Figure 3.
Epigenetic targeting selectively impairs clonogenicity in KMT2A::MLLT3 AML cells. (A) Experimental overview for the inhibition of PRC2 by UNC1999 and DNA methylation by AZA in control- and AML-HSPCs. (B) Representative immunoblot analysis and (C) quantification of relative H3K27me3 protein levels in HSPCs from control- and AML-iPSCs after 72 hours of treatment with 2 μM and 5 μM UNC1999, 2 μM UNC1999 + 1 μM AZA, or DMSO (n = 3). Histone H3 was used to calculate relative H3K27me3 levels, which were normalized to control DMSO conditions. Data are presented as mean ± SEM of relative signal and analyzed using 2-way analysis of variance (ANOVA) with Dunnett multiple comparisons test. (D) Fold cell expansion of HSPCs from control- and AML-iPSC cells lines following 72 hours of treatment with 2 μM and 5 μM UNC1999, 2 μM UNC1999 + 1 μM AZA, or DMSO relative to cell number at seeding (n = 3). Data are presented as mean ± SEM of fold cell expansion. Lines indicate comparisons within and between control and AML cells. Data were analyzed using 2-way ANOVA with Tukey multiple comparisons test. (E) CFU counts per 1000 seeded HSPCs treated with 2 μM UNC1999, 1 μM AZA, 2 μM UNC1999 + 1 μM AZA, or DMSO for 14 days (n = 6 for DMSO in AML 1.1 and control, n = 3 for others). Data are presented as mean ± SEM and analyzed using 2-way ANOVA with Dunnett multiple comparisons test. (F) CFU counts per 1000 replated AML cells from panel E after 10 days in methylcellulose without treatment (n = 3). Data are presented as mean ± SEM and analyzed using unpaired t test with Holm-Šídák multiple comparisons test. (G) CFU counts per 1000 seeded cells from leukemic cell lines treated with 2 μM UNC1999, 1 μM AZA, 2 μM UNC1999 + 1 μM AZA, or DMSO for 10 days (n = 3). Data are presented as mean ± SEM and analyzed using 1-way ANOVA with Dunnett multiple comparisons test. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001. ns, not significant.

Epigenetic targeting selectively impairs clonogenicity in KMT2A::MLLT3 AML cells. (A) Experimental overview for the inhibition of PRC2 by UNC1999 and DNA methylation by AZA in control- and AML-HSPCs. (B) Representative immunoblot analysis and (C) quantification of relative H3K27me3 protein levels in HSPCs from control- and AML-iPSCs after 72 hours of treatment with 2 μM and 5 μM UNC1999, 2 μM UNC1999 + 1 μM AZA, or DMSO (n = 3). Histone H3 was used to calculate relative H3K27me3 levels, which were normalized to control DMSO conditions. Data are presented as mean ± SEM of relative signal and analyzed using 2-way analysis of variance (ANOVA) with Dunnett multiple comparisons test. (D) Fold cell expansion of HSPCs from control- and AML-iPSC cells lines following 72 hours of treatment with 2 μM and 5 μM UNC1999, 2 μM UNC1999 + 1 μM AZA, or DMSO relative to cell number at seeding (n = 3). Data are presented as mean ± SEM of fold cell expansion. Lines indicate comparisons within and between control and AML cells. Data were analyzed using 2-way ANOVA with Tukey multiple comparisons test. (E) CFU counts per 1000 seeded HSPCs treated with 2 μM UNC1999, 1 μM AZA, 2 μM UNC1999 + 1 μM AZA, or DMSO for 14 days (n = 6 for DMSO in AML 1.1 and control, n = 3 for others). Data are presented as mean ± SEM and analyzed using 2-way ANOVA with Dunnett multiple comparisons test. (F) CFU counts per 1000 replated AML cells from panel E after 10 days in methylcellulose without treatment (n = 3). Data are presented as mean ± SEM and analyzed using unpaired t test with Holm-Šídák multiple comparisons test. (G) CFU counts per 1000 seeded cells from leukemic cell lines treated with 2 μM UNC1999, 1 μM AZA, 2 μM UNC1999 + 1 μM AZA, or DMSO for 10 days (n = 3). Data are presented as mean ± SEM and analyzed using 1-way ANOVA with Dunnett multiple comparisons test. ∗P ≤ .05; ∗∗P ≤ .01; ∗∗∗P ≤ .001. ns, not significant.

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