FBW7 inhibits myeloid differentiation in acute myeloid leukemia via GSK3-dependent ubiquitination of PU.1

GSK3β, an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including Acute Myeloid Leukemia where it promotes self-renewal, growth and survival of AML cells. Therefore, GSK3β inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte-macrophage differentiation pathway as potential GSK3β target. We demonstrate that GSK3β phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte-macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41,S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41,S140A retained its transactivation, DNA binding ability and promoted monocyte-macrophage differentiation of U937 cells even without PMA treatment. We further showed that FBW7 overexpression inhibited both PMA as well as MCSF-induced macrophage differentiation of myeloid cell lines and PBMCs from healthy volunteers respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3β-FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from leukemia patients where FBW7 over expression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared to M-CSF treatment alone. Implications: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3β inhibition and further substantiates potential of GSK3β as a therapeutic target in AML.