RESUMEN
Pancreatic ductal adenocarcinoma (PDA) is a deadly cancer characterized by complex metabolic adaptations that promote survival in a severely hypoxic and nutrient-limited tumor microenvironment (TME). Modeling microenvironmental influences in cell culture has been challenging, and technical limitations have hampered the comprehensive study of tumor-specific metabolism in vivo. To systematically interrogate metabolic vulnerabilities in PDA, we employed parallel CRISPR-Cas9 screens using in vivo and in vitro systems. This work revealed striking overlap of in vivo metabolic dependencies with those in vitro. Moreover, we identified that intercellular nutrient sharing can mask dependencies in pooled screens, highlighting a limitation of this approach to study tumor metabolism. Furthermore, metabolic dependencies were similar between 2D and 3D culture, although 3D culture may better model vulnerabilities that influence certain oncogenic signaling pathways. Lastly, our work demonstrates the power of genetic screening approaches to define in vivo metabolic dependencies and pathways that may have therapeutic utility.
Asunto(s)
Sistemas CRISPR-Cas/genética , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proliferación Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Células Tumorales Cultivadas , Microambiente Tumoral/genéticaRESUMEN
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease urgently requiring new treatments. Overexpression of the protein transporter exportin-1 (XPO1) leads to mislocalization of tumor-suppressor proteins (TSP) and their inactivation. Earlier, we showed that blocking XPO1 by CRISPR/Cas9 validated Selective Inhibitor of Nuclear Export (SINE) compounds (selinexor and analogs) restores the antitumor activity of multiple TSPs leading to suppression of PDAC in vitro and in orthotopic models. EXPERIMENTAL DESIGN: We evaluate the synergy between SINE compounds and standard-of-care treatments in preclinical models and in a PDAC Phase Ib trial. RESULTS: SINE compounds synergize with gemcitabine (GEM) and nanoparticle albumin-bound (nab)-paclitaxel leading to suppression of PDAC cellular growth and cancer stem cell (CSC) spheroids disintegration. Label-free quantitative proteome profiling with nuclear and cytoplasmic enrichment showed superior enhancement in nuclear protein fraction in combination treatment. Selinexor inhibited the growth of PDAC CSC and two patient-derived (PDX) subcutaneous xenografts. Selinexor-GEM-nab-paclitaxel blocked PDX and orthotopic tumor growth. In a phase 1b study (NCT02178436), 9 patients were exposed to selinexor (60 mg oral) with GEM (1,000 mg/m2 i.v.) and nab-paclitaxel (125 mg/m2 i.v.) on days 1, 8, and 15 of 28-day cycle. Two patients showed partial response, and 2 had stable disease. An outstanding, durable objective response was observed in one of the responders with progression-free survival of 16 months and overall survival of 22 months. CONCLUSIONS: Our preclinical and ongoing clinical study lends support to the use of selinexor-GEM-nab-paclitaxel as an effective therapy for metastatic PDAC.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carioferinas/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Albúminas/administración & dosificación , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Hidrazinas/administración & dosificación , Ratones , Ratones Endogámicos ICR , Ratones SCID , Paclitaxel/administración & dosificación , Neoplasias Pancreáticas/patología , Triazoles/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto , Gemcitabina , Proteína Exportina 1 , Neoplasias PancreáticasRESUMEN
Few therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Neoplasias/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
Emerging studies have shown that the expression of AR splice variants (ARv) lacking ligand-binding domain is associated with castrate-resistant prostate cancer (CRPC) and higher risk of tumor metastasis and recurrence. Nuclear export protein XPO1 regulates the nuclear localization of many proteins including tumor suppressor proteins. Increased XPO1 in prostate cancer is associated with a high Gleason score and bone metastasis. In this study, we found that high expression of AR splice variant 7 (AR-v7) was correlated with increased XPO1 expression. Silencing of XPO1 by RNAi or treatment with Selective Inhibitor of Nuclear Export (SINE) compounds selinexor and eltanexor (KPT-8602) down-regulated the expression of AR, AR-v7 and ARv567es at mRNA and protein levels. XPO1 silencing also inhibited the expression of AR and ARv regulators including FOXA1, Src, Vav3, MED1 and Sam68, leading to the suppression of ARv and AR target genes, UBE2C and PSA. By targeting XPO1/ARv signaling, SINE suppressed prostate cancer (PCa) growth in vitro and in vivo and potentiated the anti-cancer activity of anti-AR agents, enzalutamide and abiraterone. Therefore, XPO1 inhibition could be a novel promising agent used in combination with conventional chemotherapeutics and AR-targeted therapy for the better treatment of PCa, especially CRPC.
RESUMEN
Purpose: Human exportin-1 (XPO1) is the key nuclear-cytoplasmic transport protein that exports different cargo proteins out of the nucleus. Inducing nuclear accumulation of these proteins by inhibiting XPO1 causes cancer cell death. First clinical validation of pharmacological inhibition of XPO1 was obtained with the Selective Inhibitor of Nuclear Export (SINE) compound selinexor (KPT-330) demonstrating activity in phase-II/IIb clinical trials when dosed 1 to 3 times weekly. The second-generation SINE compound KPT-8602 shows improved tolerability and can be dosed daily. Here, we investigate and validate the drug-target interaction of KPT-8602 and explore its activity against acute lymphoblastic leukemia (ALL).Experimental Design: We examined the effect of KPT-8602 on XPO1 function and XPO1-cargo as well as on a panel of leukemia cell lines. Mutant XPO1 leukemia cells were designed to validate KPT-8602's drug-target interaction. In vivo, anti-ALL activity was measured in a mouse ALL model and patient-derived ALL xenograft models.Results: KPT-8602 induced caspase-dependent apoptosis in a panel of leukemic cell lines in vitro Using CRISPR/Cas9 genome editing, we demonstrated the specificity of KPT-8602 for cysteine 528 in the cargo-binding groove of XPO1 and validated the drug target interaction. In vivo, KPT-8602 showed potent anti-leukemia activity in a mouse ALL model as well as in patient-derived T- and B-ALL xenograft models without affecting normal hematopoiesis.Conclusions: KPT-8602 is highly specific for XPO1 inhibition and demonstrates potent anti-leukemic activity supporting clinical application of the second-generation SINE compound for the treatment of ALL. Clin Cancer Res; 23(10); 2528-41. ©2016 AACR.
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Antineoplásicos/administración & dosificación , Carioferinas/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Tiazoles/administración & dosificación , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Humanos , Carioferinas/genética , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Receptores Citoplasmáticos y Nucleares/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Exportina 1RESUMEN
Validation of drug-target interaction is essential in drug discovery and development. The ultimate proof for drug-target validation requires the introduction of mutations that confer resistance in cells, an approach that is not straightforward in mammalian cells. Using CRISPR/Cas9 genome editing, we show that a homozygous genomic C528S mutation in the XPO1 gene confers cells with resistance to selinexor (KPT-330). Selinexor is an orally bioavailable inhibitor of exportin-1 (CRM1/XPO1) with potent anticancer activity and is currently under evaluation in human clinical trials. Mutant cells were resistant to the induction of cytotoxicity, apoptosis, cell cycle arrest, and inhibition of XPO1 function, including direct binding of the drug to XPO1. These results validate XPO1 as the prime target of selinexor in cells and identify the selectivity of this drug toward the cysteine 528 residue of XPO1. Our findings demonstrate that CRISPR/Cas9 genome editing enables drug-target validation and drug-target selectivity studies in cancer cells.
Asunto(s)
Antineoplásicos/química , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Hidrazinas/química , Carioferinas/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Triazoles/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Secuencia de Bases , Puntos de Control del Ciclo Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Recombinación Homóloga , Humanos , Hidrazinas/metabolismo , Hidrazinas/farmacología , Células Jurkat , Carioferinas/genética , Carioferinas/metabolismo , Cinética , Mutación , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Triazoles/metabolismo , Triazoles/farmacología , Proteína Exportina 1RESUMEN
Infection with HIV ultimately leads to advanced immunodeficiency resulting in an increased incidence of cancer. For example primary effusion lymphoma (PEL) is an aggressive non-Hodgkin lymphoma with very poor prognosis that typically affects HIV infected individuals in advanced stages of immunodeficiency. Here we report on the dual anti-HIV and anti-PEL effect of targeting a single process common in both diseases. Inhibition of the exportin-1 (XPO1) mediated nuclear transport by clinical stage orally bioavailable small molecule inhibitors (SINE) prevented the nuclear export of the late intron-containing HIV RNA species and consequently potently suppressed viral replication. In contrast, in CRISPR-Cas9 genome edited cells expressing mutant C528S XPO1, viral replication was unaffected upon treatment, clearly demonstrating the anti-XPO1 mechanism of action. At the same time, SINE caused the nuclear accumulation of p53 tumor suppressor protein as well as inhibition of NF-κB activity in PEL cells resulting in cell cycle arrest and effective apoptosis induction. In vivo, oral administration arrested PEL tumor growth in engrafted mice. Our findings provide strong rationale for inhibiting XPO1 as an innovative strategy for the combined anti-retroviral and anti-neoplastic treatment of HIV and PEL and offer perspectives for the treatment of other AIDS-associated cancers and potentially other virus-related malignancies.