MDM2 inhibition leads to increased MHC expression and improves survival in xenograft mouse models. (A) Microarray-based analysis of the expression level of HLA class I and II in OCI-AML3 cells after treatment with DMSO, RG-7112 (1 µM), or HDM-201 (200 nM) for 24 hours is shown as 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. *indicates significant regulation (P < .05) compared with DMSO. (B-C) Representative flow cytometry histograms show the mean fluorescence intensity (MFI) for (B) HLA-DR or (C) HLA-A,B,C expression on OCI-AML3 cells after treatment with the indicated concentrations of MDM2 inhibitor RG-7112 for 72 hours. (D) The bar diagram shows the MFI for HLA-DR expression on OCI-AML3 cells after treatment with the indicated concentrations of MDM2 inhibitor RG-7112 for 72 hours. Bar graphs show the mean ± standard error of the mean (SEM) from n = 5 to 6 independent experiments. P values were calculated using the 2-sided Student unpaired t test. (E) The bar diagram shows the MFI for HLA-A,B,C expression on OCI-AML3 cells after treatment with the indicated concentrations of MDM2 inhibitor RG-7112 for 72 hours. Bar graphs show the mean ± SEM from n = 5 to 6 independent experiments. P values were calculated using the 2-sided Student unpaired t test. (F-G) Percentage survival of Rag2^−/−Il2rγ^−/−-recipient mice after the transfer of primary human AML cells from 2 different patients is shown. As indicated, mice were injected with additional human CD3⁺ T cells (isolated from the peripheral blood of an HLA nonmatched healthy donor) and treated with either vehicle or MDM2 inhibitor RG-7112. Shown are n = 10 or 8 independent animals, and P values were calculated using the 2-sided Mantel-Cox test. (H) Percentage of specific lysis of isolated, CD3/28, and IL-2 expanded human T cells in contact with OCI-AML3 cells is shown. OCI-AML3 cells were pretreated with either DMSO or the MDM2-inhibitor RG-7112, and the E:T (effector [T-cell] to target [OCI-AML3 cell]) ratio was titrated between 10:1 and 1:1 as indicated. One representative experiment of 3 independent experiments is shown. (I) Representative Western blots showing the activation of caspase-3 and loading control (β-actin) in OCI-AML3 cells. OCI-AML3 cells exposed to DMSO or RG-7112 (1 µM) were cocultured with activated T cells at an E:T ratio of 10:1 for 4 hours. (J) The bar diagram indicates the ratio of the cleaved caspase-3 to pro–caspase-3 normalized to β-actin. The values were normalized to the T-cell–only group (set as “1”). (K-L) The graph shows fold change of MFI for (K) HLA-DR and (I) HLA-A,B,C expression on WT OCI-AML3 (WT) or p53 knockdown (p53 KD) OCI-AML3 cells after treatment with RG-7112 (2 µM) 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 a 2-sided Student unpaired t test. (M) Percentage survival of Rag2^−/−Il2rγ^−/−-recipient mice after transfer of human WT or p53 knockdown (p53 KD) OCI-AML3 cells are shown. As indicated, mice were injected with additional human T cells (isolated from the peripheral blood of an HLA nonmatched healthy donor) and treated with either vehicle or MDM2-inhibitor RG-7112. Shown are n = 10, 11, or 21 independent animals per group from ≥2 independent experiments, and P values were calculated using the 2-sided Mantel-Cox test.