RESUMEN
PURPOSE: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254. EXPERIMENTAL DESIGN: LXH254 was profiled in biochemical, in vitro, and in vivo assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. RESULTS: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, in vivo, LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. CONCLUSIONS: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.
Asunto(s)
Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-raf/antagonistas & inhibidores , Animales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Células HCT116 , Humanos , Ratones , Mutación , Neoplasias/genética , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Meduloblastoma/patología , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Animales , Muerte Celular , Proliferación Celular , Supervivencia Celular , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Fibroblastos/citología , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Meduloblastoma/genética , Ratones , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/metabolismo , Ratas , Proteínas Represoras/genética , Trasplante HeterólogoRESUMEN
Advanced ovarian cancers are a leading cause of cancer-related death in women and are currently treated with surgery and chemotherapy. This standard of care is often temporarily successful but exhibits a high rate of relapse, after which, treatment options are few. Here we investigate whether biomarker-guided use of multiple targeted therapies, including small molecules and antibody-drug conjugates, is a viable alternative. A panel of patient-derived ovarian cancer xenografts (PDX), similar in genetics and chemotherapy responsiveness to human tumors, was exposed to 21 monotherapies and combination therapies. Three monotherapies and one combination were found to be active in different subsets of PDX. Analysis of gene expression data identified biomarkers associated with responsiveness to each of the three targeted therapies, none of which directly inhibits an oncogenic driver. While no single treatment had as high a response rate as chemotherapy, nearly 90% of PDXs were eligible for and responded to at least one biomarker-guided treatment, including tumors resistant to standard chemotherapy. The distribution of biomarker positivity in The Cancer Genome Atlas data suggests the potential for a similar precision approach in human patients. SIGNIFICANCE: This study exploits a panel of patient-derived xenografts to demonstrate that most ovarian tumors can be matched to effective biomarker-guided treatments.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/genética , Neoplasias Ováricas/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Antineoplásicos/farmacología , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/mortalidad , Carcinoma Epitelial de Ovario/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Terapia Molecular Dirigida/métodos , Neoplasias Ováricas/genética , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Medicina de Precisión , Prueba de Estudio ConceptualRESUMEN
Most anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung tumors initially respond to small-molecule ALK inhibitors, but drug resistance often develops. Of tumors that develop resistance to highly potent second-generation ALK inhibitors, approximately half harbor resistance mutations in ALK, while the other half have other mechanisms underlying resistance. Members of the latter group often have activation of at least one of several different tyrosine kinases driving resistance. Such tumors are not expected to respond to lorlatinib-a third-generation inhibitor targeting ALK that is able to overcome all clinically identified resistant mutations in ALK-and further therapeutic options are limited. Herein, we deployed a shRNA screen of 1,000 genes in multiple ALK-inhibitor-resistant patient-derived cells (PDCs) to discover those that confer sensitivity to ALK inhibition. This approach identified SHP2, a nonreceptor protein tyrosine phosphatase, as a common targetable resistance node in multiple PDCs. SHP2 provides a parallel survival input downstream of multiple tyrosine kinases that promote resistance to ALK inhibitors. Treatment with SHP099, the recently discovered small-molecule inhibitor of SHP2, in combination with the ALK tyrosine kinase inhibitor (TKI) ceritinib halted the growth of resistant PDCs through preventing compensatory RAS and ERK1 and ERK2 (ERK1/2) reactivation. These findings suggest that combined ALK and SHP2 inhibition may be a promising therapeutic strategy for resistant cancers driven by several different ALK-independent mechanisms underlying resistance.
Asunto(s)
Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Resistencia a Antineoplásicos/efectos de los fármacos , Reordenamiento Génico/genética , Neoplasias Pulmonares/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones Desnudos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Sulfonas/farmacología , Sulfonas/uso terapéuticoRESUMEN
Bmi-1 is a member of the Polycomb group family of proteins that function in the epigenetic silencing of genes governing self-renewal, differentiation, and proliferation. Bmi-1 was first identified through its ability to accelerate c-Myc-induced lymphomagenesis. Subsequent studies have further supported an oncogenic role for Bmi-1 in several cancers including those of the breast, lung, prostate, and brain. Using a stable and inducible shRNA system to silence Bmi-1 gene expression, we show a novel role for Bmi-1 in regulating the growth and clonogenic capacity of multiple myeloma cells both in vitro and in vivo. Moreover, to elucidate novel gene targets controlled by Bmi-1, global transcriptional profiling studies were performed in the setting of induced loss of Bmi-1 function. We found that the expression of the proapoptotic gene Bim is negatively regulated by Bmi-1 and that Bim knockdown functionally rescues the apoptotic phenotype induced upon loss of Bmi-1. Therefore, these studies not only highlight Bmi-1 as a cancer-dependent factor in multiple myeloma, but also elucidate a novel antiapoptotic mechanism for Bmi-1 function involving the suppression of Bim.
Asunto(s)
Mieloma Múltiple/patología , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
The malignant brain cancer medulloblastoma is characterized by mutations in Hedgehog (Hh) signaling pathway genes, which lead to constitutive activation of the G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor Smoothened (Smo). The Smo antagonist NVP-LDE225 inhibits Hh signaling and induces tumor regression in animal models of medulloblastoma. However, evidence of resistance was observed during the course of treatment. Molecular analysis of resistant tumors revealed several resistance mechanisms. We noted chromosomal amplification of Gli2, a downstream effector of Hh signaling, and, more rarely, point mutations in Smo that led to reactivated Hh signaling and restored tumor growth. Analysis of pathway gene expression signatures also, unexpectedly, identified up-regulation of phosphatidylinositol 3-kinase (PI3K) signaling in resistant tumors as another potential mechanism of resistance. Probing the relevance of increased PI3K signaling, we demonstrated that addition of the PI3K inhibitor NVP-BKM120 or the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor NVP-BEZ235 to the initial treatment with the Smo antagonist markedly delayed the development of resistance. Our findings may be useful in informing treatment strategies for medulloblastoma.