ABSTRACT
Allogeneic T cell expansion is the primary determinant of graft-versus-host disease (GVHD), and current dogma dictates that this is driven by histocompatibility antigen disparities between donor and recipient. This paradigm represents a closed genetic system within which donor T cells interact with peptide-major histocompatibility complexes (MHCs), though clonal interrogation remains challenging due to the sparseness of the T cell repertoire. We developed a Bayesian model using donor and recipient T cell receptor (TCR) frequencies in murine stem cell transplant systems to define limited common expansion of T cell clones across genetically identical donor-recipient pairs. A subset of donor CD4+ T cell clonotypes differentially expanded in identical recipients and were microbiota dependent. Microbiota-specific T cells augmented GVHD lethality and could target microbial antigens presented by gastrointestinal epithelium during an alloreactive response. The microbiota serves as a source of cognate antigens that contribute to clonotypic T cell expansion and the induction of GVHD independent of donor-recipient genetics.
Subject(s)
Graft vs Host Disease , Graft vs Host Disease/immunology , Graft vs Host Disease/microbiology , Animals , Mice , Mice, Inbred C57BL , CD4-Positive T-Lymphocytes/immunology , Receptors, Antigen, T-Cell/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , Microbiota/immunology , Clonal Selection, Antigen-Mediated , Transplantation, Homologous , Bayes Theorem , Stem Cell Transplantation/adverse effects , Mice, Inbred BALB C , Gastrointestinal Microbiome/immunology , Hematopoietic Stem Cell Transplantation/adverse effectsABSTRACT
The expression of a synthetic chimeric antigen receptor (CAR) to redirect antigen specificity of T cells is transforming the treatment of hematological malignancies and autoimmune diseases [1-7]. In cancer, durable efficacy is frequently limited by the escape of tumors that express low levels or lack the target antigen [8-12]. These clinical results emphasize the need for immune receptors that combine high sensitivity and multispecificity to improve outcomes. Current mono- and bispecific CARs do not faithfully recapitulate T cell receptor (TCR) function and require high antigen levels on tumor cells for recognition [13-17]. Here, we describe a novel synthetic chimeric TCR (ChTCR) that exhibits superior antigen sensitivity and is readily adapted for bispecific targeting. Bispecific ChTCRs mimic TCR structure, form classical immune synapses, and exhibit TCR-like proximal signaling. T cells expressing Bi-ChTCRs more effectively eliminated tumors with heterogeneous antigen expression in vivo compared to T cells expressing optimized bispecific CARs. The Bi-ChTCR architecture is resilient and can be designed to target multiple B cell lineage and multiple myeloma antigens. Our findings identify a broadly applicable approach for engineering T cells to target hematologic malignancies with heterogeneous antigen expression, thereby overcoming the most frequent mechanism of relapse after current CAR T therapies.
ABSTRACT
The interaction of the tumor necrosis factor receptor (TNFR) family member CD27 on naive CD8+ T (Tn) cells with homotrimeric CD70 on antigen-presenting cells (APCs) is necessary for T cell memory fate determination. Here, we examined CD27 signaling during Tn cell activation and differentiation. In conjunction with T cell receptor (TCR) stimulation, ligation of CD27 by a synthetic trimeric CD70 ligand triggered CD27 internalization and degradation, suggesting active regulation of this signaling axis. Internalized CD27 recruited the signaling adaptor TRAF2 and the phosphatase SHP-1, thereby modulating TCR and CD28 signals. CD27-mediated modulation of TCR signals promoted transcription factor circuits that induced memory rather than effector associated gene programs, which are induced by CD28 costimulation. CD27-costimulated chimeric antigen receptor (CAR)-engineered T cells exhibited improved tumor control compared with CD28-costimulated CAR-T cells. Thus, CD27 signaling during Tn cell activation promotes memory properties with relevance to T cell immunotherapy.
Subject(s)
CD28 Antigens , Gene Regulatory Networks , TNF Receptor-Associated Factor 2/genetics , TNF Receptor-Associated Factor 2/metabolism , CD28 Antigens/metabolism , Signal Transduction , Lymphocyte Activation , Receptors, Antigen, T-Cell/metabolism , Tumor Necrosis Factor Receptor Superfamily, Member 7/genetics , Tumor Necrosis Factor Receptor Superfamily, Member 7/metabolism , CD27 Ligand/genetics , CD27 Ligand/metabolism , CD8-Positive T-LymphocytesABSTRACT
The period between "successful" treatment of localized breast cancer and the onset of distant metastasis can last many years, representing an unexploited window to eradicate disseminated disease and prevent metastases. We find that the source of recurrence-disseminated tumor cells (DTCs) -evade endogenous immunity directed against tumor neoantigens. Although DTCs downregulate major histocompatibility complex I, this does not preclude recognition by conventional T cells. Instead, the scarcity of interactions between two relatively rare populations-DTCs and endogenous antigen-specific T cells-underlies DTC persistence. This scarcity is overcome by any one of three immunotherapies that increase the number of tumor-specific T cells: T cell-based vaccination, or adoptive transfer of T cell receptor or chimeric antigen receptor T cells. Each approach achieves robust DTC elimination, motivating discovery of MHC-restricted and -unrestricted DTC antigens that can be targeted with T cell-based immunotherapies to eliminate the reservoir of metastasis-initiating cells in patients.
Subject(s)
Breast Neoplasms , T-Lymphocytes , Humans , Female , Immune Evasion , Adoptive Transfer , Breast Neoplasms/therapy , ImmunotherapyABSTRACT
Activation of a conditional safety switch has the potential to reverse serious toxicities arising from the administration of engineered cellular therapies, including chimeric antigen receptor (CAR) T cells. The functionally inert, non-immunogenic cell surface marker derived from human epidermal growth factor receptor (EGFRt) is a promising safety switch that has been used in multiple clinical constructs and can be targeted by cetuximab, a clinically available monoclonal antibody. However, this approach requires high and persistent cell surface expression of EGFRt to ensure that antibody-mediated depletion of engineered cells is rapid and complete. Here we show that incorporating a short juxtamembrane sequence into the EGFRt polypeptide enhances its expression on the surface of T cells and their susceptibility to antibody-dependent cellular cytotoxicity (ADCC). Incorporating this optimized variant (EGFRopt) into bicistronic and tricistronic CAR designs results in more rapid in vivo elimination of CAR T cells and robust termination of their effector activity compared to EGFRt. These studies establish EGFRopt as a superior safety switch for the development of next-generation cell-based therapeutics.
ABSTRACT
The nature of the anti-tumor immune response changes as primary tumors progress and metastasize. We investigated the role of resident memory (Trm) and circulating memory (Tcirm) cells in anti-tumor responses at metastatic locations using a mouse model of melanoma-associated vitiligo. We found that the transcriptional characteristics of tumor-specific CD8+ T cells were defined by the tissue of occupancy. Parabiosis revealed that tumor-specific Trm and Tcirm compartments persisted throughout visceral organs, but Trm cells dominated lymph nodes (LNs). Single-cell RNA-sequencing profiles of Trm cells in LN and skin were distinct, and T cell clonotypes that occupied both tissues were overwhelmingly maintained as Trm in LNs. Whereas Tcirm cells prevented melanoma growth in the lungs, Trm afforded long-lived protection against melanoma seeding in LNs. Expanded Trm populations were also present in melanoma-involved LNs from patients, and their transcriptional signature predicted better survival. Thus, tumor-specific Trm cells persist in LNs, restricting metastatic cancer.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , Lymph Nodes/immunology , Melanoma, Experimental/immunology , Melanoma/immunology , Skin Neoplasms/immunology , Animals , Humans , Mice , Vitiligo , Melanoma, Cutaneous MalignantABSTRACT
Antibodies targeting CTLA-4 induce durable responses in some patients with melanoma and are being tested in a variety of human cancers. However, these therapies are ineffective for a majority of patients across tumor types. Further understanding the immune alterations induced by these therapies may enable the development of novel strategies to enhance tumor control and biomarkers to identify patients most likely to respond. In several murine models, including colon26, MC38, CT26, and B16 tumors cotreated with GVAX, anti-CTLA-4 efficacy depends on interactions between the Fc region of CTLA-4 antibodies and Fc receptors (FcR). Anti-CTLA-4 binding to FcRs has been linked to depletion of intratumoral T regulatory cells (Treg). In agreement with previous studies, we found that Tregs infiltrating CT26, B16-F1, and autochthonous Braf V600E Pten -/- melanoma tumors had higher expression of surface CTLA-4 (sCTLA-4) than other T-cell subsets, and anti-CTLA-4 treatment led to FcR-dependent depletion of Tregs infiltrating CT26 tumors. This Treg depletion coincided with activation and degranulation of intratumoral natural killer cells. Similarly, in non-small cell lung cancer (NSCLC) and melanoma patient-derived tumor tissue, Tregs had higher sCTLA-4 expression than other intratumoral T-cell subsets, and Tregs infiltrating NSCLC expressed more sCTLA-4 than circulating Tregs. Patients with cutaneous melanoma who benefited from ipilimumab, a mAb targeting CTLA-4, had higher intratumoral CD56 expression, compared with patients who received little to no benefit from this therapy. Furthermore, using the murine CT26 model we found that combination therapy with anti-CTLA-4 plus IL15/IL15Rα complexes enhanced tumor control compared with either monotherapy.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , CTLA-4 Antigen/antagonists & inhibitors , Interleukin-15 Receptor alpha Subunit/metabolism , Interleukin-15/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Neoplasms/immunology , Neoplasms/metabolism , Animals , CTLA-4 Antigen/genetics , CTLA-4 Antigen/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Degranulation/drug effects , Cell Degranulation/immunology , Disease Models, Animal , Gene Expression , Humans , Ipilimumab/pharmacology , Killer Cells, Natural/pathology , Lymphocyte Activation/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Mice , Neoplasms/drug therapy , Neoplasms/pathology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathology , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Xenograft Model Antitumor AssaysABSTRACT
Regulatory T cells (Treg) are critical mediators of immunosuppression in established tumors, although little is known about their role in restraining immunosurveillance during tumorigenesis. Here, we employ an inducible autochthonous model of melanoma to investigate the earliest Treg and CD8 effector T-cell responses during oncogene-driven tumorigenesis. Induction of oncogenic BRAFV600E and loss of Pten in melanocytes led to localized accumulation of FoxP3+ Tregs, but not CD8 T cells, within 1 week of detectable increases in melanocyte differentiation antigen expression. Melanoma tumorigenesis elicited early expansion of shared tumor/self-antigen-specific, thymically derived Tregs in draining lymph nodes, and induced their subsequent recruitment to sites of tumorigenesis in the skin. Lymph node egress of tumor-activated Tregs was required for their C-C chemokine receptor 4 (Ccr4)-dependent homing to nascent tumor sites. Notably, BRAFV600E signaling controlled expression of Ccr4-cognate chemokines and governed recruitment of Tregs to tumor-induced skin sites. BRAFV600E expression alone in melanocytes resulted in nevus formation and associated Treg recruitment, indicating that BRAFV600E signaling is sufficient to recruit Tregs. Treg depletion liberated immunosurveillance, evidenced by CD8 T-cell responses against the tumor/self-antigen gp100, which was concurrent with the formation of microscopic neoplasia. These studies establish a novel role for BRAFV600E as a tumor cell-intrinsic mediator of immune evasion and underscore the critical early role of Treg-mediated suppression during autochthonous tumorigenesis.Significance: This work provides new insights into the mechanisms by which oncogenic pathways impact immune regulation in the nascent tumor microenvironment. Cancer Res; 78(17); 5038-49. ©2018 AACR.
Subject(s)
Carcinogenesis/genetics , Melanoma/genetics , Proto-Oncogene Proteins B-raf/genetics , T-Lymphocytes, Regulatory/metabolism , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Line, Tumor , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Humans , Lymph Nodes/metabolism , Lymph Nodes/pathology , Melanocytes/immunology , Melanocytes/pathology , Melanoma/immunology , Melanoma/pathology , Mice , Mutation , PTEN Phosphohydrolase/genetics , Receptors, CCR4/genetics , T-Lymphocytes, Regulatory/immunology , Tumor Microenvironment/geneticsABSTRACT
The opportunistic pathogen Pseudomonas aeruginosa colonizes the lungs of susceptible individuals by deploying virulence factors targeting host defenses. The secreted factor Cif (cystic fibrosis transmembrane conductance regulator inhibitory factor) dysregulates the endocytic recycling of CFTR and thus reduces CFTR abundance in host epithelial membranes. We have postulated that the decrease in ion secretion mediated by Cif would slow mucociliary transport and decrease bacterial clearance from the lungs. To test this hypothesis, we explored the effects of Cif in cultured epithelia and in the lungs of mice. We developed a strategy to interpret the "hurricane-like" motions observed in reconstituted cultures and identified a Cif-mediated decrease in the velocity of mucus transport in vitro. Presence of Cif also increased the number of bacteria recovered at two time points in an acute mouse model of pneumonia caused by P. aeruginosa. Furthermore, recent work has demonstrated an inverse correlation between the airway concentrations of Cif and 15-epi-lipoxin A4, a proresolving lipid mediator important in host defense and the resolution of pathogen-initiated inflammation. Here, we observe elevated levels of 15-epi-lipoxin A4 in the lungs of mice infected with a strain of P. aeruginosa that expresses only an inactive form of cif compared with those mice infected with wild-type P. aeruginosa. Together these data support the inclusion of Cif on the list of virulence factors that assist P. aeruginosa in colonizing and damaging the airways of compromised patients. Furthermore, this study establishes techniques that enable our groups to explore the underlying mechanisms of Cif effects during respiratory infection.
Subject(s)
Bacterial Proteins/metabolism , Bronchi/pathology , Epithelial Cells/pathology , Pneumonia/etiology , Pseudomonas Infections/complications , Pseudomonas aeruginosa/pathogenicity , Virulence Factors/metabolism , Animals , Biological Transport , Bronchi/enzymology , Bronchi/microbiology , Cells, Cultured , Disease Models, Animal , Epithelial Cells/enzymology , Epithelial Cells/microbiology , Humans , Lipoxins/metabolism , Male , Mice , Mice, Inbred C57BL , Mucociliary Clearance , Pneumonia/metabolism , Pneumonia/pathology , Pseudomonas Infections/microbiologyABSTRACT
Tissue-resident memory T (TRM) cells have been widely characterized in infectious disease settings; however, their role in mediating immunity to cancer remains unknown. We report that skin-resident memory T cell responses to melanoma are generated naturally as a result of autoimmune vitiligo. Melanoma antigen-specific TRM cells resided predominantly in melanocyte-depleted hair follicles and were maintained without recirculation or replenishment from the lymphoid compartment. These cells expressed CD103, CD69, and CLA (cutaneous lymphocyte antigen), but lacked PD-1 (programmed cell death protein-1) or LAG-3 (lymphocyte activation gene-3), and were capable of making IFN-γ (interferon-γ). CD103 expression on CD8 T cells was required for the establishment of TRM cells in the skin but was dispensable for vitiligo development. CD103+ CD8 TRM cells were critical for protection against melanoma rechallenge. This work establishes that CD103-dependent TRM cells play a key role in perpetuating antitumor immunity.
ABSTRACT
Acquired resistance to BRAFV600E inhibitors (BRAFi) in melanoma remains a common clinical obstacle, as is the case for any targeted drug therapy that can be developed given the plastic nature of cancers. Although there has been significant focus on the cancer cell-intrinsic properties of BRAFi resistance, the impact of BRAFi resistance on host immunity has not been explored. Here we provide preclinical evidence that resistance to BRAFi in an autochthonous mouse model of melanoma is associated with restoration of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment, initially reduced by BRAFi treatment. In contrast to restoration of MDSCs, levels of T regulatory cells remained reduced in BRAFi-resistant tumors. Accordingly, tumor gene expression signatures specific for myeloid cell chemotaxis and homeostasis reappeared in BRAFi-resistant tumors. Notably, MDSC restoration relied upon MAPK pathway reactivation and downstream production of the myeloid attractant CCL2 in BRAFi-resistant melanoma cells. Strikingly, although combination checkpoint blockade (anti-CTLA-4 + anti-PD-1) was ineffective against BRAFi-resistant melanomas, the addition of MDSC depletion/blockade (anti-Gr-1 + CCR2 antagonist) prevented outgrowth of BRAFi-resistant tumors. Our results illustrate how extrinsic pathways of immunosuppression elaborated by melanoma cells dominate the tumor microenvironment and highlight the need to target extrinsic as well as intrinsic mechanisms of drug resistance. Cancer Res; 77(7); 1599-610. ©2017 AACR.
Subject(s)
Melanoma/drug therapy , Myeloid-Derived Suppressor Cells/physiology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Animals , Cell Line, Tumor , Chemokine CCL2/physiology , Drug Resistance, Neoplasm , Humans , Immunotherapy , Indoles/pharmacology , MAP Kinase Signaling System , Melanoma/immunology , Mice , Mice, Inbred C57BL , Receptors, CCR2/antagonists & inhibitors , Receptors, CCR2/physiology , Sulfonamides/pharmacology , T-Lymphocytes, Regulatory/physiology , Tumor MicroenvironmentABSTRACT
Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the ß5 and ß1 activity, but not the ß2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate ß2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the ß2 proteasome activity. LU-102 inhibited the ß2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of ß1 and ß5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, ß2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro.
Subject(s)
Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Drug Resistance, Neoplasm , Oligopeptides/pharmacology , Proteasome Inhibitors/pharmacology , Animals , Cell Line, Tumor , Disease Models, Animal , Drug Synergism , Humans , Mice , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Xenograft Model Antitumor AssaysABSTRACT
A growing body of evidence suggests that BRAF inhibitors, in addition to their acute tumor growth-inhibitory effects, can also promote immune responses to melanoma. The present study aimed to define the immunologic basis of BRAF-inhibitor therapy using the Braf/Pten model of inducible, autochthonous melanoma on a pure C57BL/6 background. In the tumor microenvironment, BRAF inhibitor PLX4720 functioned by on-target mechanisms to selectively decrease both the proportions and absolute numbers of CD4(+)Foxp3(+) regulatory T cells (Treg) and CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSC), while preserving numbers of CD8(+) effector T cells. In PLX4720-treated mice, the intratumoral Treg populations decreased significantly, demonstrating enhanced apopotosis. CD11b(+) myeloid cells from PLX4720-treated tumors also exhibited decreased immunosuppressive function on a per-cell basis. In accordance with a reversion of tumor immune suppression, tumors that had been treated with PLX4720 grew with reduced kinetics after treatment was discontinued, and this growth delay was dependent on CD8 T cells. These findings demonstrate that BRAF inhibition selectively reverses two major mechanisms of immunosuppression in melanoma and liberates host-adaptive antitumor immunity.
Subject(s)
Antineoplastic Agents/pharmacology , Indoles/pharmacology , Melanoma, Experimental/immunology , Myeloid Cells/drug effects , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Sulfonamides/pharmacology , T-Lymphocytes, Regulatory/drug effects , Animals , Cross-Priming , Enzyme Inhibitors/pharmacology , Flow Cytometry , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
Infection by Pseudomonas aeruginosa, and bacteria in general, frequently promotes acidification of the local microenvironment, and this is reinforced by pulmonary exertion and exacerbation. However, the consequence of an acidic environment on the host inflammatory response to P. aeruginosa infection is poorly understood. Here we report that the pivotal cellular and host proinflammatory interleukin-1ß (IL-1ß) response, which enables host clearance of the infection but can produce collateral inflammatory damage, is increased in response to P. aeruginosa infection within an acidic environment. Synergistic mechanisms that promote increased IL-1ß release in response to P. aeruginosa infection in an acidic environment are increased pro-IL-1ß induction and increased caspase-1 activity, the latter being dependent upon a functional type III secretion system of the bacteria and the NLRC4 inflammasome of the host. Using an in vivo peritonitis model, we have validated that the IL-1ß inflammatory response is increased in mice in response to P. aeruginosa infection within an acidic microenvironment. These data reveal novel insights into the regulation and exacerbation of inflammatory responses to P. aeruginosa.
Subject(s)
Acidosis/metabolism , Gene Expression Regulation/immunology , Interleukin-1beta/metabolism , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/physiology , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , CARD Signaling Adaptor Proteins , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase 1/metabolism , Hydrogen-Ion Concentration , Immunity, Innate , Interleukin-1beta/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , NLR Family, Pyrin Domain-Containing 3 Protein , Pseudomonas Infections/immunology , Pseudomonas Infections/metabolismABSTRACT
The proteasome inhibitor bortezomib (Velcade) is prescribed for the treatment of multiple myeloma. Clinically achievable concentrations of bortezomib cause less than 85% inhibition of the chymotrypsin-like activity of the proteasome, but little attention has been paid as to whether in vitro studies are representative of this level of inhibition. Patients receive bortezomib as an intravenous or subcutaneous bolus injection, resulting in maximum proteasome inhibition within one hour followed by a gradual recovery of activity. In contrast, most in vitro studies use continuous treatment so that activity never recovers. Replacing continuous treatment with 1 h-pulse treatment increases differences in sensitivity in a panel of 7 multiple myeloma cell lines from 5.3-fold to 18-fold, and reveals that the more sensitive cell lines undergo apoptosis at faster rates. Clinically achievable inhibition of active sites was sufficient to induce cytotoxicity only in one cell line. At concentrations of bortezomib that produced similar inhibition of peptidase activities a different extent of inhibition of protein degradation was observed, providing an explanation for the differential sensitivity. The amount of protein degraded per number of active proteasomes correlated with sensitivity to bortezomib. Thus, (i) in vitro studies of proteasome inhibitors should be conducted at pharmacologically achievable concentrations and duration of treatment; (ii) a similar level of inhibition of active sites results in a different extent of inhibition of protein breakdown in different cell lines, and hence a difference in sensitivity.
Subject(s)
Boronic Acids/therapeutic use , Multiple Myeloma/drug therapy , Pyrazines/therapeutic use , Apoptosis/drug effects , Boronic Acids/pharmacology , Bortezomib , Cell Death/drug effects , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Neoplasm Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Proteolysis/drug effects , Pyrazines/pharmacology , Time FactorsABSTRACT
Syringolins, a class of natural products, potently and selectively inhibit the proteasome and show promising antitumour activity. To gain insight in the mode of action of syringolins, the ureido structural element present in syringolins is incorporated in oligopeptide vinyl sulfones and peptide epoxyketones yielding a focused library of potent new proteasome inhibitors. The distance of the ureido linkage with respect to the electrophilic trap strongly influences subunit selectivity within the proteasome. Compounds 13 and 15 are ß5 selective and their potency exceeds that of syringolin A. In contrast, 5 may well be the most potent ß1 selective compound active in living cells reported to date.
Subject(s)
Ketones/pharmacology , Peptides/chemistry , Proteasome Inhibitors , Sulfones/pharmacology , Urea/chemistry , Cell Line , Humans , Ketones/chemistry , Sulfones/chemistryABSTRACT
Proteasomes degrade the majority of proteins in mammalian cells, are involved in the regulation of multiple physiological functions, and are established targets of anticancer drugs. The proteasome has three types of active sites. Chymotrypsin-like sites are the most important for protein breakdown and have long been considered the only suitable targets for antineoplastic drugs; however, our recent work demonstrated that inhibitors of caspase-like sites sensitize malignant cells to inhibitors of the chymotrypsin-like sites. Here, we describe the development of specific cell-permeable inhibitors and an activity-based probe of the trypsin-like sites. These compounds selectively sensitize multiple myeloma cells to inhibitors of the chymotrypsin-like sites, including antimyeloma agents bortezomib and carfilzomib. Thus, trypsin-like sites are cotargets for anticancers drugs. Together with inhibitors of chymotrypsin- and caspase-like sites developed earlier, we provide the scientific community with a complete set of tools to separately modulate proteasome active sites in living cells.