aml1 eto fusion  (Addgene inc)

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

Journal: Immune Network

Article Title: A Novel Translocation Involving RUNX1 and HOXA Gene Clusters in a Case of Acute Myeloid Leukemia with t(7;21)(p15;q22)

doi: 10.4110/in.2013.13.5.222

Figure Lengend Snippet: (A) BAC clone probes designed and HOXA gene clusters on chromosome 7p15.2. The arrow between the dotted and solid lines represents the break point area. (B) Metaphase FISH using the probe set RP11-163M21 and RP11-1148E13 showing translocation of the gene segment, including HOXA1-7 , to derivative chromosome 21. (C) Metaphase FISH using probe set RP11-838G2 and RP11-1025G19 showing translocation of the gene segment, including a part of HOXA 9-13 , to derivative chromosome 21. (D) Putative fusion gene map of t(7;21): RUNX1/HOXA9-13 .

Article Snippet: To evaluate the status of RUNX1 on 21q22, FISH was performed with a commercial LSI AML1/ETO Dual Color, Dual Fusion Translocation Probe (Vysis, Downers Grove, IL).

Techniques: Translocation Assay

Journal: Immune Network

Article Title: A Novel Translocation Involving RUNX1 and HOXA Gene Clusters in a Case of Acute Myeloid Leukemia with t(7;21)(p15;q22)

doi: 10.4110/in.2013.13.5.222

Figure Lengend Snippet: (A) Representative metaphase G-banding of the patient bone marrow aspirate showing 46,XX, t(7;21)(p22;q22). (B) Metaphase fluorescence in situ hybridization analysis using AML1/ETO dual-color, dual-fusion translocation probes demonstrate translocation of RUNX1 into chromosome 7; 2 usual-sized red signals on 2 intact chromosome 8, 1 usual-sized green signal on chromosome 21 (arrow), and tw1 small green signal each on derivative chromosome 21 (arrowhead) and derivative chromosome 7 (dotted arrow).

Article Snippet: To evaluate the status of RUNX1 on 21q22, FISH was performed with a commercial LSI AML1/ETO Dual Color, Dual Fusion Translocation Probe (Vysis, Downers Grove, IL).

Techniques: Fluorescence, In Situ Hybridization, Translocation Assay