RESUMEN
An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase III , Interferón Tipo I , Proteínas de la Membrana , Transducción de Señal , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/agonistas , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Humanos , Transducción de Señal/efectos de los fármacos , Ratones , Línea Celular Tumoral , Interferón Tipo I/metabolismo , Nucleotidiltransferasas/metabolismo , Ratones Endogámicos C57BL , Femenino , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
γ-Secretase inhibition represents a major therapeutic strategy for lowering amyloid ß (Aß) peptide production in Alzheimer's disease (AD). Progress toward clinical use of γ-secretase inhibitors has, however, been hampered due to mechanism-based adverse events, primarily related to impairment of Notch signaling. The γ-secretase inhibitor MRK-560 represents an exception as it is largely tolerable in vivo despite displaying only a small selectivity between Aß production and Notch signaling in vitro. In exploring the molecular basis for the observed tolerability, we show that MRK-560 displays a strong preference for the presenilin 1 (PS1) over PS2 subclass of γ-secretases and is tolerable in wild-type mice but causes dose-dependent Notch-related side effect in PS2-deficient mice at drug exposure levels resulting in a substantial decrease in brain Aß levels. This demonstrates that PS2 plays an important role in mediating essential Notch signaling in several peripheral organs during pharmacological inhibition of PS1 and provide preclinical in vivo proof of concept for PS2-sparing inhibition as a novel, tolerable and efficacious γ-secretase targeting strategy for AD.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Encéfalo/efectos de los fármacos , Presenilina-2/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Ratones , Presenilina-2/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sulfonamidas/farmacología , Sulfonamidas/uso terapéuticoRESUMEN
The γ-secretase complex is an appealing drug target when the therapeutic strategy is to alter amyloid-ß peptide (Aß) aggregation in Alzheimer disease. γ-Secretase is directly involved in Aß formation and determines the pathogenic potential of Aß by generating the aggregation-prone Aß42 peptide. Because γ-secretase mediates cleavage of many substrates involved in cell signaling, such as the Notch receptor, it is crucial to sustain these pathways while altering the Aß secretion. A way of avoiding interference with the physiological function of γ-secretase is to use γ-secretase modulators (GSMs) instead of inhibitors of the enzyme. GSMs modify the Aß formation from producing the amyloid-prone Aß42 variant to shorter and less amyloidogenic Aß species. The modes of action of GSMs are not fully understood, and even though the pharmacology of GSMs has been thoroughly studied regarding Aß generation, knowledge is lacking about their effects on other substrates, such as Notch. Here, using immunoprecipitation followed by MALDI-TOF MS analysis, we found that two novel, second generation GSMs modulate both Notch ß and Aß production. Moreover, by correlating S3-specific Val-1744 cleavage of Notch intracellular domain (Notch intracellular domain) to total Notch intracellular domain levels using immunocytochemistry, we also demonstrated that Notch intracellular domain is not modulated by the compounds. Interestingly, two well characterized, nonsteroidal anti-inflammatory drugs (nonsteroidal anti-inflammatory drug), R-flurbiprofen and sulindac sulfide, affect only Aß and not Notch ß formation, indicating that second generation GSMs and nonsteroidal anti-inflammatory drug-based GSMs have different modes of action regarding Notch processing.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Flurbiprofeno/farmacología , Receptores Notch/metabolismo , Sulindac/análogos & derivados , Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Femenino , Células HEK293 , Humanos , Ratones , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Estructura Terciaria de Proteína , Receptores Notch/genética , Sulindac/farmacologíaRESUMEN
γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-ß (Aß) peptides. The Aß42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aß production by targeting the APP. Here, we describe novel GSMs that are selective for Aß modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aß both in cell and cell-free systems as well as lower amyloidogenic Aß42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aß modulation and have a different mechanism of action compared with the original class of GSMs described.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Azepinas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Piranos/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Azepinas/química , Unión Competitiva , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Carbamatos/farmacología , Sistema Libre de Células , Dibenzazepinas/farmacología , Dipéptidos/farmacología , Interacciones Farmacológicas , Femenino , Flurbiprofeno/farmacología , Cobayas , Células HEK293 , Humanos , Imidazoles/farmacología , Ratones , Ratones Endogámicos C57BL , Piperidinas/farmacología , Unión Proteica , Piranos/química , Piridinas/química , Pirimidinas/química , Ratas , Receptor EphA4/metabolismo , Receptor EphB2/metabolismo , Receptores Notch/metabolismo , Sulfonamidas/farmacología , Sulindac/análogos & derivados , Sulindac/farmacologíaRESUMEN
Cancer immunotherapy based on anti-PD-1/PD-L1 blockade is particularly effective in responding to patients with hot tumors. These tumors are characterized by the accumulation of proinflammatory cytokines and T cell infiltration. In our recent report published in Science Advances, we demonstrate that targeting the autophagy-related protein Vps34 switched cold immune desert tumors into hot inflamed immune-infiltrated tumors and enhanced the efficacy of anti-PD-1/PD-L1. Our study provides the preclinical rationale to set up combination immunotherapy clinical trials using selective Vps34 inhibitors and immune checkpoint blockers in melanoma and CRC.
Asunto(s)
Inmunoterapia , Melanoma , Proteínas Relacionadas con la Autofagia , Humanos , Factores Inmunológicos , Melanoma/tratamiento farmacológicoRESUMEN
Cancer immunotherapy based on Immune checkpoint blockade (ICB) is a promising strategy to treat patients with advanced highly aggressive therapy-resistant tumors. Unfortunately, the clinical reality is that only a small number of patients benefit from the remarkable clinical remissions achieved by ICB. Experimental and clinical evidence claimed that durable clinical benefit observed using ICB depends on the immune status of tumors, notably the presence of cytotoxic effector immune cells. In our paper, we revealed that genetically targeting the autophagy-related protein PIK3C3/VPS34 in melanoma and colorectal tumor cells, or treating tumor-bearing mice with selective inhibitors of the PIK3C3/VPS34 kinase activity, reprograms cold immune desert tumors into hot, inflamed immune infiltrated tumors. Such reprograming results from the establishment of a proinflammatory signature characterized by the release of CCL5 and CXCL10 in the tumor microenvironment, and the subsequent recruitment of natural killer (NK) and CD8+ T cells into the tumor bed. Furthermore, we reported that combining pharmacological inhibitors of PIK3C3/VPS34 improves the therapeutic benefit of anti-PD-1/PD-L1 immunotherapy. Our results provided the proof-of-concept to set-up innovative clinical trials for cold ICB-unresponsive tumors by combining PIK3C3/VPS34 inhibitors with anti-PDCD1/PD-1 and anti-CD274/PD-L1.
Asunto(s)
Antígeno B7-H1 , Neoplasias , Animales , Autofagia , Proteínas Relacionadas con la Autofagia , Linfocitos T CD8-positivos , Fosfatidilinositol 3-Quinasas Clase III , Humanos , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológicoRESUMEN
One of the major challenges limiting the efficacy of anti-PD-1/PD-L1 therapy in nonresponding patients is the failure of T cells to penetrate the tumor microenvironment. We showed that genetic or pharmacological inhibition of Vps34 kinase activity using SB02024 or SAR405 (Vps34i) decreased the tumor growth and improved mice survival in multiple tumor models by inducing an infiltration of NK, CD8+, and CD4+ T effector cells in melanoma and CRC tumors. Such infiltration resulted in the establishment of a T cell-inflamed tumor microenvironment, characterized by the up-regulation of pro-inflammatory chemokines and cytokines, CCL5, CXCL10, and IFNγ. Vps34i treatment induced STAT1 and IRF7, involved in the up-regulation of CCL5 and CXCL10. Combining Vps34i improved the therapeutic benefit of anti-PD-L1/PD-1 in melanoma and CRC and prolonged mice survival. Our study revealed that targeting Vps34 turns cold into hot inflamed tumors, thus enhancing the efficacy of anti-PD-L1/PD-1 blockade.
RESUMEN
Resistance to chemotherapy is a challenging problem for treatment of cancer patients and autophagy has been shown to mediate development of resistance. In this study we systematically screened a library of 306 known anti-cancer drugs for their ability to induce autophagy using a cell-based assay. 114 of the drugs were classified as autophagy inducers; for 16 drugs, the cytotoxicity was potentiated by siRNA-mediated knock-down of Atg7 and Vps34. These drugs were further evaluated in breast cancer cell lines for autophagy induction, and two tyrosine kinase inhibitors, Sunitinib and Erlotinib, were selected for further studies. For the pharmacological inhibition of autophagy, we have characterized here a novel highly potent selective inhibitor of Vps34, SB02024. SB02024 blocked autophagy in vitro and reduced xenograft growth of two breast cancer cell lines, MDA-MB-231 and MCF-7, in vivo. Vps34 inhibitor significantly potentiated cytotoxicity of Sunitinib and Erlotinib in MCF-7 and MDA-MB-231 in vitro in monolayer cultures and when grown as multicellular spheroids. Our data suggests that inhibition of autophagy significantly improves sensitivity to Sunitinib and Erlotinib and that Vps34 is a promising therapeutic target for combination strategies in breast cancer.
Asunto(s)
Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Células MCF-7 , Ratones Endogámicos NOD , Ratones SCID , Inhibidores de Proteínas Quinasas/farmacología , Sunitinib/farmacologíaRESUMEN
Insulin-stimulated glucose uptake in muscle and adipose tissue is the result of translocation of insulin-regulated glucose transporters (GLUT4) from intracellular vesicles to the plasma membrane. Here we report that GLUT4 in the plasma membrane of 3T3-L1 adipocytes were located predominantly in caveolae invaginations: by immunogold electron microscopy of plasma membranes, 88% of GLUT4 were localized to caveolae structures and this distribution within the plasma membrane was not affected by insulin. By immunofluorescence microscopy, a major part of GLUT 4 was colocalized with caveolin. The total amount of GLUT4 in the plasma membrane increased 2.2-fold in response to insulin as determined by immunogold electron or immunofluorescence microscopy. GLUT4 were enriched in caveolae fractions isolated without detergents from plasma membranes of rat adipocytes. In these fractions, GLUT4 were largely confined to caveolin-containing membranes of the caveolae preparation isolated from insulin-stimulated cells, determined by electron microscopy. Insulin increased the amount of GLUT4 2.7-fold in this caveolae fraction. Caveolae were purified further by immunoisolation with antibodies against caveolin. The amount of GLUT4 increased to the same extent in the immunopurified caveolae as in the cruder caveolae fractions from insulin-stimulated cells. We conclude that insulin induces translocation of GLUT4 to caveolae.