Uncover TET1 Targets in MLL-Rearranged Leukemia

5-hydroxymethylcytosine (5hmC), also called the “sixth DNA base”, is involved in the DNA demethylation process which generally leads to gene activation. Formation of 5hmC is catalyzed by the Ten-Eleven-Translocation (TET) family proteins, with TET1 being the founding member. The expression of TET1 protein and the global level of its enzymatic product, 5hmC, is markedly reduced in a wide range of solid tumors, including melanoma, prostate, breast, lung, and liver cancer, suggesting that TET1 functions as a tumor suppressor in these types of cancers. However, a recent study from our group demonstrated that TET1 expression and the associated 5hmC levels are significantly up-regulated in MLL-rearranged leukemia, revealing the oncogenic role of TET1 in this type of acute myeloid leukemia (AML) (Huang H, et al. PNAS 2013; 110(29):11994-9). In support of this, another study from a different group showed that high 5hmC level is an independent predictor of poor overall survival in patients with AML (Kroeze LI, et al. Blood 2014; 124(7):1110-8). However, how TET1, as a critical methylcytosine dioxygenase, plays its oncogenic role in AML, especially in MLL-rearranged leukemia, is still unclear.

To address this issue, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. When TET1 was knocked down in MLL-ENL-estrogen receptor inducible (ERtm) mouse myeloid leukemia cells, 123 proteins were found downregulated whereas 191 were upregulated with a fold-change cutoff of 1.2. The expression changes of a set of these genes were confirmed by quantitative PCR in MLL-ENL-ERtm cells and mice samples with TET1 knock-down or depletion. After taking into account the correlation of TET1 and its candidate targets in several sets of AML patient samples, we focused on IDH1 and PSIP1, which represent the negatively- and positively-regulated targets by TET1, respectively. IDH1 encodes an isocitrate dehydrogenase whose mutations are frequently found in AML, whereas the PSIP1 protein is shown to be required for both MLL-dependent transcription and leukemic transformation. Chromatin immunoprecipitation (CHIP) assays suggest that TET1 directly binds to the CpG islands in the promoters of these two genes. Forced expression of Idh1 in leukemic bone marrow cells collected from mice developed MLL-AF9-driven AML significantly inhibited the colony-forming capacity of these cells, which mimics the effect of TET1 knock-out. We are now further investigating the functions and underlying molecular mechanisms of IDH1 and PSIP1 in AML using both in vitro and in vivo models. Considering the important roles of IDH1 and PSIP1 in AML, our findings will provide new insight into the mechanisms underlying the oncogenic role of TET1 in MLL-rearranged leukemia and may ultimately lead to the development of targeted therapy of AML.