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1.
Front Immunol ; 14: 1275085, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37965340

RESUMO

Langerhans cell histiocytosis (LCH) is a rare and clinically heterogeneous hematological disease characterized by the accumulation of mononuclear phagocytes in various tissues and organs. LCH is often characterized by activating mutations of the mitogen-activated protein kinase (MAPK) pathway with BRAFV600E being the most recurrent mutation. Although this discovery has greatly helped in understanding the disease and in developing better investigational tools, the process of malignant transformation and the cell of origin are still not fully understood. In this review, we focus on the newest updates regarding the molecular pathogenesis of LCH and novel suggested pathways with treatment potential.


Assuntos
Histiocitose de Células de Langerhans , Proteínas Proto-Oncogênicas B-raf , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Histiocitose de Células de Langerhans/genética , Histiocitose de Células de Langerhans/terapia , Mutação , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação com Ganho de Função
3.
J Vis Exp ; (193)2023 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-36971438

RESUMO

Throughout their lifetime, hematopoietic stem and progenitor cells (HSPCs) acquire somatic mutations. Some of these mutations alter HSPC functional properties such as proliferation and differentiation, thereby promoting the development of hematologic malignancies. Efficient and precise genetic manipulation of HSPCs is required to model, characterize, and better understand the functional consequences of recurrent somatic mutations. Mutations can have a deleterious effect on a gene and result in loss-of-function (LOF) or, in stark contrast, may enhance function or even lead to novel characteristics of a particular gene, termed gain-of-function (GOF). In contrast to LOF mutations, GOF mutations almost exclusively occur in a heterozygous fashion. Current genome-editing protocols do not allow for the selective targeting of individual alleles, hampering the ability to model heterozygous GOF mutations. Here, we provide a detailed protocol on how to engineer heterozygous GOF hotspot mutations in human HSPCs by combining CRISPR/Cas9-mediated homology-directed repair and recombinant AAV6 technology for efficient DNA donor template transfer. Importantly, this strategy makes use of a dual fluorescent reporter system to allow for the tracking and purification of successfully heterozygously edited HSPCs. This strategy can be employed to precisely investigate how GOF mutations affect HSPC function and their progression toward hematological malignancies.


Assuntos
Mutação com Ganho de Função , Edição de Genes , Humanos , Edição de Genes/métodos , Células-Tronco Hematopoéticas , Mutação , Sistemas CRISPR-Cas
4.
Leukemia ; 37(4): 843-853, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36813992

RESUMO

Calreticulin (CALR) mutations present the main oncogenic drivers in JAK2 wildtype (WT) myeloproliferative neoplasms (MPN), including essential thrombocythemia and myelofibrosis, where mutant (MUT) CALR is increasingly recognized as a suitable mutation-specific drug target. However, our current understanding of its mechanism-of-action is derived from mouse models or immortalized cell lines, where cross-species differences, ectopic over-expression and lack of disease penetrance are hampering translational research. Here, we describe the first human gene-engineered model of CALR MUT MPN using a CRISPR/Cas9 and adeno-associated viral vector-mediated knock-in strategy in primary human hematopoietic stem and progenitor cells (HSPCs) to establish a reproducible and trackable phenotype in vitro and in xenografted mice. Our humanized model recapitulates many disease hallmarks: thrombopoietin-independent megakaryopoiesis, myeloid-lineage skewing, splenomegaly, bone marrow fibrosis, and expansion of megakaryocyte-primed CD41+ progenitors. Strikingly, introduction of CALR mutations enforced early reprogramming of human HSPCs and the induction of an endoplasmic reticulum stress response. The observed compensatory upregulation of chaperones revealed novel mutation-specific vulnerabilities with preferential sensitivity of CALR mutant cells to inhibition of the BiP chaperone and the proteasome. Overall, our humanized model improves purely murine models and provides a readily usable basis for testing of novel therapeutic strategies in a human setting.


Assuntos
Transtornos Mieloproliferativos , Mielofibrose Primária , Humanos , Animais , Camundongos , Calreticulina/genética , Calreticulina/metabolismo , Janus Quinase 2/genética , Transtornos Mieloproliferativos/genética , Mutação , Células-Tronco Hematopoéticas/metabolismo , Mielofibrose Primária/genética , Mielofibrose Primária/metabolismo
5.
Bio Protoc ; 13(2): e4592, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36789162

RESUMO

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.

6.
EMBO Rep ; 23(4): e52904, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35156745

RESUMO

Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co-expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK-STAT signalling, TPO-independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR-dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.


Assuntos
Calreticulina , Transtornos Mieloproliferativos , Animais , Anticorpos Monoclonais , Calreticulina/genética , Calreticulina/metabolismo , Humanos , Janus Quinase 2/metabolismo , Mutação , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Ratos
7.
Genome Res ; 31(11): 2120-2130, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34407984

RESUMO

Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34+ hematopoietic stem and progenitor cells (HSPCs) and human CD3+ T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.


Assuntos
Sistemas CRISPR-Cas , Endonucleases , Endonucleases/genética , Regulação da Expressão Gênica , Genoma , RNA Guia de Cinetoplastídeos/genética , Ativação Transcricional
8.
Cancer Metab ; 8(1): 26, 2020 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-33292758

RESUMO

BACKGROUND: Aspartate biosynthesis and its delivery to the cytosol can be crucial for tumor growth in vivo. However, the impact of intracellular aspartate levels on metastasis has not been studied. We previously described that loss-of-aspartate glutamate carrier 1 (SLC25A12 or AGC1), an important component of the malate-aspartate shuttle, impairs cytosolic aspartate levels, NAD+/NADH ratio, mitochondrial respiration, and tumor growth. Here, we report the impact of AGC1-knockdown on metastasis. RESULTS: Low AGC1 expression correlates with worse patient prognosis in many cancers. AGC1-knockdown in mouse lung carcinoma and melanoma cell lines leads to increased pulmonary metastasis following subcutaneous or intravenous injections, respectively. On the other hand, conventional in vitro metastasis assays show no indication of increased metastasis capacity of AGC1-knockdown cells. CONCLUSION: This study highlights that certain branches of metabolism impact tumor growth and tumor metastasis differently. In addition, it also argues that commonly known metastasis indicators, including EMT genes, cell migration, or colony formation, do not always reflect metastatic capacity in vivo.

9.
Cancers (Basel) ; 12(5)2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-32397610

RESUMO

POU3F3 adjacent non-coding transcript 1 (PANTR1) is an oncogenic long non-coding RNA with significant influence on numerous cellular features in different types of cancer. No characterization of its role in renal cell carcinoma (RCC) is yet available. In this study, PANTR1 expression was confined to human brain and kidney tissue and was found significantly up-regulated in clear-cell renal cell carcinoma tissue (ccRCC) compared to non-cancerous kidney tissue in two independent cohorts (p < 0.001 for both cohorts). In uni- and multivariate Cox regression analysis, ccRCC patients with higher levels of PANTR1 showed significantly poorer disease-free survival in our own respective cohort (n = 175, hazard ratio: 4.3, 95% confidence interval: 1.45-12.75, p = 0.008) in accordance with significantly poorer overall survival in a large The Cancer Genome Atlas database (TCGA) cohort (n = 530, hazard ratio: 2.19, 95% confidence interval: 1.59-3.03, p ≤ 0.001). To study the underlying cellular mechanisms mediated by varying levels of PANTR1 in kidney cancer cells, we applied siRNA-mediated knock-down experiments in three independent ccRCC cell lines (RCC-FG, RCC-MF, 769-P). A decrease in PANTR1 levels led to significantly reduced cellular growth through activation of apoptosis in all tested cell lines. Moreover, as angiogenesis is a critical driver in ccRCC pathogenesis, we identified that PANTR1 expression is critical for in vitro tube formation and endothelial cell migration (p < 0.05). On the molecular level, knock-down of PANTR1 led to a decrease in Vascular Endothelial growth factor A (VEGF-A) and cell adhesion molecule laminin subunit gamma-2 (LAMC2) expression, corroborated by a positive correlation in RCC tissue (for VEGF-A R = 0.19, p < 0.0001, for LAMC2 R = 0.13, p = 0.0028). In conclusion, this study provides first evidence that PANTR1 has a relevant role in human RCC by influencing apoptosis and angiogenesis.

11.
Cancer Lett ; 420: 91-96, 2018 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-29409809

RESUMO

The Notch signaling pathway has a key role in cellular development and is involved in initiation and progression of cancer by fundamentally influencing cellular processes such as differentiation, proliferation or migration. The pathway is regulated on many stages, generating diverse outcomes depending on cellular context or signaling dose. Recent studies suggest that long non-coding RNAs (lncRNAs), a class of non-coding RNAs deregulated in many cancers, are involved in regulating the Notch pathway activity by modulating the expression of receptors or ligands on transcriptional or posttranscriptional levels. LncRNAs are also downstream targets of Notch signaling and some of these Notch-induced lncRNAs have been reported to be inducers of its oncogenic effects. This cross-talk between Notch signaling and lncRNAs makes those molecules potential biomarkers for Notch signaling activity and interesting therapeutic RNA-based targets in the future.


Assuntos
Neoplasias/genética , RNA Longo não Codificante/genética , Transdução de Sinais , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores Notch/metabolismo
12.
Int J Mol Sci ; 18(11)2017 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-29165391

RESUMO

Renal cell carcinoma (RCC) are the most common renal neoplasia and can be divided into three main histologic subtypes, among which clear cell RCC is by far the most common form of kidney cancer. Despite substantial advances over the last decade in the understanding of RCC biology, surgical treatments, and targeted and immuno-therapies in the metastatic setting, the prognosis for advanced RCC patients remains poor. One of the major problems with RCC treatment strategies is inherent or acquired resistance towards therapeutic agents over time. The discovery of microRNAs (miRNAs), a class of small, non-coding, single-stranded RNAs that play a crucial role in post-transcriptional regulation, has added new dimensions to the development of novel diagnostic and treatment tools. Because of an association between Von Hippel-Lindau (VHL) genes with chromosomal loss in 3p25-26 and clear cell RCC, miRNAs have attracted considerable scientific interest over the last years. The loss of VHL function leads to constitutional activation of the hypoxia inducible factor (HIF) pathway and to consequent expression of numerous angiogenic and carcinogenic factors. Since miRNAs represent key players of carcinogenesis, tumor cell invasion, angiogenesis, as well as in development of metastases in RCC, they might serve as potential therapeutic targets. Several miRNAs are already known to be dysregulated in RCC and have been linked to biological processes involved in tumor angiogenesis and response to anti-cancer therapies. This review summarizes the role of different miRNAs in RCC angiogenesis and their association with the VHL gene, highlighting their potential role as novel drug targets.


Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , MicroRNAs/genética , Transdução de Sinais , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Terapia de Alvo Molecular , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo
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