TET1 plays an essential oncogenic role in MLL-rearranged leukemia

Abstract

The ten-eleven translocation 1 (TET1) gene is the founding member of the TET family of enzymes (TET1/2/3) that convert 5-methylcytosine to 5-hydroxymethylcytosine. Although TET1 was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia carrying t(10,11), its definitive role in leukemia is unclear. In contrast to the frequent down-regulation (or loss-of-function mutations) and critical tumor-suppressor roles of the three TET genes observed in various types of cancers, here we show that TET1 is a direct target of MLL-fusion proteins and is significantly up-regulated in MLL-rearranged leukemia, leading to a global increase of 5-hydroxymethylcytosine level. Furthermore, our both in vitro and in vivo functional studies demonstrate that Tet1 plays an indispensable oncogenic role in the development of MLL-rearranged leukemia, through coordination with MLL-fusion proteins in regulating their critical cotargets, including homeobox A9 (Hoxa9)/myeloid ecotropic viral integration 1 (Meis1)/pre-B-cell leukemia homeobox 3 (Pbx3) genes. Collectively, our data delineate an MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL-rearranged leukemia and highlight TET1 as a potential therapeutic target in treating this presently therapy-resistant disease.

Fig. 1.

TET1 is aberrantly overexpressed in MLL-rearranged AML and is likely a direct target of MLL and particularly MLL-fusions. (A–C) Relative expression levels of TET1 (A), TET2 (B), and TET3 (C) in 12 MLL-rearranged AML, 88 non–MLL-rearranged AML, and nine normal control samples as detected by microarrays. The average expression level of each TET gene in normal controls was set as 1. Note that the gene expression values used in our analyses were log-transformed, not the original absolute values (SI Materials and Methods). (D) Relative expression levels of TET1 as detected by qPCR. The average level of TET1 expression in CD33+ cell samples was set as 1. MLL, MLL-rearranged; NC, normal control; MNC, mononuclear cells.

Fig. 2.

TET1 is a direct target of MLL and particularly MLL-fusions. (A) ChIP-qPCR assays of the enrichment of MLL-N (i.e., MLL N-terminal, representing both wild-type MLL and MLL-fusion proteins), MLL-C (i.e., MLL C-terminal, representing wild-type MLL only), and H3K79me2 at the promoter region of TET1 (sites 2 and 3) and a distal upstream region (site 1) in MONOMAC-6 and K562 cells. IgG was used as a negative control. (B) qPCR (Lower) and Western blotting (Upper) analyses of Tet1 expression in mouse normal BM progenitor (Lin⁻) cells that were transduced with MSCV-MLL-AF9, -MLL-ELL, -MLL-ENL, or empty vector (Control). Transduced cells were cultured in methylcellulose for 7 d. (C) qPCR analysis of TET1 expression in human cord blood CD34⁺ cells that were transduced with MSCV-MLL-AF9 or empty vector and cultured for a period to select transduction-positive cells (MA9-1 to -5, represent different lines of immortalized cells) (35). (D) qPCR analysis of MLL-ENL, Tet1, or Fas expression in mouse MLL-ENL-ERtm cells after withdrawal of 4-OHT. MA9, MLL-AF9. *P < 0.05; **P < 0.01, two-tailed t test. Pgk1, phosphoglycerate kinase 1.

Fig. 3.

Effects of Tet1 in MLL-AF9 (MA9)-mediated cell transformation and leukemogenesis. (A and B) Effects of depletion (A) or forced expression (B) of Tet1 on MA9-mediated cell transformation. (C and D) qPCR analysis of Tet1 expression in different passages of colony cells shown in A and B. (E) Dot blot assay of 5hmC level in genomic DNA of colony cells (passage II) shown in A and B. A similar pattern was observed in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assays. (F) Kaplan–Meier survival analysis of the BM transplantation recipient mice. The median survival of MA9, MA9+shTet1-a, MA9+shTet1-b, and MA9+shTet1-a+b mice (n = 10 for each group) is 70, 85, 108, and over 150 d, respectively; MA9+shTet1-a vs. MA9, P = 0.003; MA9+shTet1-b vs. MA9, P = 0.0003; MA9+shTet1-a+b vs. MA9, P < 0.00001; log-rank test. (G) Wright–Giemsa-stained BM cell cytospin and peripheral blood (PB) smear, and hematoxylin/eosin (H&E)-stained spleen and liver paraffin sections of transplantation recipient mice are shown. (Scale bars: 10 μm in BM/ PB and 100 μm in spleen/liver.) (H) 5hmC antibody-stained spleen. (Scale bars: 20 μm.) *P < 0.05; **P < 0.01, two-tailed t test; in A–E, MA9 samples were used as controls for the statistical comparisons. Note: “MA9” represents “MSCVneo-MA9+pGFP-V-RS-scrambled shRNA” in all of the plots except for B and D where it represents “MSCVneo-MA9+MSCVpuro”; “Control” represents “MSCVneo+MSCVpuro” or “MSCVneo+pGFP-V-RS” (see SI Materials and Methods for more details).

Fig. 4.

HOXA9, MEIS1, and PBX3 are important targets of TET1. (A–C) ChIP-qPCR assay of the binding of TET1, as well as MLL-fusion proteins, to the loci of HOXA9 (A), MEIS1 (B), and PBX3 (C). Green bars represent CpG islands. Brown-purple bars represent exons of target genes. (D and E) The effects of knockdown of TET1 by siRNA oligos (D, Left, qPCR; Right, Western blot) and of ectopic expression of mouse Tet1 (E) on expression of TET1, HOXA9, MEIS1, and PBX3 in human MONOMAC-6 leukemic cells. (F and G) Effects of knockdown of Tet1 by different shRNAs on expression of the four genes in colony cells (F) or in BM cells of transplanted mice (G, Left, qPCR; Right, Western blot); see Fig. 3 for more details about the samples. (H and I) Effects of knockdown TET1 by siRNAs with or without cotransfection of HOXA9, MEIS1, or PBX3 on apoptosis (H, Left), cell viability (H, Right), and cell growth/proliferation (I) of MLL-rearranged leukemic cells. siNC, scrambled siRNA oligos (as negative control of siTet1); +H (+M, +P, or +T) or +HOXA9 (+MEIS1, +PBX3, or +Tet1), cotransfected with MSCVpuro-HOXA9 (-MEIS1, -PBX3, or -Tet1). PGK1/Pgk1 was used as endogenous control for qPCR. *P < 0.05; **P < 0.01, t test; in A–C, K562 was used as a control for MONOMAC-6 for statistics analysis of each item. In G, MA9 group was used as the control.

Fig. 5.

Effects of Tet1-knockout and the signaling-pathway model. (A) Expression changes of three target genes in Tet1 knockout BM progenitor (i.e., Lin⁻) cells relative to wild-type controls. (B and C) Effect of Tet1 knockout on MA9-mediated cell transformation and the corresponding expression changes of the targets. (D) Kaplan–Meier survival analysis of the BM transplantation recipient mice. The median survival of Tet1-WT_MA9 (MSCVneo-MA9+MSCVpuro cotransduced into wild-type mouse BM progenitor cells; n = 8), Tet1-KO_MA9 (MSCVneo-MA9+MSCVpuro cotransduced into Tet1^−/− mouse BM progenitor cells; n = 9), and Tet1-KO_MA9+HOXA9 (MSCVneo-MA9+MSCVpuro-HOXA9 cotransduced into Tet1^−/− mouse BM progenitor cells; n = 5) is 66, >150, and 85 d, respectively; Tet1-WT_MA9 vs. Tet1-KO_MA9, P < 0.00001; Tet1-WT_MA9 vs. Tet1-KO_MA9+HOXA9, P = 0.018; Tet1-KO_MA9 vs. Tet1-KO_MA9+HOXA9, P = 0.019; log-rank test. (E) Wright–Giemsa-stained BM cell cytospin and PB smear, and hematoxylin/eosin (H&E)-stained spleen and liver paraffin sections of transplantation recipient mice are shown. (Scales bars: 10 μm in BM/PB and 100 μm in spleen/liver.) (F) The model of the MLL-fusion/Tet1/Hoxa9/Meis1/Pbx3 signaling axis in MLL-rearranged leukemia. *P < 0.05; **P < 0.01, t test.