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MDM2 inhibition in chronic lymphocytic leukemia
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Figure 6. RG7388 leads to a significant dose-dependent increase in apoptosis in p53-functional chronic lymphocytic leukemia cells. (A) Caspase 3/7 activity for three representative p53-functional chronic lymphocytic leukemia (CLL) samples (CLL 0259, CLL 0268, CLL 0276) exposed to increasing concentrations (0.1, 0.3, 1 and 3 μM) of RG7388 for 24 h. *P< 0.01; ***P< 0.0001; according to a paired t test (B) Caspase 3/7 activity of a representative p53-non-functional CLL sample (CLL 0261) exposed to increasing concentrations (0.1, 0.3, 1 and 3 μM) of RG7388 for 24 h. Caspase 3/7 activity was measured by a Caspase 3/7 Glo lumines- cence-based assay and is represented as percentage change relative to that following exposure to the dimethylsulfoxide (DMSO) solvent control. Data are presented as mean ± standard error of mean (SEM) of three repeats. P-values were calculated by a paired t-test. (C) Western immunoblot for three representative p53-functional CLL samples (CLL 0263, CLL 0264, CLL 0270) showing increased expression of cleaved poly (ADP ribosome) polymerase (PARP) induced by RG7388 treatment for 24 h. (D) Western immunoblot for a representative p53-non-functional CLL sample (CLL 0261) showing no change in either full-length or cleaved PARP (cPARP) expression after exposure to RG7388 for 24 h. Basal levels of cPARP appeared high in this sample (indicative of spontaneous apoptosis) but did not increase with RG7388 treatment. The western immunoblots show the full-length pro-form of PARP (116 kDa) and the cPARP form (89 kDa).
MDM2.45,46 Moreover, it remains unclear whether basal levels of the crucial apoptotic regulator PUMA may serve as a biomarker of response to MDM2 inhibitors. To examine whether MDM2 or PUMA basal expression influences the cytotoxic effect of RG7388, we measured the basal mRNA levels of these two transcripts by qRT- PCR. The basal Ct values of MDM2 and PUMA were generally lower, and hence expression higher, in primary CLL samples than in normal BMMC (Online Supplementary Figure S7A, B). However, mean MDM2 basal Ct values were significantly higher in CLL cells than in normal PBMC (Online Supplementary Figure S7A), whereas PUMA basal expression was comparable in CLL and normal PBMC (Online Supplementary Figure S7B). Basal MDM2 and PUMA Ct values did not differ signifi- cantly between CLL samples and CD34+ cells. The basal levels of expression of MDM2 and PUMA were also sim- ilar between RG7388-sensitive samples (LC50 <1 μM) and intermediate/resistant CLL samples (LC50 >1 μM) (Online Supplementary Figure S7C, D). Moreover, we found no
correlation between basal MDM2 or PUMA expression and RG7388 LC50 values (Online Supplementary Figure S7C, D), supporting the previous observations that varia- tion in MDM2 expression does not affect the functional activation of p53 and Nutlin 3a-induced cell death in CLL.45,46
In our cohort, the fold-changes in MDM2 and PUMA expression induced by 1 μM RG7388 at 6 h also did not, alone, correlate with the LC50 values (Online Supplementary Figure S8A, B), suggesting that additional factors are important determinants of MDM2 inhibitor-induced cyto- toxicity in CLL.
Combination treatments with RG7388
Although not the primary aim of this study, we include some initial data regarding combination treatments. Adding ABT199 (venetoclax) to RG7388 had an additive effect on response, but for ex vivo treatment there was no additional benefit of adding ibrutinib to RG7388 (Online Supplementary Figure S9).
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