RESUMO
The receptor tyrosine kinase AXL is an emerging driver of cancer recurrence, while its molecular mechanism remains unclear. In this study we investigated how AXL regulated the disease progression and poor prognosis in non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC). We performed AXL transcriptome analysis from TCGA datasets, and found that AXL expression was significantly elevated in NSCLC and TNBC correlating with poor prognosis, epithelial-mesenchymal transition (EMT) and immune-tolerant tumor microenvironment (TME). Knockdown of AXL or treatment with two independent AXL antibodies (named anti-AXL and AXL02) all diminished cell migration and EMT in AXL-high expressing NSCLC and TNBC cell lines. In a mouse model of 4T1 TNBC, administration of anti-AXL antibody substantially inhibited lung metastases formation and growth, accompanied by reduced downstream signaling activation, EMT and proliferation index, as well as an increased apoptosis and activated anti-tumor immunity. We found that AXL was abundantly activated in tumor nodule-infiltrated M2-macrophages. A specific anti-AXL antibody blocked bone marrow-derived macrophage (BMDM) M2-polarization in vitro. Targeting of AXL in M2-macrophage in addition to tumor cell substantially suppressed CSF-1 production and eliminated M2-macrophage in TME, leading to a coordinated enhancement in both the innate and adaptive immunity reflecting M1-like macrophages, mature dendritic cells, cytotoxic T cells and B cells. We generated a novel and humanized AXL-ADC (AXL02-MMAE) employing a site-specific conjugation platform. AXL02-MMAE exerted potent cytotoxicity against a panel of AXL-high expressing tumor cell lines (IC50 < 0.1 nmol/L) and suppressed in vivo growth of multiple NSCLC and glioma tumors (a minimum efficacy dose<1 mg/kg). Compared to chemotherapy, AXL02-MMAE achieved a superior efficacy in regressing large sized tumors, eliminated AXL-H tumor cell-dependent M2-macrophage infiltration with a robust accumulation of inflammatory macrophages and mature dendritic cells. Our results support AXL-targeted therapy for treatment of advanced NSCLC and TNBC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proliferação de Células , Transição Epitelial-Mesenquimal , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Linhagem Celular Tumoral , Anticorpos/metabolismo , Macrófagos/metabolismo , Microambiente TumoralRESUMO
With the wide clinical use of the third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth-generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS-106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell-free assay, LS-106 potently inhibited the kinase activities of EGFR19del/T790M/C797S and EGFRL858R/T790M/C797S with IC50 values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS-106 exhibited comparable kinase inhibitory effect to osimertinib on EGFRL858R/T790M and wild-type EGFR. Results from cellular experiments demonstrated that LS-106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR19del/T790M/C797S or EGFRL858R/T790M/C797S , and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR19del/T790M/C797S (named PC-9-OR cells) using the CRISPR/Cas9 system and found that LS-106 markedly suppressed the activation of EGFR19del/T790M/C797S and the proliferation of PC-9-OR cells. Moreover, cells harboring EGFR19del/T790M/C797S underwent remarkable apoptosis upon LS-106 treatment. In vivo experiments further demonstrated that oral administration of LS-106 caused significant tumor regression in a PC-9-OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS-106 as a novel fourth-generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S-triple-mutant tumor models.
Assuntos
Antineoplásicos , Mutação , Inibidores de Proteínas Quinases , Animais , Humanos , Camundongos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Estrutura Molecular , Mutação/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although chemotherapy and recently approved immunotherapies have improved treatment of triple-negative breast cancer (TNBC), the clinical outcome for this deadly disease remains unsatisfactory. We found that both cluster of differentiation 73 (CD73) and transforming growth factor (TGF)ß were elevated in TNBC and correlated with the epithelial-mesenchymal transition (EMT), fibrotic stroma, an immune-tolerant tumor environment, and poor prognosis. To explore the efficacy of CD73-TGFß dual-blockade, we generated a bifunctional anti-CD73-TGFß construct consisting of the CD73 antibody MEDI9447 fused with the TGFßRII extracellular-domain (termed MEDI-TGFßR). MEDI-TGFßR retained full and simultaneous blocking efficiency for CD73 and TGFß. Compared with MEDI9447 activity alone, MEDI-TGFßR demonstrated superior inhibitory activity against CD73-dependent cell migration and the EMT in CD73-high TNBC cells and effectively reduced lung metastasis in a syngeneic mouse model of TNBC. Mechanistically, the CD73-TGFß dual-blockade reverted the EMT and stromal fibrosis and induced tumor cell death, which was accompanied by the accumulation of M1-macrophages and production of tumor necrosis factor α (TNFα). The CD73-TGFß dual-blockade promoted a multifaceted inflammatory tumor microenvironment, as shown by the diminished levels of myeloid-derived suppressor cells (MDSCs) and M2-macrophages, and substantially increased levels of activated dendritic cells, cytotoxic T cells, and B cells. Collectively, our results have highlighted a novel strategy for TNBC treatment.
Assuntos
5'-Nucleotidase/metabolismo , Neoplasias de Mama Triplo Negativas , Animais , Antígenos de Diferenciação de Linfócitos B , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Antígenos de Histocompatibilidade Classe II , Camundongos , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente TumoralRESUMO
High infiltration of M2-polarized macrophages in the primary tumor indicates unfavorable prognosis and poor overall survival in the patients with triple-negative breast cancer (TNBC). Thus, reversing M2-polarized tumor-associated macrophages in the tumors has been considered as a potential therapeutic strategy for TNBC. Sphingomyelin synthase 2 (SMS2) is the key enzyme for sphingomyelin production, which plays an important role in plasma membrane integrity and function. In this study we investigated whether SMS2 inhibitor or SMS2 gene knockout could reduce macrophages M2 polarization and tumor progression in a mouse model of TNBC. We showed that SMS2 mRNA expression was linked to immunosuppressive tumor microenvironment and poor prognosis in TNBC patients. The knockout of SMS2 or application of 15w (a specific SMS2 inhibitor) markedly decreased the generation of M2-type macrophages in vitro, and reduced the tumor weight and lung metastatic niche formation in a 4T1-TNBC mouse model. We further demonstrated that the in vivo antitumor efficacy of 15w was accompanied by a multifaceted remodeling of tumor immune environment reflecting not only the suppression of M2-type macrophages but also diminished levels of regulatory T cells and myeloid-derived suppressor cells leading to a dramatically improved infiltration of antitumor CD8+ T lymphocytes. Collectively, our results reveal a novel and important role of SMS2 in the protumorigenic function and may offer a new strategy for macrophage-targeted anticancer therapy.
Assuntos
Macrófagos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Neoplasias de Mama Triplo Negativas/fisiopatologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Técnicas de Inativação de Genes , Humanos , Imunidade Celular/efeitos dos fármacos , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/fisiologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/imunologiaRESUMO
The G protein-coupled receptor (GPCR) smoothened (SMO) is a key signaling component of the sonic hedgehog (Hh) pathway and a clinically validated target for cancer treatment. The FDA-approved SMO inhibitors GDC-0449/Vismodegib and LDE225/Sonidegib demonstrated clinical antitumor efficacy. Nevertheless, relatively high percentage of treated patients would eventually develop acquired cross resistance to both drugs. Here, based on published structure and activity of GDC-0449 inhibitor class, we replaced its amide core with benzimidazole which retained bulk of the SMO-targeting activity as measured in our Hh/SMO/Gli1-reporter system. Synthesis and screening of multiple series of benzimidazole derivatives identified HH-1, HH-13, and HH-20 with potent target suppression (IC50: <0.1 µmol/L) in the reporter assays. In NIH3T3 cells stimulated with a secreted Hh (SHH), these inhibitors dose dependently reduced mRNA and protein expression of the endogenous pathway components PTCH-1, Gli1, and cyclin D1 resulting in growth inhibition via G0/G1 arrest. Mechanistically, the SMO-targeted growth inhibition involved downregulation of mTOR signaling inputs and readouts consistent with diminished mTORC1/mTORC2 functions and apoptosis. In mice, as with GDC-0449, orally administered HH inhibitors blocked paracrine activation of stromal Hh pathway in Calu-6 tumor microenvironment and attenuated growth of PTCH+/-/P53-/- medulloblastoma allograft tumors. Furthermore, HH-13 and HH-20 potently targeted the drug-resistant smoothened SMO-D473H (IC50: <0.2 µmol/L) compared to the poor inhibition by GDC-0449 (IC50: >60 µmol/L). These results identify HH-13 and HH-20 as potent inhibitors capable of targeting naïve and drug-resistant Hh/SMO-driven cancers. The current leads may be optimized to improve pharmaceutical property for potential development of new therapy for treatment of Hh pathway-driven cancers.
Assuntos
Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Células NIH 3T3 , Transdução de Sinais/genética , Receptor Smoothened/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
While cancer cell mitochondria mediate actions of many successful chemotherapeutics, little is known about mitochondrial response in mTOR-targeted anticancer therapy. We have studied mitochondrial dynamics in relation to growth suppression employing an allosteric inhibitor rapalog, a highly selective mTOR kinase inhibitor (mTOR-KI) and mTOR-ShRNA. Global targeting of mTOR increased mitochondrial membrane potential (mΔψ) and inhibited mitochondrial permeability transition pore (mPTP). Importantly, these mTOR-KI-provoked anti-survival and pro-survival effects were differentially manifested in diverse cancer cells according to intrinsic susceptibility to mTOR-targeting. The most-sensitive cells including those possessing hyperactive PI3K/AKT/mTOR and/or growth factor-dependence (LNCap, MDA361 and MG63) all displayed a dramatic increase in mΔψ, whereas the mΔψ increase was not evident in majority of resistant cancer cells. Upon mTOR-KI treatment, the resistant cells including those harboring K-Ras- or B-Raf mutation (MDA231, HT29 and HCT116) all displayed a markedly reduced mPTP opening, which paralleled a sustained AKT-hexokinase 2 (HK2) survival signaling and persistent phosphorylation (inactivation) of GSK3ß. Further studies demonstrated that the mTOR-KI-provoked mPTP closure in resistant cells was mediated through an enhanced binding of HK2 to the mitochondrial voltage-dependent anion channel (VDAC), a molecular mechanism known to promote mPTP closure and cell survival. Detaching HK2 from VDAC by an HK2-displacing peptide or methyl jasmonate specifically blocked the mTOR-KI-provoked mPTP closure and potentiated growth suppression in resistant cells. Thus, mTOR-inhibition can exert complex and differential perturbation to mitochondrial dynamics in cancer cells, which likely influence therapeutic outcome of mTOR-targeted therapy.
Assuntos
Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Poro de Transição de Permeabilidade Mitocondrial , Compostos de Fenilureia/uso terapêutico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Células Tumorais Cultivadas , Canal de Ânion 1 Dependente de Voltagem/efeitos dos fármacosRESUMO
The recently approved KRASG12C mutation-specific inhibitors sotorasib and adagrasib (KRASG12C-I) represent a promising therapy for KRASG12C-driven non-small cell lung cancer (NSCLC). However, many eligible patients do not benefit due to intrinsic or acquired drug resistance. Tissue factor (TF) is overexpressed in KRAS-mutated (KRASmut) NSCLC and is the target of the FDA-approved ADC Tivdak. Here, we employed HuSC1-39, the parent antibody of a clinical stage TF-ADC (NCT04843709), to investigate the role of TF in KRASmut NSCLC. We found that patients with TF-overexpression had poor survival, elevated P-ERK/P-AKT activity levels and low immune effector cell infiltration in the tumor. In a panel of KRASG12C cell lines, KRASG12C-I response correlated with suppression of TF mRNA, which was not observed in resistant cells. In the drug resistant cells, TF-overexpression relied on an mTORC2-mediated and proteasome-dependent pathway. Combination treatment of HuSC1-39 or mTORC1/2 inhibitor MTI-31 with KRASG12C-I each produced synergistic antitumor efficacy in cell culture and in an orthotopic lung tumor model. TF-depletion in the resistant cells diminished epithelial mesenchymal transition, reduced tumor growth and greatly sensitized KRASG12C-I response. Moreover, employing immunohistochemistry and coculture studies, we demonstrated that HuSC1-39 or MTI-31 reset the tumor microenvironment and restore KRASG12C-I sensitivity by reshaping an M1-like macrophage profile with greatly enhanced phagocytic capacity toward tumor cell killing. Thus, we have identified the TF/mTORC2 axis as a critical new mechanism for triggering immunosuppression and KRASG12C-I resistance. We propose that targeting this axis with HuSC1-39 or MTI-31 will improve KRASG12C-I response in KRAS-driven NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Tromboplastina , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Alvo Mecanístico do Complexo 2 de Rapamicina , Mutação , Microambiente TumoralRESUMO
Triple-negative breast cancer (TNBC) remains a most deadly human malignancy with limited response to chemotherapy, targeted therapy and immunotherapy. Tumor immunoenvironment plays an increasingly important role in therapy outcome. Tissue factor (TF) is the target of the FDA-approved ADC Tivdak. HuSC1-39 is the parent antibody of MRG004A, a clinical stage TF-ADC (NCT04843709). Here, we employed HuSC1-39 (termed "anti-TF") to investigate the role of TF in regulating immune-tolerance in TNBC. We found that patients with aberrant TF expression had a poor prognosis and low immune effector cell infiltration, characterizing as "cold tumor". In the 4T1 TNBC syngeneic mouse model, knockout of tumor cell TF inhibited tumor growth and increased tumor infiltration of effector T cell, which was not dependent on the clotting inhibition. In an immune-reconstituted M-NSG mouse model of TNBC, anti-TF inhibited tumor growth, which was further enhanced by a dual-targeting anti-TF&TGFßR fusion protein. There were diminished P-AKT and P-ERK signaling and profound tumor cell death in treated tumors. Transcriptome analyses and immunohistochemistry revealed a dramatically improved tumor immunoenvironment including the increase of effector T cells, decrease of Treg cells and the transformation of tumor into "hot tumor". Moreover, employing qPCR analysis and T cell culture, we further demonstrated that TF expression in tumor cells is sufficient to block the synthesis and secretion of T cell-recruiting chemokine CXCL9/10/11. Treatment of TF-high TNBC cells with anti-TF or TF-knockout all stimulated CXCL9/10/11 production, promoted T cell migration and effector function. Thus, we have identified a new mechanism of TF in TNBC tumor progression and therapy resistance.
Assuntos
Neoplasias de Mama Triplo Negativas , Animais , Camundongos , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Tromboplastina/genética , Tromboplastina/metabolismo , Evasão da Resposta Imune , Transdução de Sinais , Imunoterapia , Linhagem Celular TumoralRESUMO
Osimertinib (AZD9291), a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), has significantly improved the survival of non-small cell lung cancer (NSCLC) patients with EGFRT790M mutation, the major mechanism of acquired resistance to first-generation EGFR TKI. However, resistance to AZD9291 arises eventually and EGFRC797S mutation was reported to be a major resistance mechanism. Thus, it is highly valuable to develop novel EGFR fourth-generation inhibitors targeting C797S mutation to override the acquired resistance. In this study, we identified HCD3514 as a novel EGFR fourth-generation inhibitors targeting C797S triple mutation. It strongly inhibited EGFRL858R/T790M/C797S and EGFR19del/T790M/C797S mutations with IC50 values of 1.0 and 2.0 nM, respectively. HCD3514 dose-dependently inhibited the activation of EGFR in both engineered BaF3 cells and tumor cells harboring EGFRC797S triple mutant and thus effectively suppressed the proliferation of the cells. Moreover, HCD3514 caused a dose-dependent increase of apoptosis in C797S triple mutant cells accompanied by increased levels of cleaved caspase-3 and cleaved PARP. Furthermore, HCD3514 induced tumor growth inhibition in EGFR19del/T790M/C797S xenograft model as a single oral agent by decreasing the activation of EGFR. In addition to EGFRC797S triple mutations, HCD3514 also potently and selectively inhibited EGFRT790M double mutations (L858R/T790M and 19del/T790M). Collectively, HCD3514 is a highly selective and potent EGFR inhibitor against EGFRC797S triple mutations as well as EGFRT790M double mutations and is confirmed potently anti-tumor activity in preclinical models.
RESUMO
hSMG-1 kinase plays a dual role in a highly conserved RNA surveillance pathway termed nonsense-mediated RNA decay (NMD) and in cellular genotoxic stress response. Since deregulation of cellular responses to stress contributes to tumor growth and resistance to chemotherapy, hSMG-1 is a potential target for cancer treatment. From our screening efforts, we have identified pyrimidine derivatives as hSMG-1 kinase inhibitors. We report structure-based optimization of this pan-kinase scaffold to improve its biochemical profile and overall kinome selectivity, including mTOR and CDK, to generate the first reported selective hSMG-1 tool compound.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Pirimidinas/química , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Proteínas Serina-Treonina Quinases , Pirimidinas/síntese química , Pirimidinas/farmacologiaRESUMO
Background: Tumor-associated macrophages (TAMs) are one of the most abundant immune cells in the pancreatic cancer stroma and are related to the poor prognosis of pancreatic ductal adenocarcinoma (PDAC) patients. Therefore, targeting tumor-associated macrophages is a possible strategy for the treatment of pancreatic cancer. Purpose: We would like to investigate the role of sphingomyelin synthase 2 (SMS2) and the effect of the synthase 2 selective inhibitor YE2 in TAMs and the pancreatic tumor microenvironment. In addition, we also would like to investigate the mechanism by which YE2 attenuates macrophage M2 polarization. Methods: YE2 was utilized to treat macrophages (in vitro) and mice (in vivo). Western blotting and real-time PCR were used to detect the protein levels and mRNA levels of macrophage M2 polarization markers and their downstream signaling pathways. Sphingomyelin synthase 2 gene knockout (KO) mice and their controls were used to establish a PANC-02 orthotopic pancreatic cancer model, and immune cell infiltration in the tumor tissue was analyzed by immunohistochemistry (IHC). Results: We found that sphingomyelin synthase 2 mRNA expression is positively correlated with tumor-associated macrophages, the immunosuppressive microenvironment, and poor prognosis in pancreatic ductal adenocarcinoma patients. Sphingomyelin synthase 2 deficiency was confirmed to have an inhibitory effect on the growth of orthotopic PANC-02 tumors in vivo. The deficiency not only reduced the infiltration of tumor-associated macrophages but also regulated other immune components in the tumor microenvironment. In tissue culture, YE2 inhibited M2 polarization in both bone marrow-derived macrophages (BMDMs) and THP-1 macrophages and eliminated the protumor effect of M2 macrophages. In the mouse model, YE2 treatment reduced the infiltration of TAMs and regulated other immune components in the tumor microenvironment, slowing the progression of PANC-02 tumors. In terms of mechanism, we found that the inhibition of sphingomyelin synthase 2 could downregulate the expression of IL4Rα and CSF1R, thereby attenuating M2 polarization. Conclusion: The sphingomyelin synthase 2 inhibitor YE2 or sphingomyelin synthase 2 deficiency can prevent macrophage M2 polarization in pancreatic cancer, and sphingomyelin synthase 2 could be a new potential target for the treatment of pancreatic cancer.
RESUMO
BACKGROUND: The overall response rate to checkpoint blockade remains unsatisfactory, partially due to the immune-suppressive tumor microenvironment. A retinoic acid-related orphan receptor γt (RORγt) agonist (LYC-55716) is currently used in clinical trials combined with anti-PD-1, but how the Th17 cell transcription factor RORγt enhances antitumor immunity of PD-1 in the tumor microenvironment remains elusive. METHODS: The expression of mRNA was analyzed using qPCR assays. Flow cytometry was used to sort and profile cells. Cell migration was analyzed using Transwell assays. Biacore was used to determine the binding affinity to the RORγt protein. The RORγt GAL4 cell-based reporter gene assay was used to measure activity in the RORγt driven luciferase reporter gene expression. RESULTS: We designed a potent and selective small-molecule RORγt agonist (8-074) that shows robust antitumor efficacy in syngeneic tumor models and improves the efficacy of antiPD1 in a murine lung cancer model. RORγt agonist treatment increased intratumoral CD8+ T cells, which were correlated with CXCL10 and monocyte-derived dendritic cells (MoDCs). In addition, the RORγt agonist promoted Type 17 T cell migration by upregulating CCL20 and CCR6 expression, and Type 17 T cell tumor infiltration. CCL20 induces MoDCs migration, and CXCL10 derived from MoDCs promotes CD8+ T cell migration. CONCLUSION: Our results revealed that the RORγt agonist improved the efficacy of anti-PD-1. The RORγt agonist increased the migration of MoDCs, which increased the local levels of CXCL10, thus promoting CD8+ T cell tumor infiltration. Our findings provide the mechanistic insights implicating the RORγt agonist in immunotherapy and offer a strategy for targeting the RORγt agonist to improve PD-1 antibody efficacy in cancers.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias Pulmonares , Animais , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Células Dendríticas , Humanos , Camundongos , Monócitos/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Receptor de Morte Celular Programada 1 , Microambiente TumoralRESUMO
The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1-4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase III/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Humanos , Fosforilação , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina-Treonina Quinases TOR , Espectrometria de Massas em TandemRESUMO
We recently described several highly potent, triazine (1) and triazolopyrimidine (2) scaffold-based, dual PI3K/mTOR-inhibitors (e.g., 1, PKI-587) that were efficacious in both in vitro and in vivo models. In order to further optimize these compounds we devised a novel series, the 2-oxatriazines, which also exhibited excellent potency and good metabolic stability. Some 2-oxatriazines showed promising in vivo biomarker suppression and induced apoptosis in the MDA-MB-361 breast cancer xenograft model.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Triazinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Triazinas/química , Triazinas/metabolismoRESUMO
Homotemsirolimuses A, B, and C (2a, 2b, 2c) were found to be minor components of a temsirolimus preparation made from rapamycin. These three temsirolimus analogues are derived from the corresponding rapamycin analogues, homorapamycins A, B, and C (1a, 1b, 1c) produced by the strain Streptomyces hygroscopicus. The structures of homotemsirolimuses A, B, and C were determined by spectroscopic methods. These compounds were tested for mTOR kinase inhibition and in two proliferation assays using LNCap prostate and MDA468 breast cancer cells. The results suggested that the mTOR inhibition and antiproliferation potencies for 2a, 2b, and 2c are comparable to those of rapamycin (1) and temsirolimus (2).
Assuntos
Antineoplásicos/isolamento & purificação , Sirolimo/análogos & derivados , Streptomyces/química , Serina-Treonina Quinases TOR/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Masculino , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Sirolimo/química , Sirolimo/isolamento & purificação , Sirolimo/farmacologia , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
BACKGROUND AND PURPOSE: PD-1/PD-L1 antibodies have achieved great success in clinical treatment. However, monoclonal antibody drugs also have challenges, such as high manufacturing costs, poor diffusion, low oral bioavailability and limited penetration into tumour tissue. The development of small-molecule inhibitors of PD-1/PD-L1 interaction represents a promising perspective to overcome the above challenges in cancer immunotherapy. EXPERIMENTAL APPROACH: We explored structural activity relationships and used biochemical assays to generate a lead compound (ZE132). CD8+ T-cells killing assay and Ifng expression assay were used to verify the in vitro cellular activity of ZE132. Off-target study was performed to verify the selectivity. Syngeneic mouse models were used to verify the in vivo activity of ZE132 in tumour immune microenvironment (TIME). We also performed pharmacokinetics profiling in mice and The Cancer Genome Atlas database analysis. KEY RESULTS: ZE132 can effectively inhibit the PD-1/PD-L1 interactions in vitro, and it has a potent affinity to PD-L1. ZE132 shows robust anti-tumour effects in vivo, better than anti-PD-1 antibody. In the analysis of TIME, we found that ZE132 treatment promotes cytotoxic T-cell tumour infiltration and induces IL-2 expression. In addition, ZE132 elicits strong inhibitory effects on the mRNA expression of TGF-ß, which may serve as a potential biomarker to predict responsiveness to PD-1/PD-L1 immunotherapies. CONCLUSION AND IMPLICATIONS: We identified a new lead compound ZE132 targeting PD-1/PD-L1 interactions, not only showing favourable drug-like properties in vitro and in vivo but also showing the advantage of overcoming the barrier of TIME compared to anti-PD-1 antibody.
Assuntos
Antígeno B7-H1 , Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Antígeno B7-H1/antagonistas & inibidores , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Microambiente TumoralRESUMO
To date, the overall response rate of PD-1 blockade remains unsatisfactory, partially due to limited understanding of tumor immune microenvironment (TIME). B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, is highly expressed in cancers. By genetic depletion and pharmacological inhibition of BCL9 in tumors, we found that BCL9 suppression reduced tumor growth, promoted CD8+ T cell tumor infiltration, and enhanced response to anti-PD-1 treatment in mouse colon cancer models. To determine the underlying mechanism of BCL9's role in TIME regulation, single-cell RNA-seq was applied to reveal cellular landscape and transcription differences in the tumor immune microenvironment upon BCL9 inhibition. CD155-CD226 and CD155-CD96 checkpoints play key roles in cancer cell/CD8+ T cell interaction. BCL9 suppression induces phosphorylation of VAV1 in CD8+ T cells and increases GLI1 and PATCH expression to promote CD155 expression in cancer cells. In The Cancer Genome Atlas database analysis, we found that BCL9 expression is positively associated with CD155 and negatively associated with CD226 expression. BCL9 is also linked to adenomatous polyposis coli (APC) mutation involved in patient survival following anti-PD-1 treatment. This study points to cellular diversity within the tumor immune microenvironment affected by BCL9 inhibition and provides new insights into the role of BCL9 in regulating CD226 and CD96 checkpoints.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Neoplasias do Colo/tratamento farmacológico , Receptor de Morte Celular Programada 1/genética , Fatores de Transcrição/genética , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-vav/genética , Receptores Virais/genética , Fatores de Transcrição/antagonistas & inibidores , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Treatment of patients with triple-negative breast cancer (TNBC) has been challenging due to a lack of well-defined molecular targets. The Wnt/ß-catenin pathway is known to be activated in many TNBC patients and BCL9 and BCL9L are important transcriptional co-activators of ß-catenin, but whether inhibition of BCL9/BCL9L can suppress TNBC growth and the underlying mechanism are not fully understood. Here we demonstrate that the expression of BCL9 and BCL9L is directly correlated with malignancy in TNBC patient tumors and that BCL9 and BCL9L promote tumor cell growth, cell migration and metastasis in TNBC models. Mechanistically, we found that BCL9/BCL9L promotes tumorigenicity through both the Wnt and TGF-ß pathways. Besides, BCL9/BCL9L expression inversely correlates with CD8+ T cell infiltration in TNBC and BCL9/BCL9L inhibits the infiltration of CD8+ T cells in the tumor microenvironment. hsBCL9CT-24, an inhibitor of BCL9/ß-catenin peptides, promotes intratumoral infiltration of cytotoxic T cells, reducing regulatory T cells (Treg) and increasing dendritic cells (DCs). Inhibition of BCL9/BCL9L and TGF-ß suppresses activity of Treg. TGF-ß signaling increases tumor infiltration of cytotoxic CD8+ T cells. In accordance, genetic or pharmacological inhibition of BCL9/BCL9L synergizes with PD-1/L1 antibodies to inhibit tumor growth. In summary, these results suggest that targeting BCL9/BCL9L has a direct anti-tumor effect and also unleashes an anti-cancer immune response through inhibition of both Wnt and TGF-ß signaling, suggesting a viable therapeutic approach for TNBC treatment.
Assuntos
Proteínas de Ligação a DNA/imunologia , Fatores de Transcrição/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/fisiologia , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Microambiente TumoralRESUMO
Potent inhibitors of the mammalian target of rapamycin (mTOR) which contain the triazine scaffold and the (R)-3-methyl morpholine moiety have been identified. Such compounds also demonstrated good selectivity over the related lipid kinase PI3Kalpha. Incorporation of additional functionality at the 4-position of the arylureidophenyl ring resulted in compounds with enhanced cellular activity.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Morfolinas/química , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Triazinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR , Triazinas/químicaRESUMO
2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these compounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K. Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as highly potent (IC(50) <1nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and good potency in a cellular proliferation assay (IC(50) <50nM).