RESUMO
Oncogenic activation of fibroblast growth factor receptor 2 (FGFR2) drives multiple cancers and represents a broad therapeutic opportunity, yet selective targeting of FGFR2 has not been achieved. Although the clinical efficacy of pan-FGFR inhibitors (pan-FGFRi) validates FGFR2 driver status in FGFR2 fusion-positive intrahepatic cholangiocarcinoma, their benefit is limited by incomplete target coverage due to FGFR1- and FGFR4-mediated toxicities (hyperphosphatemia and diarrhea, respectively) and the emergence of FGFR2 resistance mutations. RLY-4008 is a highly selective, irreversible FGFR2 inhibitor designed to overcome these limitations. In vitro, RLY-4008 demonstrates >250- and >5,000-fold selectivity over FGFR1 and FGFR4, respectively, and targets primary alterations and resistance mutations. In vivo, RLY-4008 induces regression in multiple xenograft models-including models with FGFR2 resistance mutations that drive clinical progression on current pan-FGFRi-while sparing FGFR1 and FGFR4. In early clinical testing, RLY-4008 induced responses without clinically significant off-isoform FGFR toxicities, confirming the broad therapeutic potential of selective FGFR2 targeting. SIGNIFICANCE: Patients with FGFR2-driven cancers derive limited benefit from pan-FGFRi due to multiple FGFR1-4-mediated toxicities and acquired FGFR2 resistance mutations. RLY-4008 is a highly selective FGFR2 inhibitor that targets primary alterations and resistance mutations and induces tumor regression while sparing other FGFRs, suggesting it may have broad therapeutic potential. See related commentary by Tripathi et al., p. 1964. This article is featured in Selected Articles from This Issue, p. 1949.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Mutação , Colangiocarcinoma/genética , Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos/metabolismo , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Advanced systemic mastocytosis (AdvSM) is a rare hematologic neoplasm driven by the KIT D816V mutation and associated with poor survival. This phase 1 study ( NCT02561988 ) evaluated avapritinib (BLU-285), a selective KIT D816V inhibitor, in patients with AdvSM. The primary endpoints were the maximum tolerated dose, recommended phase 2 dose and safety of avapritinib. Secondary endpoints included overall response rate and changes in measures of mast cell burden. Avapritinib was evaluated at doses of 30-400 mg once daily in 86 patients, 69 with centrally confirmed AdvSM. Maximum tolerated dose was not reached, and 200 mg and 300 mg daily were studied in dose-expansion cohorts. The most frequent adverse events observed were periorbital edema (69%), anemia (55%), diarrhea (45%), thrombocytopenia (44%) and nausea (44%). Intracranial bleeding occurred in 13% overall, but in only 1% of patients without severe thrombocytopenia (platelets <50 × 109/l). In 53 response-evaluable patients, the overall response rate was 75%. The complete remission rate was 36%. Avapritinib elicited ≥50% reductions in marrow mast cells and serum tryptase in 92% and 99% of patients, respectively. Avapritinib induced deep and durable responses, including molecular remission of KIT D816V in patients with AdvSM, and was well tolerated at the recommended phase 2 dose of 200 mg daily.
Assuntos
Mastocitose Sistêmica/tratamento farmacológico , Pirazóis/uso terapêutico , Pirróis/uso terapêutico , Triazinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaios Clínicos Fase I como Assunto , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Pirazóis/farmacocinética , Pirróis/administração & dosagem , Pirróis/efeitos adversos , Pirróis/farmacocinética , Triazinas/administração & dosagem , Triazinas/efeitos adversos , Triazinas/farmacocinéticaRESUMO
Gastrointestinal stromal tumors (GIST) harboring activating mutations of PDGFRA respond to imatinib, with the notable exception of the most common mutation, D842V. Avapritinib is a novel, potent KIT/PDGFRA inhibitor with substantial clinical activity in patients with the D842V genotype. To date, only a minority of PDGFRA-mutant patients treated with avapritinib have developed secondary resistance. Tumor and plasma biopsies in 6 of 7 patients with PDGFRA primary mutations who progressed on avapritinib or imatinib had secondary resistance mutations within PDGFRA exons 13, 14, and 15 that interfere with avapritinib binding. Secondary PDGFRA mutations causing V658A, N659K, Y676C, and G680R substitutions were found in 2 or more patients each, representing recurrent mechanisms of PDGFRA GIST drug resistance. Notably, most PDGFRA-mutant GISTs refractory to avapritinib remain dependent on the PDGFRA oncogenic signal. Inhibitors that target PDGFRA protein stability or inhibition of PDGFRA-dependent signaling pathways may overcome avapritinib resistance. SIGNIFICANCE: Here, we provide the first description of avapritinib resistance mechanisms in PDGFRA-mutant GIST.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Tumores do Estroma Gastrointestinal , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Humanos , Mutação , Pirazóis , Pirróis , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , TriazinasRESUMO
Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide with no clinically confirmed oncogenic driver. Although preclinical studies implicate the FGF19 receptor FGFR4 in hepatocarcinogenesis, the dependence of human cancer on FGFR4 has not been demonstrated. Fisogatinib (BLU-554) is a potent and selective inhibitor of FGFR4 and demonstrates clinical benefit and tumor regression in patients with HCC with aberrant FGF19 expression. Mutations were identified in the gatekeeper and hinge-1 residues in the kinase domain of FGFR4 upon disease progression in 2 patients treated with fisogatinib, which were confirmed to mediate resistance in vitro and in vivo. A gatekeeper-agnostic, pan-FGFR inhibitor decreased HCC xenograft growth in the presence of these mutations, demonstrating continued FGF19-FGFR4 pathway dependence. These results validate FGFR4 as an oncogenic driver and warrant further therapeutic targeting of this kinase in the clinic. SIGNIFICANCE: Our study is the first to demonstrate on-target FGFR4 kinase domain mutations as a mechanism of acquired clinical resistance to targeted therapy. This further establishes FGF19-FGFR4 pathway activation as an oncogenic driver. These findings support further investigation of fisogatinib in HCC and inform the profile of potential next-generation inhibitors.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.
Assuntos
Carcinoma Hepatocelular/diagnóstico por imagem , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas/diagnóstico por imagem , Piranos/farmacologia , Quinazolinas/farmacologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Idoso de 80 Anos ou mais , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Transplante de Neoplasias , Domínios Proteicos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Outcomes for patients with advanced hepatocellular carcinoma (HCC) remain poor despite recent progress in drug development. Emerging data implicate FGF19 as a potential HCC driver, suggesting its receptor, FGFR4, as a novel therapeutic target. We evaluated fisogatinib (BLU-554), a highly potent and selective oral FGFR4 inhibitor, in a phase I dose-escalation/dose-expansion study in advanced HCC using FGF19 expression measured by IHC as a biomarker for pathway activation. For dose escalation, 25 patients received 140 to 900 mg fisogatinib once daily; the maximum tolerated dose (600 mg once daily) was expanded in 81 patients. Fisogatinib was well tolerated; most adverse events were manageable, grade 1/2 gastrointestinal events, primarily diarrhea, nausea, and vomiting. Across doses, the overall response rate was 17% in FGF19-positive patients [median duration of response: 5.3 months (95% CI, 3.7-not reached)] and 0% in FGF19-negative patients. These results validate FGFR4 as a targetable driver in FGF19-positive advanced HCC. SIGNIFICANCE: Fisogatinib elicited clinical responses in patients with tumor FGF19 overexpression in advanced HCC. These results validate the oncogenic driver role of the FGFR4 pathway in HCC and the use of FGF19 as a biomarker for patient selection.See related commentary by Subbiah and Pal, p. 1646.This article is highlighted in the In This Issue feature, p. 1631.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Fatores de Crescimento de Fibroblastos/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Piranos/administração & dosagem , Quinazolinas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Esquema de Medicação , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Piranos/efeitos adversos , Quinazolinas/efeitos adversos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Adulto JovemRESUMO
Targeting oncogenic kinase drivers with small-molecule inhibitors can have marked therapeutic benefit, especially when administered to an appropriate genomically defined patient population. Cancer genomics and mechanistic studies have revealed that heterogeneous mutations within a single kinase can result in various mechanisms of kinase activation. Therapeutic benefit to patients can best be optimized through an in-depth understanding of the disease-driving mutations combined with the ability to match these insights to tailored highly selective drugs. This rationale is presented for BLU-285, a clinical stage inhibitor of oncogenic KIT and PDGFRA alterations, including activation loop mutants that are ineffectively treated by current therapies. BLU-285, designed to preferentially interact with the active conformation of KIT and PDGFRA, potently inhibits activation loop mutants KIT D816V and PDGFRA D842V with subnanomolar potency and also inhibits other well-characterized disease-driving KIT mutants both in vitro and in vivo in preclinical models. Early clinical evaluation of BLU-285 in a phase 1 study has demonstrated marked activity in patients with diseases associated with KIT (aggressive systemic mastocytosis and gastrointestinal stromal tumor) and PDGFRA (gastrointestinal stromal tumor) activation loop mutations.
Assuntos
Mutação/genética , Medicina de Precisão , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/químicaRESUMO
BACKGROUND: The fibroblast growth factor receptor 4 (FGFR4) pathway is an essential regulatory component of bile acid synthesis, and its relationship with hepatocellular carcinoma (HCC) has been reported. We investigated the gene expression and clinical significance of FGFR4 and related pathways in intrahepatic cholangiocarcinoma (iCCA). RESULTS: The median age was 56 years (range 30-78) and 34 patients (74%) were male. Six patients (13%) had hepatitis B virus infection, with or without liver cirrhosis. Overall survival was significantly associated with FGFR4 (p = 0.004), FGF19 (p = 0.047), FGF21 (p = 0.04), and KLB (p = 0.03) expression. In the multivariate analysis with potential prognostic factors, high expression of FGF19, FGF21, and FGFR4 was significantly associated with better survival. In the analysis using the TCGA iCCA dataset, mRNA overexpression of at least 1 of the FGFR4-related genes was significantly associated with better disease-free survival (p = 0.02). MATERIALS AND METHODS: We assessed the expression of 98 genes in formalin-fixed paraffin embedded tumor tissue specimens from 46 patients with surgically resected iCCA using a NanoString platform. This included 10 FGF pathway genes (e.g. FGFR1-4, KLB, FGF3, 4, 19, 21, and 23), 19 distal marker genes (e.g. CYP7A1 and CYP17A1), 31 genes relevant to HCC and iCCA (e.g. AFP, TS), 18 copy number variation matched genes, and 20 control genes. Log-transformation of gene expression was performed for normalization and statistical analysis. Overall survival was correlated with gene expression (< median vs. ≥ median) using a log-rank test. The prognostic impact of FGFR4-related genes was validated using the public TCGA dataset for iCCA. CONCLUSIONS: Our results indicate that mRNA expression of FGFR4-related genes may be a biomarker to define the distinctive molecular phenotype of iCCA. Future preclinical and clinical validation is required to define the role of the FGFR4 pathway in iCCA.
Assuntos
Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/análise , Colangiocarcinoma/patologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/biossíntese , Adulto , Idoso , Neoplasias dos Ductos Biliares/mortalidade , Colangiocarcinoma/mortalidade , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/análise , TranscriptomaRESUMO
We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant.
Assuntos
Fosfatidilinositol 3-Quinases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Compostos de Boro/química , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Conformação Proteica , Células Sf9 , Transdução de Sinais , Spodoptera , Domínios de Homologia de srcRESUMO
The binding mechanism of a new class of lipid-competitive, ATP non-competitive, p110α isoform-selective PI3K (phosphoinositide 3-kinase) inhibitors has been elucidated. Using the novel technique of isoform reciprocal mutagenesis of non-conserved amino acids in the p110α and p110ß isoforms, we have identified three unique binding mechanisms for the p110α-selective inhibitors PIK-75, A-66S and J-32. Each of the inhibitor's p110α-isoform-selective binding was found to be due to interactions with different amino acids within p110. The PIK-75 interaction bound the non-conserved region 2 amino acid p110α Ser(773), A-66S bound the region 1 non-conserved amino acid p110α Gln(859), and J-32 binding had an indirect interaction with Lys(776) and Ile(771). The isoform reciprocal mutagenesis technique is shown to be an important analytical tool for the rational design of isoform-selective inhibitors.
Assuntos
Aminoácidos/metabolismo , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Prolina/análogos & derivados , Tiazóis/metabolismo , Aminoácidos/genética , Classe I de Fosfatidilinositol 3-Quinases , Classe II de Fosfatidilinositol 3-Quinases/genética , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Sequência Conservada/genética , Relação Dose-Resposta a Droga , Hidrazonas/metabolismo , Hidrazonas/farmacologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Fosfatidilinositol 3-Quinases/genética , Prolina/genética , Prolina/metabolismo , Ligação Proteica/genética , Sulfonamidas/metabolismo , Sulfonamidas/farmacologiaRESUMO
The thiazolidinedione, compound 1, has previously shown pan-inhibition of the phosphoinositide 3-kinase (PI3K) class I isoforms. We hypothesized the derivatization of the thiazolidinedione core of compound 1 could introduce isoform selectivity. We report the synthesis, characterization, and inhibitory activity of a novel series of 4-iminothiazolidin-2-ones for inhibition of the class I PI3K isoforms. Their synthesis was successfully achieved by multiple pathways described in this paper. Initial in vitro data of 28 analogues demonstrated poor inhibition of all class I PI3K isoforms. However, we identified an alternate target, the phosphodiesterases, and present preliminary screening results showing improved inhibitory activity.
RESUMO
A series of synthesized and commercially available compounds were assessed against PI3Kα for in vitro inhibitory activity and the results compared to binding calculated in silico. Using published crystal structures of PI3Kγ and PI3Kδ co-crystallized with inhibitors as a template, docking was able to identify the majority of potent inhibitors from a decoy set of 1000 compounds. On the other hand, PI3Kα in the apo-form, modeled by induced fit docking, or built as a homology model gave only poor results. A PI3Kα homology model derived from a ligand-bound PI3Kδ crystal structure was developed that has a good ability to identify active compounds. The docking results identified binding poses for active compounds that differ from those identified to date and can contribute to our understanding of structure-activity relationships for PI3K inhibitors.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Tiazolidinedionas/química , Sítios de Ligação , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Simulação por Computador , Cristalografia por Raios X , Ligação Proteica , Relação Estrutura-Atividade , Tiazolidinedionas/síntese química , Tiazolidinedionas/farmacologiaRESUMO
BACKGROUND: Gliomas frequently contain mutations in the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (IDH1) or the mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2). Several different amino acid substitutions recur at either IDH1 R132 or IDH2 R172 in glioma patients. Genetic evidence indicates that these mutations share a common gain of function, but it is unclear whether the shared function is dominant negative activity, neomorphic production of (R)-2-hydroxyglutarate (2HG), or both. METHODOLOGY/PRINCIPAL FINDINGS: We show by coprecipitation that five cancer-derived IDH1 R132 mutants bind IDH1-WT but that three cancer-derived IDH2 R172 mutants exert minimal binding to IDH2-WT. None of the mutants dominant-negatively lower isocitrate dehydrogenase activity at physiological (40 µM) isocitrate concentrations in mammalian cell lysates. In contrast to this, all of these mutants confer 10- to 100-fold higher 2HG production to cells, and glioma tissues containing IDH1 R132 or IDH2 R172 mutations contain high levels of 2HG compared to glioma tissues without IDH mutations (54.4 vs. 0.1 mg 2HG/g protein). CONCLUSIONS: Binding to, or dominant inhibition of, WT IDH1 or IDH2 is not a shared feature of the IDH1 and IDH2 mutations, and thus is not likely to be important in cancer. The fact that the gain of the enzymatic activity to produce 2HG is a shared feature of the IDH1 and IDH2 mutations suggests that this is an important function for these mutants in driving cancer pathogenesis.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Ligação Competitiva/genética , Ligação Competitiva/fisiologia , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/metabolismo , Células Cultivadas , Regulação para Baixo/genética , Regulação para Baixo/fisiologia , Ativação Enzimática/genética , Genes Dominantes/fisiologia , Glioma/enzimologia , Glioma/metabolismo , Humanos , Isocitrato Desidrogenase/metabolismo , Mutação de Sentido Incorreto/fisiologiaRESUMO
Previous genetic analyses have suggested that mutations of the genes encoding PI3Kα facilitate invasion and metastasis but have less effect on primary tumor growth. These findings have major implications for therapeutics but have not been factored into pre-clinical drug development designs. Here we show that the inhibition of PI3Kα by newly designed small molecule inhibitors prevented metastasis formation in mice but had much less effect on the growth of subcutaneous xenografts or primary intra-abdominal tumors. These data support the idea that PI3Kα plays an important role in the metastatic process and suggest a more informed strategy for selecting drugs worthy of further development for clinical application.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Hepáticas/prevenção & controle , Neoplasias Pulmonares/prevenção & controle , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Animais , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Desenho de Fármacos , Feminino , Células HCT116 , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Terapia de Alvo Molecular , Mutação , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Physiological activation of PI3Kα is brought about by the release of the inhibition by p85 when the nSH2 binds the phosphorylated tyrosine of activated receptors or their substrates. Oncogenic mutations of PI3Kα result in a constitutively activated enzyme that triggers downstream pathways that increase tumor aggressiveness and survival. Structural information suggests that some mutations also activate the enzyme by releasing p85 inhibition. Other mutations work by different mechanisms. For example, the most common mutation, His1047Arg, causes a conformational change that increases membrane association resulting in greater accessibility to the substrate, an integral membrane component. These effects are examples of the subtle structural changes that result in increased activity. The structures of these and other mutants are providing the basis for the design of isozyme-specific, mutation-specific inhibitors for individualized cancer therapies.
Assuntos
Mutação , Neoplasias/genética , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Sequência de Aminoácidos , Classe I de Fosfatidilinositol 3-Quinases , Humanos , Dados de Sequência MolecularRESUMO
The PI3K pathway is a communication hub coordinating critical cell functions including cell survival, cell growth, proliferation, motility and metabolism. Because PI3Kalpha harbors recurrent somatic mutations resulting in gains of function in human cancers, it has emerged as an important drug target for many types of solid tumors. Various PI3K isoforms are also being evaluated as potential therapeutic targets for inflammation, heart disease, and hematological malignancies. Structural biology is providing insights into the flexibility of the PI3Ks, and providing basis for understanding the effects of mutations, drug resistance and specificity.
Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/enzimologia , Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110alpha, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kalpha), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110alpha in complex with two interacting domains of its regulatory partner (p85alpha), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85alpha is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110alpha. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110alpha His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.
Assuntos
Membrana Celular/metabolismo , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Terciária de Proteína , Androstadienos/química , Androstadienos/metabolismo , Androstadienos/farmacologia , Animais , Domínio Catalítico/genética , Linhagem Celular , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Cristalização , Células HCT116 , Humanos , Ligação de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Modelos Moleculares , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , Wortmanina , Difração de Raios XRESUMO
The last few years have seen the identification of numerous small molecules that selectively inhibit specific class I isoforms of PI3K (phosphoinositide 3-kinase), yet little has been revealed about the molecular basis for the observed selectivities. Using site-directed mutagenesis, we have investigated one of the areas postulated as being critical to the observed selectivity. The residues Thr(886) and Lys(890) of the PI3Kgamma isoform project towards the ATP-binding pocket at the entrance to the catalytic site, but are not conserved. We have made reciprocal mutations between those residues in the beta isoform (Glu(858) and Asp(862)) and those in the alpha isoform (His(855) and Gln(859)) and evaluated the potency of a range of reported PI3K inhibitors. The results show that the potencies of beta-selective inhibitors TGX221 and TGX286 are unaffected by this change. In contrast, close analogues of these compounds, particularly the alpha-isoform-selective compound (III), are markedly influenced by the point mutations. The collected data suggests two distinct binding poses for these inhibitor classes, one of which is associated with potent PI3Kbeta activity and is not associated with the mutated residues, and a second that, in accord with earlier hypotheses, does involve this pair of non-conserved amino acids at the catalytic site entrance and contributes to the alpha-isoform-selectivity of the compounds studied.
Assuntos
Inibidores Enzimáticos/química , Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase , Sítios de Ligação , Catálise , Domínio Catalítico , Inibidores Enzimáticos/farmacologia , Cinética , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/genética , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Only few selected cancer cells drive tumor progression and are responsible for therapy resistance. Their specific genomic characteristics, however, are largely unknown because high-resolution genome analysis is currently limited to DNA pooled from many cells. Here, we describe a protocol for array comparative genomic hybridization (array CGH), which enables the detection of DNA copy number changes in single cells. Combining a PCR-based whole genome amplification method with arrays of highly purified BAC clones we could accurately determine known chromosomal changes such as trisomy 21 in single leukocytes as well as complex genomic imbalances of single cell line cells. In single T47D cells aberrant regions as small as 1-2 Mb were identified in most cases when compared to non-amplified DNA from 10(6) cells. Most importantly, in single micrometastatic cancer cells isolated from bone marrow of breast cancer patients, we retrieved and confirmed amplifications as small as 4.4 and 5 Mb. Thus, high-resolution genome analysis of single metastatic precursor cells is now possible and may be used for the identification of novel therapy target genes.
Assuntos
DNA de Neoplasias/análise , Metástase Neoplásica/genética , Células-Tronco Neoplásicas/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromossomos Artificiais Bacterianos , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , Feminino , Dosagem de Genes , Genômica/métodos , Humanos , Masculino , Reação em Cadeia da PolimeraseRESUMO
PIK3CA, one of the two most frequently mutated oncogenes in human tumors, codes for p110alpha, the catalytic subunit of a phosphatidylinositol 3-kinase, isoform alpha (PI3Kalpha, p110alpha/p85). Here, we report a 3.0 angstrom resolution structure of a complex between p110alpha and a polypeptide containing the p110alpha-binding domains of p85alpha, a protein required for its enzymatic activity. The structure shows that many of the mutations occur at residues lying at the interfaces between p110alpha and p85alpha or between the kinase domain of p110alpha and other domains within the catalytic subunit. Disruptions of these interactions are likely to affect the regulation of kinase activity by p85 or the catalytic activity of the enzyme, respectively. In addition to providing new insights about the structure of PI3Kalpha, these results suggest specific mechanisms for the effect of oncogenic mutations in p110alpha and p85alpha.
Assuntos
Mutação , Neoplasias/genética , Fosfatidilinositol 3-Quinases/química , Trifosfato de Adenosina , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Classe I de Fosfatidilinositol 3-Quinases , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Domínios de Homologia de srcRESUMO
Chromosomal instability in human breast cancer is known to take place before mammary neoplasias display morphological signs of invasion. We describe here the unexpected finding of a tumor cell population with normal karyotypes isolated from bone marrow of breast cancer patients. By analyzing the same single cells for chromosomal aberrations, subchromosomal allelic losses, and gene amplifications, we confirmed their malignant origin and delineated the sequence of genomic events during breast cancer progression. On this trajectory of genomic progression, we identified a subpopulation of patients with very early HER2 amplification. Because early changes have the highest probability of being shared by genetically unstable tumor cells, the genetic characterization of disseminated tumor cells provides a novel rationale for selecting patients for targeted therapies.