Molecular Mechanisms Mediating Antimyeloma Activity of An MDM2 Antagonist Nutlin.

p53 gene mutations or deletions are rare in multiple myeloma (MM). Thus, strategies to induce p53 activation in myeloma cells with wild type (wt) p53 may have therapeutic promise. However, p53 signaling and functional consequences are not clearly understood in MM. In this study we used a small molecule MDM2 antagonist; nutlin, to explore the molecular mechanisms associated with nutlin-mediated activation of the p53 signaling pathway resulting in cell cycle arrest and/or apoptosis.

MM1.S and H929 cell lines harboring wt p53, LP1 and U266 cell lines expressing mutant (mt) p53, and five primary samples from patients with MM were treated with different concentrations of nutlin or DMSO control. Nutlin-induced cells were assessed for cell viability, induction of p53 and its downstream targets, cell cycle analysis, apoptosis assays and gene expression profile at different time periods.

Treatment of MM cells harboring wt p53 with nutlin led to a dose-dependent increase in the expression of p53 and its downstream targets, p21 and MDM2. After 24 hours incubation with 10 μM nutlin, p53 protein levels increased approximately 4 to 6-fold in MM1.S and H929 cell lines and in two primary MM samples. Induction of p53 downstream targets was strictly correlated with induction of p53. In contrast, nutlin did not modulate the expression of these proteins in the cells with mt p53. Proliferation of the cells was also affected by nutlin in MM cells harboring wt p53 but not mt p53. Seventy-two hours after incubation with 10 μM nutlin, the viability of the cells (MM cell lines and primary MM samples) with wt p53 declined to 30% as assessed by MTT assay. Nutlin caused up-regulation of several pro-apoptotic targets, PUMA, Bax, and Bak, and down-regulation of two anti-apoptotic targets, Bcl2 and survivin in MM1.S cells. Furthermore, nutlin effectively arrested cell cycle progression in wt p53 MM cells, depleting the cells in S-phase compartment and increasing the cells in G1 and G2/M phase compartment, indicating G1 and G2 arrest. Annexin-V staining and flow cytometry (FCM) studies showed that there was a dose- and time-dependent increase in annexin-V binding in MM1.S cells but not in LP1 or U266 cells. These results suggest that nutlin-induced apoptosis is p53-dependent. At 72 hours following treatment with 10 μM nutlin, annexin-V binding was increased from 22% (1.0 μM) to 80% in MM1.S cells. Western blot (WB) analysis of nutlin-induced cells revealed activation of both caspase-8 and caspase-9 followed by activation of caspase-3 suggesting the association of both extrinsic and intrinsic pathways of apoptosis. In addition, direct inhibition of endogenous survivin by siRNA further enhanced nutlin-induced apoptosis, as measured by WB analysis for activation of caspase-3 and FCM assay for annexin-V binding. Forty-eight hours after nutlin treatment, 60% of MM1.S cells transfected with survivin siRNA were annexin-V positive, whereas control siRNA-transfected cells were 30% annexin-V positive. Moreover, selective blocking of p53 transcription by a p53 inhibitor, pifithrin-α (25 μM), inhibits nutlin-induced up-regulation of p53-transcriptional target genes p21, PUMA, and Bax in MM1.S cells, suggesting a transcription-dependent apoptosis. Studies by confocal microscopy and WB analysis of the fractionated samples revealed accumulation of p53 in both nuclear and cytoplasmic fractions in nutlin-treated MM1.S cells. Since transcriptional activation of p53-target genes occurs in the nucleus, while cytoplasmic p53 mediates transcription-independent apoptosis, the accumulation of nuclear and cytoplasmic p53 suggests that activated p53 used both transcription-dependent and transcription-independent pathways to induce apoptosis in MM cells. Finally, the consequence of the p53 activation in MM was validated by gene expression profiling of MM1.S cells with or without nutlin stimulation, demonstrating up-regulation of p53 and its downstream targets p21, MDM2, and BAX.

Nongenotoxic activation of the p53 pathway by nutlin sensitized MM cells harboring wt p53 to transcription-dependent and transcription-independent apoptosis. Our studies provide the preclinical framework for the evaluation of nutlin as a novel therapeutic approach in the treatment of MM.

No relevant conflicts of interest to declare.