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1.
Cancer Discov ; 13(12): 2566-2583, 2023 12 12.
Article in English | MEDLINE | ID: mdl-37728660

ABSTRACT

The tumor microenvironment (TME) restricts antitumor CD8+ T-cell function and immunotherapy responses. Cancer cells compromise the metabolic fitness of CD8+ T cells within the TME, but the mechanisms are largely unknown. Here we demonstrate that one-carbon (1C) metabolism is enhanced in T cells in an antigen-specific manner. Therapeutic supplementation of 1C metabolism using formate enhances CD8+ T-cell fitness and antitumor efficacy of PD-1 blockade in B16-OVA tumors. Formate supplementation drives transcriptional alterations in CD8+ T-cell metabolism and increases gene signatures for cellular proliferation and activation. Combined formate and anti-PD-1 therapy increases tumor-infiltrating CD8+ T cells, which are essential for enhanced tumor control. Our data demonstrate that formate provides metabolic support to CD8+ T cells reinvigorated by anti-PD-1 to overcome a metabolic vulnerability in 1C metabolism in the TME to further improve T-cell function. SIGNIFICANCE: This study identifies that deficiencies in 1C metabolism limit the efficacy of PD-1 blockade in B16-OVA tumors. Supplementing 1C metabolism with formate during anti-PD-1 therapy enhances CD8+ T-cell fitness in the TME and CD8+ T-cell-mediated tumor clearance. These findings demonstrate that formate supplementation can enhance exhausted CD8+ T-cell function. See related commentary by Lin et al., p. 2507. This article is featured in Selected Articles from This Issue, p. 2489.


Subject(s)
Neoplasms , Programmed Cell Death 1 Receptor , Humans , Programmed Cell Death 1 Receptor/metabolism , CD8-Positive T-Lymphocytes/metabolism , Neoplasms/genetics , Formates , Dietary Supplements , Tumor Microenvironment
2.
Cancer Immunol Res ; 9(2): 184-199, 2021 02.
Article in English | MEDLINE | ID: mdl-33277233

ABSTRACT

Metabolic constraints in the tumor microenvironment constitute a barrier to effective antitumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type-specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis induction by inhibitors of glutathione peroxidase 4 (GPX4) than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity-promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis but impaired antitumor CD8+ T-cell responses. Our screen also revealed high T cell-specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and endoplasmic reticulum (ER) stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of antitumor CD8+ T-cell function and potential therapeutic targets.


Subject(s)
Antineoplastic Agents/pharmacology , CD8-Positive T-Lymphocytes/drug effects , Ferroptosis/drug effects , Tumor Cells, Cultured/drug effects , Animals , Autophagy/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Endoplasmic Reticulum/drug effects , Female , Ferroptosis/genetics , Humans , Mice , Mice, Inbred C57BL , Neoplasms/drug therapy
3.
Nat Immunol ; 20(10): 1335-1347, 2019 10.
Article in English | MEDLINE | ID: mdl-31527834

ABSTRACT

CD8+ T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6+ progenitor exhausted and Tim-3+ terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8+ T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3+ cells without altering Slamf6+ numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8+ T cells enhanced Tim-3+ anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3+CD8+ T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target.


Subject(s)
Adenocarcinoma/immunology , CD8-Positive T-Lymphocytes/physiology , Colonic Neoplasms/immunology , Immunotherapy/methods , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/physiology , Lymphoid Progenitor Cells/physiology , Melanoma/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism , Skin Neoplasms/immunology , Animals , Cellular Senescence , Cytotoxicity, Immunologic , Female , Hepatitis A Virus Cellular Receptor 2/metabolism , Immune Tolerance , Interferon Type I/metabolism , Male , Melanoma, Experimental , Mice , Mice, Inbred C57BL , Mice, Transgenic , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , Signal Transduction , Signaling Lymphocytic Activation Molecule Family/metabolism
4.
Mol Cell ; 72(6): 925-941.e4, 2018 12 20.
Article in English | MEDLINE | ID: mdl-30576655

ABSTRACT

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.


Subject(s)
BRCA1 Protein/deficiency , Breast Neoplasms/enzymology , DNA Replication , DNA, Neoplasm/biosynthesis , Ubiquitin-Specific Proteases/metabolism , Uterine Cervical Neoplasms/enzymology , Animals , BRCA1 Protein/genetics , Binding Sites , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation , Cell Survival , DNA, Neoplasm/genetics , Drug Resistance , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , HeLa Cells , Humans , Mice, Nude , Mutation , Nucleic Acid Denaturation , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Protein Binding , Ubiquitin-Specific Proteases/antagonists & inhibitors , Ubiquitin-Specific Proteases/genetics , Ubiquitination , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Xenograft Model Antitumor Assays
5.
J Biol Chem ; 291(47): 24628-24640, 2016 Nov 18.
Article in English | MEDLINE | ID: mdl-27681596

ABSTRACT

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 µm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.


Subject(s)
Cell Cycle Checkpoints/drug effects , Colorectal Neoplasms/metabolism , Cyclin D1/metabolism , Endopeptidases/metabolism , Lymphoma, Mantle-Cell/drug therapy , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/metabolism , Protease Inhibitors/pharmacology , Proteolysis/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Colorectal Neoplasms/genetics , Cyclin D1/genetics , DNA Damage , DNA Repair , Endopeptidases/genetics , Humans , Lymphoma, Mantle-Cell/genetics , Lymphoma, Mantle-Cell/metabolism , Neoplasm Proteins/genetics , Protease Inhibitors/chemistry , Ubiquitin Thiolesterase
6.
Cell Stem Cell ; 18(5): 668-81, 2016 05 05.
Article in English | MEDLINE | ID: mdl-27053300

ABSTRACT

Fanconi anemia (FA) is an inherited DNA repair disorder characterized by progressive bone marrow failure (BMF) from hematopoietic stem and progenitor cell (HSPC) attrition. A greater understanding of the pathogenesis of BMF could improve the therapeutic options for FA patients. Using a genome-wide shRNA screen in human FA fibroblasts, we identify transforming growth factor-ß (TGF-ß) pathway-mediated growth suppression as a cause of BMF in FA. Blocking the TGF-ß pathway improves the survival of FA cells and rescues the proliferative and functional defects of HSPCs derived from FA mice and FA patients. Inhibition of TGF-ß signaling in FA HSPCs results in elevated homologous recombination (HR) repair with a concomitant decrease in non-homologous end-joining (NHEJ), accounting for the improvement in cellular growth. Together, our results suggest that elevated TGF-ß signaling contributes to BMF in FA by impairing HSPC function and may be a potential therapeutic target for the treatment of FA.


Subject(s)
Bone Marrow/pathology , Fanconi Anemia/pathology , Hematopoietic Stem Cells/pathology , Transforming Growth Factor beta/antagonists & inhibitors , Acetaldehyde/toxicity , Animals , Cell Survival/drug effects , DNA End-Joining Repair/drug effects , Down-Regulation/drug effects , Hematopoietic Stem Cells/drug effects , Homologous Recombination/genetics , Humans , Mice , Mice, Inbred C57BL , Mutagens/toxicity , Signal Transduction/drug effects , Stress, Physiological/drug effects , Transforming Growth Factor beta/metabolism , Up-Regulation/drug effects
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