Inhibition of the MDM2 E3 Ligase induces apoptosis and autophagy in wild-type and mutant p53 models of multiple myeloma, and acts synergistically with ABT-737

Abstract

Intracellular proteolytic pathways have been validated as rational targets in multiple myeloma with the approval of two proteasome inhibitors in this disease, and with the finding that immunomodulatory agents work through an E3 ubiquitin ligase containing Cereblon. Another E3 ligase that could be a rational target is the murine double minute (MDM) 2 protein, which plays a role in p53 turnover. A novel inhibitor of this complex, MI-63, was found to induce apoptosis in p53 wild-type myeloma models in association with activation of a p53-mediated cell death program. MI-63 overcame adhesion-mediated drug resistance, showed anti-tumor activity in vivo, enhanced the activity of bortezomib and lenalidomide, and also overcame lenalidomide resistance. In mutant p53 models, inhibition of MDM2 with MI-63 also activated apoptosis, albeit at higher concentrations, and this was associated with activation of autophagy. When MI-63 was combined with the BH3 mimetic ABT-737, enhanced activity was seen in both wild-type and mutant p53 models. Finally, this regimen showed efficacy against primary plasma cells from patients with newly diagnosed and relapsed/refractory myeloma. These findings support the translation of novel MDM2 inhibitors both alone, and in combination with other novel agents, to the clinic for patients with multiple myeloma.

Figure 1. MI-63 is cytotoxic to wild-type p53 myeloma cells.

A. Cell viability assays were performed in wild-type p53 myeloma cell lines exposed to the indicated concentrations of MI-63 for 48 hours using a tetrazolium reagent. Error bars represent standard errors of the mean from three or more replicates, and all experiments in this and later figures were repeated three times, with one representative figure shown. B. Whole cell extracts were prepared from MM1.S cells treated with vehicle or MI-63, and then subjected to immunoprecipitation with an antibody to MDM2. The precipitates were then probed by Western blotting using specific antibodies to MDM2 or p53, and densitometry was performed using Image J software. C. MM1.S cells stably infected with a Lentiviral vector expressing either a control, non-targeted shRNA or an shRNA directed at p53 were exposed to MI-63 as above, and viability was measured using the WST-1 reagent. D. Human stromal HS-5 cells or MM1.S myeloma cells were propagated in culture either alone, or co-cultured at a ratio of 20∶1, and viability was measured in the presence of MI-63. E. A murine xenograft myeloma model was developed in NOD/SCID mice, and when tumor volume reached 100 mm³, mice were treated intraperitoneally with 100 mg/kg MI-219 or vehicle, and tumor volume was monitored three times a week. Vehicle-treated mice experienced a substantial increase in the tumor burden, while MI-219 induced a significant tumor growth delay, (*p<0.05).

Figure 2. Molecular mechanisms underlying the action of MI-63.

A. Quantitative PCR was performed of selected p53 transcriptional targets in wild-type cell lines after exposure to MI-63 at the IC₅₀ for 48 hours. Values were normalized to GAPDH as a control (*p<0.005; **p<0.05). B. The indicated cell lines were treated with MI-63 at its IC₅₀ for 48 hours, and cell cycle analysis was performed after propidium iodide staining. C. Activation of apoptosis was evaluated after Annexin V staining by flow cytometry in MM1.S and MOLP-8 cells treated with MI-63 for 48 hours. D. Western blotting of cells treated for 48 hours with MI-63 at the IC₅₀ was performed to detect changes in key downstream targets, with Actin as the loading control.

Figure 3. MI-63 is also active against mutant p53 myeloma cell lines.

A. Cell viability assays were performed in a panel of mutant p53 myeloma cell lines, including ANBL-6, KAS-6/1, RPMI 8226, U266, and OPM-2 cells after exposure to MI-63 for 48 hours. B. Analysis of cell cycle distribution was performed in RPMI 8226 and U266 cells exposed to MI-63. C. Induction of apoptosis was evaluated by Annexin V staining and flow cytometry in RPMI 8226 and U266 cells treated with MI-63 at its IC₅₀ for 48 hours. D. Extracts of RPMI 8226 cells treated with MI-63 were subjected to Western blotting to detect key intermediates in apoptosis and autophagy. E. The impact of MDM2 suppression on the efficacy of MI-63 was studied in U266 cells harboring either a control shRNA, or one of two different constructs that suppressed expression of MDM2.

Figure 4. MI-63 induces autophagy in mutant p53 myeloma models.

A. Acridine orange staining and flow cytometry were used to study the appearance of acidic vacuoles in mutant p53 RPMI 8226 and U266 cells, and wild-type p53 MM1.S and MOLP-8 cells exposed to the IC₅₀ of MI-63 for 48 hours. B. Transcript levels of ATG3 and ATG5 were studied in mutant and wild-type p53 myeloma cells exposed to MI-63 (*p<0.005). C. The impact of the autophagy inhibitors chloroquine (100 nM) and 3-methyladenine (1 µM) on the activity of MI-63 against mutant and wild-type p53 cell lines was studied using viability assays (*p<0.005). D. ATG5 expression was suppressed with a Lentiviral shRNA in RPMI 8226 cells, and the effect of MI-63 was then studied in comparison to control shRNA cells. E. Beclin-1 was depleted with an shRNA, and the median inhibitory concentration of MI-63 was determined in comparison with a control shRNA. F. Activation of autophagy and apoptosis in mutant p53 myeloma cells treated with MI-63 and/or autophagy inhibitors.

Figure 5. MI-63 acts synergistically with ABT-737.

A. The combination of MI-63 and ABT-737 was studied in wild-type p53 MM1.S and MOLP-8 cells (left panel), and in mutant RPMI 8226 and U266 cells (right panel). Multiple doses of each drug were used for this experiment, with one representative condition shown. *p<0.005, ** p<0.05. B. Apoptosis was studied by staining for Annexin V in both wild-type p53 (MOLP-8; left panel) and mutant p53 (U266; right panel) cells treated with vehicle, MI-63, ABT-737, or the combination. C. Abundance of important intermediates in type I programmed cell death was studied in MOLP8 and U266 cells by Western blotting.

Figure 6. Combination studies in primary patient samples.

A. The combination of MI-63 and ABT-737 was studied against CD138⁺ primary plasma cells from a patient with multiple myeloma (left panel), and CD138⁻ marrow cells (right panel). Note that multiple doses of each drug were used for this experiment to calculate combination indices, with one representative condition shown. B–D. Additional primary plasma cells isolated from unique patients were exposed to the combination of MI-63 and ABT-737.