Journal: Experimental Hematology & Oncology

Article Title: A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

doi: 10.1186/s40164-024-00480-z

Figure Lengend Snippet: FTO promotes expression of AML1-ETO as an m 6 A demethylase by repressing YTHDF2-mediated AML1-ETO mRNA decay. A , B The m 6 A abundance in mRNA detected by m 6 A dot blotting ( A ) and the expression levels of FTO and AML1-ETO by western blotting ( B ) in SKNO-1 or Kasumi-1 AML cells with forced expression of wild-type FTO (wt-FTO), mutant FTO (mut-FTO), or mock vectors; Methylene blue (MB) represents the loading control of RNA samples. C , D The m 6 A dot blotting ( C ) and western blotting ( D ) in SKNO-1 or Kasumi-1 AML cells transfected with FTO shRNA or scramble shRNA (shNS) vectors. E , F Effects of FB23-2 treatment for 72 h on the m 6 A abundance in mRNA by m 6 A dot blotting ( E ) and expression levels of AML1-ETO detected by western blotting ( F ) in SKNO-1 and Kasumi-1 cells. G RIP-qPCR showing the interaction between AML1-ETO mRNA transcripts and FTO protein in SKNO-1 and Kasumi-1 cells. H , I The levels of m 6 A in AML1-ETO transcripts assessed by gene-specific m 6 A qPCR assay in SKNO-1 and Kasumi-1 cells transduced with wt-FTO, mut-FTO, or mock vector ( H ); or with FTO shRNA or shNS vectors ( I ). J – L The mRNA half-life (t 1/2 ) of AML1-ETO transcripts in SKNO-1 and Kasumi-1cells transduced with wt-FTO, mut-FTO, or mock vector ( J ); or with FTO shRNA or shNS vectors ( K ) or treated with DMSO or FB23-2 for 72 h ( L ). M RIP-qPCR showing the interaction between AML1-ETO mRNA transcripts and YTHDF2 protein in SKNO-1 and Kasumi-1 cells. N , O Increased expression of AML1-ETO after YTHDF2 knockdown assessed by qPCR ( N ) and western blotting ( O ) in SKNO-1 and Kasumi-1 cells. P Increased mRNA half-life (t 1/2 ) of AML1-ETO transcripts in SKNO-1 and Kasumi-1 cells after YTHDF2 knockdown

Article Snippet: The siRNA targeting the fusion site of the AML1-ETO mRNA (siAE#1, sense, 5′-CCUCGAAAUCGUACUGAGAAG-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGUU-3′; siAE#2, sense, 5′-CCUCGAAAUCGUACUGAGATT-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGTT-3′) was designed according to previous studies [ ] and was commercially synthesized by Sangon Biotech. siRNAs against human FTO (sc-75002), IGFBP2 (sc-37195), YTHDF2 (sc-78661) and YTHDF3 (sc-77724) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into cells using the 4D-Nucleofector System (Lonza, Cologne, Germany) with the SF Cell Line 4D-Nucleofector X Kit by DS-138 for SKNO-1 and CM137 for Kasumi-1.

Techniques: Expressing, Western Blot, Mutagenesis, Control, Transfection, shRNA, Transduction, Plasmid Preparation, Knockdown

Journal: Experimental Hematology & Oncology

Article Title: A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

doi: 10.1186/s40164-024-00480-z

Figure Lengend Snippet: Biological impact of FTO in t(8;21) AML cells and AML1-ETO9a mice model. A – C Effects of forced expression of wild-type FTO (wt-FTO), mutant FTO (mut-FTO) or mock vectors ( A ); or FTO shRNA or scramble vectors ( B ), or FB23-2 treatment ( C ) on the proliferation of SKNO-1 and Kasumi-1 cells by CCK-8 assays. shNS: scramble shRNA. D Effects of forced expression or knockdown of FTO on colony-forming capacity of SKNO-1 cells. E The effect of FTO knockdown on differentiation of SKNO-1 cells. The percentage of CD11b + cells was quantified (right panel). F Wright-Giemsa staining of SKNO-1 cells with or without FTO knockdown. G , H Effect of silencing the expression of FTO, confirmed by western blotting ( G ), on cell apoptosis in SKNO-1 and Kasumi-1 cells 72 h after siRNA transfection ( H ). I Western blotting of the Fto knockdown in AML1-ETO9a-driven AML mice cells. J , K Kaplan–Meier survival curves of AML1-ETO9a-driven AML mice ( n = 10 for each group) with or without Fto knockdown ( J ) or treatment with DMSO or FB23-2 ( K ). L Hematoxylin and eosin (H&E) staining of liver (left panel) and spleen (right panel) of AML1-ETO9a-driven AML mice 7 weeks after transplantation. M – O Percentage of GFP + AML1-ETO9a AML cells in the M peripheral blood (PB), N bone marrow (BM), and O spleen (SP) of the mice with or without Fto knockdown assessed by flow cytometric analysis. P – R Flow cytometric analysis of the distribution of anti-CD11b-stained GFP + AML1-ETO9a AML cells in PB ( P ), BM ( Q ), and SP ( R ) of mice with or without Fto knockdown

Article Snippet: The siRNA targeting the fusion site of the AML1-ETO mRNA (siAE#1, sense, 5′-CCUCGAAAUCGUACUGAGAAG-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGUU-3′; siAE#2, sense, 5′-CCUCGAAAUCGUACUGAGATT-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGTT-3′) was designed according to previous studies [ ] and was commercially synthesized by Sangon Biotech. siRNAs against human FTO (sc-75002), IGFBP2 (sc-37195), YTHDF2 (sc-78661) and YTHDF3 (sc-77724) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into cells using the 4D-Nucleofector System (Lonza, Cologne, Germany) with the SF Cell Line 4D-Nucleofector X Kit by DS-138 for SKNO-1 and CM137 for Kasumi-1.

Techniques: Expressing, Mutagenesis, shRNA, CCK-8 Assay, Knockdown, Staining, Western Blot, Transfection, Transplantation Assay

Journal: Experimental Hematology & Oncology

Article Title: A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

doi: 10.1186/s40164-024-00480-z

Figure Lengend Snippet: Suppression of FTO resensitizes resistant cells to Ara-C in vitro and in vivo. A Comparison of FTO mRNA expression in BM samples from AML patient with high versus poor response to Ara-C (GSE97393). B , C CCK-8 assays for SKNO-1 and Kasumi-1 cells transfected with wild-type FTO (wt-FTO), mutant FTO (mut-FTO) or mock vectors ( B ); or FTO shRNA or scramble vectors ( C ) treated with varying concentrations of Ara-C for 48 h. D CCK-8 assays for SKNO-1 and Kasumi-1 cells treated with 10 µM FB23-2 for 6 h followed by co-treatment of different concentrations of Ara-c for 48 h. E CCK-8 assays for primary BM cells collected from a relapsed t(8;21) AML patients with 10 µM FB23-2 treatment for 6 h followed by co-treatment of different concentrations of Ara-c for 48 h. F The external view of nude mice bearing SKNO-1 cell xenografts (n = 6 for each group) treated with DMSO, Ara-C, FB23-2, or a combination of Ara-C and FB23-2. G , H The growth curve of tumor volume ( G ) and the final tumor weight ( H ) for each group as indicated in F . I – L NOD/SCID/IL2rγ null immunodeficient NSG mice injected with SKNO-1 cells through tail vein treated with DMSO, Ara-C, FB23-2, or a combination of Ara-C and FB23-2 (n = 6 for each group). I Blast cells percentage in bone marrow (BM), J Wright-Giemsa staining of bone marrow and H&E staining of livers and spleens, K spleen weights and L representative external views of the spleens were shown

Article Snippet: The siRNA targeting the fusion site of the AML1-ETO mRNA (siAE#1, sense, 5′-CCUCGAAAUCGUACUGAGAAG-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGUU-3′; siAE#2, sense, 5′-CCUCGAAAUCGUACUGAGATT-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGTT-3′) was designed according to previous studies [ ] and was commercially synthesized by Sangon Biotech. siRNAs against human FTO (sc-75002), IGFBP2 (sc-37195), YTHDF2 (sc-78661) and YTHDF3 (sc-77724) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into cells using the 4D-Nucleofector System (Lonza, Cologne, Germany) with the SF Cell Line 4D-Nucleofector X Kit by DS-138 for SKNO-1 and CM137 for Kasumi-1.

Techniques: In Vitro, In Vivo, Comparison, Expressing, CCK-8 Assay, Transfection, Mutagenesis, shRNA, Injection, Staining

Journal: Experimental Hematology & Oncology

Article Title: A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

doi: 10.1186/s40164-024-00480-z

Figure Lengend Snippet: FTO promotes the expression of IGFBP2 in an m 6 A-dependent manner. A , B Expression of IGFBP2 assessed by qPCR ( A ) and western blotting ( B ) in SKNO-1 or Kasumi-1 cells transduced lentivirally with wild-type FTO (wt-FTO), mutant FTO (mut-FTO), or mock vectors. C , D Decreased expression of IGFBP2 detected by qPCR ( C ) and western blotting ( D ) in SKNO-1 and Kasumi-1 cells after knockdown of FTO by shRNAs. E , F Expression of IGFBP2 analyzed by qPCR ( E ) and western blotting ( F ) in SKNO-1 or Kasumi-1 cells after FB23-2 treatment for 72 h. G RIP-qPCR showing the interaction between IGFBP2 mRNA transcripts and FTO protein in SKNO-1 and Kasumi-1 cells. H , I The levels of m 6 A in IGFBP2 transcripts assessed by gene-specific m 6 A qPCR assay in SKNO-1 and Kasumi-1 cells transduced with wt-FTO, mut-FTO, or control vector ( H ); or with FTO shRNA or shNS vectors ( I ). J Dual luciferase reporter assays for the effect of wild-type or mutant FTO on the relative luciferase activity of psiCHECK2-IGFBP2-3′-UTR with either wild-type or mutant m 6 A sites. K , L The mRNA half-life (t 1/2 ) of IGFBP2 transcripts in SKNO-1 and Kasumi-1 cells transduced with FTO shRNA or shNS vectors ( K ) or treated with DMSO or FB23-2 for 72 h ( L ). M – O RIP-qPCR showing the interaction between IGFBP2 mRNA transcripts and YTHDF1 ( M ), YTHDF2 ( N ) and YTHDF3 ( O ) protein in SKNO-1 and Kasumi-1 cells. P , Q Increased expression of IGFBP2 after YTHDF1, YTHDF2 and YTHDF3 knockdown analyzed by qPCR ( P ) and western blotting ( Q ) in SKNO-1 and Kasumi-1 cells

Article Snippet: The siRNA targeting the fusion site of the AML1-ETO mRNA (siAE#1, sense, 5′-CCUCGAAAUCGUACUGAGAAG-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGUU-3′; siAE#2, sense, 5′-CCUCGAAAUCGUACUGAGATT-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGTT-3′) was designed according to previous studies [ ] and was commercially synthesized by Sangon Biotech. siRNAs against human FTO (sc-75002), IGFBP2 (sc-37195), YTHDF2 (sc-78661) and YTHDF3 (sc-77724) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into cells using the 4D-Nucleofector System (Lonza, Cologne, Germany) with the SF Cell Line 4D-Nucleofector X Kit by DS-138 for SKNO-1 and CM137 for Kasumi-1.

Techniques: Expressing, Western Blot, Mutagenesis, Knockdown, Transduction, Control, Plasmid Preparation, shRNA, Luciferase, Activity Assay

Journal: Experimental Hematology & Oncology

Article Title: A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

doi: 10.1186/s40164-024-00480-z

Figure Lengend Snippet: IGFBP2 promotes leukemogenesis and resistance to Ara-C. A Confirmation of IGFBP2 knockdown by shRNAs in SKNO-1 and Kasumi-1 cells using western blotting. B Effects of IGFBP2 knockdown on the proliferation of SKNO-1 and Kasumi-1 cells by CCK8 assays. C Effect of silencing IGFBP2 on cell apoptosis in SKNO-1 and Kasumi-1 cells 72 h after siRNA transfection. D The effect of IGFBP2 knockdown on differentiation of SKNO-1 and Kausmi-1 cells. The percentage of CD11b + cells was quantified (right panel). E Wright-Giemsa staining of SKNO-1 and Kausmi-1 cells with or without IGFBP2 knockdown. F CCK-8 assays for SKNO-1 and Kasumi-1 cells transfected with IGFBP2 shRNA or scramble vectors treated with varying concentrations of Ara-C for 48 h. G Western blot analysis for knockdown of Igfbp2 in AML1-ETO9a-driven AML cells. H Kaplan–Meier survival curves of AML1-ETO9a-driven AML mice (n = 10 for each group) after Igfbp2 knockdown. I Wright-Giemsa staining of bone marrow and H&E staining of livers and spleens for Igfbp2-knockdown AML1-ETO9a-driven AML mice. J – O Flow cytometric analysis of the percentage of GFP + AML1-ETO9a AML cells ( J – L ) and distribution of anti-CD11b-stained GFP + AML cells ( M – O ) in PB, BM, and SP of AML1-ETO9a-driven AML mice with and without Igfbp2 knockdown 7 weeks after transplantation

Article Snippet: The siRNA targeting the fusion site of the AML1-ETO mRNA (siAE#1, sense, 5′-CCUCGAAAUCGUACUGAGAAG-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGUU-3′; siAE#2, sense, 5′-CCUCGAAAUCGUACUGAGATT-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGTT-3′) was designed according to previous studies [ ] and was commercially synthesized by Sangon Biotech. siRNAs against human FTO (sc-75002), IGFBP2 (sc-37195), YTHDF2 (sc-78661) and YTHDF3 (sc-77724) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into cells using the 4D-Nucleofector System (Lonza, Cologne, Germany) with the SF Cell Line 4D-Nucleofector X Kit by DS-138 for SKNO-1 and CM137 for Kasumi-1.

Techniques: Knockdown, Western Blot, Transfection, Staining, CCK-8 Assay, shRNA, Transplantation Assay

Journal: Experimental Hematology & Oncology

Article Title: A novel AML1-ETO/FTO positive feedback loop promotes leukemogenesis and Ara-C resistance via stabilizing IGFBP2 in t(8;21) acute myeloid leukemia

doi: 10.1186/s40164-024-00480-z

Figure Lengend Snippet: FTO regulates leukemogenesis and sensitivity of t(8;21) AML cells to Ara-C through IGFBP2. A Western blotting for the expression of IGFBP2 in SKNO-1 and Kasumi-1 cells transduced with shNS (pLKO.1-shNS + empty pTSB), shFTO (pLKO.1-shFTO#1 + empty pTSB), shFTO + IGFBP2 (pLKO.1-shFTO#1 + pTSB-IGFBP2-CDS), or IGFBP2 (pLKO.1-shNS + pTSB-IGFBP2-CDS). shNS, scramble shRNA. B CCK-8 assays for the effects of FTO knockdown and/or overexpression of IGFBP2 on the proliferation of SKNO-1 and Kasumi-1 cells. C Effects of FTO knockdown with IGFBP2 overexpression on colony-forming capacity of SKNO-1 and Kasumi-1 cells. D CCK-8 assays for the effects of FTO knockdown and/or overexpression of IGFBP2 on the sensitivity to Ara-C in SKNO-1 and Kasumi-1 cells. E – G Effects of FTO knockdown with IGFBP2 overexpression on AML1-ETO9a-driven AML mice. Western blot analysis for Igfbp2 and Fto ( E ), Wright-Giemsa staining of bone marrow and H&E staining of livers and spleens ( F ) and flow cytometric analysis of the abundance of GFP + AML1-ETO9a AML cells in PB, BM and SP ( G ) were shown. H Proposed model depicting the regulatory interactions and role of FTO in leukemogenesis and Ara-C resistance in t(8;21) AML.

Article Snippet: The siRNA targeting the fusion site of the AML1-ETO mRNA (siAE#1, sense, 5′-CCUCGAAAUCGUACUGAGAAG-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGUU-3′; siAE#2, sense, 5′-CCUCGAAAUCGUACUGAGATT-3′, antisense, 5′-UCUCAGUACGAUUUCGAGGTT-3′) was designed according to previous studies [ ] and was commercially synthesized by Sangon Biotech. siRNAs against human FTO (sc-75002), IGFBP2 (sc-37195), YTHDF2 (sc-78661) and YTHDF3 (sc-77724) were purchased from Santa Cruz Biotechnology. siRNAs were transfected into cells using the 4D-Nucleofector System (Lonza, Cologne, Germany) with the SF Cell Line 4D-Nucleofector X Kit by DS-138 for SKNO-1 and CM137 for Kasumi-1.

Techniques: Western Blot, Expressing, Transduction, shRNA, CCK-8 Assay, Knockdown, Over Expression, Staining