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Loss of Fas function leads to resistance to FasL-induced apoptosis.

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.

Figure S1 shows MDSC profiles in chaetocin-treated tumor-bearing mice

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.

Table S1 contains oligonucleotide sequences

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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.

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.

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.

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.

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.

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.

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.

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.

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.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing‐based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

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.

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing‐based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Cumulative evidence suggests that the heat shock protein 90 (Hsp90) co-chaperone UNC-45 myosin chaperone A (UNC45A) contributes to tumorigenesis and that its expression in cancer cells correlates with proliferation and metastasis of solid tumors. However, the molecular mechanism by which UNC45A regulates cancer cell proliferation remains largely unknown. Here, using siRNA-mediated gene silencing and various human cells, we report that UNC45A is essential for breast cancer cell growth, but is dispensable for normal cell proliferation. Immunofluorescence microscopy, along with gene microarray and RT-quantitative PCR analyses, revealed that UNC45A localizes to the cancer cell nucleus, where it up-regulates the transcriptional activity of the glucocorticoid receptor and thereby promotes expression of the mitotic kinase NIMA-related kinase 7 (NEK7). We observed that UNC45A-deficient cancer cells exhibit extensive pericentrosomal material disorganization, as well as defects in centrosomal separation and mitotic chromosome alignment. Consequently, these cells stalled in metaphase and cytokinesis and ultimately underwent mitotic catastrophe, phenotypes that were rescued by heterologous NEK7 expression. Our results identify a key role for the co-chaperone UNC45A in cell proliferation and provide insight into the regulatory mechanism. We propose that UNC45A represents a promising new therapeutic target to inhibit cancer cell growth in solid tumor types., This work was supported by National Institutes of Health Grant R01 GM102443-01 and Augusta University Startup Funds (to A. C.)

// Amy V. Paschall 1, 2, 3 , Dafeng Yang 1, 3 , Chunwan Lu 1, 3 , Priscilla S. Redd 1, 2, 3 , Jeong-Hyeon Choi 2 , Christopher M. Heaton 3 , Jeffrey R. Lee 3 , Asha Nayak-Kapoor 2, 3 , Kebin Liu 1, 2, 3 1 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA 2 Georgia Cancer Center, Augusta University, Augusta, GA 30912, USA 3 Charlie Norwood VA Medical Center, Augusta, GA 30904, USA Correspondence to: Kebin Liu, email: Kliu@augusta.edu Keywords: CD133, CD24, colon cancer stem cells, 5-Fluorouracil Received: January 14, 2016 Accepted: September 12, 2016 Published: September 21, 2016 ABSTRACT The chemotherapeutic agent 5-Fluorouracil (5-FU) is the most commonly used drug for patients with advanced colon cancer. However, development of resistance to 5-FU is inevitable in almost all patients. The mechanism by which colon cancer develops 5-FU resistance is still unclear. One recently proposed theory is that cancer stem-like cells underlie colon cancer 5-FU resistance, but the phenotypes of 5-FU-resistant colon cancer stem cells are still controversial. We report here that 5-FU treatment selectively enriches a subset of CD133 + colon cancer cells in vitro . 5-FU chemotherapy also increases CD133 + tumor cells in human colon cancer patients. However, sorted CD133 + colon cancer cells exhibit no increased resistance to 5-FU, and CD133 levels exhibit no correlation with colon cancer patient survival or cancer recurrence. Genome-wide analysis of gene expression between sorted CD133 + colon cancer cells and 5-FU-selected colon cancer cells identifies 207 differentially expressed genes. CD24 is one of the genes whose expression level is lower in the CD133 + and 5-FU-resistant colon cancer cells as compared to CD133 + and 5-FU-sensitive colon cancer cells. Consequently, CD133 + CD24 lo cells exhibit decreased sensitivity to 5-FU. Therefore, we determine that CD133 + CD24 lo phenotype defines 5-FU-resistant human colon cancer stem cell-like cells.

Accumulation of myeloid-derived suppressor cells (MDSCs) is a hallmark of cancer. However, the underlying mechanism of MDSC accumulation in the tumor microenvironment (TME) remain incompletely understood. We report that MDSC accumulation is regulated by the TNFα-RIP1-mediated necroptosis. We determined that inhibition of DNMTs with Decitabine (DAC) abolished MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes (CTLs) in tumor-bearing mice. DAC-induced decrease of MDSC accumulation is correlated with increased IRF8 expression in MDSCs. However, DAC also abolished MDSC-like cell accumulation in IRF8 KO mice, indicating that DNA methylation does not regulate MDSC lineage differentiation but mediates MDSC accumulation at post differentiation stage. We determined that DAC decreased MDSC accumulation through increasing cell death and identified RIP1-dependent necroptosis as target of DNA methylation in MDSCs. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter is hypermethylated in tumor-induced MDSCs in vivo. Consequently, DAC dramatically increased TNFα level in MDSCs and neutralizing TNFα significantly decreased MDSC cell death. Furthermore, recombinant TNFα induced MDSC cell death in a does- and RIP1-dependent manner. IL6 which is expressed in MDSCs in tumor-bearing mice and human colorectal cancer patients. Our data shows that the autocrine IL6 activates the STAT3-DNMT axis to epigenetically silence the TNFα-RIP1 necroptosis pathway to sustain MDSC survival and accumulation in cancer. Targeting the TNFα-RIP1 necroptosis is potentially an effective approach to supress MDSCs to activate tumor-reactive CTLs in the TME.

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.

To evade immune clearance by cytotoxic T lymphocytes (CTLs), tumors express immunosuppressive checkpoints such as PD-L1. Tumor-expressed PD-L1 is thought to suppress CTL effector functions by binding CTL-expressed PD-1. To investigate the molecular consequences of CTL engagement with tumor PD-L1, we generated PD-L1 knockouts in murine sarcoma, breast and colorectal cancer cell lines by Crispr/Cas9. Surprisingly, PD-L1 expression conferred no protection from CTL-mediated cytotoxicity in an in vitro co-culture model. Similarly, loss of PD-L1 had no effect on in vivo growth following subcutaneous or orthotopic challenge in immunocompetent mice. However, tumor PD-L1 facilitated metastasis to the lung in both spontaneous and experimental metastasis models. Bulk RNA-seq of tumor-bearing lungs revealed global enrichment in interferon-stimulated gene expression following loss of tumor PD-L1. Flow cytometric analysis and single-cell RNA-sequencing localized this signature to an inflammatory macrophage population that expressed PD-1 and selectively expanded following injection of PD-L1 deficient cell lines. In vitro and in vivo experimentation showed that engagement of macrophage PD-1 by tumor PD-L1 reduced the expression of interferon-stimulated genes and antigen processing machinery. We propose a model by which tumor-expressed PD-L1 regulates CTL function indirectly in the lung microenvironment by suppressing the recruitment and activation of CTLs through myeloid cells.

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.

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.

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.

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&#43Gr1&#43 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.

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.

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.

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.

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.

While current immune checkpoint inhibitors (ICIs) have shown efficacy in a wide variety of human cancers, many patients and cancer types fail to respond to ICI immunotherapy, suggesting the presence of other immunoinhibitory checkpoints. We report here that a deficiency in the transcription factor interferon regulatory factor 8 (IRF8) led to a loss of function of cytotoxic T lymphocytes (CTLs) in both an allograft tumor and a peptide vaccine model. However, a T cell-specific knockout and a mixed chimera model demonstrated that IRF8 suppressed CTL activation through a CTL-extrinsic mechanism. This was found to be through IRF8-dependent suppression of the matricellular protein osteopontin (OPN). During colorectal cancer (CRC) development, IRF8 is downregulated in both tumor-associated CD11b+Ly6CloLy6Ghi myeloid cells and colon epithelial cells, where it represses OPN expression by binding to its promoter. Accordingly, in both mouse CRC models and human CRC patients, transcriptional downregulation of IRF8 was accompanied by an upregulation of OPN at the transcriptional and protein level. Enhanced levels of OPN were found to negatively correlate with patient survival. In vitro stimulation of mouse and human CTLs in the presence of OPN led to decreased activation and interferon gamma secretion. Similarly, deletion of spp1, which encodes for OPN, from murine CT26 colon adenocarcinoma cells caused increased susceptibility to CTL-mediated lysis and enhanced CTL activation. As such, tumor and myeloid-derived OPN may function as a novel immune checkpoint to restrain host CTL activation.

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.

FasL-mediated cytotoxicity is one of the mechanisms that CTLs use to kill tumor cells. However, human colon carcinoma often deregulates the Fas signaling pathway to evade host cancer immune surveillance. We aimed at testing the hypothesis that novel ceramide analogs effectively modulate Fas function to sensitize colon carcinoma cells to FasL-induced apoptosis. We used rational design and synthesized twenty ceramide analogs as Fas function modulators. Five ceramide analogs, IG4, IG7, IG14, IG17, and IG19, exhibit low toxicity and potent activity in sensitization of human colon carcinoma cells to FasL-induced apoptosis. Functional deficiency of Fas limits both FasL and ceramide analogs in the induction of apoptosis. Ceramide enhances FasL-induced activation of the MAPK, NF-κB, and caspase 8 despite induction of potent tumor cell death. Finally, a sublethal dose of several ceramide analogs significantly increased CTL-mediated and FasL-induced apoptosis of colon carcinoma cells. We have therefore developed five novel ceramide analogs that act at a sublethal dose to enhance the efficacy of tumor-specific CTLs, and these ceramide analogs hold great promise for further development as adjunct agents in CTL-based colon cancer immunotherapy.

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.

Programmed cell death-1 (PD-1) is an immunosuppressive receptor expressed on T cells upon activation. Once interacting with its ligands, 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. In fact, PD-L1 is commonly overexpressed on the surface of tumor cells as well as other cells in the tumor microenvironment, such as myeloid-derived suppressor cells. This interface has already been recognized as an effective target for cancer therapy. Blocking the PD-1:PD-L axis would unleash T cells from suppression to maintain their effector function against tumor cells. At least two anti-PD-1 and three anti-PD-L1 blocking antibodies have been approved for human cancer immunotherapy. However, these drugs are only successful in a small number of cancers. Therefore, developing inhibitors that block this axis more effectively and have a wider scale of impact is necessary. We used a molecular docking model and performed virtual chemical library screening to identify small molecule inhibitors of PD-1 and PD-L1 interactions. To this end, we have identified 175 PD-L1 small molecule inhibitors. Functional assays were established with the 4T1 murine breast cancer cell line and a tumor-specific murine T cell line in the presence or absence of the PD-L1 small molecule inhibitors to test if these small molecule PD-L1 inhibitors can increase T cell killing of tumor cells. Cell viability assays were also done to test the toxicity of these inhibitors on tumor cells in the absence of T cells. From these studies, we have identified ten novel PD-L1 small molecule inhibitors that can significantly increase the lytic activity of the tumor-specific cytotoxic T lymphocytes (CTLs) to kill 4T1 tumor cells. These ten PD-L1 small molecule inhibitors show little to no cytotoxicity to 4T1 tumor cells in the absence of the antigen-specific T cells, suggesting that these small molecule inhibitors themselves are not cytotoxic, and therefore are potentially safe as therapeutic agents. These proof of concept studies indicate that these small molecule inhibitors are potentially potent agents for blocking PD-1 and PD-L1 interaction in cancer immunotherapy. Citation Format: Priscilla S. Redd, Chunwan Lu, David Ostrov, Iryna Lebedyeva, Kebin Liu. Interference of PD-1 and PD-L1 interaction with small-molecule inhibitors enhances the efficacy of tumor-specific CTLs [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 1679.

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.

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

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.

Programmed death-ligand 1 (PD-L1) is an inhibitory ligand that binds to PD-1 to suppress T cell activation. PD-L1 is constitutively expressed and inducible in tumor cells, but the expression profiles and regulatory mechanism of PD-L1 in myeloid-derived suppressor cells (MDSCs) are largely unknown. We report that PD-L1 is abundantly expressed in tumor-infiltrating leukocytes in human patients with both microsatellite instable and microsatellite stable colon cancer. About 60% CD11b+CD33+HLA-DR− MDSCs from peripheral blood of human colon cancer patients are PD-L1+. PD-L1+ MDSCs are also significantly higher in tumor-bearing mice than in tumor-free mice. Interestingly, the highest PD-L1+ MDSCs were observed in the tumor microenvironment in which 56–71% tumor-infiltrating MDSCs are PD-L1+ in vivo. In contrast, PD-L1+ MDSCs are significantly less in secondary lymphoid organs and peripheral blood as compared to the tumor tissues, whereas bone marrow MDSCs are essentially PD-L1− in tumor-bearing mice. IFNγ is highly expressed in cells of the tumor tissues and IFNγ neutralization significantly decreased PD-L1+ MDSCs in the tumor microenvironment in vivo. However, IFNγ-activated pSTAT1 does not bind to the cd274 promoter in MDSCs. Instead, pSTAT1 activates expression of IRF1, IRF5, IRF7 and IRF8 in MDSCs, and only pSTAT1-activated IRF1 binds to a unique IRF-binding sequence element in vitro and chromatin in vivo in the cd274 promoter to activate PD-L1 transcription. Our data determine that PD-L1 is highly expressed in tumor-infiltrating MDSCs and in a lesser degree in lymphoid organs, and the pSTAT1-IRF1 axis regulates PD-L1 expression in MDSCs.

The emerging clinical success of checkpoint blockade immunotherapy in human cancer patients has highlighted the critical importance of PD-L1, a ligand for the T cell inhibitory receptor PD-1, as a molecular target in cancer immunotherapy. PD-L1 is constitutively expressed in tumor cells and is also inducible by inflammatory cytokines such as IFNgamma. It has been proposed that tumor cells may sense the elevated IFNgamma from activated host T cells as a “threat” in the tumor microenvironment and adapt it by up-regulating PD-L1. The aim of this study is to elucidate the molecular mechanism underlying IFNgamma regulation of PD-L1 expression in tumor cells. We observed that PD-L1 is constitutively expressed, albeit at low level, in multiple types of cancer cells, including pancreatic, colon, breast, and sarcoma cells. IFNgamma treatment dramatically increased PD-L1 expression level and IFNgamma up-regulates PD-L1 expression through the Jak-STAT1 signaling pathway in vitro and in vivo. Chromatin immunoprecipitation assay did not identify IFNgamma-activated pSTAT1 binding to the pd-l1 promoter. Instead, we observed that IFNgamma activates IRF1 transcription and IRF1 is required for IFNgamma-induced PD-L1 expression. Chromatin immunoprecipitation analysis shows that pSTAT1 is associated with the irf1 but not the pd-l1 promoter. Analysis of the irf1 promoter DNA sequence revealed a pSTAT1-binding consensus sequence, and electrophoretic mobility shift assay indicates that pSTAT1 directly binds to this DNA element of the irf1 promoter. Furthermore, we demonstrated that IRF1 is associated with the pd-l1 promoter chromatin near the irf1 transcription initiation site. The pd-l1 promoter region contains a putative IRF1-binding consensus sequence and electrophoretic mobility shift assay shows that IRF1 binds to this DNA element of the pd-l1 promoter region. Taken together, our data indicate that IFNgamma activates pSTAT1 that binds to the irf1 promoter to activate irf1 transcription. IFNgamma-induced IRF1 then binds to the pd-l1 promoter to activate pd-l1 transcription in tumor cells. Citation Format: Chunwan Lu, Amy V. Paschall, Priscilla S. Redd, Iryna Lebedyeva, Kebin Liu. IFNgamma regulates PD-L1 expression through activating IRF1 transcription in tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2327.