ABSTRACT
New opportunities are needed to increase immune checkpoint blockade (ICB) benefit. Whereas the interferon (IFN)γ pathway harbors both ICB resistance factors and therapeutic opportunities, this has not been systematically investigated for IFNγ-independent signaling routes. A genome-wide CRISPR/Cas9 screen to sensitize IFNγ receptor-deficient tumor cells to CD8 T cell elimination uncovered several hits mapping to the tumor necrosis factor (TNF) pathway. Clinically, we show that TNF antitumor activity is only limited in tumors at baseline and in ICB non-responders, correlating with its low abundance. Taking advantage of the genetic screen, we demonstrate that ablation of the top hit, TRAF2, lowers the TNF cytotoxicity threshold in tumors by redirecting TNF signaling to favor RIPK1-dependent apoptosis. TRAF2 loss greatly enhanced the therapeutic potential of pharmacologic inhibition of its interaction partner cIAP, another screen hit, thereby cooperating with ICB. Our results suggest that selective reduction of the TNF cytotoxicity threshold increases the susceptibility of tumors to immunotherapy.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunotherapy , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis/drug effects , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/metabolism , Cell Line, Tumor , Humans , Inhibitor of Apoptosis Proteins/metabolism , Interferon-gamma/metabolism , Kaplan-Meier Estimate , Male , Mice , Mice, Inbred C57BL , Neoplasms/mortality , Neoplasms/therapy , RNA, Guide, Kinetoplastida/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Receptors, Interferon/deficiency , Receptors, Interferon/genetics , Signal Transduction/drug effects , TNF Receptor-Associated Factor 2/deficiency , TNF Receptor-Associated Factor 2/genetics , Tumor Necrosis Factor-alpha/pharmacology , Interferon gamma ReceptorABSTRACT
By restoring tryptophan, indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors aim to reactivate anti-tumor T cells. However, a phase III trial assessing their clinical benefit failed, prompting us to revisit the role of IDO1 in tumor cells under T cell attack. We show here that IDO1 inhibition leads to an adverse protection of melanoma cells to T cell-derived interferon-gamma (IFNγ). RNA sequencing and ribosome profiling shows that IFNγ shuts down general protein translation, which is reversed by IDO1 inhibition. Impaired translation is accompanied by an amino acid deprivation-dependent stress response driving activating transcription factor-4 (ATF4)high/microphtalmia-associated transcription factor (MITF)low transcriptomic signatures, also in patient melanomas. Single-cell sequencing analysis reveals that MITF downregulation upon immune checkpoint blockade treatment predicts improved patient outcome. Conversely, MITF restoration in cultured melanoma cells causes T cell resistance. These results highlight the critical role of tryptophan and MITF in the melanoma response to T cell-derived IFNγ and uncover an unexpected negative consequence of IDO1 inhibition.
Subject(s)
Melanoma , Tryptophan , Humans , Melanoma/pathology , Interferon-gamma/metabolism , T-Lymphocytes/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/geneticsABSTRACT
Melanomas can switch to a dedifferentiated cell state upon exposure to cytotoxic T cells. However, it is unclear whether such tumor cells pre-exist in patients and whether they can be resensitized to immunotherapy. Here, we chronically expose (patient-derived) melanoma cell lines to differentiation antigen-specific cytotoxic T cells and observe strong enrichment of a pre-existing NGFRhi population. These fractions are refractory also to T cells recognizing non-differentiation antigens, as well as to BRAF + MEK inhibitors. NGFRhi cells induce the neurotrophic factor BDNF, which contributes to T cell resistance, as does NGFR. In melanoma patients, a tumor-intrinsic NGFR signature predicts anti-PD-1 therapy resistance, and NGFRhi tumor fractions are associated with immune exclusion. Lastly, pharmacologic NGFR inhibition restores tumor sensitivity to T cell attack in vitro and in melanoma xenografts. These findings demonstrate the existence of a stable and pre-existing NGFRhi multitherapy-refractory melanoma subpopulation, which ought to be eliminated to revert intrinsic resistance to immunotherapeutic intervention.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Melanoma/drug therapy , Nerve Tissue Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, Nerve Growth Factor/metabolism , Skin Neoplasms/drug therapy , T-Lymphocytes, Cytotoxic/immunology , Animals , Antineoplastic Agents, Immunological/therapeutic use , Brain-Derived Neurotrophic Factor/antagonists & inhibitors , Brain-Derived Neurotrophic Factor/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Male , Melanoma/genetics , Melanoma/immunology , Melanoma/pathology , Mice , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Nerve Tissue Proteins/antagonists & inhibitors , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , RNA-Seq , Receptors, Nerve Growth Factor/antagonists & inhibitors , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Skin Neoplasms/pathology , T-Lymphocytes, Cytotoxic/metabolism , Tumor Escape/genetics , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology , Xenograft Model Antitumor AssaysABSTRACT
Sulfated heterorhamnans produced by Gayralia oxysperma were utilized for the preparation of two homogeneous and highly sulfated Smith-degraded products (M(w) of 109 and 251 kDa), which were constituted principally by 3-linked α-L-rhamnosyl units 2- or 4-sulfate and 2-linked α-L-rhamnosyl units 4- or 3,4-sulfate, in different percentages. The homogeneous products and the crude extracts containing the sulfated heterorhamnans showed cytotoxic effect against U87MG cells. These sulfated polysaccharides induced an increase in the number of cells in G1 phase with concomitant increase of the mRNA levels of p53 and p21. The presence of 2-linked disulfated rhamnose residues together with the molecular weight could be important factors to be correlated with the inhibitory effect on human glioblastoma cells.
Subject(s)
Chlorophyta/chemistry , Deoxy Sugars/pharmacology , Mannans/pharmacology , Sulfates/chemistry , Cell Cycle/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Deoxy Sugars/chemistry , Deoxy Sugars/isolation & purification , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Mannans/chemistry , Mannans/isolation & purification , Molecular Structure , Polymerization , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Flavones have received considerable attention because of their antiproliferative properties and selective effects on cancer cells, making them good candidates for use in cancer therapy. In contrast to other flavones, little is known about the effects of the flavone core structure (2-phenyl-4H-1-benzopyran-4one) on cancer cells. Here, we report that flavone induces cell death in human hepatoma HepG2 cells. Furthermore, annexin-V+/PI- and SubG1 populations of HepG2 cells increased after flavone treatment. Exposure of HepG2 to flavone did not result in either cytochrome c release into the cytosol or changes in the mitochondrial membrane potential. Treatment of HepG2 cells with flavone for 24 h reduced the accumulation of intracellular ROS, which correlated with upregulation of Gred, CuZnSOD and MnSOD mRNA levels. Taken together, our results provided useful insights into the mechanism of cell death caused by flavones, in order to evaluate their future application in hepatocarcinoma therapy.
Subject(s)
Cell Death/drug effects , Flavones/pharmacology , Cytochromes c/metabolism , Hep G2 Cells , Humans , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolismABSTRACT
To identify factors preferentially necessary for driving tumor expansion, we performed parallel in vitro and in vivo negative-selection short hairpin RNA (shRNA) screens. Melanoma cells harboring shRNAs targeting several DNA damage response (DDR) kinases had a greater selective disadvantage in vivo than in vitro, indicating an essential contribution of these factors during tumor expansion. In growing tumors, DDR kinases were activated following hypoxia. Correspondingly, depletion or pharmacologic inhibition of DDR kinases was toxic to melanoma cells, including those that were resistant to BRAF inhibitor, and this could be enhanced by angiogenesis blockade. These results reveal that hypoxia sensitizes melanomas to targeted inhibition of the DDR and illustrate the utility of in vivo shRNA dropout screens for the identification of pharmacologically tractable targets.