Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Blood ; 144(9): 977-987, 2024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-38861668

RESUMO

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.


Assuntos
Vetores Genéticos , Imunoterapia Adotiva , Lentivirus , Receptores de Antígenos Quiméricos , Linfócitos T , Animais , Lentivirus/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Humanos , Imunoterapia Adotiva/métodos , Ligantes , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução Genética , Antígenos CD20/imunologia , Antígenos CD20/genética , Ativação Linfocitária
2.
Mod Pathol ; 32(12): 1734-1743, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31273314

RESUMO

The hyalinizing trabecular adenoma/tumor is a rare and poorly characterized follicular-derived thyroid neoplasm recently shown to harbor recurrent PAX8-GLIS1 or PAX8-GLIS3 gene fusions. Here we sought to define the repertoire of genetic alterations of hyalinizing trabecular tumors, and whether PAX8-GLIS3 fusions are pathognomonic for hyalinizing trabecular tumors. A discovery series of eight hyalinizing trabecular tumors was subjected to RNA-sequencing (n = 8), whole-exome sequencing (n = 3) or targeted massively parallel sequencing (n = 5). No recurrent somatic mutations or copy number alterations were identified in hyalinizing trabecular tumor, whereas RNA-sequencing revealed the presence of a recurrent genetic rearrangement involving PAX8 (2q14.1) and GLIS3 (9p24.2) genes in all cases. In this in-frame fusion gene, which comprised exons 1-2 of PAX8 and exons 3-11 of GLIS3, GLIS3 is likely placed under the regulation of PAX8. Reverse transcription RT-PCR and/or fluorescence in situ hybridization analyses of a validation series of 26 hyalinizing trabecular tumors revealed that the PAX8-GLIS3 gene fusion was present in all hyalinizing trabecular tumors (100%). No GLIS1 rearrangements were identified. Conversely, no PAX8-GLIS3 gene fusions were detected in a cohort of 237 control thyroid neoplasms, including 15 trabecular thyroid lesions highly resembling hyalinizing trabecular tumor from a morphological standpoint, as well as trabecular/solid follicular adenomas, solid/trabecular variants of papillary carcinoma, and Hurthle cell adenomas or carcinomas. Our data provide evidence to suggest that the PAX8-GLIS3 fusion is pathognomonic for hyalinizing trabecular tumors, and that the presence of the PAX8-GLIS3 fusion in thyroid neoplasms may be used as an ancillary marker for the diagnosis of hyalinizing trabecular tumor, thereby avoiding overtreatment in case of misdiagnoses with apparently similar malignant tumors.


Assuntos
Proteínas de Ligação a DNA/genética , Fator de Transcrição PAX8/genética , Proteínas Repressoras/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Transativadores/genética , Humanos , Proteínas de Fusão Oncogênica/genética
3.
Nat Med ; 30(5): 1406-1415, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38745011

RESUMO

GRN mutations cause progranulin haploinsufficiency, which eventually leads to frontotemporal dementia (FTD-GRN). PR006 is an investigational gene therapy delivering the granulin gene (GRN) using an adeno-associated virus serotype 9 (AAV9) vector. In non-clinical studies, PR006 transduced neurons derived from induced pluripotent stem cells of patients with FTD-GRN, resulted in progranulin expression and improvement of lipofuscin, lysosomal and neuroinflammation pathologies in Grn-knockout mice, and was well tolerated except for minimal, asymptomatic dorsal root ganglionopathy in non-human primates. We initiated a first-in-human phase 1/2 open-label trial. Here we report results of a pre-specified interim analysis triggered with the last treated patient of the low-dose cohort (n = 6) reaching the 12-month follow-up timepoint. We also include preliminary data from the mid-dose cohort (n = 7). Primary endpoints were safety, immunogenicity and change in progranulin levels in cerebrospinal fluid (CSF) and blood. Secondary endpoints were Clinical Dementia Rating (CDR) plus National Alzheimer's Disease Coordinating Center (NACC) Frontotemporal Lobar Degeneration (FTLD) rating scale and levels of neurofilament light chain (NfL). One-time administration of PR006 into the cisterna magna was generally safe and well tolerated. All patients developed treatment-emergent anti-AAV9 antibodies in the CSF, but none developed anti-progranulin antibodies. CSF pleocytosis was the most common PR006-related adverse event. Twelve serious adverse events occurred, mostly unrelated to PR006. Deep vein thrombosis developed in three patients. There was one death (unrelated) occurring 18 months after treatment. CSF progranulin increased after PR006 treatment in all patients; blood progranulin increased in most patients but only transiently. NfL levels transiently increased after PR006 treatment, likely reflecting dorsal root ganglia toxicity. Progression rates, based on the CDR scale, were within the broad ranges reported for patients with FTD. These data provide preliminary insights into the safety and bioactivity of PR006. Longer follow-up and additional studies are needed to confirm the safety and potential efficacy of PR006. ClinicalTrials.gov identifier: NCT04408625 .


Assuntos
Dependovirus , Demência Frontotemporal , Terapia Genética , Progranulinas , Humanos , Demência Frontotemporal/genética , Demência Frontotemporal/terapia , Demência Frontotemporal/líquido cefalorraquidiano , Progranulinas/genética , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Dependovirus/genética , Pessoa de Meia-Idade , Feminino , Masculino , Idoso , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/líquido cefalorraquidiano , Vetores Genéticos , Animais , Resultado do Tratamento , Pesquisa Translacional Biomédica , Camundongos , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Proteínas de Neurofilamentos/sangue
4.
J Immunother Cancer ; 11(3)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36918221

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.


Assuntos
Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Animais , Cães , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos de Linfócitos T , Leucócitos Mononucleares , Distribuição Tecidual , Engenharia Celular/métodos
5.
Breast Cancer Res ; 12(5): R84, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20946630

RESUMO

INTRODUCTION: 17-allyamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is currently in clinical trials in breast cancer. However, 17-AAG treatment often results in inhibition of tumor growth rather than shrinkage, making detection of response a challenge. Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) are noninvasive imaging methods than can be used to monitor metabolic biomarkers of drug-target modulation. This study set out to examine the MRS-detectable metabolic consequences of Hsp90 inhibition in a breast cancer model. METHODS: MCF-7 breast cancer cells were investigated, and MRS studies were performed both on live cells and on cell extracts. (31)P and (1)H MRS were used to determine total cellular metabolite concentrations and (13)C MRS was used to probe the metabolism of [1,2-(13)C]-choline. To explain the MRS metabolic findings, microarray and RT-PCR were used to analyze gene expression, and in vitro activity assays were performed to determine changes in enzymatic activity following 17-AAG treatment. RESULTS: Treatment of MCF-7 cells with 17-AAG for 48 hours caused a significant increase in intracellular levels of choline (to 266 ± 18% of control, P = 0.05) and phosphocholine (PC; to 181 ± 10% of control, P = 0.001) associated with an increase in expression of choline transporter SLC44A1 and an elevation in the de novo synthesis of PC. We also detected an increase in intracellular levels of glycerophosphocholine (GPC; to 176 ± 38% of control, P = 0.03) associated with an increase in PLA2 expression and activity. CONCLUSIONS: This study determined that in the MCF-7 breast cancer model inhibition of Hsp90 by 17-AAG results in a significant MRS-detectable increase in choline, PC and GPC, which is likely due to an increase in choline transport into the cell and phospholipase activation. (1)H MRSI can be used in the clinical setting to detect levels of total choline-containing metabolite (t-Cho, composed of intracellular choline, PC and GPC). As Hsp90 inhibitors enter routine clinical use, t-Cho could thus provide an easily detectable, noninvasive metabolic biomarker of Hsp90 inhibition in breast cancer patients.


Assuntos
Antígenos CD/biossíntese , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Colina/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Lactamas Macrocíclicas/farmacologia , Proteínas de Transporte de Cátions Orgânicos/biossíntese , Fosfolipases A2/biossíntese , Transporte Biológico/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Glicerilfosforilcolina/metabolismo , Humanos , Células MCF-7 , Espectroscopia de Ressonância Magnética , Proteínas de Membrana Transportadoras/metabolismo , Fosforilcolina/metabolismo
6.
Nat Commun ; 9(1): 3533, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30166553

RESUMO

Granular cell tumors (GCTs) are rare tumors that can arise in multiple anatomical locations, and are characterized by abundant intracytoplasmic granules. The genetic drivers of GCTs are currently unknown. Here, we apply whole-exome sequencing and targeted sequencing analysis to reveal mutually exclusive, clonal, inactivating somatic mutations in the endosomal pH regulators ATP6AP1 or ATP6AP2 in 72% of GCTs. Silencing of these genes in vitro results in impaired vesicle acidification, redistribution of endosomal compartments, and accumulation of intracytoplasmic granules, recapitulating the cardinal phenotypic characteristics of GCTs and providing a novel genotypic-phenotypic correlation. In addition, depletion of ATP6AP1 or ATP6AP2 results in the acquisition of oncogenic properties. Our results demonstrate that inactivating mutations of ATP6AP1 and ATP6AP2 are likely oncogenic drivers of GCTs and underpin the genesis of the intracytoplasmic granules that characterize them, providing a genetic link between endosomal pH regulation and tumorigenesis.


Assuntos
Tumor de Células Granulares/genética , Mutação/genética , Receptores de Superfície Celular/genética , ATPases Vacuolares Próton-Translocadoras/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Exoma , Feminino , Citometria de Fluxo , Estudos de Associação Genética , Células HEK293 , Humanos , Masculino
7.
Nat Commun ; 9(1): 1816, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29739933

RESUMO

Adenomyoepithelioma of the breast is a rare tumor characterized by epithelial-myoepithelial differentiation, whose genetic underpinning is largely unknown. Here we show through whole-exome and targeted massively parallel sequencing analysis that whilst estrogen receptor (ER)-positive adenomyoepitheliomas display PIK3CA or AKT1 activating mutations, ER-negative adenomyoepitheliomas harbor highly recurrent codon Q61 HRAS hotspot mutations, which co-occur with PIK3CA or PIK3R1 mutations. In two- and three-dimensional cell culture models, forced expression of HRASQ61R in non-malignant ER-negative breast epithelial cells with or without a PIK3CAH1047R somatic knock-in results in transformation and the acquisition of the cardinal features of adenomyoepitheliomas, including the expression of myoepithelial markers, a reduction in E-cadherin expression, and an increase in AKT signaling. Our results demonstrate that adenomyoepitheliomas are genetically heterogeneous, and qualify mutations in HRAS, a gene whose mutations are vanishingly rare in common-type breast cancers, as likely drivers of ER-negative adenomyoepitheliomas.


Assuntos
Adenomioepitelioma/genética , Adenomioepitelioma/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Genes ras , Mutação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Adenomioepitelioma/enzimologia , Biomarcadores Tumorais/genética , Mama/citologia , Mama/metabolismo , Neoplasias da Mama/enzimologia , Caderinas/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Progressão da Doença , Ativação Enzimática , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Humanos , Receptores de Estrogênio/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais , Sequenciamento do Exoma
8.
Cell Rep ; 8(4): 1077-92, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-25131208

RESUMO

γ-Secretase is composed of four proteins that are obligatory for protease activity: presenilin, nicastrin, Aph1, and Pen-2. Despite the progress toward understanding the function of these individual subunits, there is no information available pertaining to the modulation of γ-secretase in response to environmental changes in cells. Here, we show that hypoxia upregulates γ-secretase activity through a direct interaction with Hif-1α, revealing an unconventional function for Hif-1α as an enzyme subunit, which is distinct from its canonical role as a transcription factor. Moreover, hypoxia-induced cell invasion and metastasis are alleviated by either γ-secretase inhibitors or a dominant-negative Notch coactivator, indicating that γ-secretase/Notch signaling plays an essential role in controlling these cellular processes. The present study reveals a mechanism in which γ-secretase can achieve temporal control through conditional interactions with regulatory proteins, such as Hif-1α, under select physiological and pathological conditions.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Neoplasias da Mama/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Neoplasias Pulmonares/metabolismo , Receptores Notch/metabolismo , Animais , Neoplasias da Mama/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Ativação Enzimática , Feminino , Humanos , Neoplasias Pulmonares/secundário , Camundongos Nus , Transplante de Neoplasias , Ligação Proteica , Transdução de Sinais , Regulação para Cima
9.
PLoS One ; 8(4): e62610, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23626839

RESUMO

Histone deacetylase (HDAC) inhibitors have emerged as effective antineoplastic agents in the clinic. Studies from our lab and others have reported that magnetic resonance spectroscopy (MRS)-detectable phosphocholine (PC) is elevated following SAHA treatment, providing a potential noninvasive biomarker of response. Typically, elevated PC is associated with cancer while a decrease in PC accompanies response to antineoplastic treatment. The goal of this study was therefore to elucidate the underlying biochemical mechanism by which HDAC inhibition leads to elevated PC. We investigated the effect of SAHA on MCF-7 breast cancer cells using (13)C MRS to monitor [1,2-(13)C] choline uptake and phosphorylation to PC. We found that PC synthesis was significantly higher in treated cells, representing 154±19% of control. This was within standard deviation of the increase in total PC levels detected by (31)P MRS (129±7% of control). Furthermore, cellular choline kinase activity was elevated (177±31%), while cytidylyltransferase activity was unchanged. Expression of the intermediate-affinity choline transporter SLC44A1 and choline kinase α increased (144% and 161%, respectively) relative to control, as determined by mRNA microarray analysis with protein-level confirmation by Western blotting. Taken together, our findings indicate that the increase in PC levels following SAHA treatment results from its elevated synthesis. Additionally, the concentration of glycerophosphocholine (GPC) increased significantly with treatment to 210±45%. This is likely due to the upregulated expression of several phospholipase A2 (PLA2) isoforms, resulting in increased PLA2 activity (162±18%) in SAHA-treated cells. Importantly, the levels of total choline (tCho)-containing metabolites, comprised of choline, PC and GPC, are readily detectable clinically using (1)H MRS. Our findings thus provide an important step in validating clinically translatable non-invasive imaging methods for follow-up diagnostics of HDAC inhibitor treatment.


Assuntos
Neoplasias da Mama/metabolismo , Colina/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Fosforilcolina/metabolismo , Neoplasias da Mama/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicerilfosforilcolina/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Células MCF-7 , Espectroscopia de Ressonância Magnética , Vorinostat
10.
Cancer Res ; 70(4): 1296-305, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20145128

RESUMO

Numerous mechanism-based anticancer drugs that target the phosphatidylinositol 3-kinase (PI3K) pathway are in clinical trials. However, it remains challenging to assess responses by traditional imaging methods. Here, we show for the first time the efficacy of hyperpolarized (13)C magnetic resonance spectroscopy (MRS) in detecting the effect of PI3K inhibition by monitoring hyperpolarized [1-(13)C]lactate levels produced from hyperpolarized [1-(13)C]pyruvate through lactate dehydrogenase (LDH) activity. In GS-2 glioblastoma cells, PI3K inhibition by LY294002 or everolimus caused hyperpolarized lactate to drop to 42 +/- 12% and to 76 +/- 5%, respectively. In MDA-MB-231 breast cancer cells, hyperpolarized lactate dropped to 71 +/- 15% after treatment with LY294002. These reductions were correlated with reductions in LDH activity to 48 +/- 4%, 63 +/- 4%, and 69 +/- 12%, respectively, and were associated with a drop in levels of LDHA mRNA and LDHA and hypoxia-inducible factor-1alpha proteins. Supporting these findings, tumor growth inhibition achieved by everolimus in murine GS-2 xenografts was associated with a drop in the hyperpolarized lactate-to-pyruvate ratio detected by in vivo MRS imaging, whereas an increase in this ratio occurred with tumor growth in control animals. Taken together, our findings illustrate the application of hyperpolarized (13)C MRS of pyruvate to monitor alterations in LDHA activity and expression caused by PI3K pathway inhibition, showing the potential of this method for noninvasive imaging of drug target modulation.


Assuntos
Isótopos de Carbono , Inibidores Enzimáticos/uso terapêutico , Espectroscopia de Ressonância Magnética/métodos , Monitorização Fisiológica/métodos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Isótopos de Carbono/farmacocinética , Cromonas/farmacologia , Cromonas/uso terapêutico , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Everolimo , Glioblastoma/diagnóstico , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Ácido Láctico/farmacocinética , Camundongos , Camundongos Nus , Morfolinas/farmacologia , Morfolinas/uso terapêutico , Neoplasias/patologia , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA