RÉSUMÉ
BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has been successfully translated to clinical practice for the treatment of B cell malignancies. The suppressive microenvironment of many malignancies is a bottleneck preventing treatment success of CAR T cells in a broader range of tumours. Among others, the immunosuppressive metabolite adenosine is present in high concentrations within many tumours and dampens anti-tumour function of immune cells and consequently therapeutic response. METHODS: Here, we present the impact of the selective adenosine A2A and A2B receptor antagonist AB928/etrumadenant on CAR T cell cytokine secretion, proliferation, and cytotoxicity. Using phosphorylation-specific flow cytometry, we evaluated the capability of AB928 to shield CAR T cells from adenosine-mediated signalling. The effect of orally administered AB928 on CAR T cells was assessed in a syngeneic mouse model of colon carcinoma. RESULTS: We found that immunosuppressive signalling in CAR T cells in response to adenosine was fully blocked by the small molecule inhibitor. AB928 treatment enhanced CAR T cell cytokine secretion and proliferation, granted efficient cytolysis of tumour cells in vitro and augmented CAR T cell activation in vivo. CONCLUSIONS: Together our results suggest that combination therapy with AB928 represents a promising approach to improve adoptive cell therapy.
Sujet(s)
Tumeurs , Lymphocytes T , Animaux , Souris , Adénosine/pharmacologie , Cytokines , Microenvironnement tumoralRÉSUMÉ
Phosphoinositide-3-kinase γ (PI3Kγ) is highly expressed in immune cells and promotes the production and migration of inflammatory mediators. The inhibition of PI3Kγ has been shown to repolarize the tumor immune microenvironment to a more inflammatory phenotype, thereby controlling immune suppression in cancer. Herein, we report the structure-based optimization of an early lead series of pyrazolopyrimidine isoindolinones, which culminated in the discovery of highly potent and isoform-selective PI3Kγ inhibitors with favorable drug-like properties. X-ray cocrystal structure analysis, molecular docking studies, and detailed structure-activity relationship investigations resulted in the identification of the optimal amide and isoindolinone substituents to achieve a desirable combination of potency, selectivity, and metabolic stability. Preliminary in vitro studies indicate that inhibition of PI3Kγ with compound 56 results in a significant immune response by increasing pro-inflammatory cytokine gene expression in M1 macrophages.
Sujet(s)
Amides/composition chimique , Phosphatidylinositol 3-kinases de classe Ib/composition chimique , Conception de médicament , Découverte de médicament , Inhibiteurs des phosphoinositide-3 kinases/pharmacologie , Pyrimidines/composition chimique , Animaux , Humains , Mâle , Simulation de docking moléculaire , Rats , Rat Sprague-Dawley , Relation structure-activitéRÉSUMÉ
The successful application of immunotherapy in the treatment of cancer relies on effective engagement of immune cells in the tumor microenvironment. Phosphoinositide 3-kinase γ (PI3Kγ) is highly expressed in tumor-associated macrophages, and its expression levels are associated with tumor immunosuppression and growth. Selective inhibition of PI3Kγ offers a promising strategy in immuno-oncology, which has led to the development of numerous potent PI3Kγ inhibitors with variable selectivity profiles. To facilitate further investigation of the therapeutic potential of PI3Kγ inhibition, we required a potent and PI3Kγ-selective tool compound with sufficient metabolic stability for use in future in vivo studies. Herein, we describe some of our efforts to realize this goal through the systematic study of SARs within a series of 7-azaindole-based PI3Kγ inhibitors. The large volume of data generated from this study helped guide our subsequent lead optimization efforts and will inform further development of PI3Kγ-selective inhibitors for use in immunomodulation.
RÉSUMÉ
The selective inhibition of the lipid signaling enzyme PI3Kγ constitutes an opportunity to mediate immunosuppression and inflammation within the tumor microenvironment but is difficult to achieve due to the high sequence homology across the class I PI3K isoforms. Here, we describe the design of a novel series of potent PI3Kγ inhibitors that attain high isoform selectivity through the divergent projection of substituents into both the "selectivity" and "alkyl-induced" pockets within the adenosine triphosphate (ATP) binding site of PI3Kγ. These efforts have culminated in the discovery of 5-[2-amino-3-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-5-yl]-2-[(1S)-1-cyclopropylethyl]-7-(trifluoromethyl)-2,3-dihydro-1H-isoindol-1-one (4, IC50 = 0.064 µM, THP-1 cells), which displays >600-fold selectivity for PI3Kγ over the other class I isoforms and is a promising step toward the identification of a clinical development candidate. The structure-activity relationships identified throughout this campaign demonstrate that greater γ-selectivity can be achieved by inhibitors that occupy an "alkyl-induced" pocket and possess bicyclic hinge-binding motifs capable of forming more than one hydrogen bond to the hinge region of PI3Kγ.
Sujet(s)
Phosphatidylinositol 3-kinases de classe Ib/effets des médicaments et des substances chimiques , Conception de médicament , Inhibiteurs des phosphoinositide-3 kinases/pharmacologie , Animaux , Cristallographie aux rayons X , Humains , Simulation de docking moléculaire , Inhibiteurs des phosphoinositide-3 kinases/composition chimique , Inhibiteurs des phosphoinositide-3 kinases/pharmacocinétique , Rats , Relation structure-activitéRÉSUMÉ
BACKGROUND: Glucocorticoids are known to repress the expression of CC chemokine eotaxin in airway epithelial cells. We focused our study on the molecular mechanisms of the glucocorticoid, fluticasone, in the inhibition of the expression of the eotaxin gene in the cells. METHODS: The airway epithelial cell line, BEAS-2B, was stably transfected with signal transducers and activators of transcription 6 (STAT6)-expressing vector and used in the following experiments to clarify the function of STAT6. Levels of eotaxin mRNA and protein expression were determined with RT-PCR and ELISA. Mechanisms of transcriptional regulation were assessed by the electrophoretic mobility shift assay and dual luciferase assay using eotaxin promoter-luciferase reporter plasmids. RESULTS: Fluticasone significantly inhibited the induction of eotaxin protein stimulated with TNF-alpha and IL-4 in the cells. Fluticasone also repressed the induction of eotaxin mRNA with these stimuli. It partially inhibited the activity of eotaxin promoter; however, it did not inhibit the nuclear translocation and binding of transcription factors, nuclear factor-kappa B (NF-kappaB) or STAT6, to the DNA derived from the proximal promoter region of the eotaxin gene. Moreover, the inhibitory effect was also conserved in the experiments using the reporter plasmid of which the putative glucocorticoid-responsive element was mutated. CONCLUSIONS: Fluticasone inhibits the expression of eotaxin gene in airway epithelial cells in part through repression of the transcription. However, the mechanisms depend neither on the inhibition of transcription factors' translocation into nuclei nor the function of the putative glucocorticoid-responsive element in the promoter, indicating that other mechanisms would be related to the transcriptional repression of the eotaxin gene in airway epithelial cells.
Sujet(s)
Androstadiènes/pharmacologie , Bronchodilatateurs/pharmacologie , Chimiokines CC/biosynthèse , Muqueuse respiratoire/effets des médicaments et des substances chimiques , Lignée de cellules transformées , Chimiokine CCL11 , Test de retard de migration électrophorétique , Test ELISA , Fluticasone , Humains , Poumon/effets des médicaments et des substances chimiques , Poumon/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Muqueuse respiratoire/métabolisme , RT-PCR , Facteur de transcription STAT-6 , Transactivateurs/métabolisme , Transcription génétique/effets des médicaments et des substances chimiques , TransfectionRÉSUMÉ
Members of the Stat transcription factor family are specifically activated by cytokines, and each Stat mediates its biological effects through the trans-activation of a unique profile of target genes. This specificity is achieved even when Stat proteins mediating opposite transcriptional effects bind to the same palindromic Stat sites in target genes. We show here that the non-conserved sequences of Stat transcription activation domains (TADs) contribute to specificity in promoter activation. Chimeric proteins in which the Stat6 TAD was replaced by that from Stat1alpha or Stat5 exhibited normal interleukin-4-inducible DNA binding activity, but at best modest trans-activation of reporters containing Stat6 binding sites, and a failure to activate the endogenous CD23 promoter in primary B cells. The p160 coactivator nuclear coactivator-1 (Src-1) was specifically recruited by and coactivated Stat6 but not the chimeric Stat6 molecules. Strikingly, transcriptional responses exhibited distinct requirements for the nuclear coactivator-1 interaction motif of the Stat6 C terminus. Together, these findings indicate that the Stat6 TAD contributes to promoter specificity by the differential recruitment of and requirement for a p160-class coactivator.
Sujet(s)
Protéines de lait , Transactivateurs/composition chimique , Facteurs de transcription/composition chimique , Transcription génétique , Motifs d'acides aminés , Acides aminés/composition chimique , Lignée cellulaire , ADN complémentaire/métabolisme , Protéines de liaison à l'ADN/métabolisme , Histone acetyltransferases , Humains , Immunotransfert , Interleukine-4/métabolisme , Protéines nucléaires/composition chimique , Protéines nucléaires/métabolisme , Coactivateur-1 de récepteur nucléaire , Plasmides/métabolisme , Tests aux précipitines , Régions promotrices (génétique) , Liaison aux protéines , Structure tertiaire des protéines , Récepteurs aux IgE/métabolisme , Protéines de fusion recombinantes/métabolisme , Retroviridae/génétique , Facteur de transcription STAT-1 , Facteur de transcription STAT-5 , Facteur de transcription STAT-6 , Transactivateurs/métabolisme , Facteurs de transcription/métabolisme , Activation de la transcription , TransfectionRÉSUMÉ
IL-4 is a critical cytokine in the regulation of immune responses and genesis of atopy. Engagement of the IL-4R activates multiple signaling pathways, including the transcription factor Stat6. Stat6-deficient mice demonstrate the importance of this factor in lymphocyte proliferation, gene expression, and Th cell differentiation. Recently, a mutant Stat6 (Stat6VT) was generated that is transcriptionally active independent of IL-4 stimulation. To determine the ability of a constitutively active Stat6 to mimic IL-4-stimulated responses, we have generated transgenic mice expressing Stat6VT under control of the CD2 locus control region, restricting expression to lymphoid populations. The phenotype of Stat6VT transgenic mice is similar, but not identical, to IL-4 transgenic mice, suggesting a critical role for Stat6-independent signaling pathways in the generation of some IL-4 responses in vivo. The expression of a constitutively active Stat6 in vivo increases surface expression of IL-4-induced genes and increases serum levels of IgG1 and IgE, compared with nontransgenic mice. Stat6VT expression increases Th2 differentiation in vivo and in vitro. Stat6VT expression also dramatically alters homeostasis of peripheral lymphocyte populations resulting in decreased CD3(+) cells and increased B220(+) cells, compared with nontransgenic littermates. Altered T and B cell populations correlate with an activated phenotype and increased cell death in transgenic T cell, but not B cell, populations. Together these results suggest that expression of a constitutively active Stat6 has distinct effects on B and T lymphocytes.
Sujet(s)
Sous-populations de lymphocytes B/métabolisme , Homéostasie/immunologie , Transduction du signal/immunologie , Sous-populations de lymphocytes T/métabolisme , Transactivateurs/biosynthèse , Transactivateurs/génétique , Transgènes/immunologie , Animaux , Antigènes de surface/biosynthèse , Sous-populations de lymphocytes B/immunologie , Antigènes CD2/génétique , Différenciation cellulaire/génétique , Différenciation cellulaire/immunologie , Croisements génétiques , Régulation de l'expression des gènes/immunologie , Homéostasie/génétique , Commutation de classe des immunoglobulines/génétique , Immunoglobuline E/biosynthèse , Immunoglobuline G/biosynthèse , Immunophénotypage , Interleukine-4/biosynthèse , Interleukine-4/génétique , Interleukine-4/pharmacologie , Activation des lymphocytes/génétique , Souris , Souris de lignée C3H , Souris de lignée C57BL , Souris transgéniques , Facteur de transcription STAT-6 , Transduction du signal/génétique , Sous-populations de lymphocytes T/cytologie , Sous-populations de lymphocytes T/immunologie , Lymphocytes auxiliaires Th2/cytologie , Lymphocytes auxiliaires Th2/immunologie , Lymphocytes auxiliaires Th2/métabolisme , Transactivateurs/physiologie , Régulation positive/génétique , Régulation positive/immunologieRÉSUMÉ
Regulation of hematopoietic progenitor cell homeostasis is crucial for maintenance of innate immunity and the ability of the body to respond to injury and infection. In this report, we demonstrate that progenitor cell numbers and cycling status in vivo are dramatically increased in mice deficient in Stat6 and decreased in mice deficient in Stat4, targeted mutations which also alter T helper cell polarization. Experiments using mice that have T cell restricted transgenic expression of Stat4 or Stat6 or have been in vivo depleted of T cell subsets demonstrate that CD4(+) T cells regulate progenitor cell activity. Injection of the Th1 cytokine Oncostatin M but not other cytokines into Stat4-deficient mice recovers progenitor cell activity to wild-type levels. Thus, T helper cells actively regulate hematopoietic progenitor cell homeostasis.