Abstract 1154: Elevated nuclear GSK3β promotes more aggressive disease in AML
Authors: Ignatz-Hoover James J. , Mackowski Nathan , Wald David N. 
Published: July 2016

Acute myeloid leukemia (AML) is a devastating disease with a poor overall 5-yr survival rate and an antiquated and poorly tolerated therapy regimen. Despite an increasing knowledge of the molecular drivers of AML, new therapeutic targets are needed. GSK3β is a key signaling molecule that plays a role in several key oncogenic pathways including the β-catenin and NF-κB pathways, key pathways in AML and other cancers. Upregulation of GSK3β is correlated with poor prognosis in several cancers. We and others have shown GSK3β inhibition promotes AML differentiation as well as death of AML cells while sparing normal cells, suggesting GSK3β plays an important role in AML.

Here we show by western blotting and confocal microscopy that GSK3β is upregulated and more nuclear in AML cells. We utilize flow cytometry to show AML cells express elevated GSK3β compared to normal marrow lineages (Average upregulation = 6 fold). We transform mouse hematopoietic progenitor cells with the MLL-AF9 oncogene and observe GSK3β upregulation as a course of AML leukemogenesis. These data suggest that elevated and nuclear GSK3β is characteristic of AML.

To probe biological effects of GSK3β, we knocked down endogenous GSK3β and utilize a targetted, tetracycline-inducible GSK3β rescue model to show nuclear GSK3β specifically can promote AML growth. Nuclear GSK3β increases colony formation of OCI-AML3 and HL60 cell lines more potently than cytoplasmic (cyto) GSK3β in the same lines (OCI-AML3- Nuclear: 118% Cyto: 93% of control colony growth HL60- Nuclear: 117% Cyto: 100% of control colony growth, Nuclear difference- p&lt;0.01 in both lines). Utilizing a mouse xenograft model and inducing GSK3β via doxycycline water, we show inducing nuclear GSK3β shortens mouse survival compared to uninduced controls, while inducing cytoplasmic GSK3β promotes no change from controls (HL60- Nuclear: Uninduced = 35.4 days mean survival, Induced = 28.75 days mean survival Cyto: Uninduced = 38 days mean survival, Induced = 38.6 days mean survival, Induced nuclear GSK3β difference p&lt;0.01 by log rank test).

At a molecular level, when compared to cells expressing cytoplasmic GSK3β, nuclear GSK3β more potently promotes NF-κB DNA binding and NF-κB mediated transcription as measured by EMSA assay and luciferase assay (Nuclear- 18 fold increase Cyto- 3 fold increase). Nuclear GSK3β more potently promotes activation of the NF-κB subunit p65, as measured by pS536, than cytoplasmic GSK3β. Nuclear GSK3β also promotes nuclear localization of p65. These data suggest that nuclear GSK3β can promote NF-κB activation in AML.

Finally, we utilize imaging cytometry to show GSK3β is upregulated and more nuclear in clinical AML patient samples (N = 80) compared to normal bone marrow controls (N = 5). We also show nuclear localization of GSK3β and p65 correlate linearly in AML patient samples (N = 80 R2 = 0.44721 P&lt;.01). These data suggest elevated nuclear GSK3β is observed in clinical AML and may be related to NF-κB activation.

Citation Format: James J. Ignatz-Hoover, Nathan Mackowski, David N. Wald. Elevated nuclear GSK3β promotes more aggressive disease in AML. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1154.