Figure 3.
Figure 3. H3.3K27I and H3.3K27M selectively accelerate in vitro proliferation and disease latency in an AML1-ETO mouse model. (A) Experimental strategy to assess in vitro competitiveness of H3.3KM mutant–expressing cells in AML1-ETO9a (AE9a) and MLL-AF9 cells. Mouse hematopoietic stem and progenitor cells (HSPCs) transformed by either MSCV AE9a IRES GFP or MSCV MLL-AF9 IRES Puro were infected with MSCV IRES tdTomato retroviruses expressing either WT or mutant H3.3. Cells were passaged, and percentages of tdTomato+ cells were determined at each passage. (B) A proliferative advantage of H3.3K27I/M-infected cells was observed in AE9a but not in MLL-AF9 cells. Trajectories of 4 independent cultures per condition are represented by connected dots; passage numbers are indicated below. (C) Representative tdTomato FACS profiles at passages 0 and 10 indicate selection for high expression of H3.3K27I/M at the end of the culture period in AE9a cells. (D) Western analysis of AE9a cells infected with the indicated H3.3 variants. AE9a cells from the 4 replicate cultures in panel B were further expanded, lysed in Laemmli buffer, and subjected to western analysis. Reduced H3K27me2/3 and increased H3K27ac levels, but no reductions in PRC2 protein levels (Ezh1/2, Suz12), were observed. (E) Experimental strategy to assess AML progression upon H3.3KM expression in vivo. (F) Increased AML penetrance and progression upon expression of H3.3K27I/M in AE9a cells (n = 9 mice per condition). (G) Expression of H3.3K27I/M in MLL-AF9 cells leads to no change in AML progression (n = 9 mice per condition). (H) Pathological analysis of diseased AE9a recipients indicates typical AML end-stage characteristics. (I) Phenotypic analysis of bone marrow (BM) cells at disease end stage in AE9a model. Selection for cells coexpressing high levels of AE9a (GFP), H3.3 variants (tdTomato; left panels), and c-kit (middle panels). Reduced H3K27me2/3 in H3.3K27I/M-infected cells at disease end stage (right panels). H3K27me2/3 signal was normalized to H3 signal intensity and plotted.

H3.3K27Iand H3.3K27Mselectively accelerate in vitro proliferation and disease latency in an AML1-ETO mouse model. (A) Experimental strategy to assess in vitro competitiveness of H3.3KM mutant–expressing cells in AML1-ETO9a (AE9a) and MLL-AF9 cells. Mouse hematopoietic stem and progenitor cells (HSPCs) transformed by either MSCV AE9a IRES GFP or MSCV MLL-AF9 IRES Puro were infected with MSCV IRES tdTomato retroviruses expressing either WT or mutant H3.3. Cells were passaged, and percentages of tdTomato+ cells were determined at each passage. (B) A proliferative advantage of H3.3K27I/M-infected cells was observed in AE9a but not in MLL-AF9 cells. Trajectories of 4 independent cultures per condition are represented by connected dots; passage numbers are indicated below. (C) Representative tdTomato FACS profiles at passages 0 and 10 indicate selection for high expression of H3.3K27I/M at the end of the culture period in AE9a cells. (D) Western analysis of AE9a cells infected with the indicated H3.3 variants. AE9a cells from the 4 replicate cultures in panel B were further expanded, lysed in Laemmli buffer, and subjected to western analysis. Reduced H3K27me2/3 and increased H3K27ac levels, but no reductions in PRC2 protein levels (Ezh1/2, Suz12), were observed. (E) Experimental strategy to assess AML progression upon H3.3KM expression in vivo. (F) Increased AML penetrance and progression upon expression of H3.3K27I/M in AE9a cells (n = 9 mice per condition). (G) Expression of H3.3K27I/M in MLL-AF9 cells leads to no change in AML progression (n = 9 mice per condition). (H) Pathological analysis of diseased AE9a recipients indicates typical AML end-stage characteristics. (I) Phenotypic analysis of bone marrow (BM) cells at disease end stage in AE9a model. Selection for cells coexpressing high levels of AE9a (GFP), H3.3 variants (tdTomato; left panels), and c-kit (middle panels). Reduced H3K27me2/3 in H3.3K27I/M-infected cells at disease end stage (right panels). H3K27me2/3 signal was normalized to H3 signal intensity and plotted.

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