American Society of Hematology

Figure 2.

$Figure 2. H3K27 mutations are associated with globally reduced H3K27me2/3 and are subclonal. (A) H3K27me2/3 levels in AML cell lines and patient specimens. Cell lines were ethanol fixed and stained with H3K27me2/3 and pan-H3–specific primary antibodies and anti-mouse Alexa-647/anti-rabbit Alexa-350 secondary antibodies. Cell line and patient identifier in addition to the relevant genotype information are provided in each panel. Approximately 10-fold less H3K27me2/3 is detected in a subclonal population in patient 04H138 (red arrow), and a population with reduced H3K27me2/3 is detected in a subclonal population in patient 12H183 (red arrow). (B) Increased H3K27ac in H3K27me3-low AML subpopulations. Cells were treated as in panel A and costained for pan-H3 (BV421), H3K27me3 (Alexa647), and H3K27ac (PE). H3K27me3 and H3K27ac signals were normalized against H3 signals (derived parameters; Flowjo) and plotted and gated as indicated. (C) H3K27 mutations are restricted to H3K27me2/3-low subpopulations in primary AML specimens. Cells were treated and stained as in panel B. H3K27me3-high and -low populations were FACS sorted as indicated and subjected to Sanger sequencing of PCR-amplified mutated genomic DNA regions. Detected mutation sites are shaded in light blue. In both cases, the H3K27-mutant alleles were exclusively present in the H3K27me3-low fractions. (D) Distribution of the remaining mutations in the H3K27me3-high and -low subfractions of patient 04H138. Genomic DNA from panel C was used to determine the relative distribution of mutations in the H3K27me3-defined subpopulations. Mutated regions are highlighted in light blue. (E) Distribution of the ASXL2 mutation in the H3K27me3-high and -low subfractions of patient 12H183. (F) Depictions of proposed subclonal compositions and their possible evolutionary trajectories. Suggested initiating lesions are positioned at the top, followed by mutations in their suspected order of appearance. Relative sizes of subclones are assigned based on the detected frequencies of H3K27me3-low and -high subpopulations shown in panels D and E. MFI, mean fluorescence intensity.$

H3^K27mutations are associated with globally reduced H3K27me2/3 and are subclonal. (A) H3K27me2/3 levels in AML cell lines and patient specimens. Cell lines were ethanol fixed and stained with H3K27me2/3 and pan-H3–specific primary antibodies and anti-mouse Alexa-647/anti-rabbit Alexa-350 secondary antibodies. Cell line and patient identifier in addition to the relevant genotype information are provided in each panel. Approximately 10-fold less H3K27me2/3 is detected in a subclonal population in patient 04H138 (red arrow), and a population with reduced H3K27me2/3 is detected in a subclonal population in patient 12H183 (red arrow). (B) Increased H3K27ac in H3K27me3-low AML subpopulations. Cells were treated as in panel A and costained for pan-H3 (BV421), H3K27me3 (Alexa647), and H3K27ac (PE). H3K27me3 and H3K27ac signals were normalized against H3 signals (derived parameters; Flowjo) and plotted and gated as indicated. (C) H3K27 mutations are restricted to H3K27me2/3-low subpopulations in primary AML specimens. Cells were treated and stained as in panel B. H3K27me3-high and -low populations were FACS sorted as indicated and subjected to Sanger sequencing of PCR-amplified mutated genomic DNA regions. Detected mutation sites are shaded in light blue. In both cases, the H3^K27-mutant alleles were exclusively present in the H3K27me3-low fractions. (D) Distribution of the remaining mutations in the H3K27me3-high and -low subfractions of patient 04H138. Genomic DNA from panel C was used to determine the relative distribution of mutations in the H3K27me3-defined subpopulations. Mutated regions are highlighted in light blue. (E) Distribution of the ASXL2 mutation in the H3K27me3-high and -low subfractions of patient 12H183. (F) Depictions of proposed subclonal compositions and their possible evolutionary trajectories. Suggested initiating lesions are positioned at the top, followed by mutations in their suspected order of appearance. Relative sizes of subclones are assigned based on the detected frequencies of H3K27me3-low and -high subpopulations shown in panels D and E. MFI, mean fluorescence intensity.

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