TET2 Loss Dysregulates the Behavior of Mesenchymal Stem Cells and Increases Their Ability to Promote TET2−/−-Driven Myeloid Malignancy Progression

Abstract

Background: TET2 is a methylcytosine dioxygenase that regulates cytosine hydroxymethylation. Although there are extensive data implicating a pivotal role of TET2 in hematopoietic stem/progenitor cell (HSPCs), the importance of TET2 in bone marrow mesenchymal stromal cells (BMSCs) remains unknown. Aim: Tet2 loss may dysregulate the integrity of bone marrow niche, by which affects the malignant progression. Methods: Generation and maintenance of Tet2 conditional knockout MiceAnalysis of 5-hmC and 5-mC levels using dot blotFlow cytometry analysis, cell sorting, and hematopoietic progenitor cell (HPC) assayMSC Culture and long-term coculture with HSPCsReal-time PCR and RAN-Seq AnalysisMeDIP-seq to profile the genome-wide distribution of 5hmC. Results: Here, we show that while both Tet2 f/f ;Mx1Cre (conditional Tet2-inactivation in hematopoietic cells) and Tet2 f/f ;MxfCre (germ line Tet2-inactivation) developed myeloid malignancies in mice, Tet2 f/f ;MxfCre mice had a significantly shortened survival compared with Tet2 f/f ;Mx1Cre mice. Interestingly, Tet2 −/− recipient mice exhibited a higher incident of myeloid malignancies and a significantly reduced survival rate compare with WT recipient mice. These data indicate that Tet2 −/− bone marrow niche might promote the progression of myeloid malignancies in Tet2 −/− mice. Strikingly, deletion of Tet2 in 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 toward 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 with WT MSCs. Furthermore, the number of 5-hmC peaks were significantly decreased in Tet2 −/− MSCs compared with 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 with healthy controls. Conclusion: These results highlight the critical role of TET2 in the maintenance of BMSC functions and osteoblast differentiation, and provide evidence that dysregulation of epigenetic modifier in BMSCs contributes to the progression of myeloid malignancies.