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C. Ciardullo et al.
of p53 target genes (Online Supplementary Figure S2B), apoptosis (Online Supplementary Figure S2C) and moderate cytotoxicity (Online Supplementary Figure S2D).
To identify functional subgroups based on their gene expression induction after exposure to 1 μM RG7388, we performed unsupervised cluster analysis of CLL samples based on the fold-change of the 11 p53-transcriptional tar- gets. This analysis showed a significant segregation of p53-functional CLL samples into three groups (defined as groups A, B and C), with group A samples showing lower induction of p53 target compared to samples from the other groups, despite the former’s wildtype p53 genomic and functional status (Figure 4A). The three groups also showed different mean RG7388 LC50 values and, in partic- ular, the mean LC50 for group A samples was significantly higher than the mean values for samples in groups B and C (Figure 4B, C).
A
RG7388 induces a concentration-dependent cytotoxic effect on chronic lymphocytic leukemia cells
To investigate the effect of RG7388 on cell viability, 55 CLL samples (Online Supplementary Table S1) were incu- bated with RG7388 and assayed for viability after 48 h using an XTT assay. Although caspase activity, indicating the triggering of apoptosis, could be seen at 24 h, it took a further 24 h for the loss of viability to become fully evi- dent in the XTT assay (Online Supplementary Figure S1B). RG7388 induced a concentration-dependent cytotoxic effect on CLL cells exhibiting functional p53 responses (examples shown in Figure 5A) but not in those without a functional p53 response (Figure 5B). Overall, the median LC50 for TP53 wildtype samples was 0.37 μM (Figure 5C). As expected, CLL samples with mutated/deleted TP53 were much more drug-resistant (median LC50=4.1 μM) (Figure 5C, which also details the TP53 mutant allele fre-
Figure 1. p53 functional stabi- lization in chronic lymphocytic leukemia cells in response to RG7388. (A) Western immunoblots showing p53-func- tional CLL cells either untreated (DMSO) or treated with increasing concentrations of RG7388 (0.1, 0.3, 1 and 3 μM) for 6 h and 24 h. Concentration-dependent and time-dependent stabilization of p53 occurs in p53-functional chronic lymphocytic leukemia (CLL) cells after 6 h and 24 h of
BC
incubation with Representative examples of four independent experiments are shown in which both p21 and MDM2 (CLL 0263, CLL 0268), only p21 (CLL 0264) or only MDM2 (CLL 0265) were induced after treatment with RG7388. (B) Western immunoblot showing p53-non-functional CLL cells either untreated (DMSO) or treat- ed with increasing concentrations (1-3-10 μM) of RG7388 for 6 h and 24 h. Lack of stabilization of p53 or induction of MDM2 and p21 is evident in p53-non-func- tional CLL cells from patient 0255. High constitutive levels of p53, which are unchanged after treatment with RG7388, are char- acteristic of mutant, non-func- tional p53. The response of cul- tured wildtype p53 OCI-Ly3 cells to RG7388 is shown as a positive control. (C) Comparison of poten- cy between RG7388 and Nutlin- 3a for killing CLL cells, measured by an XTT assay, for four repre- sentative wildtype p53 patients’ CLL samples; The mean LC50 val- ues for 48 h of treatment were 2.4 μM for Nutlin-3a and 0.18 μM for RG7388. DMSO: dimethylsul- foxide; Nut-3a: Nutlin-3a.
RG7388.
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haematologica | 2019; 104(12)