Acid Ceramidase Inhibition Impairs Tumor Progression in a Rat Model of LGL Leukemia

Abstract

Natural killer cell large granular lymphocytic (NK-LGL) leukemia is a disorder characterized by the expansion of clonal CD3-, CD56+ cells in the peripheral blood and marrow. NK-LGL leukemia could manifest in both aggressive and chronic forms. Currently, no curative treatment is available for patients with NK-LGL leukemia. The pathogenetic mechanisms of NK-LGL leukemia are also poorly understood, which highlights the need to increase our understanding of the disorder in order to develop novel therapeutic targets. Sphingolipid dysregulation has been shown to promote tumor growth and survival in several types of cancers, including LGL leukemia. In cancer cells, the accumulation of pro-survival sphingosine-1-phosphate (S1P) throws the S1P/ceramide rheostat off balance, which leads to tumor growth and activated signaling pathways. Our laboratory previously discovered that the reversal of sphingolipid dysregulation in NK-LGL leukemia, either through targeting sphingolipid enzymes or by the addition of antiproliferative ceramide, leads to programmed cell death in leukemic cells.Acid ceramidase, a cysteine hydrolase, catalyzes the breakdown of ceramide into sphingosine and fatty acid. We found that cells from chronic NK-LGL patients have lower pro-death ceramide levels and higher S1P levels when compared to normal NK donors. Furthermore, the mRNA expression and activity of acid ceramidase (AC) were elevated in leukemic NK cells compared to normal NK cells. We further demonstrate the importance of AC in NK-LGL leukemic cells through AC knockdown in human (NKL) and rat (RNK-16) NK-LGL leukemic cell lines. AC knockdown decreased cell viability and increased ceramide levels, significantly ceramide species C16 and C24 (p<0.05), showing that AC is essential for the growth of leukemic NK cells. We further confirm that the accumulation of both C16 and C24 ceramide mediated the decreased viability of NKL and RNK-16 cells by treating the cells with C16 and C24 ceramide (6.25µM, 12.5µM and 25µM) for 24 hours.As our data showed that AC mediates the survival of NK-LGL, we did further analysis on the effects of AC knockdown on survival signals. Protein expression of AC knockdown- NKL and RNK-16 cell lines showed that AC knockdown decreased survival signaling mediators, including Bcl-2, Mcl-1 and survivin. Conversely, pro-apoptotic puma increased with AC knockdown in both cell lines. We further extended our in vitro findings in an established in vivo NK-LGL Fischer rat model. RNK-16 cells stably expressing AC shRNA (RNK-16/shRNA) or stably expressing scrambled shRNA (RNK-16/scr) were transplanted into 6-week old male rats (7 per group). The rats engrafted with RNK-16/shRNA survived significantly longer than those engrafted with RNK-16/scr (Mantel-Cox test, p<0.05). Interestingly, RNK-16/shRNA rats did not exhibit organomegaly compared to their leukemic counterparts. Splenocytes isolated from RNK-16/shRNA engrafted animals showed 50 percent reduction in AC expression compared to splenocytes from RNK-16/scr. Lipid analysis on splenocytes isolated from both RNK-16/scr and RNK-16/shRNA showed that RNK-16/shRNA cells had significant increases in total ceramide, C16 and C24 ceramide species, and a significant decrease in S1P. Collectively, our data show that targeting AC slows the progression of NK-LGL leukemia by reversing sphingolipid dysregulation and warrants further investigation as a therapeutic target in this incurable disease. DisclosuresNo relevant conflicts of interest to declare.