61 results on '"John D Klement"'
Search Results
2. WDR5-H3K4me3 epigenetic axis regulates OPN expression to compensate PD-L1 function to promote pancreatic cancer immune escape
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Jennifer L Waller, Chunwan Lu, Kebin Liu, John D Klement, Dafeng Yang, Zhuoqi Liu, Alyssa D Merting, Dakota Poschel, Thomas Albers, and Huidong Shi
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Background Despite PD-L1 (Programmed death receptor ligand-1) expression on tumor cells and cytotoxic T lymphocytes tumor infiltration in the tumor microenvironment, human pancreatic cancer stands out as one of the human cancers that does not respond to immune checkpoint inhibitor (ICI) immunotherapy. Epigenome dysregulation has emerged as a major mechanism in T cell exhaustion and non-response to ICI immunotherapy, we, therefore, aimed at testing the hypothesis that an epigenetic mechanism compensates PD-L1 function to render pancreatic cancer non-response to ICI immunotherapy.Methods Two orthotopic pancreatic tumor mouse models were used for chromatin immunoprecipitation-Seq and RNA-Seq to identify genome-wide dysregulation of H3K4me3 and gene expression. Human pancreatic tumor and serum were analyzed for osteopontin (OPN) protein level and for correlation with patient prognosis. OPN and PD-L1 cellular location were determined in the tumors using flow cytometry. The function of WDR5-H3K4me3 axis in OPN expression were determined by Western blotting. The function of H3K4me3-OPN axis in pancreatic cancer immune escape and response to ICI immunotherapy was determined in an orthotopic pancreatic tumor mouse model.Results Mouse pancreatic tumors have a genome-wide increase in H3K4me3 deposition as compared with normal pancreas. OPN and its receptor CD44 were identified being upregulated in pancreatic tumors by their promoter H3K4me3 deposition. OPN protein is increased in both tumor cells and tumor-infiltrating immune cells in human pancreatic carcinoma and is inversely correlated with pancreatic cancer patient survival. OPN is primarily expressed in tumor cells and monocytic myeloid-derived suppressor cells (M-MDSCs), whereas PD-L1 is expressed in tumor cells, M-MDSCs, polymorphonuclear MDSCs and tumor-associated macrophages. WDR5 is essential for H3K4me3-specific histone methyltransferase activity that regulates OPN expression in tumor cells and MDSCs. Inhibition of WDR5 significantly decreased OPN protein level. Inhibition of WDR5 or knocking out of OPN suppressed orthotopic mouse pancreatic tumor growth. Inhibition of WDR5 also significantly increased efficacy of anti-PD-1 immunotherapy in suppression of mouse pancreatic tumor growth in vivo.Conclusions OPN compensates PD-L1 function to promote pancreatic cancer immune escape. Pharmacological inhibition of the WDR5-H3K4me3 epigenetic axis is effective in suppressing pancreatic tumor immune escape and in improving efficacy of anti-PD-1 immunotherapy in pancreatic cancer.
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- 2021
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3. p50 suppresses cytotoxic T lymphocyte effector function to regulate tumor immune escape and response to immunotherapy
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Chunwan Lu, Kebin Liu, John D Klement, Alyssa D Smith, Dafeng Yang, and Darren D Browning
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Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Background NF-κB is a key link between inflammation and cancer. Previous studies of NF-κB have largely focused on tumor cells, and the intrinsic function of NF-κB in T cells in tumor development and response to immunotherapy is largely unknown. We aimed at testing the hypothesis that NF-κB1 (p50) activation in T cells underlies human colon cancer immune escape and human cancer non-response to anti-PD-1 immunotherapy.Methods We screened NF-κB activation in human colon carcinoma and used mouse models to determine p50 function in tumor cells and immune cells. RNA-Seq was used to identify p50 target genes. p50 binding to target gene promoters were determined by electrophoresis mobility shift assay and chromatin immunoprecipitation. A p50 activation score was generated from gene expression profiling and used to link p50 activation to T-cell activation and function pre-nivolumab and post-nivolumab immunotherapy in human patients with cancer.Results p50 is the dominant form of NF-κB that is highly activated in immune cells in the human colorectal carcinoma microenvironment and neighboring non-neoplastic colon epithelial cells. Tumor cell intrinsic p50 signaling and T-cell intrinsic p50 signaling exert opposing functions in tumor growth control in vivo. Deleting Nfkb1 in tumor cells increased whereas in T cells decreased tumor growth in preclinical mouse models. Gene expression profiling identified Gzmb as a p50 target in T cells. p50 binds directly to a previously uncharacterized κB sequence at the Gzmb promoter in T cells, resulting in repression of Gzmb expression in tumor-infiltrating cytotoxic T lymphocytes (CTLs) to induce a dysfunctional CTL phenotype to promote tumor immune escape. p50 activation is inversely correlated with both GZMB expression and T-cell tumor infiltration in human colorectal carcinoma. Furthermore, nivolumab immunotherapy decreased p50 activation and increased GZMB expression in human patients with melanoma.Conclusions Inflammation activates p50 that binds to the Gzmb promoter to repress granzyme B expression in T cells, resulting in CTL dysfunction to confer tumor immune escape and decreased response to anti-PD-1 immunotherapy.
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- 2020
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4. IRF8 Regulates Intrinsic Ferroptosis through Repressing p53 Expression to Maintain Tumor Cell Sensitivity to Cytotoxic T Lymphocytes
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Dakota B. Poschel, Mercy Kehinde-Ige, John D. Klement, Dafeng Yang, Alyssa D. Merting, Natasha M. Savage, Huidong Shi, and Kebin Liu
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ferroptosis ,IRF8 ,p53 ,tumor cell death ,cancer immunotherapy ,lipid nanoparticle gene therapy ,Cytology ,QH573-671 - Abstract
Ferroptosis has emerged as a cytotoxic T lymphocyte (CTL)-induced tumor cell death pathway. The regulation of tumor cell sensitivity to ferroptosis is incompletely understood. Here, we report that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified the ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells acquire resistance to intrinsic ferroptosis induction and IRF8-deficient tumor cells also exhibit decreased ferroptosis in response to tumor-specific CTLs. Irf8 deletion increased p53 expression in tumor cells and knocking out p53 in IRF8.KO tumor cells restored tumor cell sensitivity to intrinsic ferroptosis induction. Furthermore, IRF8.KO tumor cells grew significantly faster than WT tumor cells in immune-competent mice. To restore IRF8 expression in tumor cells, we designed and synthesized codon usage-optimized IRF8-encoding DNA to generate IRF8-encoding plasmid NTC9385R-mIRF8. Restoring IRF8 expression via a lipid nanoparticle-encapsulated NTC9385R-mIRF8 plasmid therapy suppressed established tumor growth in vivo. In human cancer patients, nivolumab responders have a significantly higher IRF8 expression level in their tumor cells as compared to the non-responders. Our data determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis.
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- 2023
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5. Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes
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Chunwan Lu, John D. Klement, Mohammed L. Ibrahim, Wei Xiao, Priscilla S. Redd, Asha Nayak-Kapoor, Gang Zhou, and Kebin Liu
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Type I interferon ,CTLs ,STAT3 ,Granzyme B ,Colon Cancer ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Abstract Background Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in cytotoxic T lymphocytes (CTLs) in the tumor microenvironment is largely unknown. Methods Tumor tissues and CTLs of human colorectal cancer patients were analyzed for interferon (alpha and beta) receptor 1 (IFNAR1) expression. IFNAR1 knock out (IFNAR-KO), mixed wild type (WT) and IFNAR1-KO bone marrow chimera mice, and mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO) were used to determine IFN-I function in T cells in tumor suppression. IFN-I target genes in tumor-infiltrating and antigen-specific CTLs were identified and functionally analyzed. Results IFNAR1 expression level is significantly lower in human colorectal carcinoma tissue than in normal colon tissue. IFNAR1 protein is also significantly lower on CTLs from colorectal cancer patients than those from healthy donors. Although IFNAR1-KO mice exhibited increased susceptibility to methylcholanthrene-induced sarcoma, IFNAR1-sufficient tumors also grow significantly faster in IFNAR1-KO mice and in mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO), suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Strikingly, tumor-infiltrating CTL levels are similar between tumor-bearing WT and IFNAR1-KO mice. Competitive reconstitution of mixed WT and IFNAR1-KO bone marrow chimera mice further determined that IFNAR1-deficient naïve CTLs exhibit no deficiency in response to vaccination to generate antigen-specific CTLs as compared to WT CTLs. Gene expression profiling determined that Gzmb expression is down-regulated in tumor-infiltrating CTLs of IFNAR1-KO mice as compared to WT mice, and in antigen-specific IFNAR1-KO CTLs as compared to WT CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that binds to the Gzmb promoter to activate Gzmb transcription in CTLs. Conclusion IFN-I induces STAT3 activation to activate Gzmb expression to enhance CTL effector function to suppress tumor development. Human colorectal carcinoma may use down-regulation of IFNAR1 on CTLs to suppress CTL effector function to evade host cancer immunosurveillance.
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- 2019
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6. Figure S2 from Loss of Fas Expression and Function Is Coupled with Colon Cancer Resistance to Immune Checkpoint Inhibitor Immunotherapy
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Kebin Liu, Natasha M. Savage, Dafeng Yang, Chunwan Lu, John D. Klement, Priscilla S. Redd, Mohammed L. Ibrahim, and Wei Xiao
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Loss of Fas function leads to resistance to FasL-induced apoptosis.
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- 2023
7. Data from Loss of Fas Expression and Function Is Coupled with Colon Cancer Resistance to Immune Checkpoint Inhibitor Immunotherapy
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Kebin Liu, Natasha M. Savage, Dafeng Yang, Chunwan Lu, John D. Klement, Priscilla S. Redd, Mohammed L. Ibrahim, and Wei Xiao
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Despite the remarkable efficacy of immune checkpoint inhibitor (ICI) immunotherapy in various types of human cancers, colon cancer, except for the approximately 4% microsatellite-instable (MSI) colon cancer, does not respond to ICI immunotherapy. ICI acts through activating CTLs that use the Fas–FasL pathway as one of the two effector mechanisms to suppress tumor. Cancer stem cells are often associated with resistance to therapy including immunotherapy, but the functions of Fas in colon cancer apoptosis and colon cancer stem cells are currently conflicting and highly debated. We report here that decreased Fas expression is coupled with a subset of CD133+CD24lo colon cancer cells in vitro and in vivo. Consistent of the lower Fas expression level, this subset of CD133+CD24loFaslo colon cancer cells exhibits decreased sensitivity to FasL-induced apoptosis. Furthermore, FasL selectively enriches CD133+CD24loFaslo colon cancer cells. CD133+CD24loFaslo colon cancer cells exhibit increased lung colonization potential in experimental metastatic mouse models and decreased sensitivity to tumor-specific CTL adoptive transfer and ICI immunotherapies. Interestingly, FasL challenge selectively enriched this subset of colon cancer cells in microsatellite-stable (MSS) but not in the MSI human colon cancer cell lines. Consistent with the downregulation of Fas expression in CD133+CD24lo cells, lower Fas expression level is significantly correlated with decreased survival in patients with human colon cancer.Implications:Our data determine that CD133+CD24loFaslo colon cancer cells are capable to evade Fas-FasL cytotoxicity of tumor-reactive CTLs and targeting this subset of colon cancer cells is potentially an effective approach to suppress colon cancer immune evasion.
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- 2023
8. Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis
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Mohammed L. Ibrahim, John D. Klement, Chunwan Lu, Priscilla S. Redd, Wei Xiao, Dafeng Yang, Darren D. Browning, Natasha M. Savage, Phillip J. Buckhaults, Herbert C. Morse, III, and Kebin Liu
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Biology (General) ,QH301-705.5 - Abstract
Summary: IL-10 functions as a suppressor of colitis and colitis-associated colon cancer, but it is also a risk locus associated with ulcerative colitis. The mechanism underlying the contrasting roles of IL-10 in inflammation and colon cancer is unknown. We report here that inflammation induces the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) that express high levels of IL-10 in colon tissue. IL-10 induces the activation of STAT3 that directly binds to the Dnmt1 and Dnmt3b promoters to activate their expression, resulting in DNA hypermethylation at the Irf8 promoter to silence IRF8 expression in colon epithelial cells. Mice with Irf8 deleted in colonic epithelial cells exhibit significantly higher inflammation-induced tumor incidence. Human colorectal carcinomas have significantly higher DNMT1 and DNMT3b and lower IRF8 expression, and they exhibit significantly higher IRF8 promoter DNA methylation than normal colon. Our data identify the MDSC-IL-10-STAT3-DNMT3b-IRF8 pathway as a link between chronic inflammation and colon cancer initiation. : Ibrahim et al. report that chronic inflammation induces colonic accumulation of myeloid-derived suppressor cells (MDSCs) that upregulates IL-10. IL-10 directly regulates STAT3 activation to upregulate DNMT3b to silence tumor suppressor IRF8 in colonic epithelial cells. The MDSC-IL-10-STAT3-DNMT3b-IRF8 pathway links chronic inflammation to colon cancer initiation. Keywords: colitis, MDSCs, IRF8, IL-10, STAT3, DNMT3b, colon cancer, chronic inflammation, colon tumorigenesis, DNA methylation
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- 2018
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9. Figure S1 from SETD1B Activates iNOS Expression in Myeloid-Derived Suppressor Cells
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Kebin Liu, Asha Nayak-Kapoor, Dafeng Yang, Amy V. Paschall, Sarah K. Sharman, John D. Klement, Mohammed L. Ibrahim, and Priscilla S. Redd
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Figure S1 shows MDSC profiles in chaetocin-treated tumor-bearing mice
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- 2023
10. Table S1 from SETD1B Activates iNOS Expression in Myeloid-Derived Suppressor Cells
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Kebin Liu, Asha Nayak-Kapoor, Dafeng Yang, Amy V. Paschall, Sarah K. Sharman, John D. Klement, Mohammed L. Ibrahim, and Priscilla S. Redd
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Table S1 contains oligonucleotide sequences
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- 2023
11. Data from Autocrine IL6-Mediated Activation of the STAT3–DNMT Axis Silences the TNFα–RIP1 Necroptosis Pathway to Sustain Survival and Accumulation of Myeloid-Derived Suppressor Cells
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Kebin Liu, Asha Nayak-Kapoor, Thomas J. Hartney, Huidong Shi, Zhuoqi Liu, Qimei Han, Dafeng Yang, Mohammed L. Ibrahim, Priscilla S. Redd, John D. Klement, Amy V. Paschall, Daniela Payne, Chunwan Lu, and Alyssa D. Smith
- Abstract
Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα–RIP1–mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3–DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation.Significance:These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.
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- 2023
12. Article marked from SETD1B Activates iNOS Expression in Myeloid-Derived Suppressor Cells
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Kebin Liu, Asha Nayak-Kapoor, Dafeng Yang, Amy V. Paschall, Sarah K. Sharman, John D. Klement, Mohammed L. Ibrahim, and Priscilla S. Redd
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marked manuscript for review only
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- 2023
13. Data from SETD1B Activates iNOS Expression in Myeloid-Derived Suppressor Cells
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Kebin Liu, Asha Nayak-Kapoor, Dafeng Yang, Amy V. Paschall, Sarah K. Sharman, John D. Klement, Mohammed L. Ibrahim, and Priscilla S. Redd
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Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO) in myeloid cells that acts as a defense mechanism to suppress invading microorganisms or neoplastic cells. In tumor-bearing mice, elevated iNOS expression is a hallmark of myeloid-derived suppressor cells (MDSC). MDSCs use NO to nitrate both the T-cell receptor and STAT1, thus inhibiting T-cell activation and the antitumor immune response. The molecular mechanisms underlying iNOS expression and regulation in tumor-induced MDSCs are unknown. We report here that deficiency in IRF8 results in diminished iNOS expression in both mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo. Strikingly, although IRF8 was silenced in tumor-induced MDSCs, iNOS expression was significantly elevated in tumor-induced MDSCs, suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathologic conditions. Furthermore, tumor-induced MDSCs exhibited diminished STAT1 and NF-κB Rel protein levels, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs showed increased SETD1B expression as compared with their cellular equivalents in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, the target of SETD1B, was enriched at the nos2 promoter in tumor-induced MDSCs, and inhibition or silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. Our results show how tumor cells use the SETD1B–H3K4me3 epigenetic axis to bypass a normal role for IRF8 expression in activating iNOS expression in MDSCs when they are generated under pathologic conditions. Cancer Res; 77(11); 2834–43. ©2017 AACR.
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- 2023
14. Supplementary Data from Autocrine IL6-Mediated Activation of the STAT3–DNMT Axis Silences the TNFα–RIP1 Necroptosis Pathway to Sustain Survival and Accumulation of Myeloid-Derived Suppressor Cells
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Kebin Liu, Asha Nayak-Kapoor, Thomas J. Hartney, Huidong Shi, Zhuoqi Liu, Qimei Han, Dafeng Yang, Mohammed L. Ibrahim, Priscilla S. Redd, John D. Klement, Amy V. Paschall, Daniela Payne, Chunwan Lu, and Alyssa D. Smith
- Abstract
Supplemental Figures 1-7. Figure S1. IRF8 expression profiles in subsets of immune cells in spleens of tumor-bearing mice. Figure S2. IRF8 expression profiles in subsets of immune cells in peripheral blood of tumor-bearing mice. Figure S3. DNA methylation and MDSC accumulation in tumor-bearing mice. Figure S4. Inhibition of DNA methylation activates antigen-specific CTLs in tumor-bearing mice. Figure S5. Neutralization of TNFα increases tumor growth and MDSC accumulation. Figure S6. RIP1 and RIP3 are expressed in MDSCs. Figure S7. IL6 expression level in human colon carcinoma and melanoma. Table S1. Colorectal cancer patient data. Table S2. Colon cancer patient data.
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- 2023
15. H3K4me3 mediates the NF-κB p50 homodimer binding to the pdcd1 promoter to activate PD-1 transcription in T cells
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Priscilla S. Redd, Chunwan Lu, John D. Klement, Mohammed L. Ibrahim, Gang Zhou, Takumi Kumai, Esteban Celis, and Kebin Liu
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pd-1 ,t cells ,nf-κb ,h3k4me3 ,p50 ,Immunologic diseases. Allergy ,RC581-607 ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
PD-1 is a co-repressive receptor that curbs T cell activation and thereby serves as a protection mechanism against autoimmunity under physiological conditions. Under pathological conditions, tumor cells express PD-L1 as an adaptive resistant mechanism to suppress PD-1+ T cells to evade host immunosurveillance. PD-1 therefore is a key target in cancer immunotherapy. Despite the extensive studies of PD-1 expression regulation, the pdcd1 transcription machinery and regulatory mechanisms are still not fully understood. We report here that the NF-κB p50 homodimer is a transcription regulator of PD-1 in activated T cells. A putative κB sequence exists at the pdcd1 promoter. All five NF-κB Rel subunits are activated in activated T cells. However, only the p50 homodimer directly binds to the κB sequence at the pccd1 promoter in CD4+ and CD8+ T cells. Deficiency in p50 results in reduced PD-1 expression in both CD4+ and CD8+ T cells in vitro. Using an in vivo mixed bone marrow chimera mouse model, we show that p50 regulates PD-1 expression in a cell-intrinsic way and p50 deficiency leads to decreased PD-1 expression in both antigen-specific CD4+ and CD8+ T cells in vivo. The expression levels of H3K4me3-specific histone methyltransferase increased significantly, resulting in a significant increase in H3K4me3 deposition at the pdcd1 promoter in activated CD4+ and CD8+ T cells. Inhibition of H3K4me3 significantly decreased p50 binding to the pdcd1 promoter and PD-1 expression in a T cell line. Our findings determine that the p50-H3K4me3 axis regulates pdcd1 transcription activation in activated T cells.
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- 2018
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16. Lipid nanoparticle delivery of Fas plasmid restores Fas expression to suppress melanoma growth in vivo
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Zeinab Y. Al Subeh, Dakota B. Poschel, Priscilla S. Redd, John D. Klement, Alyssa D. Merting, Dafeng Yang, Megh Mehta, Huidong Shi, Yolonda L. Colson, Nicholas H. Oberlies, Cedric J. Pearce, Aaron H. Colby, Mark W. Grinstaff, and Kebin Liu
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Cytotoxicity, Immunologic ,Fas Ligand Protein ,General Engineering ,General Physics and Astronomy ,Apoptosis ,Article ,Mice ,Tumor Cells, Cultured ,Humans ,Animals ,General Materials Science ,fas Receptor ,Melanoma ,T-Lymphocytes, Cytotoxic ,Plasmids - Abstract
Fas ligand (FasL), expressed on the surface of activated cytotoxic T lymphocytes (CTLs), is the physiological ligand for the cell surface death receptor, Fas. The Fas-FasL engagement initiates diverse signaling pathways, including the extrinsic cell death signaling pathway, which is one of the effector mechanisms that CTLs use to kill tumor cells. Emerging clinical and experimental data indicate that Fas is essential for the efficacy of CAR-T cell immunotherapy. Furthermore, loss of Fas expression is a hallmark of human melanoma. We hypothesize that restoring Fas expression in tumor cells reverses human melanoma resistance to T cell cytotoxicity. We observed that DNA hypermethylation, at the FAS promoter, down-regulates FAS expression and confers melanoma cell resistance to FasL-induced cell death. Forced expression of Fas in tumor cells overcomes melanoma resistance to FasL-induced cell death in vitro. Lipid nanoparticle-encapsulated mouse Fas-encoding plasmid therapy eliminates Fas(+) tumor cells and suppresses established melanoma growth in immune competent syngeneic mice. Similarly, lipid nanoparticle-encapsulated human FAS-encoding plasmid (hCOFAS01) therapy significantly increases Fas protein level on tumor cells of human melanoma patient-derived xenograft (PDX) and suppresses the established human melanoma PDX growth in humanized NSG mice. In human melanoma patients, FasL is expressed in activated and exhausted T cells, Fas mRNA level positively correlates with melanoma patient survival, and nivolumab immunotherapy increases FAS expression in tumor cells. Our data determine that hCOFAS01 is an effective immunotherapeutic agent for human melanoma therapy with dual efficacy in increasing tumor cell FAS expression and in enhancing CTL tumor infiltration.
- Published
- 2022
17. Asah2 Represses the p53–Hmox1 Axis to Protect Myeloid-Derived Suppressor Cells from Ferroptosis
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Nicolas Coant, Chunwan Lu, Priscilla S. Redd, Dafeng Yang, John D. Klement, Yusuf A. Hannun, Juan Zou, Aaron H. Colby, Dakota B. Poschel, Peng George Wang, Alyssa D. Smith, Kebin Liu, Huabin Zhu, X. Ding Liu, Mark W. Grinstaff, and David A. Ostrov
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Male ,Programmed cell death ,T-Lymphocytes ,medicine.medical_treatment ,Immunology ,Datasets as Topic ,Antineoplastic Agents ,Article ,Inhibitory Concentration 50 ,Mice ,03 medical and health sciences ,Lymphocytes, Tumor-Infiltrating ,0302 clinical medicine ,Immune system ,Cancer immunotherapy ,Downregulation and upregulation ,Cell Line, Tumor ,Neutral Ceramidase ,Tumor Microenvironment ,medicine ,Animals ,Ferroptosis ,Humans ,Immunology and Allergy ,RNA-Seq ,chemistry.chemical_classification ,Reactive oxygen species ,Tumor microenvironment ,Protein Stability ,Myeloid-Derived Suppressor Cells ,Membrane Proteins ,Molecular Docking Simulation ,Disease Models, Animal ,Ceramidase activity ,chemistry ,Colonic Neoplasms ,Cancer research ,Myeloid-derived Suppressor Cell ,Female ,Tumor Suppressor Protein p53 ,Reactive Oxygen Species ,Heme Oxygenase-1 ,030215 immunology - Abstract
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.
- Published
- 2021
18. Tumor PD-L1 engages myeloid PD-1 to suppress type I interferon to impair cytotoxic T lymphocyte recruitment
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John D. Klement, Priscilla S. Redd, Chunwan Lu, Alyssa D. Merting, Dakota B. Poschel, Dafeng Yang, Natasha M. Savage, Gang Zhou, David H. Munn, Padraic G. Fallon, and Kebin Liu
- Subjects
Cancer Research ,Oncology - Published
- 2023
19. Autocrine IL6-Mediated Activation of the STAT3–DNMT Axis Silences the TNFα–RIP1 Necroptosis Pathway to Sustain Survival and Accumulation of Myeloid-Derived Suppressor Cells
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Dafeng Yang, Alyssa D. Smith, Amy V. Paschall, Thomas J. Hartney, Qimei Han, John D. Klement, Daniela Payne, Huidong Shi, Chunwan Lu, Priscilla S. Redd, Asha Nayak-Kapoor, Zhuoqi Liu, Mohammed L. Ibrahim, and Kebin Liu
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DNA (Cytosine-5-)-Methyltransferase 1 ,STAT3 Transcription Factor ,0301 basic medicine ,Cancer Research ,Programmed cell death ,Cell Survival ,Necroptosis ,Down-Regulation ,Mice, Transgenic ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Humans ,DNA (Cytosine-5-)-Methyltransferases ,Autocrine signalling ,STAT3 ,Cells, Cultured ,Cell Proliferation ,Mice, Inbred BALB C ,Tumor microenvironment ,biology ,Interleukin-6 ,Tumor Necrosis Factor-alpha ,Chemistry ,Myeloid-Derived Suppressor Cells ,Gene Expression Regulation, Neoplastic ,Mice, Inbred C57BL ,Autocrine Communication ,030104 developmental biology ,Oncology ,Receptor-Interacting Protein Serine-Threonine Kinases ,030220 oncology & carcinogenesis ,DNA methylation ,Cancer research ,biology.protein ,Myeloid-derived Suppressor Cell ,Female ,IRF8 ,Signal Transduction - Abstract
Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα–RIP1–mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3–DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation. Significance: These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.
- Published
- 2020
20. Expression profiles and function of IL6 in polymorphonuclear myeloid-derived suppressor cells
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Dafeng Yang, Mohammed L. Ibrahim, Chunwan Lu, Alyssa D. Smith, Priscilla S. Redd, John D. Klement, and Kebin Liu
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musculoskeletal diseases ,Cancer Research ,Cell type ,medicine.medical_treatment ,Immunology ,Inflammation ,Adenocarcinoma ,Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,immune system diseases ,Tumor Microenvironment ,medicine ,Animals ,Humans ,Immunology and Allergy ,skin and connective tissue diseases ,Mice, Inbred BALB C ,Tumor microenvironment ,Interleukin-6 ,Myeloid-Derived Suppressor Cells ,Cancer ,medicine.disease ,biological factors ,Gene Expression Regulation, Neoplastic ,Cytokine ,Oncology ,Cancer research ,Myeloid-derived Suppressor Cell ,Tumor promotion ,medicine.symptom ,Colorectal Neoplasms ,Transcriptome ,Signal Transduction ,030215 immunology - Abstract
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.
- Published
- 2020
21. H3K9me3 represses G6PD expression to suppress the pentose phosphate pathway and ROS production to promote human mesothelioma growth
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Chunwan Lu, Dafeng Yang, John D. Klement, Yolonda L. Colson, Nicholas H. Oberlies, Cedric J. Pearce, Aaron H. Colby, Mark W. Grinstaff, Zhuoqi Liu, Huidong Shi, Han-Fei Ding, and Kebin Liu
- Subjects
Mesothelioma ,Pentose Phosphate Pathway ,Cancer Research ,Disease Models, Animal ,Mice ,Carcinoma ,Genetics ,Animals ,Humans ,Mice, Nude ,Glucosephosphate Dehydrogenase ,Reactive Oxygen Species ,Molecular Biology - Abstract
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.
- Published
- 2021
22. Tumor PD-L1 selectively suppresses type I interferon in myeloid cells to suppress CTL recruitment to promote lung metastasis
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Dakota B. Poschel, Zhou G, Dafeng Yang, Priscilla S. Redd, David H. Munn, Alyssa D. Merting, Kebin Liu, Chunwan Lu, and John D. Klement
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Myeloid ,business.industry ,medicine.medical_treatment ,T cell ,Immunotherapy ,medicine.disease ,Primary tumor ,CTL ,medicine.anatomical_structure ,Immune system ,Interferon ,Cancer research ,Medicine ,Cytotoxic T cell ,business ,medicine.drug - Abstract
The mechanism underlying tumor cell PD-L1 (tPD-L1) induction of immune suppression through T cell PD-1 is well-known, but the mechanism underlying tPD-L1 induction of immune suppression via an intermediate cell is incompletely understood. We report here that tPD-L1 does not suppress cytotoxic T lymphocyte (CTL) activation and lytic function when only tumor cells and CTLs are present. Strikingly, knocking out PD-L1 in tumor cells has no effect on primary tumor growth, but significantly decreases lung metastasis in a CTL-dependent manner. Depletion of myeloid cells impaired tPD-L1 promotion of lung metastasis. Single-cell RNA sequencing revealed that tPD-L1 engages myeloid PD-1 (mPD-1) to antagonize type I interferon (IFN-I) and STAT1 signaling to repress Cxcl9 and Cxcl10 expression to impair CTL recruitment to lung metastases. Human patient response to PD-1 blockade immunotherapy correlates with IFN-I response in myeloid cells. Our data determines that the tPD-L1/mPD-1/IFN-I/STAT1/Cxcl9/10 axis controls CTL tumor infiltration in lung metastasis.
- Published
- 2021
23. Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes
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Priscilla S. Redd, John D. Klement, Asha Nayak-Kapoor, Kebin Liu, Wei Xiao, Mohammed L. Ibrahim, Gang Zhou, and Chunwan Lu
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STAT3 Transcription Factor ,0301 basic medicine ,Cancer Research ,medicine.medical_treatment ,Immunology ,Receptor, Interferon alpha-beta ,Biology ,lcsh:RC254-282 ,Granzymes ,GZMB ,CTLs ,STAT3 ,03 medical and health sciences ,0302 clinical medicine ,Interferon ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Immunology and Allergy ,Cytotoxic T cell ,Type I interferon ,Mice, Knockout ,Pharmacology ,Tumor microenvironment ,Colon Cancer ,Granzyme B ,Immunotherapy ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Mice, Inbred C57BL ,Immunosurveillance ,CTL ,030104 developmental biology ,Oncology ,030220 oncology & carcinogenesis ,Interferon Type I ,Cancer research ,Molecular Medicine ,Female ,Colorectal Neoplasms ,Research Article ,Signal Transduction ,T-Lymphocytes, Cytotoxic ,medicine.drug - Abstract
Background Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in cytotoxic T lymphocytes (CTLs) in the tumor microenvironment is largely unknown. Methods Tumor tissues and CTLs of human colorectal cancer patients were analyzed for interferon (alpha and beta) receptor 1 (IFNAR1) expression. IFNAR1 knock out (IFNAR-KO), mixed wild type (WT) and IFNAR1-KO bone marrow chimera mice, and mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO) were used to determine IFN-I function in T cells in tumor suppression. IFN-I target genes in tumor-infiltrating and antigen-specific CTLs were identified and functionally analyzed. Results IFNAR1 expression level is significantly lower in human colorectal carcinoma tissue than in normal colon tissue. IFNAR1 protein is also significantly lower on CTLs from colorectal cancer patients than those from healthy donors. Although IFNAR1-KO mice exhibited increased susceptibility to methylcholanthrene-induced sarcoma, IFNAR1-sufficient tumors also grow significantly faster in IFNAR1-KO mice and in mice with IFNAR1 deficiency only in T cells (IFNAR1-TKO), suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Strikingly, tumor-infiltrating CTL levels are similar between tumor-bearing WT and IFNAR1-KO mice. Competitive reconstitution of mixed WT and IFNAR1-KO bone marrow chimera mice further determined that IFNAR1-deficient naïve CTLs exhibit no deficiency in response to vaccination to generate antigen-specific CTLs as compared to WT CTLs. Gene expression profiling determined that Gzmb expression is down-regulated in tumor-infiltrating CTLs of IFNAR1-KO mice as compared to WT mice, and in antigen-specific IFNAR1-KO CTLs as compared to WT CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that binds to the Gzmb promoter to activate Gzmb transcription in CTLs. Conclusion IFN-I induces STAT3 activation to activate Gzmb expression to enhance CTL effector function to suppress tumor development. Human colorectal carcinoma may use down-regulation of IFNAR1 on CTLs to suppress CTL effector function to evade host cancer immunosurveillance. Electronic supplementary material The online version of this article (10.1186/s40425-019-0635-8) contains supplementary material, which is available to authorized users.
- Published
- 2019
24. SUV39H1 Represses the Expression of Cytotoxic T-Lymphocyte Effector Genes to Promote Colon Tumor Immune Evasion
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Samuel K. Kulp, Cedric J. Pearce, Dafeng Yang, Mitch A. Phelps, Thomas Albers, Natasha M. Savage, Zhiliang Xie, Mark W. Grinstaff, Iryna Lebedyeva, John D. Klement, Aaron H. Colby, Nicholas H. Oberlies, Christopher C. Coss, Kebin Liu, Chunwan Lu, Il Kyu Oh, and Jennifer L. Waller
- Subjects
0301 basic medicine ,Cancer Research ,medicine.medical_treatment ,Immunology ,Antineoplastic Agents ,Article ,GZMB ,Histones ,Mice ,03 medical and health sciences ,Lymphocytes, Tumor-Infiltrating ,0302 clinical medicine ,Immune system ,Cell Line, Tumor ,Tumor Microenvironment ,medicine ,Animals ,Humans ,Cytotoxic T cell ,Enzyme Inhibitors ,Tumor microenvironment ,Effector ,Chemistry ,Methyltransferases ,Immunotherapy ,digestive system diseases ,Gene Expression Regulation, Neoplastic ,Repressor Proteins ,Disease Models, Animal ,030104 developmental biology ,030220 oncology & carcinogenesis ,Histone methyltransferase ,Colonic Neoplasms ,Cancer research ,Female ,Tumor Escape ,CD8 ,T-Lymphocytes, Cytotoxic - Abstract
Despite the presence of CTLs in the tumor microenvironment, the majority of immunogenic human colon cancer does not respond to immune checkpoint inhibitor immunotherapy, and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs were present in both MSI and microsatellite stable colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 was significantly elevated in human colon carcinoma compared with normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment have high expression of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened on the basis of the SET (suppressor of variegation 3–9, enhancer of zeste and trithorax) domain structure of the human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. On the basis of the structure of this small molecule, we modified it and chemically synthesized a small molecule, termed F5446, which has an EC50 of 0.496 μmol/L for SUV39H1 enzymatic activity. H3K9me3 was enriched in the promoters of GZMB, PRF1, FASLG, and IFNG in quiescent T cells. F5446 inhibited H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8+ CTL-dependent manner in vivo. Our data indicate that SUV39H1 represses CTL effector gene expression and, in doing so, confers colon cancer immune escape.
- Published
- 2019
25. Osteopontin Blockade Immunotherapy Increases Cytotoxic T Lymphocyte Lytic Activity and Suppresses Colon Tumor Progression
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Dafeng Yang, Alyssa D. Merting, Priscilla S. Redd, Chunwan Lu, Dakota B. Poschel, John D. Klement, and Kebin Liu
- Subjects
0301 basic medicine ,PD-L1 ,Cancer Research ,osteopontin ,Colorectal cancer ,medicine.medical_treatment ,T cell ,cytotoxic T lymphocytes ,lcsh:RC254-282 ,Article ,03 medical and health sciences ,0302 clinical medicine ,stomatognathic system ,medicine ,Cytotoxic T cell ,Osteopontin ,immune checkpoint ,MSS ,biology ,business.industry ,Immunotherapy ,medicine.disease ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Immune checkpoint ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Tumor progression ,030220 oncology & carcinogenesis ,OPN neutralization ,Cancer research ,biology.protein ,business - Abstract
Simple Summary Despite the breakthrough in human cancer immunotherapy, colorectal cancer, except for the small subset of microsatellite instable colorectal cancer (MSI, ~4% total cases), is one of the few human cancers that does not respond to current immune checkpoint inhibitor (ICI) immunotherapy. CTLs are present in both MSI and microsatellite stable (MSS) human colon carcinoma, suggesting that PD-L1-independent mechanisms may exist and suppress CTL activation in the colon tumor microenvironment. We determined that osteopontin (OPN) inhibits tumor-specific cytotoxic T lymphocyte (CTL) lytic activity to promote colon tumor growth in vivo. Accordingly, OPN blockade immunotherapy using OPN neutralization monoclonal antibodies 100D3 and 103D6 suppressed colon tumor growth in vivo. Our findings indicate that 100D3 and 103D6 has the potential to be further developed for colorectal cancer immunotherapy. Abstract 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.
- Published
- 2021
26. Osteopontin: A Key Regulator of Tumor Progression and Immunomodulation
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Dakota B. Poschel, Kebin Liu, Hannah R. Moorman, Priscilla S. Redd, John D. Klement, and Chunwan Lu
- Subjects
0301 basic medicine ,Cancer Research ,osteopontin ,integrin ,T cell ,medicine.medical_treatment ,MDSCs ,Tumor-associated macrophage ,Review ,lcsh:RC254-282 ,03 medical and health sciences ,tumor-associated macrophage ,0302 clinical medicine ,Immune system ,stomatognathic system ,medicine ,CD44 ,immune checkpoint ,immune evasion ,Tumor microenvironment ,biology ,Innate lymphoid cell ,Immunotherapy ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,Tumor progression ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research - Abstract
Simple Summary Anti-PD-1/PD-L1 and anti-CTLA-4-based immune checkpoint blockade (ICB) immunotherapy have recently emerged as a breakthrough in human cancer treatment. Durable efficacy has been achieved in many types of human cancers. However, not all human cancers respond to current ICB immunotherapy and only a fraction of the responsive cancers exhibit efficacy. Osteopontin (OPN) expression is highly elevated in human cancers and functions as a tumor promoter. Emerging data suggest that OPN may also regulate immune cell function in the tumor microenvironment. This review aims at OPN function in human cancer progression and new findings of OPN as a new immune checkpoint. We propose that OPN compensates PD-L1 function to promote tumor immune evasion, which may underlie human cancer non-response to current ICB immunotherapy. Abstract OPN is a multifunctional phosphoglycoprotein expressed in a wide range of cells, including osteoclasts, osteoblasts, neurons, epithelial cells, T, B, NK, NK T, myeloid, and innate lymphoid cells. OPN plays an important role in diverse biological processes and is implicated in multiple diseases such as cardiovascular, diabetes, kidney, proinflammatory, fibrosis, nephrolithiasis, wound healing, and cancer. In cancer patients, overexpressed OPN is often detected in the tumor microenvironment and elevated serum OPN level is correlated with poor prognosis. Initially identified in activated T cells and termed as early T cell activation gene, OPN links innate cells to adaptive cells in immune response to infection and cancer. Recent single cell RNA sequencing revealed that OPN is primarily expressed in tumor cells and tumor-infiltrating myeloid cells in human cancer patients. Emerging experimental data reveal a key role of OPN is tumor immune evasion through regulating macrophage polarization, recruitment, and inhibition of T cell activation in the tumor microenvironment. Therefore, in addition to its well-established direct tumor cell promotion function, OPN also acts as an immune checkpoint to negatively regulate T cell activation. The OPN protein level is highly elevated in peripheral blood of human cancer patients. OPN blockade immunotherapy with OPN neutralization monoclonal antibodies (mAbs) thus represents an attractive approach in human cancer immunotherapy.
- Published
- 2020
27. Restoring FAS Expression via Lipid-Encapsulated FAS DNA Nanoparticle Delivery Is Sufficient to Suppress Colon Tumor Growth In Vivo
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Alyssa D. Merting, Dakota B. Poschel, Chunwan Lu, John D. Klement, Dafeng Yang, Honglin Li, Huidong Shi, Eric Chapdelaine, Mitzi Montgomery, Michael T. Redman, Natasha M. Savage, Asha Nayak-Kapoor, and Kebin Liu
- Subjects
Cancer Research ,FAS ,colon cancer ,metastasis ,cytotoxic T lymphocyte ,cationic lipid nanoparticle ,Oncology ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Article ,RC254-282 - Abstract
Simple Summary A key feature of human colorectal tumor is loss of FAS expression. FAS is the death receptor for FASL of activated T cells. Loss of FAS expression therefore may promote tumor cell immune escape. We aimed at determining whether restoring FAS expression is sufficient to suppress colorectal tumor growth. Mouse and human FAS cDNA was synthesized and encapsulated into cationic lipid nanoparticle DOTAP-Cholesterol to formulate DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Restoring FAS expression in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro and suppressed colon-tumor growth and progression in tumor-bearing mice in vivo. Restoring FAS expression induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells in vitro. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. Tumor-selective delivery of FAS DNA nanoparticle is potentially an effective therapy for human colorectal cancer. Abstract 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.
- Published
- 2022
28. SUV39H1 regulates human colon carcinoma apoptosis and cell cycle to promote tumor growth
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Dafeng Yang, Kebin Liu, Thomas Albers, John D. Klement, Iryna Lebedyeva, Chunwan Lu, and Jennifer L. Waller
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0301 basic medicine ,Cancer Research ,Antimetabolites, Antineoplastic ,Cell cycle checkpoint ,Fas Ligand Protein ,Colorectal cancer ,Mice, Nude ,Apoptosis ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Gene expression ,medicine ,Biomarkers, Tumor ,Tumor Cells, Cultured ,Animals ,Humans ,Cell Proliferation ,biology ,Cell Cycle Checkpoints ,Methyltransferases ,Cell cycle ,medicine.disease ,Xenograft Model Antitumor Assays ,Gene Expression Regulation, Neoplastic ,Repressor Proteins ,030104 developmental biology ,Histone ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,Histone methyltransferase ,Colonic Neoplasms ,Cancer research ,biology.protein ,Female ,Fluorouracil ,Growth inhibition - Abstract
Trimethylation of histone 3 lysine 9 (H3K9me3) at gene promoters is a major epigenetic mechanism that silences gene expression. We have developed a small molecule inhibitor for the H3K9me3-specific histone methyltransferase SUV39H1. We report here that FAS expression is significantly down-regulated and SUV39H1 expression is significantly up-regulated in human colorectal carcinoma (CRC) as compared to normal colon. SUV39H1-selective inhibitor F5446 decreased H3K9me3 deposition at the FAS promoter, increased Fas expression, and increased CRC cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, inhibition of SUV39H1 altered the expression of genes with known functions in DNA replication and cell cycle in the metastatic colon carcinoma cells, which is associated with cell cycle arrest at S phase in the metastatic human colon carcinoma cells, resulting in tumor cell apoptosis and growth inhibition in a concentration-dependent manner in vitro. Moreover, F5446 increased 5-FU-resistant human CRC sensitivity to both 5-FU- and FasL-induced apoptosis and inhibited tumor cell growth in vitro. More importantly, F5446 suppressed human colon tumor xenograft growth in vivo. Our data indicate that pharmacological inhibition of SUV39H1 is an effective approach to suppress human CRC.
- Published
- 2020
29. IFNAR1 Controls Autocrine Type I IFN Regulation of PD-L1 Expression in Myeloid-Derived Suppressor Cells
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Wei Xiao, Mohammed L. Ibrahim, Chunwan Lu, John D. Klement, and Kebin Liu
- Subjects
0301 basic medicine ,Immunology ,Receptor, Interferon alpha-beta ,B7-H1 Antigen ,Article ,Interferon-gamma ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Cell Line, Tumor ,Neoplasms ,Tumor Microenvironment ,Animals ,Immunology and Allergy ,STAT1 ,Phosphorylation ,Autocrine signalling ,Receptor ,Mice, Knockout ,Mice, Inbred BALB C ,Tumor microenvironment ,biology ,Chemistry ,Myeloid-Derived Suppressor Cells ,Interferon-alpha ,Interferon-beta ,Mice, Inbred C57BL ,STAT1 Transcription Factor ,030104 developmental biology ,Cell culture ,030220 oncology & carcinogenesis ,Cancer research ,Myeloid-derived Suppressor Cell ,biology.protein ,Female ,Signal transduction - Abstract
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-β 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-β 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.
- Published
- 2018
30. Expression regulation and function of PD-1 and PD-L1 in T lymphoma cells
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Kebin Liu, Candace J. Langan, John D. Klement, Maria Y. Liu, and Jan van Riggelen
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0301 basic medicine ,Programmed Cell Death 1 Receptor ,Immunology ,Receptors, Antigen, T-Cell ,Apoptosis ,Biology ,Lymphoma, T-Cell ,B7-H1 Antigen ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,immune system diseases ,In vivo ,Cell Line, Tumor ,hemic and lymphatic diseases ,PD-L1 ,medicine ,Animals ,Humans ,Cell Proliferation ,Hyperactivation ,Cell growth ,T-cell receptor ,Neoplasms, Experimental ,medicine.disease ,In vitro ,Lymphoma ,Gene Expression Regulation, Neoplastic ,Mice, Inbred C57BL ,030104 developmental biology ,Gene Knockdown Techniques ,Cancer research ,biology.protein ,Tumor Escape ,Signal Transduction ,030215 immunology - Abstract
T lymphoma cells may constitutively express PD-1 and PD-L1. The relative role of PD-1 and PD-L1 in T lymphoma is incompletely understood. We report here that PD-1(+) PDL-1(+) human T lymphoma cells exhibit constitutive hyperactivation of the TCR signaling and do not respond to PD-L1-mediated suppression in vitro. Knocking out PD-1 or PD-L1 has no effects on T lymphoma cell apoptosis and proliferation in vitro, but significantly increased tumor-bearing mouse survival. Our findings determine that the constitutively active TCR signaling pathway maintain T lymphoma cell growth in vitro and that both PD-1 and PD-L1 promote T lymphoma growth in vivo.
- Published
- 2021
31. WDR5-H3K4me3 epigenetic axis regulates OPN expression to compensate PD-L1 function to promote pancreatic cancer immune escape
- Author
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Thomas Albers, Chunwan Lu, Alyssa D. Merting, Dakota B. Poschel, Zhuoqi Liu, John D. Klement, Kebin Liu, Jennifer L. Waller, Dafeng Yang, and Huidong Shi
- Subjects
Male ,0301 basic medicine ,Cancer Research ,medicine.medical_treatment ,B7-H1 Antigen ,Epigenesis, Genetic ,Histones ,Mice ,0302 clinical medicine ,Pancreatic tumor ,Immunology and Allergy ,Medicine ,Immune Checkpoint Inhibitors ,RC254-282 ,Clinical/Translational Cancer Immunotherapy ,biology ,Intracellular Signaling Peptides and Proteins ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,Molecular Medicine ,Female ,immunotherapy ,immune evation ,T cell ,Immunology ,03 medical and health sciences ,Immune system ,stomatognathic system ,Pancreatic cancer ,Animals ,Humans ,Pharmacology ,Tumor microenvironment ,business.industry ,CD44 ,tumor escape ,Immunotherapy ,myeloid-derived suppressor cells ,medicine.disease ,Mice, Inbred C57BL ,Pancreatic Neoplasms ,Disease Models, Animal ,HEK293 Cells ,030104 developmental biology ,Cancer research ,biology.protein ,Myeloid-derived Suppressor Cell ,Osteopontin ,business - Abstract
BackgroundDespite PD-L1 (Programmed death receptor ligand-1) expression on tumor cells and cytotoxic T lymphocytes tumor infiltration in the tumor microenvironment, human pancreatic cancer stands out as one of the human cancers that does not respond to immune checkpoint inhibitor (ICI) immunotherapy. Epigenome dysregulation has emerged as a major mechanism in T cell exhaustion and non-response to ICI immunotherapy, we, therefore, aimed at testing the hypothesis that an epigenetic mechanism compensates PD-L1 function to render pancreatic cancer non-response to ICI immunotherapy.MethodsTwo orthotopic pancreatic tumor mouse models were used for chromatin immunoprecipitation-Seq and RNA-Seq to identify genome-wide dysregulation of H3K4me3 and gene expression. Human pancreatic tumor and serum were analyzed for osteopontin (OPN) protein level and for correlation with patient prognosis. OPN and PD-L1 cellular location were determined in the tumors using flow cytometry. The function of WDR5-H3K4me3 axis in OPN expression were determined by Western blotting. The function of H3K4me3-OPN axis in pancreatic cancer immune escape and response to ICI immunotherapy was determined in an orthotopic pancreatic tumor mouse model.ResultsMouse pancreatic tumors have a genome-wide increase in H3K4me3 deposition as compared with normal pancreas. OPN and its receptor CD44 were identified being upregulated in pancreatic tumors by their promoter H3K4me3 deposition. OPN protein is increased in both tumor cells and tumor-infiltrating immune cells in human pancreatic carcinoma and is inversely correlated with pancreatic cancer patient survival. OPN is primarily expressed in tumor cells and monocytic myeloid-derived suppressor cells (M-MDSCs), whereas PD-L1 is expressed in tumor cells, M-MDSCs, polymorphonuclear MDSCs and tumor-associated macrophages. WDR5 is essential for H3K4me3-specific histone methyltransferase activity that regulates OPN expression in tumor cells and MDSCs. Inhibition of WDR5 significantly decreased OPN protein level. Inhibition of WDR5 or knocking out of OPN suppressed orthotopic mouse pancreatic tumor growth. Inhibition of WDR5 also significantly increased efficacy of anti-PD-1 immunotherapy in suppression of mouse pancreatic tumor growth in vivo.ConclusionsOPN compensates PD-L1 function to promote pancreatic cancer immune escape. Pharmacological inhibition of the WDR5-H3K4me3 epigenetic axis is effective in suppressing pancreatic tumor immune escape and in improving efficacy of anti-PD-1 immunotherapy in pancreatic cancer.
- Published
- 2021
32. Loss of Fas Expression and Function is Coupled with Colon Cancer Resistance to Immune Checkpoint Inhibitor Immunotherapy
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Chunwan Lu, Dafeng Yang, Priscilla S. Redd, Natasha M. Savage, Wei Xiao, Kebin Liu, John D. Klement, and Mohammed L. Ibrahim
- Subjects
0301 basic medicine ,Cancer Research ,Adoptive cell transfer ,Colorectal cancer ,medicine.medical_treatment ,Fas ligand ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Immune system ,Cancer stem cell ,Cell Line, Tumor ,Medicine ,Animals ,Humans ,fas Receptor ,Molecular Biology ,business.industry ,Immunotherapy ,medicine.disease ,digestive system diseases ,Disease Models, Animal ,030104 developmental biology ,Oncology ,Apoptosis ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Colonic Neoplasms ,Cancer research ,Stem cell ,business - Abstract
Despite the remarkable efficacy of immune checkpoint inhibitor (ICI) immunotherapy in various types of human cancers, colon cancer, except for the approximately 4% microsatellite-instable (MSI) colon cancer, does not respond to ICI immunotherapy. ICI acts through activating CTLs that use the Fas–FasL pathway as one of the two effector mechanisms to suppress tumor. Cancer stem cells are often associated with resistance to therapy including immunotherapy, but the functions of Fas in colon cancer apoptosis and colon cancer stem cells are currently conflicting and highly debated. We report here that decreased Fas expression is coupled with a subset of CD133+CD24lo colon cancer cells in vitro and in vivo. Consistent of the lower Fas expression level, this subset of CD133+CD24loFaslo colon cancer cells exhibits decreased sensitivity to FasL-induced apoptosis. Furthermore, FasL selectively enriches CD133+CD24loFaslo colon cancer cells. CD133+CD24loFaslo colon cancer cells exhibit increased lung colonization potential in experimental metastatic mouse models and decreased sensitivity to tumor-specific CTL adoptive transfer and ICI immunotherapies. Interestingly, FasL challenge selectively enriched this subset of colon cancer cells in microsatellite-stable (MSS) but not in the MSI human colon cancer cell lines. Consistent with the downregulation of Fas expression in CD133+CD24lo cells, lower Fas expression level is significantly correlated with decreased survival in patients with human colon cancer. Implications: Our data determine that CD133+CD24loFaslo colon cancer cells are capable to evade Fas-FasL cytotoxicity of tumor-reactive CTLs and targeting this subset of colon cancer cells is potentially an effective approach to suppress colon cancer immune evasion.
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- 2018
33. MS4A1 expression and function in T cells in the colorectal cancer tumor microenvironment
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John D. Klement, Kebin Liu, Chunwan Lu, and T. William Mudd
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Adult ,Male ,0301 basic medicine ,Databases, Factual ,T-Lymphocytes ,medicine.medical_treatment ,Programmed Cell Death 1 Receptor ,Immunology ,B7-H1 Antigen ,Article ,03 medical and health sciences ,Lymphocytes, Tumor-Infiltrating ,0302 clinical medicine ,PD-L1 ,Tumor Microenvironment ,medicine ,Humans ,Aged ,Glycoproteins ,Tumor microenvironment ,biology ,Cancer ,Immunotherapy ,Middle Aged ,Antigens, CD20 ,medicine.disease ,Immune checkpoint ,Immunosurveillance ,030104 developmental biology ,Cancer research ,biology.protein ,Female ,Nivolumab ,Colorectal Neoplasms ,CD8 ,030215 immunology - Abstract
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.
- Published
- 2021
34. p50 suppresses cytotoxic T lymphocyte effector function to regulate tumor immune escape and response to immunotherapy
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Dafeng Yang, Jennifer L. Waller, Chunwan Lu, Alyssa D. Smith, David H. Munn, Darren D. Browning, Kebin Liu, and John D. Klement
- Subjects
0301 basic medicine ,Cancer Research ,medicine.medical_treatment ,Immunology ,Inflammation ,Biology ,GZMB ,gastrointestinal neoplasms ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,medicine ,Animals ,Humans ,Immunology and Allergy ,Cytotoxic T cell ,RC254-282 ,Clinical/Translational Cancer Immunotherapy ,Pharmacology ,Melanoma ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Immunotherapy ,medicine.disease ,Granzyme B ,Disease Models, Animal ,CTL ,030104 developmental biology ,Oncology ,inflammation ,030220 oncology & carcinogenesis ,Cancer research ,CD8-positive T-lymphocytes ,Molecular Medicine ,Female ,Tumor Escape ,immunotherapy ,medicine.symptom ,T-Lymphocytes, Cytotoxic ,immune evation - Abstract
BackgroundNF-κB is a key link between inflammation and cancer. Previous studies of NF-κB have largely focused on tumor cells, and the intrinsic function of NF-κB in T cells in tumor development and response to immunotherapy is largely unknown. We aimed at testing the hypothesis that NF-κB1 (p50) activation in T cells underlies human colon cancer immune escape and human cancer non-response to anti-PD-1 immunotherapy.MethodsWe screened NF-κB activation in human colon carcinoma and used mouse models to determine p50 function in tumor cells and immune cells. RNA-Seq was used to identify p50 target genes. p50 binding to target gene promoters were determined by electrophoresis mobility shift assay and chromatin immunoprecipitation. A p50 activation score was generated from gene expression profiling and used to link p50 activation to T-cell activation and function pre-nivolumab and post-nivolumab immunotherapy in human patients with cancer.Resultsp50 is the dominant form of NF-κB that is highly activated in immune cells in the human colorectal carcinoma microenvironment and neighboring non-neoplastic colon epithelial cells. Tumor cell intrinsic p50 signaling and T-cell intrinsic p50 signaling exert opposing functions in tumor growth control in vivo. Deleting Nfkb1 in tumor cells increased whereas in T cells decreased tumor growth in preclinical mouse models. Gene expression profiling identified Gzmb as a p50 target in T cells. p50 binds directly to a previously uncharacterized κB sequence at the Gzmb promoter in T cells, resulting in repression of Gzmb expression in tumor-infiltrating cytotoxic T lymphocytes (CTLs) to induce a dysfunctional CTL phenotype to promote tumor immune escape. p50 activation is inversely correlated with both GZMB expression and T-cell tumor infiltration in human colorectal carcinoma. Furthermore, nivolumab immunotherapy decreased p50 activation and increased GZMB expression in human patients with melanoma.ConclusionsInflammation activates p50 that binds to the Gzmb promoter to repress granzyme B expression in T cells, resulting in CTL dysfunction to confer tumor immune escape and decreased response to anti-PD-1 immunotherapy.
- Published
- 2020
35. Neutral ceramidase regulates ferroptosis in myeloid-derived suppressor cells
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huabin zhu, Chunwan Lu, Dafeng Yang, Priscilla S Redd, Mohammed L. Ibrahim, John D. Klement, Alyssa D. Smith, Thomas Albers, Iryna Lebedyeva, Nicolas Coant, Yusuf Hannun, and Kebin Liu
- Subjects
Immunology ,Immunology and Allergy - Abstract
Myeloid-derived suppressor cells (MDSCs) are key targets in cancer immunotherapy. Neutral ceramidase (nCDase), a key enzyme that hydrolyzes ceramide into sphingosine, is highly expressed in colon carcinoma and regulates apoptosis and autophagy in colon carcinoma cells. We report here that nCDase is also highly expressed in tumor-infiltrating immune cells in mouse and human colon carcinoma in vivo. To target nCDase, we performed molecular docking based on human nCDase structure aganist a virtual chemical library. In vitro enzymatic inhibition analysis of top hits identified nCDC06 as a potent nCDase inhibitor. Chemical and NMR analysis determined nCDC06 structure and molecular weight (325.1 Dalton). nCDC06 has an IC50 of 10.16 to 25.91 mM for human nCDase and 18.6–30.2 mM for mouse nCDase. nCDC06 induces MDSC cell death in a dose-dependent mechanism. Inhibition of ferroptosis decreased nCDC06-induced MDSC cell death. Analysis of ultracellular structure, ROS accumulation, and glutathione revealed that nCDC06 suppresses MDSCs at least in part through a ferroptosis-dependent mechanism. Gene expression profiling revealed that nCDC06 activates the p53 pathway in MDSCs. Furthermore, nCDC06 therapy significantly inhibited tumor growth in an experimental lung metastasis mouse model. We determined that nCDase regulates MDSC ferroptosis and nCDC06 is potent in activating the p53 pathway to induce MDSC ferroptosis to suppress tumor growth in vivo.
- Published
- 2020
36. NF-kB1 induces T cell dysfunction to promote colon tumorigenesis
- Author
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Chunwan Lu, John D. Klement, Wei Xiao, Mohammed L. Ibrahim, Alyssa D. Smith, Dafeng Yang, Daren Browning, David H. Munn, and Kebin Liu
- Subjects
Immunology ,Immunology and Allergy - Abstract
Background and aims Chronic colonic inflammation drives colon tumorigenesis. Chronic infection-associated inflammation causes T cell exhaustion/dysfunction. NF-kB is a molecular link between colonic inflammation and colorectal cancer. We aimed at testing the hypothesis that the p50 NF-kB (NF-kB1) regulates T cell dysfunction to promote colon tumor immune evasion and development. Methods Nfkb1 knock out (p50 KO), Nfkb1 KO chimera mice, Nfkb1 KO colon tumor cells were used to determine p50 NF-kB function in colon tumor development. RNA-Seq, chromatin immunoprecipitation, and electrophoresis mobility shift assay were used to elucidate molecular mechanism of NF-kB1 action. Flow cytometry was used to analyze cytotoxic T lymphocytes. Results p50 KO mice showed a significantly decrease in tumorigenesis. However, mice with p50 deficiency only in immune cells also exhibited a significant decrease in tumorigenesis, and p50-sufficient colon tumor cells also grew significantly slower in p50 KO mice than in WT mice. RNA-Seq identified Gzmb as a p50 NF-kB target gene. p50 KO mice have significantly higher tumor-infiltrating granzyme B+ CTLs than WT mice, and CTL granzyme B protein level is also significantly higher in p50 KO mice than in WT mice. A kB sequence element was identified at the Gzmb promoter and p50 NF-kB directly binds to this kB element in T cells to repress Gzmb expression. Conclusion NF-kB1 has opposing functions in tumor cells and T cells in vivo. The p50 NF-kB represses Gzmb expression to confer colon tumor-infiltrating CTL a dysfunctional phenotype to promote colon tumor immune evasion, resulting in enhanced colon tumorigenesis and growth.
- Published
- 2020
37. Abstract A32: The SUV39H1-H3K9me3 pathway represses cytotoxic T lymphocyte effector expression to confer colon carcinoma immune escape
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Samuel K. Kulp, Dafeng Yang, Chunwan Lu, Mark W. Grinstaff, Mitch A. Phelps, Cedric J. Pearce, Thomas Albers, Kebin Liu, Zhiliang Xie, Natasha M. Savage, Kyu Oh, Iryna Lebedyeva, John D. Klement, Christopher C. Coss, Aaron H. Colby, Nicholas H. Oberlies, and Jennifer L. Waller
- Subjects
Cancer Research ,Tumor microenvironment ,Effector ,medicine.medical_treatment ,Immunology ,Cancer ,Immunotherapy ,Biology ,medicine.disease ,GZMB ,CTL ,Cancer research ,medicine ,Cytotoxic T cell ,CD8 - Abstract
Despite the presence of cytotoxic T lymphocytes (CTLs) in the tumor microenvironment, the majority of immunogenic human colon cancers do not respond to immune checkpoint inhibitor immunotherapy and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs are present in both MSI and microsatellite stable (MSS) colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 is significantly elevated in human colon carcinoma as compared to normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment express high levels of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened based on the SET domain structure of human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. Based on the structure of this small molecule, we modified it and chemically synthesized a novel small molecule, termed F5446, which has an EC50 of 0.496 μM for SUV39H1 enzymatic activity. H3K9me3 is enriched in the promoters of GZMB, PRF1, FASLG and IFNG in quiescent T cells. F5446 inhibits H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8+ CTL-dependent manner in vivo. Our data indicate that SUV39H1 represses CTL effector expression and, in doing so, confers colon cancer immune escape. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, II Kyu Oh, Natasha M. Savage, Jennifer L. Waller, Aaron H. Colby, Mark W. Grinstaff, Nicholas H. Oberlies, Cedric Pearce, Zhiliang Xie, Samuel Kulp, Christopher Coss, Mitch A. Phelps, Thomas Albers, Iryna O. Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 pathway represses cytotoxic T lymphocyte effector expression to confer colon carcinoma immune escape [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A32.
- Published
- 2020
38. H3K4me3 mediates the NF-κB p50 homodimer binding to the pdcd1 promoter to activate PD-1 transcription in T cells
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Kebin Liu, Chunwan Lu, John D. Klement, Gang Zhou, Priscilla S. Redd, Takumi Kumai, Mohammed L. Ibrahim, and Esteban Celis
- Subjects
lcsh:Immunologic diseases. Allergy ,0301 basic medicine ,T cell ,Immunology ,p50 ,lcsh:RC254-282 ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Transcription (biology) ,medicine ,Immunology and Allergy ,Receptor ,t cells ,nf-κb ,Original Research ,h3k4me3 ,Chemistry ,pd-1 ,NF-κB ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,In vitro ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,Histone methyltransferase ,H3K4me3 ,lcsh:RC581-607 ,CD8 - Abstract
PD-1 is a co-repressive receptor that curbs T cell activation and thereby serves as a protection mechanism against autoimmunity under physiological conditions. Under pathological conditions, tumor cells express PD-L1 as an adaptive resistant mechanism to suppress PD-1(+) T cells to evade host immunosurveillance. PD-1 therefore is a key target in cancer immunotherapy. Despite the extensive studies of PD-1 expression regulation, the pdcd1 transcription machinery and regulatory mechanisms are still not fully understood. We report here that the NF-κB p50 homodimer is a transcription regulator of PD-1 in activated T cells. A putative κB sequence exists at the pdcd1 promoter. All five NF-κB Rel subunits are activated in activated T cells. However, only the p50 homodimer directly binds to the κB sequence at the pccd1 promoter in CD4(+) and CD8(+) T cells. Deficiency in p50 results in reduced PD-1 expression in both CD4(+) and CD8(+) T cells in vitro. Using an in vivo mixed bone marrow chimera mouse model, we show that p50 regulates PD-1 expression in a cell-intrinsic way and p50 deficiency leads to decreased PD-1 expression in both antigen-specific CD4(+) and CD8(+) T cells in vivo. The expression levels of H3K4me3-specific histone methyltransferase increased significantly, resulting in a significant increase in H3K4me3 deposition at the pdcd1 promoter in activated CD4(+) and CD8(+) T cells. Inhibition of H3K4me3 significantly decreased p50 binding to the pdcd1 promoter and PD-1 expression in a T cell line. Our findings determine that the p50-H3K4me3 axis regulates pdcd1 transcription activation in activated T cells.
- Published
- 2018
39. An osteopontin/CD44 immune checkpoint controls CD8+ T cell activation and tumor immune evasion
- Author
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Esteban Celis, Dafeng Yang, Kebin Liu, Mohammed L. Ibrahim, Scott I. Abrams, Chunwan Lu, Amy V. Paschall, Keiko Ozato, John D. Klement, and Priscilla S. Redd
- Subjects
0301 basic medicine ,T cell ,medicine.medical_treatment ,T-Lymphocytes ,Biology ,CD8-Positive T-Lymphocytes ,Lymphocyte Activation ,Immune tolerance ,03 medical and health sciences ,Mice ,Immune system ,Cancer immunotherapy ,stomatognathic system ,Cell Line, Tumor ,medicine ,Tumor Microenvironment ,Immune Tolerance ,Cytotoxic T cell ,Animals ,Humans ,Mice, Knockout ,Mice, Inbred BALB C ,General Medicine ,Immunotherapy ,Immune checkpoint ,Neoplasm Proteins ,030104 developmental biology ,medicine.anatomical_structure ,Hyaluronan Receptors ,Colonic Neoplasms ,Cancer research ,Commentary ,Osteopontin ,Tumor Escape ,IRF8 ,Research Article - Abstract
Despite breakthroughs in immune checkpoint inhibitor (ICI) immunotherapy, not all human cancers respond to ICI immunotherapy and a large fraction of patients with the responsive types of cancers do not respond to current ICI immunotherapy. This clinical conundrum suggests that additional immune checkpoints exist. We report here that interferon regulatory factor 8 (IRF8) deficiency led to impairment of cytotoxic T lymphocyte (CTL) activation and allograft tumor tolerance. However, analysis of chimera mice with competitive reconstitution of WT and IRF8-KO bone marrow cells as well as mice with IRF8 deficiency only in T cells indicated that IRF8 plays no intrinsic role in CTL activation. Instead, IRF8 functioned as a repressor of osteopontin (OPN), the physiological ligand for CD44 on T cells, in CD11b(+)Ly6C(lo)Ly6G(+) myeloid cells and OPN acted as a potent T cell suppressor. IRF8 bound to the Spp1 promoter to repress OPN expression in colon epithelial cells, and colon carcinoma exhibited decreased IRF8 and increased OPN expression. The elevated expression of OPN in human colon carcinoma was correlated with decreased patient survival. Our data indicate that myeloid and tumor cell–expressed OPN acts as an immune checkpoint to suppress T cell activation and confer host tumor immune tolerance.
- Published
- 2018
40. Myeloid-Derived Suppressor Cells Produce IL10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Tumorigenesis
- Author
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Chunwan Lu, Dafeng Yang, John D. Klement, Kebin Liu, Herbert C. Morse, Mohammed L. Ibrahim, Priscilla S. Redd, and Wei Xiao
- Subjects
Colorectal cancer ,medicine.medical_treatment ,Biology ,medicine.disease ,medicine.disease_cause ,Ulcerative colitis ,digestive system diseases ,Cytokine ,medicine ,Myeloid-derived Suppressor Cell ,Cancer research ,Gene silencing ,Colitis ,IRF8 ,Carcinogenesis - Abstract
IL10 is an anti-inflammatory cytokine that suppresses colitis and colitis-associated colon cancer, but it has recently been found to be a risk locus associated with ulcerative colitis. The mechanism underlying the contrasting roles of IL10 in inflammation and colon cancer is unknown. We report here that inflammation induces accumulation of CD11b+Gr1+ myeloid-derived suppressor cells that express high level of IL10 in colon tissue. IL10 induces activation of STAT3 that directly binds to the Dnmt1 and Dnmt3b promoters to activate their expression, resulting in DNA hypermethylation at the Irf8 promoter to silence IRF8 expression in colon epithelial cells. Mice with an Irf8 knock out in colonic epithelial cells exhibit a significantly higher inflammation-induced tumor incidence. Our data identify the MDSC-IL10-STAT3-DNMT-IRF8 pathway as a link between chronic inflammation and colon cancer initiation.
- Published
- 2018
41. Abstract 2350: Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes
- Author
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Gang Zhou, Chunwan Lu, John D. Klement, Kebin Liu, Mohammed L. Ibrahim, and Priscilla S. Redd
- Subjects
Cancer Research ,Tumor microenvironment ,medicine.medical_treatment ,T cell ,Immunotherapy ,Biology ,GZMB ,Granzyme B ,Immune system ,medicine.anatomical_structure ,Oncology ,Interferon ,Cancer research ,medicine ,Cytotoxic T cell ,medicine.drug - Abstract
Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in immune cells in the tumor microenvironment is largely unknown. We determined that IFNα13 and IFNβ are selectively expressed in colon carcinoma tissues in vivo. Although IFNAR1 KO mice exhibited increased susceptibility to carcinogen and inflammation-induced sarcoma and colon tumorigenesis, IFNAR1-sufficient tumors grow significantly faster in IFNAR1 KO mice and in mice with IFNAR1 deficiency only in T cells, suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Surprisingly, unlike in an anti-viral immune response where IFN-I regulates T cell activation and clonal expansion, IFNAR1-deficient T cells exhibit no deficiency in generation of antigen-specific CTLs and IFNAR1-deficient CTLs infiltration level is similar as compared to that of WT CTLs in colon tumor-bearing mice. Gene expression profiling identified Gzmb as an IFN-I target gene in tumor-infiltrating CTLs and in antigen-specific CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that directly binds to the Gzmb promoter to activate Gzmb transcription in CTLs. IFN-I also regulates GZMB expression in human T cells, but IFNAR1 is not only significantly down-regulated in human colon carcinomas but also down-regulated in CTLs of human colon cancer patients as compared to normal colon and CTLs from healthy donors. Our data determined that IFN-I plays a more dominant role in CTLs than in tumor cells to execute its anti-tumor function, and doing so through activating the STAT3-granzyme B pathway in tumor-infiltrating CTLs. Citation Format: Chunwan Lu, John D. Klement, Mohammed L. Ibrahim, Priscilla S. Redd, Gang Zhou, Kebin Liu. Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2350.
- Published
- 2019
42. Abstract 2263: Ceramide mimetics regulation of tumor cell response to immune checkpoint inhibitor immunotherapy
- Author
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Priscilla S. Redd, Chunwan Lu, John D. Klement, Mohammed Ibrahim, Dafeng Yang, Daniela Payne, Iryna Lebedyeva, and Kebin Liu
- Subjects
Cancer Research ,Oncology - Abstract
Programmed cell death-1 (PD-1) is an immunosuppressive receptor expressed on T cells upon activation. Once interacting with its ligand, programmed death ligand-1 (PD-L1), the PD-1:PD-L pathway curbs T cell activation and thereby serves as a natural protection mechanism against autoimmunity. However, this pathway has been hijacked by cancer cells in order to evade the immune system. This interface has already been recognized as an effective target for cancer therapy leading to the development of immune checkpoint inhibitor (ICI) immunotherapy. Blocking the PD-1:PD-L axis unleashes T cells from suppression to maintain their effector function against tumor cells. ICI has durable efficacy in many types of cancer; however, colorectal cancer, except for the approximately 4% microsatellite-instable (MSI) colorectal cancer, stands out as one of the few human cancers that does not respond to ICI immunotherapy. One potential mechanism that these tumor cells may be using is resisting cell death, making them insensitive to apoptosis induction by T cells. Ceramide, the central metabolite of the sphingolipid metabolism pathway, plays a critical role in many cell signaling processes, and one of its most prevalent roles can be seen in cell death. Our lab has developed five novel ceramide mimetics (IG4, IG7, IG14, IG17, and IG19) which function to sensitize tumor cells to apoptosis and enhance T cell-mediated cell death. We have previously shown that these ceramide mimetics significantly increase FasL-induced apoptosis by perforin-deficient cytotoxic T lymphocytes. Using the murine colon carcinoma cell line CT26, we show that the ceramide mimetics induce tumor cell death in a dose-dependent manner. Interestingly, the ceramide mimetics appear to be functioning by different mechanisms in vitro. Still, we report here that these five ceramide mimetics exhibit tumor suppression activity in a colon tumor mouse model in a dose-dependent manner in vivo. Also, ceramide mimetic IG19 significantly increased the efficacy of anti-PD-1 mAb immunotherapy in suppressing colon tumor development in vivo. Our data thus indicate that ceramide mimetics are potential effective enhancers for ICI immunotherapy in colon cancer. Citation Format: Priscilla S. Redd, Chunwan Lu, John D. Klement, Mohammed Ibrahim, Dafeng Yang, Daniela Payne, Iryna Lebedyeva, Kebin Liu. Ceramide mimetics regulation of tumor cell response to immune checkpoint inhibitor immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2263.
- Published
- 2019
43. Abstract 1530: Polymorphonuclear myeloid derived suppressor cells secrete IL6 to promote colon tumorigenesis in an inflammatory vicious cycle
- Author
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Mohammed L. Ibrahim, John D. Klement, Priscilla S. Redd, Chunwan Lu, and Kebin Liu
- Subjects
Cancer Research ,Oncology - Abstract
It is widely accepted now that chronic inflammation can create a favorable environment for neoplastic transformation and tumor progression. Tumor Microenvironment consists of various innate and adaptive immune cells, which secrete numerous inflammatory mediating and immunoregulatory cytokines. Among all immune cells infiltrated to the tumor site, myeloid cells have been considered as major players in the tumor associated inflammation. Recently, a subpopulation named myeloid-derived suppressor cells (MDSCs) have gain a great attention as a potential link between inflammation and tumor progression. MDSCs are heterogeneous population of immature myeloid cells that is subdivided into monocytic (M-MDSC) and polymorphonuclear (PMN-MDSC) subsets depending on different markers expressed differentially on their surface. Both PMN-MDSC and M-MDSC have been shown to be immune-suppressive and to exert an immunoregulatory role in the tumor microenvironment. However, a clear distinction in their functional and molecular properties has remained a matter of debate. Here we report that PMN-MDSC but not M-MDSC is the major secreting immune subset of the proinflammatory and tumor-promoting cytokine Interleukin 6 (IL6) in colon cancer. IL6 has been shown in literature to function as a tumor promoter through enhancing tumor growth, invasion and chemoresistance. We observed that IL6 upregulates DNMT1 and DNMT3b in colon carcinoma cells to boost their growth and survival in a STAT3 dependent mechanism. We also observed that tumor-expressed IL6 acts as a key modulator of the immune profile in colon tumor microenvironment. We generated an IL6-overexpressing colon carcinoma cell lines. We observed that IL6-overexpressing tumor triggers a significant increment in PMN-MDSCs but not M-MDSCs infiltration and accumulation in mouse colon carcinoma xenograft model. This remarkable PMN-MDSC tumor enrichment is associated with less effector T lymphocytes infiltration and more progressive and proliferative tumor. Moreover, DSS-induced colitis murine model is characterized with higher infiltration of PMN-MDSC but not M-MDSC, which secrete significant amount of IL6. Therefore, inflammation-derived IL6 induces neoplastic transformation of colonic epithelial cells through upregulation of DNMT1 and DNMT3b. Collectively; our data indicate that PMN-MDSC is the primary IL6 producer among all tumor-infiltrated leukocytes. PMN-MDSC-derived IL6 has a dual tumor favoring function through nurturing colon tumor cells in DNA-methylation dependent mechanism, and raising an immunosuppressive context enriched with more PMN-MDSCs, which secrete more IL6 in an endless vicious cycle. Thus, targeting this circuit, may have a promising implication in colon cancer immunotherapy. Citation Format: Mohammed L. Ibrahim, John D. Klement, Priscilla S. Redd, Chunwan Lu, Kebin Liu. Polymorphonuclear myeloid derived suppressor cells secrete IL6 to promote colon tumorigenesis in an inflammatory vicious cycle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1530.
- Published
- 2019
44. Abstract 3226: The IRF8-osteopontin-CD44 axis functions as an immune checkpoint to control CD8+ T cell activation and tumor immune evasion
- Author
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Amy V. Paschall, Dafeng Yang, Keiko Ozato, Esteban Celis, Kebin Liu, Priscilla S. Redd, Chunwan Lu, Mohammed L. Ibrahim, Scott I. Abrams, and John D. Klement
- Subjects
Cancer Research ,medicine.medical_treatment ,T cell ,Immunotherapy ,Biology ,Immune checkpoint ,Immune tolerance ,Immune system ,medicine.anatomical_structure ,stomatognathic system ,Oncology ,Cancer research ,medicine ,Cytotoxic T cell ,IL-2 receptor ,IRF8 - Abstract
Despite breakthroughs in immune checkpoint inhibitor (ICI) immunotherapy, not all human cancers respond to ICI immunotherapy and only fraction of patients with responsive tumors have a durable response to current ICI immunotherapy. This clinical conundrum suggests that additional immune checkpoints may exist, particularly in cancers resistant to current ICI immunotherapy, such as colorectal cancer. We report here that interferon regulatory factor 8 (IRF8) deficiency led to impairment of cytotoxic T lymphocyte (CTL) activation in a peptide vaccine model and allowed allograft transplant tumor tolerance. These effects were associated with upregulation of the CTL surface marker CD44. However, analysis of chimeric mice with competitive reconstitution of wild type and IRF8 KO bone marrow cells as well as mice with IRF8 deficiency only in T cells indicated that IRF8 plays no intrinsic role in CTL activation. Instead, IRF8 functioned as a repressor of osteopontin (OPN), the physiological ligand for CD44 on T cells, in CD11b+Ly6CloLy6G+ myeloid cells and OPN acted as a potent T cell suppressor. In vitro stimulation of CTLs in the presence of OPN resulted in decreased expression of activation markers CD69 and CD25 and inhibited proliferation and interferon gamma (IFNg) secretion. Expression of OPN was found to be upregulated in both myeloid cells and colon epithelial cells following silencing of IRF8 expression. IRF8 bound to the Spp1 promoter, which encodes OPN, to repress OPN expression in colon epithelial cells. Correspondingly, human colon carcinoma cells exhibited decreased IRF8 and increased OPN expression. These increased OPN levels inhibited human PBMC proliferation and IFNg secretion. The elevated expression of OPN in human colon carcinoma was correlated with decreased patient survival. Our data indicates that myeloid and tumor cell-expressed OPN acts as a novel immune checkpoint to suppress T cell activation and confer host tumor immune tolerance. Blockade of this checkpoint may expand the pool of patients who may benefit from ICI immunotherapy. Citation Format: John D. Klement, Amy V. Paschall, Priscilla S. Redd, Mohammed L. Ibrahim, Chunwan Lu, Dafeng Yang, Esteban Celis, Scott I. Abrams, Keiko Ozato, Kebin Liu. The IRF8-osteopontin-CD44 axis functions as an immune checkpoint to control CD8+ T cell activation and tumor immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3226.
- Published
- 2019
45. SETD1B Activates iNOS Expression in Myeloid-Derived Suppressor Cells
- Author
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Sarah K. Sharman, Mohammed L. Ibrahim, Dafeng Yang, John D. Klement, Priscilla S. Redd, Asha Nayak-Kapoor, Amy V. Paschall, and Kebin Liu
- Subjects
0301 basic medicine ,Cancer Research ,Nitric Oxide Synthase Type II ,Nitric Oxide ,Article ,Nitric oxide ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Immune system ,Cell Line, Tumor ,Gene silencing ,Animals ,Humans ,STAT1 ,Mice, Inbred BALB C ,biology ,Myeloid-Derived Suppressor Cells ,Cell Differentiation ,Histone-Lysine N-Methyltransferase ,Cell biology ,Nitric oxide synthase ,030104 developmental biology ,Oncology ,chemistry ,Integrin alpha M ,biology.protein ,Myeloid-derived Suppressor Cell ,IRF8 - Abstract
Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO) in myeloid cells that acts as a defense mechanism to suppress invading microorganisms or neoplastic cells. In tumor-bearing mice, elevated iNOS expression is a hallmark of myeloid-derived suppressor cells (MDSC). MDSCs use NO to nitrate both the T-cell receptor and STAT1, thus inhibiting T-cell activation and the antitumor immune response. The molecular mechanisms underlying iNOS expression and regulation in tumor-induced MDSCs are unknown. We report here that deficiency in IRF8 results in diminished iNOS expression in both mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo. Strikingly, although IRF8 was silenced in tumor-induced MDSCs, iNOS expression was significantly elevated in tumor-induced MDSCs, suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathologic conditions. Furthermore, tumor-induced MDSCs exhibited diminished STAT1 and NF-κB Rel protein levels, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs showed increased SETD1B expression as compared with their cellular equivalents in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, the target of SETD1B, was enriched at the nos2 promoter in tumor-induced MDSCs, and inhibition or silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. Our results show how tumor cells use the SETD1B–H3K4me3 epigenetic axis to bypass a normal role for IRF8 expression in activating iNOS expression in MDSCs when they are generated under pathologic conditions. Cancer Res; 77(11); 2834–43. ©2017 AACR.
- Published
- 2016
46. Abstract 4966: The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion
- Author
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Dafeng Yang, Kebin Liu, Cedric J. Pearce, Thomas Albers, John D. Klement, Aaron H. Colby, Nicholas H. Oberlies, Mark W. Grinstaff, Chunwan Lu, and Iryna Lebedyeva
- Subjects
Cancer Research ,Colorectal cancer ,medicine.medical_treatment ,Intrinsic apoptosis ,Immunotherapy ,Cell cycle ,Biology ,medicine.disease ,digestive system diseases ,Fas ligand ,Oncology ,Apoptosis ,Cancer research ,medicine ,Carcinoma ,Cytotoxic T cell - Abstract
Human colon cancer, except for the small subset of microsatellite instable (MSI) colon cancer, does not respond to anti-PD-L1/PD-1 immune checkpoint inhibitor (ICI) immunotherapy. A well-established notion is that MSI colon cancer harbors various mutations that serve as neoantigens to generate tumor-specific cytotoxic T lymphocytes (CTLs). Because CTLs suppress tumor through inducing tumor cell apoptosis, we hypothesized that colon cancer cell intrinsic apoptosis resistance is potentially another mechanism underlying microsatellite stable (MSS) colon cancer nonresponse to ICI immunotherapy. If tumor cells are not sensitive to apoptosis induction, then tumor cells cannot be killed by CTLs regardless of how potent the CTLs are. To test this hypothesis, we analyzed MSI and MSS human colon carcinoma specimens and developed a small molecule that targets H3K9me3-mediated cell death pathways as a sensitizer for colon cancer ICI immunotherapy. As expected, we observed high level of CTL infiltration in all eight MSI colon carcinoma specimens examined. However, five of the nine MSS colon carcinoma specimens also exhibited high level of CTL infiltration, suggesting that CTL level is not the sole mechanism underlying MSS colon cancer non-response to ICI immunotherapy. Analysis of the TCGA database revealed that SUV39H1, a histone methyltransferase that catalyzes H3K9me3, is significantly elevated in human colon carcinoma specimens compared to normal human colon tissues. A SUV39H1 structure-based virtual chemical library screening in combination with functional assays identified a small-molecule SUV39H1 inhibitor. Further analysis of the top forty-three hits identified a potent SUV39H1 inhibitor. Structure modifications were then performed and a chemical synthesis procedure was developed to synthesize a novel SUV39H1 inhibitor, termed F5446. F5446 has an EC50 of 0.496 µM against SUV39H1 enzymatic activity and suppresses human colon carcinoma cell growth in a dose-dependent manner in vitro. F5446 suppresses colon carcinoma growth through upregulating key cell cycle regulators to induce cell cycle arrest at the S phase. Furthermore, F5446 also decreased H3K9me3 at the FAS promoter to upregulate Fas expression. Consequently, a sublethal dose of F5446 effectively overcame human colon carcinoma cell resistance to apoptosis induced by FasL, a ligand on activated CTLs in vitro. Treatment of colon carcinoma-bearing mice with F5446 and anti-PD-1 both significantly suppress tumor growth in vivo. Our data determined that F5446 is a novel SUV39H1 inhibitor that has the potential to be further developed as a sensitizer to overcome colon carcinoma resistance to ICI immunotherapy through activating the Fas-FasL pathway. Citation Format: Chunwan Lu, Dafeng Yang, John D. Klement, Aaron H. Colby, Mark W. Grinstaff, Cedric Pearce, Nicholas H. Oberlies, Thomas Albers, Iryna Lebedyeva, Kebin Liu. The SUV39H1-H3K9me3 axis mediates colon carcinoma cell intrinsic apoptosis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4966.
- Published
- 2018
47. Abstract 4682: IRF8 controls T cell development and survival to regulate T cell antitumor activity
- Author
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Chunwan Lu, Hussein Sultan, Amy V. Paschall, Keiko Ozato, Esteban Celis, Mohammed L. Ibrahim, Mary Zimmerman, Priscilla S. Redd, Kebin Liu, and John D. Klement
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Cancer Research ,Naive T cell ,T cell ,Biology ,Molecular biology ,Haematopoiesis ,medicine.anatomical_structure ,Immune system ,Oncology ,Interferon ,medicine ,Progenitor cell ,IRF8 ,B cell ,medicine.drug - Abstract
Interferon Regulatory Factor 8 (IRF8, or ICSBP1) is a member of the Interferon Regulatory transcription factor family, and functions as a key hematopoietic transcription factor. Loss of IRF8 leads to defective antigen-presenting cell activity, perturbations in B cell development and, in mouse models, an accumulation of CD11b+Gr1+ immature myeloid cells. However, the role of IRF8 in T cell development and antitumor activity remains unclear. Whole body and chimeric IRF8-knockout mice (IRF8-KO) demonstrate increased susceptibility to both allogenic transplant and carcinogen-induced tumor models. T cell function is crucial for the immune system's endogenous antitumor response. Analysis of the T cell compartment of IRF8-KO mice demonstrated a deficiency in both naïve T cell percentages and total number. Despite this peripheral decrease in T cell numbers, early T cell progenitors in both the bone marrow and thymus were significantly increased in IRF8-KO mice compared to wild-type. To further investigate the role of IRF8 in T cell development and survival, IRF8-KO:WT mixed chimera mice were generated by lethal irradiation of CD45.1+CD45.2+ recipient mice, followed by transfer of CD45.2+ IRF8-KO and CD45.1+ WT bone marrow (BM). Surprisingly, analysis of blood obtained from reconstituted mice demonstrated preferential engraftment and survival of T cells derived from WT, rather than IRF8-KO BM. This imbalanced phenotype was not rescued by increasing the proportion of IRF8-KO BM administered to mice, suggesting the effect was not due to failure of IRF8-KO BM engraftment. Furthermore, analysis of T cell populations in both primary (thymus) and secondary (spleen) lymphoid organs showed a progressive loss of IRF8-KO T cells during their maturation and development process, while WT T cells remained unaltered. Given that IRF8 has been shown in tumor cells to regulate a variety of pro- and anti-apoptotic molecules, we hypothesized that IRF8 controls the peripheral survival of T cells. To test this hypothesis, resting T cells were isolated from the spleen of mixed chimera mice and viability was measured by Annexin V/PI staining. Resting IRF8-KO cells, but not WT, demonstrated a pro-apoptotic phenotype, as shown by increased Annexin V staining. Accordingly, upon in vitro stimulation and activation, IRF8-KO T cells demonstrated increased apoptosis. Our data determine that IRF8 controls both T cell development and peripheral survival, and that loss of IRF8 impairs the T cell antitumor immune response. Citation Format: John D. Klement, Amy V. Paschall, Mary A. Zimmerman, Mohammed L. Ibrahim, Priscilla S. Redd, Chunwan Lu, Hussein Sultan, Esteban Celis, Keiko Ozato, Kebin Liu. IRF8 controls T cell development and survival to regulate T cell antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4682.
- Published
- 2018
48. Abstract 5744: Chronic inflammation activates IL6 signaling to upregulate DNMT1 and DNMT3b to promote colon tumorigenesis
- Author
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Mohammed L. Ibrahim, Kebin Liu, Priscilla S. Redd, Chunwan Lu, Daneila Payne, and John D. Klement
- Subjects
Cancer Research ,Colorectal cancer ,medicine.medical_treatment ,Cancer ,Tumor initiation ,Biology ,medicine.disease ,medicine.disease_cause ,Paracrine signalling ,Cytokine ,Oncology ,medicine ,Cancer research ,Myeloid-derived Suppressor Cell ,Carcinogenesis ,Autocrine signalling - Abstract
A strong correlation has been well documented between chronic inflammation and tumorigenesis. Colitis Associated Cancer (CAC) stands as a solid example for the inflammation-mediated tumorigenesis and is considered as the most serious complication of inflammatory bowel diseases. Pro-inflammatory cytokines are believed to regulate the neoplastic transformation of colonic epithelial cells. However, the underlying mechanism of inflammation-driven tumorigenesis is still a matter of debate. As a key player in the tumor inflammatory microenvironment, the pro-inflammatory cytokine IL6 is one of the most extensively studied cytokine in tumor biology research. IL6 is a pleiotropic cytokine secreted by different cell types including macrophages, fibroblast, epithelial cells and myeloid cells. IL6 and its downstream oncogenic transcription factor STAT3 have been shown to act as a tumor promoter through stimulating the proliferation and growth of malignant cells, inhibiting cell death and maintaining the inflammatory tumor-promoting milieu. Moreover IL6 protein and mRNA levels were found to be elevated in serum and tumor specimen from human and mice with different types of cancers including colon cancer; suggesting that IL6 may play a pro-tumorigenic function during the chronic inflammation-mediated tumorigenesis. However, the molecular mechanisms underlying IL6 tumor-promoting functions in CAC is still elusive. In this study, we aimed at determining the expression profiles and underlying mechanism of function of IL6 in the colon. Our data indicate that IL6 is significantly elevated in the colon in DSS-induced colitis mouse model and in AOM-DSS induced-CAC. Interestingly, myeloid derived suppressor cells (MDSCs) were shown to be the major source of IL6 in the lamina propria in vivo and in vitro .Given the role of IL6/STAT3/DNMTs axis in tumor promotion, we further hypothesized that IL6 may directly contribute to the inflammation-mediated colon tumorigenesis. To test our hypothesis, we generated IL6-overexpressing colon cancer cell lines and observed that IL6 overexpression is associated with STAT3 phosphorylation and resultant DNMT1 and DNMT3b upregulation in colon cancer cells. Similarly, treatment of colon cancer cells with exogenous IL6 induces STAT3 phosphorylation and up-regulation of DNMT1 and DNMT3b. Moreover, IL6-overexpression alters cell proliferation and survival. Collectively, our data highlighted a new crucial role of IL6 in the inflammation-mediated tumorigenesis through epigenetic alteration of colonic epithelial cells; suggesting that prolonged autocrine and paracrine IL6 signaling creates an inflammatory microenvironment favors tumor initiation and growth. Citation Format: Mohammed L. Ibrahim, John D. Klement, Daneila Payne, Chunwan Lu, Priscilla S. Redd, Kebin Liu. Chronic inflammation activates IL6 signaling to upregulate DNMT1 and DNMT3b to promote colon tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5744.
- Published
- 2018
49. Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis
- Author
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Dafeng Yang, Wei Xiao, Herbert C. Morse, Darren D Browning, Kebin Liu, Natasha M. Savage, Mohammed L. Ibrahim, Phillip Buckhaults, John D. Klement, Priscilla S. Redd, and Chunwan Lu
- Subjects
0301 basic medicine ,DNA (Cytosine-5-)-Methyltransferase 1 ,STAT3 Transcription Factor ,Colorectal cancer ,Carcinogenesis ,Inflammation ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Gene silencing ,Animals ,DNA (Cytosine-5-)-Methyltransferases ,Gene Silencing ,Colitis ,Promoter Regions, Genetic ,lcsh:QH301-705.5 ,Myeloid-Derived Suppressor Cells ,Epithelial Cells ,DNA Methylation ,medicine.disease ,Ulcerative colitis ,digestive system diseases ,3. Good health ,Interleukin-10 ,Up-Regulation ,Gene Expression Regulation, Neoplastic ,Mice, Inbred C57BL ,Interleukin 10 ,030104 developmental biology ,lcsh:Biology (General) ,030220 oncology & carcinogenesis ,embryonic structures ,Colonic Neoplasms ,Interferon Regulatory Factors ,Myeloid-derived Suppressor Cell ,Cancer research ,IRF8 ,medicine.symptom - Abstract
SUMMARY IL-10 functions as a suppressor of colitis and colitis-associated colon cancer, but it is also a risk locus associated with ulcerative colitis. The mechanism underlying the contrasting roles of IL-10 in inflammation and colon cancer is unknown. We report here that inflammation induces the accumulation of CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) that express high levels of IL-10 in colon tissue. IL-10 induces the activation of STAT3 that directly binds to the Dnmt1 and Dnmt3b promoters to activate their expression, resulting in DNA hypermethylation at the Irf8 promoter to silence IRF8 expression in colon epithelial cells. Mice with Irf8 deleted in colonic epithelial cells exhibit significantly higher inflammation-induced tumor incidence. Human colorectal carcinomas have significantly higher DNMT1 and DNMT3b and lower IRF8 expression, and they exhibit significantly higher IRF8 promoter DNA methylation than normal colon. Our data identify the MDSC-IL-10-STAT3-DNMT3b-IRF8 pathway as a link between chronic inflammation and colon cancer initiation., In Brief Ibrahim et al. report that chronic inflammation induces colonic accumulation of myeloid-derived suppressor cells (MDSCs) that upregulates IL-10. IL-10 directly regulates STAT3 activation to upregulate DNMT3b to silence tumor suppressor IRF8 in colonic epithelial cells. The MDSC-IL-10-STAT3-DNMT3b-IRF8 pathway links chronic inflammation to colon cancer initiation., Graphical Abstract
- Published
- 2018
50. SETD1B compensates loss of IRF8 expression to activate iNOS expression in myeloid-derived suppressor cells through an epigenetic mechanism
- Author
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Priscilla S Redd, Mohammed L Ibrahim, John D Klement, Sarah K Sharman, Amy V Paschall, Dafeng Yang, Asha Nayak-Kapoor, and Kebin Liu
- Subjects
Immunology ,Immunology and Allergy - Abstract
Inducible nitric oxide synthase (iNOS) generates nitric oxide (NO) in myeloid cells that acts as a defense mechanism to suppress invading microorganisms or neoplastic cells. In tumor-bearing mice, elevated iNOS expression is a hallmark of myeloid-derived suppressor cells (MDSCs). MDSCs use NO to nitrate the T cell receptor and STAT1 to inhibit T cell activation and the anti-tumor immune response. The molecular mechanism for iNOS expression regulation in tumor-induced MDSCs is unknown. We show that IRF8 deficiency results in diminished iNOS expression in mature CD11b+Gr1− and immature CD11b+Gr1+ myeloid cells in vivo, indicating that IRF8 is an essential transcriptional activator for iNOS expression in myeloid cells under physiological conditions. Though IRF8 is silenced in tumor-induced MDSCs, iNOS expression is dramatically elevated in tumor-induced MDSCs suggesting that the expression of iNOS is regulated by an IRF8-independent mechanism under pathological conditions. Tumor-induced MDSCs exhibit diminished STAT1 and NF-κB Rel protein level, the essential inducers of iNOS in myeloid cells. Instead, tumor-induced MDSCs show increased SETD1B expression compared to their equivalent in tumor-free mice. Chromatin immunoprecipitation revealed that H3K4me3, a product of SETD1B enzymatic activity, is enriched at the nos2 promoter in tumor-induced MDSCs. Inhibition/silencing of SETD1B diminished iNOS expression in tumor-induced MDSCs. Our data determine that IRF8 is an essential transcriptional activator of iNOS in myeloid cells under physiological conditions. Tumor cells use the SETD1B-H3K4me3 epigenetic axis to compensate for loss of IRF8 expression to activate iNOS expression in MDSCs under pathological conditions.
- Published
- 2017
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