RESUMEN
Endocrine therapies (ET) with CDK4/6 inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of 22 ER+ breast cancer patient-derived xenografts (PDXs) demonstrated that PKMYT1, a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX organoids and xenografts, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.
RESUMEN
Ovarian cancers (OVCAs) and endometrial cancers (EMCAs) with CCNE1-amplification are often resistant to standard of care treatment and represent an unmet clinical need. Previously, synthetic-lethal screening identified loss of the CDK1 regulator, PKMYT1, as synthetically lethal with CCNE1-amplification. We hypothesized that CCNE1-amplification associated replication stress will be more effectively targeted by combining the PKMYT1 inhibitor, lunresertib (RP-6306), with the ATR inhibitor, camonsertib (RP-3500/RG6526). Low dose combination RP-6306 with RP-3500 synergistically increased cytotoxicity more in CCNE1 amplified compared to non-amplified cells. Combination treatment produced durable antitumor activity and increased survival in CCNE1 amplified patient-derived and cell line-derived xenografts. Mechanistically, low doses of RP-6306 with RP-3500 increase CDK1 activation more so than monotherapy, triggering rapid and robust induction of premature mitosis, DNA damage and apoptosis in a CCNE1-dependent manner. These findings suggest that targeting CDK1 activity by combining RP-6306 with RP-3500 is a novel therapeutic approach to treat CCNE1-amplifed OVCAs and EMCAs.
RESUMEN
Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.
Asunto(s)
Isocitrato Deshidrogenasa , Leucemia Mieloide Aguda , Animales , Azacitidina/farmacología , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Mutación/genética , Células Madre/metabolismoRESUMEN
BACKGROUND: Long-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the 'cold' tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade. METHODS: An IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade. RESULTS: The HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone. CONCLUSION: The development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.
Asunto(s)
Vacunas contra el Cáncer , Glioma , Animales , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Glutaratos , Antígeno HLA-A2/genética , Antígeno HLA-DR1/genética , Humanos , Inhibidores de Puntos de Control Inmunológico , Isocitrato Deshidrogenasa/genética , Ratones , Ratones Transgénicos , Microambiente Tumoral , Regulación hacia Arriba , Vacunas de SubunidadRESUMEN
Inhibition of the S-adenosyl methionine (SAM)-producing metabolic enzyme, methionine adenosyltransferase 2A (MAT2A), has received significant interest in the field of medicinal chemistry due to its implication as a synthetic lethal target in cancers with the deletion of the methylthioadenosine phosphorylase (MTAP) gene. Here, we report the identification of novel MAT2A inhibitors with distinct in vivo properties that may enhance their utility in treating patients. Following a high-throughput screening, we successfully applied the structure-based design lessons from our first-in-class MAT2A inhibitor, AG-270, to rapidly redesign and optimize our initial hit into two new lead compounds: a brain-penetrant compound, AGI-41998, and a potent, but limited brain-penetrant compound, AGI-43192. We hope that the identification and first disclosure of brain-penetrant MAT2A inhibitors will create new opportunities to explore the potential therapeutic effects of SAM modulation in the central nervous system (CNS).
Asunto(s)
Metionina Adenosiltransferasa , Neoplasias , Encéfalo/metabolismo , Diseño de Fármacos , Humanos , Neoplasias/tratamiento farmacológico , S-Adenosilmetionina/metabolismoRESUMEN
The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was recently implicated as a synthetic lethal target in cancers with deletion of the methylthioadenosine phosphorylase (MTAP) gene, which is adjacent to the CDKN2A tumor suppressor and codeleted with CDKN2A in approximately 15% of all cancers. Previous attempts to target MAT2A with small-molecule inhibitors identified cellular adaptations that blunted their efficacy. Here, we report the discovery of highly potent, selective, orally bioavailable MAT2A inhibitors that overcome these challenges. Fragment screening followed by iterative structure-guided design enabled >10â¯000-fold improvement in potency of a family of allosteric MAT2A inhibitors that are substrate noncompetitive and inhibit release of the product, S-adenosyl methionine (SAM), from the enzyme's active site. We demonstrate that potent MAT2A inhibitors substantially reduce SAM levels in cancer cells and selectively block proliferation of MTAP-null cells both in tissue culture and xenograft tumors. These data supported progressing AG-270 into current clinical studies (ClinicalTrials.gov NCT03435250).
Asunto(s)
Inhibidores Enzimáticos/química , Metionina Adenosiltransferasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/genética , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Homocigoto , Humanos , Metionina Adenosiltransferasa/metabolismo , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Purina-Nucleósido Fosforilasa/metabolismo , S-Adenosilmetionina/metabolismo , Relación Estructura-ActividadRESUMEN
The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.
Asunto(s)
Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Metionina Adenosiltransferasa/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Purina-Nucleósido Fosforilasa/genética , Empalme del ARN/efectos de los fármacos , ARN Mensajero/genética , Animales , Línea Celular , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Daño del ADN/genética , Eliminación de Gen , Células HCT116 , Células HEK293 , Humanos , Metionina Adenosiltransferasa/genética , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Empalme del ARN/genética , S-Adenosilmetionina/metabolismoRESUMEN
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy for which new therapeutic approaches are required. One such potential therapeutic strategy is to target the ubiquitin-like modifier-activating enzyme 1 (UBA1), the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Here, we evaluated TAK-243, a first-in-class UBA1 inhibitor, in preclinical models of AML. In AML cell lines and primary AML samples, TAK-243 induced cell death and inhibited clonogenic growth. In contrast, normal hematopoietic progenitor cells were more resistant. TAK-243 preferentially bound to UBA1 over the related E1 enzymes UBA2, UBA3, and UBA6 in intact AML cells. Inhibition of UBA1 with TAK-243 decreased levels of ubiquitylated proteins, increased markers of proteotoxic stress and DNA damage stress. In vivo, TAK-243 reduced leukemic burden and targeted leukemic stem cells without evidence of toxicity. Finally, we selected populations of AML cells resistant to TAK-243 and identified missense mutations in the adenylation domain of UBA1. Thus, our data demonstrate that TAK-243 targets AML cells and stem cells and support a clinical trial of TAK-243 in this patient population. Moreover, we provide insight into potential mechanisms of acquired resistance to UBA1 inhibitors.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Nucleósidos/farmacología , Sulfonamidas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Proliferación Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Ratones , Ratones SCID , Pirazoles , Pirimidinas , Sulfuros , Células Tumorales CultivadasRESUMEN
The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.
Asunto(s)
Neoplasias/tratamiento farmacológico , Nucleósidos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonamidas/farmacología , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Humanos , Imidas/farmacología , Ratones , Neoplasias/genética , Neoplasias/patología , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Pirazoles , Pirimidinas , Sulfuros , Ubiquitina/antagonistas & inhibidores , Ubiquitina/química , Ubiquitina/genética , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Daño del ADN/efectos de los fármacos , Mitosis/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Humanos , Mitosis/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/genética , Quinasa Tipo Polo 1RESUMEN
Aurora A kinase orchestrates multiple key activities, allowing cells to transit successfully into and through mitosis. MLN8237 (alisertib) is a selective Aurora A inhibitor that is being evaluated as an anticancer agent in multiple solid tumors and heme-lymphatic malignancies. The antitumor activity of MLN8237 when combined with docetaxel or paclitaxel was evaluated in in vivo models of triple-negative breast cancer grown in immunocompromised mice. Additive and synergistic antitumor activity occurred at multiple doses of MLN8237 and taxanes. Moreover, significant tumor growth delay relative to the single agents was achieved after discontinuing treatment; notably, durable complete responses were observed in some mice. The tumor growth inhibition data generated with multiple dose levels of MLN8237 and paclitaxel were used to generate an exposure-efficacy model. Exposures of MLN8237 and paclitaxel achieved in patients were mapped onto the model after correcting for mouse-to-human variation in plasma protein binding and maximum tolerated exposures. This allowed rank ordering of various combination doses of MLN8237 and paclitaxel to predict which pair would lead to the greatest antitumor activity in clinical studies. The model predicted that 60 and 80 mg/m(2) of paclitaxel (every week) in patients lead to similar levels of efficacy, consistent with clinical observations in some cancer indications. The model also supported using the highest dose of MLN8237 that can be achieved, regardless of whether it is combined with 60 or 80 mg/m(2) of paciltaxel. The modeling approaches applied in these studies can be used to guide dose-schedule optimization for combination therapies using other therapeutic agents.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Azepinas/administración & dosificación , Neoplasias Experimentales/tratamiento farmacológico , Pirimidinas/administración & dosificación , Taxoides/administración & dosificación , Animales , Antineoplásicos/administración & dosificación , Área Bajo la Curva , Línea Celular Tumoral , Docetaxel , Esquema de Medicación , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Trasplante de Neoplasias , Paclitaxel/administración & dosificación , Investigación Biomédica TraslacionalRESUMEN
Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Antineoplásicos/administración & dosificación , Benzazepinas/administración & dosificación , Proteínas de Ciclo Celular/antagonistas & inhibidores , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tionas/administración & dosificación , Administración Oral , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Benzazepinas/farmacocinética , Benzazepinas/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Sinergismo Farmacológico , Femenino , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Rituximab , Tionas/farmacocinética , Tionas/farmacología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasa Tipo Polo 1RESUMEN
This article describes the discovery of a series of potent inhibitors of Polo-like kinase 1 (PLK1). Optimization of this benzolactam-derived chemical series produced an orally bioavailable inhibitor of PLK1 (12c, MLN0905). In vivo pharmacokinetic-pharmacodynamic experiments demonstrated prolonged mitotic arrest after oral administration of 12c to tumor bearing nude mice. A subsequent efficacy study in nude mice achieved tumor growth inhibition or regression in a human colon tumor (HT29) xenograft model.
Asunto(s)
Antineoplásicos/síntesis química , Benzazepinas/síntesis química , Proteínas de Ciclo Celular/antagonistas & inhibidores , Lactamas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tionas/síntesis química , Administración Oral , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Benzazepinas/farmacocinética , Benzazepinas/farmacología , Disponibilidad Biológica , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lactamas/farmacocinética , Lactamas/farmacología , Ratones , Ratones Desnudos , Mitosis , Modelos Moleculares , Trasplante de Neoplasias , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Tionas/farmacocinética , Tionas/farmacología , Trasplante Heterólogo , Quinasa Tipo Polo 1RESUMEN
PURPOSE: Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. EXPERIMENTAL DESIGN: We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3'-fluoro-3'-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. RESULTS: Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. CONCLUSIONS: Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors.
Asunto(s)
Azepinas/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Huso Acromático/efectos de los fármacos , Animales , Aurora Quinasa A , Aurora Quinasas , Azepinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Didesoxinucleósidos/farmacocinética , Femenino , Radioisótopos de Flúor , Células HCT116 , Células HeLa , Humanos , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Linfoma/patología , Ratones , Ratones Desnudos , Ratones SCID , Índice Mitótico , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación/efectos de los fármacos , Tomografía de Emisión de Positrones , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/química , Huso Acromático/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aurora A kinase is a serine/threonine protein kinase responsible for regulating several mitotic processes including centrosome separation, spindle assembly, and chromosome segregation. Small molecule inhibitors of Aurora A kinase are being pursued as novel anticancer agents, some of which have entered clinical trials. Despite the progress in developing these agents, terminal outcomes associated with Aurora A inhibition are not fully understood. Although evidence exists that Aurora A inhibition leads to apoptosis, other therapeutically relevant cell fates have not been reported. Here, we used the small molecule inhibitor MLN8054 to show that inhibition of Aurora A induces tumor cell senescence both in vitro and in vivo. Treatment of human tumor cells grown in culture with MLN8054 showed a number of morphologic and biochemical changes associated with senescence. These include increased staining of senescence-associated beta-galactosidase, increased nuclear and cell body size, vacuolated cellular morphology, upregulation/stabilization of p53, p21, and hypophosphorylated pRb. To determine if Aurora A inhibition induces senescence in vivo, HCT-116 xenograft-bearing animals were dosed orally with MLN8054 for 3 weeks. In the MLN8054-treated animals, increased senescence-associated beta-galactosidase activity was detected in tissue sections starting on day 15. In addition, DNA and tubulin staining of tumor tissue showed a significant increase in nuclear and cell body area, consistent with a senescent phenotype. Taken together, this data shows that senescence is a terminal outcome of Aurora A inhibition and supports the evaluation of senescence biomarkers in clinic samples.
Asunto(s)
Antineoplásicos/farmacología , Benzazepinas/farmacología , Senescencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/enzimología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/uso terapéutico , Aurora Quinasa A , Aurora Quinasas , Benzazepinas/uso terapéutico , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Tamaño de la Célula/efectos de los fármacos , Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Esquema de Medicación , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/fisiopatología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Retinoblastoma/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , beta-Galactosidasa/efectos de los fármacos , beta-Galactosidasa/metabolismoRESUMEN
Cell death is of broad physiological and pathological importance, making quantification of biochemical events associated with cell demise a high priority for experimental pathology. Fibrosis is a common consequence of tissue injury involving necrotic cell death. Using tissue specimens from experimental mouse models of traumatic brain injury, cardiac fibrosis, and cancer, as well as human tumor specimens assembled in tissue microarray (TMA) format, we undertook computer-assisted quantification of specific immunohistochemical and histological parameters that characterize processes associated with cell death. In this study, we demonstrated the utility of image analysis algorithms for color deconvolution, colocalization, and nuclear morphometry to characterize cell death events in tissue specimens: (a) subjected to immunostaining for detecting cleaved caspase-3, cleaved poly(ADP-ribose)-polymerase, cleaved lamin-A, phosphorylated histone H2AX, and Bcl-2; (b) analyzed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay to detect DNA fragmentation; and (c) evaluated with Masson's trichrome staining. We developed novel algorithm-based scoring methods and validated them using TMAs as a high-throughput format. The proposed computer-assisted scoring methods for digital images by brightfield microscopy permit linear quantification of immunohistochemical and histochemical stainings. Examples are provided of digital image analysis performed in automated or semiautomated fashion for successful quantification of molecular events associated with cell death in tissue sections.
Asunto(s)
Muerte Celular , Algoritmos , Animales , Apoptosis , Biomarcadores/metabolismo , Encéfalo/patología , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Daño del ADN , Femenino , Fibrosis , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Miocardio/metabolismo , Miocardio/patología , Trasplante de Neoplasias , Neuronas/metabolismo , Neuronas/patología , Coloración y Etiquetado , Trasplante HeterólogoRESUMEN
Synthetic triterpenoids 2-cyano-3, 12-dioxooleana-1, 9-(11)-dien-28-oic acid (CDDO) and CDDO-Me (CDDO-methyl ester) have entered clinical trials for cancer. We determined that CDDO analogues at submicromolar concentrations induce apoptosis of cultured prostate cancer cell lines, LNCaP, ALVA31, Du145, PC3, and PPC1, with lethal dose 50% approximately 1 micromol/L for CDDO-Me and an imidazole analogue (CDDO-Im). These compounds induced apoptosis of prostate cancer cells as characterized by cleavage of caspase-3, caspase-7, caspase-8, caspase-9, caspase-10, BID, and poly(ADP)ribose polymerase and by dependence on caspase activity. Moreover, triterpenoid-induced cell death was abolished by caspase-8-targeting small interfering (si) RNA. To explore the mechanism(s) involved in caspase-8 activation, we examined cell surface expression of death receptor (DR)4 and DR5 after triterpenoid treatment. Cell surface DR4 and DR5 expression was significantly up-regulated by CDDO or CDDO-Im but not by CDDO-Me. DR4 and DR5 knockdown with siRNA significantly inhibited apoptosis induced by CDDO and CDDO-Im but had no effect on CDDO-Me-induced killing, suggesting that CDDO and CDDO-Im induce apoptosis by a different mechanism than CDDO-Me. In addition to activating the caspase-8-dependent extrinsic apoptosis pathway, we observed that Bcl-X(L) overexpression inhibited triterpenoid-mediated killing of prostate cancer cell line Du145, suggesting that the intrinsic pathway (via mitochondria) also participates in triterpenoid-mediated killing. In vivo antitumor activity of CDDO-Me was shown using a Du145 tumor xenograft model in nude rats. Altogether, these findings suggest CDDO and related synthetic triterpenoids should be further evaluated as potential novel therapeutics for hormone refractory prostate cancers.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácido Oleanólico/análogos & derivados , Línea Celular Tumoral , Humanos , Masculino , Ácido Oleanólico/farmacología , Neoplasias de la Próstata/patología , Interferencia de ARN , ARN Bicatenario/efectos de los fármacos , ARN Bicatenario/genética , ARN Neoplásico/efectos de los fármacos , ARN Neoplásico/genética , ARN Interferente Pequeño/efectos de los fármacos , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: The synthetic triterpenoid 2-Cyano-3,12-Dioxooleana-1,9-Dien-28-Oic Acid (CDDO) and derivatives display anti-tumor activity against a variety of cultured tumor cell lines and in mouse xenografts. In this report, we have studied the effects of CDDO and its imidazolide derivative (CDDO-Im) on chronic lymphocytic leukemia (CLL), using patients' CLL cells and a mouse model of CLL and small B cell lymphoma (SBL). PRINCIPAL FINDINGS: CDDO and CDDO-Im efficiently induced apoptosis of malignant human and mouse B-cells ex vivo, although CDDO-Im was over 10-fold more potent than CDDO. Treating mice with CLL/SBL with liposome-formulated CDDO or CDDO-Im resulted in significant reductions of B cells in blood, spleen and lung. CDDO-Im was shown to be more potent than CDDO, while treatment with empty liposomes had no impact on disease. CDDO-Im treatment initially resulted in an increase of circulating B cells, which correlates with a reduction in resident lymphocytes in spleen, and lungs, suggesting that CDDO-Im induces mobilization of tumor cells from lymphoid organs and infiltrated tissues into the circulation. Analysis of blood cells recovered from treated mice also showed that CDDO-Im is a potent inducer of tumor cells death in vivo. Furthermore, CDDO-Im efficiently eradicated mouse CLL/SBL cells but had little effect on the viability of normal B and T cells in vivo. SIGNIFICANCE: The presented data demonstrate that triterpenoids CDDO and CDDO-Im reduce leukemia and lymphoma burden in vivo in a transgenic mouse model of CLL/SBL, and support the clinical testing of CDDO-based synthetic triterpenoids in patients with CLL.
Asunto(s)
Modelos Animales de Enfermedad , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Ácido Oleanólico/análogos & derivados , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Factor 2 Asociado a Receptor de TNF/fisiología , Animales , Linfocitos B/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Eliminación de Gen , Humanos , Técnicas para Inmunoenzimas , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Ácido Oleanólico/uso terapéutico , Linfocitos T/efectos de los fármacosRESUMEN
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIP(L), and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.