RESUMO
Natural killer (NK) cells are lymphocytes of the innate immune system. A key feature of NK cells is their ability to recognize a wide range of cells in distress, particularly tumour cells and cells infected with viruses. They combine both direct effector functions against their cellular targets and participate in the generation, shaping and maintenance of a multicellular immune response. As our understanding has deepened, several therapeutic strategies focused on NK cells have been conceived and are currently in various stages of development, from preclinical investigations to clinical trials. Here we explore in detail the complexity of NK cell biology in humans and highlight the role of these cells in cancer immunity. We also analyse the harnessing of NK cell immunity through immune checkpoint inhibitors, NK cell engagers, and infusions of preactivated or genetically modified, autologous or allogeneic NK cell products.
Assuntos
Imunoterapia Adotiva , Células Matadoras Naturais , Neoplasias , Humanos , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , Neoplasias/imunologia , Neoplasias/terapia , Imunidade InataRESUMO
The adenosinergic pathway represents an attractive new therapeutic approach in cancer immunotherapy. In this pathway, ecto-5-nucleotidase CD73 has the unique function of regulating production of immunosuppressive adenosine (ADO) through the hydrolysis of AMP. CD73 is overexpressed in many cancers, resulting in elevated levels of ADO that correspond to poor patient prognosis. Therefore, reducing the level of ADO via inhibition of CD73 is a potential strategy for treating cancers. Based on the binding mode of adenosine 5'-(α,ß-methylene)diphosphate (AOPCP) with human CD73, we designed a series of novel monophosphonate small-molecule CD73 inhibitors. Among them, OP-5244 (35) proved to be a highly potent and orally bioavailable CD73 inhibitor. In preclinical studies, 35 completely inhibited ADO production in both human cancer cells and CD8+ T cells. Furthermore, 35 lowered the ratio of ADO/AMP significantly and reversed immunosuppression in mouse models, indicating its potential as an in vivo tool compound for further development.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , Adenosina/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fatores Imunológicos/farmacologia , Nucleosídeos/farmacologia , Organofosfonatos/farmacologia , Administração Oral , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral , Cães , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Proteínas Ligadas por GPI/antagonistas & inibidores , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/síntese química , Fatores Imunológicos/farmacocinética , Macaca fascicularis , Camundongos Endogâmicos BALB C , Estrutura Molecular , Nucleosídeos/administração & dosagem , Nucleosídeos/síntese química , Nucleosídeos/farmacocinética , Organofosfonatos/administração & dosagem , Organofosfonatos/síntese química , Organofosfonatos/farmacocinética , Ratos , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Structure-based modification of mifepristone (1) led to the discovery of novel mifepristone derivatives with improved selectivity profile. Addition of a methyl group at the C10 position of the steroid has a significant impact on progesterone receptor (PR) and androgen receptor (AR) activity. Within this series, OP-3633 (15) emerged as a glucocorticoid receptor (GR) antagonist with increased selectivity against PR and AR, improved cytochrome P450 inhibition profile, and significantly improved pharmacokinetic properties compared to 1. Furthermore, 15 demonstrated substantial inhibition of GR transcriptional activity in the GR positive HCC1806 triple negative breast cancer xenograft model. Overall, compound 15 is a promising GR antagonist candidate to clinically evaluate the impact of GR inhibition in reversal or prevention of therapy resistance.
Assuntos
Mifepristona/análogos & derivados , Mifepristona/farmacologia , Receptores de Glucocorticoides/antagonistas & inibidores , Antagonistas de Receptores de Andrógenos/química , Antagonistas de Receptores de Andrógenos/farmacologia , Descoberta de Drogas , Humanos , Modelos Moleculares , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismoRESUMO
The aims of this study were to determine whether combination chemotherapeutics exhibit a synergistic effect on breast cancer cell metabolism. Palbociclib, is a selective inhibitor of cyclin-dependent kinases 4 and 6, and when patients are treated in combination with fulvestrant, an estrogen receptor antagonist, they have improved progression-free survival. The mechanisms for this survival advantage are not known. Therefore, we analyzed metabolic and transcriptomic changes in MCF-7 cells following single and combination chemotherapy to determine whether selective metabolic pathways are targeted during these different modes of treatment. Individually, the drugs caused metabolic disruption to the same metabolic pathways, however fulvestrant additionally attenuated the pentose phosphate pathway and the production of important coenzymes. A comprehensive effect was observed when the drugs were applied together, confirming the combinatory therapy's synergism in the cell model. This study also highlights the power of merging high-dimensional datasets to unravel mechanisms involved in cancer metabolism and therapy.
RESUMO
Tumors use indoleamine 2,3-dioxygenase-1 (IDO1) as a major mechanism to induce an immunosuppressive microenvironment. IDO1 expression is upregulated in many cancers and considered to be a resistance mechanism to immune checkpoint therapies. IDO1 is induced in response to inflammatory stimuli such as IFNγ and promotes immune tolerance by depleting tryptophan and producing tryptophan catabolites, including kynurenine, in the tumor microenvironment. This leads to effector T-cell anergy and enhanced Treg function through upregulation of FoxP3. As a nexus for the induction of key immunosuppressive mechanisms, IDO1 represents an important immunotherapeutic target in oncology. Here, we report the identification and characterization of the novel selective, orally bioavailable IDO1 inhibitor EOS200271/PF-06840003. It reversed IDO1-induced T-cell anergy in vitro In mice carrying syngeneic tumor grafts, PF-06840003 reduced intratumoral kynurenine levels by over 80% and inhibited tumor growth both in monotherapy and, with an increased efficacy, in combination with antibodies blocking the immune checkpoint ligand PD-L1. We demonstrate that anti-PD-L1 therapy results in increased IDO1 metabolic activity thereby providing additional mechanistic rationale for combining PD-(L)1 blockade with IDO1 inhibition in cancer immunotherapies. Supported by these preclinical data and favorable predicted human pharmacokinetic properties of PF-06840003, a phase I open-label, multicenter clinical study (NCT02764151) has been initiated.
Assuntos
Antígeno B7-H1/antagonistas & inibidores , Biocatálise , Inibidores Enzimáticos/farmacologia , Imunoterapia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indóis/farmacologia , Succinimidas/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Antígeno CTLA-4/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferon gama/metabolismo , Cinurenina/sangue , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estereoisomerismo , Especificidade por Substrato/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
The glucocorticoid receptor (GR) has been linked to therapy resistance across a wide range of cancer types. Preclinical data suggest that antagonists of this nuclear receptor may enhance the activity of anticancer therapy. The first-generation GR antagonist mifepristone is currently undergoing clinical evaluation in various oncology settings. Structure-based modification of mifepristone led to the discovery of ORIC-101 (28), a highly potent steroidal GR antagonist with reduced androgen receptor (AR) agonistic activity amenable for dosing in androgen receptor positive tumors and with improved CYP2C8 and CYP2C9 inhibition profile to minimize drug-drug interaction potential. Unlike mifepristone, 28 could be codosed with chemotherapeutic agents readily metabolized by CYP2C8 such as paclitaxel. Furthermore, 28 demonstrated in vivo antitumor activity by enhancing response to chemotherapy in the GR+ OVCAR5 ovarian cancer xenograft model. Clinical evaluation of safety and therapeutic potential of 28 is underway.
Assuntos
Descoberta de Drogas , Antagonistas de Hormônios/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Feminino , Antagonistas de Hormônios/química , Antagonistas de Hormônios/farmacocinética , Humanos , Camundongos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Ratos , Suínos , Porco Miniatura , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The CXCR4-CXCL12 axis plays an important role in the chronic lymphocytic leukemia (CLL)-microenvironment interaction. Overexpression of CXCR4 has been reported in different hematological malignancies including CLL. Binding of the pro-survival chemokine CXCL12 with its cognate receptor CXCR4 induces cell migration. CXCL12/CXCR4 signaling axis promotes cell survival and proliferation and may contribute to the tropism of leukemia cells towards lymphoid tissues and bone marrow. Therefore, we hypothesized that targeting CXCR4 with an IgG1 antibody, PF-06747143, may constitute an effective therapeutic approach for CLL. METHODS: Patient-derived primary CLL-B cells were assessed for cytotoxicity in an in vitro model of CLL microenvironment. PF-06747143 was analyzed for cell death induction and for its potential to interfere with the chemokine CXCL12-induced mechanisms, including migration and F-actin polymerization. PF-06747143 in vivo efficacy was determined in a CLL murine xenograft tumor model. RESULTS: PF-06747143, a novel-humanized IgG1 CXCR4 antagonist antibody, induced cell death of patient-derived primary CLL-B cells, in presence or absence of stromal cells. Moreover, cell death induction by the antibody was independent of CLL high-risk prognostic markers. The cell death mechanism was dependent on CXCR4 expression, required antibody bivalency, involved reactive oxygen species production, and did not require caspase activation, all characteristics reminiscent of programmed cell death (PCD). PF-06747143 also induced potent B-CLL cytotoxicity via Fc-driven antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity activity (CDC). PF-06747143 had significant combinatorial effect with standard of care (SOC) agents in B-CLL treatment, including rituximab, fludarabine (F-ara-A), ibrutinib, and bendamustine. In a CLL xenograft model, PF-06747143 decreased tumor burden and improved survival as a monotherapy, and in combination with bendamustine. CONCLUSIONS: We show evidence that PF-06747143 has biological activity in CLL primary cells, supporting a rationale for evaluation of PF-06747143 for the treatment of CLL patients.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Imunoglobulina G/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Receptores CXCR4/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Células CHO , Morte Celular/efeitos dos fármacos , Cricetulus , Feminino , Humanos , Imunoglobulina G/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos Endogâmicos BALB C , Camundongos SCID , Espécies Reativas de Oxigênio/imunologia , Receptores CXCR4/análise , Receptores CXCR4/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Células Tumorais CultivadasRESUMO
S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.
Assuntos
Metionina Adenosiltransferase/antagonistas & inibidores , Quinolinas/farmacologia , S-Adenosilmetionina/metabolismo , Triazóis/farmacologia , Sítio Alostérico/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Cinética , Metionina Adenosiltransferase/isolamento & purificação , Metionina Adenosiltransferase/metabolismo , Quinolinas/química , Relação Estrutura-Atividade , Triazóis/químicaRESUMO
The chemokine receptor CXCR4 is highly expressed and associated with poor prognosis in multiple malignancies. Upon engagement by its ligand, CXCL12, CXCR4 triggers intracellular signaling pathways that control trafficking of cells to tissues where the ligand is expressed, such as the bone marrow (BM). In hematologic cancers, CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism driving disease and therapy resistance. We developed a humanized CXCR4 immunoglobulin G1 (IgG1) antibody (Ab), PF-06747143, which binds to CXCR4 and inhibits CXCL12-mediated signaling pathways, as well as cell migration. In in vivo preclinical studies, PF-06747143 monotherapy rapidly and transiently mobilized cells from the BM into the peripheral blood. In addition, PF-06747143 effectively induced tumor cell death via its Fc constant region-mediated effector function. This Fc-mediated cell killing mechanism not only enhanced antitumor efficacy, but also played a role in reducing the duration of cell mobilization, when compared with an IgG4 version of the Ab, which does not have Fc-effector function. PF-06747143 treatment showed strong antitumor effect in multiple hematologic tumor models including non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), and multiple myeloma (MM). Importantly, PF-06747143 synergized with standard-of-care agents in a chemoresistant AML patient-derived xenograft model and in an MM model. These findings suggest that PF-06747143 is a potential best-in-class anti-CXCR4 antagonist for the treatment of hematologic malignancies, including in the resistant setting. PF-06747143 is currently in phase 1 clinical trial evaluation (registered at www.clinicaltrials.gov as #NCT02954653).
RESUMO
A new enhancer of zeste homolog 2 (EZH2) inhibitor series comprising a substituted phenyl ring joined to a dimethylpyridone moiety via an amide linkage has been designed. A preferential amide torsion that improved the binding properties of the compounds was identified for this series via computational analysis. Cyclization of the amide linker resulted in a six-membered lactam analogue, compound 18. This transformation significantly improved the ligand efficiency/potency of the cyclized compound relative to its acyclic analogue. Additional optimization of the lactam-containing EZH2 inhibitors focused on lipophilic efficiency (LipE) improvement, which provided compound 31. Compound 31 displayed improved LipE and on-target potency in both biochemical and cellular readouts relative to compound 18. Inhibitor 31 also displayed robust in vivo antitumor growth activity and dose-dependent de-repression of EZH2 target genes.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Desenho de Fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Piridonas/química , Piridonas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Ciclização , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Lactamas/química , Lactamas/farmacologia , Camundongos , Camundongos SCID , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Piridonas/uso terapêuticoRESUMO
Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Cloroquina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cloridrato de Erlotinib/farmacologia , Técnicas de Inativação de Genes , Humanos , Indóis/farmacologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Tolerância a Radiação/genética , Sunitinibe , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lactamas Macrocíclicas/administração & dosagem , Neoplasias/tratamento farmacológico , Receptores Proteína Tirosina Quinases/genética , Aminopiridinas , Quinase do Linfoma Anaplásico , Animais , Antineoplásicos/farmacologia , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Lactamas , Lactamas Macrocíclicas/farmacologia , Camundongos , Mutação , Células NIH 3T3 , Neoplasias/genética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pirazóis , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.
Assuntos
Aminoácidos/metabolismo , Epigênese Genética/fisiologia , Transportador 1 de Aminoácidos Neutros Grandes/fisiologia , Complexo Repressor Polycomb 2/fisiologia , Animais , Transporte Biológico/genética , Proliferação de Células/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células Tumorais CultivadasRESUMO
AIMS: Vorinostat (suberoylanilide hydroxamic acid; SAHA) is a histone deacetylase inhibitor (HDACi) approved in the clinics for the treatment of T-cell lymphoma and with the potential to be effective also in breast cancer. We investigated the responsiveness to SAHA in human breast primary tumors and cancer cell lines. RESULTS: We observed a differential response to drug treatment in both human breast primary tumors and cancer cell lines. Gene expression analysis of the breast cancer cell lines revealed that genes involved in cell adhesion and redox pathways, especially glutathione metabolism, were differentially expressed in the cell lines resistant to SAHA compared with the sensitive ones, indicating their possible association with drug resistance mechanisms. Notably, such an association was also observed in breast primary tumors. Indeed, addition of buthionine sulfoximine (BSO), a compound capable of depleting cellular glutathione, significantly enhanced the cytotoxicity of SAHA in both breast cancer cell lines and primary breast tumors. INNOVATION: We identify and validate transcriptional differences in genes involved in redox pathways, which include potential predictive markers of sensitivity to SAHA. CONCLUSION: In breast cancer, it could be relevant to evaluate the expression of antioxidant genes that may favor tumor resistance as a factor to consider for potential clinical application and treatment with epigenetic drugs (HDACis).
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Antineoplásicos/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/toxicidade , Oxirredução/efeitos dos fármacos , Cultura Primária de Células , VorinostatRESUMO
Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Lactamas Macrocíclicas/farmacologia , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Pirazóis/farmacologia , Piridinas/farmacologia , Aminopiridinas , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Proliferação de Células/efeitos dos fármacos , Crizotinibe , Cristalografia por Raios X , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioma/patologia , Humanos , Lactamas , Lactamas Macrocíclicas/química , Camundongos , Modelos Moleculares , Transdução de Sinais/efeitos dos fármacosRESUMO
In addition to genetic alterations, cancer cells are characterized by myriad epigenetic changes. EZH2 is a histone methyltransferase that is over-expressed and mutated in cancer. The EZH2 gain-of-function (GOF) mutations first identified in lymphomas have recently been reported in melanoma (~2%) but remain uncharacterized. We expressed multiple EZH2 GOF mutations in the A375 metastatic skin melanoma cell line and observed both increased H3K27me3 and dramatic changes in 3D culture morphology. In these cells, prominent morphological changes were accompanied by a decrease in cell contractility and an increase in collective cell migration. At the molecular level, we observed significant alteration of the axonal guidance pathway, a pathway intricately involved in the regulation of cell shape and motility. Furthermore, the aggressive 3D morphology of EZH2 GOF-expressing melanoma cells (both endogenous and ectopic) was attenuated by EZH2 catalytic inhibition. Finally, A375 cells expressing exogenous EZH2 GOF mutants formed larger tumors than control cells in mouse xenograft studies. This study not only demonstrates the first functional characterization of EZH2 GOF mutants in non-hematopoietic cells, but also provides a rationale for EZH2 catalytic inhibition in melanoma.
Assuntos
Movimento Celular , Proliferação de Células , Forma Celular , Epigênese Genética , Melanoma/genética , Mutação , Complexo Repressor Polycomb 2/genética , Neoplasias Cutâneas/genética , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma/patologia , Camundongos Nus , Terapia de Alvo Molecular , Invasividade Neoplásica , Complexo Repressor Polycomb 2/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/patologia , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted blockade of aberrantly activated signaling pathways is an attractive therapeutic strategy for solid tumors, but drug resistance is common. KRAS is a frequently mutated gene in human cancer but remains a challenging clinical target. Inhibitors against KRAS signaling mediators, namely, PI3K (phosphatidylinositol 3-kinase) and mTOR (mechanistic target of rapamycin), have limited clinical efficacy as single agents in KRAS-mutant colorectal cancer (CRC). We investigated potential bypass mechanisms to PI3K/mTOR inhibition in KRAS-mutant CRC. Using genetically engineered mouse model cells that had acquired resistance to the dual PI3K/mTOR small-molecule inhibitor PF-04691502, we determined with chemical library screens that inhibitors of the ERBB [epidermal growth factor receptor (EGFR)] family restored the sensitivity to PF-04691502. Although EGFR inhibitors alone have limited efficacy in reducing KRAS-mutant tumors, we found that PF-04691502 induced the abundance, phosphorylation, and activity of EGFR, ERBB2, and ERBB3 through activation of FOXO3a (forkhead box O 3a), a transcription factor inhibited by the PI3K to AKT pathway. PF-04691502 also induced a stem cell-like gene expression signature. KRAS-mutant patient-derived xenografts from mice treated with PF-04691502 had a similar gene expression signature and exhibited increased EGFR activation, suggesting that this drug-induced resistance mechanism may occur in patients. Combination therapy with dacomitinib (a pan-ERBB inhibitor) restored sensitivity to PF-04691502 in drug-resistant cells in culture and induced tumor regression in drug-resistant allografts in mice. Our findings suggest that combining PI3K/mTOR and EGFR inhibitors may improve therapeutic outcome in patients with KRAS-mutant CRC.
Assuntos
Neoplasias Colorretais/metabolismo , Inibidores Enzimáticos/química , Receptores ErbB/antagonistas & inibidores , Genes ras , Inibidores de Fosfoinositídeo-3 Quinase , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Sobrevivência Celular , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , Engenharia Genética , Humanos , Camundongos , Camundongos SCID , Mutação , Transplante de Neoplasias , Fosforilação , Transdução de Sinais , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , beta Catenina/metabolismo , Proteínas ras/genéticaRESUMO
The non-essential amino acid, glutamine, exerts pleiotropic effects on cell metabolism, signalling and stress resistance. Here we demonstrate that short-term glutamine restriction triggers an endoplasmic reticulum (ER) stress response that leads to production of the pro-inflammatory chemokine, interleukin-8 (IL-8). Glutamine deprivation-induced ER stress triggers colocalization of autophagosomes, lysosomes and the Golgi into a subcellular structure whose integrity is essential for IL-8 secretion. The stimulatory effect of glutamine restriction on IL-8 production is attributable to depletion of tricarboxylic acid cycle intermediates. The protein kinase, mTOR, is also colocalized with the lysosomal membrane clusters induced by glutamine deprivation, and inhibition of mTORC1 activity abolishes both endomembrane reorganization and IL-8 secretion. Activated mTORC1 elicits IL8 gene expression via the activation of an IRE1-JNK signalling cascade. Treatment of cells with a glutaminase inhibitor phenocopies glutamine restriction, suggesting that these results will be relevant to the clinical development of glutamine metabolism inhibitors as anticancer agents.
Assuntos
Interleucina-8/metabolismo , MAP Quinase Quinase 4/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Estresse do Retículo Endoplasmático , Glutamina , Humanos , Lisossomos/metabolismo , MAP Quinase Quinase 4/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
Autophagy is a cell-autonomous, catabolic process that plays context-dependent roles in tumor growth and progression. Wei et al. report that EGFR signaling promotes tumor growth through phosphorylation and functional inactivation of Beclin 1 and the consequent suppression of autophagy.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Animais , Proteína Beclina-1 , HumanosRESUMO
Mutations in the gene isocitrate dehydrogenase 1 (IDH1) are present in up to 86% of grade II and III gliomas and secondary glioblastoma. Arginine 132 (R132) mutations in the enzyme IDH1 result in excess production of the metabolite 2-hydroxyglutarate (2HG), which could be used as a biomarker for this subset of gliomas. Here, we use optimized in vivo spectral-editing and two-dimensional (2D) correlation magnetic resonance spectroscopy (MRS) methods to unambiguously detect 2HG noninvasively in glioma patients with IDH1 mutations. By comparison, fitting of conventional 1D MR spectra can provide false-positive readouts owing to spectral overlap of 2HG and chemically similar brain metabolites, such as glutamate and glutamine. 2HG was also detected using 2D high-resolution magic angle spinning MRS performed ex vivo on a separate set of glioma biopsy samples. 2HG detection by in vivo or ex vivo MRS enabled detailed molecular characterization of a clinically important subset of human gliomas. This has implications for diagnosis as well as monitoring of treatments targeting mutated IDH1.