RESUMEN
The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Factor B de Elongación Transcripcional Positiva/metabolismo , Proteínas Represoras/metabolismo , Elongación de la Transcripción Genética/efectos de los fármacos , Factores de Elongación Transcripcional/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Drosophila , Femenino , Células HCT116 , Células HEK293 , Respuesta al Choque Térmico , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Mutations and translocations within the COMPASS (complex of proteins associated with Set1) family of histone lysine methyltransferases are associated with a large number of human diseases, including cancer. Here we report that SET1B/COMPASS, which is essential for cell survival, surprisingly has a cytoplasmic variant. SET1B, but not its SET domain, is critical for maintaining cell viability, indicating a novel catalytic-independent role of SET1B/COMPASS. Loss of SET1B or its unique cytoplasmic-interacting protein, BOD1, leads to up-regulation of expression of numerous genes modulating fatty acid metabolism, including ADIPOR1 (adiponectin receptor 1), COX7C, SDC4, and COQ7 Our detailed molecular studies identify ADIPOR1 signaling, which is inactivated in both obesity and human cancers, as a key target of SET1B/COMPASS. Collectively, our study reveals a cytoplasmic function for a member of the COMPASS family, which could be harnessed for therapeutic regulation of signaling in human diseases, including cancer.
Asunto(s)
Sistema Enzimático del Citocromo P-450/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Citoplasma/enzimología , Citoplasma/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Dominios PR-SET , Subunidades de Proteína/metabolismo , Receptores de Adiponectina/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/etiología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Diffuse midline glioma (DMG) is a type of lethal brain tumor that develops mainly in children. The majority of DMG harbor the K27M mutation in histone H3. Oligodendrocyte progenitor cells (OPCs) in the brainstem are candidate cells-of-origin for DMG, yet there is no genetically engineered mouse model of DMG initiated in OPCs. Here, we used the RCAS/Tv-a avian retroviral system to generate DMG in Olig2-expressing progenitors and Nestin-expressing progenitors in the neonatal mouse brainstem. PDGF-A or PDGF-B overexpression, along with p53 deletion, resulted in gliomas in both models. Exogenous overexpression of H3.3K27M had a significant effect on tumor latency and tumor cell proliferation when compared with H3.3WT in Nestin+ cells but not in Olig2+ cells. Further, the fraction of H3.3K27M-positive cells was significantly lower in DMGs initiated in Olig2+ cells relative to Nestin+ cells, both in PDGF-A and PDGF-B-driven models, suggesting that the requirement for H3.3K27M is reduced when tumorigenesis is initiated in Olig2+ cells. RNA-sequencing analysis revealed that the differentially expressed genes in H3.3K27M tumors were non-overlapping between Olig2;PDGF-B, Olig2;PDGF-A, and Nestin;PDGF-A models. GSEA analysis of PDGFA tumors confirmed that the transcriptomal effects of H3.3K27M are cell-of-origin dependent with H3.3K27M promoting epithelial-to-mesenchymal transition (EMT) and angiogenesis when Olig2 marks the cell-of-origin and inhibiting EMT and angiogenesis when Nestin marks the cell-of-origin. We did observe some overlap with H3.3K27M promoting negative enrichment of TNFA_Signaling_Via_NFKB in both models. Our study suggests that the tumorigenic effects of H3.3K27M are cell-of-origin dependent, with H3.3K27M being more oncogenic in Nestin+ cells than Olig2+ cells.
Asunto(s)
Neoplasias Encefálicas , Glioma , Células Precursoras de Oligodendrocitos , Animales , Neoplasias Encefálicas/patología , Modelos Animales de Enfermedad , Glioma/patología , Histonas , Ratones , Mutación/genética , Nestina/genética , Células Precursoras de Oligodendrocitos/patologíaRESUMEN
Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60% of high-grade pediatric glioma cases. The median survival of this group of patients after diagnosis is â¼1 yr. Here we show that the levels of H3K27 di- and trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient samples due to the expression of the H3.3K27M mutant allele. Remarkably, we also observed that H3K27me3 and Ezh2 (the catalytic subunit of H3K27 methyltransferase) at chromatin are dramatically increased locally at hundreds of gene loci in H3.3K27M patient cells. Moreover, the gain of H3K27me3 and Ezh2 at gene promoters alters the expression of genes that are associated with various cancer pathways. These results indicate that H3.3K27M mutation reprograms epigenetic landscape and gene expression, which may drive tumorigenesis.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Glioma/genética , Histonas/genética , Histonas/metabolismo , Mutación , Línea Celular Tumoral , Genoma Humano/genética , Glioma/fisiopatología , Humanos , Metilación , Células Tumorales CultivadasRESUMEN
Medulloblastoma (MB) is the most common pediatric CNS malignancy. We identify EAG2 as an overexpressed potassium channel in MBs across different molecular and histological subgroups. EAG2 knockdown not only impairs MB cell growth in vitro, but also reduces tumor burden in vivo and enhances survival in xenograft studies. Mechanistically, we demonstrate that EAG2 protein is confined intracellularly during interphase but is enriched in the plasma membrane during late G2 phase and mitosis. Disruption of EAG2 expression results in G2 arrest and mitotic catastrophe associated with failure of premitotic cytoplasmic condensation. While the tumor suppression function of EAG2 knockdown is independent of p53 activation, DNA damage checkpoint activation, or changes in the AKT pathway, this defective cell volume control is specifically associated with hyperactivation of the p38 MAPK pathway. Inhibition of the p38 pathway significantly rescues the growth defect and G2 arrest. Strikingly, ectopic membrane expression of EAG2 in cells at interphase results in cell volume reduction and mitotic-like morphology. Our study establishes the functional significance of EAG2 in promoting MB tumor progression via regulating cell volume dynamics, the perturbation of which activates the tumor suppressor p38 MAPK pathway, and provides clinical relevance for targeting this ion channel in human MBs.
Asunto(s)
Tamaño de la Célula , Canales de Potasio Éter-A-Go-Go/metabolismo , Meduloblastoma/fisiopatología , Mitosis , Animales , Células COS , Puntos de Control del Ciclo Celular/genética , Proliferación Celular , Células Cultivadas , Chlorocebus aethiops , Activación Enzimática/genética , Canales de Potasio Éter-A-Go-Go/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Meduloblastoma/mortalidad , Ratones , Análisis de SupervivenciaRESUMEN
Despite the advances in imaging, surgery and radiotherapy, the majority of patients with brainstem gliomas die within 2 years after initial diagnosis. Factors that contribute to the dismal prognosis of these patients include the infiltrative nature and anatomic location in an eloquent area of the brain, which prevents total surgical resection and the presence of the blood-brain barrier (BBB), which reduces the distribution of systemically administered agents. The development of new therapeutic approaches which can circumvent the BBB is a potential path to improve outcomes for these children. Convection-enhanced delivery (CED) and intranasal delivery (IND) are strategies that permit direct drug delivery into the central nervous system and are an alternative to intravenous injection (IV). We treated rats bearing human brainstem tumor xenografts with nanoliposomal irinotecan (CPT-11) using CED, IND, and IV. A single treatment of CED irinotecan had a similar effect on overall survival as multiple treatments by IV route. IND CPT-11 showed significantly increased survival of animals with brainstem tumors, and demonstrated the promise of this non-invasive approach of drug delivery bypassing the BBB when combined with nanoliposomal chemotherapy. Our results indicated that using CED and IND of nanoliposomal therapy increase likelihood of practical therapeutic approach for the treatment of brainstem gliomas.
Asunto(s)
Neoplasias del Tronco Encefálico/tratamiento farmacológico , Irinotecán/administración & dosificación , Inhibidores de Topoisomerasa I/administración & dosificación , Administración Intranasal , Animales , Neoplasias del Tronco Encefálico/mortalidad , Línea Celular Tumoral , Convección , Portadores de Fármacos , Humanos , Irinotecán/farmacocinética , Liposomas , Masculino , Nanoestructuras , Ratas , Inhibidores de Topoisomerasa I/farmacocinética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BRAFV600E is a common finding in glioma (about 10-60% depending on histopathologic subclassification). BRAFV600E monotherapy shows modest preclinical efficacy against BRAFV600E gliomas and also induces adverse secondary skin malignancies. Here, we examine the molecular mechanism of intrinsic resistance to BRAFV600E inhibition in glioma. Furthermore, we investigate BRAFV600E/MEK combination therapy that overcomes intrinsic resistance to BRAFV600E inhibitor and also prevents BRAFV600E inhibitor induced secondary malignancies. Immunoblotting and Human Phospho-Receptor Tyrosine Kinase Array assays were used to interrogate MAPK pathway activation. The cellular effect of BRAFV600E and MEK inhibition was determined by WST-1 viability assay and cell cycle analysis. Flanked and orthotopic GBM mouse models were used to investigate the in vivo efficacy of BRAFV600E/MEK combination therapy and the effect on secondary malignancies. BRAFV600E inhibition leads to recovery of ERK phosphorylation. Combined BRAFV600E and MEK inhibition prevents reactivation of the MAPK signaling, which correlates with decreased cell viability and augmented cell cycle arrest. Similarly, mice bearing BRAFV600E glioma showed reduced tumor growth when treated with a combination of BRAFV600E and MEK inhibitor compared to BRAFV600E inhibition alone. Additional benefit of BRAFV600E/MEK inhibition was reflected by reduced cutaneous squamous-cell carcinoma (cSCC) growth (a surrogate for RAS-driven secondary maligancies). In glioma, recovery of MAPK signaling upon BRAF inhibition accounts for intrinsic resistance to BRAFV600E inhibitor. Combined BRAFV600E and MEK inhibition prevents rebound of MAPK activation, resulting in enhanced antitumor efficacy and also reduces the risk of secondary malignancy development.
Asunto(s)
Antineoplásicos/administración & dosificación , Glioma/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Benzamidas/administración & dosificación , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Difenilamina/administración & dosificación , Difenilamina/análogos & derivados , Modelos Animales de Enfermedad , Femenino , Glioma/tratamiento farmacológico , Glioma/genética , Humanos , Indoles/administración & dosificación , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Sulfonamidas/administración & dosificación , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Radiation (RT) is critical to the treatment of high-grade gliomas (HGGs) but cures remain elusive. The BRAF mutation V600E is critical to the pathogenesis of 10-20% of pediatric gliomas, and a small proportion of adult HGGs. Here we aim to determine whether PLX4720, a specific BRAF V600E inhibitor, enhances the activity of RT in human HGGs in vitro and in vivo. Patient-derived HGG lines harboring wild-type BRAF or BRAF V600E were assessed in vitro to determine IC50 values, cell cycle arrest, apoptosis and senescence and elucidate mechanisms of combinatorial activity. A BRAF V600E HGG intracranial xenograft mouse model was used to evaluate in vivo combinatorial efficacy of PLX4720+RT. Tumors were harvested for immunohistochemistry to quantify cell cycle arrest and apoptosis. RT+PLX4720 exhibited greater anti-tumor effects than either monotherapy in BRAF V600E but not in BRAF WT lines. In vitro studies showed increased Annexin V and decreased S phase cells in BRAF V600E gliomas treated with PLX4720+RT, but no significant changes in ß-galactosidase levels. In vivo, concurrent and sequential PLX4720+RT each significantly prolonged survival compared to monotherapies, in the BRAF V600E HGG model. Immunohistochemistry of in vivo tumors demonstrated that PLX4720+RT decreased Ki-67 and phospho-MAPK, and increased γH2AX and p21 compared to control mice. BRAF V600E inhibition enhances radiation-induced cytotoxicity in BRAF V600E-mutated HGGs, in vitro and in vivo, effects likely mediated by apoptosis and cell cycle, but not senescence. These studies provide the pre-clinical rationale for clinical trials of concurrent radiotherapy and BRAF V600E inhibitors.
Asunto(s)
Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/terapia , Quimioradioterapia , Rayos gamma , Indoles/farmacología , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Sulfonamidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Glioma , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Desnudos , Clasificación del Tumor , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although malignant astrocytomas are a leading cause of cancer-related death in children, rational therapeutic strategies are lacking. We previously identified activating mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) (BRAF(T1799A) encoding BRAF(V600E)) in association with homozygous cyclin-dependent kinase inhibitor 2A (CDKN2A, encoding p14ARF and p16Ink4a) deletions in pediatric infiltrative astrocytomas. Here we report that BRAF(V600E) expression in neural progenitors (NPs) is insufficient for tumorigenesis and increases NP cellular differentiation as well as apoptosis. In contrast, astrocytomas are readily generated from NPs with additional Ink4a-Arf deletion. The BRAF(V600E) inhibitor PLX4720 significantly increased survival of mice after intracranial transplant of genetically relevant murine or human astrocytoma cells. Moreover, combination therapy using PLX4720 plus the Cyclin-dependent kinase (CDK) 4/6-specific inhibitor PD0332991 further extended survival relative to either monotherapy. Our findings indicate a rational therapeutic strategy for treating a subset of pediatric astrocytomas with BRAF(V600E) mutation and CDKN2A deficiency.
Asunto(s)
Astrocitoma/tratamiento farmacológico , Inhibidor p16 de la Quinasa Dependiente de Ciclina/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Astrocitoma/genética , Astrocitoma/patología , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Niño , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Inmunohistoquímica , Indoles/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridinas/farmacología , Sulfonamidas/farmacologíaRESUMEN
BACKGROUND: GL261 cells are murine glioma cells that demonstrate proliferation, invasion, and angiogenesis when implanted in syngeneic C57BL/6 mice, providing a highly useful immunocompetent animal model of glioblastoma. Modification of tumor cells for luciferase expression enables non-invasive monitoring of orthotopic tumor growth, and has proven useful for studying glioblastoma response to novel therapeutics. However, tumor modification for luciferase has the potential for evoking host immune response against otherwise syngeneic tumor cells, thereby mitigating the tumor cells' value for tumor immunology and immunotherapy studies. METHODS: GL261 cells were infected with lentivirus containing a gene encoding firefly luciferase (GL261.luc). In vitro proliferation of parental (unmodified) GL261 and GL261.luc was measured on days 0, 1, 2, 4, and 7 following plating, and the expression of 82 mouse cytokines and chemokines were analyzed by RT-PCR array. Cell lines were also evaluated for differences in invasion and migration in modified Boyden chambers. GL261 and GL261.luc cells were then implanted intracranially in C57BL/6 mice, with GL261.luc tumor growth monitored by quantitative bioluminescence imaging, and all mice were followed for survival to compare relative malignancy of tumor cells. RESULTS: No difference in proliferation was indicated for GL261 vs. GL261.luc cells (p>0.05). Of the 82 genes examined by RT-PCR array, seven (9%) exhibited statistically significant change after luciferase modification. Of these, only three changed by greater than 2-fold: BMP-2, IL-13, and TGF-ß2. No difference in invasion (p=0.67) or migration (p=0.26) was evident between modified vs. unmodified cells. GL261.luc cell luminescence was detectable in the brains of C57BL/6 mice at day 5 post-implantation, and tumor bioluminescence increased exponentially to day 19. Median overall survival was 20.2 days versus 19.7 days for mice receiving implantation with GL261 and GL261.luc, respectively (p=0.62). Histopathologic analysis revealed no morphological difference between tumors, and immunohistochemical analysis showed no significant difference for staining of CD3, Ki67, or CD31 (p>0.05 for all). CONCLUSIONS: Luciferase expression in GL261 murine glioma cells does not affect GL261 proliferation, invasion, cytokine expression, or in vivo growth. Luciferase modification increases their utility for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.
Asunto(s)
Neoplasias Encefálicas/patología , Glioma/patología , Luciferasas/genética , Animales , Neoplasias Encefálicas/inmunología , División Celular , Línea Celular Tumoral , Citocinas/inmunología , Glioma/inmunología , RatonesRESUMEN
Diffuse midline glioma (DMG) H3K27-altered is one of the most malignant childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation antitumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extraterminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-Seq and the CUT&RUN (cleavage under targets and release using nuclease) analysis showed that BET bromodomain inhibitors regulated the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as potential radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.
Asunto(s)
Histonas , Tolerancia a Radiación , Humanos , Animales , Ratones , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Histonas/metabolismo , Histonas/genética , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Reparación del ADN/efectos de los fármacos , Glioma/radioterapia , Glioma/patología , Glioma/genética , Glioma/metabolismo , Glioma/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Fármacos Sensibilizantes a Radiaciones/farmacología , Factores de Transcripción/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Daño del ADN , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas que Contienen Bromodominio , ProteínasRESUMEN
High-grade gliomas (HGG) are deadly diseases for both adult and pediatric patients. Recently, it has been shown that neuronal activity promotes progression of multiple subgroups of HGG. However, epigenetic mechanisms that govern this process remain elusive. Here we report that the chromatin remodeler CHD2 regulates neuron-glioma interactions in diffuse midline glioma (DMG) characterized by onco-histone H3.1K27M. Depletion of CHD2 in H3.1K27M DMG cells compromises cell viability and neuron-to-glioma synaptic connections in vitro, neuron-induced proliferation of H3.1K27M DMG cells in vitro and in vivo, activity-dependent calcium transients in vivo, and extends the survival of H3.1K27M DMG-bearing mice. Mechanistically, CHD2 coordinates with the transcription factor FOSL1 to control the expression of axon-guidance and synaptic genes in H3.1K27M DMG cells. Together, our study reveals a mechanism whereby CHD2 controls the intrinsic gene program of the H3.1K27M DMG subtype, which in turn regulates the tumor growth-promoting interactions of glioma cells with neurons.
RESUMEN
Pediatric high-grade gliomas (pHGGs) are common malignant brain tumors without effective treatment and poor patient survival. Abnormal posttranslational modification at the histone H3 tail plays critical roles in tumor cell malignancy. We have previously shown that the trimethylation of lysine 4 at histone H3 (H3K4me3) plays a significant role in pediatric ependymoma malignancy and is associated with tumor therapeutic sensitivity. Here, we show that H3K4me3 and its methyltransferase WDR82 are elevated in pHGGs. A reduction in H3K4me3 by downregulating WDR82 decreases H3K4me3 promoter occupancy and the expression of genes associated with stem cell features, cell proliferation, the cell cycle, and DNA damage repair. A reduction in WDR82-mediated H3K4me3 increases the response of pediatric glioma cells to chemotherapy. These findings suggest that WDR82-mediated H3K4me3 is an important determinant of pediatric glioma malignancy and therapeutic response. This highlights the need for a more thorough understanding of the potential of WDR82 as an epigenetic target to increase therapeutic efficacy and improve the prognosis for children with malignant gliomas.
RESUMEN
Although tissue-resident memory T (TRM) cells specific for previously encountered pathogens have been characterized, the induction and recruitment of brain TRM cells following immune therapy has not been observed in the context of glioblastoma. Here, we show that T cells expressing fibrinogen-like 2 (FGL2)-specific single-chain variable fragments (T-αFGL2) can induce tumor-specific CD8+ TRM cells that prevent glioblastoma recurrence. These CD8+ TRM cells display a highly expanded T cell receptor repertoire distinct from that found in peripheral tissue. When adoptively transferred to the brains of either immunocompetent or T cell-deficient naïve mice, these CD8+ TRM cells reject glioma cells. Mechanistically, T-αFGL2 cell treatment increased the number of CD69+CD8+ brain-resident memory T cells in tumor-bearing mice via a CXCL9/10 and CXCR3 chemokine axis. These findings suggest that tumor-specific brain-resident CD8+ TRM cells may have promising implications for the prevention of brain tumor recurrence.
Asunto(s)
Linfocitos T CD8-positivos , Glioblastoma , Animales , Ratones , Encéfalo , Glioblastoma/terapia , Memoria Inmunológica , Células T de Memoria , Recurrencia Local de Neoplasia , Linfocitos T/inmunologíaRESUMEN
Epigenetic status-altering mutations in chromatin-modifying enzymes are a feature of human diseases, including many cancers. However, the functional outcomes and cellular dependencies arising from these mutations remain unresolved. In this study, we investigated cellular dependencies, or vulnerabilities, that arise when enhancer function is compromised by loss of the frequently mutated COMPASS family members MLL3 and MLL4. CRISPR dropout screens in MLL3/4-depleted mouse embryonic stem cells (mESCs) revealed synthetic lethality upon suppression of purine and pyrimidine nucleotide synthesis pathways. Consistently, we observed a shift in metabolic activity toward increased purine synthesis in MLL3/4-KO mESCs. These cells also exhibited enhanced sensitivity to the purine synthesis inhibitor lometrexol, which induced a unique gene expression signature. RNA-Seq identified the top MLL3/4 target genes coinciding with suppression of purine metabolism, and tandem mass tag proteomic profiling further confirmed upregulation of purine synthesis in MLL3/4-KO cells. Mechanistically, we demonstrated that compensation by MLL1/COMPASS was underlying these effects. Finally, we demonstrated that tumors with MLL3 and/or MLL4 mutations were highly sensitive to lometrexol in vitro and in vivo, both in culture and in animal models of cancer. Our results depicted a targetable metabolic dependency arising from epigenetic factor deficiency, providing molecular insight to inform therapy for cancers with epigenetic alterations secondary to MLL3/4 COMPASS dysfunction.
Asunto(s)
Neoplasias , Proteómica , Humanos , Animales , Ratones , N-Metiltransferasa de Histona-Lisina/genética , Mutación , Neoplasias/genética , Epigénesis GenéticaRESUMEN
Diffuse midline gliomas (DMGs) are lethal brain tumors characterized by p53-inactivating mutations and oncohistone H3.3K27M mutations that rewire the cellular response to genotoxic stress, which presents therapeutic opportunities. We used RCAS/tv-a retroviruses and Cre recombinase to inactivate p53 and induce K27M in the native H3f3a allele in a lineage- and spatially-directed manner, yielding primary mouse DMGs. Genetic or pharmacologic disruption of the DNA damage response kinase Ataxia-telangiectasia mutated (ATM) enhanced the efficacy of focal brain irradiation, extending mouse survival. This finding suggests that targeting ATM will enhance the efficacy of radiation therapy for p53-mutant DMG but not p53-wildtype DMG. We used spatial in situ transcriptomics and an allelic series of primary murine DMG models with different p53 mutations to identify transactivation-independent p53 activity as a key mediator of such radiosensitivity. These studies deeply profile a genetically faithful and versatile model of a lethal brain tumor to identify resistance mechanisms for a therapeutic strategy currently in clinical trials.
RESUMEN
The prognosis for diffuse infiltrating pontine gliomas (DIPG) remains extremely poor, with the majority of patients surviving less than 2 years. Here, we have adapted standard xenograft techniques to study glioma growth in the mouse brainstem, and have utilized the mouse model for studying a relevant therapeutic for treating DIPGs. bioluminescence imaging monitoring revealed a progressive increase in signal following the injection of either of two tumor cell types into the brainstem. Mice with orthotopic GS2 tumors, and receiving a single 100 mg/kg dose of temozolomide showed a lengthy period of decreased tumor luminescence, with substantially increased survival relative to untreated mice (P < 0.001). A small molecule inhibitor that targets cdk4/6 was used to test AM-38 brainstem xenograft response to treatment. Drug treatment resulted in delayed tumor growth, and significantly extended survival. Our results demonstrate the feasibility of using an orthotopic brainstem tumor model in athymic mice, and for application to testing therapeutic agents in treating DIPG.
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Antineoplásicos/uso terapéutico , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Puente/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Neoplasias del Tronco Encefálico/patología , Caspasa 3/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Dacarbazina/análogos & derivados , Dacarbazina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sustancias Luminiscentes , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias/métodos , Piperazinas/uso terapéutico , Piridinas/uso terapéutico , Proteína de Retinoblastoma/metabolismo , Temozolomida , Factores de Tiempo , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Diffuse intrinsic pontine gliomas arise almost exclusively in children, and despite advances in treatment, the majority of patients die within 2 years after initial diagnosis. Because of their infiltrative nature and anatomic location in an eloquent area of the brain, most pontine gliomas are treated without a surgical biopsy. The corresponding lack of tissue samples has resulted in a limited understanding of the underlying genetic and molecular biologic abnormalities associated with pontine gliomas, and is a substantial obstacle for the preclinical testing of targeted therapeutic agents for these tumors. We have established a human glioma cell line that originated from surgical biopsy performed on a patient with a pontine glioma. To insure sustainable in vitro propagation, tumor cells were modified with hTERT (human telomerase ribonucleoprotein reverse transcriptase), and with a luciferase reporter to enable non-invasive bioluminescence imaging. The hTERT modified cells are tumorigenic in athymic rodents, and produce brainstem tumors that recapitulate the infiltrative growth of brainstem gliomas in patients.
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Biomarcadores de Tumor/genética , Neoplasias del Tronco Encefálico/patología , Glioma/patología , Puente , Animales , Biomarcadores de Tumor/metabolismo , Western Blotting , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/terapia , Niño , Femenino , Perfilación de la Expresión Génica , Glioma/genética , Glioma/terapia , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Ratas , Ratas Desnudas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Small cell lung cancer (SCLC), accounting for around 13% of all lung cancers, often results in rapid tumor growth, early metastasis, and acquired therapeutic resistance. The POU class 2 homeobox 3 (POU2F3) is a master regulator of tuft cell identity and defines the SCLC-P subtype that lacks the neuroendocrine markers. Here, we have identified a previously uncharacterized protein, C11orf53, which is coexpressed with POU2F3 in both SCLC cell lines and patient samples. Mechanistically, C11orf53 directly interacts with POU2F3 and is recruited to chromatin by POU2F3. Depletion of C11orf53 reduced enhancer H3K27ac levels and chromatin accessibility, resulting in a reduction of POU2F3-dependent gene expression. On the basis of the molecular function of C11orf53, we renamed it as "POU Class 2 Homeobox Associating Factor 2" (POU2AF2). In summary, our study has identified a new coactivator of POU2F3 and sheds light on the therapeutic potential of targeting POU2AF2/POU2F3 heterodimer in human SCLC.
Asunto(s)
Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Línea Celular Tumoral , Cromatina/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Factores de Transcripción de Octámeros/genética , Factores de Transcripción de Octámeros/metabolismo , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
OBJECTIVE: Diffuse midline gliomas, including diffuse intrinsic pontine gliomas (DIPGs), are among the most malignant and devastating childhood brain cancers. Despite aggressive treatment, nearly all children with these tumors succumb to their disease within 2 years of diagnosis. Due to the anatomical location of the tumors within the pons, surgery is not a treatment option, and distribution of most systematically administered drugs is limited by the blood-brain barrier (BBB). New drug delivery systems that bypass the BBB are desperately needed to improve outcomes of DIPG patients. Intranasal delivery (IND) is a practical and noninvasive drug delivery system that bypasses the BBB and delivers the drugs to the brain through the olfactory and trigeminal neural pathways. In this study, the authors evaluated the efficacy of nanoliposomal (LS) irinotecan (CPT-11) and an active metabolite of CPT-11, 7-ethyl-10-hydroxycamptothecin (SN-38), using IND in DIPG patient-derived xenograft models. METHODS: In vitro responses to LS-CPT-11 and LS-SN-38 in DIPG cells were evaluated with cell viability, colony formation, and apoptosis assays. The cellular uptakes of rhodamine-PE (Rhod)-labeled LS-CPT-11 and LS-SN-38 were analyzed with fluorescence microscopy. Mice bearing DIPG patient-derived xenografts were treated with IND of LS-control (empty liposome), LS-CPT-11, or LS-SN-38 by IND for 4 weeks. In vivo responses were measured for tumor growth by serial bioluminescence imaging and animal subject survival. The concentration of SN-38 in the brainstem tumor administered by IND was determined by liquid chromatography-mass spectrometry (LC-MS). Immunohistochemical analyses of the proliferative and apoptotic responses of in vivo tumor cells were performed with Ki-67 and TUNEL staining. RESULTS: LS-SN-38 inhibited DIPG cell growth and colony formation and increased apoptosis, outperforming LS-CPT-11. Rhod-labeled LS-SN-38 showed intracellular fluorescence signals beginning at 30 minutes and peaking at 24 hours following treatment. LC-MS analysis revealed an SN-38 concentration in the brainstem tumor of 0.66 ± 0.25 ng/ml (5.43% ± 0.31% of serum concentration). IND of LS-SN-38 delayed tumor growth and significantly prolonged animal survival compared with IND of LS-control (p < 0.0001) and LS-CPT-11 (p = 0.003). IND of LS-SN-38 increased the number of TUNEL-positive cells and decreased the Ki-67-positive cells in the brainstem tumor. CONCLUSIONS: This study demonstrates that IND of LS-SN-38 bypasses the BBB and enables efficient and noninvasive drug delivery to the brainstem tumor, providing a promising therapeutic approach for treating DIPG.