ABSTRACT
Current treatments for KRAS-mutant colorectal cancers (CRCs) are often limited by cellular plasticity and rewiring responses. Here we describe a promising therapeutic strategy that simultaneously targets epigenetic and oncogenic signals. Specifically, we show that inhibitors of the histone methyltransferase, EZH2, synergize with various RAS pathway inhibitors and promote dramatic tumor regression in vivo. Together these agents cooperatively suppress WNT-driven transcription and drive CRCs into a more differentiated cell state by inducing the Groucho/TLE corepressor, TLE4, along with a network of WNT pathway inhibitors and intestinal differentiation proteins. However, these agents also induce the pro-apoptotic protein BMF, which subsequently kills these more differentiated cells. Accordingly, cell death can be prevented by activating ß-catenin, blocking differentiation, or by ablating BMF expression. Collectively, these studies reveal a new therapeutic approach for treating KRAS-mutant CRCs and illustrate a critical convergence of EZH2 and RAS on oncogenic WNT signals, intestinal differentiation, and apoptosis.
ABSTRACT
While screening and early detection have reduced mortality from prostate cancer, castration-resistant disease (CRPC) is still incurable. Here, we report that combined EZH2/HDAC inhibitors potently kill CRPCs and cause dramatic tumor regression in aggressive human and mouse CRPC models. Notably, EZH2 and HDAC both transmit transcriptional repressive signals: regulating histone H3 methylation and histone deacetylation, respectively. Accordingly, we show that suppression of both EZH2 and HDAC are required to derepress/induce a subset of EZH2 targets, by promoting the sequential demethylation and acetylation of histone H3. Moreover, we find that the induction of one of these targets, ATF3, which is a broad stress response gene, is critical for the therapeutic response. Importantly, in human tumors, low ATF3 levels are associated with decreased survival. Moreover, EZH2- and ATF3-mediated transcriptional programs inversely correlate and are most highly/lowly expressed in advanced disease. Together, these studies identify a promising therapeutic strategy for CRPC and suggest that these two major epigenetic regulators buffer prostate cancers from a lethal response to cellular stresses, thereby conferring a tractable therapeutic vulnerability.
Subject(s)
Histones , Prostatic Neoplasms, Castration-Resistant , Animals , Humans , Male , Mice , Cell Line, Tumor , Enhancer of Zeste Homolog 2 Protein/genetics , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Histones/metabolism , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/drug therapy , Histone DeacetylasesABSTRACT
The DAB2IP tumor suppressor encodes a RAS GTPase-activating protein. Accordingly, DAB2IP has been shown to be mutated or suppressed in tumor types that typically lack RAS mutations. However, here we report that DAB2IP is mutated or selectively silenced in the vast majority of KRAS and BRAF mutant colorectal cancers. In this setting, DAB2IP loss promoted tumor development by activating wild-type H- and N-RAS proteins, which was surprisingly required to achieve robust activation of RAS effector pathways in KRAS-mutant tumors. DAB2IP loss also triggered production of inflammatory mediators and the recruitment of protumorigenic macrophages in vivo. Importantly, tumor growth was suppressed by depleting macrophages or inhibiting cytokine/inflammatory mediator expression with a JAK/TBK1 inhibitor. In human tumors, DAB2IP was lost at early stages of tumor development, and its depletion was associated with an enrichment of macrophage and inflammatory signatures. Together, these findings demonstrate that DAB2IP restrains the activation of the RAS pathway and inflammatory cascades in the colon and that its loss represents a common and unappreciated mechanism for amplifying these two critical oncogenic signals in colorectal cancer. SIGNIFICANCE: DAB2IP is lost in early-stage tumors, which amplifies RAS signaling, triggers inflammatory mediators, and recruits macrophages in KRAS-mutant colon cancers.
Subject(s)
Colonic Neoplasms , Proto-Oncogene Proteins p21(ras) , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Genes, Tumor Suppressor , Colonic Neoplasms/genetics , Signal Transduction , ras GTPase-Activating Proteins/genetics , ras GTPase-Activating Proteins/metabolism , Cell Line, TumorABSTRACT
Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.
Subject(s)
Fucose , Melanoma , Humans , HLA-DRB1 Chains/genetics , HLA-DRB1 Chains/metabolism , Fucose/metabolism , Melanoma/drug therapy , Immunotherapy , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathologyABSTRACT
BACKGROUND: T cell immunoglobulin and mucin domain containing-3 (TIM-3) blocking antibodies are currently being evaluated in clinical trials for solid and hematological malignancies. Despite its identification on T cells, TIM-3 is predominantly expressed by myeloid cells, including XCR1+ type I conventional dendritic cells (cDC1s). We have recently shown that TIM-3 blockade promotes expression of CXCR3 chemokine ligands by tumor cDCs, but how this drives a CD8+ T cell-dependent response to therapy is unclear. METHODS: T cell infiltration, effector function, and spatial localization in relation to XCR1+ cDC1s were evaluated in a murine orthotopic mammary carcinoma model during response to TIM-3 blockade and paclitaxel chemotherapy. Mixed bone marrow chimeras and diphtheria toxin depletion were used to determine the role of specific genes in cDC1s during therapeutic responses. RESULTS: TIM-3 blockade increased interferon-γ expression by CD8+ T cells without altering immune infiltration. cDC1 expression of CXCL9, but not CXCL10, was required for response to TIM-3 blockade. CXCL9 was also necessary for the increased proximity observed between CD8+ T cells and XCR1+ cDC1s during therapy. Tumor responses were dependent on cDC1 expression of interleukin-12, but not MHCI. CONCLUSIONS: TIM-3 blockade increases exposure of intratumoral CD8+ T cells to cDC1-derived cytokines, with implications for the design of therapeutic strategies using antibodies against TIM-3.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Hepatitis A Virus Cellular Receptor 2/antagonists & inhibitors , Immunotherapy/methods , Interleukin-12/metabolism , Receptors, Chemokine/metabolism , Animals , Humans , Mice , Signal TransductionABSTRACT
Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.
Subject(s)
DNA/metabolism , Dendritic Cells/metabolism , Hepatitis A Virus Cellular Receptor 2/metabolism , Membrane Proteins/metabolism , Nucleotidyltransferases/metabolism , Signal Transduction/physiology , Animals , Biological Transport/physiology , Cell Line , Cell Line, Tumor , Chemokines/metabolism , Cytoplasm/metabolism , Endocytosis/physiology , Female , HEK293 Cells , Humans , Immunotherapy/methods , Mice , Mice, Inbred C57BLABSTRACT
Despite significant advances in the field of cancer immunotherapy, the majority of patients still do not benefit from treatment and must rely on traditional therapies. Dendritic cells have long been a focus of cancer immunotherapy due to their role in inducing protective adaptive immunity, but cancer vaccines have shown limited efficacy in the past. With the advent of immune checkpoint blockade and the ability to identify patient-specific neoantigens, new vaccines, and combinatorial therapies are being evaluated in the clinic. Dendritic cells are also emerging as critical regulators of the immune response within tumors. Understanding how to augment the function of these intratumoral dendritic cells could offer new approaches to enhance immunotherapy, in addition to improving the cytotoxic and targeted therapies that are partially dependent upon a robust immune response for their efficacy. Here we will discuss the role of specific dendritic cell subsets in regulating the anti-tumor immune response, as well as the current status of dendritic cell-based immunotherapies, in order to provide an overview for future lines of research and clinical trials.
Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , Cancer Vaccines/therapeutic use , Dendritic Cells/drug effects , Dendritic Cells/transplantation , Immune Checkpoint Inhibitors/therapeutic use , Immunotherapy, Adoptive , Neoplasms/therapy , Animals , Antineoplastic Agents, Immunological/adverse effects , Cancer Vaccines/adverse effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Immune Checkpoint Inhibitors/adverse effects , Immunotherapy, Adoptive/adverse effects , Neoplasms/immunology , Neoplasms/metabolism , Phenotype , Signal Transduction , Treatment OutcomeABSTRACT
Intratumoral CD103+ dendritic cells (DCs) are necessary for anti-tumor immunity. Here we evaluated the expression of immune regulators by CD103+ DCs in a murine model of breast cancer and identified expression of TIM-3 as a target for therapy. Anti-TIM-3 antibody improved response to paclitaxel chemotherapy in models of triple-negative and luminal B disease, with no evidence of toxicity. Combined efficacy was CD8+ T cell dependent and associated with increased granzyme B expression; however, TIM-3 expression was predominantly localized to myeloid cells in both human and murine tumors. Gene expression analysis identified upregulation of Cxcl9 within intratumoral DCs during combination therapy, and therapeutic efficacy was ablated by CXCR3 blockade, Batf3 deficiency, or Irf8 deficiency.
Subject(s)
Antigens, CD/immunology , Breast Neoplasms/genetics , Dendritic Cells/immunology , Hepatitis A Virus Cellular Receptor 2/genetics , Integrin alpha Chains/immunology , Animals , Basic-Leucine Zipper Transcription Factors/deficiency , Breast Neoplasms/metabolism , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Female , Humans , Interferon Regulatory Factors/immunology , Mice, TransgenicABSTRACT
Type 1 conventional dendritic cells are necessary for the development of anti-tumor immunity. In this issue of Immunity, Sharma et al. (2018) identify a phenotypically similar monocyte-derived population within inflamed tumors that promotes T cell responses during therapy.
Subject(s)
Cell Differentiation , Cells, Cultured , Dendritic Cells , Monocytes , T-LymphocytesABSTRACT
Little is known regarding how the interactions of stem cells with the immune system regulate their plasticity. A study now describes a mechanism by which normal breast and cancer stem cells utilize miR-199a to downregulate the corepressor LCOR and minimize responses to type I interferon.
Subject(s)
Interferons/metabolism , Stem Cells/metabolism , Animals , Cell Proliferation , Humans , Immune System/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Models, Biological , Neoplastic Stem Cells/pathologyABSTRACT
Etheno (ε) DNA base adducts are highly mutagenic lesions produced endogenously via reactions with lipid peroxidation (LPO) products. Cancer-promoting conditions, such as inflammation, can induce persistent oxidative stress and increased LPO, resulting in the accumulation of ε-adducts in different tissues. Using a recently described fluorescence multiplexed host cell reactivation assay, we show that a plasmid reporter bearing a site-specific 3,N4-ethenocytosine (εC) causes transcriptional blockage. Notably, this blockage is exacerbated in Cockayne Syndrome and xeroderma pigmentosum patient-derived lymphoblastoid and fibroblast cells. Parallel RNA-Seq expression analysis of the plasmid reporter identifies novel transcriptional mutagenesis properties of εC. Our studies reveal that beyond the known pathways, such as base excision repair, the process of transcription-coupled nucleotide excision repair plays a role in the removal of εC from the genome, and thus in the protection of cells and tissues from collateral damage induced by inflammatory responses.
Subject(s)
Cytosine/analogs & derivatives , DNA Adducts/metabolism , DNA Repair , Transcription, Genetic , Adenine/analogs & derivatives , Adenine/metabolism , Animals , Cell Line , Cells, Cultured , Cockayne Syndrome/genetics , Cytosine/metabolism , DNA Repair Enzymes/genetics , Humans , Mice , Mice, Knockout , Mutagenesis , RNA Polymerase II/metabolism , Xeroderma Pigmentosum/geneticsABSTRACT
Dendritic cells (DCs) are central regulators of the adaptive immune response, and as such are necessary for T-cell-mediated cancer immunity. In particular, antitumoral responses depend on a specialized subset of conventional DCs that transport tumor antigens to draining lymph nodes and cross-present antigen to activate cytotoxic T lymphocytes. DC maturation is necessary to provide costimulatory signals to T cells, but while DC maturation occurs within tumors, it is often insufficient to induce potent immunity, particularly in light of suppressive mechanisms within tumors. Bypassing suppressive pathways or directly activating DCs can unleash a T-cell response, and although clinical efficacy has proven elusive, therapeutic targeting of DCs continues to hold translational potential in combinatorial approaches.