The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276

Abstract

Background: The links between the p53/MDM2 pathway and the expression of prooncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1. Methods: Proximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype. Results: CD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control. Conclusion: Genetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

4-OI Attenuates Carbon Tetrachloride-Induced Hepatic Injury via Regulating Oxidative Stress and the Inflammatory Response

“…The concentration of protein was determined by BCA Protein Assay Kit (Beyotime Biotechnology). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed as we reported previously ( Li et al, 2020a ; Zhang et al, 2020b ). Images were recorded with the Image Lab system and analyzed using ImageJ software.…”

What do we need to know and understand about p53 to improve its clinical value?

“…Nutlin‐like compounds) has so far not only shown little obvious positive effects on cancer treatments but also causes bone marrow toxicity [27,28]. Apart from the fact that the target might not live up to expectations, there are other factors to take into account, such as a dominant MDM2 oncogenic isoform in human sarcoma might not express the N‐terminal domain of MDM2 or that Nutlin can induce PDL1 that can mediate immune suppression [29]. Mice in which p53 is activated by genetic recombination show no important toxicity outside the tumours, suggesting that in normal tissue the balance between p53 and MDM2 prevents toxicity.…”

Pharmacologic Activation of p53 Triggers Viral Mimicry Response Thereby Abolishing Tumor Immune Evasion and Promoting Antitumor Immunity

“…Several studies have described the effect of pharmacological activation of p53 on anti-tumor immunity, including IFN induction, polarization of macrophages, induction of PD-L1 and potentiation of immune checkpoint inhibitor therapy in cell culture and mouse tumor models (34,(44)(45)(46)(47). Having identified the de-repression of ERVs upon p53 activation as a mechanism that triggers IFN response, our study offers novel mechanistic insight into p53-mediated enhancement of anti-tumor immunity.…”