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MDM2 inhibition in chronic lymphocytic leukemia
cation of BAX, CKDN1A, MDM2, PUMA (BBC3), FAS, FDXR, GADD45A, TNFRSF10B, ZMAT3, TP53INP1 and WIP1/PPM1D mRNA expression was performed by real-time reverse transcrip- tase polymerase chain reaction (qRT-PCR) based on SybrGreen chemistry using an Applied Biosystems QuantStudioTM 7 Real- Time PCR System (Applied Biosystems, UK). Each sample was analyzed in triplicate using GAPDH as a housekeeping control. The relative expression of each gene, expressed as fold-change, was calculated by the 2−ΔΔCt method and the result of each sample was normalized to that of its dimethlysulfoxide-treated matched sample. Validated primer sequences are presented in Online Supplementary Table S2. The gene panel selected for this study was based on the results of a recent phase I trial of the MDM2 inhibitor RG711229 and published data from our group reporting the effect of MDM2 antagonists in different cancer cell lines.31,34
Additional analysis of a panel of anti-apoptotic genes, BCL2, MCL1 and BCL2L1 (alias BCL-XL), plus the pro-apoptotic genes PMAIP1 (alias NOXA) and BCL2L11 (alias BIM) (Online Supplementary Table S2) was also performed on a subset of samples.
Apoptosis assay
Cells (5x105/well) were seeded in 96-well plates and exposed to increasing concentrations of RG7388 for 24 h. Caspase 3/7 activity (Caspase-Glo® 3/7 Assay, Promega, UK) was assessed as detailed in the Online Supplementary Methods. Apoptosis was also deter- mined by examining cleaved poly (ADP-ribose) polymerase (PARP) by western blot.
Co-culture and stimulation of chronic lymphocytic leukemia cells with CD40L-expressing cells
CLL cells were cultured on a monolayer of CD40L-expressing mouse fibroblasts and exposed to RG7388 as detailed in the Online Supplementary Methods.
Cell cycle analysis of CD34+ hematopoietic stem cells
CD34+ cells were exposed to RG7388 for 24 h and cell cycle dis- tribution was evaluated as detailed in the Online Supplementary Methods.
Statistical analysis
Statistical analysis was performed using GraphPad Prism v6 (GraphPad Software Inc.). Statistical differences between groups were evaluated by a paired Student t-test or Mann–Whitney test. Correlations were analyzed by the Pearson rank correlation test. P-values <0.05 were considered statistically significant.
Hierarchical cluster analysis of the Euclidean distances of gene expression levels was carried out using the R pheatmap package.37 The subsequent group comparison of median lethal concentration (LC50) was performed using analysis of variance by parametric tests, applying the Holm-Sidaks correction for multiple compar- isons between groups.
Results
TP53 genomic status of chronic lymphocytic leukemia samples
Online Supplementary Table S1 provides details of the TP53 mutations, including coding region position and amino acid changes as well as 17p deletion status. The mutations detected were mostly (8/9 CLL samples) in the DNA binding domain (amino acids 102-292). The remain- ing case (CLL273) had a double mutation in the C-termi- nal tetramerization domain. All mutations were deleteri- ous, leading to loss of function.
The MDM2 inhibitor RG7388 induces functional stabilization of p53 in chronic lymphocytic leukemia cells
We assessed protein expression of p53, as well as p53- regulated downstream targets, in patient-derived CLL cells by western blot, following incubation with RG7388. Inhibition of MDM2 by RG7388 blocked ubiquitin-medi- ated degradation of p53, leading to its accumulation. In p53-functional CLL samples, RG7388 led to a concentra- tion-dependent stabilization of p53, with subsequent acti- vation of downstream proteins, p21 and MDM2 (Figure 1A). The accumulation of p53 was detectable in all p53- functional CLL samples as soon as 6 h after commence- ment of treatment and increased at 24 h (Figure 1A). In the 30 p53-functional CLL samples analyzed, RG7388 increased p21 protein expression in 77% of cases and led to a detectable auto-regulatory feedback increase in expression of MDM2 in 85% of cases. The activation of these two downstream targets occurred in a concentra- tion- and time-dependent manner (Figure 1A). Conversely, in p53-non-functional CLL samples, we did not find stabi- lization of p53 or induction of MDM2 and p21 after treat- ment with RG7388, even at concentrations of 10 μM (Figure 1B). The increased potency against CLL cells of the second-generation MDM2 inhibitor RG7388 compared with Nutlin-3a is shown in Figure 1C.
RG7388 induces a predominantly pro-apoptotic gene expression signature in chronic lymphocytic leukemia cells
We used qRT-PCR to study the expression of 11 known p53 transcriptional target genes in 26 CLL samples after treatment with RG7388. In p53-functional CLL samples, MDM2 inhibition by RG7388 led to a concentration- and time-dependent upregulation of p53-transcriptional tar- gets (exemplified by CLL 0262 and 0267) (Figure 2A). No change in gene expression was identified in p53-non-func- tional samples (exemplified by CLL 0261) (Figure 2B).
The results for the 24 p53-functional CLL samples are summarized in Figure 3A, which illustrates the concentra- tion-dependent nature of the fold-change in gene expres- sion. The results for the two p53-non-functional CLL sam- ples are shown in Figure 3B. In p53-functional samples, six genes were induced (≥2-fold expression above baseline) in response to 1 μM RG7388 for 6 h; all of these genes are known to be directly regulated by p53 (Figure 3C). We observed a mean 8.5-fold increase in PUMA, 5.1-fold in MDM2, 3.8-fold in BAX, 2.7-fold in TNFRSF10B, 2.6-fold in FAS, 2.2-fold in WIP1, and 1.6-fold in CDKN1A (Figure 3C). Thus, only a slight upregulation of CDKN1A, encod- ing the p21 cyclin-dependent kinase inhibitor, was observed and induction of pro-apoptotic genes dominat- ed. Additional analysis of a panel of anti-apoptotic genes (BCL2, MCL1 and BCL2L1 (alias BCL-XL), plus the pro- apoptotic genes PMAIP1 (alias NOXA) and BCL2L11 (alias BIM) showed no significant changes in mRNA expression compared with the large change in PUMA mRNA (Figure 3D). Western blot analysis confirmed that induction of PUMA protein by RG7388 treatment could be detected in CLL samples (Online Supplementary Figure S1A).
As would be expected on bulk analysis, CLL 0269, har- boring a small subclonal 17p deletion (22% of nuclei), but no evidence of a TP53 mutation, nevertheless showed functional stabilization of p53 by RG7388 (Online Supplementary Figure S2A) with subsequent upregulation
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