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. 2017 Mar 6;214(3):737–752. doi: 10.1084/jem.20160927

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© 2017 Behrens et al.

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Figure 9. — HHEX but not Emx2 cooperates with FLT3-ITD to induce AML. (A) Schematic representation of the experimental design is shown. HSPCs were isolated from BM cells of 5-FU–treated B6.Runx1^Δ/+, cotransduced with the indicated retroviral vectors, and transplanted into conditioned recipient mice. (B) Representative flow cytometry plots of transduced HSPCs before transplantation demonstrating co-transduction (Venus⁺/BFP⁺) of HHEX/Venus (left) or Emx2/Venus (right) together with FLT3-ITD/BFP. Co-transduction frequencies of 15 and 5.3% for HHEX/FLT3-ITD and 17 and 16.3% for Emx2/FLT3-ITD were obtained in two independent experiments. FSC, forward scatter; SSC, side scatter. (C) Two-dimensional box plots showing the median (line), 25–75th percentiles (boxed), and SD (whiskers) of Venus⁺/BFP⁺ cells in the PB of transplanted mice (n = 10 per cohort) at the indicated time points. (D) Kaplan-Meier survival curves of mice receiving FLT3-ITD/BFP and either HHEX/Venus (n = 10) or Emx2/Venus (n = 13). As parallel positive and negative controls, cohorts receiving RUNX1-ER/FLT3-ITD and treated with 4-OHT (n = 6) or GFP alone (n = 6) were examined. (E) Spleen weight and leukocyte counts of either diseased (HHEX/FLT3-ITD; n = 9) or healthy negative-control GFP (n = 6) mice are plotted. Horizontal lines indicate median value for each cohort. P-values were calculated by a nonpaired Student’s t test. **, P < 0.01; ***, P < 0.001. WBC, white blood cell. (F) Representative histological analysis of sections from sternum (left) and livers (right) from either an AML (HHEX/FLT3-ITD; n = 3) or control (GFP; n = 3) mouse. Expansion of leukemic cells in a single BM cavity is clearly observed, whereas the neighboring cavity is not impacted. Abundant and diffuse infiltrating hematopoietic cells (dark staining) were observed in the livers of AML mice leading to hepatomegaly. H&E staining was used. Bars, 300 µm. (G) Dot blot of double-positive (HHEX/Venus⁺ and FLT3-ITD/BFP⁺) cells in BM and spleen of diseased mice (n = 9) as determined by flow cytometry. Each dot represents the value for a single mouse. Horizontal lines indicate median value of all mice from two independent experiments. (H) Representative flow cytometry plots of dispersed BM cells from diseased mice demonstrating high levels of Venus⁺/BFP⁺ cells, which were consistently positive for CD11b alone (myeloid progenitor; mean 90 ± 3.9%; n = 7), as compared with normal myeloid precursors in the BM, which are double positive for both myeloid antigens (CD11b⁺/Gr1⁺; mean 88 ± 34.5%; n = 7). (I) Blood smears of mice demonstrating increased numbers of blasts and maturing cells in AML mice. Pappenheim stain was used. Bar, 20 µm. (J) Kaplan-Meier survival curves of serial transplantation of HHEX/FLT3-ITD⁺ leukemic blasts in two independent experiments. n = 6 per transplantation.