Targeting ANXA5-associated pathways reduces the inflammatory state in the BMM and prolongs MLL-AF9⁺ AML survival in ANXA5 KO mice. (A) Kaplan-Meier style survival curve of ANXA5 KO recipient mice with MLL-AF9–induced AML treated with vehicle (oil [daily until day 40]; solid line) or celecoxib (50 mg/kg daily until day 40; dashed line) starting from day 22 after transplant (P = .04; log-rank test; n = 6-7) (retroviral transduction/transplantation model). Median survival for vehicle-treated ANXA5-deficient mice was 27 days and for celecoxib-treated ANXA5-deficient mice 62 days. (B) Representative images of bone sections (left) from ANXA5 KO recipient mice with MLL-AF9–induced AML treated with vehicle (oil daily until day 40; circles) or celecoxib (50 mg/kg daily until day 40; squares) starting from day 22 after transplant, stained for reticulin. The quantification is shown on the right. Nuclei were counterstained with nuclear fast red-aluminum sulfate solution (P = .0057; t test; n = 5). The scale bar represents 50 μm. (C) Representative immunofluorescence images of bone sections (left) and quantification (right) from ANXA5 KO recipient mice with MLL-AF9–induced AML treated with vehicle (oil; daily until day 40; circles) or celecoxib (50 mg/kg daily until day 40; squares) starting from day 22 after transplantation, stained for PGE2 (red) and DAPI (blue) (P = .011; t test; n = 4-5). Bones were harvested on day 40 after transplantation. The scale bar represents 40 μm. (D) Representative images of Giemsa-stained cytospins of BM cells from ANXA5 KO recipient mice with MLL-AF9–induced AML treated with vehicle (oil; daily until day 40; circles) or celecoxib (50 mg/kg daily until day 40; squares) (left) starting from day 22 after transplant. The scale bar represents 25 μm. The quantification of the percentage of blasts of total leukocytes is shown on the right (P = .0029; t test; n = 4). (E-F) Immunofluorescence images (left) and quantification of GFP (MLL-AF9)⁺ Lin^– cells sorted (on day 40 after transplantation) from the BM of ANXA5 KO recipient mice with MLL-AF9–induced AML treated with vehicle (oil; daily until day 40; circles) or celecoxib (50 mg/kg daily; squares) starting from day 22 after transplant, stained for γH2A.X (yellow) (E) or μ-catenin (green) (F) and DAPI (blue) (P = .048; P = .0038; t test; n = 4). The scale bar represents 25 μm. For panels C,E-F, 4 different fields from 3 to 5 individual mice were imaged; each dot represents the average per mouse. γH2AX, H2A histone family member X.