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MDM2 inhibition in chronic lymphocytic leukemia
quency). Interestingly, three samples harboring a subclon- al TP53 mutation (variant allele frequency <50%) in the absence of del17p showed decreased cell viability (RG7388 LC50<1 μM). The LC50 values for all other mutant samples, including del17p cases, were >1 μM (Figure 5C). We were unable to perform DNA analysis in CLL 0255 (see Methods). This sample was functionally defective (Figure 1B) and hence included in Figure 5C in the TP53- mutant subgroup (LC50=8.4 μM).
Notably, among TP53 wildtype samples, a small subset showed an intermediate response (1 μM<LC50<10 μM, n=5) or resistance (LC50>10 μM, n=3) to RG7388 (Figure 5D). Importantly, wildtype TP53 cells from patients in dif-
A
ferent CLL risk subgroups were similarly sensitive to RG7388. There were no significant differences in LC50 between patients with Binet stage A or C (Online Supplementary Figure S3A), mutated or unmutated IGHV genes (Online Supplementary Figure S3B) or cases with high- risk cytogenetic abnormalities such as 11q deletion and trisomy 12 (Online Supplementary Figure S3C).
Given the importance of microenvironmental stimuli on survival and activation of CLL cells as well as response to therapy, we next sought to evaluate the effect of RG7388 in CD40L/IL4-stimulated CLL cells. We found that co-cul- turing CLL cells with CD40L-expressing fibroblasts and interleukin (IL)-4 significantly reduced the spontaneous
B
Figure 2. RG7388 induces mRNA upregulation of pro- apoptotic p53 target genes in chronic lymphocytic leukemia cells. (A) Real- time reverse transcriptase polymerase chain reaction (qRT-PCR) plots for two rep- resentative p53-functional samples (CLL 0262, CLL 0267) showing preferential induction of PUMA after treatment with increasing concentrations (0.1, 0.3, 1 and 3 μM) of RG7388 for 6 h and 24 h. (B) qRT-PCR plots for a representative non-functional p53 sample (CLL 0261) exposed to increasing concentrations (0.1, 0.3, 1 and 3 μM) of RG7388 for 6 h and 24 h. The results are shown as fold-induction relative to that produced by the dimethylsulfoxide (DMSO) solvent control. Genes induced above the cut-off of 2-fold were considered up- regulated by the treatment. Data are presented as mean ± standard error of mean (SEM) of three repeats. LC50: median lethal concentration.
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