RESUMO
BACKGROUND & AIMS: In gastrointestinal muscles, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) is predominantly expressed by interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor-α (PDGFRA) polypeptide is expressed by so-called fibroblast-like cells. KIT and PDGFRA have been reported to be coexpressed in ICC precursors and gastrointestinal stromal tumors (GISTs), which originate from the ICC lineage. PDGFRA signaling has been proposed to stimulate growth of GISTs that express mutant KIT, but the effects and mechanisms of selective blockade of PDGFRA are unclear. We investigated whether inhibiting PDGFRA could reduce proliferation of GIST cells with mutant KIT via effects on the KIT-dependent transcription factor ETV1. METHODS: We studied 53 gastric, small intestinal, rectal, or abdominal GISTs collected immediately after surgery or archived as fixed blocks at the Mayo Clinic and University of California, San Diego. In human GIST cells carrying imatinib-sensitive and imatinib-resistant mutations in KIT, PDGFRA was reduced by RNA interference (knockdown) or inhibited with crenolanib besylate (a selective inhibitor of PDGFRA and PDGFRB). Mouse ICC precursors were retrovirally transduced to overexpress wild-type Kit. Cell proliferation was analyzed by methyltetrazolium, 5-ethynyl-2'-deoxyuridine incorporation, and Ki-67 immunofluorescence assays; we also analyzed growth of xenograft tumors in mice. Gastric ICC and ICC precursors, and their PDGFRA(+) subsets, were analyzed by flow cytometry and immunohistochemistry in wild-type, Kit(+/copGFP), Pdgfra(+/eGFP), and NOD/ShiLtJ mice. Immunoblots were used to quantify protein expression and phosphorylation. RESULTS: KIT and PDGFRA were coexpressed in 3%-5% of mouse ICC, 35%-44% of ICC precursors, and most human GIST samples and cell lines. PDGFRA knockdown or inhibition with crenolanib efficiently reduced proliferation of imatinib-sensitive and imatinib-resistant KIT(+)ETV1(+)PDGFRA(+) GIST cells (50% maximal inhibitory concentration = 5-32 nM), but not of cells lacking KIT, ETV1, or PDGFRA (50% maximal inhibitory concentration >230 nM). Crenolanib inhibited phosphorylation of PDGFRA and PDGFRB, but not KIT. However, Kit overexpression sensitized mouse ICC precursors to crenolanib. ETV1 knockdown reduced KIT expression and GIST proliferation. Crenolanib down-regulated ETV1 by inhibiting extracellular-signal-regulated kinase (ERK)-dependent stabilization of ETV1 protein and also reduced expression of KIT and PDGFRA. CONCLUSIONS: In KIT-mutant GIST, inhibition of PDGFRA disrupts a KIT-ERK-ETV1-KIT signaling loop by inhibiting ERK activation. The PDGFRA inhibitor crenolanib might be used to treat patients with imatinib-resistant, KIT-mutant GIST.
Assuntos
Proliferação de Células/genética , Proteínas de Ligação a DNA/genética , Tumores do Estroma Gastrointestinal/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Benzamidas/metabolismo , Benzimidazóis/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Tumores do Estroma Gastrointestinal/genética , Técnicas de Silenciamento de Genes/métodos , Humanos , Mesilato de Imatinib , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Precursores de Ácido Nucleico/genética , Fosforilação/genética , Piperazinas/metabolismo , Piperidinas/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Mutations of the type III receptor tyrosine kinase FLT3 occur in approximately 30% of acute myeloid leukemia patients and lead to constitutive activation. This has made FLT3-activating mutations an attractive drug target because they are probable driver mutations of this disease. As more potent FLT3 inhibitors are developed, a predictable development of resistance-conferring point mutations, commonly at residue D835, has been observed. Crenolanib is a highly selective and potent FLT3 tyrosine kinase inhibitor (TKI) with activity against the internal tandem duplication (FLT3/ITD) mutants and the FLT3/D835 point mutants. We tested crenolanib against a panel of D835 mutant cell lines and primary patient blasts and observed superior cytotoxic effects when compared with other available FLT3 TKIs such as quizartinib and sorafenib. Another potential advantage of crenolanib is its reduced inhibition of c-Kit compared with quizartinib. In progenitor cell assays, crenolanib was less disruptive of erythroid colony growth, which may result in relatively less myelosuppression than quizartinib. Finally, correlative data from an ongoing clinical trial demonstrate that acute myeloid leukemia patients can achieve sufficient levels of crenolanib to inhibit both FLT3/ITD and resistance-conferring FLT3/D835 mutants in vivo. Crenolanib is thus an important next-generation FLT3 TKI.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/genética , Piperidinas/farmacologia , Mutação Puntual , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Antineoplásicos/farmacocinética , Benzimidazóis/farmacocinética , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Ensaio de Unidades Formadoras de Colônias , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Piperidinas/farmacocinética , Prognóstico , Proteínas Proto-Oncogênicas c-kit/metabolismo , Análise de Sequência de DNA , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
FLT3 kinase internal tandem duplication (ITD) mutations are common in acute myeloid leukemia (AML) and are associated with poor clinical outcomes. Although initial responses to FLT3 tyrosine kinase inhibitors (TKIs) are observed in FLT3-ITD-positive patients, subsequent relapse often occurs upon acquisition of secondary FLT3 kinase domain (KD) mutations, primarily at residues D835 and F691. Using biochemical assays, we determined that crenolanib, a novel TKI, demonstrates type I properties and is active against FLT3 containing ITD and/or D835- or F691-activating mutations. Potent activity was observed in FLT3-ITD-positive AML cell lines. Crenolanib delayed the outgrowth of MV4-11 cells in a xenograft mouse model, whereas in combination with the type II TKI sorafenib, a significant decrease in leukemic burden (P < .001) and prolonged survival (P < .01) was observed compared with either type I or II TKI alone. Crenolanib was active against Ba/F3 cells harboring FLT3-ITD and secondary KD mutations and sorafenib-resistant MOLM-13 cells containing FLT3-ITD/D835Y both in vitro and in vivo. In addition, crenolanib inhibited drug-resistant AML primary blasts with FLT3-ITD and D835H/Y mutations. These preclinical data demonstrate that crenolanib is effective against FLT3-ITD containing secondary KD mutations, suggesting that crenolanib may be a useful therapeutic agent for TKI-naive and drug-resistant FLT3-ITD-positive AML.
Assuntos
Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Niacinamida/administração & dosagem , Niacinamida/análogos & derivados , Compostos de Fenilureia/administração & dosagem , Piperidinas/administração & dosagem , Piperidinas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Sorafenibe , Sequências de Repetição em Tandem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Inflammatory breast cancer (IBC) is arguably the deadliest form of breast cancer due to its rapid onset and highly invasive nature. IBC carries 5- and 10-year disease-free survival rates of ~45% and <20%, respectively. Multiple studies demonstrate that in comparison with conventional breast cancer, IBC has a unique molecular identity. Here, we have identified platelet-derived growth factor receptor alpha (PDGFRA) as being uniquely expressed and active in IBC patient tumor cells. EXPERIMENTAL DESIGN: Here we focus on characterizing and targeting PDGFRA in IBC. Using gene expression, we analyzed IBC patient samples and compared them with non-IBC patient samples. Further, using IBC cells in culture, we determined the effect of small molecules inhibitors in both in vitro and in vivo assays. RESULTS: In IBC patients, we show more frequent PDGFRA activation signature than non-IBC samples. In addition, the PDGFRA activation signature is associated with shorter metastasis-free survival in both uni- and multivariate analyses. We also demonstrate that IBC cells express active PDGFRA. Finally, we show that PDGFRA targeting by crenolanib (CP-868-596), but not imatinib (STI571), two small molecule inhibitors, interferes with IBC cell growth and emboli formation in vitro and tumor growth in vivo. CONCLUSIONS: Our data suggest that PDGFRA may be a promising target for therapy in IBC.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Neoplasias Inflamatórias Mamárias/metabolismo , Piperidinas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Adulto , Idoso , Animais , Antineoplásicos/uso terapêutico , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Modelos Animais de Doenças , Feminino , Seguimentos , Expressão Gênica , Humanos , Neoplasias Inflamatórias Mamárias/tratamento farmacológico , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/patologia , Camundongos , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Piperidinas/uso terapêutico , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (FLT3-ITD) mutations are common in patients with acute myeloid leukemia (AML). These patients regularly develop resistance to FLT3 inhibitors suggesting that targeted combination drug strategies are needed to enhance AML therapy efficacy. EXPERIMENTAL DESIGN: Acquired point mutations of FLT3-ITD gene were screened using cDNA-based sequencing approach in vitro sorafenib-resistant cells, which were developed by long-term exposure of Ba/F3-ITD to increasing doses of sorafenib, and in FLT3-ITD mutated AML patients, who developed relapse following sorafenib therapy. Drug effects (e.g., proliferation inhibition, apoptosis induction, and changes in signal transduction protein expression) were assessed in AML cells harboring the point mutations in vitro and in FLT3-ITD-mutated AML patient samples. RESULTS: We identified several acquired point mutations in the tyrosine kinase domains (TKD) of the FLT3 gene in sorafenib-resistant murine leukemia cell line carrying human FLT3-ITD mutations, which were also detected in two of four sorafenib-resistant patient samples. Engineering these point mutations into Ba/F3-ITD cells generated sublines that demonstrated varying degrees of sorafenib [a type II tyrosine kinase inhibitor (TKI)] resistance. A similar pattern of resistance could be observed by exposing these sublines to the other type II TKIs AC220 and MLN518. However, these sublines retained sensitivity to the type I TKIs PKC412 or crenolanib. The combination of crenolanib with sorafenib demonstrated marked cytotoxic effects in all of the sorafenib-resistant sublines. CONCLUSIONS: These combination strategies could be clinically important in reversing acquired resistance to FLT3 inhibition in AML.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Análise Mutacional de DNA , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Duplicação Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Fenilureia/farmacologia , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas/genética , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Sequências de Repetição em Tandem , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab's potency against CLL cells. In vitro assessment of ocaratuzumab's direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P<0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1-10 ug/ml; P<0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P<0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/terapia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD20/imunologia , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/uso terapêutico , Células Matadoras Naturais/imunologia , Fagocitose/imunologia , Engenharia de Proteínas , RituximabRESUMO
A LC-ESI-MS/MS method for the determination of crenolanib (CP-868,596) in human serum was developed and validated employing d4-CP-868,596 as an internal standard (ISTD). In addition to human serum, the method was also partially validated for crenolanib determination in human cerebrospinal fluid (CSF) samples. Sample aliquots (50µl of serum or CSF) were prepared for analysis using liquid-liquid extraction (LLE) with tert-butyl methyl ether. Chromatography was performed using a phenomenex Gemini C18 column (3µm, 100mm×4.6mm I.D.) in a column heater set at 50°C and an isocratic mobile phase (methanol/water/formic acid at a volume ratio of 25/25/0.15, v/v/v). The flow rate was 0.45mL/min, and the retention time for both analyte and ISTD was less than 3.5min. Samples were analyzed with an API-5500 LC-MS/MS system (ESI) in positive ionization mode coupled to a Shimadzu HPLC system. The ion transitions monitored were m/z 444.4â373.1 and m/z 448.2â374.2 for crenolanib and ISTD, respectively. The method was linear over the range of 5-1000ng/mL for serum and 0.5-1000ng/mL for CSF. For human serum, both intra-day and inter-day precision were <4%, while intra-day and inter-day accuracy were within 8% of nominal values. Recovery was greater than 50% for both the analyte and ISTD. For CSF samples, both intra-day and inter-day precision were <9% except at the lower limit of quantification (LLOQ) which was <17%. The intra-day and inter-day accuracy were within 11% of the nominal CSF concentrations. After validation, this method was successfully applied to the analysis of serial pharmacokinetic samples obtained from a child treated with oral crenolanib.
Assuntos
Benzimidazóis/sangue , Benzimidazóis/líquido cefalorraquidiano , Cromatografia Líquida/métodos , Piperidinas/sangue , Piperidinas/líquido cefalorraquidiano , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Humanos , Reprodutibilidade dos TestesRESUMO
PURPOSE: To determine the potential of crenolanib, a potent inhibitor of PDGFRA, to treat malignancies driven by mutant PDGFRA. EXPERIMENTAL DESIGN: The biochemical activity of crenolanib was compared with imatinib using a panel of PDGFRA-mutant kinases expressed in several different cell line models, including primary gastrointestinal stromal tumors (GIST) cells. The antiproliferative activity of crenolanib was also studied in several cell lines with PDGFRA-dependent growth. RESULTS: Crenolanib was significantly more potent than imatinib in inhibiting the kinase activity of imatinib-resistant PDGFRA kinases (D842I, D842V, D842Y, DI842-843IM, and deletion I843). For example, crenolanib was 135-fold more potent than imatinib against D842V in our isogenic model system, with an IC(50) of approximately 10 nmol/L. The relative potency of crenolanib was further confirmed in BaF3 and primary GIST cells expressing PDGFRA D842V. In contrast, imatinib was at least 10-fold more potent than crenolanib in inhibiting the V561D mutation. For all other tested PDGFRA mutations, crenolanib and imatinib had comparable potency. CONCLUSIONS: Crenolanib is a potent inhibitor of imatinib-resistant PDGFRA kinases associated with GIST, including the PDGFRA D842V mutation found in approximately 5% of GISTs. The spectrum of activity of crenolanib suggests that this drug is a type I inhibitor (inhibitor of activated conformation of kinase). Based in part on these results, a phase II clinical study of this agent to treat GIST with the PDGFRA D842V mutation has been initiated.
Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Tumores do Estroma Gastrointestinal/genética , Mutação , Piperazinas/farmacologia , Pirimidinas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Benzamidas , Células CHO , Linhagem Celular Tumoral , Cricetinae , Ativação Enzimática/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Concentração Inibidora 50 , Piperidinas , Inibidores de Proteínas Quinases/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidoresRESUMO
Solid-state nanopores provide a direct means to detect and analyze DNA and proteins. In a typical setup, the DNA molecules travel through a nanopore under electrophoretic voltage bias. The nanopore is sandwiched between two chambers that are filled with ionic solution. A major challenge in using solid-state nanopores for DNA sequencing and gene detection is to improve their selectivity and detection sensitivity. To achieve these goals, one solution is to functionalize the nanopores by chemically modifying the pore walls with silanes or nucleic acids. However, little is known about molecular interactions in functionalized nanopores. This paper presents DNA translocation dynamics and the mechanism of DNA sequencing in a functionalized nanopore through a coarse-grained molecular dynamics model. The DNA nucleotide is coarse-grained into two interaction sites: one site corresponds to the base group and the other encompasses the phosphate and sugar groups. The water molecules are included in the model implicitly through Langevin dynamics. The coarse-grained model immensely improves the computational efficiency while still capturing the essential translocation dynamics. The model characterizes important physical properties of functionalized nanopores such as the effective pore diameter and effect of biasing voltage on the DNA translocation dynamics. The model reveals a nonlinear relationship between translocation speed of DNA and applied voltage. Moreover, DNA translocation in nanopores functionalized with hairpin-loop (HPL) DNA and single-stranded DNA (ss-DNA) shows significant differences: a target DNA is found to translocate through a ss-DNA coated nanopore 9 times faster than through an HPL coated one at a bias of 100 mV, putatively from lower stiffness of ss-DNA than that for HPL. The DNA translocation speed is also largely influenced by interaction potential between the DNA and surface-tethered molecules. The results reveal that such selective translocation, distinctly different translocation dynamics of target DNA molecules largely stem from the flexibility and orientation of the surface-tethered molecules. These findings can significantly impact the rational design of DNA transport experiments leading to rapid molecule-level diagnostics.