RESUMEN
Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen-specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)-associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.
Asunto(s)
Glutaminasa/antagonistas & inhibidores , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Melanoma/tratamiento farmacológico , Linfocitos T/metabolismo , Animales , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoterapia/métodos , Ratones , Microambiente TumoralRESUMEN
BACKGROUND: Myeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity. METHODS: CB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively. RESULTS: CB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers. CONCLUSIONS: These results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.
Asunto(s)
Arginasa/metabolismo , Células Mieloides/citología , Neoplasias/tratamiento farmacológico , Pirrolidinas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Microambiente Tumoral/efectos de los fármacos , Animales , Arginasa/antagonistas & inhibidores , Arginina/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Células Hep G2 , Humanos , Células K562 , Masculino , Ratones , Células Mieloides/efectos de los fármacos , Células Mieloides/enzimología , Neoplasias/inmunología , Neoplasias/metabolismo , Pirrolidinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.
Asunto(s)
Inhibidores de Proteasoma/farmacología , Quinolinas/farmacología , Transactivadores/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Quinolinas/química , Relación Estructura-Actividad , Transactivadores/metabolismoRESUMEN
The proteasome plays a crucial role in degradation of normal proteins that happen to be constitutively or inducibly unstable, and in this capacity it plays a regulatory role. Additionally, it degrades abnormal/damaged/mutant/misfolded proteins, which serves a quality-control function. Inhibitors of the proteasome have been validated in the treatment of multiple myeloma, with several FDA-approved therapeutics. Rpn11 is a Zn2+-dependent metalloisopeptidase that hydrolyzes ubiquitin from tagged proteins that are trafficked to the proteasome for degradation. A fragment-based drug discovery (FBDD) approach was utilized to identify fragments with activity against Rpn11. Screening of a library of metal-binding pharmacophores (MBPs) revealed that 8-thioquinoline (8TQ, IC50 value â¼2.5 µM) displayed strong inhibition of Rpn11. Further synthetic elaboration of 8TQ yielded a small molecule compound (35, IC50 value â¼400 nM) that is a potent and selective inhibitor of Rpn11 that blocks proliferation of tumor cells in culture.
Asunto(s)
Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Quinolinas/química , Quinolinas/farmacología , Transactivadores/antagonistas & inhibidores , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas , Humanos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transactivadores/metabolismo , Zinc/metabolismoRESUMEN
Glutamine serves as an important source of energy and building blocks for many tumor cells. The first step in glutamine utilization is its conversion to glutamate by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 had antiproliferative activity in a triple-negative breast cancer (TNBC) cell line, HCC-1806, that was associated with a marked decrease in glutamine consumption, glutamate production, oxygen consumption, and the steady-state levels of glutathione and several tricarboxylic acid cycle intermediates. In contrast, no antiproliferative activity was observed in an estrogen receptor-positive cell line, T47D, and only modest effects on glutamine consumption and downstream metabolites were observed. Across a panel of breast cancer cell lines, GAC protein expression and glutaminase activity were elevated in the majority of TNBC cell lines relative to receptor positive cells. Furthermore, the TNBC subtype displayed the greatest sensitivity to CB-839 treatment and this sensitivity was correlated with (i) dependence on extracellular glutamine for growth, (ii) intracellular glutamate and glutamine levels, and (iii) GAC (but not KGA) expression, a potential biomarker for sensitivity. CB-839 displayed significant antitumor activity in two xenograft models: as a single agent in a patient-derived TNBC model and in a basal like HER2(+) cell line model, JIMT-1, both as a single agent and in combination with paclitaxel. Together, these data provide a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in patients with TNBC and other glutamine-dependent tumors.
Asunto(s)
Antineoplásicos/administración & dosificación , Bencenoacetamidas/farmacología , Inhibidores Enzimáticos/administración & dosificación , Glutaminasa/antagonistas & inhibidores , Neoplasias Basocelulares/tratamiento farmacológico , Tiadiazoles/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Administración Oral , Animales , Antineoplásicos/uso terapéutico , Bencenoacetamidas/uso terapéutico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Neoplasias Mamarias Experimentales , Ratones , Ratones SCID , Persona de Mediana Edad , Neoplasias Basocelulares/patología , Sulfuros/administración & dosificación , Sulfuros/uso terapéutico , Tiadiazoles/administración & dosificación , Tiadiazoles/uso terapéutico , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Membrane protein biogenesis requires the coordinated movement of hydrophobic transmembrane domains (TMD) from the cytosolic vestibule of the Sec61 channel into the lipid bilayer. Molecular insight into TMD integration has been hampered by the difficulty of characterizing intermediates during this intrinsically dynamic process. In this study, we show that cotransin, a substrate-selective Sec61 inhibitor, traps nascent TMDs in the cytosolic vestibule, permitting detailed interrogation of an early pre-integration intermediate. Site-specific crosslinking revealed the pre-integrated TMD docked to Sec61 near the cytosolic tip of the lateral gate. Escape from cotransin-arrest depends not only on cotransin concentration, but also on the biophysical properties of the TMD. Genetic selection of cotransin-resistant cancer cells uncovered multiple mutations clustered near the lumenal plug of Sec61α, thus revealing cotransin's likely site of action. Our results suggest that TMD/lateral gate interactions facilitate TMD transfer into the membrane, a process that is allosterically modulated by cotransin binding to the plug. DOI: http://dx.doi.org/10.7554/eLife.01483.001.
Asunto(s)
Membrana Celular/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Animales , Sitios de Unión , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Relación Dosis-Respuesta a Droga , Células HCT116 , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Péptidos Cíclicos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Canales de Translocación SEC , Relación Estructura-Actividad , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cotransins are cyclic heptadepsipeptides that bind the Sec61 translocon to inhibit cotranslational translocation of a subset of secreted and type I transmembrane proteins. The few known cotransin-sensitive substrates are all targeted to the translocon by a cleavable signal sequence, previously shown to be a critical determinant of cotransin sensitivity. By profiling two cotransin variants against a panel of secreted and transmembrane proteins, we demonstrate that cotransin side-chain differences profoundly affect substrate selectivity. Among the most sensitive substrates we identified is the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Like all type II transmembrane proteins, TNF-α is targeted to the translocon by its membrane-spanning domain, indicating that a cleavable signal sequence is not strictly required for cotransin sensitivity. Our results thus reveal an unanticipated breadth of translocon substrates whose expression is inhibited by Sec61 modulators.
Asunto(s)
Proteínas de la Membrana/antagonistas & inhibidores , Péptidos Cíclicos/química , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Péptidos Cíclicos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Canales de Translocación SEC , Especificidad por SustratoRESUMEN
Target identification of biologically active small-molecules is often the rate-determining step in forward chemical genetics. Photo-affinity labeling (PAL) represents a useful biochemical strategy for target identification in complex protein mixtures. This unit describes the use of alkyl diazirine-based photo-affinity probes and Cu(I)-catalyzed click chemistry to covalently label and visualize the targets of biologically active small-molecules. A general method for affinity purification of probe-modified proteins, useful for identification of protein targets, is also described.
RESUMEN
Photoaffinity labeling is a powerful tool to identify protein targets of biologically active small molecules, yet is often limited by the size, chemical properties, and availability of photoreactive groups. We report an improved synthesis of photo-leucine, a diazirine-based photoreactive analogue of leucine, and demonstrate its incorporation into a cyclodepsipeptide inhibitor of cotranslational translocation. Photoaffinity labeling in a crude membrane fraction, followed by "click chemistry" with a rhodamine-azide reporter, enabled the identification of Sec61alpha, the structural core of the Sec61 translocation channel, as the inhibitor's target.
Asunto(s)
Depsipéptidos/química , Leucina/química , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Estructura Molecular , Fotoquímica , Unión Proteica , Transporte de Proteínas , Canales de Translocación SECRESUMEN
A ping-pong bi-bi kinetic mechanism ascribed to yeast orotate phosphoribosyltransferase (OPRTase) [Victor, J., Greenberg, L. B., and Sloan, D. L. (1979) J. Biol. Chem. 254, 2647-2655] has been shown to be inoperative [Witte, J. F., Tsou, R., and McClard, R. W. (1999) Arch. Biochem. Biophys. 361, 106-112]. Radiolabeled orotidine 5'-phosphate (OMP), generated in situ from [7-(14)C]-orotate and alpha-d-5-phoshorylribose 1-diphosphate (PRPP), binds tightly enough to OPRTase (a dimer composed of identical subunits) that the complex survives gel-filtration chromatography. When a sample of OMP.OPRTase is extensively dialyzed, a 1:1 (per OPRTase dimer) complex is detected by (31)P NMR. Titration of the apoenzyme with OMP yields a (31)P NMR spectrum with peaks for both free and enzyme-bound OMP when OMP is in excess; the complex maintains an OMP/enzyme ratio of 1:1 even when OMP is in substantial excess. A red shift in the UV spectrum of the OMP.OPRTase complex was exploited to measure K(d(OMP)) = 0.84 muM and to verify the 1:1 binding stoichiometry. PRPP forms a Mg(2+)-dependent 1:1 complex with the enzyme as observed by (31)P NMR. Isothermal titration calorimetry (ITC) experiments revealed 1:1 stoichiometries for both OMP and Mg(2+)-PRPP with OPRTase yielding K(d) values of 0.68 and 10 microM, respectively. The binding of either 1 equiv of OMP or PRPP is mutually exclusive. ITC experiments demonstrate that the binding of OMP is largely driven by increased entropy, suggesting substantial distal disordering of the protein. Analytical gel-filtration chromatography confirms that the OMP.OPRTase complex involves the dimeric form of enzyme. The off rate for release of OMP, determined by magnetization inversion transfer, was determined to be 27 s(-)(1). This off rate is somewhat less than the k(cat) in the biosynthetic direction (about 39 s(-)(1)); thus, the release of OMP from OMP.OPRTase may not be kinetically relevant to the steady-state reaction cycle. The body of available data can be explained in terms of alternating site catalysis with either a classical Theorell-Chance mechanism or, far more likely, a novel "double Theorell-Chance" mechanism unique to alternating site catalysis, leading us to propose co-temporal binding of orotate and the release of diphosphate as well as the binding of PRPP and the release of OMP that occur via ternary complexes in alternating site fashion across the two highly cooperative subunits of the enzyme. This novel "double Theorell-Chance" mechanism yields a steady-state rate equation indistinguishable in form from the observed classical ping-pong bi-bi kinetics.