MDM2 Inhibitor Nutlin-3a Induces p53-Dependent Apoptosis Via Transcription-Dependent and -Independent Pathways and Overcomes Fludarabine-Resistance in CLL.

Fludarabine containing combination therapies are the current standard for CLL therapy, in which p53-mediated induction of apoptosis contributes to leukemia cell killing. Although TP53 mutations occur in only 5–10% of patients with CLL, inactivation of the p53 pathway also occurs through Mdm2 overexpression or Atm deficiency. We investigate if recently developed potent and selective small-molecule antagonists of Mdm2, Nutlins, can overcome functional p53 inactivation associated with Mdm2 overexpression or Atm deficiency in CLL. Nutlin-3a caused a dose- and time-dependent increase in the percentage of Annexin V-positive cells in 20 primary CLL samples, irrespective of Mdm2 or Atm status. Samples with low Atm levels (n=3) were resistant to fludarabine. In addition to the transcriptional activation of target genes in the nucleus, cytoplasmic p53 can trigger transcription-independent apoptosis at the mitochondria. Samples with a cytoplasmic p53 localization pattern (n=7) showed a higher percentage of Nutlin-induced apoptosis than those with a nuclear pattern (n=8; P < .05). Furthermore, the inhibitory effect of cycloheximide pretreatment showed a negative correlation with the degree of Nutlin-induced apoptosis (r = - 0.617, P < .05). These findings suggest that the transcription-independent pathway may have stronger apoptogenic activity than the transcription-dependent pathway. The degree of Nutlin-3a-induced apoptosis directly correlated with apoptosis induced by the same concentrations (1–10 μM) of fludarabine in the early (24 hr) time period, and the Nutlin-3a/fludarabine combination induced synergistic apoptosis [averaged combination index (CI): 0.68]. Interestingly, this synergism was not affected by Mdm2 overexpression (CI: 0.59, n=12) or Atm deficiency (CI: 0.79, n=3). We conclude that (1) Mdm2 inhibitor Nutlin-3a efficiently induces p53-dependent apoptosis in wild-type p53 CLL cells, independent of Mdm2 overexpression or Atm deficiency, (2) transactivation-dependent apoptosis does not always play a major role in p53-dependent apoptosis and the exclusive activation of transactivation-independent pathway can fully induce apoptosis, (3) p53 activation plays a critical role in the early phase of fludarabine-induced apoptosis in vitro, and (4) Mdm2 inhibition and fludarabine synergistically induce apoptosis, which may overcome fludarabine-resistance in CLL.