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Supplementary Table III: AML patient samples tested with HDM2 inhibitor, CGM097: IC50 values for CGM097.

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient–derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML. Mol Cancer Ther; 14(10); 2249–59. ©2015 AACR.

Supplementary Figures 1-7 Supplementary Figure 1. Amplification and sequencing primers used for genotyping of p53 in AML patient samples. Supplementary Figure 2. Apoptosis data corresponding to representative bar graphs shown in Figure 1C. Supplementary Figure 3. Cell cycle analysis data corresponding to representative bar graphs shown in Figure 1D. Supplementary Figure 4. Inhibition of growth of mutant FLT3-positive AML cells by CGM097. Supplementary Figure 5. Potentiation of effects of PKC412 by CGM097 against mutant FLT3-positive AML cells. Supplementary Figure 6. Potentiation of effects of AC220 by CGM097 against mutant FLT3-positive AML cells cultured in the absence and the presence of cytoprotective SCM. Supplementary Figure 7 (A-F). Apoptosis data corresponding to representative bar graphs shown in Figure 5C. Supplementary Figure 7 (G-L). Apoptosis data corresponding to representative bar graphs shown in Figure 5C. Supplementary Figure 7 (M-R). Apoptosis data corresponding to representative bar graphs shown in Figure 5C.