Loss of epigenetic regulator TET2 and oncogenic KIT regulate myeloid cell transformation via PI3K pathway
Lakshmi Reddy Palam, … , Sophie Paczesny, Reuben Kapur
Lakshmi Reddy Palam, … , Sophie Paczesny, Reuben Kapur
Published February 22, 2018
Citation Information: JCI Insight. 2018;3(4):e94679. https://doi.org/10.1172/jci.insight.94679.
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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 9

PI3K inhibitor treatment decreases proliferation of KIT mutation-bearing mouse primary BM-derived mast cells with TET2 loss and in human SM patient samples.

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PI3K inhibitor treatment decreases proliferation of KIT mutation-bearing...
(A) BM-derived mast cells (BMMCs) derived from WT, Tet2+/–, Kit+/D814V, and Tet2+/–:Kit+/D814V mice were starved for 6 hours and cultured in the presence or absence of IL-3 (10 ng/ml) or SCF (30 ng/ml) in combination with increasing doses of GDC-0941 (p110α/δ-specific inhibitor of PI3K). After 48 hours, proliferation was evaluated by [3H] thymidine incorporation. Counts per minute (CPM) are indicated. n = 3, mean ± SD. *P < 0.05 compared with WT cells, **P < 0.05 compared with untreated Tet2+/– cells, ***P < 0.05 compared with untreated Kit+/D814V cells, ****P < 0.05 compared with untreated Tet2+/–:Kit+/D814V cells, unpaired 2-tailed Student’s t test. (B) WT and Tet2–/– BMMCs were transduced with KitD814V retroviral vector and sorted to homogeneity. These cells were treated with increasing doses of GDC-0941 (p110α/δ-specific), TGX-221 (p110β-specific), or IC87114 (p110δ-specific) inhibitors. After 48 hours, ligand-independent proliferation was evaluated by [3H] thymidine incorporation. CPM are shown. A representative experiment performed in quadruplicate is shown. Similar results were observed in 3 independent experiments. *P < 0.05 compared with untreated Tet2–/–-KitD814Vcells, **P < 0.05 compared with untreated WT-KitD814Vcells, unpaired 2-tailed Student’s t test. (C) KitD814V encoding retroviral vector was transduced into BMMCs that were deficient in the indicated catalytic subunits of PI3K. Sorted GFP-positive cells were subjected to thymidine incorporation assay in the absence of ligand stimulation. No GF, no growth factor. After 48 hours, proliferation was evaluated by [3H] thymidine incorporation. CPM are shown. Data are from a single experiment performed in triplicates. Mean ± SEM, *P < 0.05 compared with WT-KitD814V, **P < 0.05 compared with p110α–/–β–/–-KitD814V, ***P < 0.05 compared with p110α–/–δ–/–-KitD814V, 1-way ANOVA analysis performed. (D–F) Growth of mastocytosis patient-derived (2 patients bearing both TET2 and KIT mutations and 2 KIT mutations but not TET2 mutation) cells in the presence of isoform-specific inhibitors of PI3K at indicated concentrations. Experiments were performed as described in B. Numbers on the x axis represent different patient samples. Experiments were performed in triplicates. Mean ± SEM, *P < 0.05, unpaired, 2-tailed Student’s t test.