Loss Of H3K27 Trimethylation (H3K27me3) Associates With a Multi Drug Resistance Phenotype In Acute Myeloid Leukemia (AML)

Histone modifications play a crucial role in the regulation of gene expression by activating or inactivating transcription. The Polycomb Group Protein Enhancer of Zeste Homologue 2 (EZH2) mediates trimethylation of histone H3K27, thereby inducing gene silencing. Overexpression of EZH2 has been reported to be associated with metastases and cancer progression in solid tumors like breast cancer or prostate cancer. However, loss of function mutations or deletions of EZH2 occur in myeloid malignancies and T-ALL. These mutations result in a poor prognosis. The aim of this study was to analyze the relevance of histone modifications for therapy resistance in AML.

FLT3-ITD positive MV4-11 leukemic cells that were continuously cultured in media containing the kinase inhibitor PKC412 became resistant not only to PKC412 but also to standard chemotherapeutics Cytarabin (AraC) and Daunorubicin. Western blot analysis identified an almost complete loss of H3K27me3 in resistant MV4-11 cells (MV4-11R). This was accompanied by loss of EZH2 protein in the MV4-11R compared to the sensitive MV4-11. To test for acquisition of drug resistance due to reduced H3K27me3 levels, lentiviral knock-down (KD) of EZH2 was performed in the sensitive MV4-11 leading to diminished H3K27me3 levels. Knock-down cells showed resistance to the apoptosis-inducing effects of PKC412 compared to scrambled controls. Furthermore, resistance to standard chemotherapeutics AraC and Daunorubicin could be also observed in MV4-11 KD cells compared to control. To verify whether diminished levels of H3K27me3 can cause a more general, FLT3-ITD-independent drug resistance, knock-down of EZH2 was performed in FLT3-WT AML cell lines HL60, Kasumi-1 and ML-1. Again, this led to resistance to the standard chemotherapeutics AraC and Daunorubicin. In order to investigate the regulation of EZH2 in MV4-11R, promoter methylation and microRNA expression analysis was performed revealing no regulation of EZH2 expression via both mechanisms. Instead, the reduction of EZH2 protein expression was depended on posttranslational mechanisms that could be counteracted by CDK1-inhibitors. CDK1-inhibitors restored EZH2 protein and H3K27 trimethylation levels as well as drug sensitivity. By analyzing EZH2 mRNA expression of 220 primary diagnosed AML patients, a trend towards low EZH2 mRNA expression and poor overall as well as relapse free survival could be demonstrated. Thus, EZH2- and H3K27me3 protein expression were also analyzed by immunohistochemistry in bone marrow biopsies from AML patients (N=126). H3K27me3 and EZH2 protein expression correlated closely (r= 0.9, p<0.001). Low H3K27me3 levels indicated a poor prognosis with significantly decreased overall (median 11.06 vs. 38.6 months, p=0.017), event-free (median 4 vs. 19.9 months, p=0.014) and relapse-free survival (median 10 versus 31.2 months, p=0.038) compared to patients with high H3K27me3 protein expression. Similar findings were obtained for the loss of EZH2 protein. EZH2 low/absent expression was associated with a significantly decreased overall (median 9.6 vs. 47.6 months, p=0.018), event-free (median 4.06 vs. 46 months, p=0.013) and relapse-free survival (median 11.3 versus 55.3 months, p=0.47) compared to patients with high EZH2 protein expression.

Taken together, these data indicate that loss of EZH2 expression and reduction of H3K27me3 levels induce widespread therapy resistance in AML and associate with a poor prognosis in AML patients.