Tet2 Loss Dysregulates the Behavior of Mesenchymal Stem Cells and Increases Their Ability to Promote Tet2-/--Driven Myeloid Malignancy Progression

Abstract

As a hallmark of epigenetic regulation, DNA methylation plays an important role in regulating gene expression. The TET methylcytosine dioxygenase enzymes (TET1/2/3) catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and can further oxidize 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Participating in the initial steps of active DNA demethylation, TETs are important regulators of cytosine methylation in the genome. Mutations and/or deletions of the TET2 gene have been reported to frequently occur in myeloid malignancies. We and others have reported that Tet2 loss dysregulates HSC/HPC function, leading to the development of myeloid malignancies in mice. Therefore, TET2 acts as a tumor suppressor in myelopoiesis. Although the HSC/HPC-autonomous effects of Tet2 loss on the malignant process is well established, it remains to be explored whether non-hematopoietic cells contribute to Tet2 loss mediated malignant process. Specifically, Tet2loss may dysregulate the integrity of bone marrow niche, by which affects the malignant progression.Here, we show that while both Tet2f/f;Mx1Cre(conditional Tet2-inactivation in hematopoietic cells) and Tet2f/f;MxfCre (germ line Tet2-inactivation) developed myeloid malignancies in mice, Tet2f/f;MxfCremice had a significantly shortened survival compared to Tet2f/f;Mx1Cre mice. When WT and Tet2-/- recipient mice were transplanted with WT or Tet2-/- Lin-Sca-1+c-Kit+(LSK) cells, both genotypes of recipients receiving Tet2-/- but not WT LSK cells developed abnormal hematopoietic characteristics including monocytosis/neutrophilia and splenomegaly, resembling CMML and MPN. Interestingly, Tet2-/-recipient mice exhibited a higher incident of myeloid malignancies and a significantly reduced survival rate compare to WT recipient mice. These data indicate that Tet2-/- bone marrow niche might promote the progression of myeloid malignancies in Tet2-/-mice. To identify the specific niche components that are responsible for the accelerated progression of myeloid malignancies in Tet2-/-mice, we selectively deleted Tet2 from candidate niche cell components and characterized the effects on myeloid malignancy development. Deletion of Tet2 in osteoblasts (Col2.3-cre), endothelial cells (Ve-Cadherin-cre) and osteoclasts (LysM-cre) have little effects on the initiation/progression of Tet2-/--driven myeloid malignancies. Strikingly, deletion of Tet2in mesenchymal stem cells (MSCs) using Prx1-cre is associated with a significantly accelerated malignancy progression and shortened survival, suggesting that MSCs are the cell components in Tet2-/-mice play a role in the initiation/progression of Tet2-/--driven myeloid malignancies. Furthermore, Tet2-/-MSCs displayed a significantly increased self-renewal, proliferating and differentiation capability as assayed by the frequency of CFU-F and commitment towards osteoblasts. In addition, Tet2-/- but not WT MSCs exhibited a significantly increased supportive capacity to Tet2-/- HSC/HPC proliferation. RNA-sequencing analysis revealed that Tet2-/- MSCs exhibited a distinct gene expression profiles with 468 dysregulated genes as compared to WT MSCs. Quantitative real-time PCR confirmed that multiple genes critical for cell proliferation, osteoblast differentiation and stem cell pluripotency were down- (Cxcl12, Lom1, Cxcl14, Nt5e, Lfit1and Comp) or up- (Sox2, Nanog, Sp7, Bmp2, Bmp4 and Il6) regulated in Tet2-/- MSCs compared to WT MSCs. Furthermore, the number of 5-hmC peaks were significantly decreased in Tet2-/-MSCs compared to WT MSCs based on whole genomic 5-hmC profiling. The majority of TET2-dependent 5hmC modifications in MSCs are located within genes. We then examined TET2 gene expression in MSCs derived from human myeloproliferative neoplasms (MPN) patients and healthy individuals and found that TET2 and 5-hmC was moderately down-regulated in MPN MSCs as compared to healthy controls. In summary, these results indicate that Tet2loss dysregulates hydroxylation of 5-mC and gene expression in MSCs, which in turn alters their behavior and ability to promote Tet2-/--driven myeloid malignancy progression. These studies could lead to the identification of additional therapeutic targets for patients with myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.