TP53, a crucial tumor suppressor gene, is frequently mutated in human cancers. However, even in tumors with wild-type TP53, p53 pathway activity can be impaired through other mechanisms. Restoring p53 function is a major goal in cancer therapy. Inhibiting MDM2, the primary E3 ubiquitin ligase that regulates p53, has been explored, with initial success in pre-clinical studies. However, MDM2 inhibitors have shown significant dose-limiting toxicities in clinical trials, including myelosuppression, gastrointestinal issues, and cardiovascular problems, attributed to the delicate balance and negative feedback loop between MDM2 and p53. This two-edged sword effect, while beneficial in tumor cells, causes severe toxicity in normal tissues due to unregulated p53 activation. Studies with "Super-p53" mice indicate that enhanced p53 function doesn't always cause toxicity. The key seems to be maintaining MDM2's control over p53 in normal tissues. Therefore, alternative strategies to therapeutically reactivate p53 are needed. p53 is controlled by multiple pathways, and VPRBP, a corepressor interacting with p53, is overexpressed in several cancers and represents a potential therapeutic target, acting similarly to MDM2 through transcriptional repression and ubiquitin-mediated degradation but independently of MDM2.

The literature extensively documents the importance of p53 in tumor suppression and the challenges associated with targeting the p53-MDM2 pathway. While MDM2 inhibitors have shown promise in preclinical studies, their clinical application is hampered by severe toxicities. Studies highlighting the intricacies of the p53-MDM2 negative feedback loop and the importance of maintaining this regulation in normal tissues are reviewed. The role of VPRBP as an MDM2-independent regulator of p53 function, including transcriptional repression and ubiquitin-mediated degradation, is also established in the literature. The overexpression of VPRBP in various cancers makes it a promising therapeutic target. Prior research into the USP family of deubiquitinases and their role in regulating protein stability sets the stage for investigating USP2's potential involvement in VPRBP regulation.

The study utilized various in vitro and in vivo techniques. In vitro experiments included RNA interference (RNAi)-mediated knockdown of VPRBP and p53 in various human cancer cell lines (U2OS, A375, H1299) to assess the effects on p53 target gene expression and PD-L1 levels. Co-immunoprecipitation, GST pull-down assays, and luciferase reporter assays were used to study the interaction between VPRBP and IRF1, and the role of VPRBP in regulating IRF1-mediated PD-L1 transcription. Ubiquitination assays and cycloheximide chase experiments were conducted to examine VPRBP's role in PD-L1 ubiquitination and degradation. In vivo experiments involved generating mouse mammary tumor xenografts (EMT6 cells) in nude and immunocompetent (Balb/c) mice to assess the impact of VPRBP depletion on tumor growth. Combination therapy with VPRBP depletion (using shRNA) and anti-PD-1 monoclonal antibody was evaluated in immunocompetent mice. The role of USP2 in regulating VPRBP stability was investigated using USP2 inhibitors (ML364, LCAHA), CRISPR/Cas9-mediated USP2 knockout in H1299 cells, and co-immunoprecipitation assays. Complete blood counts and histopathological analysis were performed to assess the toxicity of USP2 inhibitors in mice. Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) was performed to evaluate the immune response. Statistical analyses included two-tailed unpaired t-tests and log-rank tests.

Inactivation of VPRBP increased p53 target gene expression and PD-L1 levels, independent of p53 status. VPRBP directly interacts with and represses IRF1, a transcription factor involved in PD-L1 upregulation. VPRBP also promotes PD-L1 ubiquitination and degradation through its E3 ligase activity. VPRBP depletion reduced tumor growth in a p53-dependent manner in nude mice. Combination therapy with VPRBP depletion and anti-PD-1 antibody significantly reduced tumor growth and improved survival in immunocompetent mice, an effect primarily mediated by CD8+ T cells. USP2, a deubiquitinase, stabilizes VPRBP. USP2 inhibition (using ML364) destabilized VPRBP, increasing p53 and PD-L1 levels without significantly affecting MDM2-mediated p53 regulation. ML364 treatment effectively suppressed tumor growth in combination with anti-PD-1 antibody, with no observable toxicity in normal tissues. This synergistic effect was p53-dependent.

The study successfully identifies the USP2-VPRBP axis as a novel therapeutic target for p53-based cancer therapy. The findings address the limitations of MDM2 inhibitors by offering an alternative approach to reactivate p53 without causing severe toxicity. The synergistic effect of USP2 inhibition and immune checkpoint blockade highlights a promising strategy combining targeted therapy with immunotherapy. The mechanism of action, involving both p53-dependent and p53-independent pathways, further enhances the therapeutic potential. The study's findings provide a strong rationale for clinical translation and investigation of USP2 inhibitors in combination with immune checkpoint blockade for cancer treatment. The p53-independent effects of USP2 also warrant further investigation.

This research demonstrates that targeting the USP2-VPRBP axis effectively reactivates p53's tumor suppressor function while avoiding the toxicities associated with MDM2 inhibition. The synergistic effects observed with combined USP2 inhibition and immune checkpoint blockade provide a promising novel therapeutic strategy for cancers with wild-type p53. Future studies should focus on clinical trials to evaluate the efficacy and safety of this approach in human patients. Further investigation into the p53-independent functions of USP2 is also warranted.

The study primarily used mouse models, and further research is needed to confirm the findings in human patients. The precise mechanism by which USP2 inhibition avoids the toxicity observed with MDM2 inhibition requires additional investigation. While the study provides evidence for the p53-dependent nature of the tumor regression, the contribution of p53-independent effects of USP2 inhibition warrants further exploration. The experiments were done using specific tumor cell lines and might not generalize to other tumor types.