RESUMEN
Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patient's primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term "genome shredding." Importantly, in the patient's recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. "Cancer shredding" leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Temozolomida/farmacología , Temozolomida/uso terapéutico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Glioblastoma/patología , Glioma/genética , Glioma/tratamiento farmacológico , Antineoplásicos Alquilantes/farmacologíaRESUMEN
The immune microenvironment of tumors can play a critical role in promoting or inhibiting tumor progression depending on the context. We present evidence that tumor-associated macrophages/microglia (TAMs) can promote tumor progression in the sonic hedgehog subgroup of medulloblastoma (SHH-MB). By combining longitudinal manganese-enhanced magnetic resonance imaging (MEMRI) and immune profiling of a sporadic mouse model of SHH-MB, we found the density of TAMs is higher in the ~50% of tumors that progress to lethal disease. Furthermore, reducing regulatory T cells or eliminating B and T cells in Rag1 mutants does not alter SHH-MB tumor progression. As TAMs are a dominant immune component in tumors and are normally dependent on colony-stimulating factor 1 receptor (CSF1R), we treated mice with a CSF1R inhibitor, PLX5622. Significantly, PLX5622 reduces a subset of TAMs, prolongs mouse survival, and reduces the volume of most tumors within 4 weeks of treatment. Moreover, concomitant with a reduction in TAMs the percentage of infiltrating cytotoxic T cells is increased, indicating a change in the tumor environment. Our studies in an immunocompetent preclinical mouse model demonstrate TAMs can have a functional role in promoting SHH-MB progression. Thus, CSF1R inhibition could have therapeutic potential for a subset of SHH-MB patients.
Asunto(s)
Neoplasias Cerebelosas/prevención & control , Modelos Animales de Enfermedad , Proteínas Hedgehog/fisiología , Meduloblastoma/prevención & control , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Macrófagos Asociados a Tumores/inmunología , Animales , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proliferación Celular , Neoplasias Cerebelosas/etiología , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Femenino , Humanos , Masculino , Meduloblastoma/etiología , Meduloblastoma/metabolismo , Meduloblastoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pronóstico , Células Tumorales Cultivadas , Microambiente TumoralRESUMEN
PURPOSE: Genetically engineered mouse models of sporadic cancers are critical for studying tumor biology and for preclinical testing of therapeutics. We present an MRI-based pipeline designed to produce high resolution, quantitative information about tumor progression and response to novel therapies in mouse models of medulloblastoma (MB). METHODS: Sporadic MB was modeled in mice by inducing expression of an activated form of the Smoothened gene (aSmo) in a small number of cerebellar granule cell precursors. aSmo mice were imaged and analyzed at defined time-points using a 3D manganese-enhanced MRI-based pipeline optimized for high-throughput. RESULTS: A semi-automated segmentation protocol was established that estimates tumor volume in a time-frame compatible with a high-throughput pipeline. Both an empirical, volume-based classifier and a linear discriminant analysis-based classifier were tested to distinguish progressing from nonprogressing lesions at early stages of tumorigenesis. Tumor centroids measured at early stages revealed that there is a very specific location of the probable origin of the aSmo MB tumors. The efficacy of the manganese-enhanced MRI pipeline was demonstrated with a small-scale experimental drug trial designed to reduce the number of tumor associated macrophages and microglia. CONCLUSION: Our results revealed a high level of heterogeneity between tumors within and between aSmo MB models, indicating that meaningful studies of sporadic tumor progression and response to therapy could not be conducted without an imaging-based pipeline approach.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética , Meduloblastoma/diagnóstico por imagen , Algoritmos , Animales , Cerebelo/metabolismo , Análisis Discriminante , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Imagenología Tridimensional , Modelos Lineales , Ratones , Reconocimiento de Normas Patrones Automatizadas , Transducción de Señal , Receptor Smoothened/genéticaRESUMEN
Microglia, the brain resident macrophages, critically shape forebrain neuronal circuits. However, their precise function in the cerebellum is unknown. Here we show that human and mouse cerebellar microglia express a unique molecular program distinct from forebrain microglia. Cerebellar microglial identity was driven by the CSF-1R ligand CSF-1, independently of the alternate CSF-1R ligand, IL-34. Accordingly, CSF-1 depletion from Nestin+ cells led to severe depletion and transcriptional alterations of cerebellar microglia, while microglia in the forebrain remained intact. Strikingly, CSF-1 deficiency and alteration of cerebellar microglia were associated with reduced Purkinje cells, altered neuronal function, and defects in motor learning and social novelty interactions. These findings reveal a novel CSF-1-CSF-1R signaling-mediated mechanism that contributes to motor function and social behavior.
Asunto(s)
Conducta Animal/fisiología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Microglía/metabolismo , Actividad Motora/fisiología , Células de Purkinje/metabolismo , Transducción de Señal/fisiología , Conducta Social , Animales , Humanos , Factor Estimulante de Colonias de Macrófagos/genética , Ratones , Ratones Transgénicos , Células de Purkinje/citología , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismoRESUMEN
The main cell of origin of the Sonic hedgehog (SHH) subgroup of medulloblastoma (MB) is granule cell precursors (GCPs), a SHH-dependent transient amplifying population in the developing cerebellum. SHH-MBs can be further subdivided based on molecular and clinical parameters, as well as location because SHH-MBs occur preferentially in the lateral cerebellum (hemispheres). Our analysis of adult patient data suggests that tumors with Smoothened (SMO) mutations form more specifically in the hemispheres than those with Patched 1 (PTCH1) mutations. Using sporadic mouse models of SHH-MB with the two mutations commonly seen in adult MB, constitutive activation of Smo (SmoM2) or loss-of-Ptch1, we found that regardless of timing of induction or type of mutation, tumors developed primarily in the hemispheres, with SmoM2-mutants indeed showing a stronger specificity. We further uncovered that GCPs in the hemispheres are more susceptible to high-level SHH signaling compared with GCPs in the medial cerebellum (vermis), as more SmoM2 or Ptch1-mutant hemisphere cells remain undifferentiated and show increased tumorigenicity when transplanted. Finally, we identified location-specific GCP gene-expression profiles, and found that deletion of the genes most highly expressed in the hemispheres (Nr2f2) or vermis (Engrailed1) showed opposing effects on GCP differentiation. Our studies thus provide insights into intrinsic differences within GCPs that impact on SHH-MB progression.
Asunto(s)
Neoplasias Cerebelosas/patología , Cerebelo/patología , Proteínas Hedgehog/metabolismo , Meduloblastoma/patología , Receptor Patched-1/metabolismo , Receptor Smoothened/metabolismo , Adulto , Animales , Diferenciación Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Cerebelo/metabolismo , Proteínas Hedgehog/genética , Humanos , Lactante , Meduloblastoma/genética , Meduloblastoma/metabolismo , Ratones , Receptor Patched-1/genética , Transducción de Señal , Receptor Smoothened/genética , TranscriptomaRESUMEN
Coordinate control of different classes of cyclins is fundamentally important for cell cycle regulation and tumor suppression, yet the underlying mechanisms are incompletely understood. Here we show that the PARK2 tumor suppressor mediates this coordination. The PARK2 E3 ubiquitin ligase coordinately controls the stability of both cyclin D and cyclin E. Analysis of approximately 5,000 tumor genomes shows that PARK2 is a very frequently deleted gene in human cancer and uncovers a striking pattern of mutual exclusivity between PARK2 deletion and amplification of CCND1, CCNE1 or CDK4-implicating these genes in a common pathway. Inactivation of PARK2 results in the accumulation of cyclin D and acceleration of cell cycle progression. Furthermore, PARK2 is a component of a new class of cullin-RING-containing ubiquitin ligases targeting both cyclin D and cyclin E for degradation. Thus, PARK2 regulates cyclin-CDK complexes, as does the CDK inhibitor p16, but acts as a master regulator of the stability of G1/S cyclins.
Asunto(s)
Ciclo Celular , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fase G1 , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Supresores de Tumor , Genoma Humano , Genómica , Humanos , Insectos , ARN Interferente Pequeño/metabolismo , Fase SRESUMEN
BACKGROUND: Telomeric 3' overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure. METHODS: We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels. RESULTS: Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo. CONCLUSION: This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.
Asunto(s)
Acridinas/farmacología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Acridinas/química , Animales , Antineoplásicos/química , Neoplasias Encefálicas , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cilios/efectos de los fármacos , Cilios/fisiología , Ensayos de Selección de Medicamentos Antitumorales , Células Endoteliales/efectos de los fármacos , Epéndimo/patología , G-Cuádruplex , Humanos , Concentración 50 Inhibidora , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ratas , Polimerasa Taq/antagonistas & inhibidores , Telomerasa/antagonistas & inhibidores , Telomerasa/química , Homeostasis del Telómero/efectos de los fármacosRESUMEN
Loss of neurofibromin 1 (NF1) leads to hyperactivation of RAS, which in turn signals through the RAF/MEK/ERK and phosphoinositide 3-kinase (PI3K)/mTOR pathways to regulate cell growth and survival. Because NF1-deficient acute myeloid leukemias are sensitive to MEK inhibitors, we investigated here whether NF1-deficient glioblastoma multiforme (GBM) would respond to MEK inhibition. In 19 GBM cell lines, we found that treatment with the clinically available MEK inhibitors PD0325901 or AZD6244 decreased levels of phospho-ERK, the downstream effector of MEK, regardless of NF1 status. However, growth inhibition occurred only in a subset of NF1-deficient cells, in association with decreased levels of cyclin D1, increased levels of p27, and G1 arrest. As a single agent, PD0325901 suppressed the growth of NF1-deficient, MEK inhibitor-sensitive cells in vivo as well. Mechanistically, NF1-deficient, MEK inhibitor-sensitive cells were dependent upon the RAF/MEK/ERK pathway for growth and did not activate the PI3K pathway as a mechanism of acquired resistance. Importantly, NF1-deficient cells intrinsically resistant to MEK inhibition were sensitized by the addition of the dual PI3K/mTOR inhibitor PI-103. Taken together, our findings indicate that a subset of NF1-deficient GBMs may respond to MEK inhibitors currently being tested in clinical trials.