Fusobacterium nucleatum-derived succinic acid induces tumor resistance to immunotherapy in colorectal cancer.

Immune checkpoint blockade therapy with anti-PD-1 monoclonal antibody (mAb) is a treatment for colorectal cancer (CRC). However, some patients remain unresponsive to PD-1 blockade. The gut microbiota has been linked to immunotherapy resistance through unclear mechanisms. We found that patients with metastatic CRC who fail to respond to immunotherapy had a greater abundance of Fusobacterium nucleatum and increased succinic acid. Fecal microbiota transfer from responders with low F. nucleatum, but not F. nucleatum-high non-responders, conferred sensitivity to anti-PD-1 mAb in mice. Mechanistically, F. nucleatum-derived succinic acid suppressed the cGAS-interferon-ß pathway, consequently dampening the antitumor response by limiting CD8+ T cell trafficking to the tumor microenvironment (TME) in vivo. Treatment with the antibiotic metronidazole reduced intestinal F. nucleatum abundance, thereby decreasing serum succinic acid levels and resensitizing tumors to immunotherapy in vivo. These findings indicate that F. nucleatum and succinic acid induce tumor resistance to immunotherapy, offering insights into microbiota-metabolite-immune crosstalk in CRC.

A peptidic inhibitor for PD-1 palmitoylation targets its expression and functions.

Programmed cell death protein 1 (PD-1) is a crucial anticancer target, but the relatively low response rate and acquired resistance to existing antibody drugs highlight an urgent need to develop alternative targeting strategies. Here, we report the palmitoylation of PD-1, discover the main DHHC enzyme for this modification, reveal the mechanism of its effect on PD-1 protein stability, and rationally develop a peptide for targeting PD-1 expression. Palmitoylation promoted the trafficking of PD-1 to the recycling endosome, thus preventing its lysosome-dependent degradation. Palmitoylation of PD-1, but not of PD-L1, promoted mTOR signaling and tumor cell proliferation, and targeting palmitoylation displayed significant anti-tumor effects in a three-dimensional culture system. A peptide was designed to competitively inhibit PD-1 palmitoylation and expression, opening a new route for developing PD-1 inhibitors and combinatorial cancer immunotherapy.

THADA drives Golgi residency and upregulation of PD-L1 in cancer cells and provides promising target for immunotherapy.

BACKGROUND: The abnormal upregulation of programmed death-ligand 1 (PD-L1) in cancer cells inhibits T cell-mediated cytotoxicity, but the molecular mechanisms that drive and maintain PD-L1 expression are still incompletely understood. METHODS: Combined analyses of genomes and proteomics were applied to find potential regulators of PD-L1. In vitro experiments were performed to investigate the regulatory mechanism of PD-L1 by thyroid adenoma associated gene (THADA) using human colorectal cancer (CRC) cells. The prevalence of THADA was analyzed using CRC tissue microarrays by immunohistochemistry. T cell killing assay, programmed cell death 1 binding assay and MC38 transplanted tumor models in C57BL/6 mice were developed to investigate the antitumor effect of THADA. RESULTS: THADA is critically required for the Golgi residency of PD-L1, and this non-redundant, coat protein complex II (COPII)-associated mechanism maintains PD-L1 expression in tumor cells. THADA mediated the interaction between PD-L1 as a cargo protein with SEC24A, a module on the COPII trafficking vesicle. Silencing THADA caused absence and endoplasmic reticulum (ER) retention of PD-L1 but not major histocompatibility complex-I, inducing PD-L1 clearance through ER-associated degradation. Targeting THADA substantially enhanced T cell-mediated cytotoxicity, and increased CD8+ T cells infiltration in mouse tumor tissues. Analysis on clinical tissue samples supported a potential role of THADA in upregulating PD-L1 expression in cancer. CONCLUSIONS: Our data reveal a crucial cellular process for PD-L1 maturation and maintenance in tumor cells, and highlight THADA as a promising target for overcoming PD-L1-dependent immune evasion.

Repurposing screen identifies Amlodipine as an inducer of PD-L1 degradation and antitumor immunity.

Cancer cell expression of PD-L1 leads to T cells exhaustion by transducing co-inhibitory signal, and further understanding the regulation of PD-L1 in cancer cells may provide additional therapeutic strategies. Here by drug repurposing screen, we identified amlodipine as a potent inhibitor of PD-L1 expression in cancer cells. Further survey of calcium-associated pathways revealed calpain-dependent stabilization of the PD-L1 protein. Intracellular calcium delivered an operational signal to calpain-dependent Beclin-1 cleavage, blocking autophagic degradation of PD-L1 accumulated on recycling endosome (RE). Blocking calcium flux by amlodipine depleted PD-L1 expression and increased CD8+ T-cell infiltration in tumor tissues but not in myocardium, causing dose-dependent tumor suppression in vivo. Rescuing PD-L1 expression eliminated the effects of amlodipine, suggesting the PD-L1-dependent effect of amlodipine. These results reveal a calcium-dependent mechanism controlling PD-L1 degradation, and highlight calcium flux blockade as a potential strategy for combinatorial immunotherapy.

Folded or Degraded in Endoplasmic Reticulum.

In consistent with other membrane-bound and secretory proteins, immune checkpoint proteins go through a set of modifications in the endoplasmic reticulum (ER) to acquire their native functional structures before they function at their destinations. There are various ER-resident chaperones and enzymes synergistically regulate and catalyze the glycosylation, folding and transporting of proteins. The whole processing is under the surveillance of ER quality control system which allows the correctly folded proteins to exit from the ER with the help of coat proteinII(COPII) coated vesicles, while retains the rest of terminally misfolded ones in the ER and then eliminates them via ER-associated degradation (ERAD) or ER-to-lysosomes-associated degradation (ERLAD). The dysfunction of the ER causes ER stress which triggers unfolded protein response (UPR) to restore ER proteostasis. Unsolvable prolonged ER stress ultimately results in cell death. This chapter reviews the process that proteins undergo in the ER, and the glycosylation, folding and degradation of immune checkpoint proteins as well as the associated potential immunotherapies to date.

Author Correction: Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours.

In the version of this Article originally published, 'palmitoyltransferase ZDHHC3 (DHHC3)' was incorrectly referred to as an 'acetyltransferase' rather than an as an 'acyltransferase'; this has now been corrected in five instances. In Fig. 3a, the label for the bottom row of the blots was mistakenly written as 'GAPHD'; it should have read 'GAPDH'. In the two right-most panels of Fig. 4j, the antibody labels 'α-PD-L1' for the reciprocal co-immunoprecipitation of DHHC3 were incorrect; they should have been 'α-DHHC3'. These errors have been corrected in all versions of the Article.

Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours.

Checkpoint blockade therapy targeting the programmed-death ligand 1 (PD-L1) and its receptor programmed cell death 1 promotes T-cell-mediated immunosurveillance against tumours, and has been associated with marked clinical benefit in cancer patients. Antibodies against PD-L1 function by blocking PD-L1 on the cell surface, but intracellular storage of PD-L1 and its active redistribution to the cell membrane can minimize the therapeutic benefits, which highlights the importance of targeting PD-L1 throughout the whole cell. Here, we show that PD-L1 is palmitoylated in its cytoplasmic domain, and that this lipid modification stabilizes PD-L1 by blocking its ubiquitination, consequently suppressing PD-L1 degradation by lysosomes. We identified palmitoyltransferase ZDHHC3 (DHHC3) as the main acetyltransferase required for the palmitoylation of PD-L1, and show that the inhibition of PD-L1 palmitoylation via 2-bromopalmitate, or the silencing of DHHC3, activates antitumour immunity in vitro and in mice bearing MC38 tumour cells. We also designed a competitive inhibitor of PD-L1 palmitoylation that decreases PD-L1 expression in tumour cells to enhance T-cell immunity against the tumours. These findings suggest new strategies for overcoming PD-L1-mediated immune evasion in cancer.

HIP1R targets PD-L1 to lysosomal degradation to alter T cell-mediated cytotoxicity.

Expression of programmed cell death 1 (PD-1) ligand 1 (PD-L1) protects tumor cells from T cell-mediated immune surveillance, and immune checkpoint blockade (ICB) therapies targeting PD-1 and PD-L1 have exhibited significant clinical benefits. However, the relatively low response rate and observed ICB resistance highlight the need to understand the molecular regulation of PD-L1. Here we show that HIP1R targets PD-L1 to lysosomal degradation to alter T cell-mediated cytotoxicity. HIP1R physically interacts with PD-L1 and delivers PD-L1 to the lysosome through a lysosomal targeting signal. Depletion of HIP1R in tumor cells caused PD-L1 accumulation and suppressed T cell-mediated cytotoxicity. A rationally designed peptide (PD-LYSO) incorporating the lysosome-sorting signal and the PD-L1-binding sequence of HIP1R successfully depleted PD-L1 expression in tumor cells. Our results identify the molecular machineries governing the lysosomal degradation of PD-L1 and exemplify the development of a chimeric peptide for targeted degradation of PD-L1 as a crucial anticancer target.

Cancer Cell-Intrinsic PD-1 and Implications in Combinatorial Immunotherapy.

Programmed death 1 (PD-1) and its two natural ligands PD-L1 and PD-L2 are responsible for delivering inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. In previous studies, PD-1 was found only expressed on the surface of immune cells, such as T cells and B cells while PD-1's ligands PD-L1 and PD-L2 were found expressed in some tumor cells. However, recent studies revealed intrinsic expression of PD-1 in melanoma and some other cancers. In melanoma cells, PD-1 can be activated by its ligand PD-L1 expressed by tumor cells, modulating downstream mammalian target of rapamycin signaling and promoting tumor growth independent of adaptive immunity. In addition to melanoma, PD-1 was also detected in liver cancer cells as well as in non-small lung cancer cells. Unlike its oncogenic functions in melanoma and hepatic carcinoma cells, PD-1 seemed to play a distinct role in lung cancer, as blockade of PD-1 instead promoted tumor cells proliferation. Tumor-intrinsic PD-1 expression seems to be widespread in many tumor types, according to our reanalysis on cancer transcriptomic and proteomic data. The multifaceted roles of PD-1 in tumor cells beyond immune checkpoint signaling may explain the differential therapeutic effects of anti-PD-1 and anti-PD-L1 drugs and provide crucial information when developing combinatorial approaches to enhance antitumor immunity.

Regulation of PD-L1: Emerging Routes for Targeting Tumor Immune Evasion.

Immune checkpoint blockade therapies (ICBTs) targeting programmed cell death 1 (PD-1) and its ligand programmed death ligand-1 (PD-L1/B7-H1/CD274) have exhibited momentous clinical benefits and durable responses in multiple tumor types. However, primary resistance is found in considerable number of cancer patients, and most responders eventually develop acquired resistance to ICBT. To tackle these challenges, it is essential to understand how PD-L1 is controlled by cancer cells to evade immune surveillance. Recent research has shed new light into the mechanisms of PD-L1 regulation at genetic, epigenetic, transcriptional, translational, and posttranslational levels. In this work, we systematically discuss the mechanisms that control the gene amplification, epigenetic alteration, transcription, subcellular transportation and posttranscriptional modification of PD-L1 in cancer cells. We further categorize posttranscriptional PD-L1 regulations by the molecular modification of PD-L1, including glycosylation, phosphorylation, ubiquitination, deubiquitination, and lysosomal degradation. These findings may provide new routes for targeting tumor immune escape and catalyze the development of small molecular inhibitors of PD-L1 in addition to existing antibody drugs.

A Designed Peptide Targets Two Types of Modifications of p53 with Anti-cancer Activity.

Many cancer-related proteins are controlled by composite post-translational modifications (PTMs), but prevalent strategies only target one type of modification. Here we describe a designed peptide that controls two types of modifications of the p53 tumor suppressor, based on the discovery of a protein complex that suppresses p53 (suppresome). We found that Morn3, a cancer-testis antigen, recruits different PTM enzymes, such as sirtuin deacetylase and ubiquitin ligase, to confer composite modifications on p53. The molecular functions of Morn3 were validated through in vivo assays and chemico-biological intervention. A rationally designed Morn3-targeting peptide (Morncide) successfully activated p53 and suppressed tumor growth. These findings shed light on the regulation of protein PTMs and present a strategy for targeting two modifications with one molecule.

ASAP3 regulates microvilli structure in parietal cells and presents intervention target for gastric acidity.

Gastric acidity-associated disorders such as peptic ulcer and reflux diseases are widespread, and the reported resistance and side effects of currently used medicines suggest an urgent requirement for alternative therapeutic approaches. Here we demonstrate a critical role of ASAP3 in regulating the microvilli structure of parietal cells in vivo, and reveal the feasibility of controlling gastric acidity by targeting ASAP3. Conditional knockout of ASAP3 in mice caused elongation and stacking of microvilli in parietal cells, and substantially decreased gastric acid secretion. These were associated with active assembly of F-actin caused by a higher level of GTP-bound Arf6 GTPase. Consistently, a small molecular compound QS11 inhibited ASAP3 function and significantly reduced gastric acidity in vivo. Of note, the expression of ASAP3 was positively correlated with gastric acid secretion in 90 human cases, and high expression of ASAP3 was associated with reflux disease and peptic ulcer. These results reveal for the first time that ASAP3 regulates the microvilli structures in parietal cells. Our data also suggest ASAP3 as a feasible and drugable therapeutic target for gastric acidity-associated diseases.

Scaffold Proteins in Gastrointestinal Tumors as a Shortcut to Oncoprotein Activation.

BACKGROUND: The development of cancer involves uncontrolled cell proliferation, and multiple signaling pathways that regulate cell proliferation have been found to be dysregulated in cancers. Extracellular signal-regulated protein kinase (ERK) is one of three major subtypes in the mitogen-activated protein kinase (MAPK) families. The MAPK/ERK pathway (RAS/RAF1/MEK/ERK) plays an important part in promoting cell proliferation in response to growth factors, thereby serving as a driving signal in gastrointestinal (GI) tumors. In contrast, the p53 tumor suppressor functions as a "guardian of the genome" and stops cell proliferation when oncogenic signaling is activated. SUMMARY: Both pathways constrain each other in healthy GI epithelium, facilitating controlled proliferation that is essential for tissue repair and regeneration. However, in GI tumors, the MAPK/ERK and p53 pathways are commonly dysregulated, in part due to abnormal posttranslational modifications. Hyperphosphorylation of the ERK protein causes sustained activation of cell proliferation, whereas hypoacetylation of the p53 protein impairs its transcriptional function and blocks cell apoptosis. Multiple scaffold proteins have been found to regulate the posttranslational modifications of ERK and p53 proteins in GI tumors. KEY MESSAGE: Abnormal expression of scaffold proteins may contribute to the dysregulation of the MAPK and p53 signaling pathways and thereby contribute to the development of GI tumors. PRACTICAL IMPLICATIONS: Scaffold proteins are potential biomarkers and therapeutic targets in GI tumors.

PD-L2 expression in colorectal cancer: Independent prognostic effect and targetability by deglycosylation.

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide, and immune checkpoint blockade therapy provides an opportunity for improving the outcome of CRC patients. Recent studies suggest that programmed death ligand-1 (PD-L1) is only expressed in 12% of CRCs. Here, we demonstrate that PD-L2 is expressed in approximately 40% CRCs, and its expression independently associates with poor survival of CRC patients. By detection of PD-L2 expression by immunofluorescence in 124 CRC cases with 10-y survival data, we found significant association between PD-L2 overexpression in cancer cells and worse overall survival (46.3 vs 69.1 mo; p = 0.0004). The association remained significant in multivariate COX regression analysis (hazard ratio = 2.778, 95% confidence interval [CI] = 1.668-4.627; p < 0.0001). In the validation CRC data set, significant association between PD-L2 overexpression and poor survival was supported by the univariate analysis (27.1 vs. 88.9 mo; p = 0.0002) and multivariate model (hazard ratio = 7.09, 95%CI 1.78-28.16; p = 0.005). Western Blot revealed strong induction of PD-L2 expression by interferon-γ (IFNγ) in CRC cells, and the mRNA levels of both genes were significantly correlated in CRC tissue samples. Suppression of glycosylation with tunicamycin caused a shift in molecular weight and significant decrease in the expression of PD-L2 protein. In conclusion, PD-L2 overexpression in CRC cells, under the regulation by IFNγ and glycosylation, associates with poor survival of patients with colorectal cancer. These findings highlight PD-L2 as a promising therapeutic target in CRC and suggest potential routes to control PD-L2 expression in CRC cells.