Title: Carabin Is a Negative Regulator of Cd8+ T-cell-mediated Anti-tumor Immunity
Description: The immune system plays a critical role in the prevention and eradication of cancerous lesions. Indeed, cancer immunology is a rapidly growing area of study that has already generated several FDA-approved treatments and cellular therapies to address mechanisms by which various cancers evade immune clearance. Recent studies look to expand the clinical use of these current therapies and to identify novel targets for future treatments in order to meet the unmet medical needs of the cancer patient population. Recently, multiple correlative studies have identified Carabin (Tbc1d10c) as a potential biomarker for cancer prognosis, including head and neck squamous cell carcinoma (HNSCC), breast cancer, and melanoma. Two mechanistic studies have shown that Carabin acts as a negative feedback inhibitor of canonical TCR and BCR signaling during lymphocyte activation. One group demonstrated that Carabin inhibits CD4+ T-cell activation by binding to and inhibiting the actions of Ras and calcineurin, leading to decreased NFAT and AP-1 transcriptional activity.

The second group corroborated the impact of Carabin signaling on the Ras/MAPK pathway in B cells and implicated a role for Carabin in autoimmune diseases in both mice and humans. Collectively, these studies suggest Carabin’s potential role in chronic inflammation and as a pro-tumorigenic target in human cancers. The data presented in Chapters 2-3 demonstrate an immunosuppressive role for Carabin in tumorigenesis. Using three murine tumor models, we identified a novel cancer phenotype in immune competent germline Carabin-ablated (Carabin-/-) mice: these mice showed a twofold decrease in tumor growth and an increase in tumor-free survival compared to wild-type (Carabin+/+) mice. Further assessment identified Carabin expression localized to cells of the immune lineage within the tumor microenvironment (TME), and tumor immunophenotyping showed a twofold increase in the percent of Carabin-/- total and activated CD8+ T cells infiltrating the tumors. Carabin-/- CD8+ T cells displayed an increase in TCR activation and tumor cell killing with no impact on proliferation or migration, indicating that the identified tumor outcome phenotype is due to a suppressive action on the CD8+ TCR activation pathway.

Adoptive transfer of tumor antigen-restricted CD8+ T cells into immune-deficient Rag2-/- mice led to reduction of tumor growth in mice receiving Carabin-/- CD8+ T cells. Thus, the data in Chapters 2-3 demonstrate that Carabin deficiency confers tumor resistance via increased CD8+ T-cell anti-tumor activity. This anti-tumor activity is due to an increase in basal NF-κB activity specifically within CD8+ T cells. NF-κB perturbation is the result of a twofold increase in MEKK3 (Map3k3) protein and its downstream phosphorylation of the IKK complex to activate canonical NF-κB. MEKK3 knockdown by siRNA rescued the Carabin-/- in vitro molecular and cellular phenotype without impacting Carabin+/+ CD8+ T cells, and therefore supports the assertion that Carabin signaling is mediated by downstream MEKK3 activity. The NF-κB pathway is critical for T-cell activation and effector function. NF-κB perturbation was selective to CD8+ T cells and not found in CD4+ T cells. Thus, Carabin may be a novel target to mediate NF-κB signaling specifically in CD8+ T cells to improve their effector function within the TME without simultaneously impacting NF-κB in neoplastic or immunosuppressive cells.

This is the first study to identify a causative link between Carabin and solid tumor malignancies, to demonstrate a unique mechanism for Carabin in the CD8+ T-cell response to tumorigenesis, and to suggest Carabin as a novel CD8+ T-cell-specific NF-κB inhibitor.