Figure 1.
Increased proliferation of HMC-1.2 cells after knock down of TET2.
A) HMC-1.2 cells were treated with two hairpins against TET2 (TET2 sh-1 and TET2 sh-3) or a control shRNA (ctr sh). Cell growth was calculated using the CellTiter-Glo assay from Promega. Data are presented as fold change relative to day 5 after transduction. Values represent mean ±SEM, n = 3 independent experiments. *P<.05. B) Percentage of cells in S-phase determined by BrdU incorporation in HMC-1.2 cells treated with TET2 sh-1 and sh-3 compared to a control hairpin. Values are mean ±SEM. n = 3 independent experiments, ***P<.001, ns = not significant. C) Representative FACS plots showing BrdU incorporation in relation to cell cycle stages in HMC-1.2 cells infected with control hairpin (ctr sh) compared with TET2 sh-1 and TET2 sh-3.
Figure 2.
Loss of Tet2 accentuates a Kit D814V driven mast cell phenotype.
A) Average number of mast cells per skin section across genotypes. N = 60–80 sections from 3–4 independent animals/genotype. *P<.05. B) Average number of mast cells per stomach/esophagus section across genotypes. N = 60–80 sections from 3–4 independent animals/genotype. *P<.05. For Figure 2A and 2B, numbers 1–4 indicate the following genotypes: 1 = WT ctr, 2 = Tet2+/+;Kit D814, 3 = Tet2−/−;Kit D814, 4 = Tet2−/−;Kit WT. C) Percentage of skin sections with a defined histology score from Tet2+/+;Kit D814V and Tet2−/−;Kit D814V. D) Percentage of stomach/esophagus sections with a defined histology score in Tet2+/+;Kit D814V and Tet2−/−;Kit D814V animals. For Fig 2A–2D, twenty randomly chosen and independent regions of equal thickness per animal were counted in a blinded fashion at 20× magnification, and scored according to the classification reported in Table 1. Mice were all harvested between 8 and 20 weeks after the last pI:C injection. n = 4 per genotype. E) Representative pictures of Giemsa staining performed on skin (left panels) or stomach/esophagus sections (right panels) prepared from Tet2+/+;Kit D814V and Tet2−/−;Kit D814V animals. Mast cells stain dark blue in these sections. Scale bar represents 100 µm.
Table 1.
Histology score used to classify skin and stomach/esophagus regions from animals with mastocytosis.
Figure 3.
Effects of loss of Tet2 on BMMCs derived from Kit D814V-MxCre mice.
A) Tet2 mRNA levels measured in BMMCs upon Mx1-Cre-mediated deletion of Tet2. Data are presented as fold change of Tet2 mRNA/Gapdh mRNA levels relative to Tet2+/+;Kit D814V. Data represent mean ±SEM, n = 3, *P = <05, ***P<.001. B) Phosphorylation of the Kit receptor in the absence of its ligand SCF due to the presence of a Kit activating mutation in BMMCs from Tet2+/+;Kit D814V, Tet2+/−;Kit D814V and Tet2−/−;Kit D814V animals. The western blot shown is representative of two independent experiments with similar results. Two different glycosylated forms of the Kit receptor are indicated. CG = complex glycosylation form; HM = high-mannose form. Total levels of c-KIT and β-actin are shown as loading controls. C) Proliferation of BMMCs carrying the Kit D814V mutation upon loss of Tet2. Data show average percentage ±SEM of BrdU-positive BMMCs across genotypes, n = 3, *P<.05, ns = not significant. D) Differentiation of Kit D814V positive BMMCs in the absence of Tet2. Data show average percentage ±SEM of double positive (Fcε+c-Kit+) BMMCs from Tet2+/+;Kit D814V, Tet2+/−;Kit D814V and Tet2−/−;Kit D814V (64.1±8.2) after 4 weeks in culture with mIL-3. n = 3, *P = <05, **P<.01, ns = not significant E) Representative images of Tet2+/+;Kit D814V,Tet2+/−;Kit D814V and Tet2−/−;Kit D814V BMMCs. Scale bar = 20 µm. Arrows indicate cells containing granules, which is indicative of a more differentiated phenotype.
Figure 4.
Tet2 deletion affects progression but not initiation of Kit D814V-driven ALL.
A) Leukocyte counts in moribund leukemic animals according to genotype. Each data point represents an individual animal. **P<.01. B) Leukemic infiltration of the spleen expressed as weight (in mg) across genotypes. C) Latency of disease (expressed as weeks after the last pI:C injection when mice were found moribund) across genotypes. Data in A B, C is presented as mean ±SEM (n = 3–12 per group). D) Kaplan-Meier plot demonstrating survival of recipients transplanted with Tet2+/+;Kit D814V (black line),Tet2+/−;Kit D814V (grey line) and Tet2−/−;Kit D814V (black dotted line) lymphoid leukemic blasts (median survival, 13, 13 and 11 days respectively, n = 10–15 per group, P = .009). E) Leukocyte counts in secondary moribund leukemic animals according to genotype. Each data point represents an individual animal. F) Leukemic infiltration of the spleen expressed as weight (in mg) across genotypes of secondary animals. ns = not significant.
Figure 5.
Expression of Kit D814V and loss of Tet2 in MC partially recapitulates the Mx1-Cre-mediated phenotype.
A) Cutaneous mastocytosis in mice expressing Kit D814V under the control of a mast cell-specific Cre (Mcpt5-Cre) promoter. Graph represents the average number of mast cells/skin section across listed genotypes. N = 56–96 from 3–4 independent animals per genotype. ns = not significant. Numbers 1–6 indicate the following genotypes: 1 = WT ctr, 2 = Tet2+/+;Kit D814;Mcpt5-Cre, 3 = Tet2+/−;Kit D814;Mcpt5-Cre, 4 = Tet2−/−;Kit D814V;Mcpt5-Cre, 5 = Tet2+/−;Kit WT;Mcpt5-Cre,6 = Tet2−/−;Kit WT;Mcpt5-Cre, B) Histological score of disease in affected Mcpt5-Cre animals. Each skin region was scored according to the criteria reported in Table 1. Bar graph represents the percentage of sections per genotype with a defined histology score (from 3 to >4). Mice analyzed were 9-month-old (n = 3–4 per genotype). C) Representative microphotographs showing mast cell infiltration in Tet2+/+; Kit D814V;Mcpt5-Cre (dermal lymph node), Tet2+/−;Kit D814V;Mcpt5-Cre and Tet2−/−;Kit D814V;Mcpt5-Cre animals (skin sections). Mast cells stain dark blue in these sections. Scale = 100 µm.
Figure 6.
Knock down of TET2 enhances the response of HMC-1.2 to epigenetic modifiers.
A) HMC-1.2 transduced with two hairpins against TET2 (TET2 sh-1 and TET2 sh-3) or a control shRNA (ctr sh) were treated for 72 hs with low doses of DAC (0.5 µM) or DMSO. After 72 hs, cells were washed and resuspended in media containing DMSO or DASA at a concentration of 1 µM. Annexin V staining was performed 24 hs after the addition of DASA. Bar graph shows percentage of apoptotic cells (Annexin V+ 7-AAD−) and dead cells (Annexin V+ 7-AAD+) for each treatment condition. (n = 3; data are expressed as mean ±SEM, *P<.05. B) Western blot showing cleavage of CASPASE 3 in HMC-1.2 with or without TET2 shRNA, treated with DAC and DASA or vehicle control. β-actin levels were used as a loading control. Numbers below the β-actin blot indicate the fold change to untreated of the ratio between the cleaved CASPASE 3 band and the loading control, as quantified by densitometry. One of two independent experiments with similar results is shown. C) Phosphorylation of SRC and STAT5 after treatment with DASA, with and without pretreatment with DAC. Total SRC and total STAT5 levels were determined to confirm equal loading. One of two or three independent experiments is shown.