A Selective Novel Peroxisome Proliferator-Activated Receptor (PPAR)-α Antagonist Induces Apoptosis and Inhibits Proliferation of CLL Cells In Vitro and In Vivo

Abstract

Tumor-specific metabolic changes can reveal new therapeutic targets. Our findings implicate a supporting role for fatty acid metabolism in chronic lymphocytic leukemia (CLL) cell survival. Peroxisome proliferator-activated receptor (PPAR)-α, a major transcriptional regulator of fatty acid oxidation, was recently shown to be upregulated in CLL. To evaluate PPARα as a potential therapeutic target, we developed a highly selective, potent small molecule antagonist of PPARα, NXT629. NXT629 inhibited agonist-induced transcription of PPARα-regulated genes, demonstrating target engagement in CLL cells. Furthermore, NXT629 induced apoptosis of CLL cells even in the presence of a protective microenvironment. To mimic the proliferative lymphoid compartment of CLL, we examined the activity of NXT629 on CLL cells that were stimulated to proliferate in vitro. NXT629 reduced the number of leukemia cells undergoing cell division. In addition, in two xenograft mouse models of CLL (one a model for nondividing and one for dividing CLL), NXT629 reduced the number of viable CLL cells in vivo. Overall, these results suggest that fatty acid metabolism promotes survival and proliferation of primary CLL cells and that inhibiting PPARα gene regulation could be a new therapeutic approach to treating CLL.

Figure 1

Target engagement in CLL cells. (A) Purified CLL cells were incubated with increasing doses of antagonist or vehicle control for 2 h. Subsequently, the synthetic PPARα agonist GW590735 (purchased from GlaxoSmithKline) was added at 1 μmol/L for 48 h. Cells were harvested for RNA isolation. PDK4 (a PPARα target gene) expression was measured by real-time PCR. Data are mean ± standard error of the mean (SEM) from six independent experiments using six different CLL donors. Significant difference: *p < 0.05, unpaired Student t test. (B) Purified CLL cells were preincubated with vehicle control (veh), NXT629 or the control compound NXT962, both at 30 μmol/L for 2 h. Subsequently, GW590735 was added at 1 μmol/L for 48 h. Cells were analyzed as above. One representative result of two independent experiments using two different CLL donors is shown. Significant difference: *p < 0.05, **p < 0.005, unpaired Student t test. n.s., Nonsignificant. (C, D) Purified CLL cells were incubated in serum- and glucose-free RPMI with NXT629 for 2 h. Subsequently, the natural agonist OEA was added at 10 μmol/L for an additional 4 h. Cells were analyzed for PDK4 (C) or CPT1A (D) as above. Data are the mean ± standard deviation (SD) from independent experiments using four different CLL donors. Significant difference: *p < 0.05, unpaired Student t test.

Figure 2

PPARα antagonist is cytotoxic to CLL cells, even in the presence of the microenvironment. (A) CLL cells were cultured in the presence of the PPARα antagonist NXT629 or DMSO control, added once at the beginning of the culture. CLL cells were harvested after 4 d and stained with DiOC₆/PI and analyzed by flow cytometry. The percentage of viable cells as determined by gating on DiOC₆ bright and PI-negative cells is shown. Data are mean ± SD for five different experiments with five different CLL donors. The IC₅₀ was calculated using GraphPad Prism software. (B) CLL cells were cultured alone or in the presence of mitomycin C–treated J774 macrophages. NXT629 (10 μmol/L) or DMSO was added once at the beginning of the coculture. Cell viability was measured at d 6 as above. Data are mean ± SD from two independent experiments using two different CLL donors. Significant difference: ***p < 0.0005, unpaired Student t test. CLL cells were cultured in the presence (C) or absence (D) of mitomycin C–treated J774 macrophages. DMSO or fludarabine was added once at the beginning of the coculture. Cell viability was measured at d 6 as above. Data are mean ± SEM from two independent experiments using two different CLL donors. Significant difference: ***p < 0.0005, unpaired Student t test. n.s., Nonsignificant. (E) CLL cells were culture alone or in the presence of preadipocytes (OP9) cells. NXT629 (10 μmol/L) or DMSO was added once at the beginning of the coculture. Cell viability was assessed at d 6 as above. Data are mean ± SEM from two independent experiments using two different CLL donors. Significant difference: **p < 0.005, unpaired Student t test.

Figure 3

PPARα antagonist inhibits CLL proliferation. (A) To demonstrate CLL proliferation under the described culture conditions, CLL cells were CFSE-labeled and cocultured with activated allogeneic T cells as described in Materials and Methods. Five days after coculture, CLL cells were stained with CD19-APC and proliferation was assessed by flow cytometry. The histograms to the right show CFSE profile gated on CD19⁺ cells. (B) CLL cells were incubated with either PPARα antagonist NXT629 or the negative control compound NXT962 for 2 h. Subsequently, T cells were added and CLL proliferation was assessed after 5 d. The number of viable cells determined by gating on DiOC₆ bright and PI negative cells with the use of Accuri software is depicted. Data are mean ± SD from three independent experiments using CLL cells from the same donor. Significant difference: *p < 0.05, **p < 0.005, unpaired Student t test. (C) CLL cells were incubated with either PPARα antagonist NXT629 or vehicle control for 2 h. Subsequently, T cells were added, and CLL proliferation was assessed after 8 d. The number of viable cells (DiOC₆ bright and PI negative) was quantified by flow cytometry and normalized to the vehicle control, set as 100%. Data are mean ± SEM from 10 different CLL patient samples. (D) CLL cells were incubated as in (C) and, after 8 d, cell cycle analysis was performed using PI staining as described in Materials and Methods. Percentage of cells in G2/M and G0/1 phase was normalized to vehicle control. Data are mean ± SEM from four different CLL donors. Significant difference: *p < 0.05, **p < 0.005, unpaired Student t test.

Figure 4

CLL mouse model. (A) CLL PBMCs from two patients were labeled with CFSE and randomized among the groups. The 10⁸ CFSE-labeled cells were injected intravenously into NSG mice (lacking T, B and NK cells). Groups of five mice received daily dosing of saline: NXT629 at 30 mg/kg IP or fludarabine at 50 mg/kg IP. Mice were killed 4 wks after engraftments, and the splenocytes were stained with hCD19 and analyzed by flow cytometry. Data are hCD19⁺/CFSE⁺ cells normalized to vehicle control. (B) Proliferative model. Groups of mice received daily dosing of saline, NXT629 at 30 mg/kg IP. Mice were killed 2 wks after engraftments of CLL cells. CLL cells in spleen of NSG mice were quantified by gating on human CD45⁺ cells, and a second gate was applied to determine CD19⁺/CD5⁺ CLL cells. Representative flow cytometry plots are depicted to the left. The absolute number of CLL cells is significantly decreased in NXT629-treated animals. Data are mean ± SEM. Significant difference: *p < 0.05, unpaired Student t test. n.s., Nonsignificant.