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.

中文翻译：

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STAT3介导慢性淋巴细胞性白血病中C6-神经酰胺诱导的细胞死亡。

慢性淋巴细胞性白血病（CLL）的发病机理知之甚少，目前仍无法治愈。先前我们已经表明，纳米脂质体C6-神经酰胺（CNL）在CLL的体内鼠模型中是一种有效的疗法。然而，介导CLL中CNL诱导的细胞死亡的关键信号通路仍然未知。我们假设CNL靶向STAT3，这是造血生物学的关键调节因子。我们观察到CNL处理可减少CLL细胞系和患者细胞中Y705和S727残基处的磷酸化STAT3。反过来，这降低了STAT3转录活性，并降低了STAT3依赖性生存关键因子（如Mcl-1和survivin）的表达。离体进一步证实了CNL对STAT3的作用如从用CNL治疗的小鼠获得的异种移植肿瘤中STAT3磷酸化水平降低所表明的。CNL分别通过降低BTK活性和MEK1 / 2激酶/ PKC活性来抑制Y705和S727处的STAT3磷酸化。此外，在与CNL和BTK抑制剂依鲁替尼共同治疗时，观察到CLL细胞活力的协同降低。STAT3的致癌形式的表达赋予了对CNL的部分抗性，从而证实STAT3介导了CNL诱导的细胞死亡。综上所述，这些发现为证明神经酰胺调节STAT3磷酸化提供了第一批证据。这些结果也是第一个证明神经酰胺对BTK有影响的结果，