Tumor immune microenvironment permissive to metastatic progression of ING4-deficient breast cancer.

Deficiencies in the ING4 tumor suppressor are associated with advanced stage tumors and poor patient survival in cancer. ING4 was shown to inhibit NF-kB in several cancers. As NF-kB is a key mediator of immune response, the ING4/NF-kB axis is likely to manifest in tumor-immune modulation but has not been investigated. To characterize the tumor immune microenvironment associated with ING4-deficient tumors, three approaches were employed in this study: First, tissue microarrays composed of 246 primary breast tumors including 97 ING4-deficient tumors were evaluated for the presence of selective immune markers, CD68, CD4, CD8, and PD-1, using immunohistochemical staining. Second, an immune-competent mouse model of ING4-deficient breast cancer was devised utilizing CRISPR-mediated deletion of Ing4 in a Tp53 deletion-derived mammary tumor cell line; mammary tumors were evaluated for immune markers using flow cytometry. Lastly, the METABRIC gene expression dataset was evaluated for patient survival related to the immune markers associated with Ing4-deleted tumors. The results showed that CD68, CD4, CD8, or PD-1, was not significantly associated with ING4-deficient breast tumors, indicating no enrichment of macrophages, T cells, or exhausted T cell types. In mice, Ing4-deleted mammary tumors had a growth rate comparable to Ing4-intact tumors but showed increased tumor penetrance and metastasis. Immune marker analyses of Ing4-deleted tumors revealed a significant increase in tumor-associated macrophages (Gr-1loCD11b+F4/80+) and a decrease in granzyme B-positive (GzmB+) CD4+ T cells, indicating a suppressive and/or less tumoricidal immune microenvironment. The METABRIC data analyses showed that low expression of GZMB was significantly associated with poor patient survival, as was ING4-low expression, in the basal subtype of breast cancer. Patients with GZMB-low/ING4-low tumors had the worst survival outcomes (HR = 2.80, 95% CI 1.36-5.75, p = 0.0004), supportive of the idea that the GZMB-low immune environment contributes to ING4-deficient tumor progression. Collectively, the study results demonstrate that ING4-deficient tumors harbor a microenvironment that contributes to immune evasion and metastasis.

Distinct sets of molecular characteristics define tumor-rejecting neoantigens.

Challenges in identifying tumor-rejecting neoantigens limit the efficacy of neoantigen vaccines to treat cancers, including cutaneous squamous cell carcinoma (cSCC). A minority of human cSCC tumors shared neoantigens, supporting the need for personalized vaccines. Using a UV-induced mouse cSCC model which recapitulated the mutational signature and driver mutations found in human disease, we found that CD8 T cells constrain cSCC. Two MHC class I neoantigens were identified that constrained cSCC growth. Compared to the wild-type peptides, one tumor-rejecting neoantigen exhibited improved MHC binding and the other had increased solvent accessibility of the mutated residue. Across known neoantigens that do not impact MHC binding, structural modeling of the peptide/MHC complexes indicated that increased solvent accessibility, which will facilitate TCR recognition of the neoantigen, distinguished tumor-rejecting from non-immunogenic neoantigens. This work reveals characteristics of tumor-rejecting neoantigens that may be of considerable importance in identifying optimal vaccine candidates in cSCC and other cancers.

Major histocompatibility complex class II in the tumor microenvironment: functions of nonprofessional antigen-presenting cells.

Major histocompatibility complex class-II-restricted presentation by nonprofessional antigen-presenting cells in the tumor microenvironment can regulate antitumor T-cell responses. In murine models, tumor cell-specific MHC class II expression decreases in vivo tumor growth, dependent on T cells. Tumor cell-specific MHC class II expression is associated with improved survival and response to immune checkpoint inhibitors in human cancers. Antigen-presenting cancer-associated fibroblasts (apCAF) present MHC class-II-restricted antigens and activate CD4 T cells. The role of MHC class II on apCAFs depends on the cell of origin. MHC class II on tumoral lymphatic endothelial cells leads to expansion of regulatory T cells and increased in vivo tumor growth.

High GILT Expression Is Associated with Improved Survival in Metastatic Melanoma Patients Treated with Immune Checkpoint Inhibition.

Gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II restricted presentation of multiple melanoma antigens. There is variable GILT protein expression in malignant melanocytes in melanoma specimens. High GILT mRNA expression in melanoma specimens is associated with improved overall survival, before the advent of immune checkpoint inhibitors (ICI). However, the association of GILT in metastatic melanoma with survival in patients treated with ICI and the cell type expressing GILT associated with survival have not been determined. Using RNA sequencing datasets, high GILT mRNA expression in metastatic melanoma specimens was associated with improved progression-free and overall survival in patients treated with ICI. A clinical dataset of metastatic melanoma specimens was generated and annotated with clinical information. Positive GILT immunohistochemical staining in antigen presenting cells and melanoma cells was observed in 100% and 65% of metastatic melanoma specimens, respectively. In the subset of patients treated with ICI in the clinical dataset, high GILT protein expression within melanoma cells was associated with improved overall survival. The association of GILT mRNA and protein expression with survival was independent of cancer stage. These studies support that high GILT mRNA expression in bulk tumor samples and high GILT protein expression in melanoma cells is associated with improved survival in ICI-treated patients. These findings support further investigation of GILT as a biomarker to predict the response to ICI.

Physachenolide C induces complete regression of established murine melanoma tumors via apoptosis and cell cycle arrest.

Melanoma is an aggressive skin cancer that metastasizes to other organs. While immune checkpoint blockade with anti-PD-1 has transformed the treatment of advanced melanoma, many melanoma patients fail to respond to anti-PD-1 therapy or develop acquired resistance. Thus, effective treatment of melanoma still represents an unmet clinical need. Our prior studies support the anti-cancer activity of the 17ß-hydroxywithanolide class of natural products, including physachenolide C (PCC). As single agents, PCC and its semi-synthetic analog demonstrated direct cytotoxicity in a panel of murine melanoma cell lines, which share common driver mutations with human melanoma; the IC50 values ranged from 0.19-1.8 µM. PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. In vivo treatment with PCC alone caused the complete regression of established melanoma tumors in all mice, with a durable response in 33% of mice after discontinuation of treatment. T cell-mediated immunity did not contribute to the therapeutic efficacy of PCC or prevent tumor recurrence in YUMM2.1 melanoma model. In addition to apoptosis, PCC treatment induced G0-G1 cell cycle arrest of melanoma cells, which upon removal of PCC, re-entered the cell cycle. PCC-induced cycle cell arrest likely contributed to the in vivo tumor recurrence in a portion of mice after discontinuation of treatment. Thus, 17ß-hydroxywithanolides have the potential to improve the therapeutic outcome for patients with advanced melanoma.