HO-1 Promotes Resistance to EZH2 Inhibitor through the Prb-E2F Pathway: Corelated with the Conversion of Myelodysplastic Syndrome to Acute Myeloid Leukemia

Myelodysplastic syndrome (MDS) patients would have a chance to become acute myeloid leukemia (AML) when undergoing chemotherapy or waiting for the hematopoietic stem cell transplantation. These patients were not sensitive to demethylation therapy and we should explore deeper mechanisms. According to the WPSS scoring system, we divided 58 MDS patients into four different groups. Real-time PCR results showed the expression of EZH2 or HO-1 in MDS patients were higher than that in normal donors (P<0.05). Even HO-1 and EZH2 were simultaneously increased in some patients, especially in high-risk and extremely very high-risk groups. The linear correlation analysis result of them was 0.42 (P<0.05). In addition, Laser scanning confocal microscopy results also indicated that they were both present in the nucleus of tumor cells. Therefore, we speculated that there was a correlation between EZH2 and HO-1 in MDS patients. Using the High-throughput sequencing to analyze MDS cells, we found that the conversion of MDS to AML may be related to EZH2. The EZH2 in converted MDS patients were significantly higher than that of other MDS patients (P<0.05). Among these patients, we also found that HO-1 and EZH2 were also positively correlated. We found the new EZH2 inhibitor JQEZ5 could significantly promote tumor cells apoptosis in this part of patients. When the concentration of JQEZ5 was 10 umol/mL, the apoptosis rate of tumor cells reached 46.7% after 24 hours (P<0.05). Apoptosis rate was positively correlated with the concentration of JQEZ5 (P<0.05). And tumor cells were significantly inhibited in the G0/G1 cell phase. Cell cycle regulatory genes (CDK4 and CDK6) and apoptosis regulatory genes (Caspase-3 and Caspase-9) would changed. The expression of P15 and P53 would also be affected. In order to verify the malignant degree of MDS cells whether be related to the expression of EZH2. We injected MDS cells into 10 mice. Compared to the control group, MDS cells that highly express EZH2 infiltrated the spleen of experimental mice. Interestingly, the spleens of the experimental group were significantly reduced (0.2CM-0.4CM) and the spleens weight of the experimental group was reduced by 0.028g-0.12g compared to the control group spleens weight. These cells also significantly shortened the survival days of mice and reduced their body weight. Although control mice could survived for 30 days without disease, the time of the experimental mice was significantly shortened (18-25 days). Even One of them survived only 15 days. The results of immunohistochemistry indicated that EZH2 was related to the pRB-E2F pathway. Using the E2F inhibitor HLM006474, we proved HO-1 could regulated EZH2 through the pRB-E2F pathway. Our previous experiments indicated that HO-1 could help leukemia cells resistance and proliferation. The effect of JQEZ5 would be affected when we used hemin and zinc protoporphyrin to regulate HO-1 in MDS cells. The EZH2 was significantly inhibited by JQEZ5 after HO-1 was silenced by siRNAs. Also, the apoptosis rate of MDS cells increased and the cell cycle was arrested in the G0/G1 phase. However, when HO-1 expression was up-regulated by lentivirus, the effects of JQEZ5 were attenuated.

MDS patients are frequently in a state of HO-1 enrichment during chemotherapy. HO-1 stimulates MDS patients to transcribe and activate excess EZH2 through pRB-E2F pathway, which increases the chances of becoming AML. Therefore, the conversion of MDS may be attributed to EZH2. In addition, considering HO-1 could promote the expression of EZH2, HO-1 may be a target for enhancing the effects of EZH2 inhibitors on MDS and the influence of HO-1 should be considered in the treatment of patients with high-risk and extremely very high-risk MDS.