Bcl-2 inhibition combined with PPARα activation synergistically targets leukemic stem cell-like cells in acute myeloid leukemia

Abstract

Persistence of leukemic stem cells (LSCs) is one of the determining factors to acute myeloid leukemia (AML) treatment failure and responsible for the poor prognosis of the disease. Hence, novel therapeutic strategies that target LSCs are crucial for treatment success. We investigated if targeting Bcl-2 and peroxisome proliferator activated receptor α (PPARα), two distinct cell survival regulating mechanisms could eliminate LSCs. This study demonstrate that the Bcl-2 inhibitor venetoclax combined with the PPARα agonist chiglitazar resulted in synergistic killing of LSC-like cell lines and CD34⁺ primary AML cells while sparing their normal counterparts. Furthermore, the combination regimen significantly suppressed AML progression in patient-derived xenograft (PDX) mouse models. Mechanistically, chiglitazar-mediated PPARα activation inhibited the transcriptional activity of the PIK3AP1 gene promoter and down-regulated the PI3K/Akt signaling pathway and anti-apoptotic Bcl-2 proteins, leading to cell proliferation inhibition and apoptosis induction, which was synergized with venetoclax. These findings suggest that combinatorial Bcl-2 inhibition and PPARα activation selectively eliminates AML cells in vivo and vitro, representing an effective therapy for patients with relapsed and refractory AML.

Fig. 1. Venetoclax combined with chiglitazar synergistically inhibited the proliferation of LSC-like cells in vitro.

A Representative FACS analysis for determining the percentage of CD34⁺CD38⁻ cells enriched from KG-1α and Kasumi-1 cells. B, C Living cells of KG-1α and Kasumi-1 were counted with trypan blue staining B and a CCK-8 assay was used to detect the anti-proliferative ability C. D Combination index (CI) plots showing venetoclax/chiglitazar combinatorial treatment in KG-1α and Kasumi-1 cells. Values represent mean ± SD for three replicates. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 2. Venetoclax combined with chiglitazar synergistically induced the apoptosis and inhibited clone formation of LSC-like cells in vitro.

A, B Representative flow cytometric data for Annexin V/PI staining of KG-1α A and percentages of apoptotic cells B were determined. C, D Representative flow cytometric data C and percentages of apoptotic D of Kasumi-1. E, F Clonogenic capacity identification of KG-1α and Kasumi-1 cells was performed by determining the percentage of CFUs. Values represent mean ± SD for three replicates. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 3. Molecular mechanisms of venetoclax combined with chiglitazar to synergistically target LSC-like cells.

A Heat map of differentially expressed genes in KG-1α cells in response to chiglitazar for 24 h. B The mRNA levels of PPARα in KG-1α and Kasumi-1 cells treated with venetoclax and/or chiglitazar for 24 h. C Western blot analysis of PI3K/Akt signaling pathway and its downstream apoptotic proteins in KG-1α and Kasumi-1 cells treated with venetoclax and/or chiglitazar for 24 h. D The mRNA levels of PIK3AP1 in KG-1α and Kasumi-1 cells treated with venetoclax and/or chiglitazar for 24 h. E Western blot analysis of the Raf-MEK-ERK pathway and c-MYC protein in KG-1α and Kasumi-1 cells treated with venetoclax and/or chiglitazar for 24 h. F Venetoclax combined with chiglitazar synergistically inhibited the mRNA expression of proliferation-related target genes (c-Myc, c-fos, c-jun, and ETS1) downstream of p-ERK1/2. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 4. Chiglitazar-mediated PPARα activation inhibited PIK3AP1 promoter activity in LSC-like cells.

A The human PIK3AP1 promoter was cloned into pGL3 and co-transfected with PPARα into HEK293T cells for dual luciferase assays. B KG-1α and Kasumi-1 cells were transfected with the indicated reporters with or without increasing amounts of PPARα or chiglitazar for dual luciferase assays and western blotting. C The binding sites for PPARα on the PIK3AP1 promoter region was predicted using the JASPAR database. The human PIK3AP1 promoter with deletion mutations at PPRE1 site (M1), PPRE2 site (M2), and PPRE1 and PPRE2 sites (DM) were constructed. D KG-1α and Kasumi-1 cells were transfected with these reporters with or without increasing amounts of PPARα or 10 μM chiglitazar for dual luciferase assays and western blotting. E ChIP assay analyzed the recruitment of PPARα to PPRE sites of the PIK3AP1 gene promoter in KG-1α and Kasumi-1 cells, the chiglitazar at the concentration of 15 μM. F Overexpression of PIK3AP1 in chiglitazar-treated KG-1α and Kasumi-1 cells and the experssion of PI3K/Akt phosphorylation and its downstream apoptotic proteins were analyzed by western blotting. G KG-1α and Kasumi-1 cells were treated with 20 μM Z-VAD-fmk for 3 h, followed by treatment with venetoclax (160 nM) or chiglitazar (16 μM) or the combination for 24 h, and then the percentages of apoptotic cells were determined by flow cytometry. H Western blot analysis of the BCL2 like proteins and Bim in chiglitazar-treated KG-1α and Kasumi-1 cells with/without Z-VAD-fmk. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 5. Venetoclax combined with chiglitazar preferentially targets CD34⁺ AML cells in vivo.

A Primary AML cells were exposed to venetoclax (50 nM) or chiglitazar (10 μM) alone or in combination for 24 h, then apoptosis and survive were measured using flow cytometric analysis and CCK-8 assays (n = 22). B Healthy donors HSC cells (n = 9). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 6. Venetoclax combined with chiglitazar suppresses tumor progression in PDX models of AML in vivo.

A Schematic outline of the animal study. B, C Images and weights of spleens from the sacrificed mice (n = 4). D Detection of hCD45⁺ and mCD45^- cells in spleen was performed using flow cytometry (n = 4). E Body weight was monitored daily during treatment (n = 4). F The Kaplan-Meier survival curve was used to analyze animal survival (n = 5). G Representative data for H&E staining of the spleen and immunohistochemical staining for hCD34, hCD45, p-PI3K, p-Akt1 and p-ERK1/2 in the BM (n = 4). HE 200×, scale bar: 50 μm; IHC 400×, scale bar: 25 μm. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 7. Mechanism of action of the combined treatment.

A schematic model for the mechanism by which venetoclax synergistically interacts with chiglitazar to inhibit AML progression.