FOXM1/DEPDC1 feedback loop promotes hepatocarcinogenesis and represents promising targets for cancer therapy.

Forkhead box M1 (FOXM1) is a key regulator of mitosis and is identified as an oncogene involved in several kinds of human malignancies. However, how it induces carcinogenesis and related therapeutic approaches remains not fully understood. In this study, we aimed to identify a regulatory axis involving FOXM1 and its target gene DEP domain containing 1 (DEPDC1) and investigate their biological functions. FOXM1 bound to the promoter and transcriptionally induced DEPDC1 expression, in turn, DEPDC1 physically interacted with FOXM1, promoted its nuclear translocation, and reinforced its transcriptional activities. The FOXM1/DEPDC1 axis was indispensable for cancer cells, as evidenced by the fact that DEPDC1 rescued cell growth inhibition caused by FOXM1 knockdown, and silencing DEPDC1 efficiently attenuated tumor growth in a murine hepatocellular carcinoma model. Furthermore, strong positive associations between FOXM1/DEPDC1 axis and poor clinical outcome were observed in human hepatocellular carcinoma samples, further indicating their significance for hepatocarcinogenesis. Finally, we attempted to exploit immunotherapy approaches to target the FOXM1/DEPDC1 axis. Several HLA-A24:02-restricted T-cell epitopes targeting FOXM1 or DEPDC1 were identified through bioinformatic analysis. Then, T cell receptor (TCR)-engineered T cells targeting FOXM1262-270 or DEPDC1294-302 were successfully established and proved to efficiently eradicate tumor cells. Our findings highlight the significance of the FOXM1/DEPDC1 axis in the process of oncogenesis and indicate their potential as immunotherapy targets.

Rapid generation of genetically engineered T cells for the treatment of virus-related cancers.

Adoptive transfer of T cell receptor (TCR)-engineered T cells targeting viral epitopes represents a promising approach for treating virus-related cancers. However, the efficient identification of epitopes for T cells and the corresponding TCR remains challenging. Here, we report a workflow permitting the rapid generation of human papillomavirus (HPV)-specific TCR-T cells. Six epitopes of viral proteins belonged to HPV16 or HPV18 were predicted to have high affinity to A11:01 according to bioinformatic analysis. Subsequently, CTL induction were performed with these six antigen peptides separately, and antigen-specific T cells were sorted by FACS. TCR clonotypes of these virus-specific T cells were determined using next-generation sequencing. To improve the efficiency of TCRαß pair validation, a lentiviral vector library containing 116 TCR constructs was generated that consisted of predominant TCRs according to TCR repertoire analysis. Later, TCR library transduced T cells were simulated with peptide pool-pulsed antigen-presenting cells, then CD137-positive cells were sorted and subjected to TCR repertoire analysis. The top-hit TCRs and corresponding antigen peptides were deduced and validated. Through this workflow, a TCR targeting the E692-101 of HPV16 was identified. These HPV16-specific TCR-T cells showed high activity towards HPV16-positive human cervical cancer cells in vitro and efficiently repressed tumor growth in a murine model. This study provides a HPV16-specific TCR fitted to the HLA-A11:01 population, and exemplifies an efficient approach that can be applied in large-scale screening of virus-specific TCRs, further encouraging researchers to exploit the therapeutic potential of the TCR-T cell technique in treating virus-related cancers.

Generation of neoantigen-specific T cells for adoptive cell transfer for treating head and neck squamous cell carcinoma.

Adoptive cell therapy using TCR-engineered T cells (TCR-T cells) represents a promising strategy for treating relapsed and metastatic cancers. We previously established methods to identify neoantigen-specific TCRs based on patients' PBMCs. However, in clinical practice isolation of PBMCs from advanced-stage cancer patients proves to be difficult. In this study, we substituted blood-derived T cells for tumor-infiltrating lymphocytes (TILs) and used an HLA-matched cell line of antigen-presenting cells (APCs) to replace autologous dendritic cells. Somatic mutations were determined in head and neck squamous cell carcinoma resected from two patients. HLA-A*02:01-restricted neoantigen libraries were constructed and transferred into HLA-matched APCs for stimulation of patient TILs. TCRs were isolated from reactive TIL cultures and functionality was tested using TCR- T cells in vitro and in vivo. To exemplify the screening approach, we identified the targeted neoantigen leading to recognition of the minigene construct that stimulated the strongest TIL response. Neoantigen peptides were used to load MHC-tetramers for T cell isolation and a TCR was identified targeting the KIAA1429D1358E mutation. TCR-T cells were activated, exhibited cytotoxicity, and secreted cytokines in a dose-dependent manner, and only when stimulated with the mutant peptide. Furthermore, comparable to a neoantigen-specific TCR that was isolated from the patient's PBMCs, KIAA1429D1358E-specific TCR T cells destroyed human tumors in mice. The established protocol provides the required flexibility to methods striving to identify neoantigen-specific TCRs. By using an MHC-matched APC cell line and neoantigen-encoding minigene libraries, autologous TILs can be stimulated and screened when patient PBMCs and/or tumor material are not available anymore. Abbreviations: Head and neck squamous cell carcinoma (HNSCC); adoptive T cell therapy (ACT); T cell receptor (TCR); tumor-infiltrating lymphocytes (TIL); cytotoxic T lymphocyte (CTL); peripheral blood mononuclear cell (PBMC); dendritic cell (DC); antigen-presenting cells (APC).