The Role of TET2 in Macrophage Differentiation and Polarization
The epigenetic regulator Tet methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), leading to DNA demethylation. Inactivating TET2 mutations are common in myeloid cancers such as chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS). Little is known about the role of TET2 in disease-relevant monocytes/macrophages (MΦs). Therefore, our goals for this project were to characterize Tet expression during normal murine MΦ differentiation and polarization, examine the effect of Tet2-deficiency on broader MΦ polarization gene signatures, and determine the translatability of our murine findings to human MDS/CMML. In this thesis, we characterized the expression of Tet2 in murine MΦ differentiation and polarization. Using murine bone marrow-derived MΦs (BMMΦ), we observed that Tet2 was highly expressed in differentiated MΦs. Interestingly, differentiation-associated gene expression was not altered in Tet2-knockout BMMΦs and peritoneal MΦs (PMΦs), suggesting that Tet2 is dispensable for MΦ development. MΦ response to polarizing stimuli was then explored. Tet2 transcript levels increase early in the LPS response. The NFκB inhibitor compound BAY11-7082 was shown to abolish LPS-mediated Tet2 induction, indicating that this gene is NFκB-responsive. Exploring Tet2-deficiency, we characterized the phenotype of Tet2-knockout MΦs using NanoString gene expression analysis. Constitutive expression of LPS-induced genes was observed in resting state Tet2-deficient PMΦs. In particular, we noted increased Il1β and Il6, as well as the anti-inflammatory gene Arginase 1 (Arg1), in resting Tet2-deficient PMΦs and in LPS-treated Tet2-knockout BMMΦs. These results implicated Tet2 loss in the establishment of aberrant inflammation, raising the question of whether TET2-mutant MΦs participate in altering the immune microenvironment. Assessing immune dysregulation in TET2-mutant cells, we assayed IL1β and IL6 mRNA expression in human MDS/CMML patient samples as well as MDS/CMML-derived human MΦs. While levels of both targets were variable, some TET2-mutant cells had high IL6 expression. We also assessed ARG1 expression in MDS/CMML BM biopsies using immunohistochemical analysis. TET2 and/or DNMT3A mutations were correlated with high ARG1 staining, providing a link between ARG1 overexpression and TET2 mutations in humans. Overall, the work presented in this thesis provides a novel connection that ties TET2 lesions to the development of an inflammatory MDS/CMML local microenvironment.