Treatment failure and relapse in acute lymphoblastic leukemia (ALL) is promoted by bone marrow stromal cells that provide growth stimulation under steady-state conditions and anti-apoptotic signals when the ALL cells are treated with chemotherapy. In the previous period of support, this project has made significant advances in understanding how ALL cells respond when they are simultaneously challenged by drug treatment and provided with stromal support. Modification of cell surface molecules through sialylation regulates interactions of immune cells with the microenvironment and modulates signal transduction strength. Our preliminary studies show abnormally high levels of sialic acid 9-O-acetylation on both mouse and human ALL cells, which is consistently and significantly increased as they develop drug resistance. Strikingly, human ALL cells proliferating in the protective bone marrow microenvironment of transplanted mice were eradicated when 9-O-acetylation was removed through activation of a specific esterase that cleaves the R9 acetyl group from sialic acid. We hypothesize that ALL cells generate 9-O-acetylated, sialic acid-modified glycoproteins and glycolipids as a mechanism of protection against spontaneous and drug- induced apoptosis. To address this, Aim 1 will investigate intracellular pathways activated during death due to 9-O-acetylation removal from Sias in pre-B ALL cells, by expression of inducible 9-O-acetyl esterases in human pre-B ALL and mouse wild type and GD3-/- pre-B ALL cells in co-culture with protective stroma.
Aim 2 will determine how increased 9-O-acetylation levels of Sias contribute to drug resistance development by comparing drug sensitivity and resistance in pre-B ALL cells with different 9-O-acetylated sialic acid levels in vitro and in mous models. The specific contribution of GD3 will be examined in st8sia1-/- pre-B ALL cells.
Aim 3 will determine how acetylation of Sias changes the reactivity of sialoglycoproteins or of GD3 to a conformation favorable for ALL cell survival. The effect of the oxidative state of GD3 on its pro-apoptotic activity will be evaluated in st8sia1-/- pre-B ALL cells that lack endogenous GD3. We will also characterize critical sialoglycoproteins that are 9-O-acetylated in human and mouse ALL cells for subcellular location, induction kinetics upon drug treatment and development of drug resistance using the C. antennarius lectin that specifically detects this modification. Immunoaffinity purification and mass spectrometry will be used to identify the core proteins that are modified by 9-O-acetyl sialylation and are upregulated as ALL cells develop drug resistance in the presence of stromal support in vitro. 9-O-sialylation has not been examined in drug resistance in ALL but our data show it is extremely relevant to their growth and survival. These innovative studies will yield new insight into the mechanisms through which ALL cells withstand drug treatment and lead to novel targets and approaches to treat this and other types of cancer.
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