Abstract 3336: ST6Gal-I glycosyltransferase promotes an undifferentiated cell phenotype and enhances c-kit signaling

Acute myelogenous leukemia (AML) is characterized by an increased number of myeloid cells in the bone marrow. These cells are arrested in their maturation process, frequently resulting in hematopoietic insufficiency. Recent advancements in molecular genetics have identified new markers that may be useful in diagnosing AML, including mutant c-kit (CD117). c-kit is a receptor tyrosine kinase and activating mutations in this receptor have been identified as a functional driver of AML and other myeloproliferative diseases. c-kit also plays a major role in maintaining normal stem cell pools. Prior studies suggest that ST6Gal-I, a unique glycosyltransferase upregulated in many cancers, promotes a stem-like cell phenotype in epithelial tumor cells. ST6Gal-I is a sialyltransferase that adds a negatively charged sialic acid in an α2-6 linkage to membrane glycoproteins. In this study, we show c-kit to be a substrate of ST6Gal-I, with increased sialylation correlating to increased c-kit activation. In order to study the effects of sialylation on signaling, we forced constitutive expression of ST6Gal-I in the U937 monocytic leukemia cell line. c-kit signaling was then induced by treating U937 cells with stem cell factor (SCF), the c-kit ligand. Here we show enhanced SCF-mediated c-kit activation, indicated by increased levels of phospho-c-kit in ST6Gal-I overexpressing cells. This, in turn, is associated with increased activation of downstream effecters of c-kit signaling, phospho-Erk and phospho-Akt. We further hypothesize that ST6Gal-I might play a role in the cell differentiation process. To test the effects of sialylation on differentiation, we used a well-established model to induce monocyte cell differentiation, treatment with the phorbol ester, PMA. PMA is known to induce macrophage differentiation in U937 cells, evidenced by exit from the cell cycle and upregulation of differentiation markers, including CD11b. Macrophage differentiation induces downregulation of endogenous ST6Gal-I, thereby reducing surface sialylation of specific receptors. However, forced overexpression of ST6Gal-I inhibits PMA-induced exit from the cell cycle, suggesting that ST6Gal-I acts to maintain cells in an undifferentiated state. To further understand the effects of receptor sialylation on PMA-induced differentiation, we used flow cytometric analysis to evaluate surface markers on U937 cells with or without ST6Gal-I overexpression. We show that forced expression of ST6Gal-I inhibits PMA-induced upregulation of CD11b, whereas c-kit expression is elevated in ST6Gal-I overexpressing cells relative to parental cells. These results suggest a novel mechanism by which ST6Gal-I contributes to an undifferentiated state in part through the regulation of the c-kit receptor. Citation Format: Kaitlyn A. Dorsett, Susan L. Bellis. ST6Gal-I glycosyltransferase promotes an undifferentiated cell phenotype and enhances c-kit signaling. [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 3336.