UBE2D3 is an Intracellular Checkpoint in Pancreatic Ductal Carcinoma that Restricts CD8+ T-Cell Antitumor Immunity
Ontology highlight
ABSTRACT: Ubiquitylation is pivotal in regulating cellular responses, with its aberration implicated in tumor immune evasion. However, the impact of ubiquitin-conjugating enzymes (E2s) on this evasion remains unclear. Here, we employ a systematic approach to demonstrate that in pancreatic ductal carcinoma (PDAC), the inflammatory microenvironment induces overexpression of the E2 enzyme UBE2D3, contributing to tumor progression through non-oncogene codependent disorders. Through gene expression analyses and functional investigations, we elucidate a mechanism wherein cancer cells evade T cell immune responses by UBE2D3 binding to the ubiquitin ligase KLHL13 to co-ubiquitinate TAP2. This K63-linkage ubiquitination at the lysine 245 site of TAP2 impedes antigenic peptide transport by the TAP1/TAP2 complex, hindering p-MHC assembly and presentation in cancer cells. We demonstrate that genetic inhibition of UBE2D3 enhances tumor-specific CD8+ T cell proliferation and extends effector-memory-like phenotypes. Building on this, we develop a small-molecule inhibitor, QX-6, targeting the active site of UBE2D3 to disrupt its function. Pharmacologic inhibition of UBE2D3 blocks the ubiquitylation of antigen presentation-related substrates, leading to increased p-MHC presentation by cancer cells and reduced T cell exhaustion. Using immunocompetent and humanized models, we elucidate the therapeutic efficacy of targeting UBE2D3. Furthermore, we evaluate the synergistic anti-tumor effects of QX-6 in combination with KRAS-targeted TCR-T cell immunotherapy. In summary, our study reveals a post-translational modification mechanism wherein the intracellular checkpoint UBE2D3 regulates the TAP2 switch to control cancer cell evasion of CTLs, presenting a potential immunotherapeutic strategy to improve PDAC treatment outcomes.
Project description:Ubiquitylation is pivotal in regulating cellular responses, with its aberration implicated in tumor immune evasion. However, the impact of ubiquitin-conjugating enzymes (E2s) on this evasion remains unclear. Here, we employ a systematic approach to demonstrate that in pancreatic ductal carcinoma (PDAC), the inflammatory microenvironment induces overexpression of the E2 enzyme UBE2D3, contributing to tumor progression through non-oncogene codependent disorders. Through gene expression analyses and functional investigations, we elucidate a mechanism wherein cancer cells evade T cell immune responses by UBE2D3 binding to the ubiquitin ligase KLHL13 to co-ubiquitinate TAP2. This K63-linkage ubiquitination at the lysine 245 site of TAP2 impedes antigenic peptide transport by the TAP1/TAP2 complex, hindering p-MHC assembly and presentation in cancer cells. We demonstrate that genetic inhibition of UBE2D3 enhances tumor-specific CD8+ T cell proliferation and extends effector-memory-like phenotypes. Building on this, we develop a small-molecule inhibitor, QX-6, targeting the active site of UBE2D3 to disrupt its function. Pharmacologic inhibition of UBE2D3 blocks the ubiquitylation of antigen presentation-related substrates, leading to increased p-MHC presentation by cancer cells and reduced T cell exhaustion. Using immunocompetent and humanized models, we elucidate the therapeutic efficacy of targeting UBE2D3. Furthermore, we evaluate the synergistic anti-tumor effects of QX-6 in combination with KRAS-targeted TCR-T cell immunotherapy. In summary, our study reveals a post-translational modification mechanism wherein the intracellular checkpoint UBE2D3 regulates the TAP2 switch to control cancer cell evasion of CTLs, presenting a potential immunotherapeutic strategy to improve PDAC treatment outcomes.
Project description:Ubiquitylation is pivotal in regulating cellular responses, with its aberration implicated in tumor immune evasion. However, the impact of ubiquitin-conjugating enzymes (E2s) on this evasion remains unclear. Here, we employ a systematic approach to demonstrate that in pancreatic ductal carcinoma (PDAC), the inflammatory microenvironment induces overexpression of the E2 enzyme UBE2D3, contributing to tumor progression through non-oncogene codependent disorders. Through gene expression analyses and functional investigations, we elucidate a mechanism wherein cancer cells evade T cell immune responses by UBE2D3 binding to the ubiquitin ligase KLHL13 to co-ubiquitinate TAP2. This K63-linkage ubiquitination at the lysine 245 site of TAP2 impedes antigenic peptide transport by the TAP1/TAP2 complex, hindering p-MHC assembly and presentation in cancer cells. We demonstrate that genetic inhibition of UBE2D3 enhances tumor-specific CD8+ T cell proliferation and extends effector-memory-like phenotypes. Building on this, we develop a small-molecule inhibitor, QX-6, targeting the active site of UBE2D3 to disrupt its function. Pharmacologic inhibition of UBE2D3 blocks the ubiquitylation of antigen presentation-related substrates, leading to increased p-MHC presentation by cancer cells and reduced T cell exhaustion. Using immunocompetent and humanized models, we elucidate the therapeutic efficacy of targeting UBE2D3. Furthermore, we evaluate the synergistic anti-tumor effects of QX-6 in combination with KRAS-targeted TCR-T cell immunotherapy. In summary, our study reveals a post-translational modification mechanism wherein the intracellular checkpoint UBE2D3 regulates the TAP2 switch to control cancer cell evasion of CTLs, presenting a potential immunotherapeutic strategy to improve PDAC treatment outcomes.
Project description:Loss of MHC class I (MHC-I) antigen presentation in cancer cells can lead to immunotherapy resistance. Using a genome-wide CRISPR/Cas9 screen we identify a critical role for polycomb repressive complex 2 (PRC2) in the coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP). This evolutionarily conserved function of PRC2 promotes evasion of T-cell mediated immunity, enabling tumor transmission to non-histocompatible recipients in small cell lung cancer (SCLC) and Tasmanian Devil Facial Tumor. MHC-I APP gene promoters in MHC-I low cancers harbour bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine induced MHC-I APP gene upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological silencing of MHC-I expression highlights a conserved mechanism by which cancers arising from these primitive tissues coopt PRC2 activity to enable immune evasion.
Project description:The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using two novel preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, which are similar to tumor-infiltrating lymphocytes of human PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical to maintain immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.
Project description:Activated SUMOylation is a hallmark of aggressive cancers. Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionary conserved function of activated SUMOylation, which attenuates the immunogenicity of tumor cells. Activated SUMOylation allows cancer cells to evade CD8+ T-cell immunosurveillance by repressing the MHC-I antigen processing and presentation machinery (APM). While loss of the MHC-I APM is a frequent cause of resistance to cancer immunotherapies, the pharmacological inhibition of SUMOylation (SUMOi) restored the expression of the MHC-I APM and enhanced the susceptibility of tumor cells to CD8+ T-cell mediated killing. Importantly, SUMOi also triggered the activation of CD8+ T-cells itself and thereby drives a feed-forward loop amplifying the specific anti-tumor immune response. In summary, we show that activated SUMOylation converts tumor cells into a state of immune evasion, and identify SUMOi as rational therapeutic strategy for enhancing the efficacy of cancer immunotherapies.
Project description:Oncolytic viruses (OVs), known for their cancer-killing characteristics, overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro we found that reovirus induces the presentation of tumor MHC-I ligands in cancer cells. Concurrent multiplexed quantitative proteomics revealed that the changes in tumor MHC-I ligand presentation were mostly independent of reovirus-induced alterations of their source proteins. In an in vivo model, tumor MHC-I ligands were induced by reovirus in tumors but also, more importantly, analysis of spleens (a source of antigen-presenting cells) showed exclusive induction of most MHC-I ligands occurred in tumor-bearing mice. Finally, IFNγ assays demonstrated immunogenicity of the reovirus-induced MHC-I ligands. OV-induced MHC-I responses may be exploited in combinatorial approaches to promote the efficacy of cancer immunotherapies.
Project description:Loss of MHC class I (MHC-I) antigen presentation in cancer cells can lead to immunotherapy resistance. Using a genome-wide CRISPR/Cas9 screen we identify a critical role for polycomb repressive complex 2 (PRC2) in the coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP). This evolutionarily conserved function of PRC2 promotes evasion of T-cell mediated immunity, enabling tumour transmission to non-histocompatible recipients in small cell lung cancer (SCLC) and Tasmanian Devil Facial Tumour. MHC-I APP gene promoters in MHC-I low cancers harbour bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine induced MHC-I APP gene upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological silencing of MHC-I expression highlights a conserved mechanism by which cancers arising from these primitive tissues coopt PRC2 activity to enable immune evasion.
Project description:Effective immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex Class I (MHC-I). Recent developments in proteomics have improved the identification of peptides that are naturally presented by MHC-I, collectively known as the “immunopeptidome”. Current approaches to profile tumor immunopeptidomes have been limited to in vitro investigation, which fails to capture the in vivo repertoire of MHC-I peptides, or bulk tumor lysates, which are obscured by the lack of cancer cell-specific MHC-I isolation. To overcome these limitations, we report here the engineering of a Cre recombinase-inducible affinity tag into the endogenous mouse MHC-I gene and targeting of this allele to the KrasLSL-G12D/+; p53fl/fl (KP) mouse model (KP/KbStrep). This novel approach has allowed us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. With this powerful analytical tool, we were able to profile the evolution of the LUAD immunopeptidome from the alveolar type 2 cell-of-origin through late-stage disease. Differential peptide presentation in LUAD is not driven by increased mRNA expression or translation rate and is likely driven by post-translational mechanisms. Vaccination of mice with peptides presented by LUAD in vivo provoked CD8 T-cell responses in naïve and tumor bearing mice. Many peptides unique to LUAD, including immunogenic peptides, exhibited very low expression of the cognate mRNA provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance. Beyond cancer, the KbStrep allele is compatible with a broad range of Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.
Project description:Impaired expression of MHC class I constitutes a major mechanism of immune evasion of cancers, leading to poor prognosis and resistance to checkpoint blockade therapies. Existing drugs for MHC class I have limited applicability due to severe side effects. Here we show a novel approach of robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA), NLRC5, using a CRISPR/Cas9 based gene-specific targeted demethylaion (TDM) system and targeted demethylation and activation (TDMa) system. The TDMa system specifically recruits a demethylating enzyme and transcriptional activators, providing efficient demethylation and transactivation of the NLRC5 promoter. TDMa in mouse and human cancer cells induced MHC class I antigen presentation and accelerated CD8+ T cell activation with tumor suppression effects both in vitro and in vivo. Moreover, enhanced immunogenicity by NLRC5 TDMa boosted efficacy of anti-PD1 therapy. Therefore, NLRC5 targeting by the TDMa system confers an attractive therapeutic approach against cancer.
Project description:Immune evasion is an important hallmark of cancer ensured by diverse strategies, including immunosuppression and downregulation of antigen presentation. Here, to restore immunogenicity of cancer cells, we employed the minimal gene regulatory network of highly immunogenic type 1 conventional dendritic cells (cDC1) to reprogram cancer cells into professional antigen presenting cells (APCs). We showed that enforced expression of PU.1, IRF8 and BATF3 (PIB) was sufficient to induce cDC1 phenotype in 33 cell lines derived from human and mouse hematological and solid tumors. PIB gradually modified the cancer cell transcriptional and epigenetic program imposing global antigen presentation and cDC1 gene signatures within 9 days. cDC1 reprogramming restored the expression of antigen presentation complexes as well as co-stimulatory molecules at the cell surface, leading to the presentation of endogenous antigens on MHC-I, and to CD8+ T cell mediated killing. Functionally, tumor- APCs acquired the ability to uptake and process exogenous proteins and dead cells, secreted inflammatory cytokines and cross-presented antigens to naïve CD8+ T cells. Importantly, tumor-APCs were efficiently generated at the single cell level from primary cancer cells of 7 solid tumors that presented antigens to memory and naïve T-cells, as well as to activated patient-specific intra-tumoral lymphocytes. Alongside antigen presentation, tumor-APCs harboring TP53, KRAS and PTEN mutations showed impaired tumorigenicity in vitro and in vivo. Finally, using in vivo mouse models of melanoma, we showed that intra-tumoral injection of tumor-APCs promoted lymphoid infiltration, delayed tumor growth and increased survival. The anti-tumor immunity elicited by tumor-APCs was synergistic with immune checkpoint inhibitors enabling tumor eradication. Our approach combines cDC1’s antigen processing and presenting abilities with endogenous generation of tumor antigens and serves as a platform for the development of novel immunotherapies based on endowed antigen presentation in cancer cells.