Loss of epigenetic regulator TET2 and oncogenic KIT regulate myeloid cell transformation via PI3K pathway
Lakshmi Reddy Palam, Raghuveer Singh Mali, Baskar Ramdas, Sridhar Nonavinkere Srivatsan, Valeria Visconte, Ramon V. Tiu, Bart Vanhaesebroeck, Axel Roers, Alexander Gerbaulet, Mingjiang Xu, Sarath Chandra Janga, Clifford M. Takemoto, Sophie Paczesny, Reuben Kapur
Lakshmi Reddy Palam, Raghuveer Singh Mali, Baskar Ramdas, Sridhar Nonavinkere Srivatsan, Valeria Visconte, Ramon V. Tiu, Bart Vanhaesebroeck, Axel Roers, Alexander Gerbaulet, Mingjiang Xu, Sarath Chandra Janga, Clifford M. Takemoto, Sophie Paczesny, Reuben Kapur
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Research Article Hematology

Loss of epigenetic regulator TET2 and oncogenic KIT regulate myeloid cell transformation via PI3K pathway

Abstract

Mutations in KIT and TET2 are associated with myeloid malignancies. We show that loss of TET2-induced PI3K activation and -increased proliferation is rescued by targeting the p110α/δ subunits of PI3K. RNA-Seq revealed a hyperactive c-Myc signature in Tet2–/– cells, which is normalized by inhibiting PI3K signaling. Loss of TET2 impairs the maturation of myeloid lineage–derived mast cells by dysregulating the expression of Mitf and Cebpa, which is restored by low-dose ascorbic acid and 5-azacytidine. Utilizing a mouse model in which the loss of TET2 precedes the expression of oncogenic Kit, similar to the human disease, results in the development of a non–mast cell lineage neoplasm (AHNMD), which is responsive to PI3K inhibition. Thus, therapeutic approaches involving hypomethylating agents, ascorbic acid, and isoform-specific PI3K inhibitors are likely to be useful for treating patients with TET2 and KIT mutations.

Authors

Lakshmi Reddy Palam, Raghuveer Singh Mali, Baskar Ramdas, Sridhar Nonavinkere Srivatsan, Valeria Visconte, Ramon V. Tiu, Bart Vanhaesebroeck, Axel Roers, Alexander Gerbaulet, Mingjiang Xu, Sarath Chandra Janga, Clifford M. Takemoto, Sophie Paczesny, Reuben Kapur

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Figure 3

Loss of Tet2 alters mast cell differentiation, development, and proliferation in a gene dosage-dependent manner.

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Loss of Tet2 alters mast cell differentiation, development, and prolifer...
(A) BM cells were harvested from WT, Tet2+/–, and Tet2–/– mice, and BMMCs were generated. Cells were harvested after 1 week, 2 weeks, or 3 weeks of culture and stained with antibodies that recognize KIT and IgE receptor followed by flow cytometry. The percentage of cells double positive for KIT and IgE receptor is shown in top right quadrant of dot plot. Dot plot for 1 of 3 representative experiments is shown. (B) Flow cytometric quantification of IgE receptor–positive or KIT receptor–positive or IgE receptor and KIT receptor double-positive cells for WT, Tet2+/–, and Tet2–/– genotypes is shown. *P < 0.05, n = 3, mean ± SD. (C) Cytospin images of BMMCs from the indicated genotypes (original magnification, ×400). (D) Tet2, Cebpa, c-Myc, and Kit mRNA levels relative to β-actin mRNA levels in WT and Tet2–/– BMMCs were measured using QRT-PCR. *P < 0.05, n = 3, mean ± SD. (E) BMMCs from WT, Tet2+/–, and Tet2–/– mice were starved of serum/cytokines for 6 hours and cultured in the presence or absence of IL-3 for 48 hours, and cell proliferation was evaluated by [3H] thymidine incorporation. Counts per minute (CPM) are shown. n = 3, mean ± SD, *P < 0.05. Similar results were observed in 3 independent experiments. (F) BMMCs from WT and Tet2–/– mice were cultured in the absence of serum/cytokines for 18 hours or 48 hours, and these cells were stained with annexin V and 7-AAD followed by flow cytometry analysis. Representative dot plot of 3 independent experiments is shown. Flow cytometry quantification of percentage cell survival (annexin V and 7-AAD double negative) after 48 hours of cytokine withdrawal is shown. n = 3, mean ± SD, *P < 0.05. One-way ANOVA analysis (B) and unpaired, 2-tailed Student’s t test (D–F) methods were used for statistical analysis.