RESUMO
There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Sondas Moleculares/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Compostos de Espiro/farmacologia , Linhagem Celular Tumoral , Quinase 8 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Humanos , Modelos Moleculares , Sondas Moleculares/química , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Piridinas/química , Compostos de Espiro/químicaRESUMO
In a high-throughput screening campaign for c-Met kinase inhibitors, a thiadiazinone derivative with a carbamate group was identified as a potent in vitro inhibitor. Subsequent optimization guided by c-Met-inhibitor X-ray structures furnished new compound classes with excellent in vitro and in vivo profiles. The thiadiazinone ring of the HTS hit was first replaced by a pyridazinone followed by an exchange of the carbamate hinge binder with a 1,5-disubstituted pyrimidine. Finally an optimized compound, 22 (MSC2156119), with excellent in vitro potency, high kinase selectivity, long half-life after oral administration and in vivo anti-tumor efficacy at low doses, was selected as a candidate for clinical development.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Piridazinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridazinas/síntese química , Piridazinas/química , Relação Estrutura-AtividadeRESUMO
N-terminal processing by methionine aminopeptidases (MetAP) is a crucial step in the maturation of proteins during protein biosynthesis. Small-molecule inhibitors of MetAP2 have antiangiogenic and antitumoral activity. Herein, we characterize the structurally novel MetAP2 inhibitor M8891. M8891 is a potent, selective, reversible small-molecule inhibitor blocking the growth of human endothelial cells and differentially inhibiting cancer cell growth. A CRISPR genome-wide screen identified the tumor suppressor p53 and MetAP1/MetAP2 as determinants of resistance and sensitivity to pharmacologic MetAP2 inhibition. A newly identified substrate of MetAP2, translation elongation factor 1-alpha-1 (EF1a-1), served as a pharmacodynamic biomarker to follow target inhibition in cell and mouse studies. Robust angiogenesis and tumor growth inhibition was observed with M8891 monotherapy. In combination with VEGF receptor inhibitors, tumor stasis and regression occurred in patient-derived xenograft renal cell carcinoma models, particularly those that were p53 wild-type, had Von Hippel-Landau gene (VHL) loss-of-function mutations, and a mid/high MetAP1/2 expression score.
Assuntos
Aminopeptidases , Carcinoma de Células Renais , Neoplasias Renais , Humanos , Animais , Camundongos , Proteína Supressora de Tumor p53/genética , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Células Endoteliais/metabolismo , Metaloendopeptidases/metabolismo , Inibidores Enzimáticos , Inibidores da Angiogênese/farmacologia , Neoplasias Renais/tratamento farmacológicoRESUMO
MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.
Assuntos
Adenocarcinoma/enzimologia , Autofagia/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Neoplasias Gástricas/enzimologia , Adenina/análogos & derivados , Adenina/farmacologia , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , Piridazinas/farmacologia , Pirimidinas/farmacologia , Sulfonas/farmacologia , Enzimas Ativadoras de Ubiquitina/genéticaRESUMO
Over half of all cancer patients undergo radiation therapy but there is an unmet need for more efficacious combination strategies with molecular targeted drugs. DNA damage response has emerged as an important intervention point for improving anti-tumor effects of radiation and several inhibitors are currently in development. Ataxia telangiectasia mutated (ATM) kinase is a key regulator of cellular response to DNA double strand breaks and a potential target for radiosensitization. We recently reported two new potent and selective ATM inhibitors, M3541 and M4076, that effectively sensitize cancer cells to radiation and regress human xenografts in clinically relevant animal models. Here, we dive deeper into the cellular events in irradiated cancer cells exposed to ATM inhibitors. Suppression of ATM activity inhibited radiation-induced ATM signaling and abrogated G1 checkpoint activation resulting in enhanced cell death. Our data indicated that entry into mitosis with gross structural abnormalities in multiple chromosomes is the main mechanism behind the increased cell killing. Misalignment and mis-segregation led to formation of multiple micronuclei and robust activation of the interferon response and inflammatory signaling via the cGAS/STING/TBK1 pathway. Cancer cells exposed to radiation in the presence of M3541 were more susceptible to killing in co-culture with NK cells from healthy donors. In addition, strong upregulation of PD-L1 expression was observed in the surviving irradiated cancer cells exposed to M3541. Simultaneous activation of the STING pathway and PD-L1 suggested that combination of radiation, ATM inhibitors and PD-L1 targeted therapy may offer a novel approach to radio-immunotherapy of locally advanced tumors.
Assuntos
Ataxia Telangiectasia , Neoplasias , Animais , Humanos , Antígeno B7-H1/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Fosforilação , Morte Celular , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Neoplasias/tratamento farmacológico , Neoplasias/radioterapiaRESUMO
Ataxia telangiectasia and Rad3-related protein (ATR) kinase regulate a key cell regulatory node for maintaining genomic integrity by preventing replication fork collapse. ATR inhibition has been shown to increase replication stress resulting in DNA double-strand breaks (DSBs) and cancer cell death, and several inhibitors are under clinical investigation for cancer therapy. However, activation of cell-cycle checkpoints controlled by ataxia telangiectasia-mutated (ATM) kinase could minimize the lethal consequences of ATR inhibition and protect cancer cells. Here, we investigate ATR-ATM functional relationship and potential therapeutic implications. In cancer cells with functional ATM and p53 signaling, selective suppression of ATR catalytic activity by M6620 induced G1-phase arrest to prevent S-phase entry with unrepaired DSBs. The selective ATM inhibitors, M3541 and M4076, suppressed both ATM-dependent cell-cycle checkpoints, and DSB repair lowered the p53 protective barrier and extended the life of ATR inhibitor-induced DSBs. Combination treatment amplified the fraction of cells with structural chromosomal defects and enhanced cancer cell death. ATM inhibitor synergistically potentiated the ATR inhibitor efficacy in cancer cells in vitro and increased ATR inhibitor efficacy in vivo at doses that did not show overt toxicities. Furthermore, a combination study in 26 patient-derived xenograft models of triple-negative breast cancer with the newer generation ATR inhibitor M4344 and ATM inhibitor M4076 demonstrated substantial improvement in efficacy and survival compared with single-agent M4344, suggesting a novel and potentially broad combination approach to cancer therapy.
Assuntos
Ataxia Telangiectasia , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteínas Mutadas de Ataxia Telangiectasia , Reparo do DNA , Proteínas de Ciclo Celular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Dano ao DNA , Quinase 1 do Ponto de Checagem/genéticaRESUMO
The mesenchymal-epithelial transition factor (MET) proto-oncogene encodes the MET receptor tyrosine kinase. MET aberrations drive tumorigenesis in several cancer types through a variety of molecular mechanisms, including MET mutations, gene amplification, rearrangement, and overexpression. Therefore, MET is a therapeutic target and the selective type Ib MET inhibitor, tepotinib, was designed to potently inhibit MET kinase activity. In vitro, tepotinib inhibits MET in a concentration-dependent manner irrespective of the mode of MET activation, and in vivo, tepotinib exhibits marked, dose-dependent antitumor activity in MET-dependent tumor models of various cancer indications. Tepotinib penetrates the blood-brain barrier and demonstrates strong antitumor activity in subcutaneous and orthotopic brain metastasis models, in-line with clinical activity observed in patients. MET amplification is an established mechanism of resistance to EGFR tyrosine kinase inhibitors (TKI), and preclinical studies show that tepotinib in combination with EGFR TKIs can overcome this resistance. Tepotinib is currently approved for the treatment of adult patients with advanced or metastatic non-small cell lung cancer harboring MET exon 14 skipping alterations. This review focuses on the pharmacology of tepotinib in preclinical cancer models harboring MET alterations and demonstrates that strong adherence to the principles of the Pharmacological Audit Trail may result in a successful discovery and development of a precision medicine.
Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-met , Adulto , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Nephronophthisis is the most common genetic cause of end-stage renal failure during childhood and adolescence. Genetic studies have identified disease-causing mutations in at least 11 different genes (NPHP1-11), but the function of the corresponding nephrocystin proteins remains poorly understood. The two evolutionarily conserved proteins nephrocystin-1 (NPHP1) and nephrocystin-4 (NPHP4) interact and localize to cilia in kidney, retina, and brain characterizing nephronophthisis and associated pathologies as result of a ciliopathy. Here we show that NPHP4, but not truncating patient mutations, negatively regulates tyrosine phosphorylation of NPHP1. NPHP4 counteracts Pyk2-mediated phosphorylation of three defined tyrosine residues of NPHP1 thereby controlling binding of NPHP1 to the trans-Golgi sorting protein PACS-1. Knockdown of NPHP4 resulted in an accumulation of NPHP1 in trans-Golgi vesicles of ciliated retinal epithelial cells. These data strongly suggest that NPHP4 acts upstream of NPHP1 in a common pathway and support the concept of a role for nephrocystin proteins in intracellular vesicular transport.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cílios/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas/fisiologia , Tirosina/química , Linhagem Celular , Proteínas do Citoesqueleto , Complexo de Golgi/metabolismo , Humanos , Doenças Renais Císticas/metabolismo , Modelos Biológicos , Mutação , Fosforilação , Ligação Proteica , Distribuição TecidualRESUMO
We demonstrate a role for protein kinase casein kinase 2 (CK2) in the phosphorylation and regulation of the M3-muscarinic receptor in transfected cells and cerebellar granule neurons. On agonist occupation, specific subsets of receptor phosphoacceptor sites (which include the SASSDEED motif in the third intracellular loop) are phosphorylated by CK2. Receptor phosphorylation mediated by CK2 specifically regulates receptor coupling to the Jun-kinase pathway. Importantly, other phosphorylation-dependent receptor processes are regulated by kinases distinct from CK2. We conclude that G protein-coupled receptors (GPCRs) can be phosphorylated in an agonist-dependent fashion by protein kinases from a diverse range of kinase families, not just the GPCR kinases, and that receptor phosphorylation by a defined kinase determines a specific signalling outcome. Furthermore, we demonstrate that the M3-muscarinic receptor can be differentially phosphorylated in different cell types, indicating that phosphorylation is a flexible regulatory process where the sites that are phosphorylated, and hence the signalling outcome, are dependent on the cell type in which the receptor is expressed.
Assuntos
Caseína Quinase II/fisiologia , Receptor Muscarínico M3/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Caseína Quinase II/antagonistas & inibidores , Células Cultivadas , Sequência Consenso , Cricetinae , Cricetulus , Humanos , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Receptor Muscarínico M3/química , Transdução de Sinais , Quinases de Receptores Adrenérgicos beta/metabolismoRESUMO
Radiotherapy and chemical DNA-damaging agents are among the most widely used classes of cancer therapeutics today. Double-strand breaks (DSB) induced by many of these treatments are lethal to cancer cells if left unrepaired. Ataxia telangiectasia-mutated (ATM) kinase plays a key role in the DNA damage response by driving DSB repair and cell-cycle checkpoints to protect cancer cells. Inhibitors of ATM catalytic activity have been shown to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiotherapy and other DSB-inducing modalities. Here, we describe the pharmacological activities of two highly potent and selective ATM inhibitors from a new chemical class, M3541 and M4076. In biochemical assays, they inhibited ATM kinase activity with a sub-nanomolar potency and showed remarkable selectivity against other protein kinases. In cancer cells, the ATM inhibitors suppressed DSB repair, clonogenic cancer cell growth, and potentiated antitumor activity of ionizing radiation in cancer cell lines. Oral administration of M3541 and M4076 to immunodeficient mice bearing human tumor xenografts with a clinically relevant radiotherapy regimen strongly enhanced the antitumor activity, leading to complete tumor regressions. The efficacy correlated with the inhibition of ATM activity and modulation of its downstream targets in the xenograft tissues. In vitro and in vivo experiments demonstrated strong combination potential with PARP and topoisomerase I inhibitors. M4076 is currently under clinical investigation.
Assuntos
Ataxia Telangiectasia , Neoplasias , Animais , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Radiotherapy is the most widely used cancer treatment and improvements in its efficacy and safety are highly sought-after. Peposertib (also known as M3814), a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, effectively suppresses the repair of radiation-induced DNA double-strand breaks (DSB) and regresses human xenograft tumors in preclinical models. Irradiated cancer cells devoid of p53 activity are especially sensitive to the DNA-PK inhibitor, as they lose a key cell-cycle checkpoint circuit and enter mitosis with unrepaired DSBs, leading to catastrophic consequences. Here, we show that inhibiting the repair of DSBs induced by ionizing radiation with peposertib offers a powerful new way for improving radiotherapy by simultaneously enhancing cancer cell killing and response to a bifunctional TGFß "trap"/anti-PD-L1 cancer immunotherapy. By promoting chromosome misalignment and missegregation in p53-deficient cancer cells with unrepaired DSBs, DNA-PK inhibitor accelerated micronuclei formation, a key generator of cytosolic DNA and activator of cGAS/STING-dependent inflammatory signaling as it elevated PD-L1 expression in irradiated cancer cells. Triple combination of radiation, peposertib, and bintrafusp alfa, a fusion protein simultaneously inhibiting the profibrotic TGFß and immunosuppressive PD-L1 pathways was superior to dual combinations and suggested a novel approach to more efficacious radioimmunotherapy of cancer. IMPLICATIONS: Selective inhibition of DNA-PK in irradiated cancer cells enhances inflammatory signaling and activity of dual TGFß/PD-L1 targeted therapy and may offer a more efficacious combination option for the treatment of locally advanced solid tumors.
Assuntos
Neoplasias , Inibidores de Proteínas Quinases , Antígeno B7-H1/metabolismo , DNA , Humanos , Imunoterapia , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Inibidores de Proteínas Quinases/farmacologia , Piridazinas , Quinazolinas , Fator de Crescimento Transformador betaRESUMO
Nicotinic acid (niacin), a vitamin of the B complex, has been used for almost 50 years as a lipid-lowering drug. The pharmacological effect of nicotinic acid requires doses that are much higher than those provided by a normal diet. Its primary action is to decrease lipolysis in adipose tissue by inhibiting hormone-sensitive triglyceride lipase. This anti-lipolytic effect of nicotinic acid involves the inhibition of cyclic adenosine monophosphate (cAMP) accumulation in adipose tissue through a G(i)-protein-mediated inhibition of adenylyl cyclase. A G-protein-coupled receptor for nicotinic acid has been proposed in adipocytes. Here, we show that the orphan G-protein-coupled receptor, 'protein upregulated in macrophages by interferon-gamma' (mouse PUMA-G, human HM74), is highly expressed in adipose tissue and is a nicotinic acid receptor. Binding of nicotinic acid to PUMA-G or HM74 results in a G(i)-mediated decrease in cAMP levels. In mice lacking PUMA-G, the nicotinic acid-induced decrease in free fatty acid (FFA) and triglyceride plasma levels was abrogated, indicating that PUMA-G mediates the anti-lipolytic and lipid-lowering effects of nicotinic acid in vivo. The identification of the nicotinic acid receptor may be useful in the development of new drugs to treat dyslipidemia.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Hipolipemiantes/metabolismo , Niacina/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Nicotínicos/metabolismo , Tecido Adiposo/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Ácidos Graxos não Esterificados/metabolismo , Genes Reporter , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Ensaio Radioligante , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Receptores Nicotínicos/genética , Distribuição Tecidual , Triglicerídeos/metabolismoRESUMO
Peposertib (M3814) is a potent and selective DNA-PK inhibitor in early clinical development. It effectively blocks non-homologous end-joining repair of DNA double-strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By suppressing DNA-PK catalytic activity in the presence of DNA DSB, M3814 potentiates ATM/p53 signaling leading to enhanced p53-dependent antitumor activity in tumor cells. Here, we investigated the therapeutic potential of M3814 in combination with DSB-inducing agents in leukemia cells and a patient-derived tumor. We show that in the presence of IR or topoisomerase II inhibitors, M3814 boosts the ATM/p53 response in acute leukemia cells leading to the elevation of p53 protein levels as well as its transcriptional activity. M3814 synergistically sensitized p53 wild-type, but not p53-deficient, AML cells to killing by DSB-inducing agents via p53-dependent apoptosis involving both intrinsic and extrinsic effector pathways. The antileukemic effect was further potentiated by enhancing daunorubicin-induced myeloid cell differentiation. Further, combined with the fixed-ratio liposomal formulation of daunorubicin and cytarabine, CPX-351, M3814 enhanced the efficacy against leukemia cells in vitro and in vivo without increasing hematopoietic toxicity, suggesting that DNA-PK inhibition could offer a novel clinical strategy for harnessing the anticancer potential of p53 in AML therapy.
Assuntos
Quebras de DNA de Cadeia Dupla , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/patologia , Piridazinas/farmacologia , Quinazolinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose , Proliferação de Células , Reparo do DNA , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
High-quality dose predictions based on a good understanding of target engagement is one of the main translational goals in drug development. Here, we systematically evaluate active human dose predictions for 15 Merck KGaA/EMD Serono assets spanning several modalities and therapeutic areas. Using case studies, we illustrate the value of adhering to the translational best practices of having an exposure-response relationship in an appropriate animal model; having validated, translatable pharmacodynamic (PD) biomarkers measurable in Phase I populations in the right tissue; having a deeper understanding of biology; and capturing uncertainties in predictions. Given the gap in publications on the subject, we believe that the learnings from this unique diverse data set, which are generic to the industry, will trigger actions to improve future predictions.
Assuntos
Relação Dose-Resposta a Droga , Animais , Biomarcadores/metabolismo , Desenvolvimento de Medicamentos/métodos , Indústria Farmacêutica/métodos , HumanosRESUMO
Despite significant advances in the treatment of acute myeloid leukemia (AML) the long-term prognosis remains relatively poor and there is an urgent need for improved therapies with increased potency and tumor selectivity. Mylotarg is the first AML-targeting drug from a new generation of antibody drug conjugate (ADC) therapies aiming at the acute leukemia cell compartment with increased specificity. This agent targets leukemia cells for apoptosis with a cytotoxic payload, calicheamicin, carried by a CD33-specific antibody. Calicheamicin induces DNA double strand breaks (DSB) which, if left unrepaired, lead to cell cycle arrest and apoptosis in cancer cells. However, repair of DSB by the non-homologous end joining pathway driven by DNA-dependent protein kinase (DNA-PK) can reduce the efficacy of calicheamicin. M3814 is a novel, potent and selective inhibitor of DNA-PK. This compound effectively blocks DSB repair, strongly potentiates the antitumor activity of ionizing radiation and DSB-inducing chemotherapeutics and is currently under clinical investigation. Suppressing DSB repair with M3814 synergistically enhanced the apoptotic activity of calicheamicin in cultured AML cells. Combination of M3814 with Mylotarg in two AML xenograft models, MV4-11 and HL-60, demonstrated increased efficacy and significantly improved survival benefit without elevated body weight loss. Our results support a new application for pharmacological DNA-PK inhibitors as enhancers of Mylotarg and a potential new combination treatment option for AML patients.
RESUMO
Tepotinib is an oral MET inhibitor approved for metastatic non-small cell lung cancer (NSCLC) harboring MET exon 14 (METex14) skipping mutations. Examining treatment-naive or tepotinib-resistant cells with MET amplification or METex14 skipping mutations identifies other receptor tyrosine kinases (RTKs) that co-exist in cells prior to tepotinib exposure and become more prominent upon tepotinib resistance. In a small cohort of patients with lung cancer with MET genetic alterations treated with tepotinib, gene copy number gains of other RTKs were found at baseline and affected treatment outcome. An Src homology 2 domain-containing phosphatase 2 (SHP2) inhibitor delayed the emergence of tepotinib resistance and synergized with tepotinib in treatment-naive and tepotinib-resistant cells as well as in xenograft models. Alternative signaling pathways potentially diminish the effect of tepotinib monotherapy, and the combination of tepotinib with an SHP2 inhibitor enables the control of tumor growth in cells with MET genetic alterations.
RESUMO
Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability.
Assuntos
Dano ao DNA , Epitélio/patologia , Mesoderma/patologia , Fosfoproteínas/metabolismo , Proteômica , Animais , Linhagem Celular Tumoral , Reparo do DNA/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Epitélio/efeitos da radiação , Feminino , Humanos , Mesoderma/efeitos da radiação , Camundongos , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos da radiação , Radiação Ionizante , Reprodutibilidade dos Testes , Especificidade por Substrato/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/radioterapia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/radioterapia , Inibidores de Proteínas Quinases/farmacologia , Piridazinas/farmacologia , Quinazolinas/farmacologia , Radiação Ionizante , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Nus , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibition of DNA double-strand break (DSB) repair in cancer cells has been proposed as a new therapeutic strategy for potentiating the anticancer effects of radiotherapy. M3814 is a novel, selective pharmacologic inhibitor of the serine/threonine kinase DNA-dependent protein kinase (DNA-PK), a key driver of nonhomologous end-joining, one of the main DSB-repair pathways, currently under clinical investigation. Here, we show that M3814 effectively blocks the repair of radiation-induced DSBs and potently enhances p53 phosphorylation and activation. In p53 wild-type cells, ataxia telangiectasia-mutated (ATM) and its targets, p53 and checkpoint kinase 2 (CHK2), were more strongly activated by combination treatment with M3814 and radiation than by radiation alone, leading to a complete p53-dependent cell-cycle block and premature cell senescence. Cancer cells with dysfunctional p53 were unable to fully arrest their cell cycle and entered S and M phases with unrepaired DNA, leading to mitotic catastrophe and apoptotic cell death. Isogenic p53-null/wild-type A549 and HT-1080 cell lines were generated and used to demonstrate that p53 plays a critical role in determining the response to ionizing radiation and M3814. Time-lapse imaging of cell death and measuring apoptosis in panels of p53 wild-type and p53-null/mutant cancer lines confirmed the clear differences in cell fate, dependent on p53 status. IMPLICATIONS: Our results identify p53 as a possible biomarker for response of cancer cells to combination treatment with radiation and a DNA-PK inhibitor and suggest that p53 mutation status should be considered in the design of future clinical trials. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/17/12/2457/F1.large.jpg.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Pulmonares/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Células A549 , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinase do Ponto de Checagem 2/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Inibidores de Proteínas Quinases/farmacologia , Radiação Ionizante , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/radioterapiaRESUMO
Poor oxygenation is a common hallmark of solid cancers that strongly associates with aggressive tumor progression and treatment resistance. While a hypoxia-inducible factor 1α (HIF-1α)-associated transcriptional overexpression of the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK) MET has been previously documented, any regulation of the HIF-1α system through MET downstream signaling in hypoxic tumors has not been yet described. By using MET-driven in vitro as well as ex vivo tumor organotypic fresh tissue models we report that MET targeting results in depletion of HIF-1α and its various downstream targets. Mechanistically, we provide evidence that MET regulates HIF-1α levels through a protein translation mechanism that relies on phosphorylation modulation of the eukaryotic initiation factor 4G1 (eIF4G1) on serine 1232 (Ser-1232). Targeted phosphoproteomics data demonstrate a significant drop in eIF4G1 Ser-1232 phosphorylation following MET targeting, which is linked to an increased affinity between eIF4G1 and eIF4E. Since phosphorylation of eIF4G1 on Ser-1232 is largely mediated through mitogen-activated protein kinase (MAPK), we show that expression of a constitutively active K-RAS variant is sufficient to abrogate the inhibitory effect of MET targeting on the HIF-1α pathway with subsequent resistance of tumor cells to MET targeting under hypoxic conditions. Analysis of The Cancer Genome Atlas data demonstrates frequent co-expression of MET, HIF-1α and eIF4G1 in various solid tumors and its impact on disease-free survival of non-small cell lung cancer patients. Clinical relevance of the MET-eIF4G1-HIF-1α pathway is further supported by a co-occurrence of their expression in common tumor regions of individual lung cancer patients.