The Wdr5-H3K4me3 Epigenetic Axis Regulates Pancreatic Tumor Immunogenicity and Immune Suppression.

The WDR5/MLL1-H3K4me3 epigenetic axis is often activated in both tumor cells and tumor-infiltrating immune cells to drive various cellular responses in the tumor microenvironment and has been extensively studied in hematopoietic cancer, but its respective functions in tumor cells and immune cells in the context of tumor growth regulation of solid tumor is still incompletely understood. We report here that WDR5 exhibits a higher expression level in human pancreatic tumor tissues compared with adjacent normal pancreas. Moreover, WDR5 expression is negatively correlated with patients' response to chemotherapy or immunotherapy in human colon cancer and melanoma. However, WDR5 expression is positively correlated with the HLA level in human cancer cells, and H3K4me3 enrichment is observed at the promoter region of the HLA-A, HLA-B, and HLA-C genes in pancreatic cancer cells. Using mouse tumor cell lines and in vivo tumor models, we determined that WDR5 deficiency or inhibition significantly represses MHC I expression in vitro and in vivo in pancreatic tumor cells. Mechanistically, we determine that WDR5 deficiency inhibits H3K4me3 deposition at the MHC I (H2K) promoter region to repress MHC I (H2K) transcription. On the other hand, WDR5 depletion leads to the effective downregulation of immune checkpoints and immunosuppressive cytokines, including TGFß and IL6, in the pancreatic tumor microenvironments. Our data determine that WDR5 not only regulates tumor cell immunogenicity to suppress tumor growth but also activates immune suppressive pathways to promote tumor immune evasion. Selective activation of the WDR5-MHC I pathway and/or selective inhibition of the WDR5-immune checkpoint and WDR5-cytokine pathways should be considered in WDR5-based epigenetic cancer immunotherapy.

Asah2 Represses the p53-Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis.

Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that massively accumulate under pathological conditions to suppress T cell immune response. Dysregulated cell death contributes to MDSC accumulation, but the molecular mechanism underlying this cell death dysregulation is not fully understood. In this study, we report that neutral ceramidase (N-acylsphingosine amidohydrolase [ASAH2]) is highly expressed in tumor-infiltrating MDSCs in colon carcinoma and acts as an MDSC survival factor. To target ASAH2, we performed molecular docking based on human ASAH2 protein structure. Enzymatic inhibition analysis of identified hits determined NC06 as an ASAH2 inhibitor. Chemical and nuclear magnetic resonance analysis determined NC06 as 7-chloro-2-(3-chloroanilino)pyrano[3,4-e][1,3]oxazine-4,5-dione. NC06 inhibits ceramidase activity with an IC50 of 10.16-25.91 µM for human ASAH2 and 18.6-30.2 µM for mouse Asah2 proteins. NC06 induces MDSC death in a dose-dependent manner, and inhibition of ferroptosis decreased NC06-induced MDSC death. NC06 increases glutathione synthesis and decreases lipid reactive oxygen species to suppress ferroptosis in MDSCs. Gene expression profiling identified the p53 pathway as the Asah2 target in MDSCs. Inhibition of Asah2 increased p53 protein stability to upregulate Hmox1 expression to suppress lipid reactive oxygen species production to suppress ferroptosis in MDSCs. NC06 therapy increases MDSC death and reduces MDSC accumulation in tumor-bearing mice, resulting in increased activation of tumor-infiltrating CTLs and suppression of tumor growth in vivo. Our data indicate that ASAH2 protects MDSCs from ferroptosis through destabilizing p53 protein to suppress the p53 pathway in MDSCs in the tumor microenvironment. Targeting ASAH2 with NC06 to induce MDSC ferroptosis is potentially an effective therapy to suppress MDSC accumulation in cancer immunotherapy.

MS4A1 expression and function in T cells in the colorectal cancer tumor microenvironment.

The majority of human colorectal cancer remains resistant to immune checkpoint inhibitor (ICI) immunotherapy, but the underlying mechanism is incompletely understood. We report here that MS4A1, the gene encoding B cell surface marker CD20, is significantly downregulated in human colorectal carcinoma. Furthermore, MS4A1 expression level in colorectal carcinoma is positively correlated with patient survival. Analysis of scRNA-Seq dataset from public database revealed that MS4A1 is also expressed in subsets of T cells. A CD8+CD20+ subset of T cells exists in the neighboring non-neoplastic colon but disappears in tumor in human colorectal carcinoma. Furthermore, analysis of a published nivolumab treatment dataset indicated that nivolumab-bound T cells from human patients during anti-PD-1 immunotherapy exhibit significantly higher MS4A1 expression. Our findings indicate that CD8+CD20+ T subset functions in host cancer immunosurveillance and tumor microenvironment suppresses this T subset through a PD-L1-dependent mechanism.

Expression profiles and function of IL6 in polymorphonuclear myeloid-derived suppressor cells.

IL6 is an inflammatory cytokine with pleiotropic functions in both immune and nonimmune cells, and its expression level is inversely correlated with disease prognosis in patients with cancer. However, blocking IL6 alone has only yielded minimal efficacy in human cancer patients. We aimed at defining IL6 expression profiles under inflammatory conditions and cancer, and elucidating the mechanism underlying IL6 intrinsic signaling in colon carcinoma. We report here that colonic inflammation induces IL6 expression primarily in the CD11b+Ly6G+Ly6Clo polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in colon. Although both tumor cells, T cells and myeloid cells all express IL6, PMN-MDSCs are the primary cell type that express IL6 in colon carcinoma, suggesting that IL6 up-regulation is a response to inflammation in colon epithelium and tumor microenvironment. Furthermore, we determined that IL6 activates STAT3 to up-regulate DNMT1 and DNMT3b expression in colon tumor cells, thereby revealing an epigenetic mechanism that mediates the IL6-STAT3 signaling pathway in colon carcinoma. Surprisingly, knocking out IL6 in colon tumor cells did not significantly alter tumor growth in WT mice. Conversely, IL6-sufficient colon and pancreatic tumor grow at similar rate in WT and IL6-deficient mice. However, overexpression of IL6 in colon tumor cells significantly increases tumor growth in vivo. Our findings determine that a high tumor local IL6 threshold is essential for IL6 function in colon tumor promotion and targeting the IL6-expressing PMN-MDSCs is potentially an effective approach to suppress colon tumor growth in vivo.

IFNAR1 Controls Autocrine Type I IFN Regulation of PD-L1 Expression in Myeloid-Derived Suppressor Cells.

Tumor cells respond to IFN-γ of activated T cells to upregulate programmed death-ligand 1 (PD-L1) in the tumor microenvironment as an adaptive immune resistance mechanism. Tumor cells also express oncogene-driven PD-L1. PD-L1 is also expressed on myeloid-derived suppressor cells (MDSCs). It is known that both type I and II IFNs upregulate PD-L1 expression in MDSCs. However, the molecular mechanism underlying PD-L1 expression in MDSCs is still largely unknown. We report in this article that MDSCs exhibit constitutive STAT1 phosphorylation in vitro without exogenous IFNs, indicating a constitutive active JAK-STAT signaling pathway in mouse MDSCs in vitro. Furthermore, IFN-α and IFN-ß but not IFN-γ are endogenously expressed in the MDSC cell line in vitro and in tumor-induced MDSCs in vivo. Neutralizing type I IFN or inhibiting the JAK-STAT signaling pathway significantly decreased constitutive PD-L1 expression in MDSCs in vitro. However, neither IFN-α expression level nor IFN-ß expression level is correlated with PD-L1 expression level in MDSCs; instead, the level of IFN receptor type I (IFNAR1) is correlated with PD-L1 expression levels in MDSCs. Consequently, knocking out IFNAR1 in mice diminished PD-L1 expression in tumor-induced MDSCs. Therefore, we determined that 1) PD-L1 expression in MDSCs is activated by type I IFN through an autocrine manner and 2) the expression level of PD-L1 is controlled at least in part by the IFNAR1 level on MDSCs. Our data indicate that MDSCs may maintain their PD-L1 expression via autocrine type I IFN to exert their suppressive activity in the absence of IFN-γ from the suppressed T cells in the tumor microenvironment.

Contrasting roles of H3K4me3 and H3K9me3 in regulation of apoptosis and gemcitabine resistance in human pancreatic cancer cells.

BACKGROUND: Pancreas ductal adenocarcinoma (PDAC) has the most dismal prognosis among all human cancers since it is highly resistant to chemotherapy, radiotherapy and immunotherapy. The anticipated consequence of all therapies is induction of tumor apoptosis. The highly resistance nature of PDACs to all therapies suggests that the intrinsic tumor cell factors, likely the deregulated apoptosis pathway, are key mechanisms underlying PDAC non-response to these therapies, rather than the therapeutic agents themselves. The aim of this study is to test the hypothesis that epigenetic dysregulation of apoptosis mediators underlies PDAC resistance to gemcitabine, the standard chemotherapy for human PDAC. METHODS: PDAC cells were analyzed for apoptosis sensitivity in the presence of a selective epigenetic inhibitor. The epigenetic regulation of apoptosis regulators was determined by Western Blotting and quantitative PCR. The specific epigenetic modification of apoptosis regulator promoter chromatin was determined by chromatin immunoprecipitation in PDAC cells. RESULTS: Inhibition of histone methyltransferase (HMTase) by a selective HMTase inhibitor, verticillin A, significantly increased human PDAC cell sensitivity to gemcitabine-induced growth suppression. Verticillin A treatment decreased FLIP, Mcl-1, Bcl-x and increased Bak, Bax and Bim protein level in the tumor cells, resulting in activation of caspases, elevated cytochrome C release and increased apoptosis as determined by upregulated PARP cleavage in tumor cells. Analysis of human PDAC specimens indicated that the expression levels of anti-apoptotic mediators Bcl-x, Mcl-1, and FLIP were significantly higher, whereas the expression levels of pro-apoptotic mediators Bim, Bak and Bax were dramatically lower in human PDAC tissues as compared to normal pancreas. Verticillin A downregulated H3K4me3 levels at the BCL2L1, CFLAR and MCL-1 promoter to decrease Bcl-x, FLIP and Mcl-1 expression level, and inhibited H3K9me3 levels at the BAK1, BAX and BCL2L11 promoter to upregulate Bak, Bax and Bim expression level. CONCLUSION: We determined that PDAC cells use H3K4me3 to activate Bcl-x, FLIP and Mcl-1, and H3K9me3 to silence Bak, Bax and Bim to acquire an apoptosis-resistant phenotype. Therefore, selective inhibition of H3K4me3 and H3K9me3 is potentially an effective approach to overcome PDAC cells resistance to gemcitabine.

UFBP1, a Key Component of the Ufm1 Conjugation System, Is Essential for Ufmylation-Mediated Regulation of Erythroid Development.

The Ufm1 conjugation system is an ubiquitin-like modification system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. The biological importance of this system is highlighted by its essential role in embryogenesis and erythroid development, but the underlying mechanism is poorly understood. UFBP1 (Ufm1 binding protein 1, also known as DDRGK1, Dashurin and C20orf116) is a putative Ufm1 target, yet its exact physiological function and impact of its ufmylation remain largely undefined. In this study, we report that UFBP1 is indispensable for embryonic development and hematopoiesis. While germ-line deletion of UFBP1 caused defective erythroid development and embryonic lethality, somatic ablation of UFBP1 impaired adult hematopoiesis, resulting in pancytopenia and animal death. At the cellular level, UFBP1 deficiency led to elevated ER (endoplasmic reticulum) stress and activation of unfolded protein response (UPR), and consequently cell death of hematopoietic stem/progenitor cells. In addition, loss of UFBP1 suppressed expression of erythroid transcription factors GATA-1 and KLF1 and blocked erythroid differentiation from CFU-Es (colony forming unit-erythroid) to proerythroblasts. Interestingly, depletion of Uba5, a Ufm1 E1 enzyme, also caused elevation of ER stress and under-expression of erythroid transcription factors in erythroleukemia K562 cells. By contrast, knockdown of ASC1, a newly identified Ufm1 target that functions as a transcriptional co-activator of hormone receptors, led to down-regulation of erythroid transcription factors, but did not elevate basal ER stress. Furthermore, we found that ASC1 was associated with the promoters of GATA-1 and Klf1 in a UFBP1-dependent manner. Taken together, our findings suggest that UFBP1, along with ASC1 and other ufmylation components, play pleiotropic roles in regulation of hematopoietic cell survival and differentiation via modulating ER homeostasis and erythroid lineage-specific gene expression. Modulating the activity of this novel ubiquitin-like system may represent a novel approach to treat blood-related diseases such as anemia.

H3K9 Trimethylation Silences Fas Expression To Confer Colon Carcinoma Immune Escape and 5-Fluorouracil Chemoresistance.

The Fas-FasL effector mechanism plays a key role in cancer immune surveillance by host T cells, but metastatic human colon carcinoma often uses silencing Fas expression as a mechanism of immune evasion. The molecular mechanism under FAS transcriptional silencing in human colon carcinoma is unknown. We performed genome-wide chromatin immunoprecipitation sequencing analysis and identified that the FAS promoter is enriched with H3K9me3 in metastatic human colon carcinoma cells. The H3K9me3 level in the FAS promoter region is significantly higher in metastatic than in primary cancer cells, and it is inversely correlated with Fas expression level. We discovered that verticillin A is a selective inhibitor of histone methyltransferases SUV39H1, SUV39H2, and G9a/GLP that exhibit redundant functions in H3K9 trimethylation and FAS transcriptional silencing. Genome-wide gene expression analysis identified FAS as one of the verticillin A target genes. Verticillin A treatment decreased H3K9me3 levels in the FAS promoter and restored Fas expression. Furthermore, verticillin A exhibited greater efficacy than decitabine and vorinostat in overcoming colon carcinoma resistance to FasL-induced apoptosis. Verticillin A also increased DR5 expression and overcame colon carcinoma resistance to DR5 agonist drozitumab-induced apoptosis. Interestingly, verticillin A overcame metastatic colon carcinoma resistance to 5-fluorouracil in vitro and in vivo. Using an orthotopic colon cancer mouse model, we demonstrated that tumor-infiltrating cytotoxic T lymphocytes are FasL(+) and that FasL-mediated cancer immune surveillance is essential for colon carcinoma growth control in vivo. Our findings determine that H3K9me3 of the FAS promoter is a dominant mechanism underlying FAS silencing and resultant colon carcinoma immune evasion and progression.

Targeting translation: eIF4E as an emerging anticancer drug target.

The translation initiation factor eIF4E mediates a rate-limiting process that drives selective translation of many oncongenic proteins such as cyclin D1, survivin and VEGF, thereby contributing to tumour growth, metastasis and therapy resistance. As an essential regulatory hub in cancer signalling network, many oncogenic signalling pathways appear to converge on eIF4E. Therefore, targeting eIF4E-mediated cap-dependent translation is considered a promising anticancer strategy. This paper reviews the strategies that can be used to target eIF4E, highlighting agents that target eIF4E activity at each distinct level.

The activating transcription factor 3 protein suppresses the oncogenic function of mutant p53 proteins.

Mutant p53 proteins (mutp53) often acquire oncogenic activities, conferring drug resistance and/or promoting cancer cell migration and invasion. Although it has been well established that such a gain of function is mainly achieved through interaction with transcriptional regulators, thereby modulating cancer-associated gene expression, how the mutp53 function is regulated remains elusive. Here we report that activating transcription factor 3 (ATF3) bound common mutp53 (e.g. R175H and R273H) and, subsequently, suppressed their oncogenic activities. ATF3 repressed mutp53-induced NFKB2 expression and sensitized R175H-expressing cancer cells to cisplatin and etoposide treatments. Moreover, ATF3 appeared to suppress R175H- and R273H-mediated cancer cell migration and invasion as a consequence of preventing the transcription factor p63 from inactivation by mutp53. Accordingly, ATF3 promoted the expression of the metastasis suppressor SHARP1 in mutp53-expressing cells. An ATF3 mutant devoid of the mutp53-binding domain failed to disrupt the mutp53-p63 binding and, thus, lost the activity to suppress mutp53-mediated migration, suggesting that ATF3 binds to mutp53 to suppress its oncogenic function. In line with these results, we found that down-regulation of ATF3 expression correlated with lymph node metastasis in TP53-mutated human lung cancer. We conclude that ATF3 can suppress mutp53 oncogenic function, thereby contributing to tumor suppression in TP53-mutated cancer.

The NF-κB p65 and p50 homodimer cooperate with IRF8 to activate iNOS transcription.

BACKGROUND: Inducible nitric oxide synthase (iNOS) metabolizes L-arginine to produce nitric oxide (NO) which was originally identified in myeloid cells as a host defense mechanism against pathogens. Recent studies, however, have revealed that iNOS is often induced in tumor cells and myeloid cells in the tumor microenvironment. Compelling experimental data have shown that iNOS promotes tumor development in certain cellular context and suppresses tumor development in other cellular conditions. The molecular mechanisms underlying these contrasting functions of iNOS is unknown. Because iNOS is often induced by inflammatory signals, it is therefore likely that these contrasting functions of iNOS could be controlled by the inflammatory signaling pathways, which remains to be determined. METHODS: iNOS is expressed in colon carcinoma and myeloid cells in the tumor microenvironment. Colon carcinoma and myeloid cell lines were used to elucidate the molecular mechanisms underlying iNOS expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay were used to determine the IFNγ-activated pSTAT1 and NF-κB association with the chromatin DNA of the nos2 promoter. RESULTS: We show here that iNOS is dramatically up-regulated in inflammed human colon tissues and in human colon carcinoma as compared to normal colon tissue. iNOS is expressed in either the colon carcinoma cells or immune cells within the tumor microenvironment. On the molecular level, the proinflammatory IFNγ and NF-κB signals induce iNOS expression in human colon cancer cells. We further demonstrate that NF-κB directly binds to the NOS2 promoter to regulate iNOS expression. Although neither the IFNγ signaling pathway nor the NF-κB signaling pathway alone is sufficient to induce iNOS expression in myeloid cells, IFNγ and NF-κB synergistically induce iNOS expression in myeloid cells. Furthermore, we determine that IFNγ up-regulates IRF8 expression to augment NF-κB induction of iNOS expression. More interestingly, we observed that the p65/p65 and p50/p50 homodimers, not the canonical p65/p50 heterodimer, directly binds to the nos2 promoter to regulate iNOS expression in myeloid cells. CONCLUSIONS: IFNγ-induced IRF8 acts in concert with NF-κB to regulate iNOS expression in both colon carcinoma and myeloid cells. In myeloid cells, the NF-κB complexes that bind to the nos2 promoter are p65/p65 and p50/p50 homodimers.

Type I Interferon Activates PD-1 Expression through Activation of the STAT1-IRF2 Pathway in Myeloid Cells.

PD-1 (Programmed cell death protein 1) regulates the metabolic reprogramming of myeloid-derived suppressor cells and myeloid cell differentiation, as well as the type I interferon (IFN-I) signaling pathway in myeloid cells in the tumor microenvironment. PD-1, therefore, is a key inhibitory receptor in myeloid cells. However, the regulation of PD-1 expression in myeloid cells is unknown. We report that the expression level of PDCD1, the gene that encodes the PD-1 protein, is positively correlated with the levels of IFNB1 and IFNAR1 in myeloid cells in human colorectal cancer. Treatment of mouse myeloid cell lines with recombinant IFNß protein elevated PD-1 expression in myeloid cells in vitro. Knocking out IFNAR1, the gene that encodes the IFN-I-specific receptor, diminished the inductive effect of IFNß on PD-1 expression in myeloid cells in vitro. Treatment of tumor-bearing mice with a lipid nanoparticle-encapsulated IFNß-encoding plasmid (IFNBCOL01) increased IFNß expression, resulting in elevated PD-1 expression in tumor-infiltrating myeloid cells. At the molecular level, we determined that IFNß activates STAT1 (signal transducer and activator of transcription 1) and IRFs (interferon regulatory factors) in myeloid cells. Analysis of the cd279 promoter identified IRF2-binding consensus sequence elements. ChIP (chromatin immunoprecipitation) analysis determined that the pSTAT1 directly binds to the irf2 promoter and that IRF2 directly binds to the cd279 promoter in myeloid cells in vitro and in vivo. In colon cancer patients, the expression levels of STAT1, IRF2 and PDCD1 are positively correlated in tumor-infiltrating myeloid cells. Our findings determine that IFNß activates PD-1 expression at least in part by an autocrine mechanism via the stimulation of the pSTAT1-IRF2 axis in myeloid cells.

Delivery of Interferon ß-Encoding Plasmid via Lipid Nanoparticle Restores Interferon ß Expression to Enhance Antitumor Immunity in Colon Cancer.

Type I interferon (IFN-I) plays a critical role in host cancer immunosurveillance, but its expression is often impaired in the tumor microenvironment. We aimed at testing the hypothesis that cationic lipid nanoparticle delivery of interferon ß (IFNß)-encoding plasmid to tumors is effective in restoring IFNß expression to suppress tumor immune evasion. We determined that IFN-I function in tumor suppression depends on the host immune cells. IFN-I activates the expression of Cxcl9 and Cxcl10 to enhance T cell tumor infiltration. RNA-Seq detected a low level of IFNα13 and IFNß in colon tumor tissue. scRNA-Seq revealed that IFNß is expressed in immune cell subsets in non-neoplastic human tissues and to a lesser degree in human colon tumor tissues. Forced expression of IFNα13 and IFNß in colon tumor cells up-regulates major histocompatibility complex I (MHC I) expression and suppresses colon tumor growth in vivo. In human cancer patients, IFNß expression is positively correlated with human leukocyte antigen (HLA) expression, and IFN-I signaling activation correlates with the patient response to PD-1 blockade immunotherapy. To translate this finding to colon cancer immunotherapy, we formulated a 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-cholesterol-encapsulated IFNß-encoding plasmid (IFNBCOL01). IFNBCOL01 transfects colon tumor cells to express IFNß to increase the level of MHC I expression. IFNBCOL01 therapy transfects tumor cells and tumor-infiltrating immune cells to produce IFNß to activate MHC I and granzyme B expression and inhibits colon tumor growth in mice. Our data determine that lipid nanoparticle delivery of IFNß-encoding plasmid DNA enhances tumor immunogenicity and T cell effector function to suppress colon tumor growth in vivo.

5-Fluorouracil Suppresses Colon Tumor through Activating the p53-Fas Pathway to Sensitize Myeloid-Derived Suppressor Cells to FasL+ Cytotoxic T Lymphocyte Cytotoxicity.

Myelosuppression is a major adverse effect of 5-fluorouracil (5-FU) chemotherapy. However, recent findings indicate that 5-FU selectively suppresses myeloid-derived suppressor cells (MDSCs), to enhance antitumor immunity in tumor-bearing mice. 5-FU-mediated myelosuppression may thus have a beneficial effect for cancer patients. The molecular mechanism underlying 5-FU's suppression of MDSCs is currently unknown. We aimed at testing the hypothesis that 5-FU suppresses MDSCs through enhancing MDSC sensitivity to Fas-mediated apoptosis. We observed that, although FasL is highly expressed in T cells, Fas is weakly expressed in myeloid cells in human colon carcinoma, indicating that downregulation of Fas is a mechanism underlying myeloid cell survival and accumulation in human colon cancer. 5-FU treatment upregulated expression of both p53 and Fas, and knocking down p53 diminished 5-FU-induced Fas expression in MDSC-like cells, in vitro. 5-FU treatment also increased MDSC-like cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, we determined that 5-FU therapy increased expression of Fas on MDSCs, suppressed MDSC accumulation, and increased CTL tumor infiltration in colon tumor-bearing mice. In human colorectal cancer patients, 5-FU chemotherapy decreased MDSC accumulation and increased CTL level. Our findings determine that 5-FU chemotherapy activates the p53-Fas pathway, to suppress MDSC accumulation, to increase CTL tumor infiltration.

Tumor PD-L1 engages myeloid PD-1 to suppress type I interferon to impair cytotoxic T lymphocyte recruitment.

The cellular and molecular mechanisms underlying tumor cell PD-L1 (tPD-L1) function in tumor immune evasion are incompletely understood. We report here that tPD-L1 does not suppress cytotoxic T lymphocyte (CTL) activity in co-cultures of tumor cells and tumor-specific CTLs and exhibits no effect on primary tumor growth. However, deleting tPD-L1 decreases lung metastasis in a CTL-dependent manner in tumor-bearing mice. Depletion of myeloid cells or knocking out PD-1 in myeloid cells (mPD-1) impairs tPD-L1 promotion of tumor lung metastasis in mice. Single-cell RNA sequencing (scRNA-seq) reveals that tPD-L1 engages mPD-1 to activate SHP2 to antagonize the type I interferon (IFN-I) and STAT1 pathway to repress Cxcl9 and impair CTL recruitment to lung metastases. Human cancer patient response to PD-1 blockade immunotherapy correlates with IFN-I response in myeloid cells. Our findings determine that tPD-L1 engages mPD-1 to activate SHP2 to suppress the IFN-I-STAT1-CXCL9 pathway to impair CTL tumor recruitment in lung metastasis.

H3K9me3 represses G6PD expression to suppress the pentose phosphate pathway and ROS production to promote human mesothelioma growth.

The role of glucose-6-phosphate dehydrogenase (G6PD) in human cancer is incompletely understood. In a metabolite screening, we observed that inhibition of H3K9 methylation suppressed aerobic glycolysis and enhances the PPP in human mesothelioma cells. Genome-wide screening identified G6PD as an H3K9me3 target gene whose expression is correlated with increased tumor cell apoptosis. Inhibition of aerobic glycolysis enzyme LDHA and G6PD had no significant effects on tumor cell survival. Ablation of G6PD had no significant effect on human mesothelioma and colon carcinoma xenograft growth in athymic mice. However, activation of G6PD with the G6PD-selective activator AG1 induced tumor cell death. AG1 increased tumor cell ROS production and the resultant extrinsic and intrinsic death pathways, mitochondrial processes, and unfolded protein response in tumor cells. Consistent with increased tumor cell death in vitro, AG1 suppressed human mesothelioma xenograft growth in a dose-dependent manner in vivo. Furthermore, AG1 treatment significantly increased tumor-bearing mouse survival in an intra-peritoneum xenograft athymic mouse model. Therefore, in human mesothelioma and colon carcinoma, G6PD is not essential for tumor growth. G6PD acts as a metabolic checkpoint to control metabolic flux towards the PPP to promote tumor cell apoptosis, and its expression is repressed by its promotor H3K9me3 deposition.

Restoring FAS Expression via Lipid-Encapsulated FAS DNA Nanoparticle Delivery Is Sufficient to Suppress Colon Tumor Growth In Vivo.

A hallmark of human colorectal cancer is lost expression of FAS, the death receptor for FASL of cytotoxic T lymphocytes (CTLs). However, it is unknown whether restoring FAS expression alone is sufficient to suppress csolorectal-cancer development. The FAS promoter is hypermethylated and inversely correlated with FAS mRNA level in human colorectal carcinomas. Analysis of single-cell RNA-Seq datasets revealed that FAS is highly expressed in epithelial cells and immune cells but down-regulated in colon-tumor cells in human colorectal-cancer patients. Codon usage-optimized mouse and human FAS cDNA was designed, synthesized, and encapsulated into cationic lipid to formulate nanoparticle DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Overexpression of codon usage-optimized FAS in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro, suppressed colon tumor growth, and increased the survival of tumor-bearing mice in vivo. Overexpression of codon-optimized FAS-induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. DOTAP-Chol-mFAS therapy exhibited no significant liver toxicity. Our data determined that tumor-selective delivery of FAS DNA nanoparticles is sufficient for suppression of human colon tumor growth in vivo.

G6PD functions as a metabolic checkpoint to regulate granzyme B expression in tumor-specific cytotoxic T lymphocytes.

BACKGROUND: Granzyme B is a key effector of cytotoxic T lymphocytes (CTLs), and its expression level positively correlates with the response of patients with mesothelioma to immune checkpoint inhibitor immunotherapy. Whether metabolic pathways regulate Gzmb expression in CTLs is incompletely understood. METHODS: A tumor-specific CTL and tumor coculture model and a tumor-bearing mouse model were used to determine the role of glucose-6-phosphate dehydrogenase (G6PD) in CTL function and tumor immune evasion. A link between granzyme B expression and patient survival was analyzed in human patients with epithelioid mesothelioma. RESULTS: Mesothelioma cells alone are sufficient to activate tumor-specific CTLs and to enhance aerobic glycolysis to induce a PD-1hi Gzmblo CTL phenotype. However, inhibition of lactate dehydrogenase A, the key enzyme of the aerobic glycolysis pathway, has no significant effect on tumor-induced CTL activation. Tumor cells induce H3K9me3 deposition at the promoter of G6pd, the gene that encodes the rate-limiting enzyme G6PD in the pentose phosphate pathway, to downregulate G6pd expression in tumor-specific CTLs. G6PD activation increases acetyl-coenzyme A (CoA) production to increase H3K9ac deposition at the Gzmb promoter and to increase Gzmb expression in tumor-specific CTLs converting them from a Gzmblo to a Gzmbhi phenotype, thus increasing CTL tumor lytic activity. Activation of G6PD increases Gzmb+ tumor-specific CTLs and suppresses tumor growth in tumor-bearing mice. Consistent with these findings, GZMB expression level was found to correlate with increased survival in patients with epithelioid mesothelioma. CONCLUSION: G6PD is a metabolic checkpoint in tumor-activated CTLs. The H3K9me3/G6PD/acetyl-CoA/H3K9ac/Gzmb pathway is particularly important in CTL activation and immune evasion in epithelioid mesothelioma.

Effect of PDCA-based nursing intervention on activities of daily living, neurological function and self-management in acute cerebral stroke.

OBJECTIVE: This study explored and analyzed the effects of PDCA-based nursing intervention on the activities of daily living, neurological function and self-management of patients with acute cerebral stroke. METHODS: A total of 137 patients with acute cerebral stroke who were hospitalized from March 2018 to March 2020 were enrolled and divided into the observation-group (n = 70) and the control-group (n = 67). The control-group was given routine care, while those subjects in the observation group were provided with nursing intervention under the optimization of PDCA cycling. The activities of daily living (ADL), NIHSS score, self-management ability and life quality were compared between these two groups. RESULTS: ADL scores of the two groups after intervention were much higher than those without intervention (P<0.05), and the observation-group had apparently higher scores than the control-group (P<0.05). After intervention, the NIHSS scores of the two groups were much lower than before intervention (P<0.05), and the score of the observation-group was remarkably lower than the control-group (P<0.05). After intervention, the scores of each dimension of self-management behavior in both groups increased substantially compared to prior-intervention, and the score was higher in observation-group than in control-group (P<0.05). In addition, the two groups had increased scores in each dimension of life quality in post-intervention (P<0.05), and the observation-group had evidently higher scores than the control-group (P<0.05). CONCLUSION: PDCA-based nursing intervention can substantially enhance the daily living ability, neurological function, and self-management ability of patients, thus contributing to improve the clinical prognosis of patients and as such is worth popularizing.

Osteopontin Blockade Immunotherapy Increases Cytotoxic T Lymphocyte Lytic Activity and Suppresses Colon Tumor Progression.

Human colorectal cancers are mostly microsatellite-stable with no response to anti-PD-1 blockade immunotherapy, necessitating the development of a new immunotherapy. Osteopontin (OPN) is elevated in human colorectal cancer and may function as an immune checkpoint. We aimed at elucidating the mechanism of action of OPN and determining the efficacy of OPN blockade immunotherapy in suppression of colon cancer. We report here that OPN is primarily expressed in tumor cells, myeloid cells, and innate lymphoid cells in human colorectal carcinoma. Spp1 knock out mice exhibit a high incidence and fast growth rate of carcinogen-induced tumors. Knocking out Spp1 in colon tumor cells increased tumor-specific CTL cytotoxicity in vitro and resulted in decreased tumor growth in vivo. The OPN protein level is elevated in the peripheral blood of tumor-bearing mice. We developed four OPN neutralization monoclonal antibodies based on their efficacy in blocking OPN inhibition of T cell activation. OPN clones 100D3 and 103D6 increased the efficacy of tumor-specific CTLs in killing colon tumor cells in vitro and suppressed colon tumor growth in tumor-bearing mice in vivo. Our data indicate that OPN blockade immunotherapy with 100D3 and 103D6 has great potential to be further developed for colorectal cancer immunotherapy and for rendering a colorectal cancer response to anti-PD-1 immunotherapy.