Combined Loss of Tet1 and Tet2 Promotes B-Cell, but Not Myeloid Malignancies in Mice

Objective: Tet1/2/3 are methylcytosine dioxygenases regulating cytosine methylation in the genome. Tet1 and Tet2 are abundantly expressed in HSC/HPCs and implicated in hematological malignancies. Tet2 -deletion in mice causes myeloid malignancies, while Tet1 -null mice are overtly normal early in life. Here, we investigated the overlapping and non-redundant functions of Tet1/Tet2 in HSC maintenance and hematological malignancies using Tet1/2 double knockout (DKO) mice.

Methods: 1) Kinetic analysis of the hematologicalparameters on WT, Tet1^-/-, Tet2^-/- and DKO mice; 2) Analysis of HSC, myeloid and lymphoid progenitors and various maturation stages of B-cell populations; 3) Competitive bone marrow reconstitution assay; 4) RAN-Seq on LK cells and B220⁺ cells from young/undiseased and diseased DKO mice respectively; 5) Chemical labeling and affinity purification method coupled with high-throughput sequencing (hMe-Seal) to profile the genome-wide distribution of 5hmC, and methylated DNA immunoprecipitation coupled with high-throughput sequencing (MeDIP-seq) to profile 5mC in BM LK cells from young WT, Tet2^-/- and DKO mice; 6) q-PCR analysis of the mRNA expression levels of Tet1 and Tet2 on BM CD19⁺ cells from B-ALL patients and compared to that of CD19⁺ B-cells from healthy controls.

Results: We found that T et 1 and T et 2 are often concomitantly down-regulatedin patients with B-ALL. Therefore, it is important to investigate the effects of combined loss of Tet1 and Tet2 on the hematopoietic phenotype and development of hematological malignancies in vivo. The LSK and CMP/GMP/MEP cell populations are comparable in yound WT, Tet1^-/- and DKO mice, while were significantly increasedin Tet2^-/- mice. When a replating assay was performed using LSK cells, Tet2^-/- LSK cell cultures had a significant higher colony formation in each round of replating, while Tet1^-/- and DKO LSK cell cultures only exhibited a moderate increase in the number of colonies at P2, but not P3 and P4. Furthermore, young DKO mice had an increased percentage of CLP, BLP and Pro-/Pre-/Immature-B cell populations in their BM as compared to WT, Tet1^-/- and Tet2^-/- mice. Consistent to the B-lineage phenotypic analysis, DKO BM cells contained higher pre-B cell colony forming cells than the three genotypes of control mice. Interestingly, DKO mice exhibited a strikingly decreased incidence and delayed onset of myeloid malignancies compared to Tet2^-/- mice and in contrast developed lethal B-cell malignancies, most closely resembling B-ALL. The loss of Tet2 or DKO leads to genome-wide alterations of both 5mC and 5hmC. Significant overlaps between the differential hydroxymethylated regions (DhMRs) or differential methylated regions (DMRs) of two genotypes of LK cells were observed. However, intriguingly, the overlaps between DhMRs and DMRs within each genotype of LK cells were minimal, indicating that DhMRs and DMRs might represent distinct loci with altered epigenetic modifications under these conditions. When the expression of a pool of 654 genes that are known to be involved in regulating hematopoietic cell development and/or promoting leukemogenesis were overlap with DhMRs and DMRs identified above, we observed significant numbers of these genes with altered either 5hmC or 5mC modifications which however did not alter their gene expression. Furthermore, RNA-Seq on B-ALL DKO B220⁺ cells showed alteration of a set of genes involved in B-cell development and B-cell lymphoma/leukemogenesis.

Conclusion: Using Tet1/2 double knockout mice, we found that Tet1 is required for Tet2 -deletion mediated HSC dysregulation, myeloid skewing and myeloid malignancy, indicating distinct roles of the two enzymes. Tet1 loss modulates the Tet2 -deletion mediated disease phenotype, not only decreasing the incidence and delaying the onset of myeloid malignancies, but also promoting the pathogenesis of B-cell malignancies. Furthermore, our observations highlight the roles of distinct cytosine modifications, particularly 5hmC, could play in marking the specific genes and enabling cells to fate decision change upon stimulation signals. These findings provide a pathological framework for further elucidating the molecular mechanisms and critical cross talks between Tet1 and Tet2 in the pathogenesis of hematological malignancies.