Journal: Cell Research

Article Title: Vulnerabilities in mIDH2 AML confer sensitivity to APL-like targeted combination therapy

doi: 10.1038/s41422-019-0162-7

Figure Lengend Snippet: mIDH2 leukaemia are sensitive to ATRA and ATO. a Schematic representation of the metastable state of HoxA9/Meis1a/mIDH2 cells: LSD1 activation sensitizes to ATRA, while the effect of this pro-differentiating stimuli is counteracted by high PIN1 levels. Treatment of mIDH2 cells with pharmacological doses of ATRA targets PIN1 for degradation and enables full LSD1 activation to promote differentiation. b–c, Cytospin images and quantitation of human TF1 cell line stably overexpressing the mutant variant R140Q of IDH2 (mIDH2) and the respective controls (CTRL) that have been treated with pharmacological concentrations of ATRA (10–6M) or vehicle (DMSO). Data are means ± SD; t-test; *p ≤ 0.05. d Colony forming assay of mouse leukaemia cells harbouring mutant or wildtype IDH2 and treated with the PIN1 inhibitor (Juglone, 1 μM) or respective vehicle (VHL). Colonies were quantitated 12 days after plating. Data are means ± SD; one-way anova; ***p ≤ 0.001. e Colony forming assay of TF1 cells overexpressing the mIDH2 or respective control (CTRL) and treated with the PIN1 inhibitor (Juglone, 1 μM) or respective vehicle (VHL). Colonies were quantitated after 7 days from plating. Data are means ± SD; one-way anova; ***p ≤ 0.001. f Schematic representation of the metastable state of HoxA9/Meis1a/mIDH2 cells. Increased one-carbon (1C-) metabolism and oxidative stress (ROS) sensitize cells to pro-oxidant agents such as arsenic trioxide (ATO). Excessive and impaired scavenging of ROS induces DNA damage and leads to cell death. The induction of 1C-metabolism and glutathione synthesis is essential to ensure survival of leukaemia cells. Thus, block of this antioxidant effect by treatment with ATO pushes cells towards collapse and death. g Fold induction of cell death (Annexin V/7AAD positive cells) in mouse leukaemia cells isolated from second or third recipients and treated in vitro with ATO (0.5 μM) for 96 h. Data are means ± SD; t-test; *p ≤ 0.05. h Fold induction of cell death (Annexin V/7AAD positive cells) in TF1 leukaemia cells overexpressing IDH2R140Q and treated with ATO (0.5 μM) for 96 h. Data are means ± SD; t-test; ns not significant, *p ≤ 0.05. i Representative images of methylcellulose colony forming assay and colony quantification of mouse leukaemia cells isolated from second transplants (early stage) and treated in vitro with pharmacological concentrations of ATRA (1 μM), ATO (0.5 μM), a combination of both (ATRA + ATO), or vehicle (VHL) as a control. Treatments were performed in either the presence or absence of the IDH mutation (CTRL). Data are mean ± SD, one-way anova; **p ≤ 0.01, ***p ≤ 0.001. j Representative images of colony forming assay of HoxA9/Meis1A/mIDH2 mouse leukaemia cells isolated from third recipients (late stage) and treated with ATRA (1 μM), ATO (0.5 μM), a combination of both (ATRA + ATO), or vehicle (VHL) as a control. Histograms show the quantification of colony forming ability. Leukaemia cells were maintained on doxycycline (DOX ON) to favour the expression of the mIDH2 and increased 2HG levels or out of doxycycline (DOX OFF) to evaluate sensitivity in the context of mIDH-independence. Data are mean ± SD, one-way anova; ***p ≤ 0.001. k Quantification of colony forming ability of mouse leukaemia cells harbouring mutations in IDH2 (IDH2R140Q) or IDH1 (IDH1R132C) in association with multiple combinatorial genetic driver such as NPMc+ or FLT3ITD and treated with pharmacological concentrations of ATRA (1 μM), ATO (0.5 μM), a combination of both (ATRA + ATO), or vehicle (VHL) as a control. Non-mutant IDH2 cells such as MLL-AF9 or NPMc+/FLT3ITD were also included. Data are mean ± SD; one-way anova; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. See also Supplementary Information, Figs. S4–S7

Article Snippet: Vectors and constructs To generate stable lines for mutant IDH expression, TF1 and U937 cells were infected with lenti-virus pLVX-IRES –neo vector (Clontech #6321810) or pCIG3 (pCMV-IRES_GFP, from Felicia Goodrum, Addgene plasmid # 78264), or retro-virus pBABE-GFP (from William Hahn, Addgene plasmid # 10668) containing full length IDH2/R140Q, IDH1/R132C or respective empty vectors as controls.

Techniques: Activation Assay, Quantitation Assay, Stable Transfection, Mutagenesis, Variant Assay, Blocking Assay, Isolation, In Vitro, Expressing