The identification of TET2 mutations in myeloid neoplasms suggested that TET2 function could be both an oncogenic trigger and a regulator of physiological hematopoiesis. As mentioned previously, mutational analysis suggested that the majority of these mutations severely affect enzymatic activity by either inhibiting gene expression or the function of essential catalytic domains of the protein. These studies underlined the need for models of Tet2 deficiency in the hematopoietic system, to directly address the function of this gene in both physiological differentiation and leukemic transformation.
Rao and colleagues initially used RNAi-mediated Tet2 silencing to address such questions. They reported that Tet2 knockdown in early hematopoietic progenitors affects myelopoiesis as it promotes expansion of monocyte-macrophage cells in the presence of G-CSF and GM-CSF, which promote granulocyte and granulocyte/monocyte differentiation respectively. No such effects were noted in the presence of M-CSF, a cytokine promoting the development of monocytic progenitors (Ko et al., 2010
). More recently similar knock-down studies were performed using human progenitor cells (Pronier et al., 2011
). TET2 silencing led to skewed differentiation towards the myelo-monocytic lineage, at the expense of lymphopoiesis and erythropoiesis. Interestingly, monocytic cell differentiation was specifically favored, providing a possible explanation for the high frequency of TET2 mutations in CMML, a disease associated with the expansion of the monocytic compartment.
A different outcome was reported in another recent study in which primary bone marrow cells were infected with viruses expressing either Tet2 shRNAs or mutant IDH2 molecules (thus affecting Tet2 enzymatic activity). After culturing the cells on both methylcellulose and liquid cultures, a higher percentage of more immature c-Kit+ cells were observed (Figueroa et al., 2010
). Higher c-kit levels suggested that Tet2 silencing is able to suppress stem/progenitor cell differentiation and retain the cells in a more immature state. In agreement with these studies, Moran-Crusio and colleagues have shown that Tet2 silencing by shRNA and by genetic deletion not only increases the c-Kit+ fraction but increases the replating ability of the cells in vitro
, suggesting direct effects on self-renewal (Moran-Crusio et al., 2011
Although these studies provided valuable information of the role of Tet2, hematopoiesis and leukemogenesis are dynamic processes that can be optimally studied in vivo
. Three recent studies have reported the generation and analysis of Tet2 deficient animals (Li, 2011
; Moran-Crusio et al., 2011
; Quivoron et al., 2011
). Interestingly, four different Tet2-/- models were described: a)
a “gene-trapped” Tet2 allele targeting a lacZ/GFP cassette at the transcriptional start of Tet2, b)
a “gene-trapped” allele targeting a lacZ cassette at the intron between exons 9 and 10, c)
a conditional allele targeting the first (and larger) coding exon (exon 3) of the gene and d)
a conditional allele targeting Exon 11 which encodes for the catalytic domain of the enzyme. Although one of the “gene-trapped” alleles resulted in a hypomorphic allele and different Cre recombinase-deleter strains were used for the deletion of the gene, all four mice led to highly similar phenotypes, underlining the reproducibility of the findings and mimicking the diverse mutational targets found in human disease.
Tet2 deletion led to the progressive enlargement of the stem/progenitor compartment (Lineage-Sca1+c-Kit+, LSK cells) however no compartment (defined by SLAM marker staining) was specifically enlarged. Strikingly, these mice developed significant extramedullary hematopoiesis, with the appearance of both LSK and myelo-erythroid progenitors in peripheral lymphoid organs. These include bona fide HSCs as shown by transplantation assays. Moreover, in addition to their relative enlargement in absolute numbers, Tet2-/- HSCs have increased self-renewing abilities as proven by both in vitro re-plating assays and in vivo competitive transplantation experiments. Indeed, Tet2-/- total bone marrow and purified LSK cells have the ability to serially replate. These cells express high levels of c-Kit and remain arrested at a common myeloid progenitor-like (CMP) stage. Whole-transcriptome analysis has shown that the replating pre-myeloblasts have downregulated expression of several genes characteristic of myeloid differentiation and upregulated expression of genes indicative of stem cell self-renewal (c-Kit, Evi1, Meis1). Interestingly, previous studies of DNA methylation in TET2WT and TET2MUT patient samples, identified the Evi1 promoter as differentially methylated in these two cell types, suggesting that Tet2-mediated differential methylation of specific gene-targets could lead to restrained stem cell self-renewal in normal HSPCs. Future studies that will map whole-genome Tet2, 5hmC and 5mC enrichment in distinct gene loci during HSC differentiation will identify which TET2-regulated genes are in fact direct Tet2 targets. We should mention here that in all three studies Tet2 deletion led to the loss of 5hmC consistent with the suggested enzymatic activity of the protein.
One of the most intriguing findings was the progressive development of myeloid disease, in all the animal models, starting at 4-6 months after the deletion of the gene. Although there were slight differences in the disease onset and the kinetics of progression, most likely due to different backgrounds and efficiency/mode of gene deletion, Tet2 deficient mice developed myeloid neoplasms and predominantly CMML-like disease in agreement with the high prevalence of TET2 mutations in human CMML (Abdel-Wahab et al., 2009
). Indeed, Tet2-/-
mice developed progressive leukocytosis, splenomegaly, neutrophilia, blood monocytosis and myeloid dysplasia. Although CMML-like disease was the most predominant, other types of neoplasms, including MDS with erythroid predominance and MPD-like disease were detected in a fraction of the animals (Li et al., 2011
). These studies demonstrated that Tet2 functions as a bona fide tumor suppressor and its deletion is sufficient to initiate myeloid transformation. However, the kinetics of disease induction suggests that additional genetic lesions can co-operate with Tet2 loss to induce leukemia. Indeed, Tet2 mutations co-occur with a wide range of genetic lesions in both myeloid leukemia (Klinakis et al., 2011
) and lymphocytic neoplasms (Quivoron et al., 2011
). It is intriguing to speculate that these additional genetic lesions not only co-operate with Tet2 but also influence the generation of distinct types of blood neoplasms. Further experiments are required to address these issues.
Finally, all three studies demonstrated that Tet2+/- mice also develop similar types of disease albeit with slower kinetics. These findings suggest a haploinsufficient tumor suppressor function for Tet2 and are clinically important as the majority of patients carry heterozygous TET2 mutations or deletions. However, Tet2+/- mice develop disease with longer latency and milder phenotype suggesting gene-dosage effects. Interestingly all three TET family proteins are expressed in hematopoietic progenitors and deletion of Tet2 does not lead to the upregulation of the expression of Tet1 and Tet3. It is thus startling that reduction of the dosage of one of the three genes (Tet2) is sufficient to induce significant 5hmC changes, a skewing in hematopoietic differentiation and development of myeloid neoplasms. Further studies should focus on the relative redundancy between the three proteins and putative interactions between them in developing hematopoietic progenitor cells.