Inhibition of TET2 Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine Disturbs Myelopoiesis and Granulo-Monocytic Differentiation.

Abstract

1584 TET2, a gene frequently mutated in myeloid malignancies, encodes an oxygenase that may convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in hematopoietic cells. Using high performance liquid chromatography coupled to tandem mass sprectrometry (HPLC-MS-MS), we found lower levels of 5hmC in erythroblasts from patients with myeloproliferative neoplasms and TET2 mutations than in erythroblasts from non mutated patients. To study the function of TET2 during myelopoiesis, we used lentiviruses expressing short hairpin RNA (shRNA) designed to inhibit TET2 expression in CD34+ cells from normal bone marrow or umbilical cord blood. TET2 mRNA was knocked down by 56 to 74% in several human leukemia cell lines expressing shRNA-TET2, compared to cell lines expressing scramble shRNA. TET2 expression was then assessed in these cell lines by western blot, which showed a 49% reduction of TET2 protein in TF1 cell line, and 80 to 90% reduction in Kasumi, Uke1, and Mo7E cell lines. In addition, knock down of TET2 led to a 73% decrease in 5hmC in Mo7E cell line DNA whereas 5mC and cytosine contents remained unchanged as measured by HPLC-MS-MS. These results indicate that TET2 has a role in the hydroxylation of 5mC in human cells of hematopoietic origin. We then studied the consequences of TET2 knock-down in umbilical cord blood CD34+ cells in vitro. We observed a skewing of CD34+CD38- progenitor differentiation toward the myeloid lineage (52 +/− 4% of the TET2 knock down cells versus 35 +/− 3% of the control cells, n=3, p=0.001) at the expense of lympho-myeloid development and B cell and natural killer (NK) lymphoid lineages in a B/myeloid/NK culture condition. Methylcellulose in the presence of EPO, IL3, SCF, G-CSF showed greater numbers of CFU-G/GM (66 +/− 4 versus 58 +/− 3 colonies per 1000 input CD34+ cells) and lower numbers of BFU-E (60 +/−19 versus 78 +/− 19) in TET2 knock-down samples than in control samples (n= 4, p<0.05) indicating that there was also a skewing toward the granulo-monocytic differentiation at the expense of the erythroid lineage. In addition, in presence of G-CSF, IL3, and FLT3-ligand granulocytic differentiation was delayed in TET2 knock-down cells with a relative excess of monocytic cells at day 10 of culture as assessed by the analysis of cell morphology (47 +/− 4% monocytic cells versus 37 +/− 3%, n=5, p=0.006) and immunophenotype (52 +/− 3% of CD14+ cells versus 39+/−8%, n=5, p=0.002). We then analyzed the expression of a set of transcription factors at the mRNA level that confirmed that some transcription factors specific to granulocytic differentiation were under-expressed in TET2 knocked down cells between day 5 and day 10 of culture. Together, our results show that TET2 participates to the conversion of 5mC to 5hmC in hematopoietic cells, and suggest that TET2 inactivation may have a role in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation. Disclosures: No relevant conflicts of interest to declare.

TET2 Deficiency Inhibits Mesoderm and Hematopoietic Differentiation in Human Embryonic Stem Cells

“…Short hairpin (sh)RNA strategy to regulate TET2 expression based on lentivirus was used as previously described . Sequences for human shTET2 and shTET2.b were (5′GGGTAAGCCAAGAAAGAAA3′) and (5′AAACAAAGAGCAAGA GATT3′), respectively.…”

“…Western blot analysis was done using an anti‐TET1 or anti‐TET2 rabbit polyclonal (produced in house) or an anti‐TET2 mouse serum against the C‐terminal part of the protein (produced in house) or anti‐TET3 (CliniSciences, Santa‐Cruz, Nanterre, France). Normalization was done using HSC70 antibody (Enzo Life Sciences, Villeurbanne, France, http://www.enzolifesciences.com/contact-us/).…”

“…We and others have identified alterations in the TET2 gene in various hematological malignancies . A decrease in 5‐hmC levels was found in TET2 ‐mutated patients displaying myeloid disorders associated with hypomethylated or hypermethylated genomic DNA . TET2 knockdown affects both murine and human adult hematopoiesis by allowing the self‐renewing potential of hematopoietic stem cells and favoring monocytic differentiation at the expense of granulopoiesis .…”

“…A decrease in 5‐hmC levels was found in TET2 ‐mutated patients displaying myeloid disorders associated with hypomethylated or hypermethylated genomic DNA . TET2 knockdown affects both murine and human adult hematopoiesis by allowing the self‐renewing potential of hematopoietic stem cells and favoring monocytic differentiation at the expense of granulopoiesis . With age, tet2 +/ − and tet2 − / − mice develop a myeloid malignancy resembling human myelomonocytic leukemia .…”

The hypomethylating agent Decitabine causes a paradoxical increase in 5-hydroxymethylcytosine in human leukemia cells

“…Aberrant DNA methylation, such as hypermethylation of tumor suppressor genes, is a hallmark of cancer and a testament to the role of epigenetics in oncogenesis 1 2 3 . Epigenetic alterations on genes that regulate the differentiation of hematopoietic stem cells (HSCs) into matured blood cells, clinically christened hematopoiesis, are frequently observed in myeloid malignancies 4 5 6 7 8 . The recent discovery of the 5mC derivative, 5hmC 9 10 , in addition to its sub-derivatives, 5fC and 5caC 11 ( Figure 1a ), has produced new players that could participate in epigenetic regulation of transcription 10 12 13 14 15 .…”