Figure 6.
The birinapant/MDR1i combination is well tolerated, efficiently kills murine LSCs, and prolongs survival in murine AML models. (A) LSK HSPCs from wild-type (WT) C57BL/6 mice were treated with Bir (0.5 μM), with or without Tari (1 μM) or Ara-c (0.5 and 10 μM), for 48 hours (n = 3-5; 3 independent experiments). (B) MLL-AF9 blasts and LSCs were pretreated with Rho-123 (250 nM), with or without Tari (1 μM). Rho-123 fluorescence determined by flow cytometry.Histograms with different shades of gray represent control cells untreated (dark) and treated (light) with MDR1i in the absence of Rho-123. (C) MLL-AF9 blasts and LSCs were treated with Bir (500 nM), with or without Tari (1 μM), for 10 minutes. Whole-cell lysates were probed with the indicated antibodies, with actin used as the loading control. M# indicates individual membranes. MLL-AF9 AML blasts (D) and LSCs (E) were treated with Bir (0.001, 0.016, 0.08, 0.4, and 2 μM), with or without Tari or Zosu (1 μM), for 24 hours (n = 4-7). P values were obtained by comparison of Bir alone with Bir/Tari (red) or Bir/Zosu (green) treatment at the indicated time points. (F) C57BL/6 mice receiving MDR1H MLL-AF9-GFP AML cell transplants were treated with Ara-c (50 mg/kg) for 5 consecutive days, followed by either Bir single-agent treatment (10 mg/kg) or Bir/tTari (10 mg/kg) combination therapy, given 2 or 3 times a week (Ara-c+2×Combo and Ara-c+3×Combo, respectively; n = 9-13; 4 independent experiments). P values were obtained by comparison of Ara-c single-agent treatment and Bir/ Tari treatments and determined by log-rank (Mantel-Cox) test. Histology of spleens and livers (G) and percentages of GFP+ cells in the bone marrow (H) of mice. The weights of livers (I) and spleens (J) as a percentage of body weight (n = 6-11) were also determined. Cell survival was measured by flow cytometry analysis of PI exclusion. P values were obtained by comparison of Ara-c+Bir and Ara-c+2×Combo or Ara-c+3×Combo. Data are the mean ± SEM throughout. *P < .05; **P < .01; ***P < .001; ****P < .0001. FSC, forward scatter.

The birinapant/MDR1i combination is well tolerated, efficiently kills murine LSCs, and prolongs survival in murine AML models. (A) LSK HSPCs from wild-type (WT) C57BL/6 mice were treated with Bir (0.5 μM), with or without Tari (1 μM) or Ara-c (0.5 and 10 μM), for 48 hours (n = 3-5; 3 independent experiments). (B) MLL-AF9 blasts and LSCs were pretreated with Rho-123 (250 nM), with or without Tari (1 μM). Rho-123 fluorescence determined by flow cytometry.Histograms with different shades of gray represent control cells untreated (dark) and treated (light) with MDR1i in the absence of Rho-123. (C) MLL-AF9 blasts and LSCs were treated with Bir (500 nM), with or without Tari (1 μM), for 10 minutes. Whole-cell lysates were probed with the indicated antibodies, with actin used as the loading control. M# indicates individual membranes. MLL-AF9 AML blasts (D) and LSCs (E) were treated with Bir (0.001, 0.016, 0.08, 0.4, and 2 μM), with or without Tari or Zosu (1 μM), for 24 hours (n = 4-7). P values were obtained by comparison of Bir alone with Bir/Tari (red) or Bir/Zosu (green) treatment at the indicated time points. (F) C57BL/6 mice receiving MDR1H MLL-AF9-GFP AML cell transplants were treated with Ara-c (50 mg/kg) for 5 consecutive days, followed by either Bir single-agent treatment (10 mg/kg) or Bir/tTari (10 mg/kg) combination therapy, given 2 or 3 times a week (Ara-c+2×Combo and Ara-c+3×Combo, respectively; n = 9-13; 4 independent experiments). P values were obtained by comparison of Ara-c single-agent treatment and Bir/ Tari treatments and determined by log-rank (Mantel-Cox) test. Histology of spleens and livers (G) and percentages of GFP+ cells in the bone marrow (H) of mice. The weights of livers (I) and spleens (J) as a percentage of body weight (n = 6-11) were also determined. Cell survival was measured by flow cytometry analysis of PI exclusion. P values were obtained by comparison of Ara-c+Bir and Ara-c+2×Combo or Ara-c+3×Combo. Data are the mean ± SEM throughout. *P < .05; **P < .01; ***P < .001; ****P < .0001. FSC, forward scatter.

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