RESUMO
Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs1. Genetic, transcriptional and translational heterogeneity contributes to this dynamic process2,3. Metabolic heterogeneity has also been observed4, yet its role in cancer progression is less explored. Here we find that the loss of phosphoglycerate dehydrogenase (PHGDH) potentiates metastatic dissemination. Specifically, we find that heterogeneous or low PHGDH expression in primary tumours of patients with breast cancer is associated with decreased metastasis-free survival time. In mice, circulating tumour cells and early metastatic lesions are enriched with Phgdhlow cancer cells, and silencing Phgdh in primary tumours increases metastasis formation. Mechanistically, Phgdh interacts with the glycolytic enzyme phosphofructokinase, and the loss of this interaction activates the hexosamine-sialic acid pathway, which provides precursors for protein glycosylation. As a consequence, aberrant protein glycosylation occurs, including increased sialylation of integrin αvß3, which potentiates cell migration and invasion. Inhibition of sialylation counteracts the metastatic ability of Phgdhlow cancer cells. In conclusion, although the catalytic activity of PHGDH supports cancer cell proliferation, low PHGDH protein expression non-catalytically potentiates cancer dissemination and metastasis formation. Thus, the presence of PHDGH heterogeneity in primary tumours could be considered a sign of tumour aggressiveness.
Assuntos
Neoplasias da Mama , Metástase Neoplásica , Fosfoglicerato Desidrogenase , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Feminino , Inativação Gênica , Humanos , Camundongos , Fosfoglicerato Desidrogenase/genética , Serina/metabolismoRESUMO
Plasmodium parasites cause malaria, a global health disease that is responsible for more than 200 million clinical cases and 600 000 deaths each year. Most deaths are caused by various complications, including malaria-associated acute respiratory distress syndrome (MA-ARDS). Despite the very rapid and efficient killing of parasites with antimalarial drugs, 15% of patients with complicated malaria succumb. This stresses the importance of investigating resolution mechanisms that are involved in the recovery from these complications once the parasite is killed. To study the resolution of MA-ARDS, P. berghei NK65-infected C57BL/6 mice were treated with antimalarial drugs after onset of symptoms, resulting in 80% survival. Micro-computed tomography revealed alterations of the lungs upon infection, with an increase in total and non-aerated lung volume due to edema. Whole body plethysmography confirmed a drastically altered lung ventilation, which was restored during resolution. Single-cell RNA sequencing indicated an increased inflammatory state in the lungs upon infection, which was accompanied by a drastic decrease in endothelial cells, consistent with CD8+ T cell-mediated killing. During resolution, anti-inflammatory pathways were upregulated and proliferation of endothelial cells was observed. MultiNicheNet interactome analysis identified important changes in the ligand-receptor interactions during disease resolution that warrant further exploration in order to develop new therapeutic strategies. In conclusion, our study provides insights in pro-resolving pathways that limit inflammation and promote endothelial cell proliferation in experimental MA-ARDS. This information may be useful for the design of adjunctive treatments to enhance resolution after Plasmodium parasite killing by antimalarial drugs.
Assuntos
Antimaláricos , Malária , Síndrome do Desconforto Respiratório , Humanos , Animais , Camundongos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Células Endoteliais/metabolismo , Microtomografia por Raio-X/efeitos adversos , Camundongos Endogâmicos C57BL , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/metabolismo , Malária/parasitologia , Análise de Sequência de RNA , Plasmodium bergheiRESUMO
BACKGROUND: Lymphangiogenesis, the formation of lymphatic vessels, is tightly linked to the development of the venous vasculature, both at the cellular and molecular levels. Here, we identify a novel role for Sorbs1, the founding member of the SoHo family of cytoskeleton adaptor proteins, in vascular and lymphatic development in the zebrafish. RESULTS: We show that Sorbs1 is required for secondary sprouting and emergence of several vascular structures specifically derived from the axial vein. Most notably, formation of the precursor parachordal lymphatic structures is affected in sorbs1 mutant embryos, severely impacting the establishment of the trunk lymphatic vessel network. Interestingly, we show that Sorbs1 interacts with the BMP pathway and could function outside of Vegfc signaling. Mechanistically, Sorbs1 controls FAK/Src signaling and subsequently impacts on the cytoskeleton processes regulated by Rac1 and RhoA GTPases. Inactivation of Sorbs1 altered cell-extracellular matrix (ECM) contacts rearrangement and cytoskeleton dynamics, leading to specific defects in endothelial cell migratory and adhesive properties. CONCLUSIONS: Overall, using in vitro and in vivo assays, we identify Sorbs1 as an important regulator of venous and lymphatic angiogenesis independently of the Vegfc signaling axis. These results provide a better understanding of the complexity found within context-specific vascular and lymphatic development.
Assuntos
Vasos Linfáticos , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Vasos Linfáticos/metabolismo , Linfangiogênese/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citoesqueleto/metabolismoRESUMO
Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of hematological cancers arising from the malignant transformation of mature T cells. In a cohort of 28 PTCL cases, we identified recurrent overexpression of MYCN, a member of the MYC family of oncogenic transcription factors. Approximately half of all PTCL cases was characterized by a MYC expression signature. Inducible expression of MYCN in lymphoid cells in a mouse model caused T-cell lymphoma that recapitulated human PTCL with an MYC expression signature. Integration of mouse and human expression data identified EZH2 as a key downstream target of MYCN. Remarkably, EZH2 was found to be an essential cofactor for the transcriptional activation of the MYCN-driven gene expression program, which was independent of methyltransferase activity but dependent on phosphorylation by CDK1. MYCN-driven T-cell lymphoma was sensitive to EZH2 degradation or CDK1 inhibition, which displayed synergy with US Food and Drug Administration-approved histone deacetylase (HDAC) inhibitors.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Linfoma de Células T Periférico , Proteína Proto-Oncogênica N-Myc , Humanos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Linfoma de Células T Periférico/genética , Proteína Proto-Oncogênica N-Myc/genéticaRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of leukemia caused by accumulation of multiple genetic alterations in T-cell progenitors. However, for many genes it remains unknown how their mutations contribute to disease development. Therefore, we performed two single-cell CRISPR screens in primary pro-T cells ex vivo to study the transcriptional impact of loss-of-function alterations in T-ALL and correlate this with effects on cell fitness. The various perturbations were clustered based on their effects on E2F/MYC or STAT/NOTCH signatures, which play a defining role in driving T-cell proliferation. Many of the perturbations resulted in positive effects on the STAT and NOTCH signatures and were predicted to behave as haploinsufficient tumor suppressors in T-ALL. Additionally, Spi1 was identified as an essential gene for pro-T cell survival, associated with deregulation of the MYC signature and epigenetic consequences. In contrast, Bcl11b was identified a strong tumor suppressor gene in immature T lymphocytes, associated with deregulation of NF-kB and JAK/STAT signaling. We found a correlation between BCL11B expression level and JAK/STAT pathway mutations in T-ALL patients and demonstrated oncogenic cooperation between Bcl11b inactivation and JAK3 hyperactivation in pro-T cells. Altogether, these single-cell CRISPR screens in pro-T cells provide fundamental insights in the mechanisms of transcriptional deregulation caused by genetic alterations in T-ALL.
RESUMO
Tight regulation of IL-7Rα expression is essential for normal T-cell development. IL-7Rα gain-of-function mutations are known drivers of T-cell acute lymphoblastic leukemia (T-ALL). Although a subset of patients with T-ALL display high IL7R messenger RNA levels and cases with IL7R gains have been reported, the impact of IL-7Rα overexpression, rather than mutational activation, during leukemogenesis remains unclear. In this study, overexpressed IL-7Rα in tetracycline-inducible Il7r transgenic and Rosa26 IL7R knockin mice drove potential thymocyte self-renewal, and thymus hyperplasia related to increased proliferation of T-cell precursors, which subsequently infiltrated lymph nodes, spleen, and bone marrow, ultimately leading to fatal leukemia. The tumors mimicked key features of human T-ALL, including heterogeneity in immunophenotype and genetic subtype between cases, frequent hyperactivation of the PI3K/Akt pathway paralleled by downregulation of p27Kip1 and upregulation of Bcl-2, and gene expression signatures evidencing activation of JAK/STAT, PI3K/Akt/mTOR and Notch signaling. Notably, we also found that established tumors may no longer require high levels of IL-7R expression upon secondary transplantation and progressed in the absence of IL-7, but remain sensitive to inhibitors of IL-7R-mediated signaling ruxolitinib (Jak1), AZD1208 (Pim), dactolisib (PI3K/mTOR), palbociclib (Cdk4/6), and venetoclax (Bcl-2). The relevance of these findings for human disease are highlighted by the fact that samples from patients with T-ALL with high wild-type IL7R expression display a transcriptional signature resembling that of IL-7-stimulated pro-T cells and, critically, of IL7R-mutant cases of T-ALL. Overall, our study demonstrates that high expression of IL-7Rα can promote T-cell tumorigenesis, even in the absence of IL-7Rα mutational activation.
Assuntos
Carcinogênese , Regulação Leucêmica da Expressão Gênica , Mutação , Proteínas de Neoplasias , Neoplasias Experimentais , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptores de Interleucina-7 , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptores de Interleucina-7/biossíntese , Receptores de Interleucina-7/genética , Transdução de Sinais , Timócitos/metabolismoRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia that is most frequent in children and is characterized by the presence of few chromosomal rearrangements and 10 to 20 somatic mutations in protein-coding regions at diagnosis. The majority of T-ALL cases harbor activating mutations in NOTCH1 together with mutations in genes implicated in kinase signaling, transcriptional regulation, or protein translation. To obtain more insight in the level of clonal heterogeneity at diagnosis and during treatment, we used single-cell targeted DNA sequencing with the Tapestri platform. We designed a custom ALL panel and obtained accurate single-nucleotide variant and small insertion-deletion mutation calling for 305 amplicons covering 110 genes in about 4400 cells per sample and time point. A total of 108 188 cells were analyzed for 25 samples of 8 T-ALL patients. We typically observed a major clone at diagnosis (>35% of the cells) accompanied by several minor clones of which some were less than 1% of the total number of cells. Four patients had >2 NOTCH1 mutations, some of which present in minor clones, indicating a strong pressure to acquire NOTCH1 mutations in developing T-ALL cells. By analyzing longitudinal samples, we detected the presence and clonal nature of residual leukemic cells and clones with a minor presence at diagnosis that evolved to clinically relevant major clones at later disease stages. Therefore, single-cell DNA amplicon sequencing is a sensitive assay to detect clonal architecture and evolution in T-ALL.
Assuntos
Evolução Clonal , DNA de Neoplasias/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Análise de Célula Única/métodos , Células Sanguíneas/química , Células da Medula Óssea/química , Criança , Humanos , Mutação INDEL , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Neoplasia Residual/diagnóstico , PTEN Fosfo-Hidrolase/genética , Filogenia , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/genética , Receptor Notch1/fisiologia , Recidiva , Terapia de Salvação , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
TAL1 is ectopically expressed in about 30% of T-cell acute lymphoblastic leukemia (T-ALL) due to chromosomal rearrangements leading to the STIL-TAL1 fusion genes or due to non-coding mutations leading to a de novo enhancer driving TAL1 expression. Analysis of sequence data from T-ALL cases demonstrates a significant association between TAL1 expression and activating mutations of the PI3K-AKT pathway. We investigated the oncogenic function of TAL1 and the possible cooperation with PI3K-AKT pathway activation using isogenic pro-T-cell cultures ex vivo and in vivo leukemia models. We found that TAL1 on its own suppressed T-cell growth, in part by affecting apoptosis genes, while the combination with AKT pathway activation reduced apoptosis and was strongly driving cell proliferation ex vivo and leukemia development in vivo. As a consequence, we found that TAL1+AKTE17K transformed cells are more sensitive to PI3K-AKT pathway inhibition compared to AKTE17K transformed cells, related to the negative effect of TAL1 in the absence of activated PI3K-AKT signaling. We also found that both TAL1 and PI3K-AKT signaling increased the DNA-repair signature in T cells resulting in synergy between PARP and PI3K-AKT pathway inhibition. In conclusion, we have developed a novel mouse model for TAL1+AKTE17K driven T-ALL development and have identified a vulnerability of these leukemia cells to PI3K-AKT and PARP inhibitors.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , DNA , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Linfócitos T/metabolismoRESUMO
Acute lymphoblastic leukemia (ALL) is a malignancy that can be subdivided into distinct entities based on clinical, immunophenotypic and genomic features, including mutations, structural variants (SVs), and copy number alterations (CNA). Chromosome banding analysis (CBA) and Fluorescent In-Situ Hybridization (FISH) together with Multiple Ligation-dependent Probe Amplification (MLPA), array and PCR-based methods form the backbone of routine diagnostics. This approach is labor-intensive, time-consuming and costly. New molecular technologies now exist that can detect SVs and CNAs in one test. Here we apply one such technology, optical genome mapping (OGM), to the diagnostic work-up of 41 ALL cases. Compared to our standard testing pathway, OGM identified all recurrent CNAs and SVs as well as additional recurrent SVs and the resulting fusion genes. Based on the genomic profile obtained by OGM, 32 patients could be assigned to one of the major cytogenetic risk groups compared to 23 with the standard approach. The latter identified 24/34 recurrent chromosomal abnormalities, while OGM identified 33/34, misinterpreting only 1 case with low hypodiploidy. The results of MLPA were concordant in 100% of cases. Overall, there was excellent concordance between the results. OGM increased the detection rate and cytogenetic resolution, and abrogated the need for cascade testing, resulting in reduced turnaround times. OGM also provided opportunities for better patient stratification and accurate treatment options. However, for comprehensive cytogenomic testing, OGM still needs to be complemented with CBA or SNP-array to detect ploidy changes and with BCR::ABL1 FISH to assign patients as soon as possible to targeted therapy.
Assuntos
Aberrações Cromossômicas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Mapeamento Cromossômico/métodos , Variações do Número de Cópias de DNA , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fluxo de TrabalhoRESUMO
The polycomb repressive complex 2, with core components EZH2, SUZ12, and EED, is responsible for writing histone 3 lysine 27 trimethylation histone marks associated with gene repression. Analysis of sequence data from 419 T-cell acute lymphoblastic leukemia (T-ALL) cases demonstrated a significant association between SUZ12 and JAK3 mutations. Here we show that CRISPR/Cas9-mediated inactivation of Suz12 cooperates with mutant JAK3 to drive T-cell transformation and T-ALL development. Gene expression profiling integrated with ChIP-seq and ATAC-seq data established that inactivation of Suz12 led to increased PI3K/mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), and WNT signaling. Moreover, a drug screen revealed that JAK3/Suz12 mutant leukemia cells were more sensitive to histone deacetylase (HDAC)6 inhibition than JAK3 mutant leukemia cells. Among the broad genome and gene expression changes observed on Suz12 inactivation, our integrated analysis identified the PI3K/mTOR, VEGF/VEGF receptor, and HDAC6/HSP90 pathways as specific vulnerabilities in T-ALL cells with combined JAK3 and SUZ12 mutations.
Assuntos
Transformação Celular Neoplásica/genética , Complexo Repressor Polycomb 2/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Transdução de Sinais/fisiologia , Animais , Humanos , Janus Quinase 3/genética , Camundongos , Mutação , Proteínas de Neoplasias , Fatores de TranscriçãoRESUMO
The Janus kinase 3 (JAK3) tyrosine kinase is mutated in 10% to 16% of T-cell acute lymphoblastic leukemia (T-ALL) cases. JAK3 mutants induce constitutive JAK/STAT signaling and cause leukemia when expressed in the bone marrow cells of mice. Surprisingly, we observed that one third of JAK3-mutant T-ALL cases harbor 2 JAK3 mutations, some of which are monoallelic and others that are biallelic. Our data suggest that wild-type JAK3 competes with mutant JAK3 (M511I) for binding to the common γ chain and thereby suppresses its oncogenic potential. We demonstrate that JAK3 (M511I) can increase its limited oncogenic potential through the acquisition of an additional mutation in the mutant JAK3 allele. These double JAK3 mutants show increased STAT5 activation and increased potential to transform primary mouse pro-T cells to interleukin-7-independent growth and were not affected by wild-type JAK3 expression. These data extend our insight into the oncogenic properties of JAK3 mutations and provide an explanation of why progression of JAK3-mutant T-ALL cases can be associated with the accumulation of additional JAK3 mutations.
Assuntos
Janus Quinase 3/genética , Mutação Puntual , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Transdução de Sinais , Alelos , Linhagem Celular Tumoral , Humanos , Janus Quinase 3/metabolismo , Modelos Moleculares , Taxa de Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismoRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive childhood leukemia that is caused by the accumulation of multiple genomic lesions resulting in transcriptional deregulation and increased cell proliferation and survival. Through analysis of gene expression data, we provide evidence that the hedgehog pathway is activated in 20% of T-ALL samples. Hedgehog pathway activation is associated with ectopic expression of the hedgehog ligands Sonic hedgehog (SHH) or Indian hedgehog (IHH), and with upregulation of the transcription factor GLI1 Ectopic expression of SHH or IHH in mouse T cells in vivo caused hedgehog pathway activation in both lymphoid and epithelial cells in the thymus and resulted in increased expression of important T-cell stimulatory ligands (Dll4, Il7, and Vegf) by thymic epithelial cells. In T-ALL cell lines, pharmacological inhibition or short interfering RNA-mediated knockdown of SMO or GLI1 led to decreased cell proliferation. Moreover, primary T-ALL cases with high GLI1 messenger RNA levels, but not those with low or undetectable GLI1 expression, were sensitive to hedgehog pathway inhibition by GANT61 or GDC-0449 (vismodegib) using ex vivo cultures and in vivo xenograft models. We identify the hedgehog pathway as a novel therapeutic target in T-ALL and demonstrate that hedgehog inhibitors approved by the US Food and Drug Administration could be used for the treatment of this rare leukemia.
Assuntos
Anilidas/farmacologia , Proteínas Hedgehog/metabolismo , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Animais , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Smoothened/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
JAK3 is a tyrosine kinase that associates with the common γ chain of cytokine receptors and is recurrently mutated in T-cell acute lymphoblastic leukemia (T-ALL). We tested the transforming properties of JAK3 pseudokinase and kinase domain mutants using in vitro and in vivo assays. Most, but not all, JAK3 mutants transformed cytokine-dependent Ba/F3 or MOHITO cell lines to cytokine-independent proliferation. JAK3 pseudokinase mutants were dependent on Jak1 kinase activity for cellular transformation, whereas the JAK3 kinase domain mutant could transform cells in a Jak1 kinase-independent manner. Reconstitution of the IL7 receptor signaling complex in 293T cells showed that JAK3 mutants required receptor binding to mediate downstream STAT5 phosphorylation. Mice transplanted with bone marrow progenitor cells expressing JAK3 mutants developed a long-latency transplantable T-ALL-like disease, characterized by an accumulation of immature CD8(+) T cells. In vivo treatment of leukemic mice with the JAK3 selective inhibitor tofacitinib reduced the white blood cell count and caused leukemic cell apoptosis. Our data show that JAK3 mutations are drivers of T-ALL and require the cytokine receptor complex for transformation. These results warrant further investigation of JAK1/JAK3 inhibitors for the treatment of T-ALL.
Assuntos
Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Janus Quinase 1/metabolismo , Janus Quinase 3/genética , Leucemia de Células T/genética , Camundongos , Doença Aguda , Animais , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Ativação Enzimática/efeitos dos fármacos , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/metabolismo , Leucemia de Células T/tratamento farmacológico , Leucemia de Células T/metabolismo , Leucemia de Células T/patologia , Masculino , Camundongos/genética , Camundongos/metabolismo , Camundongos Endogâmicos BALB C , Mutação , Piperidinas/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Recurrent deletions of the long arm of chromosome 5 were detected in 23/200 cases of T-cell acute lymphoblastic leukemia. Genomic studies identified two types of deletions: interstitial and terminal. Interstitial 5q deletions, found in five cases, were present in both adults and children with a female predominance (chi-square, P=0.012). Interestingly, these cases resembled immature/early T-cell precursor acute lymphoblastic leukemia showing significant down-regulation of five out of the ten top differentially expressed genes in this leukemia group, including TCF7 which maps within the 5q31 common deleted region. Mutations of genes known to be associated with immature/early T-cell precursor acute lymphoblastic leukemia, i.e. WT1, ETV6, JAK1, JAK3, and RUNX1, were present, while CDKN2A/B deletions/mutations were never detected. All patients had relapsed/resistant disease and blasts showed an early differentiation arrest with expression of myeloid markers. Terminal 5q deletions, found in 18 of patients, were more prevalent in adults (chi-square, P=0.010) and defined a subgroup of HOXA-positive T-cell acute lymphoblastic leukemia characterized by 130 up- and 197 down-regulated genes. Down-regulated genes included TRIM41, ZFP62, MAPK9, MGAT1, and CNOT6, all mapping within the 1.4 Mb common deleted region at 5q35.3. Of interest, besides CNOT6 down-regulation, these cases also showed low BTG1 expression and a high incidence of CNOT3 mutations, suggesting that the CCR4-NOT complex plays a crucial role in the pathogenesis of HOXA-positive T-cell acute lymphoblastic leukemia with terminal 5q deletions. In conclusion, interstitial and terminal 5q deletions are recurrent genomic losses identifying distinct subtypes of T-cell acute lymphoblastic leukemia.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 5 , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Adolescente , Adulto , Biomarcadores , Criança , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Estudos de Associação Genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Fenótipo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Adulto JovemRESUMO
Despite therapeutic improvements, a sizable number of patients with T-cell acute lymphoblastic leukemia still have a poor outcome. To unravel the genomic background associated with refractoriness, we evaluated the transcriptome of 19 cases of refractory/early relapsed T-cell acute lymphoblastic leukemia (discovery cohort) by performing RNA-sequencing on diagnostic material. The incidence and prognostic impact of the most frequently mutated pathways were validated by Sanger sequencing on genomic DNA from diagnostic samples of an independent cohort of 49 cases (validation cohort), including refractory, relapsed and responsive cases. Combined gene expression and fusion transcript analyses in the discovery cohort revealed the presence of known oncogenes and identified novel rearrangements inducing overexpression, as well as inactivation of tumor suppressor genes. Mutation analysis identified JAK/STAT and RAS/PTEN as the most commonly disrupted pathways in patients with chemorefractory disease or early relapse, frequently in association with NOTCH1/FBXW7 mutations. The analysis on the validation cohort documented a significantly higher risk of relapse, inferior overall survival, disease-free survival and event-free survival in patients with JAK/STAT or RAS/PTEN alterations. Conversely, a significantly better survival was observed in patients harboring only NOTCH1/FBXW7 mutations: this favorable prognostic effect was abrogated by the presence of concomitant mutations. Preliminary in vitro assays on primary cells demonstrated sensitivity to specific inhibitors. These data document the negative prognostic impact of JAK/STAT and RAS/PTEN mutations in T-cell acute lymphoblastic leukemia and suggest the potential clinical application of JAK and PI3K/mTOR inhibitors in patients harboring mutations in these pathways.
Assuntos
Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Análise de Sequência de RNA , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Ciclo Celular/metabolismo , Criança , Análise por Conglomerados , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD , Feminino , Perfilação da Expressão Gênica , Humanos , Janus Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Fusão Oncogênica/genética , PTEN Fosfo-Hidrolase/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/terapia , Prognóstico , Receptor Notch1/genética , Receptor Notch1/metabolismo , Recidiva , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Análise de Sobrevida , Resultado do Tratamento , Ubiquitina-Proteína Ligases/metabolismo , Adulto Jovem , Proteínas ras/metabolismoRESUMO
T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia.
Assuntos
Epigênese Genética , Janus Quinases/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Receptores de Interleucina-7/genética , Adulto , Criança , Evolução Clonal/genética , Variações do Número de Cópias de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Estudos de Associação Genética , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Janus Quinases/metabolismo , Masculino , Polimorfismo de Nucleotídeo Único , Leucemia-Linfoma Linfoblástico de Células T Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Prognóstico , Receptores de Interleucina-7/metabolismo , Reprodutibilidade dos Testes , Transdução de SinaisRESUMO
The CRISPR genome editing technology has revolutionized the way gene function is studied. Genome editing can be achieved in single genes or for thousands of genes simultaneously in sensitive genetic screens. While conventional genetic screens are limited to bulk measurements of cell behavior, recent developments in single-cell technologies make it possible to combine CRISPR screening with single-cell profiling. In this way, cell behavior and gene expression can be monitored simultaneously, with the additional possibility of including data on chromatin accessibility and protein levels. Moreover, the availability of various Cas proteins leading to inactivation, activation, or other effects on gene function further broadens the scope of such screens. The integration of single-cell multi-omics approaches with CRISPR screening open the path to high-content information on the impact of genetic perturbations at single-cell resolution. Current limitations in cell throughput and data density need to be taken into consideration, but new technologies are rapidly evolving and are likely to easily overcome these limitations. In this review, we discuss the use of bulk CRISPR screening in hematology research, as well as the emergence of single-cell CRISPR screening and its added value to the field.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Testes GenéticosRESUMO
Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.
Assuntos
Lisina Acetiltransferases , NF-kappa B , Camundongos , Animais , NF-kappa B/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Acetilação , Acetilcoenzima A/metabolismo , Palmitatos , Lisina Acetiltransferases/metabolismoRESUMO
Acute lymphoblastic leukemia (ALL) is characterized by the presence of chromosomal changes, including numerical changes, translocations, and deletions, which are often associated with additional single-nucleotide mutations. In this study, we used single cell-targeted DNA sequencing to evaluate the clonal heterogeneity of B-ALL at diagnosis and during chemotherapy treatment. We designed a custom DNA amplicon library targeting mutational hotspot regions (in 110 genes) present in ALL, and we measured the presence of mutations and small insertions/deletions (indels) in bone marrow or blood samples from 12 B-ALL patients, with a median of 7973 cells per sample. Nine of the 12 cases showed at least 1 subclonal mutation, of which cases with PAX5 alterations or high hyperdiploidy (with intermediate to good prognosis) showed a high number of subclones (1 to 7) at diagnosis, defined by a variety of mutations in the JAK/STAT, RAS, or FLT3 signaling pathways. Cases with RAS pathway mutations had multiple mutations in FLT3, NRAS, KRAS, or BRAF in various clones. For those cases where we detected multiple mutational clones at diagnosis, we also studied blood samples during the first weeks of chemotherapy treatment. The leukemia clones disappeared during treatment with various kinetics, and few cells with mutations were easily detectable, even at low frequency (<0.1%). Our data illustrate that about half of the B-ALL cases show >2 subclones at diagnosis and that even very rare mutant cells can be detected at diagnosis or during treatment by single cell-targeted DNA sequencing.