Abstract 761: Acid ceramidase promotes drug resistance in acute myeloid leukemia through P-gp upregulation mediated by NF- kB activation

Abstract

Abstract Background. Acute myeloid leukemia (AML) is the most common form of adult leukemia that affects the differentiation and survival of myeloid precursors. AML patients have elevated immature leukemic cells known as blasts. Failure to achieve remission commonly revolves around the drug resistance advantage of AML blasts. P-glycoprotein (P-gp), an ATP-binding cassette transporter (MDR1/ABCB1), is highly expressed in AML blasts. However, P-gp inhibitors have failed to increase overall survival in AML patients. Sphingolipids are a class of lipids that have a wide variety of functions in cellular processes. Acid ceramidase (AC), an enzyme in the sphingolipid network, catalyzes ceramide breakdown to generate sphingosine. Sphingosine can then undergo phosphorylation by sphingosine kinases to form sphingosine -1-phosphate (S1P). Aims. Our aims were to determine whether AC is elevated in AML, is essential for AML drug resistance and to identify the mechanism through which AC maintains drug resistance in AML. Methods. We used microarray and fluorogenic AC substrate to quantify AC expression and activity levels. Quantitative real-time PCR, Western blotting and flow cytometry were used to determine P-gp expression and activity. AC inhibitor LCL204 and AC shRNA were used in human AML cell lines HL-60, HL-60/VCR and HL-60/ABT to uncover the role of AC in drug resistance. We created an AC-overexpressing HL-60 cell line to study AC-mediated drug resistance. NF-kB inhibitor JSH-23 was used to test the pathway's involvement in AC-mediated drug resistance in AML. Results. We demonstrate that AC expression and activity are elevated in AML patient samples compared to normal donors. P-gp expression is also elevated in AML patient samples compared to normal PBMC and cord blood. Using human HL-60 cells and drug-resistant derivatives HL-60/VCR and HL-60/ABT as our model, we found that P-gp expression is high in both HL-60/VCR and HL-60/ABT. AC activity in both drug resistant cell lines is also higher than the parental HL-60. Targeting AC with pharmacological inhibitor LCL204 in HL-60/VCR cells decreases P-gp levels and increases sensitivity to both chemotherapeutic drugs mitoxantrone and daunorubicin. Lentiviral AC overexpression in HL-60 upregulates P-gp expression and confers resistance to mitoxantrone and daunorubicin. Conversely, AC knockdown in HL-60/VCR and HL-60/ABT cells decreased P-gp expression. AC overexpression in HL-60 increases P-gp mRNA levels, which suggests involvement of a transcriptional regulatory mechanism. NF-κB inhibition by JSH-23 decreases p65, P-gp and AC expression and warrants further investigation into the mechanism for AC-mediated regulation of P-gp expression. Summary. Our data supports an important role for AC in regulating P-gp expression in AML and presents a novel target to overcome drug resistance in AML. Citation Format: Su-Fern Tan, Xin Liu, Kenichiro Doi, Hong-Gang Wang, Myles Cabot, David Feith, Thomas P. Loughran. Acid ceramidase promotes drug resistance in acute myeloid leukemia through P-gp upregulation mediated by NF- kB activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 761. doi:10.1158/1538-7445.AM2014-761