The Role of TET Methylcytosine Dioxygenase 2 in Myeloid Malignancies
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AbstractTET methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate stage in the DNA demethylation processthat controls the transcription of genes. TET2 is highly expressed in the hematopoietic system and is suggestedto regulate the maintenance and differentiation of hematopoietic stem/progenitors. Mutations in TET2 has been found in different hematological malignancies including Acute Myeloid Leukemia (AML), Chronic Myelomonocytic Leukemia (CMML), Myeloproliferative Neoplasms (MPN), Myelodysplastic Syndrome (MDS), etc. However, the mechanisms by which TET2 controls hematopoiesis and contributes to myeloidmalignancies remainunknown. The direct targets of TET2 have not been elucidated yet. In order to identify the direct targets of TET2, we have performed chromatin immunoprecipitation using TET2 specific antibody followed by genome-wide sequencing. We have found enrichment in binding of TET2 in the promoters of SHP1, SOCS3 and PLZF among other targets. Interestingly, the expression of these genes is also significantly downregulated in the hematopoietic progenitors of TET2 deficient mice. Furthermore, we have found that CMML patients with TET2 mutations also have decreased expression of these genes. Knockdown of TET2 resulted in downregulation of these genes in leukemic Molm14 and murine Ba/F3 cells. Conversely, overexpression of TET2 in monocytic U937 cells increased the expression of these genes. Using methylation specific PCR, we also have observed increased methylation in the promoters of SHP1, SOCS3 and PLZF in TET2 deficient Molm14 and Ba/F3 cells. Also, using methyl/hydroxymethyl-DNA immunoprecipitation, we observed an increase in the 5mC level and decrease in the 5hmC level in the promoter region of these genes suggesting that TET2 directly regulates the expression of these genes by regulating the methyl and hydroxymethyl level of the promoter of these genes. Although loss of function of TET2 has been associated with multiple hematopoietic malignancies, TET2 is most commonly mutated in CMML with almost 50% patients bearing TET2 mutations. Interestingly, TET2 mutations are frequently associated with CBL mutations in CMML. In order to assess the concurrent effects of TET2 and CBL deficiencies, we generated TET2 CBL double knockout mice. We observed that simultaneous deletion of TET2 and CBL resulted in increased leukocytes and neutrophil and enhanced splenomegaly compared to control mice. The double knockout mice showed increase in the granulocyte macrophage progenitors and a significant expansion of the stem progenitor cell population. The overall survival of these mice also reduced substantially. It suggests that concurrent deletion of TET2 and CBL increased the severity of the CMML like disease in mice and thus TET2 and CBL deletion may cooperate in the pathogenesis of CMML. Although most studies suggest a tumor suppressor function of TET2, we also have found a tumor promoter function, especially in MLL rearranged leukemia. We have found that knockdown of TET2 resulted in decreased proliferation in MLL-AF9 positive Molm14 leukemia cells and murine Ba/F3 MLL-AF9 expressing cells. Conversely overexpression of TET2 significantly increased the proliferation level of Molm14 cells. In orderto understand the in vivo role of TET2 in MLL-AF9 mediated leukemia, we performed a retroviral BMT experiment. Whereas the expression of MLL-AF9 in wild type BM resulted in marked increase in WBC and NE and splenomegaly, the deletion of TET2 reduced the white blood cell and neutrophil count and also caused reduction in the spleen size. MLL-AF9 overexpression resulted in the increase in hematopoietic stem/progenitor cells and granulocyte macrophage progenitors and granulocyte/monocyte precursors which was significantly reduced in TET2 KO MLL-AF9 mice. There was a significant reduction of the hematopoietic colony formation ability mediated by MLL-AF9 in TET2 KO mice. The overall survival of the knockout mice was markedly improved compared to the control MLL-AF9 mice. Together these results suggest a tumor promoter role of TET2. Taken together, all the results indicate a dual role of TET2 in myeloid leukemia.
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