A Novel Mechanism of Nilotinib-Induced Apoptosis; Sphingolipids.

Abstract 4246

Chronic myeloid leukemia (CML) is a hematological malignancy resulting from the reciprocal translocation of chromosomes 9 and 22 that generates BCR/ABL oncogene. Nilotinib is a rationally designed, specific BCR/ABL tyrosine kinase inhibitor. Ceramide is a novel regulator of cell growth and proliferation, differentiation, senescence, cell cycle and also acts a strong apoptotic molecule while its conversion to antiapoptotic glucosyle ceramide (GC) and sphingosine-1-phosphate (S1P) by glucosyle ceramide synthase (GCS) and sphingosine kinase-1 (SK-1) enzymes result in more aggressive and resistant cancers. In this study, we studied the roles of ceramide metabolising genes in nilotinib induced apoptosis and possibility of increasing the sensitivity of BCR/ABL positive K562 and Meg-01 cells to nilotinib through targeting ceramide metabolism.

The cytotoxicity analyses of nilotinib, C8:ceramide to induce de novo generation of ceramides, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) to inhibit GCS and SK1 inhibitor were conducted by XTT cell proliferation assay. The changes in caspase-3 enzyme activity and mitochondrial membrane potential (MMP) were measured by caspase-3 colorimetric assay and JC-1 MMP detection kit, respectively. Expression analyses of ceramide synthase (LASS) genes, SK-1 and GCS genes were performed by RT-PCR.

We have shown that nilotinib induces apoptosis and inhibits cell-cycle progression in K562 and Meg-01 cells in a dose dependent manner. We have shown significant synergistic apoptotic effects of nilotinib in combination with C8:ceramide or PDMP or SK-1 inhibitor by XTT cell proliferation assay in addition to the changes in caspase-3 enzyme activity and changes in mitochondrial membrane potential, as compared to any agent alone. These results revealed that increasing de novo generation of ceramides or inhibiting conversion of ceramides to antiapoptotic GC or S1P increased sensitivity of BCR/ABL CML cells to nilotinib. More importantly, RT-PCR results revealed that there were significant decreases in expression levels of SK1 in response to increasing concentrations of nilotinib. On the other hand increases in expression levels of LASS2, -4, -5, and -6 ceramide synthase genes were determined in a dose dependent manner as compared to untreated controls.

It was shown for the first time by this study that targeting ceramide metabolism in addition to inhibition of BCR/ABL by nilotinib induces apoptosis synergistically in BCR/ABL positive K562 and Meg-01 CML cells.

This study was supported by The Scientific and Technological Council of Turkey