The transcription factor TCF-1 enforces commitment to the innate lymphoid cell lineage.

Innate lymphoid cells (ILCs) play important functions in immunity and tissue homeostasis, but their development is poorly understood. Through the use of single-cell approaches, we examined the transcriptional and functional heterogeneity of ILC progenitors, and studied the precursor-product relationships that link the subsets identified. This analysis identified two successive stages of ILC development within T cell factor 1-positive (TCF-1+) early innate lymphoid progenitors (EILPs), which we named 'specified EILPs' and 'committed EILPs'. Specified EILPs generated dendritic cells, whereas this potential was greatly decreased in committed EILPs. TCF-1 was dispensable for the generation of specified EILPs, but required for the generation of committed EILPs. TCF-1 used a pre-existing regulatory landscape established in upstream lymphoid precursors to bind chromatin in EILPs. Our results provide insight into the mechanisms by which TCF-1 promotes developmental progression of ILC precursors, while constraining their dendritic cell lineage potential and enforcing commitment to ILC fate.

CD47-Dependent Regulation of Immune Checkpoint Gene Expression and MYCN mRNA Splicing in Murine CD8 and Jurkat T Cells.

Elevated expression of CD47 in some cancers is associated with poor survival related to its function as an innate immune checkpoint when expressed on tumor cells. In contrast, elevated CD47 expression in cutaneous melanomas is associated with improved survival. Previous studies implicated protective functions of CD47 expressed by immune cells in the melanoma tumor microenvironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell function that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was upregulated in CD47-deficient cells but downregulated in CD47-deficient cells following activation. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms, as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified a significant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Thrombospondin-1 inhibited the induction of TIGIT, CD40LG, and MCL1 mRNAs following T cell activation in vitro. Increased mRNA expression of these T cell transcripts and MYCN in melanomas was associated with improved overall survival.

5-Azacytidine prevents relapse and produces long-term complete remissions in leukemia xenografts treated with Moxetumomab pasudotox.

Moxetumomab pasudotox (Moxe) is a chimeric protein composed of an anti-CD22 Fv fused to a portion of Pseudomonas exotoxin A and kills CD22-expressing leukemia cells. It is very active in hairy-cell leukemia, but many children with relapsed/refractory acute lymphoblastic leukemia (ALL) either respond transiently or are initially resistant. Resistance to Moxe in cultured cells is due to low expression of diphthamide genes (DPH), but only two of six ALL blast samples from resistant patients had low DPH expression. To develop a more clinically relevant model of resistance, we treated NSG mice bearing KOPN-8 or Reh cells with Moxe. More than 99.9% of the cancer cells were killed by Moxe, but relapse occurred from discrete bone marrow sites. The resistant cells would no longer grow in cell culture and showed major chromosomal changes and changes in phenotype with greatly decreased CD22. RNA deep sequencing of resistant KOPN-8 blasts revealed global changes in gene expression, indicating dedifferentiation toward less-mature B cell precursors, and showed an up-regulation of myeloid genes. When Moxe was combined with 5-azacytidine, resistance was prevented and survival increased to over 5 months in the KOPN-8 model and greatly improved in the Reh model. We conclude that Moxe resistance in mice is due to a new mechanism that could not be observed using cultured cells and is prevented by treatment with 5-azacytidine.

Control of Regulatory T Cell Differentiation by the Transcription Factors Thpok and LRF.

The CD4+ lineage-specific transcription factor Thpok is required for intrathymic CD4+ T cell differentiation and, together with its homolog LRF, supports CD4+ T cell helper effector responses. However, it is not known whether these factors are needed for the regulatory T cell (Treg) arm of MHC class II responses. In this study, by inactivating in mice the genes encoding both factors in differentiated Tregs, we show that Thpok and LRF are redundantly required to maintain the size and functions of the postthymic Treg pool. They support IL-2-mediated gene expression and the functions of the Treg-specific factor Foxp3. Accordingly, Treg-specific disruption of Thpok and Lrf causes a lethal inflammatory syndrome similar to that resulting from Treg deficiency. Unlike in conventional T cells, Thpok and LRF functions in Tregs are not mediated by their repression of the transcription factor Runx3. Additionally, we found that Thpok is needed for the differentiation of thymic Treg precursors, an observation in line with the fact that Foxp3+ Tregs are CD4+ cells. Thus, a common Thpok-LRF node supports both helper and regulatory arms of MHC class II responses.

Detailed longitudinal sampling of glioma stem cells in situ reveals Chr7 gain and Chr10 loss as repeated events in primary tumor formation and recurrence.

Intratumoral heterogeneity at the genetic, epigenetic, transcriptomic, and morphologic levels is a commonly observed phenomenon in many aggressive cancer types. Clonal evolution during tumor formation and in response to therapeutic intervention can be predicted utilizing reverse engineering approaches on detailed genomic snapshots of heterogeneous patient tumor samples. In this study, we developed an extensive dataset for a GBM case via the generation of polyclonal and monoclonal glioma stem cell lines from initial diagnosis, and from multiple sections of distant tumor locations of the deceased patient's brain following tumor recurrence. Our analyses revealed the tissue-wide expansion of a new clone in the recurrent tumor and chromosome 7 gain and chromosome 10 loss as repeated genomic events in primary and recurrent disease. Moreover, chromosome 7 gain and chromosome 10 loss produced similar alterations in mRNA expression profiles in primary and recurrent tumors despite possessing other highly heterogeneous and divergent genomic alterations between the tumors. We identified ETV1 and CDK6 as putative candidate genes, and NFKB (complex), IL1B, IL6, Akt and VEGF as potential signaling regulators, as potentially central downstream effectors of chr7 gain and chr10 loss. Finally, the differences caused by the transcriptomic shift following gain of chromosome 7 and loss of chromosome 10 were consistent with those generally seen in GBM samples compared to normal brain in large-scale patient-tumor data sets.

Differential regulation by CD47 and thrombospondin-1 of extramedullary erythropoiesis in mouse spleen.

Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in cd47-/- spleens but significantly depleted in thbs1-/- spleens. Single cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in thbs1-/- spleens relative to basal levels in wild type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.

Effects of a humanized CD47 antibody and recombinant SIRPα proteins on triple negative breast carcinoma stem cells.

Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.

Aire drives steroid hormone biosynthesis by medullary thymic epithelial cells.

Thymic epithelial cells (TECs) produce glucocorticoids, which antagonize negative selection of autoreactive thymocytes and promote a competent T cell antigen-specific repertoire. To characterize their source, we generated a knock-in reporter mouse in which endogenous Cyp11b1, the final enzyme in de novo production of active glucocorticoids, was fluorescently tagged with mScarlet. Here, we find that Cyp11b1 is expressed in medullary TECs (mTECs) but not cortical TECs or other cells in the thymus. A distinct characteristic of mTECs is the presence of Aire, a transcription factor that drives expression of tissue-restricted antigens (TRAs) important for establishing immune tolerance. Cyp11b1 expression was highest in Aire+ mTECs, lower in post-Aire mTECs, and absent in mTECs of Aire-deficient mice. Transcriptomic analyses found that multiple enzymatic biosynthetic pathways are expressed specifically in mTECs and are also Aire dependent. In particular, we found that the thymus expresses messenger RNA for enzymes that catalyze production of many bioactive steroids and that glucocorticoids and sex steroids were secreted by cultured thymi. Expression of the transcripts for these genes and production of their final steroid products were markedly reduced in the absence of Aire. Thus, in addition to its well-established role in inducing TRAs that promote negative selection, Aire has an additional and contrary function of inducing glucocorticoids that antagonize negative selection, which together may expand and enhance the TCR repertoire. Furthermore, because Aire drives expression of multiple enzymes responsible for production of other non-gene-encoded bioactive molecules, it might have yet other roles in thymus development and function.

Tumors produce glucocorticoids by metabolite recycling, not synthesis, and activate Tregs to promote growth.

Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, encoded by Hsd11b1). Here, we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth, but reduced in vivo tumor progression, which corresponded with increased frequencies of CD8+ tumor-infiltrating lymphocytes (TILs) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of conventional T cell activation in tumor-infiltrating Tregs. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11ß-HSD1 reduced tumor growth to the same degree as gene knockout and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11ß-HSD1 is well tolerated in clinical studies, these data suggest that targeting 11ß-HSD1 may be a beneficial adjunct in cancer therapy.

Development of a bicistronic anti-CD19/CD20 CAR construct including abrogation of unexpected nucleic acid sequence deletions.

To address CD19 loss from lymphoma after anti-CD19 chimeric antigen receptor (CAR) T cell therapy, we designed a bicistronic construct encoding an anti-CD19 CAR and an anti-CD20 CAR. We detected deletions from the expected bicistronic construct sequence in a minority of transcripts by mRNA sequencing. Loss of bicistronic construct transgene DNA was also detected. Deletions of sequence were present at much higher frequencies in transduced T cell mRNA versus gamma-retroviral vector RNA. We concluded that these deletions were caused by intramolecular template switching of the reverse transcriptase enzyme during reverse transcription of gamma-retroviral vector RNA into transgene DNA of transduced T cells. Intramolecular template switching was driven by repeated regions of highly similar nucleic acid sequence within CAR sequences. We optimized the sequence of the bicistronic CAR construct to reduce repeated regions of highly similar sequences. This optimization nearly eliminated sequence deletions. This work shows that repeated regions of highly similar nucleic acid sequence must be avoided in complex CAR constructs. We further optimized the bicistronic construct by lengthening the linker of the anti-CD20 single-chain variable fragment. This modification increased CD20-specific interleukin-2 release and reduced CD20-specific activation-induced cell death. We selected an optimized anti-CD19/CD20 bicistronic construct for clinical development.

IL-27 induces an IFN-like signature in murine macrophages which in turn modulate colonic epithelium.

Mucosal delivery of IL-27 has been shown to have a therapeutic benefit in murine models of inflammatory bowel disease (IBD). The IL-27 effect was associated with phosphorylated STAT1 (pSTAT1), a product of IL27 receptor signaling, in bowel tissue. To determine whether IL-27 acted directly on colonic epithelium, murine colonoids and primary intact colonic crypts were shown to be unresponsive to IL-27 in vitro and to lack detectable IL-27 receptors. On the other hand, macrophages, which are present in inflamed colon tissue, were responsive to IL-27 in vitro. IL-27 induced pSTAT1 in macrophages, the transcriptome indicated an IFN-like signature, and supernatants induced pSTAT1 in colonoids. IL-27 induced anti-viral activity in macrophages and MHC Class II induction. We conclude that the effects of mucosal delivery of IL-27 in murine IBD are in part based on the known effects of IL27 inducing immunosuppression of T cells mediated by IL-10. We also conclude that IL-27 has potent effects on macrophages in inflamed colon tissue, generating mediators that in turn act on colonic epithelium.

HIV vaccine candidate efficacy in female macaques mediated by cAMP-dependent efferocytosis and V2-specific ADCC.

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14+ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14+ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

Loss of CD47 alters CD8+ T cell activation in vitro and immunodynamics in mice.

CD47 has established roles in the immune system for regulating macrophage phagocytosis and lymphocyte activation, with growing evidence of its cell-intrinsic regulatory roles in natural killer and CD8+ T cells. CD47 limits antigen-dependent cytotoxic activities of human and murine CD8+ T cells, but its role in T cell activation kinetics remains unclear. Using in vitro and in vivo models, we show here that CD47 differentially regulates CD8+ T cell responses to short- versus long-term activation. Although CD47 was not required for T cell development in mice and early activation in vitro, short-term stimuli elevated pathogen-reactive gene expression and enhanced proliferation and the effector phenotypes of Cd47-deficient relative to Cd47-sufficient CD8+ T cells. In contrast, persistent TCR stimulation limited the effector phenotypes of Cd47 -/- CD8+ T cells and enhanced their apoptosis signature. CD8+ T cell expansion and activation in vivo induced by acute lymphocytic choriomeningitis virus (LCMV) infection did not differ in the absence of CD47. However, the frequency and effector phenotypes of Cd47-/- CD8+ T cells were constrained in chronic LCMV-infected as well as in mice bearing B16 melanoma tumors. Therefore, CD47 regulates CD8+ T cell activation, proliferation, and fitness in a context-dependent manner.

Remodeling the tumor microenvironment via blockade of LAIR-1 and TGF-ß signaling enables PD-L1-mediated tumor eradication.

Collagens in the extracellular matrix (ECM) provide a physical barrier to tumor immune infiltration, while also acting as a ligand for immune inhibitory receptors. Transforming growth factor-ß (TGF-ß) is a key contributor to shaping the ECM by stimulating the production and remodeling of collagens. TGF-ß activation signatures and collagen-rich environments have both been associated with T cell exclusion and lack of responses to immunotherapy. Here, we describe the effect of targeting collagens that signal through the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) in combination with blockade of TGF-ß and programmed cell death ligand 1 (PD-L1). This approach remodeled the tumor collagenous matrix, enhanced tumor infiltration and activation of CD8+ T cells, and repolarized suppressive macrophage populations, resulting in high cure rates and long-term tumor-specific protection across murine models of colon and mammary carcinoma. The results highlight the advantage of direct targeting of ECM components in combination with immune checkpoint blockade therapy.

Urine Single-Cell RNA Sequencing in Focal Segmental Glomerulosclerosis Reveals Inflammatory Signatures.

INTRODUCTION: Individuals with focal segmental glomerular sclerosis (FSGS) typically undergo kidney biopsy only once, which limits the ability to characterize kidney cell gene expression over time. METHODS: We used single-cell RNA sequencing (scRNA-seq) to explore disease-related molecular signatures in urine cells from subjects with FSGS. We collected 17 urine samples from 12 FSGS subjects and captured these as 23 urine cell samples. The inflammatory signatures from renal epithelial and immune cells were evaluated in bulk gene expression data sets of FSGS and minimal change disease (MCD) (The Nephrotic Syndrome Study Network [NEPTUNE] study) and an immune single-cell data set from lupus nephritis (Accelerating Medicines Partnership). RESULTS: We identified immune cells, predominantly monocytes, and renal epithelial cells in the urine. Further analysis revealed 2 monocyte subtypes consistent with M1 and M2 monocytes. Shed podocytes in the urine had high expression of marker genes for epithelial-to-mesenchymal transition (EMT). We selected the 16 most highly expressed genes from urine immune cells and 10 most highly expressed EMT genes from urine podocytes as immune signatures and EMT signatures, respectively. Using kidney biopsy transcriptomic data from NEPTUNE, we found that urine cell immune signature and EMT signature genes were more highly expressed in FSGS biopsies compared with MCD biopsies. CONCLUSION: The identification of monocyte subsets and podocyte expression signatures in the urine samples of subjects with FSGS suggests that urine cell profiling might serve as a diagnostic and prognostic tool in nephrotic syndrome. Furthermore, this approach may aid in the development of novel biomarkers and identifying personalized therapies targeting particular molecular pathways in immune cells and podocytes.

Using pre-existing microarray datasets to increase experimental power: application to insulin resistance.

Although they have become a widely used experimental technique for identifying differentially expressed (DE) genes, DNA microarrays are notorious for generating noisy data. A common strategy for mitigating the effects of noise is to perform many experimental replicates. This approach is often costly and sometimes impossible given limited resources; thus, analytical methods are needed which increase accuracy at no additional cost. One inexpensive source of microarray replicates comes from prior work: to date, data from hundreds of thousands of microarray experiments are in the public domain. Although these data assay a wide range of conditions, they cannot be used directly to inform any particular experiment and are thus ignored by most DE gene methods. We present the SVD Augmented Gene expression Analysis Tool (SAGAT), a mathematically principled, data-driven approach for identifying DE genes. SAGAT increases the power of a microarray experiment by using observed coexpression relationships from publicly available microarray datasets to reduce uncertainty in individual genes' expression measurements. We tested the method on three well-replicated human microarray datasets and demonstrate that use of SAGAT increased effective sample sizes by as many as 2.72 arrays. We applied SAGAT to unpublished data from a microarray study investigating transcriptional responses to insulin resistance, resulting in a 50% increase in the number of significant genes detected. We evaluated 11 (58%) of these genes experimentally using qPCR, confirming the directions of expression change for all 11 and statistical significance for three. Use of SAGAT revealed coherent biological changes in three pathways: inflammation, differentiation, and fatty acid synthesis, furthering our molecular understanding of a type 2 diabetes risk factor. We envision SAGAT as a means to maximize the potential for biological discovery from subtle transcriptional responses, and we provide it as a freely available software package that is immediately applicable to any human microarray study.

Tumour-targeted interleukin-12 and entinostat combination therapy improves cancer survival by reprogramming the tumour immune cell landscape.

Poorly inflamed carcinomas do not respond well to immune checkpoint blockade. Converting the tumour microenvironment into a functionally inflamed immune hub would extend the clinical benefit of immune therapy to a larger proportion of cancer patients. Here we show, by using comprehensive single-cell transcriptome, proteome, and immune cell analysis, that Entinostat, a class I histone deacetylase inhibitor, facilitates accumulation of the necrosis-targeted recombinant murine immune-cytokine, NHS-rmIL12, in experimental mouse colon carcinomas and poorly immunogenic breast tumours. This combination therapy reprograms the tumour innate and adaptive immune milieu to an inflamed landscape, where the concerted action of highly functional CD8+ T cells and activated neutrophils drive macrophage M1-like polarization, leading to complete tumour eradication in 41.7%-100% of cases. Biomarker signature of favourable overall survival in multiple human tumor types shows close resemblance to the immune pattern generated by Entinostat/NHS-rmIL12 combination therapy. Collectively, these findings provide a rationale for combining NHS-IL12 with Entinostat in the clinical setting.

The thymoproteasome hardwires the TCR repertoire of CD8+ T cells in the cortex independent of negative selection.

The thymoproteasome expressed specifically in thymic cortical epithelium optimizes the generation of CD8+ T cells; however, how the thymoproteasome contributes to CD8+ T cell development is unclear. Here, we show that the thymoproteasome shapes the TCR repertoire directly in cortical thymocytes before migration to the thymic medulla. We further show that the thymoproteasome optimizes CD8+ T cell production independent of the thymic medulla; independent of additional antigen-presenting cells, including medullary thymic epithelial cells and dendritic cells; and independent of apoptosis-mediated negative selection. These results indicate that the thymoproteasome hardwires the TCR repertoire of CD8+ T cells with cortical positive selection independent of negative selection in the thymus.

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

Induction of DNMT3B by PGE2 and IL6 at Distant Metastatic Sites Promotes Epigenetic Modification and Breast Cancer Colonization.

Current cancer treatments are largely based on the genetic characterization of primary tumors and are ineffective for metastatic disease. Here we report that DNA methyltransferase 3B (DNMT3B) is induced at distant metastatic sites and mediates epigenetic reprogramming of metastatic tumor cells. Multiomics analysis and spontaneous metastatic mouse models revealed that DNMT3B alters multiple pathways including STAT3, NFκB, PI3K/Akt, ß-catenin, and Notch signaling, which are critical for cancer cell survival, apoptosis, proliferation, invasion, and colonization. PGE2 and IL6 were identified as critical inflammatory mediators in DNMT3B induction. DNMT3B expression levels positively correlated with human metastatic progression. Targeting IL6 or COX-2 reduced DNMT3B induction and improved chemo or PD1 therapy. We propose a novel mechanism linking the metastatic microenvironment with epigenetic alterations that occur at distant sites. These results caution against the "Achilles heel" in cancer therapies based on primary tumor characterization and suggests targeting DNMT3B induction as new option for treating metastatic disease. SIGNIFICANCE: These findings reveal that DNMT3B epigenetically regulates multiple pro-oncogenic signaling pathways via the inflammatory microenvironment at distant sites, cautioning the clinical approach basing current therapies on genetic characterization of primary tumors.