STING agonist 8803 reprograms the immune microenvironment and increases survival in preclinical models of glioblastoma.

STING agonists can reprogram the tumor microenvironment to induce immunological clearance within the central nervous system. Using multiplexed sequential immunofluorescence (SeqIF) and the Ivy Glioblastoma Atlas, STING expression was found in myeloid populations and in the perivascular space. The STING agonist 8803 increased median survival in multiple preclinical models of glioblastoma, including QPP8, an immune checkpoint blockade-resistant model, where 100% of mice were cured. Ex vivo flow cytometry profiling during the therapeutic window demonstrated increases in myeloid tumor trafficking and activation, alongside enhancement of CD8+ T cell and NK effector responses. Treatment with 8803 reprogrammed microglia to express costimulatory CD80/CD86 and iNOS, while decreasing immunosuppressive CD206 and arginase. In humanized mice, where tumor cell STING is epigenetically silenced, 8803 therapeutic activity was maintained, further attesting to myeloid dependency and reprogramming. Although the combination with a STAT3 inhibitor did not further enhance STING agonist activity, the addition of anti-PD-1 antibodies to 8803 treatment enhanced survival in an immune checkpoint blockade-responsive glioma model. In summary, 8803 as a monotherapy demonstrates marked in vivo therapeutic activity, meriting consideration for clinical translation.

Novel murine glioblastoma models that reflect the immunotherapy resistance profile of a human disease.

BACKGROUND: The lack of murine glioblastoma models that mimic the immunobiology of human disease has impeded basic and translational immunology research. We, therefore, developed murine glioblastoma stem cell lines derived from Nestin-CreERT2QkL/L; Trp53L/L; PtenL/L (QPP) mice driven by clinically relevant genetic mutations common in human glioblastoma. This study aims to determine the immune sensitivities of these QPP lines in immunocompetent hosts and their underlying mechanisms. METHODS: The differential responsiveness of QPP lines was assessed in the brain and flank in untreated, anti-PD-1, or anti-CTLA-4 treated mice. The impact of genomic landscape on the responsiveness of each tumor was measured through whole exome sequencing. The immune microenvironments of sensitive (QPP7) versus resistant (QPP8) lines were compared in the brain using flow cytometry. Drivers of flank sensitivity versus brain resistance were also measured for QPP8. RESULTS: QPP lines are syngeneic to C57BL/6J mice and demonstrate varied sensitivities to T cell immune checkpoint blockade ranging from curative responses to complete resistance. Infiltrating tumor immune analysis of QPP8 reveals improved T cell fitness and augmented effector-to-suppressor ratios when implanted subcutaneously (sensitive), which are absent on implantation in the brain (resistant). Upregulation of PD-L1 across the myeloid stroma acts to establish this state of immune privilege in the brain. In contrast, QPP7 responds to checkpoint immunotherapy even in the brain likely resulting from its elevated neoantigen burden. CONCLUSIONS: These syngeneic QPP models of glioblastoma demonstrate clinically relevant profiles of immunotherapeutic sensitivity and potential utility for both mechanistic discovery and evaluation of immune therapies.

ATR-mediated CD47 and PD-L1 up-regulation restricts radiotherapy-induced immune priming and abscopal responses in colorectal cancer.

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited significantly elevated postirradiation CD47 levels. The combination of RT, anti-SIRPα, and anti-PD-1 reversed adaptive immune resistance and drove efficient TAA cross-presentation, resulting in robust TAA-specific CD8 T cell priming, functional activation of T effectors, and increased T cell clonality and clonal diversity. We observed significantly higher complete response rates to RT/anti-SIRPα/anti-PD-1 in both irradiated and abscopal tumors and prolonged survival in three distinct murine CRC models, including a cecal orthotopic model. The efficacy of triple combination therapy was STING dependent as knockout animals lost most benefit of adding anti-SIRPα and anti-PD-1 to RT. Despite activation across the myeloid stroma, the enhanced dendritic cell function accounts for most improvements in CD8 T cell priming. These data suggest ATR-mediated CD47 and PD-L1 up-regulation as a key mechanism restraining radiation-induced immune priming. RT combined with SIRPα and PD-1 blockade promotes robust antitumor immune priming, leading to systemic tumor regressions.

p73 regulates epidermal wound healing and induced keratinocyte programming.

p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (ΔNp73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by ΔNp73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function.