Abstract
Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα–RIP1–mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3–DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation.
These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.
Introduction
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells (IMC) that are induced under various pathologic conditions, including cancer (1). In mice, MDSCs are defined as CD11b+Gr1+ IMC that can be further divided into monocytic MDSCs (M-MDSC) and polymorphonuclear MDSCs (PMN-MDSC; ref. 2). In humans, the phenotype of MDSCs is less defined, but a phenotype of CD11b+CD33+HLA-DR− in combination with other markers is often used to identify MDSCs in patients with human cancer (2). A key function of MDSCs is immune suppression. MDSCs use various mechanisms to suppress activation and function of T cells and NK cells to enable tumor immune escape (3). Furthermore, MDSCs also promote tumor growth through secreting cytokines and growth factors to modulate tumor angiogenesis and to silence tumor suppressors by an epigenetic mechanism (4, 5). Therefore, MDSCs are key targets in cancer immunotherapy.
Immune cell homeostasis is controlled by the balance of cell lineage differentiation and cell death rate. MDSCs are induced from myeloid progenitor cells by proinflammatory mediators in the tumor microenvironment (6). It was also well-documented that transcription factor IRF8 regulates lineage-specific differentiation of CD11b+Gr1+ immature cells from myeloid progenitor cells (7). Regulation of MDSC turnover is not well understood. The fact that MDSCs massively accumulate in tumor-bearing host suggests that MDSCs could have a decreased cell death rate (8–11). Indeed, several cellular pathways have been shown to play important roles in MDSC survival and accumulation (8–10). The TNFR2 signaling upregulates c-FLIP and inhibits caspase-8 activation to promote MDSC survival (12). The IL4Ra signaling is also pivotal for MDSC survival and accumulation (13). Moreover, MDSCs tends to downregulate the death receptor Fas and downstream apoptosis-regulatory mediators Bax and Bcl-xL to escape from CTL-mediated cytotoxicity (14, 15). Autophagy plays opposing role in MDSC survival. A ceramide mimetic has been shown to activate lysosomal cathepsin B and cathepsin D to attenuate autophagy and induces ER stress to suppress MDSCs (16), but autophagy also promote MDSC survival and function (8, 17). On the other hand, MDSCs have increased levels of death receptor TRAIL-R as compared with mature myeloid cells such as neutrophils and monocytes (9), and TRAIL-R2 agonist antibody immunotherapy eliminated MDSCs in tumor-bearing mice and patients with human cancer (18). We report here the identification of DNA methylation as a mechanism of MDSC survival and accumulation. Specifically, we determined that DNA methylation sustains MDSC accumulation through protecting MDSCs from necroptosis at the post lineage differentiation phase in vivo. Mechanistically, we determined that MDSC autocrine IL6 activates the STAT3–DNMT intrinsic signaling pathway to hypermethylate the Tnf promoter to silence Tnf expression, resulting in impaired RIP1-mediated necroptosis to promote MDSC survival and accumulation.
Materials and Methods
Mice and human specimens
IRF8 KO and IRF8-GFP mice were as described (7, 19). C57BL/6 and BALB/c mice were obtained from Jackson Laboratory (Bar Harbor, ME). Use of mice was performed according to approved protocols by institutional animal use and care committee of Augusta University (protocol no. 2008-0162). Peripheral blood specimens from healthy donors were provided by Shepeard Community Blood Bank. Peripheral blood specimens of colorectal cancer patients were collected from Georgia Cancer Center and Charlie Norwood VA Medical Center according to approved protocols by Augusta University Institutional Review Board (protocol no. 1354508-1) and Charlie Norwood VA Medical Center Institutional Review Board (protocol no. 1314554-4).
Mouse tumor models
Colon carcinoma cell line CT26 were obtained directly from ATCC. ATCC has characterized this cell line by morphology, immunology, DNA fingerprint, and cytogenetics. J774M cells were sorted from J774 cells and established as CD11b+Gr1+ cell line. J774M cells were phenotypically and functionally characterized as described previously (20). The AT3 cell line was derived from C57BL/6 mice and was kindly provided by Dr. Scott Abrams (Roswell Park Cancer Institute, Buffalo, NY) and was characterized as described previously (21). All cell lines were stored in aliquots in liquid nitrogen and all cell lines were used in less than 30 passages after obtaining them. These cell lines were not further authenticated by the authors. All cell lines were tested for Mycoplasma every 2 monthly and all cells used in this study were negative for Mycoplasma. AT3 cells were injected to the mammary gland of female C57BL/6 to establish orthotopic tumor. CT26 cells were injected to the right flank of BALB/c mice (2 × 105 cells/mouse) to establish subcutaneous tumor or surgically injected to cecal wall of BALB/c mice (1 × 104 cells/mouse) to establish orthotopic colon tumor.
Reagents
Decitabine (DAC), Necrostatin-1, and Ferrostostatin-1 (Fer-1) were obtained from Sigma-Aldrich. Necrosulfonamide (NSA) was obtained from Calbiochem. Z-DEVD-FMK was obtained from BD Pharmingen. Recombinant mouse TNFα was purchased from R&D Systems. Mouse TNFα neutralization mAb (clone XT3.11) was obtained from Bio X cell. Anti-mouse CD11b, Gr1, CD11c, CD4, CD8, CD19, NK1.1, F4/80, CD45.2, TNFα mAbs, Annexin V, Zobbie Violet, and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from BioLegend. Anti-pSTAT3 was obtained from Cell Signaling Technology. Anti-STAT3 was obtained from BD Biosciences. Propidium iodide (PI) was obtained from MP Biomedicals.
Flow cytometry
Tissue collection and analysis by flow cytometry was performed as described previously (5, 22). Flow cytometry analysis was done on the LSRFortessa for multicolor panels, on the BD FACSCalibur for two color panels, and on the BD Accuri C6 flow cytometer for three color panels.
Targeted CpG site bisulfite DNA sequencing of the Irf8 DNA promoter region in MDSCs
Genomic DNA samples were bisulfite converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) according to the manufacturer's instructions. Eight PCR amplicons were designed to cover the 2,000 bp upstream of the mouse Irf8 gene according to the coordinates for mm10 from NCBI. The covered sequence was slightly extended into the first exon to cover the full CpG island. The resulting amplicons were pooled for harvesting and subsequent barcoding. After barcoding, samples were purified and then prepared for massively parallel sequencing using a MiSeq V2 300bp Reagent Kit (Illumina) and paired-end sequencing protocol according to the manufacturer's guidelines. Sequence reads were identified using standard Illumina base-calling software and then analyzed using a Zymo Research proprietary analysis pipeline by Zymo Research. Low-quality nucleotides and adapter sequences were trimmed off. Sequence reads were aligned back to the reference genome using Bismark (Galaxy tool version: 0.22.1). The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. The genomic coordinates, The methylated CpG count and total CpG count for CpG sites were then calculated and tracked to the genomic coordinates using University of California, Santa Cruz (UCSC) genomic browser.
Genome-wide bisulfite DNA methylation sequencing
CD11b+Gr1+ MDSCs were purified from spleens of AT3 tumor-bearing mice using CD11b MicroBeads and MACS separation column according to the manufacturer's instructions (Miltenyi Biotec Inc.). The purity of CD11b+Gr1+ cells is greater than 95%. Genomic DNA was extracted from the purified MDSCs using Quick-DNA Miniprep Plus Kit (Zymo Research). The genomic DNA was then fragmented, ligated with an adaptor, subjected to bisulfite conversion, size selected for library construction. The library was sequenced using the Illumina next generation high-through sequencing. The bisulfite sequences alignment and methylation call were carried out using Galaxy/Europe Server (usegalaxy.eu). The raw reads were first analyzed by FastQC (Galaxy tool version: 0.72) program to obtain basic statistics of the sequencing runs. The raw reads were then trimmed with Trimmomatic (Galaxy tool version: 0.36.5) to filter out adaptor contents and low-quality reads. The cleaned reads were mapped to the mouse genome (mm10) using Bismark Mapper. PCR duplicates were removed using Bismark Deduplicate (Galaxy tool version: 0.22.1). The methylation call was performed using MethylDeckel (Galaxy tool version: 0.3.0.2). The circos plot was generated using R package circlize (version 0.4.6). Heatmaps were plotted using R package EnrichedHeatmap (version 1.14.0). The entire dataset is deposited in GEO database (accession no. GSE144649).
The Cancer Genome Atlas database genomic data mining
IL6 expression datasets in human colon carcinoma and melanoma were extracted from The Cancer Genome Atlas (TCGA) Colon Cancer (COAD) and melanoma (Melanoma) ploy A+ IlluminaHiSeq pancan normalized RNA-seq dataset using UCSC Xena Cancer Genomics Browser.
Induction of MDSCs from bone marrow
Bone marrow (BM) cells (5 × 105 cells/mL) were cultured with RPMI1640 medium containing 10% (v/v) FCS and supplemented with either 20 ng/mL recombinant mouse GM-CSF or 50% (v/v) of tumor cell–conditioned medium for 5 days.
IL6 protein measurement
Cell culture medium was collected and analyzed for IL6 protein levels using multiplex LEGENDplex Mouse Inflammation Panel (BioLegend) according to the manufacturer's instructions. Data were collected on FACSCalibur two-laser flow cytometer (BD Biosciences) and analyzed using LEGENDplex Data Analysis Software (BioLegend).
Statistical analysis
Statistical analysis was performed using Student t test. A P < 0.05 was taken as statistically significant.
Results
IRF8 is selectively silenced in MDSCs among immune cells in tumor-bearing mice
IRF8 is a transcriptional factor that controls lineage differentiation of monocytes and dendritic cells in humans. Global deletion of Irf8 in mice leads to accumulation of CD11b+Gr1+ IMCs (7) that phenotypically and functionally resemble tumor-induced MDSCs (21). To determine IRF8 expression profiles in immune cells in tumor-bearing host, we made use of the IRF8-GFP reporter mice (19). As expected, CD11b+Gr1+ MDSCs massively accumulate in spleen, blood, and tumor in tumor-bearing mice (Fig. 1A–D; Supplementary Figs. S1 and S2). IRF8 expression level is significantly decreased in MDSCs and F4/80 macrophage cells, but not in other subset of myeloid cells, T cells, B cells, and NK cells (Supplementary Fig. S1A–S1H and S2A–S2H).
Inhibition of DNA methylation reduces MDSC accumulation and increased antigen-specific CTL activation in vivo
IRF8 is an essential regulator of myeloid cell lineage differentiation from hematopoietic stem and myeloid progenitor cells (7). DNA methylation is a key regulator of myeloid cell differentiation from hematopoietic stem and progenitor cells (23) and IRF8 is regulated by its promoter DNA methylation in myeloid cells (24). We therefore aimed at testing the hypothesis that tumor cells use DNA methylation to silence IRF8 expression to induce MDSC accumulation. The chemotherapeutic agent DAC was used as a DNMT inhibitor here. Tumor-bearing mice were treated with DAC and analyzed for MDSC accumulation. DAC treatment at a dose of 1 mg/kg body weight significantly decreased MDSC levels in spleens but not in the tumors in the tumor-bearing mice (Supplementary Fig. S3A and S3B). However, increasing DAC dose to 2 mg/kg body weight significantly decreased MDSC levels in spleens, peripheral blood, and tumors in the mammary carcinoma AT3 tumor-bearing mice (Fig. 1A–C). The colon carcinoma CT26 tumor model was then used as a complementary model in mice that were then treated with DAC. As observed in the AT3 tumor-bearing mice, DAC therapy at a dose of 2 mg/kg body weight also significantly decreased MDSC accumulation in the tumor (Fig. 1D). These findings indicate that DNA methylation maintains MDSC accumulation in tumor-bearing mice.
The CT26 tumor-bearing mice were then analyzed for T-cell activation. DAC therapy significantly increased tumor-infiltrating CD8+ T cells (Supplementary Fig. S4A). CT26 tumor cells harbor the viral gp70 protein, which may serve as an antigen to generate antigen-specific T cells. The tumors were analyzed with a tetramer that is specific for an epitope (AH1) of the gp70 protein. It is clear that DAC therapy significantly and dramatically increased the tumor-infiltrating antigen-specific CTLs in the tumor-bearing mice (Supplementary Fig. S4A and S4B). These findings indicate that DAC therapy is effective in suppressing MDSC accumulation and increasing antigen-specific CTL activation.
DNA methylation regulates MDSC accumulation at the post-differentiation phase
To determine whether inhibition of DNA methylation activates IRF8 expression to suppress MDSC differentiation, we analyzed the Irf8 promoter DNA methylation. The mouse Irf8 promoter contains a CpG island (Fig. 2A). Bisulfite DNA sequencing of tumor-induced MDSCs revealed that the CpG island region and a region upstream of the Irf8 transcription start site are methylated (Fig. 2A) and DAC treatment induced the Irf8 promoter demethylation (Fig. 2B). Consistent with the methylation status of tumor-induced MDSCs, DAC treatment significantly increased IRF8+ MDSC cells in the tumor microenvironment (Fig. 2C). We then treated IRF8 KO mice with DAC. The rationale is that if DAC targets MDSC accumulation at the differentiation stage through upregulating IRF8, then DAC therapy should not affect the CD11b+Gr1+ IMC accumulation in IRF8 KO mice since IRF8 expression and function is lost in IRF8 KO mice. However, DAC therapy still significantly decreased MDSC accumulation in IRF8 KO mice (Fig. 2D). These findings indicate that DNA methylation regulates MDSC accumulation post-MDSC lineage differentiation.
DNA methylation maintains tumor-induced MDSC survival in vivo
The above finding that DNA methylation regulates MDSC accumulation at the after lineage differentiation stage suggests that DNA methylation might regulate MDSC survival. To test this hypothesis, the AT3 tumor-bearing and CT26 tumor-bearing mice were treated with DAC. Spleen cells from the tumor-bearing mice were then analyzed for cell death. Cell death was measured by % PI+ or DAPI+ and % Annexin V+ cells of the gated CD11b+Gr1+ cells. DAC therapy significantly increased % DAPI+, % Annexin V+DAPI−, and % Annexin V+DAPI+ cells in the CD11b+Gr1+ spleen cells in both AT3 tumor-bearing (Fig. 3A and B) and CT26 tumor-bearing (Fig. 3C and D) mice. The effect of DAC on tumor-infiltrating MDSC cell death was not measured due to technical difficulties. These observations indicate that DNA methylation regulates MDSC accumulation through enhancing MDSC survival.
DNA methylation impairs necroptosis to promote MDSC survival
To determine which cell death pathway underlies DNA methylation-dependent MDSC survival, we made use of the MDSC-like J774M cells. J774M cells have a CD11b+Gr1+ phenotype and exhibit potent suppressive activity against CTL activation and proliferation (20), and thus phenotypically and functionally resemble MDSCs. Treatment of J774M cells with DNA methylation inhibitor DAC induced cell death in a dose-dependent manner in vitro (Fig. 4A and B). To determine which cell death pathways are activated by DAC, we treated cells with DAC in the presence of cell death pathway-selective inhibitors. Only RIP1-specific inhibitor significantly decreased DAC-induced cell death in J774M cells (Fig. 4C and D). This observation indicates that DAC induces J774M cell death through RIP1-mediated necroptosis in vitro, suggesting that DNA methylation regulates MDSC necroptosis (Fig. 4C and D).
DNA methylation and necroptosis in tumor-induced MDSCs
We next performed genome-wide DNA methylation analysis of tumor-induced MDSCs. Purified MDSCs from spleens of AT3 tumor-bearing mice were used for genomic DNA preparation that was subjected to bisulfite conversion and genome-wide high throughout DNA sequencing. DNA hypermethylation occurs throughout the entire genome in tumor-induced MDSCs (Fig. 5A). The promoter regions of several genes with known functions in cell death were hypermethylated (Fig. 5B). Particularly, the promoters of Tnf, Ripk1, and Ripk3 were hypermethylated (Fig. 5B). Analysis of the promoter DNA sequences revealed that the Tnf promoter lacks a CpG island, whereas both Ripk1 and Ripk3 promoters contains CpG islands (Fig. 5C). However, all three promoters are hypermethylated (Fig. 5D).
DNA methylation sustains MDSC survival through impairing the TNFα–RIP1 necroptosis pathway
The above findings suggest that DNA methylation maintain MDSC survival at least in part through silencing the RIP1-dependent necroptosis pathway. TNFα is a potent necroptosis inducer (25) and its promoter DNA is hypermethylated in tumor-induced MDSCs (Fig. 5D). We then analyzed TNFα expression in J774M cells. Consistent with the observation that the Tnf promoter is hypermethylated (Fig. 5B and D), DAC treatment significantly increased TNFα expression level in J774M cells (Fig. 6A). Furthermore, TNFα protein induced J774M cell death in a dose-dependent manner (Fig. 6B), and neutralizing TNFα diminished DAC-induced cell death (Fig. 6C). To determine whether this in vitro finding can be extended to in vivo MDSC survival regulation, tumor-bearing mice with treated with IgG control and TNFα neutralization mAb, respectively. Neutralizing TNFα significantly increased tumor growth (Supplementary Fig. S5A) and increased MDSC accumulation in the tumor (Supplementary Fig. S5B and S5C). Although the promoters of Ripk1 and Ripk3 are hypermethylated (Fig. 5B–D), DAC treatment failed to increase RIP1 expression levels in J774M cells (Supplementary Figs. S6A and S6B), suggesting that DNA methylation is not a major mechanism underlying RIP1 expression regulation in MDSCs.
IL6 intrinsic signaling silences the TNFα–RIP1 necroptosis pathway
DNMT1 and DNMT3b are transcriptionally regulated by activated STAT3 (5, 26). IL6 is one of the STAT3 inducers (27, 28). It is therefore likely that MDSCs may express IL6 to activate DNMTs to hypermethylate TNFα. To extend these findings to myeloid cells, we analyzed IL6 expression levels in tumor-infiltrating MDSCs in vivo. Although only weak IL6 expression was detected in tumor cells, the tumor-infiltrating MDSCs express relatively high level of IL6 (Fig. 7A and B). To determine the human relevance, we collected peripheral blood specimens from healthy donors and colorectal cancer patients (Supplementary Tables S1 and S2). The CD11b+CD33+HLA-DR− MDSCs were gated and analyzed for IL6 level. As expected, patients with human colorectal cancer have a significantly higher level of MDSC accumulation than healthy donors (Fig. 7C, D, and F). IL6 protein level is significantly higher in MDSCs from patients with colorectal cancer as compared with health donors (Fig. 7C, E, and G). Analysis of the human colon cancer dataset revealed that IL6 expression level is significantly higher in human colon carcinomas than in the normal colon tissues (Supplementary Fig. S7A). No enough metastatic colon carcinoma data are available from the TCGA database, but analysis of human melanoma IL6 expression level indicates that there is no significant difference in IL6 expression between primary and metastatic human melanoma (Supplementary Fig. S7B).
Analysis of culture supernatant revealed that the MDSC-like J774M cells secrete abundant IL6 protein under in vitro culture conditions, whereas the in vitro cultured tumor cells have undetectable IL6 protein under in vitro culture conditions (Fig. 7H). However, bone marrow-derived MDSCs induced tumor cell-conditioned medium and GM-CSF also secrete abundant IL6 protein in vitro (Fig. 7H). IL6 is a potent activator of STAT3 (29), and STAT3 is known to upregulate DNMT1 and DNMT3b expression (5, 26–28). The high IL6 protein level in MDSCs suggest that MDSCs may use autocrine IL6 to activate STAT3 to upregulate DNMT1 and DNMT3b. To test this hypothesis, J774M cells were treated with IL6 and analyzed STAT3 activation and expression of DNMT1 and DNMT3b. Indeed, IL6 treatment induced STAT3 activation (Fig. 7I) and significantly increased DNMT1 and DNMT3b expression in J774M cells (Fig. 7J). At the same time, IL6 treatment also decreased TNFα production in J774M cells (Fig. 7K) and increased J774M cell proliferation (Fig. 7L). Taken together, our data indicate that MDSC autocrine IL6–STAT3–DNMT–TNFα–RIP1 pathway promotes MDSC survival and accumulation.
Discussion
IRF8 is an essential lineage-specific transcription factor for myeloid cell differentiation and maturation. Loss of IRF8 expression or function leads to interrupted myelopoiesis in humans (30) and accumulation of IMCs in mice with a CD11b+Gr1+ phenotype (7). IRF8 therefore functions as a suppressor of CD11b+Gr1+ IMCs under physiologic conditions (7). One phenotype of tumor-bearing mice is also the accumulation of CD11b+Gr1+ MDSCs (31) and IRF8 is silenced in the tumor-induced MDSCs (21, 32). IRF8 therefore also acts as a suppressor of CD11b+Gr1+ MDSC differentiation under pathologic conditions (21). In addition, IRF8 regulates expression of apoptosis regulatory factors, such as caspase-3, Bcl2, Bcl-xL, Bax, and Fas, in myeloid cells and MDSCs to promote myeloid cells/MDSC apoptosis (14, 32–34). IRF8 therefore functions in regulation of both myeloid cell lineage differentiation and apoptosis. In this study, we determined that the Irf8 promoter DNA is hypermethylated in MDSCs and DAC treatment decreased the Irf8 promoter DNA methylation in tumor-bearing mice in vivo. However, inhibition of DNA also diminished CD11b+Gr1+ cell accumulation in IRF8 KO mice in which IRF8 function is lost. Our findings thus suggest that DNA methylation regulates MDSC accumulation not at the stage of MDSC differentiation from myeloid progenitor cells but rather at the stage post-MDSC lineage differentiation. Our findings also indicate that DNA methylation regulates MDSC death through an IRF8-independent mechanism.
Cellular turnover plays a key role in MDSC accumulation and function (8, 9, 11, 12, 35). Although apoptosis and autophagy are known to be involved in MDSC survival (9, 14, 32), these cell death mechanisms involve extrinsic ligands such as TRAIL and FasL (9, 14). In this study, we extended the regulation of MDSC cell death to an epigenetic mechanism. We determined that DNA methylation is essential for MDSC survival and accumulation in tumor-bearing mice. Furthermore, we extended the cell death pathways to necroptosis in MDSCs. We determined that MDSCs can be induced to die in a RIP1-dependent manner. It is known that TNFα is a potent inducer of RIP1-dependent necroptosis (36, 37). Consistent with this phenomenon, we observed that inhibition of DNA methylation significantly increased TNFα expression in MDSCs and MDSC-produced TNFα induces MDSC necroptosis through an autocrine mechanism. We should emphasize that the CD11b+Gr1+ MDSCs are still sensitive to cell death induction, such as to TRAIL- and FasL-induced apoptosis (9, 18) and TNFα-induced necroptosis as observed in this study. However, CD11b+Gr1+ myeloid cells in tumor-bearing hosts and in IRF8 KO mice exhibit significantly less spontaneous apoptosis in vivo and are less sensitive to FasL-induced apoptosis as compared with CD11b+Gr1+ myeloid cells in tumor-free and wild-type mice (32). This relative decrease in sensitivity to cell death induction may have a significant impact on MDSC accumulation. Several cell death pathways regulate MDSC turnover (8–16). The TNFα-induced necroptosis is therefore one of the mechanisms that regulate MDSC turnover. The relative contribution of this TNFα–RIP1 necroptosis pathway in overall MDSC turnover in the tumor microenvironment requires further study.
Apoptosis and necroptosis attenuate each other in mammalian cell death regulation (38). In this study, we observed that inhibition of DNA methylation induced Annexin V+ cells in MDSCs and RIP1-selective inhibitor necropstain-1 also decreased this Annexin V+ population in DAC-treated cells. It was recently proposed that cells can die through RIP1-dependent apoptosis (RDA) (39). It is therefore possible that this Annexin V+ cells might undergo RDA, which also require further study.
IL6 is well known to promote MDSC differentiation (6) and tumor cell-produced IL6 exhibits immune suppressive function and promotes tumor stemness (40–42). Here, we observed that MDSCs is the major producer of IL6 and express much higher level of IL6 than tumor cells in tumor-bearing mice. Furthermore, IL6 expression level is significantly elevated in MDSCs from cancer patients. Although STAT3 can be activated by several cytokines (43, 44), it is known that IL6 activates STAT3 and the activated STAT3 activates both histone methyltransferase and DNA methyltransferases to induce an epigenetic silencing program (27–29, 45–47). We extended this phenomenon to MDSCs. We determined that IL6 is dramatically upregulated in MDSCs and autocrine IL6 intrinsic signaling activates STAT3 to upregulated DNMT1 and DNMT3b in MDSCs. Therefore, we conclude that the IL6–STAT3–DNMT1/3b epigenetic pathway silencing the TNFα–RIP1 necroptosis pathway to increase MDSC survival to maintain MDSC accumulation in the tumor-bearing host.
A recent genome-wide CRISPR/Cas9 screen uncovered the TNFα level is low in tumor and TNF antitumor activity is only limited in tumors at baseline and in immune checkpoint blockade immunotherapy (ICB) nonresponders (48). It is possible that TNFα is silenced by its promoter DNA methylation in the tumor microenvironment. Pharmacologic sensitization of tumor cells to the TNFα–RIP1 pathway significantly increased the efficacy of ICB immunotherapy (48). Furthermore, a multicenter phase III clinical trial revealed that DAC induced suppression of MDSC-like neutrophils (49). Pathologically activated neutrophils are a subset of PMN-MDSCs that contribute to the failure of cancer therapies and are associated with poor clinical outcomes (50). Therefore, DAC-mediated epigenetic reactivation of TNFα may have dual efficacies: activate the MDSC intrinsic TNFα–RIP1 pathway to suppress MDSC accumulation and enhance the tumor intrinsic TNFα signaling pathway to augment the sensitivity of tumor cells to ICB immunotherapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
A. Smith: Conceptualization, data curation, investigation, visualization, methodology, writing-original draft, writing-review and editing. C. Lu: Conceptualization, data curation, investigation. D. Payne: Investigation, methodology. A. Paschall: Conceptualization, formal analysis, funding acquisition, investigation, methodology. J. Klement: Investigation, methodology. P. Redd: Investigation, methodology. M. Ibrahim: Investigation, methodology. D. Yang: Investigation, methodology, project administration. Q. Han: Formal analysis. Z. Liu: Formal analysis, investigation. H. Shi: Conceptualization, formal analysis, writing-review and editing. T. Hartney: Conceptualization, writing-review and editing. A. Nayak-Kapoor: Conceptualization, resources, data curation, writing-review and editing. K. Liu: Conceptualization, formal analysis, supervision, funding acquisition, writing-original draft, project administration, writing-review and editing.
Acknowledgments
We thank Dr. Roni J. Bollag at the Georgia Cancer Center Biorepository for advice and for providing human blood specimens. We also thank Dr. Rafal Pacholczyk for advice and assistance in cytokine analysis. Grant support from US Department of Veterans Affairs Merit Review Award (I01 CX001364 to K. Liu) and NIH (R01 CA133085, R01 CA182518, and R01 CA227433 to K. Liu; 1F30CA236436 to J. Klement; and 1F31 AI120487 to A. Paschall).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.