Abstract
Abstract Novel approaches to enhancing the delivery, uptake, and therapeutic effect of sphingolipids are of interest for the treatment of neoplasms. We attempt to promote cellular ceramide accumulation in murine and human models of acute myelogenous leukemia (AML) by modifying ceramide metabolism with either the sphingosine kinase1 inhibitor safingol (Lip-Saf), or the acid ceramidase inhibitor LCL204, preventing ceramide glycosylation by inhibition of p-glycoprotein (P-GP) activity with tamoxifen (Lip-Tam), and supplementing cells with exogenous C6 ceramide (Lip-C6) via liposomal delivery. Ceramide is widely recognized as an apoptotic signal and blocking its catabolism to sphingosisne-1-phosphate has been shown to induce autophagy. Safingol and LCL204 both modify ceramide catabolism. Supplementation of tamoxifen has been shown to promote apoptosis via ceramide accumulation by inhibiting the activity of P-GP at the Golgi membrane, which can participate in ceramide glycosylation. C1498, HL-60, HL-60/VCR, and KG-1 leukemia cell line viability was quantified following treatment with Lip-C6, Lip-Saf, or a 2:1 combination to measure synergy. Likewise, cell line viability was evaluated using Lip-Tam and Lip-C6, independently and in a 1:1 combination to study synergy. C1498 viability was also quantified with increasing concentrations of LCL204. CalcuSyn software was used to analyze dose-response and synergistic therapeutic efficacy. Colony-forming capacity was assessed using a poor-prognosis AML patient sample treated with Lip-C6, Lip-Saf, or the combination of both. Autophagy and apoptosis were further evaluated using flow cytometry with primary AML samples and leukemia cell lines. A reduction in IC50 of the drugs used in combination was noted in several cell lines. Dramatic IC50 shifts were observed in KG-1, HL-60, and the vincristine-resistant HL-60/VCR cell lines after exposure to a combination of Lip-C6 and Lip-Saf in a 2:1 ratio. Flow cytometry confirmed Lip-Saf enhances autophagy, whereas Lip-C6 exerts an effect primarily through apoptosis in C1498 and primary AML samples. Therefore, the observed cell death synergism is likely due to induction of both autophagy and apoptosis. Additionally, it was shown that the anti-AML efficacy of Lip-Tam was dramatically enhanced in combination with Lip-C6, demonstrating that inhibition of ceramide glycosylation is also a valid combinatorial therapeutic approach. Lastly, inhibition of acid ceramidase via LCL204 demonstrated efficacy as a cytotoxic agent both in vitro and in vivo using C1498 cells alone or engrafted into C57BL/6J mice. These studies verify inhibition of ceramide metabolism as an effective strategy to enhance the therapeutic efficacy of ceramide in the treatment of AML. Citation Format: Timothy J. Brown, Aileen M. Garcia, Su-Fern Tan, Lindsey N. Kissinger, David J. Feith, Brian M. Barth, David F. Claxton. Improving the anti-AML efficacy of liposomal ceramide by targeting sphingolipid metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3396. doi:10.1158/1538-7445.AM2013-3396
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