Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules.

Protein O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis of O-GlcNAcylation has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of the O-GlcNAcylation challenging. To address this, we have developed heterobifunctional small molecules, named O-GlcNAcylation TArgeting Chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in living cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 in cellulo by recruiting FKBP12F36V-fused O-GlcNAc transferase (OGT), with temporal, magnitude, and reversible control. Overall, the OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

Targeting PPAR-gamma counteracts tumour adaptation to immune-checkpoint blockade in hepatocellular carcinoma.

OBJECTIVE: Therapy-induced tumour microenvironment (TME) remodelling poses a major hurdle for cancer cure. As the majority of patients with hepatocellular carcinoma (HCC) exhibits primary or acquired resistance to antiprogrammed cell death (ligand)-1 (anti-PD-[L]1) therapies, we aimed to investigate the mechanisms underlying tumour adaptation to immune-checkpoint targeting. DESIGN: Two immunotherapy-resistant HCC models were generated by serial orthotopic implantation of HCC cells through anti-PD-L1-treated syngeneic, immunocompetent mice and interrogated by single-cell RNA sequencing (scRNA-seq), genomic and immune profiling. Key signalling pathway was investigated by lentiviral-mediated knockdown and pharmacological inhibition, and further verified by scRNA-seq analysis of HCC tumour biopsies from a phase II trial of pembrolizumab (NCT03419481). RESULTS: Anti-PD-L1-resistant tumours grew >10-fold larger than parental tumours in immunocompetent but not immunocompromised mice without overt genetic changes, which were accompanied by intratumoral accumulation of myeloid-derived suppressor cells (MDSC), cytotoxic to exhausted CD8+ T cell conversion and exclusion. Mechanistically, tumour cell-intrinsic upregulation of peroxisome proliferator-activated receptor-gamma (PPARγ) transcriptionally activated vascular endothelial growth factor-A (VEGF-A) production to drive MDSC expansion and CD8+ T cell dysfunction. A selective PPARγ antagonist triggered an immune suppressive-to-stimulatory TME conversion and resensitised tumours to anti-PD-L1 therapy in orthotopic and spontaneous HCC models. Importantly, 40% (6/15) of patients with HCC resistant to pembrolizumab exhibited tumorous PPARγ induction. Moreover, higher baseline PPARγ expression was associated with poorer survival of anti-PD-(L)1-treated patients in multiple cancer types. CONCLUSION: We uncover an adaptive transcriptional programme by which tumour cells evade immune-checkpoint targeting via PPARγ/VEGF-A-mediated TME immunosuppression, thus providing a strategy for counteracting immunotherapeutic resistance in HCC.

Eliminating METTL1-mediated accumulation of PMN-MDSCs prevents hepatocellular carcinoma recurrence after radiofrequency ablation.

BACKGROUND AND AIMS: Radiofrequency ablation (RFA) is an important curative therapy in hepatocellular carcinoma (HCC), but recurrence rate remains as high as all the other HCC therapeutic modalities. Methyltransferase 1 (METTL1), an enzyme for m 7 G tRNA modification, was reported to promote HCC development. Here, we assessed the role of METTL1 in shaping the immunosuppressive tumor microenvironment after insufficient RFA (iRFA). APPROACH AND RESULTS: By immunohistochemistry and multiplex immunofluorescence (mIF) staining, we showed that METTL1 expression was enhanced in post-RFA recurrent HCC, accompanied by increased CD11b + CD15 + polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and decreased CD8 + T cells. Mechanistically, heat-mediated METTL1 upregulation enhanced TGF-ß2 translation to form the immunosuppressive environment by induction of myeloid-derived suppressor cell. Liver-specific overexpression or knockdown of Mettl1 significantly affected the accumulation of PMN-MDSCs and subsequently affected CD8 + T cell infiltration. Complete RFA successfully eliminated the tumor, whereas iRFA-treated mice exhibited enhanced tumor growth and metastasis with increased PMN-MDSC accumulation and decreased CD8 + T cells compared to sham surgery. Interrupting METTL1-TGF-ß2-PMN-MDSC axis by anti-Ly6G antibody, or knockdown of hepatoma-intrinsic Mettl1 or Tgfb2 , or TGF-ß signaling blockade significantly mitigated tumor progression induced by iRFA and restored CD8 + T cell population. CONCLUSIONS: Our study sheds light on the pivotal role of METTL1 in modulating an immunosuppressive microenvironment and demonstrated that interrupting METTL1-TGF-ß2-PMN-MDSC axis could be a therapeutic strategy to restore antitumor immunity and prevent HCC recurrence after RFA treatment, meriting further clinical studies.

Fibrotic immune microenvironment remodeling mediates superior anti-tumor efficacy of a nano-PD-L1 trap in hepatocellular carcinoma.

The local microenvironment where tumors develop can shape cancer progression and therapeutic outcome. Emerging evidence demonstrate that the efficacy of immune-checkpoint blockade (ICB) is undermined by fibrotic tumor microenvironment (TME). The majority of hepatocellular carcinoma (HCC) develops in liver fibrosis, in which the stromal and immune components may form a barricade against immunotherapy. Here, we report that nanodelivery of a programmed death-ligand 1 (PD-L1) trap gene exerts superior efficacy in treating fibrosis-associated HCC when compared with the conventional monoclonal antibody (mAb). In two fibrosis-associated HCC models induced by carbon tetrachloride and a high-fat, high-carbohydrate diet, the PD-L1 trap induced significantly larger tumor regression than mAb with no evidence of toxicity. Mechanistic studies revealed that PD-L1 trap, but not mAb, consistently reduced the M2 macrophage proportion in the fibrotic liver microenvironment and promoted cytotoxic interferon gamma (IFNγ)+tumor necrosis factor α (TNF-α)+CD8+T cell infiltration to the tumor. Moreover, PD-L1 trap treatment was associated with decreased tumor-infiltrating polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) accumulation, resulting in an inflamed TME with a high cytotoxic CD8+T cell/PMN-MDSC ratio conductive to anti-tumor immune response. Single-cell RNA sequencing analysis of two clinical cohorts demonstrated preferential PD-L1 expression in M2 macrophages in the fibrotic liver, thus supporting the translational potential of nano-PD-L1 trap for fibrotic HCC treatment.

Isoformic PD-1-mediated immunosuppression underlies resistance to PD-1 blockade in hepatocellular carcinoma patients.

OBJECTIVE: Immune checkpoint blockade (ICB) has improved cancer treatment, yet why most hepatocellular carcinoma (HCC) patients are resistant to PD-1 ICB remains elusive. Here, we elucidated the role of a programmed cell death protein 1 (PD-1) isoform, Δ42PD-1, in HCC progression and resistance to nivolumab ICB. DESIGN: We investigated 74 HCC patients in three cohorts, including 41 untreated, 28 treated with nivolumab and 5 treated with pembrolizumab. Peripheral blood mononuclear cells from blood samples and tumour infiltrating lymphocytes from tumour tissues were isolated for immunophenotyping. The functional significance of Δ42PD-1 was explored by single-cell RNA sequencing analysis and validated by functional and mechanistic studies. The immunotherapeutic efficacy of Δ42PD-1 monoclonal antibody was determined in HCC humanised mouse models. RESULTS: We found distinct T cell subsets, which did not express PD-1 but expressed its isoform Δ42PD-1, accounting for up to 71% of cytotoxic T lymphocytes in untreated HCC patients. Δ42PD-1+ T cells were tumour-infiltrating and correlated positively with HCC severity. Moreover, they were more exhausted than PD-1+ T cells by single T cell and functional analysis. HCC patients treated with anti-PD-1 ICB showed effective PD-1 blockade but increased frequencies of Δ42PD-1+ T cells over time especially in patients with progressive disease. Tumour-infiltrated Δ42PD-1+ T cells likely sustained HCC through toll-like receptors-4-signalling for tumourigenesis. Anti-Δ42PD-1 antibody, but not nivolumab, inhibited tumour growth in three murine HCC models. CONCLUSION: Our findings not only revealed a mechanism underlying resistance to PD-1 ICB but also identified anti-Δ42PD-1 antibody for HCC immunotherapy.

Accurate identification of structural variations from cancer samples.

Structural variations (SVs) are commonly found in cancer genomes. They can cause gene amplification, deletion and fusion, among other functional consequences. With an average read length of hundreds of kilobases, nano-channel-based optical DNA mapping is powerful in detecting large SVs. However, existing SV calling methods are not tailored for cancer samples, which have special properties such as mixed cell types and sub-clones. Here we propose the Cancer Optical Mapping for detecting Structural Variations (COMSV) method that is specifically designed for cancer samples. It shows high sensitivity and specificity in benchmark comparisons. Applying to cancer cell lines and patient samples, COMSV identifies hundreds of novel SVs per sample.

Development of Alkylated Hydrazides as Highly Potent and Selective Class I Histone Deacetylase Inhibitors with T cell Modulatory Properties.

Histone deacetylases (HDACs) are epigenetic regulators and additionally control the activity of non-histone substrates. We recently demonstrated that inhibition of HDAC8 overexpressed in various of cancers reduces hepatocellular carcinoma tumorigenicity in a T cell-dependent manner. Here, we present alkylated hydrazide-based class I HDAC inhibitors in which the n-hexyl side chain attached to the hydrazide moiety shows HDAC8 selectivity in vitro. Analysis of the mode of inhibition of the most promising compound 7d against HDAC8 revealed a substrate-competitive binding mode. 7d marked induced acetylation of the HDAC8 substrates H3K27 and SMC3 but not tubulin in CD4+ T lymphocytes, and significantly upregulated gene expressions for memory and effector functions. Furthermore, intraperitoneal injection of 7d (10 mg/kg) in C57BL/6 mice increased interleukin-2 expression in CD4+ T cells and CD8+ T cell proportion with no apparent toxicity. This study expands a novel chemotype of HDAC8 inhibitors with T cell modulatory properties for future therapeutic applications.

The mitotic regulator RCC2 promotes glucose metabolism through BACH1-dependent transcriptional upregulation of hexokinase II in glioma.

Weighted gene co-expression network analysis (WGCNA) identified a cell-cycle module that is associated with poor prognosis and aggressiveness of glioma. One of the core members, Regulator of chromatin condensation 2 (RCC2) is a component of the chromosome passenger complex. Accumulating evidence suggests that RCC2 plays a vital role in the mitotic process and that abnormal RCC2 expression is involved in cancer development. Gene silencing experiments show that RCC2 is required for glioma cell proliferation and migration. RNA-Sequencing analysis reveals a dual role of RCC2 in both the cell cycle and metabolism. Specifically, RCC2 regulates G2/M progression via CDC2 phosphorylation at Tyrosine 15. Metabolomic analysis identifies a role for RCC2 in promoting the glycolysis and pentose phosphate pathway. RCC2 exerts effects on metabolism by stabilizing the transcription factor BACH1 at its C-terminus leading to the transcriptional upregulation of hexokinase 2 (HK2). These findings elucidate a novel PTEN/RCC2/BACH1/HK2 signaling axis that drives glioma progression through the dual regulation of mitotic cell cycle and glycolytic events.

Single-Hit Inactivation Drove Tumor Suppressor Genes Out of the X Chromosome during Evolution.

Cancer-related genes are under intense evolutionary pressure. In this study, we conjecture that X-linked tumor suppressor genes (TSG) are not protected by the Knudson's two-hit mechanism and are therefore subject to negative selection. Accordingly, nearly all mammalian species exhibited lower TSG-to-noncancer gene ratios on their X chromosomes compared with nonmammalian species. Synteny analysis revealed that mammalian X-linked TSGs were depleted shortly after the emergence of the XY sex-determination system. A phylogeny-based model unveiled a higher X chromosome-to-autosome relocation flux for human TSGs. This was verified in other mammals by assessing the concordance/discordance of chromosomal locations of mammalian TSGs and their orthologs in Xenopus tropicalis. In humans, X-linked TSGs are younger or larger in size. Consistently, pan-cancer analysis revealed more frequent nonsynonymous somatic mutations of X-linked TSGs. These findings suggest that relocation of TSGs out of the X chromosome could confer a survival advantage by facilitating evasion of single-hit inactivation. SIGNIFICANCE: This work unveils extensive trafficking of TSGs from the X chromosome to autosomes during evolution, thus identifying X-linked TSGs as a genetic Achilles' heel in tumor suppression.

Vitamin D3 and carbamazepine protect against Clostridioides difficile infection in mice by restoring macrophage lysosome acidification.

Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea. TcdB is a major C. difficile exotoxin that activates macrophages to promote inflammation and epithelial damage. Lysosome impairment is a known trigger for inflammation. Herein, we hypothesize that TcdB could impair macrophage lysosomal function to mediate inflammation during CDI. Effects of TcdB on lysosomal function and the downstream pro-inflammatory SQSTM1/p62-NFKB (nuclear factor kappa B) signaling were assessed in cultured macrophages and in a murine CDI model. Protective effects of two lysosome activators (i.e., vitamin D3 and carbamazepine) were assessed. Results showed that TcdB inhibited CTNNB1/ß-catenin activity to downregulate MITF (melanocyte inducing transcription factor) and its direct target genes encoding components of lysosomal membrane vacuolar-type ATPase, thereby suppressing lysosome acidification in macrophages. The resulting lysosomal dysfunction then impaired autophagic flux and activated SQSTM1-NFKB signaling to drive the expression of IL1B/IL-1ß (interleukin 1 beta), IL8 and CXCL2 (chemokine (C-X-C motif) ligand 2). Restoring MITF function by enforced MITF expression or restoring lysosome acidification with 1α,25-dihydroxyvitamin D3 or carbamazepine suppressed pro-inflammatory cytokine expression in vitro. In mice, gavage with TcdB-hyperproducing C. difficile or injection of TcdB into ligated colon segments caused prominent MITF downregulation in macrophages. Vitamin D3 and carbamazepine lessened TcdB-induced lysosomal dysfunction, inflammation and histological damage. In conclusion, TcdB inhibits the CTNNB1-MITF axis to suppress lysosome acidification and activates the downstream SQSTM1-NFKB signaling in macrophages during CDI. Vitamin D3 and carbamazepine protect against CDI by restoring MITF expression and lysosomal function in mice.Abbreviations: ATP6V0B: ATPase H+ transporting V0 subunit b; ATP6V0C: ATPase H+ transporting V0 subunit c; ATP6V0E1: ATPase H+ transporting V0 subunit e1; ATP6V1H: ATPase H+ transporting V1 subunit H; CBZ: carbamazepine; CDI: C. difficile infection; CXCL: chemokine C-X-X motif ligand; IL: interleukin; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; LEF: lymphoid enhancer binding factor 1; MITF: melanocyte inducing transcription factor; NFKB: nuclear factor kappa B; PMA: phorbol 12-myristate 13-acetate; TcdA: Clostridial toxin A; TcdB: Clostridial toxin B; TFE3: transcription factor E3; TFEB: transcription factor EB.

Smad3 Promotes Cancer-Associated Fibroblasts Generation via Macrophage-Myofibroblast Transition.

Cancer-associated fibroblasts (CAFs) are important in tumor microenvironment (TME) driven cancer progression. However, CAFs are heterogeneous and still largely underdefined, better understanding their origins will identify new therapeutic strategies for cancer. Here, the authors discovered a new role of macrophage-myofibroblast transition (MMT) in cancer for de novo generating protumoral CAFs by resolving the transcriptome dynamics of tumor-associated macrophages (TAM) with single-cell resolution. MMT cells (MMTs) are observed in non-small-cell lung carcinoma (NSCLC) associated with CAF abundance and patient mortality. By fate-mapping study, RNA velocity, and pseudotime analysis, existence of novel macrophage-lineage-derived CAF subset in the TME of Lewis lung carcinoma (LLC) model is confirmed, which is directly transited via MMT from M2-TAM in vivo and bone-marrow-derived macrophages (BMDM) in vitro. Adoptive transfer of BMDM-derived MMTs markedly promote CAF formation in LLC-bearing mice. Mechanistically, a Smad3-centric regulatory network is upregulated in the MMTs of NSCLC, where chromatin immunoprecipitation sequencing(ChIP-seq) detects a significant enrichment of Smad3 binding on fibroblast differentiation genes in the macrophage-lineage cells in LLC-tumor. More importantly, macrophage-specific deletion and pharmaceutical inhibition of Smad3 effectively block MMT, therefore, suppressing the CAF formation and cancer progression in vivo. Thus, MMT may represent a novel therapeutic target of CAF for cancer immunotherapy.

A selective HDAC8 inhibitor potentiates antitumor immunity and efficacy of immune checkpoint blockade in hepatocellular carcinoma.

Insufficient T cell infiltration into noninflamed tumors, such as hepatocellular carcinoma (HCC), restricts the effectiveness of immune-checkpoint blockade (ICB) for a subset of patients. Epigenetic therapy provides further opportunities to rewire cancer-associated transcriptional programs, but whether and how selective epigenetic inhibition counteracts the immune-excluded phenotype remain incompletely defined. Here, we showed that pharmacological inhibition of histone deacetylase 8 (HDAC8), a histone H3 lysine 27 (H3K27)-specific isozyme overexpressed in a variety of human cancers, thwarts HCC tumorigenicity in a T cell-dependent manner. The tumor-suppressive effect of selective HDAC8 inhibition was abrogated by CD8+ T cell depletion or regulatory T cell adoptive transfer. Chromatin profiling of human HDAC8-expressing HCCs revealed genome-wide H3K27 deacetylation in 1251 silenced enhancer-target gene pairs that are enriched in metabolic and immune regulators. Mechanistically, down-regulation of HDAC8 increased global and enhancer acetylation of H3K27 to reactivate production of T cell-trafficking chemokines by HCC cells, thus relieving T cell exclusion in both immunodeficient and humanized mouse models. In an HCC preclinical model, selective HDAC8 inhibition increased tumor-infiltrating CD8+ T cells and potentiated eradication of established hepatomas by anti-PD-L1 therapy without evidence of toxicity. Mice treated with HDAC8 and PD-L1 coblockade were protected against subsequent tumor rechallenge as a result of the induction of memory T cells and remained tumor-free for greater than 15 months. Collectively, our study demonstrates that selective HDAC8 inhibition elicits effective and durable responses to ICB by co-opting adaptive immunity through enhancer reprogramming.

A proof-of-concept study for the pathogenetic role of enhancer hypomethylation of MYBPHL in multiple myeloma.

Enhancer DNA methylation and expression of MYBPHL was studied in multiple myeloma (MM). By bisulfite genomic sequencing, among the three CpGs inside the MYBPHL enhancer, CpG1 was significantly hypomethylated in MM cell lines (6.7-50.0%) than normal plasma cells (37.5-75.0%) (P = 0.007), which was negatively correlated with qPCR-measured MYBPHL expression. In RPMI-8226 and WL-2 cells, bearing the highest CpG1 methylation, 5-azadC caused enhancer demethylation and expression of MYBPHL. In primary samples, higher CpG1 methylation was associated with lower MYBPHL expression. By luciferase assay, luciferase activity was enhanced by MYBPHL enhancer compared with empty vector control, but reduced by site-directed mutagenesis of each CpG. RNA-seq data of newly diagnosed MM patients showed that MYBPHL expression was associated with t(11;14). MOLP-8 cells carrying t(11;14) express the highest levels of MYBPHL, and its knockdown reduced cellular proliferation and increased cell death. Herein, as a proof-of-concept, our data demonstrated that the MYBPHL enhancer, particularly CpG1, was hypomethylated and associated with increased MYBPHL expression in MM, which was implicated in myelomagenesis.

Chemotherapy-induced recruitment of myeloid-derived suppressor cells abrogates efficacy of immune checkpoint blockade.

BACKGROUND & AIMS: Immune checkpoint blockade (ICB) has been approved for treatment of hepatocellular carcinoma (HCC). However, many patients with advanced HCC are non-responders to ICB monotherapy. Cytotoxic chemotherapy has been proposed to modulate the tumor microenvironment (TME) and sensitize tumors to ICB. Thus, we aimed to study the combination of cytotoxic chemotherapy and ICB in an orthotopic HCC model. METHODS: Preclinical orthotopic HCC mouse models were used to elucidate the efficacy of 5-fluorouracil (5-FU) and ICB. The mice were intrahepatically injected with RIL-175 or Hepa1-6 cells, followed by treatment with 5-FU and anti-programmed cell death ligand 1 (PD-L1) antibody. Myeloid-derived suppressor cells (MDSCs) were depleted to validate their role in attenuating sensitivity to immunotherapy. Flow cytometry-based immune profiling and immunofluorescence staining were performed in mice and patient samples, respectively. RESULTS: 5-FU could induce intratumoral MDSC accumulation to counteract the infiltration of T lymphocytes and natural killer cells, thus abrogating the anti-tumor efficacy of PD-L1 blockade. In clinical samples, MDSCs accumulated and CD8+ T cell numbers decreased following transarterial chemoembolization. CONCLUSION: 5-FU can trigger the accumulation of immunosuppressive MDSCs, impairing the response to PD-L1 blockade in HCC. Our data suggest that the combination of specific chemotherapy and ICB may impair anti-tumor immune responses, warranting further study in preclinical models and consideration in clinical settings. LAY SUMMARY: Our findings suggest that some chemotherapies may impair the anti-tumor efficacy of immunotherapy. Further studies are required to uncover the specific effects of different chemotherapies on the immunological profile of tumors. This data will be critical for the rational design of combination immunotherapy strategies for patients with hepatocellular carcinoma.

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis.

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.

Autism-associated PTEN missense mutation leads to enhanced nuclear localization and neurite outgrowth in an induced pluripotent stem cell line.

Germline mutation in the PTEN gene is the genetic basis of PTEN hamartoma tumor syndrome with the affected individuals harboring features of autism spectrum disorders. Characterizing a panel of 14 autism-associated PTEN missense mutations revealed reduced protein stability, catalytic activity, and subcellular distribution. Nine out of 14 (64%) PTEN missense mutants had reduced protein expression with most mutations confined to the C2 domain. Selected mutants displayed enhanced polyubiquitination and shortened protein half-life, but that did not appear to involve the polyubiquitination sites at lysine residues at codon 13 or 289. Analyzing their intrinsic lipid phosphatase activities revealed that 78% (11 out of 14) of these mutants had twofold to 10-fold reduction in catalytic activity toward phosphatidylinositol phosphate substrates. Analyzing the subcellular localization of the PTEN missense mutants showed that 64% (nine out of 14) had altered nuclear-to-cytosol ratios with four mutants (G44D, H123Q, E157G, and D326N) showing greater nuclear localization. The E157G mutant was knocked-in to an induced pluripotent stem cell line and recapitulated a similar nuclear targeting preference. Furthermore, iPSCs expressing the E157G mutant were more proliferative at the neural progenitor cell stage but exhibited more extensive dendritic outgrowth. In summary, the combination of biological changes in PTEN is expected to contribute to the behavioral and cellular features of this neurodevelopmental disorder.

Targeting monocyte-intrinsic enhancer reprogramming improves immunotherapy efficacy in hepatocellular carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy. DESIGN: Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined. RESULTS: Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model. CONCLUSION: Our results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy.

Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis.

BACKGROUND: Growth arrest-specific gene 2 (GAS2) plays a role in modulating in reversible growth arrest cell cycle, apoptosis, and cell survival. GAS2 protein is universally expressed in most normal tissues, particularly in the liver, but is depleted in some tumor tissues. However, the functional mechanisms of GAS2 in hepatocellular carcinoma (HCC) are not fully defined. AIM: To investigate the function and mechanism of GAS2 in HCC. METHODS: GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting. Cell proliferation was analyzed by counting, MTS, and colony formation assays. Cell cycle analysis was performed by flow cytometry. Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting. RESULTS: GAS2 protein expression was lower in HCC than in normal tissues. Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53, while knockdown of GAS2 promoted the proliferation of hepatocytes (P < 0.05). Furthermore, GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells, particularly the elevation of sub G1 (P < 0.01). Apoptosis induced by GAS2 was dependent on p53, which was increased by etoposide addition. The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated, but became diminished upon downregulation of GAS2. In the clinic specimen, GAS2 was downregulated in more than 60% of HCCs. The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues (P < 0.05). CONCLUSION: GAS2 plays a vital role in HCC cell proliferation and apoptosis, possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

Metabolic enzyme PDK3 forms a positive feedback loop with transcription factor HSF1 to drive chemoresistance.

Background & Aims: Dysregulation of metabolism plays an important role in the development and progression of cancers, while the underlying mechanisms remain largely unknown. This study aims to explore the regulation and relevance of glycolysis in chemoresistance of gastric cancer. Methods: Biochemical differences between chemoresistant and chemosensitive cancer cells were determined by metabolism profiling, microarray gene expression, PCR or western blotting. Cancer cell growth in vitro or in vivo were analyzed by viability, apoptosis and nude mice assay. Immunoprecipation was used to explore the interaction of proteins with other proteins or DNAs. Results: By metabolic and gene expression profiling, we found that pyruvate dehydrogenase kinase 3 (PDK3) was highly expressed to promote glycolysis in chemoresistant cancer cells. Its genetic or chemical inhibition reverted chemoresistance in vitro and in vivo. It was transcriptionally regulated by transcription factor HSF1 (Heat shock factor 1). Interestingly, PDK3 can localize in the nucleus and interact with HSF1 to disrupt its phosphorylation by GSK3ß. Since HSF1 was subjected to FBXW7-catalyzed polyubiquitination in a phosphorylation-dependent manner, PDK3 prevented HSF1 from proteasomal degradation. Thus, metabolic enzyme PDK3 and transcription factor HSF1 forms a positive feedback loop to promote glycolysis. As a result, inhibition of HSF1 impaired enhanced glycolysis and reverted chemoresistance both in vitro and in vivo. Conclusions: PDK3 forms a positive feedback loop with HSF1 to drive glycolysis in chemoresistance. Targeting this mitonuclear communication may represent a novel approach to overcome chemoresistance.