RESUMEN
Neurofibromatosis Type 1 (NF1) is a common cancer predisposition syndrome, caused by heterozygous loss of function mutations in the tumor suppressor gene NF1. Individuals with NF1 develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell linage after somatic loss of the wild type NF1 allele, some of which progress further to malignant peripheral nerve sheath tumors (MPNST). There is only one FDA approved targeted therapy for symptomatic plexiform neurofibromas and none approved for MPNST. The genetic basis of NF1 syndrome makes associated tumors ideal for using synthetic drug sensitivity approaches to uncover therapeutic vulnerabilities. We developed a drug discovery pipeline to identify therapeutics for NF1-related tumors using isogeneic pairs of NF1-proficient and deficient immortalized human Schwann cells. We utilized these in a large-scale high throughput screen (HTS) for drugs that preferentially kill NF1-deficient cells, through which we identified 23 compounds capable of killing NF1-deficient Schwann cells with selectivity. Multiple hits from this screen clustered into classes defined by method of action. Four clinically interesting drugs from these classes were tested in vivo using both a genetically engineered mouse model of high-grade peripheral nerve sheath tumors and human MPNST xenografts. All drugs tested showed single agent efficacy in these models as well as significant synergy when used in combination with the MEK inhibitor selumetinib. This HTS platform yielded novel therapeutically relevant compounds for the treatment of NF1-associated tumors and can serve as a tool to rapidly evaluate new compounds and combinations in the future.
RESUMEN
Neurofibromatosis type 1 (NF1) is characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. We identified the G-protein-coupled receptor (GPCR) P2ry14 in human neurofibromas, neurofibroma-derived SC precursors (SCPs), mature SCs, and mouse SCPs. Mouse Nf1-/- SCP self-renewal was reduced by genetic or pharmacological inhibition of P2ry14. In a mouse model of NF1, genetic deletion of P2ry14 rescued low cAMP signaling, increased mouse survival, delayed neurofibroma initiation, and improved SC Remak bundles. P2ry14 signals via Gi to increase intracellular cAMP, implicating P2ry14 as a key upstream regulator of cAMP. We found that elevation of cAMP by either blocking the degradation of cAMP or by using a P2ry14 inhibitor diminished NF1-/- SCP self-renewal in vitro and neurofibroma SC proliferation in in vivo. These studies identify P2ry14 as a critical regulator of SCP self-renewal, SC proliferation, and neurofibroma initiation.
Asunto(s)
AMP Cíclico/metabolismo , Neurofibroma , Neurofibromatosis 1 , Receptores Purinérgicos P2Y/metabolismo , Animales , Autorrenovación de las Células , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Ratones , Neurofibroma/genética , Neurofibroma/metabolismo , Neurofibroma/patología , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Células de Schwann/metabolismoRESUMEN
Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft-tissue sarcomas that cause significant mortality in adults with neurofibromatosis type 1. We compared gene expression of growth factors in normal human nerves to MPNST and normal human Schwann cells to MPNST cell lines. We identified WNT5A as the most significantly upregulated ligand-coding gene and verified its protein expression in MPNST cell lines and tumors. In many contexts WNT5A acts as an oncogene. However, inhibiting WNT5A expression using shRNA did not alter MPNST cell proliferation, invasion, migration, or survival in vitro. Rather, shWNT5A-treated MPNST cells upregulated mRNAs associated with the remodeling of extracellular matrix and with immune cell communication. In addition, these cells secreted increased amounts of the proinflammatory cytokines CXCL1, CCL2, IL6, CXCL8, and ICAM1. Versus controls, shWNT5A-expressing MPNST cells formed larger tumors in vivo. Grafted tumors contained elevated macrophage/stromal cells, larger and more numerous blood vessels, and increased levels of Mmp9, Cxcl13, Lipocalin-1, and Ccl12. In some MPNST settings, these effects were mimicked by targeting the WNT5A receptor ROR2. These data suggest that the non-canonical Wnt ligand WNT5A inhibits MPNST tumor formation by modulating the MPNST microenvironment, so that blocking WNT5A accelerates tumor growth in vivo.
Asunto(s)
Proliferación Celular/genética , Neoplasias de la Vaina del Nervio/genética , Microambiente Tumoral/genética , Proteína Wnt-5a/genética , Línea Celular Tumoral , Movimiento Celular/genética , Matriz Extracelular/genética , Humanos , Neoplasias de la Vaina del Nervio/patología , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Neurofibrosarcoma/genética , Neurofibrosarcoma/patología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Células de Schwann/patologíaRESUMEN
Plexiform neurofibromas are benign nerve sheath Schwann cell tumors characterized by biallelic mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene. Atypical neurofibromas show additional frequent loss of CDKN2A/Ink4a/Arf and may be precursor lesions of aggressive malignant peripheral nerve sheath tumors (MPNST). Here we combined loss of Nf1 in developing Schwann cells with global Ink4a/Arf loss and identified paraspinal plexiform neurofibromas and atypical neurofibromas. Upon transplantation, atypical neurofibromas generated genetically engineered mice (GEM)-PNST similar to human MPNST, and tumors showed reduced p16INK4a protein and reduced senescence markers, confirming susceptibility to transformation. Superficial GEM-PNST contained regions of nerve-associated plexiform neurofibromas or atypical neurofibromas and grew rapidly on transplantation. Transcriptome analyses showed similarities to corresponding human tumors. Thus, we recapitulated nerve tumor progression in NF1 and provided preclinical platforms for testing therapies at each tumor grade. These results support a tumor progression model in which loss of NF1 in Schwann cells drives plexiform neurofibromas formation, additional loss of Ink4a/Arf contributes to atypical neurofibromas formation, and further changes underlie transformation to MPNST. SIGNIFICANCE: New mouse models recapitulate the stepwise progression of NF1 tumors and will be useful to define effective treatments that halt tumor growth and tumor progression in NF1.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Neurofibroma/genética , Neurofibroma/patología , Neurofibrosarcoma/genética , Neurofibrosarcoma/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Genes de Neurofibromatosis 1 , Ratones , Ratones Mutantes , Neurofibromatosis 1/genética , Neurofibromatosis 1/patologíaRESUMEN
Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are two dominantly inherited disorders that cause tumors in Schwann cells. NF1 patients have a high risk for malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. NF2 patients have a high risk for schwannomas. To identify potential therapeutic targets in these two tumors, we screened the NF1 MPNST cell line, ST88-14, and the NF2 schwannoma cell line, HEI-193, against ~2000 drugs of known mechanisms of action (including ~600 cancer relevant drugs), and also screened the cell lines against an siRNA library targeting most protein kinases. Both the drug screen and the siRNA screen identified Polo-like kinase 1 (PLK1) among the most potent hits in both cell lines. Since PLK1 acts on the cell cycle primarily at the G2/M transition, the same stage where aurora kinase (AURKA) acts, we explored PLK1 and its relationship to aurora kinase in MPNST. Quantitative profiling of PLK1 inhibitors against a panel of 10 neurofibromatosis cell lines found that they were potent inhibitors and, unlike AURKA inhibitors, were not more selective for NF1 over NF2 tumor cells. Furthermore, one PLK1 inhibitor, BI6727 stabilized tumor volume in MPNST xenografts. We conclude that PLK1 is a therapeutic target for MPNSTs and schwannomas, but inhibitors may have a narrow therapeutic index that limits their use as a single agent.
RESUMEN
PURPOSE: In neurofibromatosis type 1 (NF1) and in highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), constitutively active RAS-GTP and increased MAPK signaling are important in tumorigenesis. Dual specificity phosphatases (DUSPs) are negative regulators of MAPK signaling that dephosphorylate p38, JNK, and ERK in different settings. Although often acting as tumor suppressors, DUSPs may also act as oncogenes, helping tumor cells adapt to high levels of MAPK signaling. We hypothesized that inhibiting DUSPs might be selectively toxic to cells from NF1-driven tumors. EXPERIMENTAL DESIGN: We examined DUSP gene and protein expression in neurofibroma and MPNSTs. We used small hairpin RNA (shRNA) to knock down DUSP1 and DUSP6 to evaluate cell growth, downstream MAPK signaling, and mechanisms of action. We evaluated the DUSP inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), in MPNST cell lines and in cell-line and patient-derived MPNST xenografts. RESULTS: DUSP1 and DUSP6 are expressed in NF1-deleted tumors. Knockdown of DUSP1 and DUSP6, alone or in combination, reduced MPNST cell growth and led to ERK and JNK hyperactivation increasing downstream TP53 and p-ATM. The DUSP inhibitor, BCI, diminished the survival of NF1-deleted Schwann cells and MPNST cell lines through activation of JNK. In vivo, treatment of an established cell-line xenograft or a novel patient-derived xenograft (PDX) of MPNSTs with BCI increased ERK and JNK activation, caused tumor necrosis and fibrosis, and reduced tumor volume in one model. CONCLUSIONS: Targeting DUSP1 and DUSP6 genetically or with BCI effectively inhibits MPNST cell growth and promotes cell death, in vitro and in xenograft models. The data support further investigation of DUSP inhibition in MPNSTs.
Asunto(s)
Antineoplásicos/farmacología , Fosfatasa 1 de Especificidad Dual/antagonistas & inhibidores , Fosfatasa 6 de Especificidad Dual/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/patología , Inhibidores de Proteínas Quinasas/farmacología , Animales , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Neurofibromatosis 1/genética , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Normal Schwann cells (SCs) are quiescent in adult nerves, when ATP is released from the nerve in an activity dependent manner. We find that suppressing nerve activity in adult nerves causes SC to enter the cell cycle. In vitro, ATP activates the SC G-protein coupled receptor (GPCR) P2Y2. Downstream of P2Y2, ß-arrestin-mediated signaling results in PP2-mediated de-phosphorylation of AKT, and PP2 activity is required for SC growth suppression. NF1 deficient SC show reduced growth suppression by ATP, and are resistant to the effects of ß-arrestin-mediated signaling, including PP2-mediated de-phosphorylation of AKT. In patients with the disorder Neurofibromatosis type 1, NF1 mutant SCs proliferate and form SC tumors called neurofibromas. Elevating ATP levels in vivo reduced neurofibroma cell proliferation. Thus, the low proliferation characteristic of differentiated adult peripheral nerve may require ongoing, nerve activity-dependent, ATP. Additionally, we identify a mechanism through which NF1 SCs may evade growth suppression in nerve tumors.
Asunto(s)
Adenosina Trifosfato/metabolismo , Arrestina/metabolismo , Neurofibromina 1/deficiencia , Neuroglía/metabolismo , Proteína Fosfatasa 2/metabolismo , Nervio Ciático/citología , Animales , Bupivacaína/farmacología , Calcio/metabolismo , Células Cultivadas , Embrión de Mamíferos , Ganglios Espinales/citología , Humanos , Hidróxidos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurofibromina 1/genética , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Dimensión del Dolor , Neuropatía Ciática , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Malignant peripheral nerve sheath tumor (MPNST) and neuroblastoma models respond to the investigational small molecule Aurora A kinase inhibitor, alisertib. We previously reported that MPNST and neuroblastomas are also susceptible to oncolytic herpes virus (oHSV) therapy. Herein, we show that combination of alisertib and HSV1716, a virus derived from HSV-1 and attenuated by deletion of RL1, exhibits significantly increased antitumor efficacy compared to either monotherapy. Alisertib and HSV1716 reduced tumor growth and increased survival in two xenograft models of MPNST and neuroblastoma. We found the enhanced antitumor effect was due to multiple mechanisms that likely each contribute to the combination effect. First, oncolytic herpes virus increased the sensitivity of uninfected cells to alisertib cytotoxicity, a process we term virus-induced therapeutic adjuvant (VITA). Second, alisertib increased peak virus production and slowed virus clearance from tumors, both likely a consequence of it preventing virus-mediated increase of intratumoral NK cells. We also found that alisertib inhibited virus-induced accumulation of intratumoral myeloid derived suppressor cells, which normally are protumorigenic. Our data suggest that clinical trials of the combination of oHSV and alisertib are warranted in patients with neuroblastoma or MPNST.
Asunto(s)
Antineoplásicos/administración & dosificación , Azepinas/administración & dosificación , Neurilemoma/patología , Neuroblastoma/patología , Viroterapia Oncolítica/métodos , Pirimidinas/administración & dosificación , Animales , Aurora Quinasa A/antagonistas & inhibidores , Western Blotting , Línea Celular Tumoral , Terapia Combinada , Citotoxicidad Inmunológica/inmunología , Femenino , Citometría de Flujo , Herpesvirus Humano 1 , Humanos , Inmunidad Innata/inmunología , Inmunohistoquímica , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Malignant peripheral nerve sheath tumors (MPNST) are rare soft tissue sarcomas that are a major source of mortality in neurofibromatosis type 1 (NF1) patients. To identify MPNST driver genes, we performed a lentiviral short hairpin (sh) RNA screen, targeting all 130 genes up-regulated in neurofibroma and MPNSTs versus normal human nerve Schwann cells. NF1 mutant cells show activation of RAS/MAPK signaling, so a counter-screen in RAS mutant carcinoma cells was performed to exclude common RAS-pathway driven genes. We identified 7 genes specific for survival of MPSNT cells, including MEIS1. MEIS1 was frequently amplified or hypomethylated in human MPSNTs, correlating with elevated MEIS1 gene expression. In MPNST cells and in a genetically engineered mouse model, MEIS1 expression in developing nerve glial cells was necessary for MPNST growth. Mechanistically, MEIS1 drives MPNST cell growth via the transcription factor ID1, thereby suppressing expression of the cell cycle inhibitor p27(Kip) and maintaining cell survival.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Vaina del Nervio/patología , ARN Interferente Pequeño/metabolismo , Neoplasias de los Tejidos Blandos/patología , Animales , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Modelos Animales de Enfermedad , Puntos de Control de la Fase G1 del Ciclo Celular , Genotipo , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Neoplasias de la Vaina del Nervio/genética , Neoplasias de la Vaina del Nervio/metabolismo , Neoplasias de la Vaina del Nervio/mortalidad , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/mortalidad , Neurofibromatosis 1/patología , Plásmidos/genética , Plásmidos/metabolismo , Interferencia de ARN , Células de Schwann/citología , Células de Schwann/metabolismo , Transducción de Señal , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/metabolismo , Neoplasias de los Tejidos Blandos/mortalidadRESUMEN
Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that are a major cause of mortality of Neurofibromatosis type 1 (NF1) patients. MPNST patients have few therapeutic options available and only complete surgical resection can be curative. MPNST formation and survival are dependent on activated ß-catenin signaling. The goal of this study was to determine if inhibition of the CK2 enzyme can be therapeutically exploited in MPNSTs, given CK2's role in mainta ining oncogenic phenotypes including stabilization of ß-catenin. We found that CK2α is over-expressed in MPNSTs and is critical for maintaining cell survival, as the CK2 inhibitor, CX-4945 (Silmitasertib), and shRNA targeting CK2α each significantly reduce MPNST cell viability. These effects were preceded by loss of critical signaling pathways in MPNSTs, including destabilization of ß-catenin and TCF8. CX-4945 administration in vivo slowed tumor growth and extends survival time. We conclude that CK2 inhibition is a promising approach to blocking ß-catenin in MPNST cells, although combinatorial therapies may be required for maximal efficacy.