Fig 2: Evaluation of ALKBH5 and FTO knockout. (A) Schematic representation of guide RNA (gRNA) locations. Two gRNAs targeting the third exon of FTO (for nickase CRISPR/Cas9) and two gRNAs targeting the first and third exons of ALKBH5 (for CRISPR/Cas9) are shown. The coordinates are according to the hg38 genome assembly. (B) Western blot of wild type and ΔALKBH5ΔFTO HEK293T cells using anti-ALKBH5, anti-FTO, and anti-ACTB as a loading control.

Fig 3: Decreased m6A Level is Mediated by FTO Upregulation in NPM1-mutated AML. (A) m6A dot blot analysis of the global m6A abundance in NPM1-mutated AML, NPM1-unmutated AML, and healthy donor samples. Methylene blue staining served as a loading control. (B) Heatmaps of m6A-related genes in AML patients with NPM1 mutations and without NPM1 mutations from TCGA (n=65), GSE14468 (n=187), and GSE15434 (n=251) datasets. (C) The expression patterns of FTO transcript in NPM1-mutated AML samples compared to NPM1-unmutated AML samples from the above three public datasets. (D–F) Survival analysis based on FTO, ALKBH5, METTL3 mRNA expression in TCGA primary NPM1-mutated AML samples. Survival analysis was performed using Kaplan–Meier method. (G) The expression patterns of FTO transcript in NPM1-mutated AML samples from Beat AML dataset (n=42). (H) Univariate Cox regression analyses of the association between clinicopathological factors (including FTO expression) and overall survival of NPM1-mutated AML patients from Beat AML dataset (n=42). (I) Multivariate Cox regression analyses of the association between clinicopathological factors (including FTO expression) and overall survival of NPM1-mutated AML patients from Beat AML dataset (n=42). (J, K) qRT-PCR and western blot analysis of the FTO expression in a panel of human acute myeloid leukemia cell lines. (L, M) qRT-PCR and western blot analysis of the FTO mRNA and protein level in OCI-AML3 cells infected with two independent shRNAs targeting FTO or a control shRNA. ß-actin was used as the internal control. The bar graph showed the relative level of FTO protein. (N) m6A dot blot analysis of the global m6A abundance in the FTO-silenced OCI-AML3 cells. Methylene blue staining served as a loading control. (O) Schematic presentation of the FTO domains and point mutations. (P, Q) qRT-PCR and western blot analysis of the FTO mRNA and protein level in OCI-AML3 cells following transfection of the HA-FTO-WT and HA-FTO-MUT plasmids. ß-actin was used as the internal control. The bar graph showed the relative level of FTO protein. (R) m6A dot blot analysis of the global m6A abundance in OCI-AML3 cells following transfection of the HA-FTO-WT and HA-FTO-MUT plasmids. Methylene blue staining served as a loading control. Data are presented as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001.

Fig 4: NPM1-mA Upregulates FTO Expression to Reduce the m6A Level through Impeding the Proteasome Pathway. (A, C) Western blot analysis of the FTO, NPM1-mA, and NPM1-wt protein level in the NPM1-mA-silenced OCI-AML3, NPM1-wt-enforced OCI-AML2, and NPM1-mA-enforced OCI-AML2 cells. The bar graph showed the relative level of protein. (B, D) qRT-PCR analysis of the FTO, NPM1-mA, and NPM1-wt mRNA level in the NPM1-mA-silenced OCI-AML3, NPM1-wt-enforced OCI-AML2, and NPM1-mA-enforced OCI-AML2 cells. (E) OCI-AML3 cells were treated with the ubiquitin-proteasome pathway inhibitor MG132 (20 µM), the autophagic-lysosomal pathway inhibitor CQ (20 µM), and activator rapamycin (20 µM) for 8 h. The bar graph showed the relative level of FTO protein. (F) Western blot analysis of the FTO protein level in the NPM1-silenced OCI-AML3 cells following treatment with MG132 (20 µM) for 8h. The bar graph showed the relative level of FTO protein. (G) Western blot analysis of the FTO and NPM1-mA protein level in the NPM1-mA-silenced OCI-AML3 cells following treatment with the protein synthesis inhibitor CHX (100 µg/mL) for the indicated time. The line graph showed the degradation rate of FTO protein. (H) Ubiquitination analysis of the ubiquitinated FTO level in the NPM1-mA-silenced OCI-AML3 cells. ß-actin was used as the internal control. (I, J) m6A dot blot analysis of the global m6A abundance in the NPM1-mA-silenced OCI-AML3, NPM1-wt-enforced OCI-AML2, and NPM1-mA-enforced OCI-AML2 cells. Methylene blue staining served as a loading control. (K) Western blot analysis of the FTO protein level in the NPM1-mA-silenced OCI-AML3 cells transfected with the HA-FTO plasmids. The bar graph showed the relative level of FTO protein. ß-actin was used as the internal control. (L) m6A dot blot analysis of the global m6A abundance in the NPM1-mA-silenced OCI-AML3 cells transfected with the HA-FTO plasmids. Methylene blue staining served as a loading control. Data were presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001; N.S. indicated not significant.

Fig 5: The Oncogenic Function of FTO is Dependent on m6A RNA Demethylase Activity. (A) CCK-8 analysis of cell proliferation in OCI-AML3 cells following transfection of the HA-FTO-WT and HA-FTO-MUT plasmids. (B) m6A dot blot analysis of the m6A level of global RNAs in OCI-AML3 cells with MA or DMSO treatment for 24 h. Methylene blue staining served as a loading control. (C) Dose-dependent effect of MA on cell viability. CCK-8 analysis of cell proliferation in OCI-AML3 cells exposed to 0, 10, 25, 50, and 100 µM MA for 24 h. (D) Time-dependent effect of MA on cell viability. CCK-8 analysis of cell proliferation in OCI-AML3 cells exposed to 100 µM MA for 0, 24, and 48 h. (E) EdU analysis of cell proliferation in OCI-AML3 cells upon 50 µM and 100 µM MA treatments of 24 h. The bar graph showed the percentage of EdU positive cells. Scale bar: 50 µm. (F) Flow cytometric analysis of the cell cycle distribution in OCI-AML3 cells upon 50 µM and 100 µM MA treatments of 24 h. The bar graph showed the percentage of G0/G1, S, and G2/M phase cells. (G) Cell apoptosis was measured by flow cytometric analysis of the Annexin V/DAPI stained cells. The bar graph showed the cell apoptosis rate. Data were presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.