The p53-MDM2 Antagonist RG7388 Activates p53 and Induces a Predominantly Pro-Apoptotic Gene Expression Signature in Chronic Lymphocytic Leukemia

Introduction. Current treatment protocols for chronic lymphocytic leukemia (CLL), including FCR (fludarabine, cyclophosphamide and rituximab), have dramatically improved response rates. However, since none of these therapies is curative, continued preclinical studies on innovative therapeutic strategies are warranted. In particular, the identification of new agents that interfere with the survival of CLL cells by promoting their apoptosis is one approach to improve therapeutic outcome. Taking into account that p53 deletions and/or mutations in CLL are restricted to 10%-15% of patients at diagnosis, we have investigated the efficacy of the second-generation MDM2 antagonist RG7388, in primary CLL samples, examining its ability to disrupt the p53-MDM2 interaction, resulting in expression of p53 transcriptional targets that trigger apoptosis.

Methods. Heparinized peripheral blood samples were obtained from CLL patients (n=42) with informed consent, in accordance with institutional guidelines and the Declaration of Helsinki. Mononuclear cells were purified by Lymphoprep density-gradient centrifugation. Cells were resuspended in RPMI 1640 medium supplemented with 10% FCS and 1% penicillin/streptomycin. Cells were treated with increasing concentrations of RG7388 and incubated at 37°C, 5% CO₂. Ex vivo cytotoxicity was assessed in all samples 48 hours after treatment using an XTT assay. A pilot time-course experiment identified 6 hours after treatment as the most informative time point for gene expression analysis. Therefore, in 8 samples cells were treated for 6 hours and RNA was extracted using standard methods. qRT-PCR on a panel of nine p53 transcriptional targets (CDKN1A, MDM2, PUMA, BAX, FAS, FDXR, ZMAT3, TP53INP1, DR5) was performed using SYBR Green chemistry. The functional integrity of p53 was assessed by western blot following treatment with MDM2 antagonists in 20 samples. Apoptosis was assessed using a Caspase 3/7 luminescent assay.

Results. Treatment with RG7388 significantly decreased cell viability (LC₅₀ < 1µM) in 28/42 CLL samples (mean LC₅₀ = 0.40 ± 0.05 µM). As expected, the 8 CLL samples that displayed non functional p53 on western blot and/or mutated TP53 by sequencing were more drug-resistant (mean LC₅₀ = 5.22 ± 1.13 µM) than those sensitive responders (LC50 < 1µM) with a functional p53 (mean LC₅₀ = 0.39 ± 0.05 µM, n=12). Interestingly, among p53 functional samples, we identified a small subset that showed intermediate response (1 µM <LC₅₀< 10 µM, n=2) or resistance (LC₅₀ >10 µM, n=3) to RG7388 treatment.

RG7388 significantly upregulated the mRNA expression of MDM2 and of p53 transcriptional targets involved in the intrinsic pathway of apoptosis (PUMA and BAX), and the extrinsic pathway of apoptosis (death receptor 5 (DR5)) (Figure 1A, n=5 samples). Interestingly, only a slight upregulation of CDKN1A was observed and upregulation of pro-apoptotic genes dominated. Overall, in p53 functional CLL samples, exposure to 3 µM RG7388 for 6 hours led to a 7.8-fold increase in MDM2, a 7-fold increase in PUMA, a 4.9-fold increase in DR5, a 4.5-fold increase in BAX, and a 3.3-fold increase in CDKN1A (Figure 1A). Conversely, no significant upregulation of p53 target genes was seen in p53 non-functionalCLL samples (Figure 1B, n=3 samples).

Treatment with RG7388 led to a concentration-dependent increase in caspase 3/7 activity in p53 functional CLL samples (Figure 1C). No significant difference in caspase 3/7 activity was observed in p53 non-functional samples after RG7388 treatment (Figure 1D).

Interestingly, when we analysed samples taken two years apart from a fludarabine- and chlorambucil-resistant CLL patient, we found that treatment with RG7388 decreased cell viability, induced p53 accumulation and upregulation of p53-target genes, suggesting that inhibiting the p53-MDM2 interaction might be a promising treatment strategy in chemo-resistant CLL patients that show a functional p53.

Conclusion. Taken together, our data indicate that RG7388 induces a characteristic dominantly pro-apoptotic gene expression profile of p53-target genes in primary p53-functional CLL samples. RG7388 showed a consequent potent apoptotic effect on CLL cells, which was dependent on the presence of a functional p53. Identification of patients in whom functional p53 activation drives apoptosis may translate into improved outcomes for patients treated with non-genotoxic MDM2 antagonists.

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