Figure 6.
Lack of GADD45A influences the response of primary human AML cells to ferroptosis induction. (A) Percentages of viable cells (DAPI negative) in GADD45A-KO vs CRISPR-Ctr mCherry+ human AML PDX cells, following ex vivo treatment with 3 μM RSL3 for 24 hours. n = 3 independent experiments. Data are given as mean ± SD. ∗P < .05, ∗∗∗P < .0005, unpaired t-test. (B) Percentages of viable (DAPI-negative) mCherry+ human AML cells (n = 3, mean ± SD) and quantitative polymerase chain reaction (qPCR) of GADD45A expression (n = 6, mean ± SEM) in CRISPR-Ctr PDX bone marrow (BM) cells, pretreated ex vivo for 18 hours with 5 μM ferrostatin-1 (Fer-1; ferroptosis inhibitor) and subsequently treated with 3 μM RSL3 (ferroptosis inducer) for an additional 24 hours. ∗P < .05, ∗∗∗∗P < .0001; NS, not significant (P > .05). One-way ANOVA. (C) qPCR showing relative expression levels of GADD45A in primary specimens from patients with AML (n = 6), compared with remission samples (n = 2). Data are given as mean ± SD. n = 3 replicates. ∗∗∗P < .0005, ∗∗∗∗P < .0001, one-way ANOVA. Note: two paired diagnostic/remission samples: AML-NK_1/remission_1 and AML-NK_2/remission_2, showing higher levels of GADD45A at remission than paired diagnosis, consistent with higher expression of GADD45A in normal human BM and CD34+ cells than in MLL-rearranged patients with AML (supplemental Figure 8). (D) Schematic overview of AML patient specimens responding to ferroptosis inducer RSL3 ex vivo. (E) Intracellular ferrous iron (Fe2+) was detected using the fluorescent turn-off sensor Phen Green (PG) SK that is quenched on binding iron, while ROS levels were measured using the lipid peroxidation sensor C11-BODIPY (581/591) that shifts its fluorescence from red (∼590 nm) to green (∼530 nm) on oxidation in hCD34+ primary AML patient specimens, following ex vivo treatment with 3 μM RSL3 for 24 hours. ΔPG-SK revealed the reversed value of PG-SK fluorescence quenching, showing an increased intracellular Fe2+ in hCD34+ cells from a patient with AML with acute promyelocytic leukemia (APL) but not 9p deletion. Data are given as mean ± SD. ∗∗P < .005, ∗∗∗P < .0005; NS, not significant (P > .05). Unpaired t-test. Also see supplemental Figure 9 for additional patient samples examined. (F) Percentages of viable cells (n = 4 replicates, mean ± SD) tested using the alamarBlue assay in primary AML patient specimens pretreated ex vivo for 18 hours with 5 μM Fer-1, followed by treatment with 3 μM RSL3 for an additional 24 hours. ∗∗∗P < .0005, ∗∗∗∗P < .0001; NS, not significant (P > .05). One-way ANOVA. (G) qPCR of GADD45A expression (mean ± SEM) in primary AML patient specimens pretreated ex vivo for 18 hours with 5 μM Fer-1, followed by treatment with 3 μM RSL3 for an additional 24 hours. ∗∗∗P < .0005; NS, not significant (P > .05). One-way ANOVA.