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MDM2 inhibition in chronic lymphocytic leukemia
We showed that RG7388 activates p53 and restores p53- transcriptional activity, inducing a characteristic dominant pro-apoptotic gene expression signature of p53-target genes selectively in CLL cells. Overall, no significant induction of transcriptional targets was observed in p53- non-functional samples, consistent with the specificity of RG7388 for p53 wildtype cells. However, a CLL sample harboring a subclonal 17p deletion in 22% of nuclei showed functional activation of p53 and induction of cell death in response to RG7388. This suggests that in the presence of low subclonal levels of p53 loss, the predomi- nant p53-functional cell population can still respond to non-genotoxic activation of p53 and patients with sub- clonal TP53 abnormalities could still benefit from treat- ment with new-generation MDM2 inhibitors, especially in combination with other p53-independent targeted ther- apies.
Moreover, RG7388 triggered apoptosis in CLL cells. This effect was dependent, in the majority of samples, on the presence of functional p53. CLL samples with pre- dominantly mutated, non-functional p53 did not show induction of apoptosis. As a consequence of upregulation
of apoptotic genes and activation of apoptosis, RG7388 significantly decreased the cell viability of p53-functional CLL samples, but CLL samples that displayed non-func- tional p53 on western blot and mutated/deleted TP53 showed greater resistance. However, in the TP53-mutant subgroup, three samples harboring subclonal TP53 muta- tions showed LC50 values lower than 1 μM, indicating sig- nificantly decreased cell viability upon exposure to RG7388. This finding is in line with the results of a recent phase I clinical trial evaluating the effect of the earlier-gen- eration MDM2 inhibitor RG7112 in leukemia.29 This clin- ical study included a small number of heavily pre-treated CLL patients and in this subgroup RG7112 showed clinical activity, with evidence of induction of PUMA and apopto- sis in a patient with CLL whose white blood count decreased by >50%.29 Among RG7112-treated patients, the investigators reported two patients with TP53 mutant leukemic cell samples who exhibited a clinical response.29
Interestingly, among TP53 wildtype CLL samples, we identified a small subset that showed an intermediate response or resistance to RG7388 treatment, suggesting that TP53 mutational status is not the only determinant of
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Figure 8. RG7388 selectively induces PUMA expression and cell death in chronic lymphocytic leukemia cells compared with normal hematopoietic cells. (A-C) Fold changes in mRNA of PUMA (A), MDM2 (B) and CDKN1A (C) measured by real-time reverse transcriptase polymerase chain reaction in p53-functional CLL samples (n=24), p53-non-functional chronic lymphocytic leukemia (CLL) samples (n=2), normal bone marrow mononuclear cells (BMMC) (n=5), normal peripheral blood mononuclear cells (PBMC) (n=6) and normal CD34+ samples (n=3) exposed to 1 μM RG7388 for 6 h. (D) Cytotoxic response comparison of normal BMMC, normal PBMC, normal CD34+ samples and p53-functional CLL samples exposed to RG7388 (0.03, 0.1, 0.3, 1 and 3 μM) for 48 h. RG7388 markedly decreased cell viability, assessed by an XTT assay, of p53-functional CLL cells but to a much lesser extent in normal cells.
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