Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease.

Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically synthesized siRNAs. Transfection of rhabdomyosarcoma (RD) cells with siRNA targeting the 3'UTR, 2C, 3C, or 3D region significantly alleviated cytopathic effects of EV71. The inhibitory effect was dosage-dependent with a corresponding decrease in viral RNA, viral proteins, and plaque formations by EV71. Viral inhibition of siRNA transfected RD cells was still evident after 48 h. In addition, no significant adverse off-target silencing effects were observed. These results demonstrated the potential and feasibility for the use of siRNA as an antiviral therapy for EV71 infections.

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These results provide evidence for the existence of cancer stem-like cells in the NPC cell line C666-1 cells.

Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published mouse model CRISPR screen suggested that targeting an E3 ligase (STUB1) involved with protein homeostasis, may overcome ICB resistance but the molecular basis behind this observation is unclear. Using the ICB-resistant and poorly immunogenic B16-F10 murine melanoma model, we reveal an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Deletion of Stub1 in tumour cells increased IFNGR1 abundance on cellular surface, thus lowering the stimulating threshold of IFNγ. These outcomes translated to IFNγ-enhanced antigen presentation and upregulation of the immunoproteasome complexes. Through proteomics and gene expression profiling, we confirmed STUB1 as a negative regulator of the IFNγ signaling pathway. To block the function of STUB1 in tumour cells, we stably expressed a rationally designed inhibitory biologic, which recapitulated the Stub1-null phenotypes in both murine and human tumour cells. Overall, our findings elucidate STUB1 as a barrier for IFNγ sensing and offer a roadmap to pursue STUB1 inhibitors, which may improve tumour response to checkpoint inhibitory therapy.

Immune checkpoint blockade (ICB) leads to durable and complete tumour regression in some patients but in others gives temporary, partial or no response. Accordingly, significant efforts are underway to identify tumour-intrinsic mechanisms underlying ICB resistance. Results from a published CRISPR screen in a mouse model suggested that targeting STUB1, an E3 ligase involved in protein homeostasis, may overcome ICB resistance but the molecular basis of this effect remains unclear. Herein, we report an under-appreciated role of STUB1 to dampen the interferon gamma (IFNγ) response. Genetic deletion of STUB1 increased IFNGR1 abundance on the cell surface and thus enhanced the downstream IFNγ response as showed by multiple approaches including Western blotting, flow cytometry, qPCR, phospho-STAT1 assay, immunopeptidomics, proteomics, and gene expression profiling. Human prostate and breast cancer cells with STUB1 deletion were also susceptible to cytokine-induced growth inhibition. Furthermore, blockade of STUB1 protein function recapitulated the STUB1-null phenotypes. Despite these encouraging in vitro data and positive implications from clinical datasets, we did not observe in vivo benefits of inactivating Stub1 in mouse syngeneic tumour models—with or without combination with anti-PD-1 therapy. However, our findings elucidate STUB1 as a barrier to IFNγ sensing, prompting further investigations to assess if broader inactivation of human STUB1 in both tumors and immune cells could overcome ICB resistance.

Background: Immunotherapy has so far had limited success in colorectal cancer (CRC), with its efficacy restricted to a subset of microsatellite instability high (MSI-H) tumors. A comprehensive interrogation of the CRC tumor immune microenvironment (TME) is urgently needed. We present here an ongoing multi-platform study on early stage colon and rectal cancers, where immuno-genomic profiling of tumors and patient-derived cell models of tumor epithelia, cancer-associated fibroblasts and tumor-infiltrating lymphocytes (TIL) complement each other, with opportunities for mutual cross-validation between experimental and bioprofiling data. Methods: At the time of writing, 21 of a planned 50 early stage CRC patients have been recruited. Surgically resected tumors are processed for 4 broad classes of analyses: 1) Bulk tissue profiling by RNA and whole exome sequencing; 2) High resolution protein and transcriptome analysis comprising scRNA-seq and flow cytometry/CyToF; 3) H&E analysis and multiplex immunohistochemistry for TME-specific proteins; 4) Culture of epithelial, fibroblast, and TIL lines, and generation of patient-derived xenografts for functional studies. Results: Four tumors were MSI-H and 17 were microsatellite stable (MSS), with 1 POLE-mutant MSS patient harboring over 6000 mutations. Bulk genomics analysis revealed the most common mutations to be in TP53, APC, MUC17, and TGFBR2. The most frequently altered pathways were WNT, followed in order by p53, TGFβ, PI3K, and RAS-MAPK. scRNA-seq and flow cytometry/CyToF analyses are being performed to examine immune phenotypes, mediators of cell migration, and immune suppressive populations, which complement data on transcriptomic profile, histopathology, and spatial localization of TME cellular components. Of the 3 cultured cell types, 16 patients have lines of at least one cell compartment established in vitro. Characteristics of individual models will be reported at the meeting. Establishing the 3 cultured cell types from the same patient will enable us to develop an autologous patient-derived co-culture system to evaluate all 3 pairwise interactions, including TIL cytotoxicity toward epithelial cells, mutual modulation by fibroblast and epithelial cells in co-culture and their phenotypic alterations, and fibroblast regulation of TIL cytotoxic function. Autologous co-cultures with all 3 cell types are also planned. Conclusions: The generation of well-annotated multi-platform profiling data from CRC tumors, complemented by matched tri-compartment patient-derived cell cultures, enables mutual cross-validation between experimental models of the TME and bioprofiling data. Citation Format: Lindsay H. Kua, Fiona Y. Lee, Christine L. Eng, Harini Srinivasan, Rahul Nahar, Janice H. Oh, Nicole Ann L. Gunn, Kai Xian Thng, Ashley S. Yong, Adrian C. Sim, Rebecca Lim, Nicole Boo, Simeen Malik, Michael T. Wong, Tze Guan Tan, Shu Wen S. Ho, Shirleen Soh, Wan Jun Lim, Macalinao Dominique Camat, Joe P. Yeong, Clarinda W. Chua, Wei Qiang Leow, Ramanuj DasGupta, Si-Lin Koo, Lewis Hong, Brian Henry, Tony K. Lim, Iain B. Tan. Tri-compartment (epithelial, immune, fibroblast) patient-derived models of the tumor microenvironment from an immuno-genomic profiled cohort of early stage colorectal cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3990.