Abstract
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.
Highlights
Chronic lymphocytic leukemia (CLL) is a B-cell malignancy characterized by the clonal expansion and accumulation of neoplastic B lymphocytes expressing CD5, CD19, CD20 and CD23 in the bone marrow, peripheral blood and often the lymph nodes.[1]
The final western blot image was created by grouping different parts of the same film of the same gel as indicated by the black dividing line. (c) STAT3 inhibition reduces viability of CLL cell lines and patient cells. (i) Peripheral blood mononuclear cells (PBMCs) from three normal donors and three CLL patients, (ii) JVM-3 cells and (iii) Mec-2 cells were treated with Stattic for 24 h and cell viability was determined by the MTS assay. (ii) Western blotting analysis in JVM-3 confirms the effectiveness of Stattic treatment
Several studies have reported that STAT3 is constitutively phosphorylated on S727 and not Y705 in CLL.[17,18]
Summary
Chronic lymphocytic leukemia (CLL) is a B-cell malignancy characterized by the clonal expansion and accumulation of neoplastic B lymphocytes expressing CD5, CD19, CD20 and CD23 in the bone marrow, peripheral blood and often the lymph nodes.[1] Depending on the degree of somatic hypermutation and chromosomal abnormalities, the clinical course of CLL ranges from slow progression to rapid disease progression.[1,2] The standard treatment regimen of fludarabine, cyclophosphamide and rituximab has an overall response rate of ~ 90% and complete remission of 72%.3,4. A large body of evidence has demonstrated that ceramide potentiates signaling cascades leading to cell death. Intracellular delivery of ceramide remains a challenge due to limited solubility and cannot be delivered by conventional methods.[5,6] Our laboratory has developed a nanoliposomal formulation of C6-
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