STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

The pathogenesis of chronic lymphocytic leukemia (CLL) is poorly understood and it remains incurable with current therapies. We have previously shown that nanoliposomal C6-ceramide (CNL) is an effective therapy in an in vivo murine model of CLL. However, the key signaling pathways mediating CNL-induced cell death in CLL remains unknown. We hypothesized that CNL targets STAT3, a critical regulator of hematopoietic biology. We observed that CNL treatment reduced phosphorylated STAT3 at both Y705 and S727 residues in CLL cell lines and patient cells. This, in turn, reduced STAT3 transcriptional activity and expression of critical STAT3-dependent survival factors like Mcl-1 and Survivin. The effect of CNL on STAT3 was further confirmed ex vivo as shown by reduced STAT3 phosphorylation in xenograft tumors obtained from mice treated with CNL. CNL suppressed STAT3 phosphorylation at Y705 and S727 through reduction in Btk activity and MEK1/2 kinase/PKC activities, respectively. Moreover, a synergistic reduction in CLL cell viability was observed on co-treatment with CNL and the Btk Inhibitor, ibrutinib. Expression of an oncogenic form of STAT3 conferred partial resistance to CNL, providing confirmation that STAT3 mediates CNL-induced cell death. Taken together, these findings provide the first body of evidence demonstrating ceramide regulation of STAT3 phosphorylation. These results are also the first to demonstrate an effect of ceramide on Btk, a critical kinase mediating the B-cell receptor signaling in CLL cells and suggest a novel and synergistic combination of CNL and Btk inhibitors for CLL treatment.