(A) Schematic depicting the strategy for generation of an inducible IDH2^R140Q allele in the mouse. Following Flp-mediated recombination, the IDH2^R140Q cDNA flanked by a tetracycline response element (TRE) and a protamine-1 poly-A cassette is integrated into the ColA1 locus. The PGK promoter drives hygromycin resistance, allowing selection of integrants.
(B) 2-HG in BM MNCs measured by LC-MS following 8 weeks of transgene induction.
(C) Representative flow cytometry analysis of bone marrow mononuclear cells and splenocytes from mice treated with doxycycline for 8 weeks or 7 months. Numbers indicate cells within the gate as a percentage of all living cells in the sample.
(D) Spleen weights of mice following 8 weeks of doxycycline treatment.
(E) Percentage of cKit⁺Sca1⁺Lin⁻ (KSL) cells in the spleens of mice following 8 weeks of doxycycline treatment.
(F) Percentage of KSL cells in the bone marrow of mice following 7 months of doxycycline treatment.
(G) Percentage of CD48⁻CD150⁺KSL hematopoietic stem cells in the bone marrow of mice following 7 months of doxycycline treatment.
Error bars, mean ± SEM; p value calculated using a two-tailed unpaired Student’s t test. See also .

(A) Methylcellulose CFU-GEMM assay using KSL cells sorted from IDH2^R140Q–t or control mice following 8 weeks of doxycycline treatment. Doxycycline was added to the media to maintain transgene expression throughout the differentiation process (n = 7 biological replicates/group).
(B) Methylcellulose CFU-GEMM assay done as in (A) but with or without adding doxycycline to the medium (n = 3 biological replicates/group).
(C and D) Methylcellulose CFU-GEMM assay done as in (A), with doxycycline in the medium and with or without the mutant IDH2 inhibitor AGI-6780. 2-HG was quantified by LC-MS in cells collected from the assay after 7 days of culture. b.q.l., below quantitative limit (C). Colonies were scored (n = 4 biological replicates/ group) in (D).
Error bars, mean ± SEM; p value calculated using a two-tailed unpaired Student’s t test. See also .

(A and B) Microarray analysis of cells cultured for 3 days in the methylcellulose CFU-GEMM assay (n ≥ 3 biological replicates/group). Hierarchical clustering of significantly altered genes (p < 0.05, fold change >1.5) is shown. Green, downregulated in IDH2^R140Q–t cells; red, upregulated in IDH2^R140Q–t cells (A). Pathway enrichment analysis shows genes that are downregulated in IDH2^R140Q–t cells (green) or upregulated (red) in IDH2^R140Q–t cells. The significance of the enrichment for each pathway (based on the false discovery rate q value) is proportional to the size of the gray box (i.e., the larger the gray box, the more significant the enrichment) (B).
(C) qRT-PCR analysis of cells cultured as in (A) and (B) but with or without AGI-6780. For each of the transcripts examined, pharmacological inhibition of 2-HG production at least partially reverses transcriptional changes in IDH2^R140Q–t cells. Error bars, mean ± SEM.
See also .

(A) Schematic outlining the strategy for randomized deinduction experiments.
(B) Representative FACS plots of bone marrow from secondary recipients untreated or treated with doxycycline. Continued expression of IDH2^R140Q is essential for proliferation and/or maintenance of GFP/YFP⁺ leukemic cells and the onset of leukemia.
(C) Percentage of GFP/YFP⁺ cells in the bone marrow of secondary recipients untreated or treated with doxycycline (n = 5 recipients on doxycycline, n = 3 recipients off doxycycline).
(D) PCR on genomic DNA isolated from BM MNCs of doxycycline-treated and -untreated recipients using primers specific for MSCV-HoxA9-GFP and MSCV-Meis1a–YFP. Vector (i.e., MSCV-HoxA9-GFP or MSCV-Meis1a–YFP, respectively) DNA (V) and wild-type C57BL/6 genomic DNA (G) were used as controls.
(E) Percentage of GFP/YFP⁺ cells in the bone marrow of secondary recipients untreated or treated with doxycycline at various time points following transplantation. Randomized deinduction was performed 2 weeks after transplantation (n = 2–3 recipients/time point/treatment).
(F) Percentage of blasts in the bone marrow of secondary recipients untreated or treated with doxycycline at various time-points following transplantation. Randomized deinduction was performed 2 weeks after transplantation (n = 2–3 recipients/time point/treatment). Blasts were scored on May-Grunwald-Giemsa-stained bone marrow cytospins.
See also .

(A) Representative H&E staining of tissue sections from IDH2^R140Q–t;Flt3^ITD compound transgenic animals and an Flt3^ITD control animal. Compound transgenic animals develop acute myeloid (AML-like) and lymphoid (ALL-like) malignancies whereasFlt3^ITD animals develop myeloproliferative disease (MPD). Left panels: a compound transgenic animal diagnosed with AML-like disease. Apoptotic debris and numerous tingible body macrophages (magnified in inset, upper left panel) characteristic of highly proliferative disease are evident throughout. Infiltration of intermediate-sized blast-like immature mononuclear cells (magnified in inset, upper right panel) is evident in the red pulp of the spleen, the liver, and kidney. Middle panels: a compound transgenic animal diagnosed with ALL-like disease. T lymphoblasts (confirmed by immunohistochemical staining using anti-CD3ε antibody, not shown) with high-grade morphology are present in the thymus, spleen, liver, and lung. Numerous mitoses are present, characteristic of a rapidly proliferative disease. Right panels: MPD in an Flt3^ITD animal with a hypercellular bone marrow with myeloid maturation and increased myeloid cells in the spleen. Original magnification, 600×.
(B) Examples of FACS plots demonstrating the types of acute leukemia diagnosed in IDH2^R140Q–t;Flt3^ITD compound transgenic animals. Left panels: presence of CD3ε⁺cKit⁺ lymphoblasts in the thymus and bone marrow; cells in the thymus are exclusively CD8⁺CD4⁻. Right panels: cells in the bone marrow are almost exclusively Mac1^+/low and a population of Mac1^lowcKit⁺ immature cells is evident; in the thymus of the same animal, CD3ε⁻CD8⁺ lymphoblasts are present.
(C and D) Primary AML (BM MNCs from donor #5517) and ALL (thymocytes from donor #12063) cells recapitulate a lethal leukemia in transplanted recipients treated with doxycycline. FACS plots demonstrate the overwhelming majority of donor-derived (CD45.2⁺) cells in the bone marrow of moribund recipients and the loss of lineage markers (Mac1 and CD3ε, respectively) upon serial transplantation.
See also .

(A) Schematic outlining the strategy for randomized deinduction experiments using AML cells from recipient #5517.
(B) May-Grunwald-Giemsa-stained bone marrow cytospins showing presence of leukemic blasts and differentiated myeloid cells in doxycycline-treated and -untreated recipients, respectively.
(C) Examples of FACS plots showing reduced donor chimerism in untreated versus doxycycline-treated recipients based on differential staining for the cell surface markers CD45.1/CD45.2.
(D and E) Genetic deinduction of IDH2^R140Q expression by withdrawal of doxycycline has a profound effect on the proliferation and/or differentiation of leukemic blasts in vivo. Percentage of donor-derived (CD45.2⁺) cells (D) and blasts (E) in the bone marrow of primary, secondary, or tertiary recipients untreated or treated with doxycycline is shown. Blasts were scored on May-Grunwald-Giemsa-stained bone marrow cytospins. Randomized deinduction was performed 3 days after transplantation.
(F) Model for the role of IDH2^R140Q in primary HSPCs and in the maintenance of leukemia.
See also .