Figure 2.
MDM2 inhibition enhances GVL effects in syngeneic AML models and TRAIL-R1/2 upregulation on leukemia cells. (A) Percentage survival of BALB/c recipient mice after transfer of AML WEHI-3B cells (BALB/c background) and allogeneic C57BL/6 BM is shown. Mice were injected with additional allogeneic T cells (Tc; C57BL/6) and/or treated with either vehicle or MDM2 inhibitor RG-7112 when indicated. A total of n = 9 to 10 independent animals per group are shown, and P values were calculated using the 2-sided Mantel-Cox test. (B) Percentage survival of C57BL/6-recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c) and/or treated with either vehicle or MDM2-inhibitor RG-7112 when indicated. A total of n = 10 biologically independent animals from 2 experiments are shown, and P values were calculated using the 2-sided Mantel-Cox test. (C) Representative bioluminescence imaging (BLI) on day 14 after transplantation with LUC+ MOLM-13 leukemia cells showing the expansion of the leukemia cells in Rag2−/−Il2rγ−/− mice. When indicated, mice received human CD3+ T cells (isolated from the peripheral blood of an HLA nonmatched healthy donor) and were treated with either vehicle or MDM2 inhibitor RG-7112. Images are of a representative mouse from each of the groups. (D) The graph shows the quantification of the BLI signal at day 14 after allo-HCT for each treatment group. Data from 3 independent experiments were pooled. The mean ± standard error of the mean (SEM) for each treatment group is shown. The P values were calculated using the 2-sided Mann-Whitney U test. (E) The graph shows the percent of human CD45+CD3−CD8− cells (representing the MOLM-13 AML cells) of all viable cells in the BM of Rag2−/−Il2rγ−/− mice 23 days after transplantation with LUC+ MOLM-13 leukemia cells and human CD3+ T cells. Data are shown as mean ± SEM from n = 3 independent experiments. P values were calculated using a 2-sided Student unpaired t test. (F) Microarray-based analysis of the expression level of TRAIL-R1 and TRAIL-R2 (TNFRSF10A and TNFRSF10B) in OCI-AML3 cells after treatment with DMSO, RG-7112 (1 µM), or HDM-201 (200 nM) for 24 hours is shown as a tile display from Robust Multichip Average (RMA) signal values (Z-score), n = 6 biologically independent samples per group. The adjusted P value was calculated using the Benjamini & Hochberg method. For both genes, a significant upregulation (both P < .001) compared with DMSO was observed. (G) The graph shows the fold change of MFI for TRAIL-R1 expression on OCI-AML3 cells after treatment with the indicated concentrations of MDM2-inhibitor RG-7112 for 72 hours. Data are shown as mean ± SEM from n = 3 independent experiments. P values were calculated using a 2-sided Student unpaired t test. (H) The graph shows the fold change of MFI for TRAIL-R2 expression on OCI-AML3 cells after treatment with the indicated concentrations of MDM2 inhibitor RG-7112 for 72 hours as mean ± SEM from n = 5 independent experiments. P values were calculated using a 2-sided Student unpaired t test. (I-J) The graph shows fold change of MFI for (I) TRAIL-R1 or (J) TRAIL-R2 expression on WT OCI-AML3 or p53 KD OCI-AML3 cells after treatment with the indicated concentrations of MDM2 inhibitor RG-7112 for 72 hours as mean ± SEM from n = 4 independent experiments. MFI of control-treated cells was set as 1.0. P values were calculated using the 2-sided Student unpaired t test.

MDM2 inhibition enhances GVL effects in syngeneic AML models and TRAIL-R1/2 upregulation on leukemia cells. (A) Percentage survival of BALB/c recipient mice after transfer of AML WEHI-3B cells (BALB/c background) and allogeneic C57BL/6 BM is shown. Mice were injected with additional allogeneic T cells (Tc; C57BL/6) and/or treated with either vehicle or MDM2 inhibitor RG-7112 when indicated. A total of n = 9 to 10 independent animals per group are shown, and P values were calculated using the 2-sided Mantel-Cox test. (B) Percentage survival of C57BL/6-recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c) and/or treated with either vehicle or MDM2-inhibitor RG-7112 when indicated. A total of n = 10 biologically independent animals from 2 experiments are shown, and P values were calculated using the 2-sided Mantel-Cox test. (C) Representative bioluminescence imaging (BLI) on day 14 after transplantation with LUC+ MOLM-13 leukemia cells showing the expansion of the leukemia cells in Rag2−/−Il2rγ−/− mice. When indicated, mice received human CD3+ T cells (isolated from the peripheral blood of an HLA nonmatched healthy donor) and were treated with either vehicle or MDM2 inhibitor RG-7112. Images are of a representative mouse from each of the groups. (D) The graph shows the quantification of the BLI signal at day 14 after allo-HCT for each treatment group. Data from 3 independent experiments were pooled. The mean ± standard error of the mean (SEM) for each treatment group is shown. The P values were calculated using the 2-sided Mann-Whitney U test. (E) The graph shows the percent of human CD45+CD3−CD8− cells (representing the MOLM-13 AML cells) of all viable cells in the BM of Rag2−/−Il2rγ−/− mice 23 days after transplantation with LUC+ MOLM-13 leukemia cells and human CD3+ T cells. Data are shown as mean ± SEM from n = 3 independent experiments. P values were calculated using a 2-sided Student unpaired t test. (F) Microarray-based analysis of the expression level of TRAIL-R1 and TRAIL-R2 (TNFRSF10A and TNFRSF10B) in OCI-AML3 cells after treatment with DMSO, RG-7112 (1 µM), or HDM-201 (200 nM) for 24 hours is shown as a tile display from Robust Multichip Average (RMA) signal values (Z-score), n = 6 biologically independent samples per group. The adjusted P value was calculated using the Benjamini & Hochberg method. For both genes, a significant upregulation (both P < .001) compared with DMSO was observed. (G) The graph shows the fold change of MFI for TRAIL-R1 expression on OCI-AML3 cells after treatment with the indicated concentrations of MDM2-inhibitor RG-7112 for 72 hours. Data are shown as mean ± SEM from n = 3 independent experiments. P values were calculated using a 2-sided Student unpaired t test. (H) The graph shows the fold change of MFI for TRAIL-R2 expression on OCI-AML3 cells after treatment with the indicated concentrations of MDM2 inhibitor RG-7112 for 72 hours as mean ± SEM from n = 5 independent experiments. P values were calculated using a 2-sided Student unpaired t test. (I-J) The graph shows fold change of MFI for (I) TRAIL-R1 or (J) TRAIL-R2 expression on WT OCI-AML3 or p53 KD OCI-AML3 cells after treatment with the indicated concentrations of MDM2 inhibitor RG-7112 for 72 hours as mean ± SEM from n = 4 independent experiments. MFI of control-treated cells was set as 1.0. P values were calculated using the 2-sided Student unpaired t test.

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