Lyn regulates creatine uptake in an imatinib-resistant CML cell line

Abstract

Background: Imatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells.

Methods: Stable isotope metabolic labeling, media creatine depletion, and Na⁺/K⁺-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na⁺/K⁺-ATPase and creatine uptake.

Results: Inhibition of the Na⁺/K⁺-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na⁺/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na⁺/K⁺-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na⁺/K⁺-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na⁺/K⁺-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells.

Conclusions: These data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na⁺/K⁺-ATPase pump activity.

General significance: These studies identify kinase regulation of the Na⁺/K⁺-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.

Keywords: (1)H NMR spectroscopy; Apoptosis; Cell viability; Chronic myelogenous leukemia; Creatine; Creatine transporter; Cyclocreatine; Drug resistance; Imatinib; Kinase; Lyn; Metabolites; Metabolomics; Phosphocreatine; Ponatinib; Sodium/potassium-ATPase.