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C. Ciardullo et al.
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Figure 5. Effect of RG7388 on p53-functional and p53-non-functional chronic lymphocytic leukemia cell viability ex vivo. (A) Cytotoxicity curves for three represen- tative p53-functional chronic lymphocytic leukemia (CLL) samples (CLL 0260, CLL 0262, CLL 0268) exposed to increasing concentrations (0.1, 0.3, 1 and 3 μM) of RG7388 for 48 h. RG7388 markedly decreased cell viability, as assessed by an XTT assay. (B) Cytotoxicity curves for two representative p53-non-functional CLL sam- ples (CLL 0261, CLL 0255) exposed to RG7388 for 48 h. RG7388 showed no impact on cell viability. (C) Dot-plot of median lethal concentration (LC50) values for n=45 TP53 wildtype and n=10 TP53 mutant CLL samples exposed to RG7388 for 48 h. TP53 status of these samples was assessed by next-generation sequencing and fluorescence in situ hybridization and/or multiplex ligation-dependent probe amplification. The size of the dots indicates the variant allele frequency (VAF). Horizontal bars represent the median. The P-value was assessed by the Mann-Whitney test. *** P value <0.0001 (D) Dot-plot of LC50 concentrations for n=45 TP53 wildtype CLL samples exposed to RG7388 for 48 h and classified according to their cytotoxic response as sensitive responders (LC50 <1 μM), intermediate responders (1 μM <LC50 <10 μM) and resistant (LC50 >10 μM).
We then compared the data obtained from CLL cells (Figures 3-6) with the effects seen in normal cells. Treatment with 1 μM RG7388 for 6 h induced the pro- apoptotic gene PUMA in p53-functional CLL cells but not in p53-non-functional CLL or normal BMMC. Only a rel- atively small induction of PUMA was observed in normal PBMC and CD34+ cells (Figure 8A). However, for MDM2, induction was highest in normal CD34+ cells and lower, but comparable, in normal PBMC and p53-functional CLL cells (Figure 8B). Furthermore and strikingly, MDM2 upregulation dominated over the other target genes in nor- mal cells (Figure 7) in contrast to the dominance of PUMA in CLL cells (Figure 2). Of additional importance, the mean induction of CDKN1A was higher in normal PBMC than in p53-functional CLL cells (Figure 8C), suggesting that the reactivation of p53 in normal circulating blood cells by MDM2 inhibitors does not activate a cell-death signal.
Importantly, the RG7388 LC50 values were always >3 μM for normal PBMC and BMMC, and >2 μM for CD34+ cells (Figure 8D), whereas the LC50 values were <0.4 μM for p53-functional CLL cells (Figures 5C and 8D). We also found that when normal BMMC and PBMC were treated
with RG7388, the increase of caspase 3/7 activity was sig- nificantly lower than that observed in p53-functional CLL cells (Online Supplementary Figure S5). The small amount of caspase activity and cell killing induced by RG7388 in PBMC likely represents the effect on the small component of normal B cells, while T cells remain unaffected, as pre- viously reported for the response to Nutlin-3a.42
Also of note, positively-selected CD34+ cells (Online Supplementary Figure S6A, B) incubated with RG7388 for 24 h showed a reduced proportion of cells in S-phase, together with an increase of those in G0/G1 (Online Supplementary Figure S6C). There was also a small increase of cells in the subG1 phase of the cell cycle (Online Supplementary Figure S6D).
RG7388 induces cytotoxicity independently of MDM2 and PUMA basal expression or upregulation
MDM2 has been reported to be overexpressed in 50- 70% of CLL cases.43,44 However, the role of MDM2 over- expression in p53 dysfunction remains controversial, and it has been suggested that p53 activation in CLL cells is largely unaffected by variations in basal levels of
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haematologica | 2019; 104(12)