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Nutritional metal ions play critical roles in many important immune processes. Hence, the effective modulation of metal ions may open up new forms of immunotherapy, termed as metalloimmunotherapy. Here, we demonstrate a prototype of cancer metalloimmunotherapy using cyclic dinucleotide (CDN) stimulator of interferon genes (STING) agonists and Mn2+. We screened various metal ions and discovered specific metal ions augmented STING agonist activity, wherein Mn2+ promoted a 12- to 77-fold potentiation effect across the prevalent human STING haplotypes. Notably, Mn2+ coordinated with CDN STING agonists to self-assemble into a nanoparticle (CDN–Mn2+ particle, CMP) that effectively delivered STING agonists to immune cells. The CMP, administered either by local intratumoural or systemic intravenous injection, initiated robust anti-tumour immunity, achieving remarkable therapeutic efficacy with minute doses of STING agonists in multiple murine tumour models. Overall, the CMP offers a new platform for local and systemic cancer treatments, and this work underscores the great potential of coordination nanomedicine for metalloimmunotherapy. The stimulation of interferon genes (STING) pathway with STING agonists such as cyclic dinucleotides (CDNs) has emerged as a promising immunotherapeutic approach. Here, the authors show that Mn2+ can amplify the STING-promoted anti-tumour immune response in challenging murine tumour models by coordinating with CDNs and self-assembling into nanoparticles that can be delivered locally and systemically.

Head and neck squamous cell carcinoma (HNSCC) are immunosuppressed solid tumors often diagnosed at advanced stages of the disease. Most HNSCC patients have a history of oral epithelial dysplasia; however, there are limited diagnostic criteria used to determine which lesions will progress to cancer. There is an unmet clinical need to determine what cellular events occur during tumor initiation that enables anti-tumor immune evasion and heightened disease activity. Amplification of SOX2, a transcription factor that regulates stem cell pluripotency and fate, is a high-risk event that correlates to cancer initiation in squamous cell carcinomas. We have previously shown that SOX2 amplification results in dampened STING-type I interferon (IFN-I) signaling, a crucial pathway for innate immune sensing of cancers. Our clinical data from a longitudinal study following SOX2-positive patients with oral epithelial dysplasia and then HNSCC diagnosis confirmed infiltration of suppressive myeloid subsets and exclusion of effector T cells upon malignant transformation. Subcutaneous implantation of Sox2-expressing tumors exhibited expansion of myeloid-derived suppressor cells and loss of infiltrating CD8+ T cells. Furthermore, tumor microenvironment displayed secretion of cytokines involved in myeloid recruitment including Csf-2 and IL1a. Moreover, expression of SOX2 in HNSCC cell lines and human-derived epithelial progenitor cells enhanced IL-1α expression. To recapitulate tumor formation in the oral cavity, we engineered a keratin-5-restricted, tamoxifen-inducible Cre to trigger Sox2 expression in the buccal mucosa of mice. We observed that immune landscape substantially shifted before the HNSCC histology occurred. Upon analysis of immune infiltrates at 2-weeks and 4-weeks post tamoxifen painting, we found the recruitment of a distinct myeloid population following malignant transformation. These early myeloid cells display a dichotomous IFN-I-low and IL-1α-high signature. Upon culturing bone marrow-derived macrophages (BMDMs) with recombinant IL-1α and then waiting four days to challenge with agonists to induce IFN-I signaling, we show that priming with IL-1α and IL-6 but not IL-10 desensitizes myeloid cells to STING stimulation. We then found that blockade of IL-1/IL1r1 signaling in our pre-clinical model delayed the influx of myeloid cells and the onset of Sox2-driven cancer initiation. From this, we propose that Sox2-mediated epithelial transformation results in expression of IL-1α which primes myeloid cells to generate an immune-tolerant niche. We employed pharmacological and genetic approaches to show that blockade of IL1 signaling during the cancer initiation stage delays the onset of Sox2-driven HNSCC. Overall, we identified a novel cancer-immune cell interaction axis where Sox2 in cancer cells drives IL1α-mediated STING suppression. Citation Format: Hulya F. Taner, Wong Gong, Luke Broses, Wanqing Chen, Jung Kuczura, Sashider Rajesh, Yee Sun Tan, Yuying Xie, Yu Leo Lei. Resolving an immune tolerogenic niche at the earliest phase of oral cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2874.

Background: Progression from noninvasive to muscle-invasive and then metastatic bladder cancer causes >17,000 deaths each year in the United States. Unfortunately, the molecular drivers of progression remain poorly understood and no strategies to prevent progression exist. We have identified an E3 ubiquitin ligase, TRIM29, which is highly expressed in 70% of muscle invasive bladder cancers (MIBC). Global overexpression of TRIM29 in a genetically engineered mouse model (GEMM) is sufficient to induce urothelial cancer initiation and invasive progression. TRIM29 has also been shown to bind and ubiquitinate STING (Stimulator of Interferon Genes), a regulator of anti-tumor cytokine production. However, the role of TRIM29-mediated suppression of STING and innate immune responses in invasive progression was not previously understood. Hypothesis: Given the importance of Trim29 in invasive progression demonstrated in our GEMM, we hypothesized TRIM29-mediated degradation of STING creates an immune-privileged niche required for muscle invasion. Methods: To determine the requirement for Trim29-mediated immune suppression and bladder cancer invasive progression, we generated a urothelial specific tamoxifen inducible Trim29 knockout (KO) GEMM. To induce bladder tumorigenesis, N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), a urothelial specific chemical carcinogen with similarities to tobacco, was delivered to mice via drinking water. Results: As expected, BBN promoted development of muscle-invasive urothelial tumors in 50% of mice after 24 weeks. Tamoxifen-induced Trim29 KO completely blocked progression to muscle-invasion, although these mice did demonstrate development of low grade, non-muscle invasive tumors. BBN upregulated Trim29 expression in the urothelium, but only modestly increased Sting and infiltrating macrophage and T cells. In contrast, Trim29 KO mice, which did not develop invasive tumors, harbored substantially elevated Sting levels in the urothelium and substantially increased macrophage and T cell infiltration in the bladder and tumor. In vitro, TRIM29 promoted STING ubiquitination and TRIM29 knockdown increased STING protein levels in bladder cancer cell lines. TRIM29 KO in these cells also resulted in increased expression of innate immune cytokines. Conclusion: These results suggest that TRIM29-mediated downregulation of STING in the bladder results in decreased immune surveillance in the tumor microenvironment, contributing to the development of an immune privileged niche required for MIBC progression. Citation Format: Alan Kelleher, Yin Wang, Luke Broses, Yu Leo Lei, Phillip Palmbos. TRIM29-mediated immune evasion in bladder cancer invasive progression [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A27.

Radiotherapy promotes tumor cell death and senescence through the induction of oxidative damage. Recent work has highlighted the importance of lipid peroxidation for radiotherapy efficacy. Excessive lipid peroxidation can promote ferroptosis, a regulated form of cell death. In this review, we address the evidence supporting a role of ferroptosis in response to radiotherapy and discuss the molecular regulators that underlie this interaction. Finally, we postulate on the clinical implications for the intersection of ferroptosis and radiotherapy.

Background: Innate immune pathway of stimulator of interferon genes (STING) orchestrates antitumor immune responses and can elicit potent antitumor efficacy. However, clinical development of cyclic-dinucleotide (CDN) STING agonists is mostly limited to intratumoral administration because of the undesirable pharmacological properties of CDN. This limits their applications in treating metastasis, hematologic malignancies, and hard-to-reach solid tumor. Here, we aim to develop a pharmacological formulation that facilitate systemic delivery of CDN STING agonist to broaden its applications for cancer treatment. Methods: Nutritional metal ions physiologically mediate various immune processes, which could potentially be utilized for modulating the STING pathway. By screening different nutritional metal ions, we discovered manganese ion (Mn2+) and cobalt ion (Co2+) synergize with CDN STING agonists and significantly enhance type-I interferon (IFN-I) responses. As Mn2+ has been used in FDA approved drugs, we further developed the formulation to enable systemic codelivery of Mn2+ and CDN. Results: Mn2+ could significantly potentiate the STING activation by up to 77-fold. In addition, Mn2+ interact with CDNs and self-assemble into nanocrystals that could be stabilized as uniform nanoparticles (~120 nm) through lipid layer coating (CDN-Mn Particle, CMP). After either intratumoral or intravenous administrations, CMP effectively elevate the serum levels of inflammatory cytokines (IFN-β, TNF-α, CXCL-9, and CXCL-10), mitigates the suppressive functions of myeloid cells, relieve the immunosuppressive tumor microenvironment (TME), and induce antigen-specific CD8+ T cells expansion. Minute dose of CMP given intravenously exert remarkable antitumor efficacy in CT26, B16F10, and immune checkpoint blocker (ICB)-resistance tobacco-associated NOOC1 murine tumor models, leading to efficient tumor regression. Furthermore, the systemic biodistribution of CMP could be modulated by changing the composition of the lipid layer coating, allowing for organ-specific delivery of CMP to target tumor malignancies in certain organs. Conclusions: CMP provides a novel and versatile platform for local and systemic cancer treatment. More importantly, it highlights the potential of harnessing metal ions in treating various immune-mediated diseases. Citation Format: Xingwu Zhou, Xiaoqi Sun, Wang Gong, Yu Leo Lei, James J. Moon. Amplifying STING activation by cyclic dinucleotide-manganese particles for systemic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 314.

Type-I interferon (IFN-I) signaling is critical to maintaining antigen-presenting cell function for anti-tumor immunity. However, recent studies have suggested that IFN-I signaling may also contribute to more aggressive phenotypes, raising the possibility that IFN-I downstream signaling in cancer and myeloid cells may exert dichotomous functions.We analyzed the clinicopathologic correlation of cancer-specific IFN-I activation in 195 head and neck squamous cell carcinoma patients. We also characterized the immune impact of IFN-I receptor (IFNAR1)-deficiency in syngeneic tumor models using biochemistry, flow cytometry, and single-cell RNA-Seq. We stained HNSCC tissue microarrays with a sensitive IFN-I downstream signaling activation marker, MX1, and quantitated cancer cell-specific MX1 staining. Kaplan-Meier analysis revealed that MX1-high tumors exhibited worse survival, a phenotype that depends on the number of CD8+ intratumoral T-cells. We found that cancer-specific IFNAR1 engagement promotes cancer stemness and higher expression levels of suppressive immune checkpoint receptor ligands in cancer-derived exosomes. Notably, mice bearing Ifnar1-deficient tumors exhibited lower tumor burden, increased T-cell infiltration, reduced exhausted CD4+PD1high T-cells, and increased effector population CD8+IFN-γ+ T-cells. Then, we performed single-cell RNA-sequencing and discovered that cancer-specific IFN-I signaling not only restricts effector cells expansion but also dampens their functional fitness.The beneficial role of IFN-I activation is largely dependent on the myeloid compartment. Cancer-specific IFN-I receptor engagement promotes cancer stemness and the release of cancer-derived exosomes with high expression levels of immune checkpoint receptor ligands. Cancer-specific IFN-I activation is associated with poor immunogenicity and worse clinical outcomes in HNSCC.