TET1 Modulates DNA Replication in Leukemia Cells Via a Catalytic-Independent Mechanism through Cooperating with KAT8

TET1 was first identified as a fusion partner of the histone H3 Lys4 (H3K4) methyltransferase MLL (mixed-lineage leukemia) in acute myeloid leukemia (AML), and then was discovered as the founding member of the Ten-Eleven-Translocation (TET) family of DNA hydroxylases which are capable of converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Our group has previously demonstrated that TET1 plays an oncogenic role in MLL-rearranged AML (Huang H, et al. PNAS 2013; 110(29):11994-9) and also other TET1-overexpressing AMLs (e.g., t(8;21) AML and AMLs carrying FLT3-ITD and/or NPM1 mutations) (Jiang X, et al. Nature Communications. 2017; 8(1):2099). The expression of the TET1 protein and the global level of its enzymatic product, 5hmC, are significantly up-regulated in MLL-rearranged leukemia, whereas the opposite has been reported in other cancers where TET1 functions as a tumor suppressor. Therefore, a comprehensive identification of all critical targets of TET1 in AML is important for us to better understand the role and underlying molecular mechanism of TET1 in AML.

To this end, we cultured murine inducible MLL-ENL cells and performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling in parallel with RNA-seq to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. Gene ontology (GO) analysis of target genes indicated enrichment in genes associated with DNA replication (FDR<0.001) and cell cycle progression (FDR<0.001). Interestingly, the six main minichromosome maintenance genes, including MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, showed marked downregulation when TET1 expression was depleted. We further showed that not only the total levels of the MCM2-7 proteins, but also their binding to chromatin, were decreased upon TET1 knockdown in human AML cell lines. Consistent with the roles of MCMs in cell cycle regulation, knockdown of TET1 led to a significant decrease in the S phase population, attributing to the inhibition on DNA replication, as shown by reduced 5-ethynyl-2'-deoxyuridine (EdU) incorporation into newly synthesized DNA upon TET1 knockdown. Furthermore, DNA combing assays suggest that TET1 knockdown inhibits new origin firing but does not influence replication fork speed.

Chromatin immunoprecipitation (ChIP) assays demonstrated that TET1 binds directly to the CpG islands in the promoters of the MCM genes, suggesting that the regulation of these genes by TET1 may occur at the transcriptional level. However, 5hmC sequencing revealed low abundance of 5hmC around these genomic regions, and more importantly, the abundance was not influenced by TET1 knockdown. In addition, catalytically inactive TET1 showed similar effects to wild-type TET1 on promoting cell cycle progression and DNA replication, suggesting that TET1 regulates MCM genes and cell cycle in a manner independent of its catalytic activity. Interestingly, we found that KAT8, an acetyltransferase that specifically catalyzes histone H4 lysine 16 acetylation (H4K16Ac) and was reported to bind to TET1, plays a role in the regulation of TET1 on transcription of MCM genes. Direct binding of KAT8 with TET1 was confirmed in AML cells. Depletion of TET1 reduced H4K16Ac abundance in the MCM promoters where TET1 bind, suggesting that the binding of KAT8 to such genomic regions is owing to its recruitment by TET1. Similar to TET1 knockdown, KAT8 knockdown also decreased MCM genes expression at both RNA and protein levels and resulted in defects in cell cycle progression and DNA replication.

Based on the above data, we speculated that combined inhibition of TET1 and KAT8 could exert potent inhibitory effect on DNA replication in TET1-overexpressing AML cells. Indeed, Tet1 knockout sensitized MLL-AF9-transformed mouse bone marrow (BM) progenitor cells to MG149, a KAT8 inhibitor. In addition, TET1 inhibitor (U514321) synergized with MG149 in inhibiting the viability and growth of human AML cells. Furthermore, co-treatment of MG149 and U514321 exhibited synergistic effect on inhibiting colony-forming ability of MLL-AF9-transformed mouse BM progenitors. Collectively, our findings reveal a catalytic-independent novel function of TET1 on regulating DNA replication in AML cells through cooperating with KAT8 and highlight the therapeutic implication of targeting both TET1 and KAT8 in treating TET1-overexpressing AMLs.