Interplay between hypertriglyceridemia and acute promyelocytic leukemia mediated by the cooperation of peroxisome proliferator-activated receptor-α with the PML/RAR α fusion protein on super-enhancers

Abstract

Patients with newly diagnosed acute promyelocytic leukemia (APL) are often obese or overweight, accompanied by metabolic disorders, such as dyslipidemia. However, the link between dyslipidemia and leukemia is obscure. Here, we conducted a retrospective study containing 1,412 cases (319 newly diagnosed APL patients, 393 newly diagnosed non-APL acute myeloid leukemia patients, and 700 non-tumor controls) and found that APL patients had higher triglyceride levels than non- APL and control groups. Using clinical data, we revealed that hypertriglyceridemia served as a risk factor for early death in APL patients, and there was a positive correlation between triglyceride levels and leukocyte counts. RNA sequencing analysis of APL patients having high or normal triglyceride levels highlighted the contribution of peroxisome proliferatoractivated receptor-α (PPARα), a crucial regulator of cell metabolism and a transcription factor involved in cancer development. The genome-wide chromatin occupancy of PPARα revealed that PPARα co-existed with PML/RARα within the super-enhancer regions to promote cell proliferation. PPARα knockdown affected the expression of target genes responsible for APL proliferation, including FLT3, and functionally inhibited the proliferation of APL cells. Moreover, in vivo results in mice having high fat diet-induced high triglyceride levels supported the connection between high triglyceride levels and the leukemic burden, as well as the involvement of PPARα-mediated-FLT3 activation in the proliferation of APL cells. Our findings shed light on the association between APL proliferation and high triglyceride levels and provide a genetic link to PPARα-mediated hyperlipidemia in APL.

Figure 1.

Flow chart showing the inclusions and exclusions of the primary data set. ^*For the body mass index (BMI) analysis, cases with underlying diseases that may affect BMI or lipid, e.g., diabetes, hypertension, coronary heart disease, and hyperlipidemia, were excluded.

Figure 2.

Comparison of blood lipid levels in acute promyelocytic leukemia, non-acute promyelocytic leukemia acute myeloid leukemia patients, and non-tumor controls. (A) The proportions of the acute promyelocytic leukemia (APL), non-APL acute mye-loid leukemia (AML), and control groups with body mass index (BMI) ≥ 25. (B to E) Serum triglyceride (B) total cholesterol (C) low-density lipoprotein (D) and high-density lipoprotein (E) concentrations in the APL, non-APL AML, and non-tumor control groups. TG: triglyceride; TC: total cholesterol; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; ULN: upper limit of normal; LLN: lower limit of normal. Clinical reference ranges:TG, normal 0.34-1.71 mmol/L; TC, normal 3-5.18 mmol/L; LDL-C <3.37 mmol/L; HDL-C>1.04 mmol/L. *P<0.05; **P<0.01; ****P<0.0001; NS: not significant.

Figure 3.

A high triglyceride level was associated with higher white blood cell counts and was a risk factor for early death in acute promyelocytic leukemia patients. (A) Acute promyelocytic leukemia (APL) patients with high triglyceride levels had higher white blood cell (WBC) counts than APL patients with normal triglyceride levels. (B) Linear regression analysis indicated a positive correlation between triglyceride levels and WBC counts at diagnosis. (C) The ROC curves analysis of early death prediction using factors described in Table 2. TG: triglyceride; PLT: platelets; AUC: area under the curve.

Figure 4.

The PPAR-signaling pathway was enriched in hypertriglyceridemia acute promyelocytic leukemia samples. (A) Volcano plot of differential pathways in APL patients with high triglycerides vs. normal triglycerides. The 2 horizontal dashed lines denote a P-value cutoff of 0.05 and a false discovery rate (FDR) cutoff of 0.3. Two-sided P-values were adjusted for multiple hypothesis testing using the Benjamini-Hochberg correction. (B) The top 10 significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in APL patients with high triglycerides levels compared with APL with normal triglycerides levels. (C) Gene set enrichment analysis plot of the PPAR signaling signature genes in patients with high triglycerides levels vs. patients with normal triglycerides. (D) Violin plots showing the expression levels of PPAR in patients with high triglycerides and normal triglyceride levels. TG: triglyceride.

Figure 5.

PPARα was required for the proliferation of acute promyelocytic leukemia cells by co-existing with PML/RARα to control super-enhancer regulation. (A) Heatmap shows the binding sites of PPARα and PML/RARα. (B) PPARα tended to bind at enhancers with higher H3K27ac signals. (C) Venn diagram of the binding sites of PML/RARα, PPARα, and super-enhancer (SE). (D) Bubble diagram of enriched GO terms. (E) Downregulation of target genes co-bound by PML/RARα and PPARα upon PPARα knockdown using small interfering RNA (siRNA). (F) Repression of the protein levels of FLT3 expression and its downstream gene STAT5 upon PPARα knockdown. (G) Inhibition of cell growth in NB4 cells upon PPARα knockdown using siRNA. (H) Upregulation of target genes co-bound by PML/RARα and PPARα upon PPARα activation with GW7647 treatment. Data are shown as the means ± standard deviation of triplicates. *P<0.05; **P<0.01; ***P<0.001 ****P<0.0001; NC: negative control.

Figure 6.

PML/RARα-transgenic mice with high triglyceride levels had increased tumor burden and exhibited increased levels of PPARα and FLT3. (A) Body weights of mice fed with a high-fat diet (HFD) or a normal diet (ND). (B) Serum triglyceride and total cholesterol concentrations were measured in mice fed with HFD or ND for 6 weeks. (C) Relative leukocyte counts (x10⁹/L) in the HFD and ND groups of FVB/APL mice at 6, 13, and 17 days after transplantation. (D) GFP-positive ratios in peripheral blood cells of HFD and ND groups of FVB/APL mice at 6, 13, and 17 days after transplantation. (E) High triglyceride mice were more likely to develop acute promyelocytic leukemia (APL). We used a Kaplan-Meier analysis to estimate the survival of FVB/APL mice. (F) The spleen/body weight ratios showed that the spleens of the HFD group were swollen. (G) quantitative polymerase chain reaction showing the expression of PPARα and FLT3 in mice fed with HFD and ND for 6 weeks.