RESUMEN
Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.
Asunto(s)
Resistencia a Antineoplásicos/genética , Lactamas Macrocíclicas/farmacología , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pirazoles/farmacología , Piridinas/farmacología , Aminopiridinas , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Proliferación Celular/efectos de los fármacos , Crizotinib , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Glioma/patología , Humanos , Lactamas , Lactamas Macrocíclicas/química , Ratones , Modelos Moleculares , Transducción de Señal/efectos de los fármacosRESUMEN
The expression of endogenous retrotransposable elements, including long interspersed nuclear element 1 (LINE-1 or L1) and human endogenous retrovirus, accompanies neoplastic transformation and infection with viruses such as HIV. The ability to engender immunity safely against such self-antigens would facilitate the development of novel vaccines and immunotherapies. In this article, we address the safety and immunogenicity of vaccination with these elements. We used immunohistochemical analysis and literature precedent to identify potential off-target tissues in humans and establish their translatability in preclinical species to guide safety assessments. Immunization of mice with murine L1 open reading frame 2 induced strong CD8 T cell responses without detectable tissue damage. Similarly, immunization of rhesus macaques with human LINE-1 open reading frame 2 (96% identity with macaque), as well as simian endogenous retrovirus-K Gag and Env, induced polyfunctional T cell responses to all Ags, and Ab responses to simian endogenous retrovirus-K Env. There were no adverse safety or pathological findings related to vaccination. These studies provide the first evidence, to our knowledge, that immune responses can be induced safely against this class of self-antigens and pave the way for investigation of them as HIV- or tumor-associated targets.
Asunto(s)
Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Elementos Transponibles de ADN/inmunología , Retrovirus Endógenos/inmunología , Vacunas contra el SIDA/genética , Adulto , Secuencia de Aminoácidos , Animales , Vacunas contra el Cáncer/genética , Elementos Transponibles de ADN/genética , Modelos Animales de Enfermedad , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Femenino , Humanos , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The chemokine receptor CXCR4 mediates cell anchorage in the bone marrow (BM) microenvironment and is overexpressed in 25-30% of patients with acute myeloid leukemia (AML). Here we have shown that a new CXCR4 receptor antagonist IgG1 antibody (PF-06747143) binds strongly to AML cell lines and to AML primary cells inhibiting their chemotaxis in response to CXCL12. PF-06747143 also induced cytotoxicity in AML cells via Fc-effector function. To characterize the effects of PF-06747143 on leukemia progression, we used two different patient-derived xenograft (PDX) models: Patient 17CXCR4-low and P15CXCR4-high models, characterized by relatively low and high CXCR4 expression, respectively. Weekly administration of PF-06747143 to leukemic mice significantly reduced leukemia development in both models. Secondary transplantation of BM cells from PF-06747143-treated or IgG1 control-treated animals showed that leukemic progenitors were also targeted by PF-06747143. Administration of a single dose of PF-06747143 to PDX models induced rapid malignant cell mobilization into the peripheral blood (PB). These findings support evaluation of this antibody in AML therapy, with particular appeal to patients resistant to chemotherapy and to unfit patients, unable to tolerate intensive chemotherapy.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Leucemia Mieloide Aguda/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Modelos Animales de Enfermedad , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/mortalidad , Ratones , Terapia Molecular Dirigida , Cultivo Primario de Células , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The tolerability, pharmacodynamic effects, and pharmacokinetics of belimumab (LymphoStat-B) were evaluated in cynomolgus monkeys. Belimumab is a fully human IgG1lambda antibody directed against B-lymphocyte stimulator (BLyS) protein. BLyS is a TNF family member that supports B-lymphocyte maturation and survival and has been implicated in the pathogenesis of autoimmune diseases and B-lymphocyte malignancies. Belimumab was developed to antagonize BLyS activity in autoimmune diseases and B-lymphocyte malignancies, where undesirable effects of B-lymphocyte activity may cause or contribute to disease. Pharmacodynamic effects of belimumab were monitored by immunophenotyping of peripheral blood. Pathology end points, including tissue immunophenotyping, are described after 13 and 26 weeks of treatment and after a 34-week treatment-free (recovery) period. Belimumab was safe and well tolerated in repeat-dose toxicology studies at 5-50 mg/kg for up to 26 weeks. Monkeys exposed to belimumab had significant decreases in peripheral blood B lymphocytes by 13 weeks of exposure, continuing into the recovery period, despite total lymphocyte counts similar to the controls. There were concomitant decreases in spleen and lymph node B-lymphocyte representation after 13 or 26 weeks of treatment with belimumab. Microscopically, monkeys treated with belimumab for 13 or 26 weeks had decreases in the number and size of lymphoid follicles in the white pulp of the spleen. All findings were generally reversible within a 34-week recovery period. These data confirm the specific pharmacologic activity of belimumab in reducing B lymphocytes in the cynomolgus monkey. The favorable safety profile and lack of treatment-related infections also support continued clinical development of belimumab.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Linfocitos B/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Antígenos CD20/inmunología , Factor Activador de Células B , Femenino , Inmunoglobulinas/sangre , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/crecimiento & desarrollo , Ganglios Linfáticos/inmunología , Recuento de Linfocitos , Macaca fascicularis , Masculino , Tamaño de los Órganos/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/crecimiento & desarrollo , Bazo/inmunologíaRESUMEN
Crizotinib (Xalkori) is a tyrosine kinase inhibitor of both anaplastic lymphoma kinase (ALK) and mesenchymal-epithelial transition factor (c-Met). Though not predicted from standard nonclinical toxicological evaluation, visual disturbance became a frequently observed adverse event in humans. To understand the possible mechanism of this vision effect, an in vivo electroretinogram (ERG) study was conducted to assess retinal functional changes following oral administration of crizotinib. Immunohistochemical (IHC) staining of ALK and c-Met in the neural retinas of human, non-human primate, dog, rat, and mouse was used to aid in the animal model selection. ALK IHC staining was identified predominantly in the ganglion cell and inner nuclear layers of most species evaluated, in the inner plexiform layer in human and rodent, and in the nerve fiber layer in human and rat only. There was no apparent staining of any layer of the neural retina for c-Met in any of the species evaluated. ERG measurements identified a significant reduction in b-wave amplitude during the initial phase of dark adaptation in the crizotinib-treated rats. ERGs were also taken following oral administration of PF-06463922 (an ALK-selective inhibitor), for an understanding of potential kinase involvement. ERG effects were not observed in PF-06463922-treated animals when comparable exposures in the vitreous humor were achieved. Collectively, our results suggest that the ERG b-wave amplitude decreases during dark adaption following crizotinib administration may be related to signaling changes within the retina in rats, likely independent of ALK inhibition.
Asunto(s)
Adaptación a la Oscuridad/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Retina/efectos de los fármacos , Administración Oral , Quinasa de Linfoma Anaplásico , Animales , Crizotinib , Perros , Electrorretinografía , Potenciales Evocados , Humanos , Inmunohistoquímica , Luz , Macaca fascicularis , Masculino , Ratones , Estimulación Luminosa , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirazoles/administración & dosificación , Piridinas/administración & dosificación , Ratas Long-Evans , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/metabolismo , Retina/enzimología , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2-6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Histonas/metabolismo , Humanos , Intestino Delgado/patología , Ratones , Ratones SCID , Mitosis/efectos de los fármacos , Fosforilación , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/toxicidad , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Trasplante HeterólogoRESUMEN
We report the preclinical evaluation of PF-06463922, a potent and brain-penetrant ALK/ROS1 inhibitor. Compared with other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK-driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors because of secondary ALK kinase domain mutations and/or brain metastases.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/secundario , Resistencia a Antineoplásicos/efectos de los fármacos , Lactamas Macrocíclicas/administración & dosificación , Neoplasias/tratamiento farmacológico , Proteínas Tirosina Quinasas Receptoras/genética , Aminopiridinas , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/farmacología , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Lactamas , Lactamas Macrocíclicas/farmacología , Ratones , Mutación , Células 3T3 NIH , Neoplasias/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Metastatic bladder cancer is a serious condition with a 5-year survival rate of approximately 14 %, a rate that has remained unchanged for almost three decades. Thus, there is a profound need to identify the driving mutations for these aggressive tumors to better determine appropriate treatments. Mutational analyses of clinical samples suggest that mutations in either the phosphoinositide-3 kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) or RAS/MEK/ERK pathways drive bladder cancer progression, although it remains to be tested whether the inhibition of either (or both) of these pathways can arrest PI3K/mTOR- or Ras-driven proliferation. METHODS: Herein, we used several bladder cancer cell lines to determine drug sensitivity according to genetic background and also studied mouse models of engrafted UM-UC-3 cells and patient-derived xenografts (PDXs) to test PI3K/mTOR and MEK inhibition in vivo. RESULTS: Inhibition of these pathways utilizing PF-04691502, a PI3K and mTOR inhibitor, and PD-0325901, a MEK inhibitor, slowed the tumor growth of PDX models of bladder cancer. The growth inhibitory effect of combination therapy was similar to that of the clinical maximum dose of cisplatin; mechanistically, this appeared to predominantly occur via drug-induced cytostatic growth inhibition as well as diminished vascular endothelial growth factor secretion in the tumor models. Kinase arrays of tumors harvested after treatment demonstrated activated p53 and Axl as well as STAT1 and STAT3. CONCLUSION: Taken together, these results indicate that clinically relevant doses of PF-04691502 and PD-0325901 can suppress bladder tumor growth in PDX models, thus offering additional potential treatment options by a precision medicine approach.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Anciano de 80 o más Años , Animales , Benzamidas/administración & dosificación , Benzamidas/farmacología , Difenilamina/administración & dosificación , Difenilamina/análogos & derivados , Difenilamina/farmacología , Modelos Animales de Enfermedad , Femenino , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridonas/administración & dosificación , Piridonas/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Distribución Aleatoria , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Vejiga Urinaria/enzimología , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
We performed immunohistochemistry for macrophage colony-stimulating factor 1 receptor (also known as c-fms proto-oncogene product) on tissue microarrays of human nontumor lung, pulmonary squamous cell carcinomas (SCC) and adenocarcinomas (ADC), and breast and ovarian carcinomas using a commercially available anti-cFMS antibody. The specificity of the antibody was validated by Western blot and mass spectrometry analysis. Staining of cFMS was restricted to stromal fibroblasts in pulmonary SCC and ADC specimens and was not identified in tumor epithelium or epithelium and stromal cells of nontumor lung. Evaluation of pulmonary SCC (n=63) and ADC (n=71) specimens revealed stromal fibroblast cFMS staining in 60% (38 of 63) and 35% (25 of 71) of the tumor samples, respectively. A similar pattern of stromal fibroblast cFMS staining was observed in breast (n=21) and ovarian (n=50) carcinomas. It was reported that glucocorticoids induced cFMS expression in breast carcinomas and choriocarcinomas. To investigate whether stromal cFMS expression in lung cancers was associated with glucocorticoid signaling, glucocorticoid receptor protein distribution was evaluated in lung tissue microarrays by immunohistochemistry. Stromal fibroblast glucocorticoid receptor staining was only observed in 18% (2 of 11) of pulmonary SCC and 6% (1 of 17) of ADC specimens, suggesting that cFMS expression may not be directly mediated by glucocorticoids in stromal fibroblasts of lung cancers. The tumor stromal cell expression of cFMS in certain tumor types (lung, ovarian, and breast) suggests the potential for more diverse tumor therapeutic options and presents an attractive target for drug development.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Carcinoma de Células Escamosas/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmón/metabolismo , Neoplasias Ováricas/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Adenocarcinoma/patología , Anciano , Neoplasias de la Mama/patología , Carcinoma de Células Escamosas/patología , Femenino , Humanos , Inmunohistoquímica , Pulmón/patología , Neoplasias Pulmonares/patología , Masculino , Terapia Molecular Dirigida , Neoplasias Ováricas/patología , Proto-Oncogenes Mas , Análisis de Matrices TisularesRESUMEN
Noninvasive imaging has been widely applied for monitoring antiangiogenesis therapy in cancer drug discovery. In this report, we used different imaging modalities including high-frequency ultrasound (HFUS), dynamic contrast enhanced-MR (DCE-MR), and fluorescence molecular tomography (FMT) imaging systems to monitor the changes in the tumor vascular properties after treatment with γ-secretase inhibitor PF-03084014. Sunitinib was tested in parallel for comparison. In the MDA-MB-231Luc model, we demonstrated that antiangiogenesis was one of the contributing mechanisms for the therapeutic effect of PF-03084014. By immunohistochemistry and FITC-lectin perfusion assays, we showed that the vascular defects upon treatment with PF-03084014 were associated with Notch pathway modulation, evidenced by a decrease in the HES1 protein and by the changes in VEGFR2 and HIF1α levels, which indicates down-stream effects. Using a 3D power Doppler scanning method, ultrasound imaging showed that the% vascularity in the MDA-MB-231Luc tumor decreased significantly at 4 and 7 days after the treatment with PF-03084014. A decrease in the tumor vessel function was also observed through contrast-enhanced ultrasound imaging with microbubble injection. These findings were consistent with the PF-03084014-induced functional vessel changes measured by suppressing the K(trans) values using DCE-MRI. In contrast, the FMT imaging with the AngioSence 680EX failed to detect any treatment-associated tumor vascular changes. Sunitinib demonstrated an outcome similar to PF-03084014 in the tested imaging modalities. In summary, ultrasound and DCE-MR imaging successfully provided longitudinal measurement of the phenotypic and functional changes in tumor vasculature after treatment with PF-03084014 and sunitinib.
Asunto(s)
Imagen por Resonancia Magnética , Neovascularización Patológica/diagnóstico por imagen , Neovascularización Patológica/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Inhibidores de la Angiogénesis/administración & dosificación , Línea Celular Tumoral , Medios de Contraste/química , Humanos , Indoles/administración & dosificación , Imagen Molecular , Neovascularización Patológica/patología , Pirroles/administración & dosificación , Radiografía , Sunitinib , Tetrahidronaftalenos/administración & dosificación , Ultrasonografía , Valina/administración & dosificación , Valina/análogos & derivados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (µCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available µCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. µCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4-ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative µCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced µCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.
Asunto(s)
Medios de Contraste , Modelos Animales de Enfermedad , Neoplasias Pulmonares/diagnóstico por imagen , Microtomografía por Rayos X/métodos , Animales , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.
Asunto(s)
Antineoplásicos/farmacología , Fosfatidilinositol 3-Quinasa Clase I/genética , Neoplasias Colorrectales/tratamiento farmacológico , Mutación , Células Madre Neoplásicas/efectos de los fármacos , Piridonas/farmacología , Pirimidinas/farmacología , Administración Oral , Adulto , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Humanos , Masculino , Ratones SCID , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recent studies in several tumor models indicated that treatment with angiogenic inhibitors may trigger induction of metastasis to other organs. Here we investigated modes of resistance and invasion in several tumor cell lines including 4T1 (breast), H460 (lung) and Colo205 (colorectal) using sunitinib at doses comparable to clinically utilized regimen. In comparison with vehicle-treated tumors, sunitinib increased metastasis to lung in 4T1 tumors and to peritoneal lymph node in Colo205 tumors. However, the same treatment did not induce invasiveness in H460 tumors, further suggesting that accelerating metastasis during treatment with angiogenic inhibitors is tumor cell-type dependent. Interestingly, Crizotinib (a dual inhibitor of c-Met and ALK pathways) as single agent or in combination with sunitinib reduced metastasis in all models tested suggesting a role for c-Met/HGF pathway in intrinsic- or sunitinib-induced-metastasis. Moreover, ELISA data showed that while c-Met is highly enriched in tumor cells, HGF is secreted mainly by the stroma (mouse HGF) suggesting a paracrine fashion for c-Met pathway activation in the tumors. In conclusion, our findings indicate that sunitinib-induced metastasis is tumor cell-type dependent and further supports a rationale for combination of anti-angiogenics and c-Met inhibition in the clinic.
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Inhibidores de la Angiogénesis/efectos adversos , Neoplasias de la Mama/patología , Neoplasias Colorrectales/patología , Factor de Crecimiento de Hepatocito/metabolismo , Indoles/efectos adversos , Neoplasias Pulmonares/secundario , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirroles/efectos adversos , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Indoles/uso terapéutico , Neoplasias Pulmonares/metabolismo , Metástasis Linfática , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Neovascularización Patológica/tratamiento farmacológico , Pirroles/uso terapéutico , Transducción de Señal/efectos de los fármacos , SunitinibRESUMEN
The primary target organ for uptake of systemically administered phosphorothioate oligonucleotides is the kidney cortex and the proximal tubular epithelium in particular. To determine the effect of oligonucleotide uptake on renal function, a detailed renal physiology study was performed in cynomolgus monkeys treated with 10-40 mg/kg/week ISIS 113715 for 4 weeks. The concentrations of oligonucleotide in the kidney cortex ranged from 1400 to 2600 µg/g. These concentrations were associated with histologic changes in proximal tubular epithelial cells that ranged from the appearance of cytoplasmic basophilic granules to atrophic and degenerative changes at higher concentrations. However, there were no renal functional abnormalities as determined by the typical measurements of blood urea nitrogen, serum creatinine, creatinine clearance, or urine specific gravity. Nor were there changes in glomerular filtration rate, or renal blood flow. Specific urinary markers of tubular epithelial cell damage, such as N-acetyl-glucosaminidase, and α-glutathione-s-transferase were not affected. Tubular function was further evaluated by monitoring the urinary excretion of amino acids, ß(2)-microglobulin, or glucose. Renal function was challenged by administering a glucose load and by examining concentrating ability after a 4-h water deprivation. Neither challenge produced any evidence of change in renal function. The only change observed was a low incidence of increased urine protein/creatinine ratio in monkeys treated with ≥40 mg/kg/week which was rapidly reversible. Collectively, these data indicate that ISIS 113715-uptake by the proximal tubular epithelium has little or no effect on renal function at concentrations of 2600 µg/g.
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Células Epiteliales/efectos de los fármacos , Corteza Renal/metabolismo , Túbulos Renales Proximales/patología , Oligorribonucleótidos/farmacocinética , Animales , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Creatinina/orina , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Células Epiteliales/patología , Glucosa/metabolismo , Pruebas de Función Renal , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/efectos de los fármacos , Macaca fascicularis , Masculino , Oligorribonucleótidos/administración & dosificación , Oligorribonucleótidos/toxicidad , Proteínas/metabolismo , Distribución TisularRESUMEN
The role of PI3K and MAPK pathways in tumor initiation and progression is well established; hence, several inhibitors of these pathways are currently in different stages of clinical trials. Recent studies identified a PI3K/mTOR (PF-04691502) and a MEK inhibitor (PD-0325901) with strong potency and efficacy in different cell lines and tumor models. PD-0325901, however, showed adverse effects when administered at or above MTD (maximum tolerated dose) in the clinic. Here, we show in preclinical models that PD-0325901 at doses well below MTD (sub-MTD 1.5 mg/kg SID) is still a potent compound as single agent or in combination with PF-04691502. We first observed that PD-0325901 at 1.5 mg/kg SID and in combination with PF-04691502 (7.5 mg/kg; SID) significantly inhibited growth of H460 (carry Kras and PIK3CA mutations) orthotopic lung tumors. Additionally, we tested efficacy of PD-0325901 in Kras(G12D-LSL) conditional GEMMs (genetically engineered mouse models) which are a valuable tool in translational research to study tumor progression. Intranasal delivery of adenoviruses expressing Cre recombinase (Adeno-Cre) resulted in expression of mutant Kras leading to development of tumor lesions in lungs including adenomatous hyperplasia, large adenoma, and adenocarcinoma. Similar to H460 tumors, PD-0325901 as single agent or in combination with PF-04691502 significantly inhibited growth of tumor lesions in lungs in Kras(G12D-LSL) mice when treatment started at adenocarcinoma stage (at 14 weeks post-Adeno-Cre inhalation). In addition, immunohistochemistry showed inhibition of pS6 (phosphorylated ribosomal S6) in the treated animals particularly in the combination group providing a proof of mechanism for tumor growth inhibition. Finally, m-CT imaging in live Kras(G12D-LSL) mice showed reduction of tumor burdens in PD-0325901-treated animals at sub-MTD dose. In conclusion, our data suggest that PD-0325901 at doses below MTD is still a potent compound capable of tumor growth inhibition where Kras and/or PI3K are drivers of tumor growth and progression.
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Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas p21(ras)/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adenocarcinoma/enzimología , Adenocarcinoma del Pulmón , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Benzamidas/administración & dosificación , Línea Celular Tumoral , Difenilamina/administración & dosificación , Difenilamina/análogos & derivados , Relación Dosis-Respuesta a Droga , Heterocigoto , Humanos , Neoplasias Pulmonares/enzimología , Dosis Máxima Tolerada , Ratones , Ratones Mutantes , Trasplante de Neoplasias , Piridonas/administración & dosificación , Pirimidinas/administración & dosificaciónRESUMEN
Osteopontin (OPN), also known as SPP1 (secreted phosphoprotein), is an integrin binding glyco-phosphoprotein produced by a variety of tissues. In cancer patients expression of OPN has been associated with poor prognosis in several tumor types including breast, lung, and colorectal cancers. Despite wide expression in tumor cells and stroma, there is limited evidence supporting role of OPN in tumor progression and metastasis. Using phage display technology we identified a high affinity anti-OPN monoclonal antibody (hereafter AOM1). The binding site for AOM1 was identified as SVVYGLRSKS sequence which is immediately adjacent to the RGD motif and also spans the thrombin cleavage site of the human OPN. AOM1 efficiently inhibited OPNa binding to recombinant integrin αvß3 with an IC50 of 65 nM. Due to its unique binding site, AOM1 is capable of inhibiting OPN cleavage by thrombin which has been shown to produce an OPN fragment that is biologically more active than the full length OPN. Screening of human cell lines identified tumor cells with increased expression of OPN receptors (αvß3 and CD44v6) such as mesothelioma, hepatocellular carcinoma, breast, and non-small cell lung adenocarcinoma (NSCLC). CD44v6 and αvß3 were also found to be highly enriched in the monocyte, but not lymphocyte, subset of human peripheral blood mononuclear cells (hPBMCs). In vitro, OPNa induced migration of both tumor and hPBMCs in a transwell migration assay. AOM1 significantly blocked cell migration further validating its specificity for the ligand. OPN was found to be enriched in mouse plasma in a number of pre-clinical tumor model of non-small cell lung cancers. To assess the role of OPN in tumor growth and metastasis and to evaluate a potential therapeutic indication for AOM1, we employed a Kras(G12D-LSL)p53(fl/fl) subcutaneously implanted in vivo model of NSCLC which possesses a high capacity to metastasize into the lung. Our data indicated that treatment of tumor bearing mice with AOM1 as a single agent or in combination with Carboplatin significantly inhibited growth of large metastatic tumors in the lung further supporting a role for OPN in tumor metastasis and progression.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/secundario , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Osteopontina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Factores Quimiotácticos/química , Factores Quimiotácticos/inmunología , Factores Quimiotácticos/metabolismo , Modelos Animales de Enfermedad , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Integrinas/metabolismo , Ratones , Ratones SCID , Datos de Secuencia Molecular , Osteopontina/química , Osteopontina/inmunología , Unión Proteica , Estabilidad Proteica , Trombina/metabolismoRESUMEN
PURPOSE: We aimed to assess the biologic activity of PF-03084014 in breast xenograft models. The biomarkers for mechanism and patient stratification were also explored. EXPERIMENTAL DESIGN: The in vitro and in vivo properties of PF-03084014 were investigated. The mRNA expressions of 40 key Notch pathway genes at baseline or after treatment were analyzed to link with the antitumor efficacy of PF-03084014 in a panel of breast cancer xenograft models. RESULTS: In vitro, PF-03084014 exhibited activity against tumor cell migration, endothelial cell tube formation, and mammosphere formation. In vivo, we observed apoptosis, antiproliferation, reduced tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity after the treatment of PF-03084014. PF-03084014 treatment displayed significant antitumor activity in 10 of the 18 breast xenograft models. However, the antitumor efficacy in most models did not correlate with the in vitro antiproliferation results in the corresponding cell lines, suggesting the critical involvement of tumor microenvironment during Notch activation. In the tested breast xenograft models, the baseline expressions of the Notch receptors, ligands, and the cleaved Notch1 failed to predict the antitumor response to PF-03084014, whereas several Notch pathway target genes, including HEY2, HES4, and HES3, strongly corresponded with the response with a P value less than 0.01. Many of the best molecular predictors of response were also significantly modulated following PF-03084014 treatment. CONCLUSIONS: PF-03084014 showed antitumor and antimetastatic properties via pleiotropic mechanisms. The Notch pathway downstream genes may be used to predict the antitumor activity of PF-03084014 and enrich for responders among breast cancer patients.
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Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Tetrahidronaftalenos/farmacología , Valina/análogos & derivados , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Antineoplásicos/administración & dosificación , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Metástasis de la Neoplasia/tratamiento farmacológico , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Tetrahidronaftalenos/administración & dosificación , Valina/administración & dosificación , Valina/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: [(18)F]FLT (3'-Fluoro-3' deoxythymidine)-PET imaging was proposed as a tool for measuring in vivo tumor cell proliferation. The aim of this article was to validate the use of [(18)F]FLT-PET imaging for measuring xenograft proliferation and subsequent monitoring of targeted therapy. EXPERIMENTAL DESIGN: In exponentially growing xenografts, factors that could impact the outcome of [(18)F]FLT-PET imaging, such as nucleoside transporters, thymidine kinase 1, the relative contribution of DNA salvage pathway, and the ratio of FLT to thymidine, were evaluated. The [(18)F]FLT tracer avidity was compared with other proliferation markers. RESULTS: In a panel of proliferating xenografts, [(18)F]FLT or [(3)H]thymidine tracer avidity failed to reflect the tumor growth rate across different tumor types, despite the high expressions of Ki67 and TK1. When FLT was injected at the same dose level as used in the preclinical [(18)F]FLT-PET imaging, the plasma exposure ratio of FLT to thymidine was approximately 1:200. Thymidine levels in different tumor types seemed to be variable and exhibited an inverse relationship with the FLT tracer avidity. In contrast, high-dose administration of bromdeoxyuridine (BrdUrd; 50 mg/kg) yielded a plasma exposure of more than 4-fold higher than thymidine and leads to a strong correlation between the BrdUrd uptake and the tumor proliferation rate. In FLT tracer-avid models, [(18)F]FLT-PET imaging as a surrogate biomarker predicted the therapeutic response of CDK4/6 inhibitor PD-0332991. CONCLUSIONS: Tumor thymidine level is one of the factors that impact the correlation between [(18)F]FLT uptake and tumor cell proliferation. With careful validation, [(18)F]FLT-PET imaging can be used to monitor antiproliferative therapies in tracer-avid malignancies.
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Didesoxinucleósidos , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Bromodesoxiuridina/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Didesoxinucleósidos/farmacocinética , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Piperazinas/administración & dosificación , Piperazinas/farmacología , Piridinas/administración & dosificación , Piridinas/farmacología , Timidina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: To evaluate the safety and persistence of injectable polymethylmethacrylate (PMMA) microspheres as a long-lasting urethral bulking agent in pigs. PMMA microspheres of 2 different diameters (40 and 125 µm) were tested to investigate the potential for migration and dislocation after injection. A similar product containing 40-µm PMMA microspheres has been used as an injectable wrinkle filler for >25 years and received Food and Drug Administration approval in 2006 (ArteFill). METHODS: A total of 22 female pigs received 4 submucosal implantations of PMMA microspheres, using either a cystoscope or a newly developed urethral injection device (UroScope). At death and necropsy at 8 days and 1, 3, and 6 months, the urethral injection site, liver, lung, spleen, and pelvic and iliac lymph nodes were processed for histologic examination and microsphere count using organ dissolution and microscopy. RESULTS: All injected submucosal blebs were still present at 6 months and showed no signs of inflammation. Tissue dissolution of the local lymph nodes and major organs demonstrated the transport of some of the 40-µm microspheres to the local lymph nodes and lung but not to the liver or spleen. In contrast, no 125-µm microspheres were detected in any distant organ. CONCLUSIONS: The submucosal implantation of 125-µm PMMA microspheres into the urethra provided a safe and persistent bulking effect in pigs. The positive results of the present study encourage additional investigation of 125-µm PMMA microspheres as a long-lasting bulking agent for the treatment of female stress urinary incontinence. Furthermore, a newly developed urethral injection device (UroScope) proved beneficial and cost-effective to facilitate the transurethral injections of 125-µm PMMA microspheres.
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Microesferas , Polimetil Metacrilato/uso terapéutico , Incontinencia Urinaria de Esfuerzo/terapia , Animales , Materiales Biocompatibles , Femenino , Reacción a Cuerpo Extraño/patología , Inyecciones , Ensayo de Materiales , Tamaño de la Partícula , Polimetil Metacrilato/administración & dosificación , Porcinos , Porcinos EnanosRESUMEN
Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.