MDM2/MDMX inhibition by Sulanemadlin synergizes with anti-Programmed Death 1 immunotherapy in wild-type p53 tumors.

Immunotherapy has revolutionized cancer treatment but its efficacy depends on a robust immune response in the tumor. Silencing of the tumor suppressor p53 is common in tumors and can affect the recruitment and activation of different immune cells, leading to immune evasion and poor therapy response. We found that the p53 activating stapled peptide MDM2/MDMX inhibitor Sulanemadlin (ALRN-6924) inhibited p53 wild-type cancer cell growth in vitro and in vivo. In mice carrying p53 wild-type CT26.WT tumors, monotherapy with the PD-1 inhibitor DX400 or Sulanemadlin delayed tumor doubling time by 50% and 37%, respectively, while combination therapy decreased tumor doubling time by 93% leading to an increased median survival time. Sulanemadlin treatment led to increased immunogenicity and combination treatment with PD-1 inhibition resulted in an increased tumor infiltration of lymphocytes. This combination treatment strategy could potentially turn partial responders into responders of immunotherapy, expanding the patient target group for PD-1-targeting immunotherapy.

MDM2 inhibitors, nutlin-3a and navtemadelin, retain efficacy in human and mouse cancer cells cultured in hypoxia.

Activation of p53 by small molecule MDM2 inhibitors can induce cell cycle arrest or death in p53 wildtype cancer cells. However, cancer cells exposed to hypoxia can develop resistance to other small molecules, such as chemotherapies, that activate p53. Here, we evaluated whether hypoxia could render cancer cells insensitive to two MDM2 inhibitors with different potencies, nutlin-3a and navtemadlin. Inhibitor efficacy and potency were evaluated under short-term hypoxic conditions in human and mouse cancer cells expressing different p53 genotypes (wild-type, mutant, or null). Treatment of wild-type p53 cancer cells with MDM2 inhibitors reduced cell growth by > 75% in hypoxia through activation of the p53-p21 signaling pathway; no inhibitor-induced growth reduction was observed in hypoxic mutant or null p53 cells except at very high concentrations. The concentration of inhibitors needed to induce the maximal p53 response was not significantly different in hypoxia compared to normoxia. However, inhibitor efficacy varied by species and by cell line, with stronger effects at lower concentrations observed in human cell lines than in mouse cell lines grown as 2D and 3D cultures. Together, these results indicate that MDM2 inhibitors retain efficacy in hypoxia, suggesting they could be useful for targeting acutely hypoxic cancer cells.

The MDM2 Inhibitor Navtemadlin Arrests Mouse Melanoma Growth In Vivo and Potentiates Radiotherapy.

The tumor suppressor protein p53 is mutated in close to 50% of human tumors and is dysregulated in many others, for instance by silencing or loss of p14ARF. Under steady-state conditions, the two E3 ligases MDM2/MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 in tumors with wild-type (WT) p53 has therefore been considered a therapeutic strategy. Moreover, studies indicate that p53 reactivation may synergize with radiation and increase tumor immunogenicity. In vivo studies of most MDM2 inhibitors have utilized immunodeficient xenograft mouse models, preventing detailed studies of action of these molecules on the immune response. The mouse melanoma cell line B16-F10 carries functional, WT p53 but does not express the MDM2 regulator p19ARF. In this study, we tested a p53-MDM2 protein-protein interaction inhibitor, the small molecule Navtemadlin, which is currently being tested in phase II clinical trials. Using mass spectrometry-based proteomics and imaging flow cytometry, we identified specific protein expression patterns following Navtemadlin treatment of B16-F10 melanoma cells compared with their p53 CRISPR-inactivated control cells. In vitro, Navtemadlin induced a significant, p53-dependent, growth arrest but little apoptosis in B16-F10 cells. When combined with radiotherapy, Navtemadlin showed synergistic effects and increased apoptosis. In vivo, Navtemadlin treatment significantly reduced the growth of B16-F10 melanoma cells implanted in C57Bl/6 mice. Our data highlight the utility of a syngeneic B16-F10 p53+/+ mouse melanoma model for assessing existing and novel p53-MDM2/MDM4 inhibitors and in identifying new combination therapies that can efficiently eliminate tumors in vivo. Significance: The MDM2 inhibitor Navtemadlin arrests mouse tumor growth and potentiates radiotherapy. Our results support a threshold model for apoptosis induction that requires a high, prolonged p53 signaling for cancer cells to become apoptotic.

CRISPR/Cas9-Induced DNA Damage Enriches for Mutations in a p53-Linked Interactome: Implications for CRISPR-Based Therapies.

Inactivating p53 mutations are the most abundant genetic alterations found in cancer. Here we show that CRISPR/Cas9-induced double-stranded DNA breaks enrich for cells deficient in p53 and in genes of a core CRISPR-p53 tumor suppressor interactome. Such enrichment could predispose to cancer development and thus pose a challenge for clinical CRISPR use. Transient p53 inhibition could suppress the enrichment of cells with these mutations. The level of DNA damage response induced by an sgRNA influenced the enrichment of p53-deficient cells and could be a relevant parameter in sgRNA design to limit cellular enrichment. Furthermore, a dataset of >800 human cancer cell lines identified additional factors influencing the enrichment of p53-mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, these data provide details about p53 biology in the context of CRISPR-induced DNA damage and identify strategies to enable safer CRISPR use. SIGNIFICANCE: CRISPR-mediated DNA damage enriches for cells with escape mutations in a core CRISPR-p53 interactome, which can be suppressed by transient inhibition of p53.

CD11c-CD8 Spatial Cross Presentation: A Novel Approach to Link Immune Surveillance and Patient Survival in Soft Tissue Sarcoma.

Checkpoint inhibitors are slowly being introduced in the care of specific sarcoma subtypes such as undifferentiated pleomorphic sarcoma, alveolar soft part sarcoma, and angiosarcoma even though formal indication is lacking. Proper biomarkers to unravel potential immune reactivity in the tumor microenvironment are therefore expected to be highly warranted. In this study, intratumoral spatial cross presentation was investigated as a novel concept where immune cell composition in the tumor microenvironment was suggested to act as a proxy for immune surveillance. Double immunohistochemistry revealed a prognostic role of direct spatial interactions between CD11c+ antigen-presenting cells (APCs) and CD8+ cells in contrast to each marker alone in a soft tissue sarcoma (STS) cohort of 177 patients from the Karolinska University Hospital (MFS p = 0.048, OS p = 0.025). The survival benefit was verified in multivariable analysis (MFS p = 0.012, OS p = 0.004). Transcriptomics performed in the TCGA sarcoma cohort confirmed the prognostic value of combining CD11c with CD8 (259 patients, p = 0.005), irrespective of FOXP3 levels and in a CD274 (PD-LI)-rich tumor microenvironment. Altogether, this study presents a histopathological approach to link immune surveillance and patient survival in STS. Notably, spatial cross presentation as a prognostic marker is distinct from therapy response-predictive biomarkers such as immune checkpoint molecules of the PD-L1/PD1 pathway.

Combined proinflammatory cytokine and cognate activation of invariant natural killer T cells enhances anti-DNA antibody responses.

Invariant natural killer T (iNKT) cells serve as early rapid responders in the innate immune response to self-derived autoantigens and pathogen-derived danger signals and antigens. iNKT cells can serve both as helpers for effector B cells and negatively regulate autoreactive B cells. Specifically, iNKT cells drive B cell proliferation, class switch, and antibody production to induce primary antigen-specific immune responses. On the other hand, inflammasome-mediated activation drives accumulation of neutrophils, which license iNKT cells to negatively regulate autoreactive B cells via Fas ligand (FasL). This positions iNKT cells at an apex to support or inhibit B cell responses in inflammation. However, it is unknown which effector mechanism dominates in the face of cognate glycolipid activation during chronic inflammation, as might result from glycolipid vaccination or infection during chronic autoimmune disease. We stimulated iNKT cells by cognate glycolipid antigen α-galactosylceramide (αGalCer) and measured B cell activation during interleukin 18 (IL-18)-induced chronic inflammation. Moreover, glycolipid-activated iNKT cells increased the serum concentration of autoantibodies, frequency of germinal center (GC) B cells, and antigen-specific plasma cells induced during chronic IL-18-mediated inflammation, as compared with IL-18 alone. Further, activation of iNKT cells via cognate glycolipid during IL-18-mediated inflammation overrides the licensing function of neutrophils, instead inducing iNKT follicular helper (iNKTfh) cells that in turn promote autoimmunity. Thus, our data demonstrate that glycolipids which engage iNKT cells support antigen-specific B cell help during inflammasome-mediated inflammation.