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 5

Deficiency of TET2 in BMMCs induces c-MYC upregulation via PI3K activation.

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Deficiency of TET2 in BMMCs induces c-MYC upregulation via PI3K activati...
(A) c-MYC protein levels in BMMCs from WT, Tet2–/–, and Tet2–/–:p85α–/– mice were assessed. A representative experiment from 3 independent experiments is shown. Cell lysates collected from Tet2–/– cells that were treated with or without 0.5 μM GDC-0941 (p110αδ-specific PI3K inhibitor) for 12 hours were subjected to Western blot analysis using an anti–c-MYC antibody. A representative experiment from 3 experiments is shown. (B and D) c-Myc, Eno1, Bcat1, Shmt1, Npm1, Id2, Odc1, Tet2, Fos, and JunB mRNA levels in BMMCs from WT, Tet2–/–, and Tet2–/–:p85α–/– mice were measured using QRT-PCR. *P < 0.05, n = 3, mean ± SD. (C) c-Myc mRNA knockdown in Tet2–/– BMMCs was achieved using Myc shRNA. Luciferase shRNA was used as a control. Shown are c-Myc knockdown levels, as assessed by QRT-PCR. These Myc-shRNA– and Luc-shRNA–harboring cells were starved for 6 hours and cultured in the presence or absence of IL-3. After 48 hours, cell proliferation was evaluated by [3H] thymidine incorporation. Counts per minute (CPM) are shown. n = 3, mean ± SD, **P < 0.05. (E) Retroviral JunB-GFP construct was transduced into Tet2–/– BMMCs and sorted for GFP-positive cells, and c-Myc mRNA levels were measured using QRT-PCR. mRNA levels were assessed relative to β-actin mRNA by QRT-PCR assay (B–E). One-way ANOVA (B and D) and unpaired, 2-tailed Student’s t test (C and E) were used for statistical analysis.