Interactions of the Mdm2/p53 and Proteasome Pathways: Implications for Combination Strategies to Enhance the Anti-Myeloma Activity of Bortezomib
Authors: Ooi Melissa G. , Hayden Patrick J. , McMillin Douglas W. , Negri Joseph M. , Delmore Jake , Raje Noopur S , Munshi Nikhil C. , Chauhan Dharminder , Hideshima Teru , Richardson Paul G. , Mitsiades Constantine S. , Anderson Kenneth C , Mitsiades Nicholas 
Published: April 2020

Abstract
The “guardian of the genome” p53 is inactivated in most human cancers via missense mutations in its DNA binding core domain or via overexpression of the human homolog of Mdm2 (Hdm2), an E3 ubiquitin ligase that binds and ubiquitinates p53 thereby leading to its degradation via the ubiquitin/proteasome pathway. Direct inhibition of Mdm2 function could potentially stabilize p53 and activate the p53 apoptotic pathway, which could be a useful non-genotoxic approach for the treatment of cancer. Nutlin-3, a cis-imidazoline small molecule with affinity for the p53-binding pocket of Mdm2 and capable of disrupting the p53-Mdm2 interaction, can activate p53 and induce apoptosis in vitro in many malignancies, including multiple myeloma (MM) cells. We hypothesized that suppression of Mdm2-mediated p53 ubiquitination may synergize with accumulation of p53 triggered by bortezomib. To address this question, we evaluated the response of MM cells vs. select models of epithelial cancers to bortezomib and its combination with nutlin-3. We observed that breast (MDA-MB-231-luc); prostate (DU145); thyroid (SW579, FRO, WRO, TT) and colon (ARO/HT-29) cancer cell lines exhibited synergistic apoptotic response to the combination of sublethal concentrations of bortezomib plus nutlin-3. This synergistic killing effect was associated with synergistic increase in the expression of p53, p21, Mdm2, Bax, Noxa, PUMA and the cleavage of caspase-3, caspase-9 and PARP. In contrast, MM cell lines (MM-1S, MM-1R, NCI-H929, KMS-11) exhibited mostly an additive effect when treated with the combination of bortezomib plus nutlin-3. To further probe the biological relevance of these findings in the context of the bone microenvironment in MM, we compared the transcriptional profile of p53 target genes as well as the response to nutlin-3 in MM cells cultured alone vs. in the presence of bone marrow stromal cells (BMSCs). We observed that co-culture with BMSCs attenuates the response of MM cells to single-agent nutlin-3 and is associated, in p53 wild-type cells, with increased amplitude of the transcriptional signature of genes suppressed by activated p53, suggesting that BMSCs suppress p53 activity in MM cells. Overall, these results suggest a complex correlation between the Mdm2/p53 and proteasome pathways: many p53 mutant epithelial cancer models can become more bortezomib-responsive by Mdm2 inhibition, while MM cells, with higher baseline responsiveness to bortezomib and nutlin-3, typically do not exhibit significant increases in their response to the combination of these 2 drug classes compared to the single-agent treatments. These observations suggest that concurrent Mdm2 inhibition may extend the spectrum of bortezomib applications to tumor types with currently limited single-agent response to proteasome inhibition. In MM, p53 mutations have been historically considered to be present in only late stage disease (e.g. plasma cell leukemia/extramedullary MM), but their prevalence may increase in the future as more patients with advanced MM survive longer thanks to recently introduced drug classes (thalidomide, bortezomib, lenalidomide). In those MM patients who harbor p53 pathway lesions and have developed bortezomib-resistance/refractoriness, combinations of bortezomib with Mdm2 inhibitors may also represent in the future an intriguing potential therapeutic option that merits further preclinical and clinical evaluation.