Peroxisome Proliferator-Activated Receptors: Targets for the Treatment of Philadelphia Chromosome-Positive Leukemia Cells

Introduction: The efficacy of chemotherapy in patients with Philadelphia chromosome (Ph)-positive leukemia has been dramatically improved by the development of small-molecule ABL tyrosine kinase inhibitors (TKIs), such as imatinib. However, resistance to ABL-TKI can develop in patients with chronic myeloid leukemia (CML) because of the presence of leukemia stem cells (LSCs). Thus, a new approach against LSCs may improve the outcome of patients with Ph-positive leukemia. Peroxisome proliferator-activated receptors (PPARs) constitute a superfamily of nuclear hormone receptors and regulate the transcription of target genes. Three subtypes of PPARs (α, γ, and δ/β) have been identified. PPARγ is expressed in both adipose tissue and cancer cells. A PPARγ agonist, pioglitazone, is used in the treatment of diabetes patients. It has recently been reported that PPARγ agonists eradicate leukemia stem cells in patients with CML. We hypothesized that targeting PPARγ, in combination with ABL-TKIs, would result in enhanced therapeutic activity in Ph-positive leukemia cells.

Materials and Methods: We investigated the effect of a PPARγ agonist, pioglitazone, on Ph-positive leukemia cell lines and primary samples.

Results: We examined the expression of PPARs in Ph-positive leukemia cells and found that PPARα and PPARγ were expressed in Ph-positive leukemia cells, including primary samples. However, we could not detect PPARα and PPARγ expressions in normal CD34-positive cells by immunoblot analysis; this suggests that PPARα and PPARγ may be targets of Ph-positive leukemia cells. Next, we examined the efficacy of pioglitazone. Pioglitazone treatment for 72 h inhibited the growth of Ph-positive cells, including T315I-mutant cells. In contrast, pioglitazone did not inhibit normal CD34-positive cells. We then examined intracellular signaling. The phosphorylation of BCR-ABL and Crk-L was not reduced after pioglitazone treatment. In contrast, the phosphorylation of AMP-activated protein kinase (AMPK) was increased, whereas the phosphorylation of S6 ribosomal protein and signal transducer and activator of transcription 5 (STAT5) was reduced. The combined treatment of Ba/F3 T315I cells with ponatinib and 30 µM pioglitazone resulted in greater cytotoxicity. Phosphorylation of S6 ribosomal protein and STAT5 was reduced, whereas AMPK phosphorylation was increased. Cleaved poly (ADP-ribose) polymerase (PARP) was increased after treatment with ponatinib and pioglitazone. Thereafter, we investigated anti-angiogenic effects on human umbilical vein endothelial cells (HUVECs) because expression of PPARα and PPARγ was reported in HUVECs in previous studies. Pioglitazone inhibited tube formation of HUVECs through vascular endothelial growth factor (VEGF) in the extracellular matrix gel, suggesting that pioglitazone inhibits angiogenesis in vitro . Further, we found that the treatment with ponatinib or imatinib and/or pioglitazone inhibited the growth of primary samples, including CD34-positive CML cells; however, these drugs did not affect the normal cells. The phosphorylation of STAT5 was reduced and AMPK phosphorylation was increased. We next conducted a case study. A 61-year-old patient was treated with nilotinib (800 mg/day), and BCR-ABL levels in the peripheral blood cell samples of the patient were monitored using real-time polymerase chain reaction (PCR); however, the international scale (IS) of BCR-ABL remained elevated. Oral pioglitazone (15 mg, once daily) was administered because the patient had high blood glucose levels for more than 1 year. Initiation of pioglitazone therapy resulted in reduction of IS% of BCR-ABL.

Conclusion: Our study indicated that administration of the PPARγ agonist, pioglitazone, may enhance the effects of ABL-TKI and is effective in suppressing angiogenesis mediated by VEGF. Thus, PPARγ agonists may possess promising clinical relevance as a candidate therapeutic agent for Ph-positive leukemia in the future.