Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: ( A ) Expression analysis of FTO and ALKBH5 among 57 primary patients with T-ALL and 21 normal T cells from GSE33469 and GSE33470 . The expression values are presented as box-and-whisker plots with a log2 scale. ( B ) Experimental scheme for intracellular NOTCH1 (ICN1)–induced T-ALL model. Bone marrow (BM) lineage-negative (Lin − ) cells were isolated from the mice as depicted and infected with MigR1-GFP-ICN1 retrovirus and then transplanted into irradiated recipient mice. ( C and D ) Percentage of GFP + leukemia cells in peripheral blood (PB) of recipient mice at the indicated time after BM transplantation (BMT). Recipient mice were transplanted with ICN1-transduced Fto fl/fl ( n = 14), Fto −/− ( n = 16) (C), Alkbh5 fl/fl ( n = 12), or Alkbh5 −/− ( n = 14) (D) BM Lin − cells as indicated. ( E ) Representative images of Wright-Giemsa staining of PB from recipient mice as indicated. Mice were euthanized at the same time when the Fto fl/fl or Alkbh5 fl/fl control became moribund. The scale bar represents 20 μm. ( F ) Spleen image (left) and flow cytometry analysis of GFP + leukemia cells in the spleen (right) are shown. ( G ) Hematoxylin and eosin (H&E) staining (left) and PCNA immunohistological images (right) in spleen sections from indicated mice. The scale bar represents 20 μm. PCNA, proliferating cell nuclear antigen. ( H and I ) Kaplan-Meier survival curves for recipient mice transplanted with ICN1-transduced BM Lin − cells from Fto fl/fl ( n = 14), Fto −/− ( n = 13) (H), Alkbh5 fl/fl ( n = 11), or Alkbh5 −/− ( n = 13) (I). Data are represented as means (±SD). Data were analyzed by Welch t test (A), Mann-Whitney test [(C) and (D)], and log-rank test [(H) and (I)]. ** P < 0.01 and *** P < 0.001. n.s., nonsignificant.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: Expressing, Whisker Assay, Isolation, Infection, Irradiation, Transplantation Assay, Staining, Control, Flow Cytometry, MANN-WHITNEY

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: ( A ) Heatmap showing differentially expressed genes in KOPTK1 cells with or without FTO shRNA expression. Differential genes are derived from RNA-seq data and defined as |fold change| ≥ 1.5, P value <0.05. ( B ) KEGG enrichment analysis of down-regulated transcripts in FTO -depleted KOPTK1 cells. ( C ) Cumulative distribution function representing the log2 peak intensity of m 6 A-modified sites in KOPTK1 cells with or without FTO knockdown. The P value was calculated using two-tailed Kolmogorov-Smirnov test. ( D ) Top consensus sequence motif identified within m 6 A peaks by hypergeometric optimization of motif enrichment (HOMER) in KOPTK1 cells with or without FTO knockdown. ( E ) Metagene profiles of m 6 A peak distribution along a normalized transcript composed of three rescaled nonoverlapping segments: 5′UTR, CDS, and 3′UTR in KOPTK1 cells with or without FTO knockdown. ( F ) Distribution of genes exhibiting a significant change in both m 6 A level and overall transcript level in FTO -depleted KOPTK1 cells. ( G ) KEGG pathway enrichment analysis with the 634 hyperdown genes shown in (F). ( H ) Bubble-rank plot of hyperdown transcripts in (F). The genes encoding transcription factors are highlighted in red. ( I ) Growth curves of FTO -depleted KOPTK1 cells with or without the ectopic expression of ELK3 (left). Immunoblots of FTO and ELK3 are shown on the right. Data are presented as means (±SD) from two biological replicates, each performed with three technical replicates. Data were analyzed by two-way ANOVA with Bonferroni’s multiple comparisons. *** P < 0.001.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: shRNA, Expressing, Derivative Assay, RNA Sequencing, Modification, Knockdown, Two Tailed Test, Sequencing, Western Blot

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: ( A ) Genome browser tracks showing the mRNA abundance and m 6 A peaks of ELK3 transcripts in KOPTK1 cells with or without FTO depletion. ( B ) qPCR analysis of ELK3 expression in FTO knockdown T-ALL cells as indicated. ( C ) mRNA (top) and protein (bottom) levels of Elk3 in primary murine T-ALL cells from Fto fl/fl or Fto −/− BMT as shown in fig. S2G ( n = 4 per group). ( D ) qPCR (left) and immunoblot (right) analysis of ELK3 expression in FTO -depleted KOPTK1 cells rescued with WT or MUT FTO. ( E ) Gene-specific m 6 A-qPCR analysis of the ELK3 mRNA in T-ALL cells as indicated. ( F ) FTO-RIP qPCR validation of FTO interaction with the ELK3 mRNA in T-ALL cells. ( G ) The mRNA half-life ( t 1/2 ) of ELK3 in FTO knockdown KOPTK1 cells with enforced expression of WT or MUT FTO. ( H ) Relative luciferase activity showing the WT or MUT ELK3 exon4 firefly luciferase reporter in 293T cells transduced with Control or FTO shRNA. ( I ) YTHDF2-RIP qPCR showing the binding of YTHDF2 to ELK3 mRNA in FTO knockdown leukemia cells. ( J ) The ELK3 mRNA level in FTO -deficient T-ALL cells with or without YTHDF2 shRNA expression. ( K ) Scatterplot showing the correlation of FTO and ELK3 in 108 T-ALL samples from databases of National Omics Data Encyclopedia (NODE) (OEP002748). The expression levels were log 2 (TPM). Pearson correlation coefficient ( R ) and P value ( P ) are marked. Data are presented as means (±SD) of technical triplicates, and the experiments were independently repeated at least twice with the similar results [(B) and (D) to (J)]. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons [(B), (D), and (J)], two-way ANOVA with Bonferroni’s multiple comparisons (H), and unpaired two-tailed Student’s t test [(C), (E), (F), and (I)]. *** P < 0.001, n.s., nonsignificant.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: Expressing, Knockdown, Western Blot, Biomarker Discovery, Luciferase, Activity Assay, Transduction, Control, shRNA, Binding Assay, Two Tailed Test

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: ( A ) Growth curves of ELK3 -depleted T-ALL cell lines (CUTLL1 and KOPTK1) and primary T-ALL cells. Data are presented as the mean ± SD from two biological replicates, each with three technical replicates. ( B ) KEGG pathway analysis of down-regulated genes in ELK3 -depleted KOPTK1 cells. ( C ) GSEA plot showing enrichment of gene sets of hallmark glycolysis in ELK3 knockdown CUTLL1 cells. ( D ) Glycolytic proton efflux rate (glycoPER) analyzed in ELK3 -depleted CUTLL1 cells using a Seahorse extracellular flux analyzer. ( E ) Heatmap presentation of glycolysis-related metabolites from metabolomic analysis in KOPTK1 cells ( n = 3 per group). ( F ) qPCR analysis of glycolytic gene expression in ELK3 -depleted KOPTK1 cells. ( G ) Density plot of ELK3 (red) and H3K27ac (blue) ( GSE76783 ) occupancy around the transcription start site (TSS) of genes significantly down-regulated in ELK3-depleted KOPTK1 cells. ChIP-seq signal normalized with input. ( H ) Genome browser tracks of ELK3 and H3K27ac ChIP-seq signals at the promoters of PGK1 (top) and PFKFB4 (bottom). ( I ) ChIP-qPCR validation of ELK3 and H3K27ac binding to PGK1 and PFKFB4 promoters in KOPTK1 cells. ( J ) qPCR analysis of glycolysis-related genes in FTO -depleted KOPTK1 cells with or without the overexpression of ELK3. ( K ) Glycolytic proton efflux rate (glycoPER) analyzed in FTO -depleted CUTLL1 cells with or without the overexpression of ELK3 . Data are presented as means (±SD) of technical triplicates, and the experiments were independently repeated at least twice with the similar results [(D), (F), (I), (J), and (K)]. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons (J), two-way ANOVA with Bonferroni’s multiple comparisons (A), and unpaired two-tailed Student’s t test [(D), (F), and (I)]. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: Knockdown, Gene Expression, ChIP-sequencing, ChIP-qPCR, Biomarker Discovery, Binding Assay, Over Expression, Two Tailed Test

Journal: Science Advances

Article Title: FTO regulates ELK3-mediated metabolic rewiring and represents a unique therapeutic target in T cell leukemia

doi: 10.1126/sciadv.adq3052

Figure Lengend Snippet: Elevated FTO expression in T-ALL maintains low levels of m 6 A methylation on many transcripts including the ELK3 mRNA, which prevents the YTHDF2-mediated degradation. Augmented ELK3 expression in turn up-regulates glycolysis-related genes like PGK1 and PFKFB4 , thereby enhancing glycolysis. Selective FTO inhibitor Dac51 leads to increased m 6 A modification on ELK3 mRNA, resulting in decreased ELK3 expression, ultimately lowering glycolytic capacity and suppressing T-ALL progression. See more details in the text.

Article Snippet: Antibodies used in the experiments include FTO (Proteintech, 27226-1-AP), ELK3 (OriGene, TA503603; NOVUS, NBP2-01264), ALKBH5 (Abcam, ab195377), and β-ACTIN (Abclonal, AC026).

Techniques: Expressing, Methylation, Modification