Moloud Sooreshjani

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.

Cytokines play an important role in regulating the immune response. Although there is great interest in exploiting cytokines for cancer immunotherapy, their clinical potential is limited by their pleiotropic properties and instability. A variety of cancer cell-intrinsic and extrinsic characteristics pose a barrier to effective treatments including cytokines. Recent studies using gene and cell therapy offer new opportunities for targeting cytokines or their receptors, demonstrating that they are actionable targets. Current efforts such as virotherapy, systemic cytokine therapy, and cellular and gene therapy have provided novel strategies that incorporate cytokines as potential therapeutic strategies for glioblastoma. Ongoing research on characterizing the tumor microenvironment will be informative for prioritization and combinatorial strategies of cytokines for future clinical trials. Unique therapeutic opportunities exist at the convergence of cytokines that play a dual role in tumorigenesis and immune modulation. Here, we discuss the underlying strategies in pre- and clinical trials aiming to enhance treatment outcomes in glioblastoma patients.

Despite being the leading cause of cancer-related childhood mortality, pediatric gliomas have been relatively understudied, and the repurposing of immunotherapies has not been successful. Whole-transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapeutic strategy that could be applied to multiple preclinical glioma models. MAPK-driven pediatric gliomas have a higher IFN signature relative to other molecular subgroups. Single-cell sequencing identified an activated and cytotoxic microglia (MG) population designated MG-Act in BRAF-fused, MAPK-activated pilocytic astrocytoma (PA), but not in high-grade gliomas or normal brain. T cell immunoglobulin and mucin domain 3 (TIM3) was expressed on MG-Act and on the myeloid cells lining the tumor vasculature but not normal brain vasculature. TIM3 expression became upregulated on immune cells in the PA microenvironment, and anti-TIM3 reprogrammed ex vivo immune cells from human PAs to a proinflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven, low-grade gliomas, anti-TIM3 treatment increased median survival over IgG- and anti-PD-1-treated mice. Single-cell RNA-Seq data during the therapeutic window of anti-TIM3 revealed enrichment of the MG-Act population. The therapeutic activity of anti-TIM3 was abrogated in mice on the CX3CR1 MG-KO background. These data support the use of anti-TIM3 in clinical trials of pediatric low-grade, MAPK-driven gliomas.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have recently been demonstrated to extract and express cognate tumor antigens through trogocytosis. This process may contribute to tumor antigen escape, T cell exhaustion, and fratricide, which plays a central role in CAR dysfunction. We sought to evaluate the importance of this effect in epidermal growth factor receptor variant III (EGFRvIII) specific CAR T cells targeting glioma. METHODS: EGFRvIII-specific CAR T cells were generated from various donors and analyzed for cytotoxicity, trogocytosis, and in vivo therapeutic activity against intracranial glioma. Tumor autophagy resulting from CAR T cell activity was evaluated in combination with an autophagy inducer (verteporfin) or inhibitor (bafilomycin A1). RESULTS: CAR T cell products derived from different donors induced markedly divergent levels of trogocytosis of tumor antigen as well as PD-L1 upon engaging target tumor cells correlating with variability in efficacy in mice. Pharmacological facilitation of CAR induced-autophagy with verteporfin inhibits trogocytic expression of tumor antigen on CARs and increases CAR persistence and efficacy in mice. CONCLUSION: These data propose CAR-induced autophagy as a mechanism counteracting CAR-induced trogocytosis and provide a new strategy to innovate high-performance CARs through pharmacological facilitation of T cell-induced tumor death.

Abstract Purpose: Trials of immunotherapy in diffuse glioma patients have been mostly unsuccessful. We therefore sought to determine the combined effects of the signal transducer and activator of transcription 3 (STAT3) inhibitor WP1066 and the STING agonist IACS-8803 in a preclinical model of glioma. Experimental Design: C57BL/6 mice (n=9-10/group) with orthotopically engrafted GL261 cells were treated orally with WP1066, a blood-brain-barrier penetrant inhibitor of STAT3, in combination with the STING agonist IACS-8803 administered directly into the tumor. Analysis of treatment effects included immunoblots, ubiquitination, multiplex immunohistochemistry, and NanoString immune phenotyping of glioma-infiltrating immune cells. Results: The STING agonist 8803 at 2.5µg/mouse, in combination with WP1066 dosed at 30mg/kg with a 12-hour delay, increased animal subject median survival (MS) to 58 days, in contrast to monotherapy (WP1066=25 days, 8803=29 days) or control (25 days) (p=0.002 of combo relative to all other groups). Dose escalation of WP1066 to 60 mg/kg, administered simultaneously with 8803, completely abrogated the therapeutic combinatorial effect. Five ubiquitin-binding domains were predicted on STING using the UbPred program; concentrations of 5µM or higher of WP1066 induced polyubiquitination of phosphorylated STING. Immune profiling during the therapeutic window demonstrated a multi-pathway induction of anti-tumor immunity. Conclusions: Co-administration of a reduced effective dose of WP1066 is necessary with STING agonist 8803 to effectively enhance the anti-glioma immune reactivity in the tumor microenvironment. Citation Format: Hinda Najem, Shashwat Tripathi, Moloud Sooreshjani, Lisa Hurley, Corey Dussold, Victor Arrieta, Martina Ott, Jun Wei, Michael Curran, Anantha Marisetty, Waldemar Priebe, Leondias C. Platanias, Maciej S. Lesniak, Charles D. James, Craig Horbinski, David M. Ashley, Amy B. Heimberger. Establishment of the effective dose of the STAT3 inhibitor WP1066 used in combination with STING activation for reprograming the preclinical glioma microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB335.