Tet1 is not required for myeloid leukemogenesis by MLL-ENL in novel mouse models

Abstract

The Ten Eleven Translocation 1 (TET1) gene encodes an epigenetic modifying molecule that is involved in demethylation of 5-methylcytosine. In hematological malignancies, loss-of-function mutations of TET2, which is one of the TET family genes including TET1, are frequently found, while the mutations of TET1 are not. However, clinical studies have revealed that TET1 is highly expressed in some cases of the hematological malignancies including acute myeloid leukemia. Indeed, studies by mouse models using conventional Tet1 knockout mice demonstrated that Tet1 is involved in myeloid leukemogenesis by Mixed Lineage Leukemia (MLL) fusion gene or TET2 mutant. Meanwhile, the other study showed that Tet1 is highly expressed in hematopoietic stem cells (HSCs), and that deletion of Tet1 in HSCs enhances potential self-renewal capacity, which is potentially associated with myeloid leukemogenesis. To examine the role of Tet1 in myeloid leukemogenesis more precisely, we generated novel conditional Tet1-knockout mice, which were used to generate the compound mutant mice by crossing with the inducible MLL-ENL transgenic mice that we developed previously. The leukemic immortalization in vitro was not critically affected by conditional ablation of Tet1 in HSCs with the induced expression of MLL-ENL or in hematopoietic progenitor cells retrovirally transduced with MLL-ENL. In addition, the leukemic phenotypes caused by the induced expression of MLL-ENL in vivo was not also critically affected in the compound mutant mouse model by conditional ablation of Tet1, although we found that the expression of Evi1, which is one of critical target genes of MLL fusion gene, in tumor cells was remarkably low under Tet1-ablated condition. These results revealed that Tet1 was dispensable for the myeloid leukemogenesis by MLL-ENL, suggesting that the therapeutic application of Tet1 inhibition may need careful assessment.

Bahcc1 is critical for the aberrant epigenetic program in a mouse model of MLL-ENL–mediated leukemia

“…Compound C57BL/6 (B6) mice conditionally expressing cytomegalovirus early enhancer / chicken β actin ( CAG ) promoter–driven MLL-ENL by inducible Cre-estrogen receptor α chain (CreER) fusion protein expressed from the Rosa26 locus (C/Tg mice) have been previously described. 9 , 26 All animal studies were approved by the Animal Care Committee of Mie University (no. 24-34-re5).…”

“…LT-HSCs (CD150 + /CD48 − KSL), ST-HSCs (CD150 − /CD48 − KSL), MPP2 (CD150 + /CD48 + KSL) cells, and MPP3/4 (CD150 − /CD48 + KSL) cells of C/Tg mice, and KSL and c-Kit high Sca-1 − Lin − (hereafter designated MP) cells of wild-type B6 mice were purified from the respective bone marrow (BM) cells using a fluorescence-activated cell sorting (FACS) Aria II (BD Biosciences, Franklin Lakes, NJ). 9 , 26 In brief, lineage-depleted cells were isolated from BM mononuclear cells labeled with Lineage Cell Detection Cocktail-Biotin using a MACS cell separation system (Miltenyi Biotec, Auburn, CA). For purification of LT-HSCs, ST-HSCs, MPP2 cells, and MPP3/4 cells derived from C/Tg mice, lineage-depleted cells were stained with peridinin chlorophyll protein (PerCP)-Cy5.5-conjugated streptavidin, and phycoerythrin (PE)–conjugated anti-c-Kit (2B8), PE-Cy7–conjugated anti-Sca-1 (D7), allophycocyanin (APC)–conjugated anti-CD150 (TC15-12FL12.2), and brilliant violet 421 (BV421)–conjugated anti-CD48 (HM48-1) monoclonal antibodies.…”

“…Myeloid immortalization assays via serial replating were performed as described elsewhere. 26 In brief, purified LT-HSCs, ST-HSCs, MPP2 cells, and MPP3/4 cells (150 cells) were directly sorted into 1.5 mL of methylcellulose medium prepared from M3234 (StemCell Technologies, Vancouver, Canada) according to the manufacturer’s protocol, supplemented with 25 ng/mL mouse stem cell factor, 10 ng/mL each of human interleukin 6, mouse interleukin 3, and mouse granulocyte-macrophage colony–stimulating factor (Miltenyi Biotec), and 0.05 μM of 4-hydroxytamoxifen (Sigma-Aldrich, St Louis, MO) dissolved in ethanol or an equal amount of ethanol as a vehicle control. Sorted cells in 1 mL of the mixture were immediately plated in 35-mm dishes.…”

Role of TET dioxygenases in the regulation of both normal and pathological hematopoiesis

“…Tet1 is involved in MLL-AML development by promoting the expression of oncogenic target genes such as Hoxa9, Meis1, and Pbx3 [95]. However, a recent study demonstrated that Tet1 is not required for AML pathogenesis in an MLL-ENL mouse model [116]. Such a discrepancy might be explained by a difference in the MLL-fusion gene or the techniques used for fusion gene induction.…”