represent a novel therapeutic strategy for AML. Future experiments will further define the role of RSK in proliferation and survival of AML cells and normal hematopoietic cells.
Disclosures
No relevant conflicts of interest to declare.
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