Publications by authors named "John D Klement"

17 Publications

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Expression regulation and function of PD-1 and PD-L1 in T lymphoma cells.

Cell Immunol 2021 Aug 17;366:104397. Epub 2021 Jun 17.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cellimm.2021.104397DOI Listing
August 2021

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

Cancers (Basel) 2021 Feb 28;13(5). Epub 2021 Feb 28.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.

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.
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http://dx.doi.org/10.3390/cancers13051006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957528PMC
February 2021

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

J Immunol 2021 Mar 5;206(6):1395-1404. Epub 2021 Feb 5.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912;

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 IC 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.
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http://dx.doi.org/10.4049/jimmunol.2000500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946776PMC
March 2021

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

Cell Immunol 2021 02 14;360:104260. Epub 2020 Dec 14.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Medical College of Georgia, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. Electronic address:

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 CD8CD20 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 CD8CD20 T subset functions in host cancer immunosurveillance and tumor microenvironment suppresses this T subset through a PD-L1-dependent mechanism.
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http://dx.doi.org/10.1016/j.cellimm.2020.104260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855947PMC
February 2021

Osteopontin: A Key Regulator of Tumor Progression and Immunomodulation.

Cancers (Basel) 2020 Nov 15;12(11). Epub 2020 Nov 15.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA.

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.
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http://dx.doi.org/10.3390/cancers12113379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698217PMC
November 2020

p50 suppresses cytotoxic T lymphocyte effector function to regulate tumor immune escape and response to immunotherapy.

J Immunother Cancer 2020 10;8(2)

Department of Biochemistry and Molecular Biology, Augusta University, Augusta, Georgia, United States

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 in tumor cells increased whereas in T cells decreased tumor growth in preclinical mouse models. Gene expression profiling identified as a p50 target in T cells. p50 binds directly to a previously uncharacterized κB sequence at the promoter in T cells, resulting in repression of 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 expression and T-cell tumor infiltration in human colorectal carcinoma. Furthermore, nivolumab immunotherapy decreased p50 activation and increased expression in human patients with melanoma.

Conclusions: Inflammation activates p50 that binds to the 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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http://dx.doi.org/10.1136/jitc-2020-001365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555101PMC
October 2020

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.

Cancer Res 2020 08 17;80(15):3145-3156. Epub 2020 Jun 17.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.

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 promoter was hypermethylated in tumor-induced MDSCs . DAC treatment dramatically increased TNFα levels in MDSC , and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice . 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. , 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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http://dx.doi.org/10.1158/0008-5472.CAN-19-3670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416440PMC
August 2020

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

Cancer Immunol Immunother 2020 Nov 1;69(11):2233-2245. Epub 2020 Jun 1.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, 1410 Laney Walker Blvd, Augusta, GA, 30912, USA.

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 CD11bLy6GLy6C 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.
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http://dx.doi.org/10.1007/s00262-020-02620-wDOI Listing
November 2020

SUV39H1 regulates human colon carcinoma apoptosis and cell cycle to promote tumor growth.

Cancer Lett 2020 04 12;476:87-96. Epub 2020 Feb 12.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, 30912, USA; Georgia Cancer Center, Medical College of Georgia, Augusta, GA, 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA, 30904, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.canlet.2020.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138631PMC
April 2020

Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes.

J Immunother Cancer 2019 06 22;7(1):157. Epub 2019 Jun 22.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, 30912, USA.

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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http://dx.doi.org/10.1186/s40425-019-0635-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589175PMC
June 2019

SUV39H1 Represses the Expression of Cytotoxic T-Lymphocyte Effector Genes to Promote Colon Tumor Immune Evasion.

Cancer Immunol Res 2019 03 4;7(3):414-427. Epub 2019 Jan 4.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.

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 EC of 0.496 μmol/L for SUV39H1 enzymatic activity. H3K9me3 was enriched in the promoters of , and 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 Our data indicate that SUV39H1 represses CTL effector gene expression and, in doing so, confers colon cancer immune escape.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397681PMC
March 2019

Myeloid-Derived Suppressor Cells Produce IL-10 to Elicit DNMT3b-Dependent IRF8 Silencing to Promote Colitis-Associated Colon Tumorigenesis.

Cell Rep 2018 12;25(11):3036-3046.e6

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta University, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. Electronic address:

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 CD11bGr1 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.
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http://dx.doi.org/10.1016/j.celrep.2018.11.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319669PMC
December 2018

Loss of Fas Expression and Function Is Coupled with Colon Cancer Resistance to Immune Checkpoint Inhibitor Immunotherapy.

Mol Cancer Res 2019 02 14;17(2):420-430. Epub 2018 Nov 14.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.

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 CD133CD24 colon cancer cells and . Consistent of the lower Fas expression level, this subset of CD133CD24Fas colon cancer cells exhibits decreased sensitivity to FasL-induced apoptosis. Furthermore, FasL selectively enriches CD133CD24Fas colon cancer cells. CD133CD24Fas 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 CD133CD24 cells, lower Fas expression level is significantly correlated with decreased survival in patients with human colon cancer. IMPLICATIONS: Our data determine that CD133CD24Fas 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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http://dx.doi.org/10.1158/1541-7786.MCR-18-0455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359951PMC
February 2019

An osteopontin/CD44 immune checkpoint controls CD8+ T cell activation and tumor immune evasion.

J Clin Invest 2018 12 5;128(12):5549-5560. Epub 2018 Nov 5.

Department of Biochemistry and Molecular Biology, and.

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+Ly6CloLy6G+ 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.
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http://dx.doi.org/10.1172/JCI123360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264631PMC
December 2018

H3K4me3 mediates the NF-κB p50 homodimer binding to the promoter to activate PD-1 transcription in T cells.

Oncoimmunology 2018;7(9):e1483302. Epub 2018 Jul 23.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA.

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 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 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 promoter in CD4 and CD8 T cells. Deficiency in p50 results in reduced PD-1 expression in both CD4 and CD8 T cells . Using an 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 . The expression levels of H3K4me3-specific histone methyltransferase increased significantly, resulting in a significant increase in H3K4me3 deposition at the promoter in activated CD4 and CD8 T cells. Inhibition of H3K4me3 significantly decreased p50 binding to the promoter and PD-1 expression in a T cell line. Our findings determine that the p50-H3K4me3 axis regulates transcription activation in activated T cells.
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http://dx.doi.org/10.1080/2162402X.2018.1483302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140591PMC
July 2018

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

J Immunol 2018 07 11;201(1):264-277. Epub 2018 May 11.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912;

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.
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http://dx.doi.org/10.4049/jimmunol.1800129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008224PMC
July 2018

SETD1B Activates iNOS Expression in Myeloid-Derived Suppressor Cells.

Cancer Res 2017 06 5;77(11):2834-2843. Epub 2017 Apr 5.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.

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 CD11bGr1 and immature CD11bGr1 myeloid cells 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. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-2238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495112PMC
June 2017
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