Exploring the therapeutic effects of p53 modulation in cancer and immune cell biology

Abstract: The tumor suppressor protein p53 is dysregulated in almost all cancer. In around 50% of cancer p53 retains wild type activity but its inactivation by the E3 ligase MDM2 in a heterodimer complex with MDMX may be amplified, leading to loss of p53 activity. As p53 activity is crucial for regulation of cell cycle progression, DNA repair and apoptosis, p53 dysfunction has severe consequences, enabling uncontrolled tumor cell growth. Apart from its function as the guardian of the genome, p53 has also been shown to be important in the regulation of many other cellular processes and functions, such as metabolism, autophagy, angiogenesis and inflammation. Importantly, the p53 status of the tumor cells can affect the composition and functionality of the tumor microenvironment. Immune cell function has also been shown to be regulated directly by p53 activity. Several potent small molecule MDM2 inhibitors, such as Navtemadlin and Siremadlin, and one stapled peptide MDM2/MDMX inhibitor, Sulanemadlin, are currently being evaluated in clinical trials. However, most pre-clinical studies investigating these compounds were performed in xenograft models in immunocompromised mice, limiting the available knowledge on the treatment effect on all components of tumors. Further, there is a lack of knowledge in how these compounds are affected by the features of the tumor microenvironment, such as hypoxia. Neither are the cellular effects of these drugs clear. For instance, it is not known how p53 activation regulates cell fate. Further, very little is known about how p53 activation affects immune cell function. Therefore, it is challenging to anticipate how these drugs influence the tumor cells and the immune cells of the tumor microenvironment during treatment. To this end, this thesis aimed to investigate the therapeutic effect of p53 modulation on tumor cells and on immune cells. In paper I we developed a syngeneic B16-F10 malignant melanoma mouse model where we compared the effect of Navtemadlin on B16-F10 p53+/+ tumor cells with the effect on B16-F10 p53-/- cells in vitro and in vivo. Contrary to previous reports, we found that Navtemadlin was efficient at reducing tumor growth, p53 dependently, in murine cells. Navtemadlin monotreatment mainly inhibited cell growth by inducing cell cycle arrest in this model. However, when treatment was conducted over a prolonged time and combined with radiation therapy, Navtemadlin induced apoptosis. These results support an apoptotic threshold model where p53 activity needs to be sustained over a prolonged period of time and possibly benefits from being induced through several pathways, such as DNA damage through radiation, to promote apoptosis over reversible cell cycle arrest. In paper II we investigated whether the effects of Navtemadlin and the pre-clinical small molecule MDM2 inhibitor Nutlin-3a were affected by a hypoxic environment. Our results demonstrate that Navtemadlin and Nutlin-3a retain efficacy during hypoxia, by inducing p53 downstream targets and cell cycle arrest, both in p53 wild-type human and murine cell lines. Soft tissue sarcoma tumors are typically characterized by relatively low mutational burden and low immune cell infiltration. It is however common, especially for liposarcoma tumors, to have MDM2 amplifications. Even though prognostic markers that may indicate benefits from immune checkpoint therapy are lacking, both immune checkpoint therapy and MDM2 inhibitors are currently being evaluated as treatment options for subgroups of soft tissue sarcoma patients in clinical trials. In paper III we studied if intra-tumoral interactions between CD11c+ cells and CD8+ cells, indicative of cross-presentation, correlated with patient outcome in soft tissue sarcoma. Indeed, we found that spatial cross-presentation within tumors correlated with increased overall survival, independently of tumor grade or expression of regulatory functions, such as Foxp3 and CD274, encoding PD-L1. Hence, our data suggests that intra-tumoral interactions between CD11c+ and CD8+ cells can be used as an easily evaluated prognostic marker in soft tissue sarcoma. In paper IV we evaluated the efficacy of Sulanemadlin and Siremadlin in combination with PD-1 blocking monoclonal antibodies. In vivo, in a CT26.WT colon carcinoma syngeneic mouse model, Sulanemadlin increased the immunogenicity of tumors and tumor infiltration of lymphocytes. Further, Sulanemadlin but not Siremadlin, synergized with anti-PD-1 treatment and drastically improved survival. As different treatment strategies are combined with MDM2 inhibitors in clinical trials, it is of great importance to understand how these combinations may affect treatment outcome. We here show that it is of great importance for treatment success to maintain a high level of p53 activity over a prolonged period of time. We also demonstrate that MDM2 inhibitors continue to function during hypoxia. We show that active cross- presentation within tumors is more important in controlling tumor growth and promoting patient survival that the expression of immune regulating genes such as Foxp3 and CD274, encoding PD-L1. Finally, we reveal that MDM2/MDMX inhibition is superior to MDM2 inhibition in combination with PD-1 blocking monoclonal antibody treatment. Sulanemadlin can increase immunogenicity and promote lymphocyte infiltration into tumors.