Journal: Cancer Science

Article Title: Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells

doi: 10.1111/cas.15994

Figure Lengend Snippet: Intracellular 2‐hydroxyglutarate‐dependent signal transducer and activator of transcription (STAT) phosphorylation promoted cell proliferation in IDH mutant (mut) acute myeloid leukemia (AML) cells. (A) Mass cytometric analysis of the IDH WT and IDH1/2 mut bone marrow cells of AML patients. The viSNE analysis identified genotype‐specific dominant cell populations. The phosphorylation of STAT1/3/5 proteins in each population is shown. (B) The live cell number of IDH2 WT or mut TF‐1 cells cultured under cytokine‐free conditions treated without (DMSO) or with the mut IDH2‐specific inhibitor (AG‐221) in vitro ( n = 6 from two independent experiments). (C) Concentrations of intracellular 2‐hydroxyglutaric acid in IDH2 WT and IDH2 mut TF‐1 cells treated without (DMSO) or with AG‐221 for 10 days. ( n = 3). (D) Western blot images of total and phosphorylated STAT5 in cell lysates of IDH2 WT and IDH2 mut TF‐1 cells treated without (DMSO) or with AG‐221 for 10 days. β‐Actin (ACTB) was used as a loading control. (E) Concentration of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in the cell culture supernatant of IDH2 WT and IDH2 mut TF‐1 cells under cytokine‐free conditions treated without (DMSO) or with AG‐221 for 10 days. * p < 0.05, ** p < 0.01, *** p < 0.001 (two‐tailed t ‐test). N.D., not detected.

Article Snippet: TF‐1 cells (CRL‐2003; ATCC) and TF‐1 IDH2 mut cells (CRL‐2003IG; ATCC), in which the homozygous c.419G > A knock‐in mutation encoding the IDH2R140Q protein was induced by CRISPR/Cas9 technology, were purchased from the ATCC.

Techniques: Phospho-proteomics, Mutagenesis, Cell Culture, In Vitro, Western Blot, Control, Concentration Assay, Two Tailed Test

Journal: Cancer Science

Article Title: Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells

doi: 10.1111/cas.15994

Figure Lengend Snippet: Downregulation of phospholipase C (PLC) expression contributed to the growth advantage in AG‐221‐treated IDH2 mutant (mut) acute myeloid leukemia cells. (A) Principal component (PC) analysis plot of a metabolome analysis of IDH2 WT and IDH2 mut TF‐1 cells treated without (DMSO) or with AG‐221 for 10 days. (B) Metabolite Set Enrichment Analysis plots of the metabolome dataset derived from IDH2 WT and IDH2 mut TF‐1 cells treated with AG‐221 were compared. The top 25 significantly enriched metabolite sets are shown. (C) Drug screening using a metabolic inhibitor library: IDH2 mut TF‐1 cells were maintained with 2 ng/mL thrombopoietin and treated with each drug or the DMSO control, followed by an evaluation of cell growth. Candidate drugs that suppressed or promoted cell growth (fold change >2 or <0.5 from the DMSO control) are shown ( n = 3). (D) Gene Ontology (GO) analysis of biological processes significantly enriched in IDH2 mut TF‐1 cells compared to IDH2 WT TF‐1 cells ( p < 0.05 Fisher's test, >5‐fold enrichment; blue, downregulated). (E) Gene Set Enrichment Analysis comparing IDH2 WT and IDH2 mut TF‐1 cells in the indicated GO terms. (F) mRNA expression level of PLCB1 and PLCG1 in IDH2 WT and IDH2 mut TF‐1 cells treated without (DMSO) or with AG‐221 for 10 days. Expression level is represented in reads per kilobase of exon per million mapped reads (RPKM). (G) Western blot images of PLCG1 protein in IDH2 WT and IDH2 mut TF‐1 cells treated without (DMSO) or with AG‐221 for 10 days. β‐Actin (ACTB) was used as a loading control. (H) Methylation analysis of control (Ctrl) and IDH2 mut‐overexpressed TF‐1 cells treated without (DMSO) or with mut IDH2‐specific inhibitor (AGI‐6780) for 7 days. Published dataset (GSE51352) was reanalyzed. * p < 0.05, ** p < 0.01, *** p < 0.001 (two‐tailed t ‐test). n.s., not significant.

Article Snippet: TF‐1 cells (CRL‐2003; ATCC) and TF‐1 IDH2 mut cells (CRL‐2003IG; ATCC), in which the homozygous c.419G > A knock‐in mutation encoding the IDH2R140Q protein was induced by CRISPR/Cas9 technology, were purchased from the ATCC.

Techniques: Expressing, Mutagenesis, Derivative Assay, Drug discovery, Control, Western Blot, Methylation, Two Tailed Test

Journal: Cancer Science

Article Title: Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells

doi: 10.1111/cas.15994

Figure Lengend Snippet: Apoptosis resistance in IDH2 mutant (mut) acute myeloid leukemia (AML) cells through the downregulation of intracellular arachidonic acid. (A) Scheme of phospholipase C (PLC)‐mediated arachidonic acid release from the phospholipid bilayer. DAG, diacylglycerol. (B) Quantities of intracellular arachidonic acid measured with gas chromatography–mass spectrometry in IDH2 WT and mut TF‐1 cells treated without (DMSO) or with AG‐221 for 10 days. Values relative to IDH2 WT TF‐1 cells treated with DMSO are depicted. Statistical analyses compared with DMSO‐treated IDH2 WT TF‐1 cells were carried out with the paired two‐tailed t ‐test ( n = 4–5 from four to five independent experiments). (C) Representative FACS histogram of JC‐1 red and geometric mean fluorescent intensity (MFI) of JC‐1 red in IDH2 WT and mut TF‐1 cells treated without (DMSO) or with AG‐221 for 10 days ( n = 5–6 from two independent experiments). (D) Western blot images of cytochrome c in cytosolic fraction of IDH2 WT and IDH2 mut TF‐1 cells treated without (DMSO) or with AG‐221 for 10 days. β‐Actin (ACTB) was used as a loading control. (E) The percentage of annexin V + cells in IDH2 WT and mut TF‐1 cells treated without (DMSO) or with AG‐221 for 3 days ( n = 6 from two independent experiments). (F) The percentage of annexin V + cells in IDH2 WT and mut TF‐1 cells cultured with erythropoietin (EPO) treated without (DMSO) or with AG‐221 for 7 days ( n = 5–6 from two independent experiments). (G) mRNA expression levels of the indicated genes in the bone marrow mononuclear cells of AML patients with IDH2 gene mutations and IDH WT AML patients. p adj, adjusted p value. (H) Gene Ontology analysis of biological processes significantly enriched in IDH2 mut TF‐1 cells compared to IDH2 WT TF‐1 cells ( p < 0.05 Fisher's test, >5‐fold enrichment; red, upregulated). * p < 0.05, ** p < 0.01, *** p < 0.001 (two‐tailed t ‐test).

Article Snippet: TF‐1 cells (CRL‐2003; ATCC) and TF‐1 IDH2 mut cells (CRL‐2003IG; ATCC), in which the homozygous c.419G > A knock‐in mutation encoding the IDH2R140Q protein was induced by CRISPR/Cas9 technology, were purchased from the ATCC.

Techniques: Mutagenesis, Gas Chromatography, Mass Spectrometry, Two Tailed Test, Western Blot, Control, Cell Culture, Expressing

Journal: Cancer Science

Article Title: Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells

doi: 10.1111/cas.15994

Figure Lengend Snippet: Anti‐inflammatory drug treatment combined with the inhibition of mutant (mut) IDH2 induced apoptosis in IDH2 mut acute myeloid leukemia cells by normalizing intracellular arachidonic acid levels in vitro. (A) Scheme of anti‐inflammatory drug therapy targeting the arachidonic acid metabolic pathway. The inhibition of COX2 and 5‐lipoxygenase (5‐LOX) with celecoxib and zileuton, respectively, led to the intracellular accumulation of arachidonic acid, which induced cellular apoptosis. (B) Live cell numbers of IDH2 WT or IDH2 mut TF‐1 cells cultured under cytokine‐free conditions treated with AG‐221, celecoxib, and zileuton at the indicated combinations in vitro ( n = 6 from two independent experiments). (C, D) Representative FACS plots of (C) annexin V staining and (D) the percentage of annexin V + cells in IDH2 WT and IDH2 mut TF‐1 cells treated with AG‐221, celecoxib, and zileuton at the indicated combinations for 10 days ( n = 6 from two independent experiments). (E) Quantities of intracellular arachidonic acid measured with gas chromatography–mass spectrometry in IDH2 WT and IDH2 mut TF‐1 cells treated with AG‐221, celecoxib, and zileuton at the indicated combinations for 10 days. Relative values compared to IDH2 WT TF‐1 cells treated with DMSO are depicted ( n = 4–5 from four or five independent experiments). (F) The live cell number and (G) the percentage of annexin V + cells of IDH2 mut TF‐1 cells cultured with erythropoietin (EPO) treated with AG‐221, celecoxib, and zileuton at the indicated combinations in vitro for 10 days. ( n = 6 from two independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001 (two‐tailed t ‐test). AA, arachidonic acid; DAG, diacylglycerol; LT, leukotriene; PG, prostaglandin; PLC, phospholipase C.

Article Snippet: TF‐1 cells (CRL‐2003; ATCC) and TF‐1 IDH2 mut cells (CRL‐2003IG; ATCC), in which the homozygous c.419G > A knock‐in mutation encoding the IDH2R140Q protein was induced by CRISPR/Cas9 technology, were purchased from the ATCC.

Techniques: Inhibition, Mutagenesis, In Vitro, Cell Culture, Staining, Gas Chromatography, Mass Spectrometry, Two Tailed Test

Journal: Cancer Science

Article Title: Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells

doi: 10.1111/cas.15994

Figure Lengend Snippet: Anti‐inflammatory drug treatment combined with the inhibition of mutant (mut) IDH2 reduced tumor burden by inducing apoptosis in IDH2 mut acute myeloid leukemia cells in vivo. (A) Scheme of the in vivo drug treatment experiment on a xenograft model: IDH2 mut TF‐1 cells were precultured in cytokine‐free medium for 10 days and transplanted into the left femur of sublethally (2.5 Gy)‐irradiated MSTRG mice. Seven weeks after transplantation, transplanted mice were treated with drugs by oral gavage for 3 weeks. Peripheral blood was examined every week during the drug treatment and bone marrow (BM) was analyzed at the end of the drug treatment. (B) The chimerism of human CD45 + cells and (C) the percentage of annexin V + cells within the human CD45 + fraction in BM cells derived from the left femur of drug‐treated mice ( n = 5–6 from three independent experiments). (D) Schematic summary of the molecular basis underlying the acquisition of growth advantage by IDH2 mutant AML and its cancelation: IDH2 gene mutations induce signal transducer and activator of transcription (STAT) phosphorylation dependent on intracellular 2‐hydroxyglutarate level and apoptosis resistance driven by phospholipid metabolic adaptation. A treatment with a mut IDH2‐specific inhibitor blocks the STAT‐mediated growth advantage in IDH2 mut cells, while maintaining the survival advantage by resistance to apoptosis through phospholipid metabolic adaptation. An additional treatment with COX2 and 5‐lipoxygenase (5‐LOX) inhibitors targeting the metabolism of arachidonic acid cancels resistance to apoptosis and eradicates IDH2 mut AML cells. * p < 0.05, ** p < 0.01 (two‐tailed t ‐test). 2‐HG , 2‐hydroxyglutarate; AA, arachidonic acid; Ctrl, control; n.s., not significant; PB, peripheral blood.

Article Snippet: TF‐1 cells (CRL‐2003; ATCC) and TF‐1 IDH2 mut cells (CRL‐2003IG; ATCC), in which the homozygous c.419G > A knock‐in mutation encoding the IDH2R140Q protein was induced by CRISPR/Cas9 technology, were purchased from the ATCC.

Techniques: Inhibition, Mutagenesis, In Vivo, Irradiation, Transplantation Assay, Derivative Assay, Phospho-proteomics, Two Tailed Test, Control