AML cells exposed to an ANXA5-deficient BMM show increased blasts and DNA damage. (A) Representative immunofluorescence images (left) and quantification (right) of bone sections from WT (red) or ANXA5 KO (blue) recipient mice with MLL-AF9–induced AML (retroviral transduction/transplantation model), stained for PGE2 (red) and DAPI (blue), on day 40 after transplantation (P = .038; t test; n = 4-5). The scale bar represents 40 μm. Four different fields from 4 individual mice were imaged; each dot represents the average per mouse. (B) Percentage of cells positive for phagocytosed, phycoerythrin-conjugated pHrodo Escherichia coli bioparticles of GFP (MLL-AF9)⁺ Gr1⁺ cells. The AML cells were taken from the BM of WT (red) or ANXA5 KO (blue) recipient mice with MLL-AF9–induced AML on day 40 after transplantation (P = .024; n = 12-13). (C) Representative images of Giemsa-stained cytospins of BM cells from WT (red) or ANXA5 KO (blue) recipient mice with MLL-AF9–induced AML (left) on day 40 after transplantation and quantification of the respective percentage of blasts of total leukocytes (right) (P = .023; t test; n = 4-5). The scale bar represents 25 μm. Four different fields from 4 to 5 individual mice were imaged; each dot represents the average per mouse. (D) Percentage of cells in the G0, G1, and G2/S phases of the cell cycle of GFP (MLL-AF9)⁺ Lin^– cells. Cells were derived from the BM of WT (red) or ANXA5 KO (blue) recipient mice with MLL-AF9–induced AML on day 40 after transplantation (P = .039; P = .021; 2-way ANOVA; Tukey test; n = 9-10). (E) Median fluorescence intensity (MFI) of γH2A.X in GFP (MLL-AF9)⁺ Lin^– cells in the BM (left) or spleen (right) of WT (red) or ANXA5 KO (blue) recipient mice with MLL-AF9–induced AML on day 40 after transplantation (P = .035; t test; n = 6-7). (F) Representative immunofluorescence images of nuclear β-catenin levels of GFP (MLL-AF9)⁺ Lin^– cells sorted from the BM of WT or ANXA5 KO recipient mice with MLL-AF9–induced AML, stained for β-catenin (green) and DAPI (blue) on day 40 after transplantation (n = 4). The scale bar represents 25 μm. Four different fields from 4 individual mice were imaged; each dot represents the average per mouse. (G) Schematic representation of the proposed interaction between AML cells and cells of the BMM. AML cell–derived TNF-α stimulates increased production of PGE2 by cells of the BMM. PGE2 leads to increased expression of β-catenin and HIF-1α via its binding to the prostaglandin E receptor 4. (H) Correlation of the messenger RNA (mRNA) levels of TNF and CTNNB1 (β-catenin) in patients with AML. (I) Correlation of the mRNA levels of PTGER4 and HIF1A (HIF-1α) in patients with AML. (J) Correlation of the mRNA levels of TNF and HIF1A (HIF-1α) in patients with AML. The correlation analyses were performed using the data from the Oregon Health and Science University study by Verma et al,²⁹ which was accessed via the cBioPortal. EP4, prostaglandin E2 receptor EP4; RNA seq, RNA sequencing; RPKM, reads per kilobase of transcript per million mapped reads.