Acute myeloid leukemia (AML) is an aggressive hematologic cancer resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy, relapse caused by intrinsic or acquired drug resistance remains a major hurdle in the treatment of AML. Recently, we found KDM6A as a novel relapse-associated gene in a cohorte of 50 cytogenetically normal AML patients. KDM6A (or UTX) is a histone 3 lysine 27 (H3K27)-specific demethylase and a member of the COMPASS (complex of proteins associated with Set1)-like complex, which is important for chromatin enhancer activation. KDM6A is targeted by inactivating mutations in a variety of cancer types with frequency of occurrence ranging from 0.7 to 4% in AML.
In this study, we used matched diagnosis and relapse samples from AML patients, patient-derived xenografts (PDX), and myeloid leukemia cell lines to investigate the status of KDM6A during disease progression and the implications of KDM6A loss regarding chemotherapy resistance. We found three AML patients with enrichment of KDM6A mutations at relapse and mutation-independent, relapse-specific loss of KDM6A expression in three additional AML patients. KDM6A mutations comprise deletions and point mutations and appear to be mainly loss-of-function mutations. In addition, we examined the mutation profile and KDM6A expression in patient-derived xenograft (PDX) samples from 8 relapsed AML patients. In 4/8 samples, KDM6A protein levels were low or completely lost. Due to the fact that all patients had received induction therapy including single or combination treatment with agents such as cytarabine (AraC), daunorubicin (DNR), and 6-thioguanine (6-TG), we hypothesized that loss of KDM6A confers resistance to chemotherapy. To exclude gender-specific effects (KDM6A escapes X inactivation leading to higher levels in females), we compared male KDM6A knockout (KO) with WT leukemia cell lines and found increased AraC resistance in the KDM6A KO cells (unpaired, two-tailed Student's t-test; P=0.0441). In addition, we treated two relapsed PDX AML cells of the same gender, AML 491 (KDM6A WT and strong expression) and AML 393 (KDM6A mutation and weak expression) with AraC for 72h in vitro and found significantly increased AraC resistance in the KDM6A-mutant PDX AML 393 cells (P=0.016). To further investigate whether reduced expression or loss of KDM6A leads to increased resistance towards multiple drugs, we silenced KDM6A expression by shRNA or CRISPR/Cas9 in K562 and MM-1 cells. Compared to control, KDM6A knockdown (KD) and KO K562 cells showed a strong proliferative advantage after AraC and DNR but not 6-TG treatment. A similar drug resistance phenotype was observed in KDM6A KO MM-1 cells.
To unravel the mechanism of drug resistance, we performed RNA-Seq analysis in K562 cells treated with siRNA or shRNA against KDM6A under native conditions and after AraC (150nM) treatment for 72h. We compared these differentially expressed genes with known key candidate genes in AraC, DNR, and 6-TG metabolic pathway and found that ENT1 was consistently downregulated in KDM6A KD cells in both siRNA- and shRNA-mediated RNA-Seq screenings. Decreased ENT1 levels were also detected in KDM6A KO K562 single cell clones. ENT1 (also known as SLC29A1) is a membrane transporter relevant for the cellular uptake of nucleosides and its analogues. Competitive inhibition of ENT1 by the small molecule antagonist NBMPR lead to decreased sensitivity towards AraC but not DNR and 6-TG suggesting that increased AraC resistance in KDM6A KO cells is caused, at least partially, by downregulation of ENT1.
To elucidate the mechanism of ENT1 regulation by KDM6A, we performed ChIP-seq analysis for H3K27me3 and H3K27ac in the sister cell lines MM-1 (KDM6A WT) and MM-6 (KDM6A KO). ChIP-seq for H3K27me3 showed no enrichment on the ENT1 locus, but we detected differential H3K27ac peaks in the promoter and a putative enhancer region of ENT1 in MM-1 compared to MM-6. These data suggest that increased ENT1 expression may function through direct or indirect effects of KDM6A on enhancer regions, independent of its H3K27 demethylase activity.
In conclusion, our results show that mutations in KDM6A are associated with the outgrowth of drug-resistant clones and highlight KDM6A as a novel biomarker of drug resistance in AML.
Hiddemann:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.
Asterisk with author names denotes non-ASH members.