Figure 2.
Enhanced AR expression increases the aggressiveness of AML donor cells. (A) Viability of 1° WT high AR–expressing AML cells treated with ARN509 at 0, 50, 100, and 150 nM for 24 hours (n = 4). The frequency of CD45.1+ AML cells was assessed by flow cytometry. (B) The frequency of the Annexin V+7AAD– population in 1° WT high AR–expressing AML cells treated with ARN509 at 0 or 150 nM for 24 hours (n = 5) pregated for the identification of CD45.1 by flow cytometry. (C) Cell cycle analysis of 1° WT high AR–expressing AML cells treated with ARN509 at 0 or 150 nM for 24 hours (n = 5). The cells were first gated on Forward Scatter-Area/Height, FSC-A/FSC-H, and FSC-A and Side Scatter-Area, SSC-A, to acquire singlets. AML cells were identified as the CD45.1+ population and further evaluated for cell cycle phase using the Dean-Jet-Fox model in the FlowJo software program. (D) Primary WT high AR–expressing AML cells were plated in methylcellulose medium (2500 cells per well, 4 replicates) with/without 150 nM ARN509. The CFUs were counted on day 8. (E) Secondary transplantation was done retro-orbitally with 1° CD45.1 WT high AR–expressing AML donor cells to CD45.2 WT female recipients; at 1 week after transplantation, the mice were administered ARN509 daily at 10 mg/kg or 25 mg/kg intraperitoneally for 2 weeks. Mice were euthanized at 3 weeks after transplantation; the blood, bone marrow, and spleen were sampled (n = 5-7 in each group). (F) Complete blood count (CBC) analysis of recipient mice in panel E at the end point. (G) Spleen weights (milligram) of recipient mice in panel E at the end point. (H-I) Counts of AML cells in the Lin– (H) and LIC (I) populations in the spleen of recipient mice in panel E at the end point. (J) Count of platelets in the periphery of recipient mice in panel E at the end point. (K) Body weight change expressed as the ratio of body weight before the treatment and at the end point in panel E. (L) A Kaplan-Meier curve was generated to estimate the survival of female recipients transplanted with 1° WT high AR–expressing AML cells after in vitro culture in the absence or presence of 150 nM ARN509. Survival was followed up for 60 days after transplantation. ∗P < .05; ∗∗P < .01, as determined by a log-rank test. (M) Illustration showing the targeted knockdown of Ar by a CRISPR interference technique that targets the promoter of Ar. (N) Knockdown of AR was assessed by western blot. (O) Survival analysis of 2° male and female recipients transplanted with WT high AR–expressing or AR KD AML cells; ∗P < .05; ∗∗P < .01, as determined by a log-rank test. Panels A-D, error bars represent the mean ± SEM of the replicates. ∗P< .05; ∗∗P < .01, as determined by a 1-tailed unpaired Student t test. Panels G-K, error bars represent the mean ± SEM of replicates. ∗P < .05; ∗∗P < .01, as determined by a 1-way analysis of variance (ANOVA) test. BA, basophil; Ctl, control; CFU, colony forming unit; EO, eosinophil; LY, lymphocyte; MO, monocyte; NE, neutrophil; PBS, phosphate-buffered saline.

Enhanced AR expression increases the aggressiveness of AML donor cells. (A) Viability of 1° WT high AR–expressing AML cells treated with ARN509 at 0, 50, 100, and 150 nM for 24 hours (n = 4). The frequency of CD45.1+ AML cells was assessed by flow cytometry. (B) The frequency of the Annexin V+7AAD– population in 1° WT high AR–expressing AML cells treated with ARN509 at 0 or 150 nM for 24 hours (n = 5) pregated for the identification of CD45.1 by flow cytometry. (C) Cell cycle analysis of 1° WT high AR–expressing AML cells treated with ARN509 at 0 or 150 nM for 24 hours (n = 5). The cells were first gated on Forward Scatter-Area/Height, FSC-A/FSC-H, and FSC-A and Side Scatter-Area, SSC-A, to acquire singlets. AML cells were identified as the CD45.1+ population and further evaluated for cell cycle phase using the Dean-Jet-Fox model in the FlowJo software program. (D) Primary WT high AR–expressing AML cells were plated in methylcellulose medium (2500 cells per well, 4 replicates) with/without 150 nM ARN509. The CFUs were counted on day 8. (E) Secondary transplantation was done retro-orbitally with 1° CD45.1 WT high AR–expressing AML donor cells to CD45.2 WT female recipients; at 1 week after transplantation, the mice were administered ARN509 daily at 10 mg/kg or 25 mg/kg intraperitoneally for 2 weeks. Mice were euthanized at 3 weeks after transplantation; the blood, bone marrow, and spleen were sampled (n = 5-7 in each group). (F) Complete blood count (CBC) analysis of recipient mice in panel E at the end point. (G) Spleen weights (milligram) of recipient mice in panel E at the end point. (H-I) Counts of AML cells in the Lin– (H) and LIC (I) populations in the spleen of recipient mice in panel E at the end point. (J) Count of platelets in the periphery of recipient mice in panel E at the end point. (K) Body weight change expressed as the ratio of body weight before the treatment and at the end point in panel E. (L) A Kaplan-Meier curve was generated to estimate the survival of female recipients transplanted with 1° WT high AR–expressing AML cells after in vitro culture in the absence or presence of 150 nM ARN509. Survival was followed up for 60 days after transplantation. ∗P < .05; ∗∗P < .01, as determined by a log-rank test. (M) Illustration showing the targeted knockdown of Ar by a CRISPR interference technique that targets the promoter of Ar. (N) Knockdown of AR was assessed by western blot. (O) Survival analysis of 2° male and female recipients transplanted with WT high AR–expressing or AR KD AML cells; ∗P < .05; ∗∗P < .01, as determined by a log-rank test. Panels A-D, error bars represent the mean ± SEM of the replicates. ∗P< .05; ∗∗P < .01, as determined by a 1-tailed unpaired Student t test. Panels G-K, error bars represent the mean ± SEM of replicates. ∗P < .05; ∗∗P < .01, as determined by a 1-way analysis of variance (ANOVA) test. BA, basophil; Ctl, control; CFU, colony forming unit; EO, eosinophil; LY, lymphocyte; MO, monocyte; NE, neutrophil; PBS, phosphate-buffered saline.

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