JMJD1c, a Histone Demethylase, Is Required for the Maintenance of MLL-AF9 AML Leukemic Stem Cells

While most leukemic cells are initially sensitive to chemo- and radiotherapy, leukemic stem cells (LSC) persist and are therefore considered to be the basis for disease relapse in acute myeloid leukemia (AML) (Nat Biotechnol, 25: 1315-1321, 2007). The discovery of epigenetic mutations being the cause of a number of cancers has increased interest in their therapeutic potential (Nat Rev Genet, 7:21-33, 2006; Cell, 128: 683-692, 2007). Although the clinical importance of epigenetic dysregulation has been recognised in various cancers, including MLL-rearranged AML, this topic is currently a novel area of study.

Our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified JMJD1c, a histone demethylase, as a potential LSC-specific target. An analysis of a comprehensive patient outcome database showed that high levels of JMJD1c were associated with significantly poorer survival in patients with AML (P = 0.0029), making it clinically relevant and emphasising the therapeutic potential of JMJD1c. Furthermore, the JMJD1c mRNA level was significantly higher in MLL-rearranged than in non-MLL AML patient xenograft samples.

Western blot and quantitative PCR confirmed the microarray results, where JMJD1c was significantly upregulated in MLL-AF9 LSC compared to Hoxa9/Meis1a derived LSC and HSC. Overexpression of JMJD1c in Hoxa9/Meis1a pre-LSC significantly increased the clonogenic activity and proliferation compared to control cells. Subsequent microarray experiment identified Hox genes as downstream targets of this epigenetic regulator (fold change > 2, P < 0.05), suggesting the regulatory role of JMJD1c in the self-renewal pathway. Consistent with in vitro observations, there was a significant increase in disease progression as shown by leukemia onset in mice with Hoxa9/Meisa1 pre-LSC transduced with JMJD1c cDNA compared to control (P < 0.0001; at least 12 mice per cohort). Stable knockdown of JMJD1c in MLL-AF9 cells using shRNA significantly impaired the proliferation, clonogenic activity and induced cell differentiation in vitro. In vivo survival studies in mice showed that JMJD1c knockdown severely impaired the maintenance of MLL-AF9 induced leukemia (P < 0.0001; 12 mice per cohort).

In conclusion, our studies have identified JMJD1c as an important driver in leukemia maintenance and strongly suggest that JMJD1c possesses an oncogenic role in MLL-AF9 leukemogenesis. Targeting this oncogene may enable us to directly target LSC as a strategy for overcoming drug resistance in AML patients.