Elijah F. Edmondson

Estrogen receptor-negative (ER-) breast tumors are aggressive subtypes with limited therapeutic options. Cyclooxygenase-2 promotes chronic inflammation, immune suppression, and therapeutic resistance. Whether COX2 signaling organizes tumor microenvironments across oxygen gradients remains unclear. Restricted Exchange Environment Chambers (REECs) were used to explore the COX2 impact on spatial tumor architecture across oxygen gradients. Baseline COX2+, B7H4+, and CD44v6+ cells exhibited distinct distributions, with CD44v6 predominance. Inflammatory stimulation increased and spatially reorganized COX2+, B7H4+, and COX2+CD44v6+ populations. While COX2+ cells occupied oxygenated-to-intermediate zones, B7H4+ populations were enriched in intermediate-to-hypoxic regions, and CD44v6 remained broadly distributed. Diclofenac disrupted this organization and reduced clustering of COX2+, B7H4+, and COX2+CD44v6+ phenotypes. Poor-outcome patients showed high COX2 at the tumor-stroma interface as well as altered COX2+, B7H4+, and CD44v6+ clustering phenotypes. These findings identify COX2-dependent microenvironments as clinically relevant features of therapeutic resistance. Diclofenac preferentially depleted hypoxic compartments preserved following cisplatin treatment alone, supporting the targeting of spatially organized inflammatory niches to improve therapeutic response.

Despite the advent of immunotherapy, human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) carries a high morbidity and mortality rate, thus novel therapies are urgently needed. Suppressor Of Variegation 4-20 Homolog 1 (SUV420H1) is a protein lysine methyltransferase that writes H4K20me3. Approximately 35% of HPV-negative HNSCC tumors carry gains/amplifications of SUV420H1 . Gene Set Enrichment Analysis (GSEA) showed enrichment of proliferation-, epithelial-mesenchymal transition (EMT)- and immune-response pathways in SUV420H1 -overexpressing HPV-negative HNSCC tumors. Depletion of SUV420H1 led to decreased proliferation, cell cycling and invasion in human HPV-negative HNSCC cell lines, while enzymatic inhibition decreased the invasive capacity but not the proliferation and cell cycling of HPV-negative HNSCC cells, supporting the presence of catalytically-independent and -dependent functions of SUV420H1. In a syngeneic mouse model of mouse oral carcinoma 1 tumors (MOC1), Suv420h1 knockout (KO) in MOC1 cancer cells halted tumor growth and synergized with anti-PD-1 therapy. In the tumor immune microenvironment (TIME) of Suv420h1 KO MOC1 tumors, a significant decrease in the protumorigenic granulocytic myeloid derived suppressor cells (gMDSCs) with concurrent decrease in Cxcl2 and enrichment in type I IFN response pathways was observed. Genome-wide mapping of SUV420H1-mediated H4K20me3 revealed enrichment of EMT-, IFN-response, and myeloid-attracting chemokine genes. This work provides rationale for the depletion/degradation of SUV420H1 as a novel therapeutic strategy to hinder proliferation and invasion, and to impart sensitization to anti-PD-1 immunotherapy for patients with HPV-negative HNSCC tumors with SUV420H1 gain/amplification.

Background: Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with poor prognosis and limited curative treatment options. While chimeric antigen receptor (CAR) T cells have shown some promise in solid tumors, ACC remains largely unexplored in this context. Here, we used patient-derived xenograft (PDX) models of ACC to identify immunotherapeutic targets and develop novel CAR T-cell strategies. Methods: Target identification and tumor microenvironment (TME) profiling were conducted using publicly available bulk and single-cell RNA sequencing data from patients with ACC samples. Surface proteomic analysis and flow cytometry of PDXs were conducted to validate antigen candidates. CAR T cells were engineered and tested for cytotoxicity in vitro and in vivo and profiled using flow cytometry and cytokine analysis. Results: We identified ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3) as a shared immunotherapy target in 4/7 (57%) ACC PDXs. ENPP3-targeted CAR T-cells eradicated >85% of ENPP3 ⁺ ACC cells in vitro but showed attenuated efficacy in PDX mouse models. Restrained ENPP3 CAR T-cell activity correlated with immunosuppressive features of the TME, particularly the presence of CD206 ⁺ tumor-associated macrophages (TAMs). Co-treatment with an agent modulating CD206 ⁺ TAMs restored CAR T-cell function and improved antitumor responses in ENPP3 ^high and ENPP3 ^low PDX models (difference between mean tumor weights −270.1 mg±117.4; p < 0.05). Conclusion: ENPP3 is a novel target for CAR T-cell therapy in ACC. ENPP3 CAR T cells, when combined with CD206 modulation to overcome immune suppression within the TME, have therapeutic efficacy in ACC by mediating robust tumor growth suppression. This combinatorial strategy, which includes CAR T cells alongside therapies that recalibrate immunosuppressive CD206 ⁺ TAMs, may be a novel approach for improved immunotherapy of solid cancers beyond ACC.

Most cancer immunotherapy strategies are focused on direct tumor killing by immune cells, especially T lymphocytes. Clinical and conceptual limitations of these approaches create a need for additional strategies. We identified a tumor stroma–targeting mechanism in which tumor antigen–specific CD4 ⁺ T cells inhibit tumor growth through myeloid cell and tumor necrosis factor (TNF)–dependent vascular damage. Multiplex immunofluorescence and single-cell and tissue transcriptomics showed that CD4 ⁺ T cells trigger the formation of perivascular myeloid cell clusters containing “classically activated” macrophages that produce TNF in response to T cell–derived interleukin-3. TNF causes intratumoral endothelial damage and blood supply disruption, which are associated with localized tumor cell death. Thus, intratumoral antigen-triggered T cell activation can mediate antitumor effects without direct recognition of living tumor cells, thereby avoiding many of the inhibitory mechanisms that limit anti-tumor immunity.

Sotorasib sensitivity is dependent on KRAS(G12C) expression

Mass spectrometry analysis of RAS(G12C) protein covalent modification.

Figure S4 contains cytotoxicity assay and immunoblot data from the MCF7 and MCF7 DpVp300 cell lines showing that expression of METTL7A confers resistance.

Supplemental Table S2 contains information about the cross-resistance profile of MCF7 DpVp300 cells for multiple drugs

Establishment of a xenograft model of NRAS(G12C)-mutant leukemia