Abstract 3295: Combining GSK3 inhibition and Vitamin D as a differentiation strategy for AML.

Abstract

Abstract Vitamin D has widely been considered a promising therapy for cancer, particularly Acute Myeloid Leukemia (AML), due to its ability to terminally differentiate leukemic cells. Despite the promise of Vitamin D in preclinical studies, clinical trials have been disappointing likely due to dose limiting hypercalcemia that occurs at relatively low doses. We have identified that the kinase GSK3 is a major regulator of the Vitamin D receptor (VDR) and that targeting GSK3 can sensitize leukemic cells to low concentrations of Vitamin D that may be achievable clinically. Utilizing cell and animal based models, we found that GSK3 inhibition can significantly enhance the anti-leukemic effects of Vitamin D. By inhibiting GSK3 with both small molecules and shRNA, we found that targeting GSK3 leads to a significant sensitization in AML cells derived from multiple AML subtypes to Vitamin D-mediated differentiation. Differentiation induction can be seen through immunophenotyping for cell surface markers, functional differentiation as measured by NBT reduction, and changes in morphology. For example, treatment of HL-60 cells with low doses of Vitamin D (5nM) or a GSK3 inhibitor SB415286 (10uM) leads to only moderate differentiation (<50%) while the combination of these agents leads to greater than 95% differentiation as measured by the upregulation of the differentiation associated marker, CD11b. In addition to differentiation induction, GSK3 inhibition also significantly enhanced the ability of Vitamin D to cause irreversible growth inhibition as measured by colony assays and slowed the progression of AML in a mouse model system. Mechanistic studies have revealed significant insights into how GSK3 modulates Vitamin D signaling. GSK3 was found to bind and phosphorylate VDR which negatively regulates VDR activity as measured by luciferase based reporter assays. The mechanism through which GSK3 appears to regulate VDR activity is through impacting its interactions with co-repressors and co-activators. These co-modulators are known to play a predominant role in regulating VDR activity. Utilizing co-immunoprecipitation and mammalian two hybrid assays, we determined that GSK3 inhibition can impair the interaction of VDR with the co-repressor NcoR, and enhance its interaction with the co-activator, SRC-3. Overall, our studies suggest the potential of Vitamin D and GSK3 inhibition as a strategy to improve the efficacy of AML treatment and provide a mechanistic framework to explain the promising activity of this combination regimen. . Citation Format: Kalpana Gupta, Tammy Stefan, David N. Wald. Combining GSK3 inhibition and Vitamin D as a differentiation strategy for AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3295. doi:10.1158/1538-7445.AM2013-3295