HO-1 promotes resistance to an EZH2 inhibitor through the pRB-E2F pathway: correlation with the progression of myelodysplastic syndrome into acute myeloid leukemia
Myelodysplastic syndrome (MDS) can progress to acute myeloid leukemia (AML), and conventional chemotherapy (decitabine) does not effectively inhibit tumor cells. Enhancer of zeste homologue 2 (EZH2) and Heme oxygenase-1 (HO-1) are two key factors in patients resistance and deterioration.
In total, 58 MDS patients were divided into four groups. We analyzed the difference in HO-1 and EZH2 expression among the groups by real-time PCR. After treatment with Hemin or Znpp IX, flow cytometry was used to detect apoptosis and assess the cell cycle distribution of tumor cells. Following injection of mice with very high-risk MDS cells, spleen and bone marrow samples were studied by immunohistochemistry (IHC) and hematoxylin and eosin (H&E) staining. MDS cells overexpressing EZH2 and HO-1 were analyzed by high-throughput sequencing. The effect of HO-1 on the pRB-E2F pathway was analyzed by Western blotting. The effects of decitabine on P15INK4B and TP53 in MDS cells after inhibiting HO-1 were detected by Western blotting.
Real-time PCR results showed that EZH2 and HO-1 expression levels were higher in MDS patients than in normal donors. The levels of HO-1 and EZH2 were simultaneously increased in the high-risk and very high-risk groups. Linear correlation analysis and laser scanning confocal microscopy results indicated that EZH2 was related to HO-1. MDS cells that highly expressed EZH2 and HO-1 infiltrated the tissues of experimental mice. IHC results indicated that these phenomena were related to the pRB-E2F pathway. High-throughput sequencing indicated that the progression of MDS to AML was related to EZH2. Using the E2F inhibitor HLM006474 and the EZH2 inhibitor JQEZ5, we showed that HO-1 could regulate EZH2 expression. HO-1 could stimulate the transcription and activation of EZH2 through the pRB-E2F pathway in MDS patients during chemotherapy, which reduced TP53 and P15INK4B expression.
EZH2 was associated with HO-1 in high-risk and very high-risk MDS patients. HO-1 could influence MDS resistance and progression to AML.
KeywordsMyelodysplastic syndrome Acute myeloid leukemia pRB-E2F JQEZ5 HO-1 EZH2
acute myeloid leukemia
enhancer of zeste homologue 2
WHO prognostic scoring system
hematopoietic stem cell transplantation
refractory with multilineage dysplasia
RA with excess of blasts
RA with excess of blasts in transformation
- H&E staining
hematoxylin and eosin staining
Kyoto Encyclopedia of Genes and Genomes
small interfering RNA
empty plasmid vectors
lentivirus encoding HO-1
small interfering RNA specific for HO-1
World Health Organization
Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder that can progress to acute myeloid leukemia (AML) . The pathogenesis of MDS is controversial, with epigenetic regulation being one of the main pathogenic mechanisms . We grouped patients according to the World Health Organization (WHO) prognostic scoring system (WPSS). Currently, some high-risk and very high-risk MDS patients are resistant to decitabine and therefore have a poor prognosis . A study indicates that the median survival time of these patients is less than 0.8 years, which was significantly less than the median survival time of low-risk patients (5.3 years). 25% of high-risk patients convert to AML within 1.4 years . We noticed that enhancer of zeste homologue 2 (EZH2) could affect the self-renewal and differentiation of hematopoietic stem cells  and may be an independent prognostic factor  related to a poor prognosis . EZH2 expression is also abnormally upregulated in AML. A study indicated that combining inhibition of EZH2 and LSD1 resulted in synergistic activity against AML in vitro and in vivo. This synergy was mechanistically correlated with upregulation of H3K4me1/2 and H3K9Ac and downregulation of H3K27me3, leading to a decrease in the level of the antiapoptotic protein Bcl-2 . Due to the role of EZH2 in the treatment of AML, we speculated that this gene may contribute to MDS progression to AML and the rise of drug-resistant AML and MDS phenotypes. Other studies have also shown that EZH2 can increase cancer cell aggressiveness [9, 10, 11]. It is not known whether MDS patients can easily progress to AML following EZH2 overexpression. It is also unclear whether EZH2 inhibition has inhibitory effects on high-risk or very high-risk MDS progression.
Heme oxygenase-1 (HO-1) is an antioxidant enzyme involved in many cellular processes, including oxidative stress and apoptosis regulation. Several studies have indicated that HO-1 can affect leukemic cells [12, 13], but the mechanisms involved require further investigation . HO-1 has antiapoptotic effects on MDS cells [15, 16]. It is worth noting that EZH2 and HO-1 share similar functions . For example, overexpression of HO-1 or EZH2 can significantly inhibit patients’ response to decitabine which is related to patients’ prognosis. To date, the effects of the HO-1 gene on inhibitors of EZH2 have never been reported. In recent years, the development of MDS therapies has been slow, and we hope to provide a new perspective. In this study, we mainly explored MDS patient models that were different from the models in previous studies.
Materials and methods
Our research was approved by the Committee on Animal Protection and use of the Animal Management Board of the United States. Conducting relevant animal experiments according to approved guidelines, we follow all applicable international, national and institutional guidelines for animal care and use. The experiment was strictly followed the Helsinki declaration and passed the ethical examination of animal experiments in Guizhou Medical University. (Ethical approval number: 2017-13)
Cells and culture conditions
At present, many famous biotech companies have not cultivated MDS cell lines recognized by blood experts. MDS cells are also unstable. So we choose to extract patient cells. All human MDS cells and AML cells were preserved in our laboratory. We collected MDS cells and MDS cells that progress to AML. All cells are clearly grouped and divided into treated and untreated. All cells were cultured with 15% fetal bovine serum (Gibco BRL; Life Technologies, Carlsbad, CA, USA), penicillin of 100 μ/ml and RPMI-1640 culture of streptomycin in 100 μg/ml. All cells were kept at 37 °C incubator with humidity of 95% and CO2 content of 5%. RPMI-1640 medium was purchased from Invitrogen (Carlsbad, CA, USA).
WPSS risk group
Self-prepared sequences containing human HO-1 and small interfering RNA targeting human HO-1 were selected with Invitrogen designer software. Retroviruses were generated by transfecting empty plasmid vectors containing the enhanced green fluorescence protein (EGFP) or vectors containing human HO-1-EGFP/siRNA-HO-1-EGFP into 293FT packaging cells, using the FuGENE HD6. Lentiviral stocks were concentrated using Lenti-X concentrator, and titers were determined with Lenti-X qRT-PCR titration kit (Shanghai Innovation Biotechnology Co., Ltd., China). Finally, 4 recombinant lentiviral vectors were constructed: lentivirus-V5-D-TOPO-HO-1-EGFP (L-HO-1), lentivirus-V5-D-TOPO-EGFP (TOPO-EGFP), lentivirus-pRNAi-u6.2-EGFP-siHO-1 (siHO-1), and lentivirus-pRnai-u6.2-egfP (RNAi-EGFP). For transduction, cells were plated onto 12-well plates at the density of 2.5 × 105/well, infected with the lentiviral stocks at a multiplicity of infection of 10 in the presence of polybrene (10 µg/ml), and then analyzed by fluorescence microscopy (Olympus, Tokyo, Japan) and Western blotting 48 h after transduction. MDS cells were transduced with L-HO-1, siHO-1, RNAi-EGFP and TOPO-EGFP, respectively. Titer: L-HO-1: 4.0 × 109TU/ml, si-HO-1: 8.0 × 108TU/ml, EV1-siHO-1: 6.0 × 108TU/ml, EV2-L-HO-1: 4.0 × 108TU/ml.
RNA isolation and quantitative PCR
Total RNAs from cells were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Quantitative PCR was performed by using SYBR Green PCR Master Mix (TianGen, Biotech, Beijing, China) and the PRISM 7500 real-time PCR detection system (ABI, USA). cDNA samples, primers and SYBR Master Mix were mixed with a total volume of 20ul. The thermal cycling conditions used in the protocol were 1 min at 94 °C, followed by 40 cycles at 94 °C for 10 s and at 60 °C for 15 s.
HO-1 enhancer Hemin was purchased from Sigma (St Louis, MO, USA). HO-1 inhibitor Znpp IX was purchased from Cayman Chemical (Ann Arbor, MI, USA). EZH2 inhibitor JQEZ5 and E2F inhibitor HLM006474 were purchased from MCE (Shanghai, China). The drugs were dissolved in a small amount of DMSO and stored in − 20 °C. Before using, we used serum-free RPMI-1640 to dilute it. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection Kit was purchased from BD (San Diego, CA, USA). qPCR primers such as HO-1 and EZH2 were synthesized by Sangon Biotech (Shanghai, China). Western-blot was probed with primary antibodies, including antibodies against HO-1, EZH2, pRB. Secondary antibodies were purchased from Li-Cor Corp (Lincoln, Nebraska, USA). The TRIzol of total RNA extracted from cells and mononuclear cells was purchased from Invitrogen (Carlsbad, CA, USA).
After PI staining, cell apoptosis was detected by flow cytometer (BD Biosciences, San Jose, CA, USA). The cells were washed with phosphate buffer saline (PBS), resuspended in 100 μl of binding buffer containing 5 μl of annexin V and finally stained with 5 μl of PI at room temperature in dark for 15 min.
Cell cycle analysis
Cell cycles were detected by flow cytometry. The cells were washed with PBS, fixed in 70% cool ethanol for 2 h, washed with PBS again, and resuspended in PBS containing PI (Sigma), DNase free RNase (Citomed) and Triton X-100 for 1 h. Finally, the cells were collected by FACS Calibur flow cytometer (Becton-Dickinson), and cell cycle-related data were analyzed by FlowJo software (Tree Star, Inc., Ashland, OR, USA).
Western blot analysis
Western blot was employed to detect the protein expressions of related genes in patients with different MDS risks, as well as in the cells transfected by siRNA or lentivirus in combination with JQEZ5 and decitabine treatment. The expression of proteins in blood samples or treated cells were analyzed by Western-blot. PBS were lysed by sonication in RIPA buffer (the cells were lysed sonication in RIPA buffer). The cells were fully mixed and transferred to the new EP tube, and then were centrifuged at 12,000*g for 10 min at 4 °C. After centrifugation, the supernatant was mixed with loading buffer and stored at − 80 °C. After loading the same amount of protein (50–100 μg) with 10% SDS-PAGE, electrophoresis was separated and then was transferred to the PVDF membrane (Millipore Corporation, Milford, MA, USA). The protein PVDF was transferred to the TRIS buffer which contained 5% skim milk powder overnight. The membrane was blotted with relevant primary antibodies (1:1500) for 2 h. After being washed with PBS and 0.1% Tween-20, the blot was incubated with secondary antibody (1:2000). The expression level of related proteins was determined by enhanced chemiluminescence (7sea Biotech, Shanghai, China). Each experiments was conducted more than 3 times.
Animals and treatments
Male C57BL/6Ly5.2 mice weighing 20–21 g were purchased from the Institute of Laboratory Animal Sciences (PUMC, Beijing, China). Mice were cultured in SPF class (SPF, Specific Pathogen Free) animal laboratory. After being adapted to the environment, the 10 mice were divided into two groups randomly. One group of five mice were served as control group and were only injected culture medium. The remaining groups of mice were experimental group. (each mice was injected 3 × 107 U266 cells). All mice were injected via tail vein every 2 days for 4 weeks. The loss of weight and survival time of mice were recorded and analyzed. immunohistochemistry (IHC) and hematoxylin and eosin (HE) staining were used to detect MM cell infiltration in liver, spleen, kidney. All experiments were conducted at least three times.
Each experiment was repeated at least 3 times and the most representative example was given. Statistical analysis of experimental data was performed by using GraphPad Prism 5 software (GraphPad Software Inc, San Diego, CA, USA). All data were represented as mean ± standard error. Statistical analyses were performed by using analysis of variance and the t test. Results were considered statistically significant if P < 0.05 and data were represented as mean ± standard deviation (SD) of three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001).
EZH2 and HO-1 are relevant in some high-risk and very high-risk MDS patients
WPSS risk group
No. of monocytes
0.8 × 109/L
0.7 × 109/L
JQEZ5 significantly promotes MDS cell apoptosis
MDS cells overexpressing EZH2 and HO-1 inhibit the expression of P15INK4B and TP53 in mice
Characteristics of MDS cells overexpressing EZH2 and HO-1
HO-1 regulates EZH2 via the pRB-E2F pathway
Studies have shown that the pRB-E2F pathway is involved in the regulation of tumor cell function . EZH2 is a gene downstream of the pRB-E2F pathway in MDS and is involved in the activation of oncogenes . Inhibiting this pathway is therefore very important in cancer treatment [35, 36]. If high-risk or very high-risk MDS cells have an activated pRB-E2F pathway and EZH2 overexpression, the patients from whom the cells were isolated progress to AML. In our in vivo experiments, this phenotype also led to MDS cells infiltrating the mouse bone marrow in a short period of time. We suggest that these tumor cells destroyed the normal hematopoietic environment in the mice and consumed various growth factors. The pRB-E2F pathway can affect cell proliferation and apoptosis by regulating P53 and P15. A study found that P53 could also counteract pRB-E2F . The balance between P53 and pRB-E2F is key to the normal growth of cells. HO-1 breaks the balance between them and makes it difficult for chemotherapeutic drugs to eliminate tumor cells.
Our experiments did not detail the characteristics of low-risk MDS patients because the expression of HO-1 and EZH2 in low-risk MDS is not significantly different from that in normal donors. Decitabine is not a preferred treatment for low-risk MDS patients, and the prognosis of low-risk MDS patients is generally better than that of high-risk MDS patients . In total, 25% of patients with low-risk MDS progress to AML within 10.8 years . Currently, the Food and Drug Administration (FDA) suggests that high-risk or very high-risk MDS patients be treated with decitabine. However, the results are unsatisfactory [39, 40]. For some high-risk or very high-risk MDS patients, EZH2 can be suppressed by demethylation drugs. Nevertheless, we found that if high HO-1 expression remained unchanged in patients, HO-1 functioned as an EZH2 catalyst. Thus, EZH2 expression in patients could be increased again when they were treated with demethylation drugs. In addition, HO-1 expression would abnormally increased after chemotherapy, which formed a cycle. Although JQEZ5 can significantly inhibit EZH2 expression in vitro, HO-1 expression in tumor cells still guarantees their survival. In summary, HO-1 regulated EZH2 expression via the pRB-E2F pathway, which was responsible for MDS progression to AML. High-throughput sequencing helped discover many pathways that may play a role in disease progression. However, we chose factors that were overlooked and then conducted in-depth research. If we do not pay attention to the mechanisms of drug resistance and overcome them, new drugs will not improve the prognosis of high-risk patients. Our experiments suggest that HO-1 is a potential target for the treatment of MDS. However, there is not enough clinical evidence to show that targeting HO-1 is absolutely safe in vivo. The collateral damage associated with targeting HO-1 is not clear. Many chemotherapeutic drugs inevitably have adverse effects on patients . Our research aimed to provide a new perspective for MDS therapy. Increasingly people are paying attention to the impact of HO-1 on the treatment of leukemia, and thus, this molecule may be a target for preventing MDS progression to AML and the development of resistance.
In conclusion, we demonstrated that EZH2 expression was associated with HO-1 expression in high-risk and very high-risk MDS patients. HO-1 could influence MDS drug resistance and progression into AML. The regulation of EZH2 might be mediated by the activation of the pRB-E2F pathway. Our study provides an important clue to the role of HO-1 to facilitate the development of EZH2-directed diagnostics and therapeutics for MDS.
ZCH and LLR designed the experiments. ZCH, SYZ and DM performed the experiments. LPY and SXS analyzed the experimental data. YC, JSW, LLR, and QF provided technical and material support. ZCH and SXS wrote and reviewed the manuscript. All authors read and approved the final manuscript.
This research was supported in part by the National Natural Science Foundation of China (#81360501, #81470006, and #81660616), the International Cooperation Project of Guizhou Province (#2011-7010), the Social Project of Guizhou Province (#2011-3012), and the Provincial Governor Special Fund of Guizhou Province (#2010-84).
Ethics approval and consent to participate
Our research was approved by the Committee on Animal Protection and Use of the Animal Management Board of the United States. Relevant animal experiments were conducted according to approved guidelines, and we followed all applicable international, national and institutional guidelines for animal care and use. The experiment was strictly followed the Helsinki declaration and passed the ethical examination of animal experiments at Guizhou Medical University, (Ethical approval number: 2017-13). According to the Helsinki Declaration, the informed consent was first obtained in writing. All patients volunteered to participate in this experiment. All patient information has been kept confidential.
Consent for publication
This study consists of animal and human data.
The authors declare that they have no competing interests.
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