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Acute myeloid leukemia (AML) with nucleophosmin 1 (NPM1) mutations exhibits distinct biological and clinical features, accounting for approximately one-third of AML. Recently, the N6-methyladenosine (m6A) RNA modification has emerged as a new epigenetic modification to contribute to tumorigenesis and development. However, there is limited knowledge on the role of m6A modifications in NPM1-mutated AML. In this study, the decreased m6A level was first detected and high expression of fat mass and obesity-associated protein (FTO) was responsible for the m6A suppression in NPM1-mutated AML. FTO upregulation was partially induced by NPM1 mutation type A (NPM1-mA) through impeding the proteasome pathway. Importantly, FTO promoted leukemic cell survival by facilitating cell cycle and inhibiting cell apoptosis. Mechanistic investigations demonstrated that FTO depended on its m6A RNA demethylase activity to activate PDGFRB/ERK signaling axis. Our findings indicate that FTO-mediated m6A demethylation plays an oncogenic role in NPM1-mutated AML and provide a new layer of epigenetic insight for future treatments of this distinctly leukemic entity.

Additional file 12 : Figure S7. HOTAIRM1 expression in the different transfected cells. a-b HOTAIRM1 expression in HOTAIRM1-silenced (a) and HOTAIRM1-enforced OCI-AML3 cells (b). c-d HOTAIRM1 expression in HOTAIRM1-silenced (c) as well as HOTAIRM1-enforced OCI-AML2 + NPM1-mA cells (d). e HOTAIRM1 expression in HOTAIRM1-enforced OCI-AML2 cells.

Additional file 10 : Figure S5. Enrichment of KLF5 on the E2 fragment of the HOTAIRM1 promoter was measured by ChIP assays in leukemia cells.

Additional file 17 : Figure S12. Screening for the ubiquitin ligase of EGR1. a Network view of the predicted E3 ubiquitin ligase for EGR1. b Recognition motif for the potential E3 ubiquitin ligase MDM2 in the EGR1 sequence.

Additional file 4 : Table S4. The top 10 predicted factors bound to HOTAIRM1 promoter by JASPAR database analysis.

Additional file 16 : Figure S11. EGR1 does not affect the expression of HOTAIRM1. a The efficiency of EGR1 overexpression in OCI-AML3 cells was validated by western blotting. b qRT-PCR was used to detect the expression of HOTAIRM1 in EGR1-enforced OCI-AML3 cells. The data are presented as the mean ± SD of three independent experiments. n.s. indicates no significant difference.

Additional file 3 : Table S3. Primers used for ChIP-qPCR.

Additional file 11 : Figure S6. Co-IP assays were used to evaluate the binding of NPM1 and KLF5, NPM1-wt and WWP1 in OCI-AML3 cells. a-b The interaction between NPM1-mA (a) or NPM1-wt (b) and KLF5 in OCI-AML3 cells were determined by IP assays. c IP assays were used to determine the binding of WWP1 to NPM1-wt in OCI-AML3 cells.

Additional file 19 : Figure S14. Cytoplasmic HOTAIRM1 promotes leukemia cell autophagy and proliferation through ULK3. a, c Western blot analysis of LC3-II, p62 and ULK3 levels in HOTAIRM1-silenced OCI-AML3 cells following ULK3 overexpression (a) and HOTAIRM1-overexpressed OCI-AML2 cells following ULK3 knockdown (c). b, d CCK-8 assays were used to analyze cell proliferation in transfected OCI-AML3 (b) and OCI-AML2 cells (d). The data are presented as the mean ± SD of three independent experiments. *P

Additional file 1 : Table S1. Clinical characteristics of newly diagnosed AML patients.

Additional file 6 : Figure S1. Stable NPM1-mA-GFP expressing OCI-AML2 cells (OCI-AML2 + NPM1-mA) were generated. a Cytoplasmic localization of NPM1-mA in the OCI-AML2 stably expressing Flag-GFP tagged NPM1-mA. Cytospun cells were fixed and stained with DAPI. b The level of NPM1-mA in leukemia cells were measured by western blot analysis.

Additional file 13 : Figure S8. HOTAIRM1 promotes autophagy and proliferation in transfected OCI-AML2 + NPM1-mA leukemia cells. a, i Western blot analysis of LC3-II and p62 levels in OCI-AML2 + NPM1-mA cells after transfection of shHOTAIRM1 (a) and HOTAIRM1 expression vector (i). b, j Representative microscopy images of LC3 puncta in HOTAIRM1-silenced OCI-AML2 + NPM1-mA (b) and HOTAIRM1-enforced OCI-AML2 + NPM1-mA (j). The bar graphs showed the quantification of the fluorescent puncta data. Scale bar: 25 μm. c, k Evaluation of cell viability in OCI-AML2 + NPM1-mA cells transfected with shHOTAIRM1 (c) or HOTAIRM1 plasmid (k) for indicated by CCK-8 assays. d, l Evaluation of cell proliferation in HOTAIRM1-silenced (d) and HOTAIRM1-enforced OCI-AML + NPM1-mA cells (l) for indicated by EdU assays. The bar graphs showed the percentage of EdU positive cells. Scale bar: 100 μm. e, m Flow cytometry was performed to assess cell cycle of HOTAIRM1-silenced (e) and HOTAIRM1-enforced OCI-AML2 + NPM1-mA cells (m). The bar graph shows the percentages of G0/G1-, S-, and G2/M-phase cells. f, n Flow cytometry was used to detect apoptosis of HOTAIRM1-silenced (f) and HOTAIRM1-enforced OCI-AML2 + NPM1-mA cells (n). LL, dead cells; UL, viable cells; LR, early apoptotic cells; UR, late apoptotic cells. g, o Flow cytometric analysis was performed to analyze apoptosis of OCI-AML2 + NPM1-mA cells after transfection with shHOTAIRM1 (g) or HOTAIRM1 plasmid (o), followed by treatment with 200 nM Ara-C for 48 h. h, p The protein levels of Cyclin D1, CDK4, Bax and Bcl-2 in HOweTAIRM1-silenced (h) and HOTAIRM1-enforced OCI-AML2 + NPM1-mA cells (p). The data are presented as the mean ± SD of three independent experiments. **P

Additional file 5 : Table S5. The top 10 proteins with specific binding to HOTAIRM1 identified by mass spectrometry analysis.

Additional file 18 : Figure S13. Nuclear HOTAIRM1 promotes cell cycle progression and inhibits apoptosis in leukemia cells through EGR1. a, c Flow cytometry was used to determine and compare the differences in cell cycle progression in differently transfected groups of OCI-AML3 (a) and OCI-AML2 cells (c). b, d Flow cytometry was used to determine and compare differences in apoptosis in differently transfected groups of OCI-AML3 (b) and OCI-AML2 cells (d). The data are presented as the mean ± SD of three independent experiments. *P

Additional file 7 : Figure S2. The expression levels of HOTAIRM1 in the nucleus and cytoplasm of leukemia cells were measured by qRT-PCR.

Additional file 20 : Figure S15. Cytoplasmic HOTAIRM1 promotes cell cycle progression and inhibits apoptosis in leukemia cells through ULK3. a-b Flow cytometry was used to analyze the changes in cell the cycle (a) and apoptosis (b) in HOTAIRM1-silenced OCI-AML3 cells following ULK3 upregulation. c-d Flow cytometry was used to analyze the change in the cell cycle (c) and apoptosis (d) in HOTAIRM1-overexpressed OCI-AML2 cells following ULK3 downregulation. The data are presented as the mean ± SD of three independent experiments. *P

Additional file 8 : Figure S3. NPM1-mA expression in leukemia cells in the different treatment groups. a The efficiency of RNA interference-mediated knockdown of NPM1-mA expression was determined by qRT-PCR. b The efficiency of RNA interference-mediated knockdown of NPM1-mA expression was determined by western blotting. c NPM1-mA protein levels in NPM1-mA enforced OCI-AML2 cells. d Western blot analysis of NPM1-mA in OCI-AML3 cells treated with KPT-330. β-tubulin as the cytoplasmic control and PCNA as the nuclear control. The data are presented as the mean ± SD of three independent experiments.

Additional file 14 : Figure S9. HOTAIRM1 promotes autophagy and proliferation in transfected OCI-AML2 leukemia cells. a Western blot analysis of LC3-II and p62 levels in OCI-AML2 cells after transfection of HOTAIRM1 expression vector. b Representative microscopy images of LC3 puncta in HOTAIRM1-enforced OCI-AML2 cells. The bar graphs showed the quantification of the fluorescent puncta data. Scale bar: 25 μm. c Evaluation of cell viability in OCI-AML2 cells transfected with HOTAIRM1 plasmid for indicated by CCK-8 assays. d Evaluation of cell proliferation in HOTAIRM1-enforced OCI-AML2 cells for indicated by EdU assays. The bar graphs showed the percentage of EdU positive cells. Scale bar: 100 μm. e Flow cytometry was performed to assess cell cycle of HOTAIRM1-enforced OCI-AML2 cells. The bar graph shows the percentages of G0/G1-, S-, and G2/M-phase cells. f Flow cytometry was used to detect apoptosis of HOTAIRM1-enforced OCI-AML2 cells. LL, dead cells; UL, viable cells; LR, early apoptotic cells; UR, late apoptotic cells. g The protein levels of Cyclin D1, CDK4, Bax and Bcl-2 in HOTAIRM1-enforced OCI-AML2 cells. The data are presented as the mean ± SD of three independent experiments. **P

Additional file 2 : Table S2. Primers used for qRT-PCR.

Additional file 15 : Figure S10. HOTAIRM1 expression in HOTAIRM1-silenced OCI-AML3 cells transfected with different vectors.