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1.
Blood ; 139(11): 1670-1683, 2022 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-34871371

RESUMEN

Pediatric and young adult (YA) patients with acute myeloid leukemia (AML) who relapse after allogeneic hematopoietic cell transplantation (HCT) have an extremely poor prognosis. Standard salvage chemotherapy and donor lymphocyte infusions (DLIs) have little curative potential. Previous studies showed that natural killer (NK) cells can be stimulated ex vivo with interleukin-12 (IL-12), -15, and -18 to generate memory-like (ML) NK cells with enhanced antileukemia responses. We treated 9 pediatric/YA patients with post-HCT relapsed AML with donor ML NK cells in a phase 1 trial. Patients received fludarabine, cytarabine, and filgrastim followed 2 weeks later by infusion of donor lymphocytes and ML NK cells from the original HCT donor. ML NK cells were successfully generated from haploidentical and matched-related and -unrelated donors. After infusion, donor-derived ML NK cells expanded and maintained an ML multidimensional mass cytometry phenotype for >3 months. Furthermore, ML NK cells exhibited persistent functional responses as evidenced by leukemia-triggered interferon-γ production. After DLI and ML NK cell adoptive transfer, 4 of 8 evaluable patients achieved complete remission at day 28. Two patients maintained a durable remission for >3 months, with 1 patient in remission for >2 years. No significant toxicity was experienced. This study demonstrates that, in a compatible post-HCT immune environment, donor ML NK cells robustly expand and persist with potent antileukemic activity in the absence of exogenous cytokines. ML NK cells in combination with DLI present a novel immunotherapy platform for AML that has relapsed after allogeneic HCT. This trial was registered at https://clinicaltrials.gov as #NCT03068819.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Niño , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Células Asesinas Naturales , Leucemia Mieloide Aguda/terapia , Trasplante Homólogo , Donante no Emparentado
3.
Proc Natl Acad Sci U S A ; 117(6): 3123-3134, 2020 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-31996479

RESUMEN

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a-/- bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a-/- hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a-/- mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic "state" created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.


Asunto(s)
Células de la Médula Ósea/metabolismo , ADN (Citosina-5-)-Metiltransferasas , Metilación de ADN/genética , Expresión Génica/genética , Animales , Trasplante de Médula Ósea , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Hematopoyesis/genética , Humanos , Ratones , Ratones Transgénicos , Mutación/genética
4.
N Engl J Med ; 379(24): 2330-2341, 2018 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-30380364

RESUMEN

BACKGROUND: As consolidation therapy for acute myeloid leukemia (AML), allogeneic hematopoietic stem-cell transplantation provides a benefit in part by means of an immune-mediated graft-versus-leukemia effect. We hypothesized that the immune-mediated selective pressure imposed by allogeneic transplantation may cause distinct patterns of tumor evolution in relapsed disease. METHODS: We performed enhanced exome sequencing on paired samples obtained at initial presentation with AML and at relapse from 15 patients who had a relapse after hematopoietic stem-cell transplantation (with transplants from an HLA-matched sibling, HLA-matched unrelated donor, or HLA-mismatched unrelated donor) and from 20 patients who had a relapse after chemotherapy. We performed RNA sequencing and flow cytometry on a subgroup of these samples and on additional samples for validation. RESULTS: On exome sequencing, the spectrum of gained and lost mutations observed with relapse after transplantation was similar to the spectrum observed with relapse after chemotherapy. Specifically, relapse after transplantation was not associated with the acquisition of previously unknown AML-specific mutations or structural variations in immune-related genes. In contrast, RNA sequencing of samples obtained at relapse after transplantation revealed dysregulation of pathways involved in adaptive and innate immunity, including down-regulation of major histocompatibility complex (MHC) class II genes ( HLA-DPA1, HLA-DPB1, HLA-DQB1, and HLA-DRB1) to levels that were 3 to 12 times lower than the levels seen in paired samples obtained at presentation. Flow cytometry and immunohistochemical analysis confirmed decreased expression of MHC class II at relapse in 17 of 34 patients who had a relapse after transplantation. Evidence suggested that interferon-γ treatment could rapidly reverse this phenotype in AML blasts in vitro. CONCLUSIONS: AML relapse after transplantation was not associated with the acquisition of relapse-specific mutations in immune-related genes. However, it was associated with dysregulation of pathways that may influence immune function, including down-regulation of MHC class II genes, which are involved in antigen presentation. These epigenetic changes may be reversible with appropriate therapy. (Funded by the National Cancer Institute and others.).


Asunto(s)
Genes MHC Clase II/fisiología , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Mutación , Adolescente , Adulto , Anciano , Regulación hacia Abajo , Epigénesis Genética , Femenino , Citometría de Flujo , Humanos , Inmunidad/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , ARN Neoplásico/análisis , Recurrencia , Análisis de Secuencia de ARN , Linfocitos T/inmunología , Trasplante Homólogo , Secuenciación del Exoma
5.
N Engl J Med ; 375(21): 2023-2036, 2016 11 24.
Artículo en Inglés | MEDLINE | ID: mdl-27959731

RESUMEN

BACKGROUND: The molecular determinants of clinical responses to decitabine therapy in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) are unclear. METHODS: We enrolled 84 adult patients with AML or MDS in a single-institution trial of decitabine to identify somatic mutations and their relationships to clinical responses. Decitabine was administered at a dose of 20 mg per square meter of body-surface area per day for 10 consecutive days in monthly cycles. We performed enhanced exome or gene-panel sequencing in 67 of these patients and serial sequencing at multiple time points to evaluate patterns of mutation clearance in 54 patients. An extension cohort included 32 additional patients who received decitabine in different protocols. RESULTS: Of the 116 patients, 53 (46%) had bone marrow blast clearance (<5% blasts). Response rates were higher among patients with an unfavorable-risk cytogenetic profile than among patients with an intermediate-risk or favorable-risk cytogenetic profile (29 of 43 patients [67%] vs. 24 of 71 patients [34%], P<0.001) and among patients with TP53 mutations than among patients with wild-type TP53 (21 of 21 [100%] vs. 32 of 78 [41%], P<0.001). Previous studies have consistently shown that patients with an unfavorable-risk cytogenetic profile and TP53 mutations who receive conventional chemotherapy have poor outcomes. However, in this study of 10-day courses of decitabine, neither of these risk factors was associated with a lower rate of overall survival than the rate of survival among study patients with intermediate-risk cytogenetic profiles. CONCLUSIONS: Patients with AML and MDS who had cytogenetic abnormalities associated with unfavorable risk, TP53 mutations, or both had favorable clinical responses and robust (but incomplete) mutation clearance after receiving serial 10-day courses of decitabine. Although these responses were not durable, they resulted in rates of overall survival that were similar to those among patients with AML who had an intermediate-risk cytogenetic profile and who also received serial 10-day courses of decitabine. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT01687400 .).


Asunto(s)
Antimetabolitos Antineoplásicos/administración & dosificación , Azacitidina/análogos & derivados , Médula Ósea/patología , Leucemia Mieloide Aguda/tratamiento farmacológico , Mutación , Síndromes Mielodisplásicos/tratamiento farmacológico , Proteína p53 Supresora de Tumor/genética , 5-Metilcitosina/análisis , Adulto , Anciano , Anciano de 80 o más Años , Antimetabolitos Antineoplásicos/efectos adversos , Azacitidina/administración & dosificación , Azacitidina/efectos adversos , Biomarcadores de Tumor/análisis , Médula Ósea/química , Decitabina , Exoma , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Estudios Prospectivos , Factores de Riesgo , Tasa de Supervivencia
6.
N Engl J Med ; 376(8): 797-8, 2017 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-28225682
8.
Proc Natl Acad Sci U S A ; 108(45): E1089-98, 2011 Nov 08.
Artículo en Inglés | MEDLINE | ID: mdl-21734149

RESUMEN

Survival of yeast during starvation has been shown to depend on the nature of the missing nutrient(s). In general, starvation for "natural" nutrients such as sources of carbon, phosphate, nitrogen, or sulfate results in low death rates, whereas starvation for amino acids or other metabolites in auxotrophic mutants results in rapid loss of viability. Here we characterized phenotype, gene expression, and metabolite abundance during starvation for methionine. Some methionine auxotrophs (those with blocks in the biosynthetic pathway) respond to methionine starvation like yeast starving for natural nutrients such as phosphate or sulfate: they undergo a uniform cell cycle arrest, conserve glucose, and survive. In contrast, methionine auxotrophs with defects in the transcription factors Met31p and Met32p respond poorly, like other auxotrophs. We combined physiological and gene expression data from a variety of nutrient starvations (in both respiratory competent and incompetent cells) to show that successful starvation response is correlated with expression of genes encoding oxidative stress response and nonrespiratory mitochondrial functions, but not respiration per se.


Asunto(s)
Mitocondrias/metabolismo , Estrés Oxidativo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Glucosa/metabolismo , Metionina/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Transcripción Genética
9.
Cancer Discov ; 14(6): 1106-1131, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38416133

RESUMEN

Recent clinical trials have highlighted the limited efficacy of T cell-based immunotherapy in patients with glioblastoma (GBM). To better understand the characteristics of tumor-infiltrating lymphocytes (TIL) in GBM, we performed cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing with paired V(D)J sequencing, respectively, on TILs from two cohorts of patients totaling 15 patients with high-grade glioma, including GBM or astrocytoma, IDH-mutant, grade 4 (G4A). Analysis of the CD8+ TIL landscape reveals an enrichment of clonally expanded GZMK+ effector T cells in the tumor compared with matched blood, which was validated at the protein level. Furthermore, integration with other cancer types highlights the lack of a canonically exhausted CD8+ T-cell population in GBM TIL. These data suggest that GZMK+ effector T cells represent an important T-cell subset within the GBM microenvironment and may harbor potential therapeutic implications. SIGNIFICANCE: To understand the limited efficacy of immune-checkpoint blockade in GBM, we applied a multiomics approach to understand the TIL landscape. By highlighting the enrichment of GZMK+ effector T cells and the lack of exhausted T cells, we provide a new potential mechanism of resistance to immunotherapy in GBM. This article is featured in Selected Articles from This Issue, p. 897.


Asunto(s)
Linfocitos T CD8-positivos , Glioblastoma , Linfocitos Infiltrantes de Tumor , Humanos , Glioblastoma/inmunología , Glioblastoma/terapia , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias Encefálicas/inmunología , Microambiente Tumoral/inmunología
10.
Nat Commun ; 15(1): 478, 2024 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-38216553

RESUMEN

Vestibular schwannomas (VS) are benign tumors that lead to significant neurologic and otologic morbidity. How VS heterogeneity and the tumor microenvironment (TME) contribute to VS pathogenesis remains poorly understood. In this study, we perform scRNA-seq on 15 VS, with paired scATAC-seq (n = 6) and exome sequencing (n = 12). We identify diverse Schwann cell (SC), stromal, and immune populations in the VS TME and find that repair-like and MHC-II antigen-presenting SCs are associated with myeloid cell infiltrate, implicating a nerve injury-like process. Deconvolution analysis of RNA-expression data from 175 tumors reveals Injury-like tumors are associated with larger tumor size, and scATAC-seq identifies transcription factors associated with nerve repair SCs from Injury-like tumors. Ligand-receptor analysis and in vitro experiments suggest that Injury-like VS-SCs recruit myeloid cells via CSF1 signaling. Our study indicates that Injury-like SCs may cause tumor growth via myeloid cell recruitment and identifies molecular pathways that may be therapeutically targeted.


Asunto(s)
Neuroma Acústico , Humanos , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , Neuroma Acústico/patología , Ecosistema , Multiómica , Células de Schwann/metabolismo , Transducción de Señal/fisiología , Análisis de la Célula Individual , Microambiente Tumoral
11.
Proc Natl Acad Sci U S A ; 107(15): 6946-51, 2010 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-20335538

RESUMEN

Oscillations in patterns of expression of a large fraction of yeast genes are associated with the "metabolic cycle," usually seen only in prestarved, continuous cultures of yeast. We used FISH of mRNA in individual cells to test the hypothesis that these oscillations happen in single cells drawn from unsynchronized cultures growing exponentially in chemostats. Gene-expression data from synchronized cultures were used to predict coincident appearance of mRNAs from pairs of genes in the unsynchronized cells. Quantitative analysis of the FISH results shows that individual unsynchronized cells growing slowly because of glucose limitation or phosphate limitation show the predicted oscillations. We conclude that the yeast metabolic cycle is an intrinsic property of yeast metabolism and does not depend on either synchronization or external limitation of growth by the carbon source.


Asunto(s)
División Celular , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Metabolismo Energético , Perfilación de la Expresión Génica , Genes Fúngicos , Hibridación Fluorescente in Situ , Modelos Biológicos , Oscilometría , ARN Mensajero/metabolismo
12.
J Clin Invest ; 133(13)2023 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-37279078

RESUMEN

Since the T-box transcription factors (TFs) T-BET and EOMES are necessary for initiation of NK cell development, their ongoing requirement for mature NK cell homeostasis, function, and molecular programming remains unclear. To address this, T-BET and EOMES were deleted in unexpanded primary human NK cells using CRISPR/Cas9. Deleting these TFs compromised in vivo antitumor response of human NK cells. Mechanistically, T-BET and EOMES were required for normal NK cell proliferation and persistence in vivo. NK cells lacking T-BET and EOMES also exhibited defective responses to cytokine stimulation. Single-cell RNA-Seq revealed a specific T-box transcriptional program in human NK cells, which was rapidly lost following T-BET and EOMES deletion. Further, T-BET- and EOMES-deleted CD56bright NK cells acquired an innate lymphoid cell precursor-like (ILCP-like) profile with increased expression of the ILC-3-associated TFs RORC and AHR, revealing a role for T-box TFs in maintaining mature NK cell phenotypes and an unexpected role of suppressing alternative ILC lineages. Our study reveals the critical importance of sustained EOMES and T-BET expression to orchestrate mature NK cell function and identity.


Asunto(s)
Inmunidad Innata , Proteínas de Dominio T Box , Humanos , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Células Asesinas Naturales/metabolismo , Factores de Transcripción/metabolismo , Citocinas/metabolismo
13.
Exp Hematol ; 111: 50-65, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35429619

RESUMEN

The molecular events responsible for decitabine responses in myelodysplastic syndrome and acute myeloid leukemia patients are poorly understood. Decitabine has a short serum half-life and limited stability in tissue culture. Therefore, theoretical pharmacologic differences may exist between patient molecular changes in vitro and the consequences of in vivo treatment. To systematically identify the global genomic and transcriptomic alterations induced by decitabine in vivo, we evaluated primary bone marrow samples that were collected during patient treatment and applied whole-genome bisulfite sequencing, RNA-sequencing, and single-cell RNA sequencing. Decitabine induced global, reversible hypomethylation after 10 days of therapy in all patients, which was associated with induction of interferon-induced pathways, the expression of endogenous retroviral elements, and inhibition of erythroid-related transcripts, recapitulating many effects seen previously in in vitro studies. However, at relapse after decitabine treatment, interferon-induced transcripts remained elevated relative to day 0, but erythroid-related transcripts now were more highly expressed than at day 0. Clinical responses were not correlated with epigenetic or transcriptional signatures, although sample size and interpatient variance restricted the statistical power required for capturing smaller effects. Collectively, these data define global hypomethylation by decitabine and find that erythroid-related pathways may be relevant because they are inhibited by therapy and reverse at relapse.


Asunto(s)
Decitabina , Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Decitabina/uso terapéutico , Humanos , Interferones , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Recurrencia
14.
Blood Cancer Discov ; 3(1): 32-49, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-35019859

RESUMEN

To better understand clonal and transcriptional adaptations after relapse in patients with acute myeloid leukemia (AML), we collected presentation and relapse samples from six normal karyotype AML cases. We performed enhanced whole-genome sequencing to characterize clonal evolution, and deep-coverage single-cell RNA sequencing on the same samples, which yielded 142,642 high-quality cells for analysis. Identifying expressed mutations in individual cells enabled us to discriminate between normal and AML cells, to identify coordinated changes in the genome and transcriptome, and to identify subclone-specific cell states. We quantified the coevolution of genetic and transcriptional heterogeneity during AML progression, and found that transcriptional changes were significantly correlated with genetic changes. However, transcriptional adaptation sometimes occurred independently, suggesting that clonal evolution does not represent all relevant biological changes. In three cases, we identified cells at diagnosis that likely seeded the relapse. Finally, these data revealed a conserved relapse-enriched leukemic cell state bearing markers of stemness, quiescence, and adhesion. SIGNIFICANCE: These data enabled us to identify a relapse-enriched leukemic cell state with distinct transcriptional properties. Detailed case-by-case analyses elucidated the complex ways in which the AML genome, transcriptome, and immune microenvironment interact to evade chemotherapy. These analyses provide a blueprint for evaluating these factors in larger cohorts.This article is highlighted in the In This Issue feature, p. 1.


Asunto(s)
Leucemia Mieloide Aguda , Evolución Clonal , Humanos , Cariotipo , Leucemia Mieloide Aguda/diagnóstico , Mutación , Recurrencia , Microambiente Tumoral
15.
Genome Med ; 14(1): 49, 2022 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-35534852

RESUMEN

BACKGROUND: Recent investigations of the meninges have highlighted the importance of the dura layer in central nervous system immune surveillance beyond a purely structural role. However, our understanding of the meninges largely stems from the use of pre-clinical models rather than human samples. METHODS: Single-cell RNA sequencing of seven non-tumor-associated human dura samples and six primary meningioma tumor samples (4 matched and 2 non-matched) was performed. Cell type identities, gene expression profiles, and T cell receptor expression were analyzed. Copy number variant (CNV) analysis was performed to identify putative tumor cells and analyze intratumoral CNV heterogeneity. Immunohistochemistry and imaging mass cytometry was performed on selected samples to validate protein expression and reveal spatial localization of select protein markers. RESULTS: In this study, we use single-cell RNA sequencing to perform the first characterization of both non-tumor-associated human dura and primary meningioma samples. First, we reveal a complex immune microenvironment in human dura that is transcriptionally distinct from that of meningioma. In addition, we characterize a functionally diverse and heterogenous landscape of non-immune cells including endothelial cells and fibroblasts. Through imaging mass cytometry, we highlight the spatial relationship among immune cell types and vasculature in non-tumor-associated dura. Utilizing T cell receptor sequencing, we show significant TCR overlap between matched dura and meningioma samples. Finally, we report copy number variant heterogeneity within our meningioma samples. CONCLUSIONS: Our comprehensive investigation of both the immune and non-immune cellular landscapes of human dura and meningioma at single-cell resolution builds upon previously published data in murine models and provides new insight into previously uncharacterized roles of human dura.


Asunto(s)
Neoplasias Meníngeas , Meningioma , Animales , Células Endoteliales/patología , Humanos , Inmunidad , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patología , Meninges/patología , Meningioma/genética , Meningioma/patología , Ratones , Microambiente Tumoral
16.
Sci Transl Med ; 14(633): eabm1375, 2022 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-35196021

RESUMEN

Natural killer (NK) cells are innate lymphoid cells that eliminate cancer cells, produce cytokines, and are being investigated as a nascent cellular immunotherapy. Impaired NK cell function, expansion, and persistence remain key challenges for optimal clinical translation. One promising strategy to overcome these challenges is cytokine-induced memory-like (ML) differentiation, whereby NK cells acquire enhanced antitumor function after stimulation with interleukin-12 (IL-12), IL-15, and IL-18. Here, reduced-intensity conditioning (RIC) for HLA-haploidentical hematopoietic cell transplantation (HCT) was augmented with same-donor ML NK cells on day +7 and 3 weeks of N-803 (IL-15 superagonist) to treat patients with relapsed/refractory acute myeloid leukemia (AML) in a clinical trial (NCT02782546). In 15 patients, donor ML NK cells were well tolerated, and 87% of patients achieved a composite complete response at day +28, which corresponded with clearing high-risk mutations, including TP53 variants. NK cells were the major blood lymphocytes for 2 months after HCT with 1104-fold expansion (over 1 to 2 weeks). Phenotypic and transcriptional analyses identified donor ML NK cells as distinct from conventional NK cells and showed that ML NK cells persisted for over 2 months. ML NK cells expressed CD16, CD57, and high granzyme B and perforin, along with a unique transcription factor profile. ML NK cells differentiated in patients had enhanced ex vivo function compared to conventional NK cells from both patients and healthy donors. Overall, same-donor ML NK cell therapy with 3 weeks of N-803 support safely augmented RIC haplo-HCT for AML.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Humanos , Inmunidad Innata , Interleucina-15 , Células Asesinas Naturales , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia
17.
Nat Commun ; 12(1): 6321, 2021 11 03.
Artículo en Inglés | MEDLINE | ID: mdl-34732716

RESUMEN

The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.


Asunto(s)
Unión Competitiva/fisiología , Neoplasias Encefálicas/genética , Glioblastoma/genética , Factores de Transcripción SOXB1/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Dominio B30.2-SPRY , Unión Competitiva/genética , Femenino , Técnicas de Silenciamiento del Gen , Glioblastoma/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteómica , Factores de Transcripción SOXB1/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
18.
Clin Cancer Res ; 27(12): 3339-3350, 2021 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-33832946

RESUMEN

PURPOSE: N-803 is an IL15 receptor superagonist complex, designed to optimize in vivo persistence and trans-presentation, thereby activating and expanding natural killer (NK) cells and CD8+ T cells. Monoclonal antibodies (mAbs) direct Fc receptor-bearing immune cells, including NK cells, to recognize and eliminate cancer targets. The ability of IL15R agonists to enhance tumor-targeting mAbs in patients has not been reported previously. PATIENTS AND METHODS: Relapsed/refractory patients with indolent non-Hodgkin lymphoma were treated with rituximab and intravenous or subcutaneous N-803 on an open-label, dose-escalation phase I study using a 3+3 design (NCT02384954). Primary endpoint was maximum tolerated dose. Immune correlates were performed using multidimensional analysis via mass cytometry and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) which simultaneously measures protein and single-cell RNA expression. RESULTS: This immunotherapy combination was safe and well tolerated and resulted in durable clinical responses including in rituximab-refractory patients. Subcutaneous N-803 plus rituximab induced sustained proliferation, expansion, and activation of peripheral blood NK cells and CD8 T cells, with increased NK cell and T cells present 8 weeks following last N-803 treatment. CITE-seq revealed a therapy-altered NK cell molecular program, including enhancement of AP-1 transcription factor. Furthermore, the monocyte transcriptional program was remodeled with enhanced MHC expression and antigen-presentation genes. CONCLUSIONS: N-803 combines with mAbs to enhance tumor targeting in patients, and warrants further investigation in combination with immunotherapies.


Asunto(s)
Interleucina-15 , Linfoma no Hodgkin , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Linfocitos T CD8-positivos/patología , Humanos , Interleucina-15/uso terapéutico , Linfoma no Hodgkin/patología , Proteínas Recombinantes de Fusión , Rituximab
19.
Nat Commun ; 10(1): 3660, 2019 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-31413257

RESUMEN

Virtually all tumors are genetically heterogeneous, containing mutationally-defined subclonal cell populations that often have distinct phenotypes. Single-cell RNA-sequencing has revealed that a variety of tumors are also transcriptionally heterogeneous, but the relationship between expression heterogeneity and subclonal architecture is unclear. Here, we address this question in the context of Acute Myeloid Leukemia (AML) by integrating whole genome sequencing with single-cell RNA-sequencing (using the 10x Genomics Chromium Single Cell 5' Gene Expression workflow). Applying this approach to five cryopreserved AML samples, we identify hundreds to thousands of cells containing tumor-specific mutations in each case, and use the results to distinguish AML cells (including normal-karyotype AML cells) from normal cells, identify expression signatures associated with subclonal mutations, and find cell surface markers that could be used to purify subclones for further study. This integrative approach for connecting genotype to phenotype is broadly applicable to any sample that is phenotypically and genetically heterogeneous.


Asunto(s)
Leucemia Mieloide Aguda/genética , ARN/genética , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Perfilación de la Expresión Génica/métodos , Genómica , Genotipo , Humanos , Mutación , Fenotipo , Polimorfismo de Nucleótido Simple , Secuenciación Completa del Genoma
20.
Sci Rep ; 8(1): 6553, 2018 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-29700310

RESUMEN

Although blood-based liquid biopsies have emerged as a promising non-invasive method to detect biomarkers in various cancers, limited progress has been made for brain tumors. One major obstacle is the blood-brain barrier (BBB), which hinders efficient passage of tumor biomarkers into the peripheral circulation. The objective of this study was to determine whether FUS in combination with microbubbles can enhance the release of biomarkers from the brain tumor to the blood circulation. Two glioblastoma tumor models (U87 and GL261), developed by intracranial injection of respective enhanced green fluorescent protein (eGFP)-transduced glioblastoma cells, were treated by FUS in the presence of systemically injected microbubbles. Effect of FUS on plasma eGFP mRNA levels was determined using quantitative polymerase chain reaction. eGFP mRNA were only detectable in the FUS-treated U87 mice and undetectable in the untreated U87 mice (maximum cycle number set to 40). This finding was replicated in GL261 mice across three different acoustic pressures. The circulating levels of eGFP mRNA were 1,500-4,800 fold higher in the FUS-treated GL261 mice than that of the untreated mice for the three acoustic pressures. This study demonstrated the feasibility of FUS-enabled brain tumor liquid biopsies in two different murine glioma models across different acoustic pressures.


Asunto(s)
Neoplasias Encefálicas/diagnóstico , Biopsia Guiada por Imagen , Biopsia Líquida , Ultrasonografía , Animales , Biomarcadores de Tumor , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Genes Reporteros , Glioblastoma/diagnóstico , Xenoinjertos , Biopsia Guiada por Imagen/métodos , Inmunohistoquímica , Biopsia Líquida/métodos , Ratones , Ultrasonografía/métodos
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