Abstract
Ceramides are naturally occurring cellular sphingolipids which are increased following various effective cancer therapies. Bioavailable forms of ceramide are under development as therapeutic tools (Anticancer Agents Med Chem. 2011 Nov;11(9):911-9). Short side chain (C6) ceramide has, in pegylated nanoliposomal (NL) formulations, shown excellent cellular uptake and significant anti-tumor activity in a variety of tumor types and models. Cellular autophagy is an active microsomal process implicated in ceramide metabolism and potentially in the detoxification of C6-ceramide. We have previously shown synergistic combination therapy with Lip-C6-ceramide and vinblastine and its association with autophagy dysfunction in hepatocarcinoma and colorectal cancer models (Cancer Lett. 2013 Sep 1;337(2):254-65.).
We explored the therapeutic activity of nano-liposomally delivered C6-ceramide (Lip-C6) in cell lines and primary human acute myelogenous leukemia (hAML). Lip-C6 cytotoxicity was tested in multiple human and murine AML lines (in short term viability assays) and in multiple cryopreserved primary human AML cases (in clonogenic assays - colony formation in methylcellulose). Lip-C6 demonstrated anti-AML activity in these cells with an EC50 of 2-23uM Lip-C6. In order to enhance this efficacy the interplay between ceramide induced apoptosis and autophagy was explored. The AML line KG1 was found to be relatively resistant to Lip-C6 (EC50 23uM). KGI showed supra-additive sensitivity to the combination of Lip-C6 with either vinblastine (VBL) or vincristine, both microtubule binding agents with the ability to reduce autophageic flux. KG1 sub-clones with knockdown of ATG5 demonstrated increased sensitivity to Lip-C6 relative to control or scrambled knockdown transfectants, consistent with the hypothesis that autophagy “rescues” this line from ceramide cytotoxicity. To further confirm these findings we examined the interaction of Lip-C6 (20uM) with VBL (5uM) or chloroquine (25uM) in a series of 13 fresh, patient derived AML cases (Figure: A-F). After 48 hours of co-culture, cells were examined for apoptosis of whole leukemic(E) and putative leukemia “stem cell” (LSC - F) fractions by flow cytometry. We also studied the in-vivo activity of Ghost (control) NLs, Lip-C6, VBL-NLs and combinatorial Lip-C6+VBL-NLs in NSG xenografts bearing a single poor prognosis (inversion 3) hAML (data are shown in the figure G.)
Panels A-D: AML Cells of patient #661 gated on CD34+/CD38-ve (“LSC”) fraction examined for viability with 7AAD and apoptosis with AnnexinV – A Ghost Liposomes, B –Lip-C6, C, VBL, D Lip-C6+VBL. Panels E and F – whole cell populations and CD34+/CD38-ve respectively – results of this study of 13 AMLs with Lip-C6, VBL and Chloroquine as shown. In nearly all cases at least additive efficacy was seen with VBL or chloroquine added to Lip-C6, but this was particularly dramatic in cells with the LSC phenotype. Panel G shows human AML in the marrow of xenografted NSG mice treated with IV combinatorial nanoliposomes(NL). AML was quantitated by flow cytometry for human specific myeloid antigens. Lip-C6 alone shows minimal activity and ∼50% of AML growth is inhibited by the VBL only NL, whereas the Lip-C6 and VBL combinatorial NLs yield optimal inhibition of hAML growth.
Panels A-D: AML Cells of patient #661 gated on CD34+/CD38-ve (“LSC”) fraction examined for viability with 7AAD and apoptosis with AnnexinV – A Ghost Liposomes, B –Lip-C6, C, VBL, D Lip-C6+VBL. Panels E and F – whole cell populations and CD34+/CD38-ve respectively – results of this study of 13 AMLs with Lip-C6, VBL and Chloroquine as shown. In nearly all cases at least additive efficacy was seen with VBL or chloroquine added to Lip-C6, but this was particularly dramatic in cells with the LSC phenotype. Panel G shows human AML in the marrow of xenografted NSG mice treated with IV combinatorial nanoliposomes(NL). AML was quantitated by flow cytometry for human specific myeloid antigens. Lip-C6 alone shows minimal activity and ∼50% of AML growth is inhibited by the VBL only NL, whereas the Lip-C6 and VBL combinatorial NLs yield optimal inhibition of hAML growth.
We conclude that VBL and chloroquine both yield significant enhancement of Lip-C6 ceramide anti-hAML cytotoxicity. Available evidence suggests that this effect is mediated by inhibition of autophagy in these ceramide stressed cells. These data suggest that the combination of Lip-6 with autophagy inhibitors delivered simultaneously or in combinatorial formulations may prove clinically useful for these disorders.
Kester:Keystone Nano Inc.: Membership on an entity’s Board of Directors or advisory committees, Penn state research foundation has licensed ceramide nanoliposome technology to keystone nano Inc. For commercialization, MK is co-founder and CMO of Keystone Nano., Penn state research foundation has licensed ceramide nanoliposome technology to keystone nano Inc. For commercialization, MK is co-founder and CMO of Keystone Nano. Patents & Royalties.
