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1.
Elife ; 122024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38635416

RESUMO

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/ß. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.


Assuntos
Proteína AIRE , Elementos de DNA Transponíveis , Camundongos , Humanos , Animais , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Timócitos/metabolismo , Células Epiteliais/metabolismo , Diferenciação Celular/genética , Camundongos Endogâmicos C57BL
2.
Blood Adv ; 8(1): 112-129, 2024 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-37729615

RESUMO

ABSTRACT: Acute megakaryoblastic leukemia (AMKL) is a rare, developmentally restricted, and highly lethal cancer of early childhood. The paucity and hypocellularity (due to myelofibrosis) of primary patient samples hamper the discovery of cell- and genotype-specific treatments. AMKL is driven by mutually exclusive chimeric fusion oncogenes in two-thirds of the cases, with CBFA2T3::GLIS2 (CG2) and NUP98 fusions (NUP98r) representing the highest-fatality subgroups. We established CD34+ cord blood-derived CG2 models (n = 6) that sustain serial transplantation and recapitulate human leukemia regarding immunophenotype, leukemia-initiating cell frequencies, comutational landscape, and gene expression signature, with distinct upregulation of the prosurvival factor B-cell lymphoma 2 (BCL2). Cell membrane proteomic analyses highlighted CG2 surface markers preferentially expressed on leukemic cells compared with CD34+ cells (eg, NCAM1 and CD151). AMKL differentiation block in the mega-erythroid progenitor space was confirmed by single-cell profiling. Although CG2 cells were rather resistant to BCL2 genetic knockdown or selective pharmacological inhibition with venetoclax, they were vulnerable to strategies that target the megakaryocytic prosurvival factor BCL-XL (BCL2L1), including in vitro and in vivo treatment with BCL2/BCL-XL/BCL-W inhibitor navitoclax and DT2216, a selective BCL-XL proteolysis-targeting chimera degrader developed to limit thrombocytopenia in patients. NUP98r AMKL were also sensitive to BCL-XL inhibition but not the NUP98r monocytic leukemia, pointing to a lineage-specific dependency. Navitoclax or DT2216 treatment in combination with low-dose cytarabine further reduced leukemic burden in mice. This work extends the cellular and molecular diversity set of human AMKL models and uncovers BCL-XL as a therapeutic vulnerability in CG2 and NUP98r AMKL.


Assuntos
Antineoplásicos , Leucemia Megacarioblástica Aguda , Humanos , Criança , Pré-Escolar , Animais , Camundongos , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Proteômica , Fatores de Transcrição , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras
3.
J Clin Invest ; 134(1)2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-37906288

RESUMO

Hormone receptor-positive breast cancer (HR+) is immunologically cold and has not benefited from advances in immunotherapy. In contrast, subsets of triple-negative breast cancer (TNBC) display high leukocytic infiltration and respond to checkpoint blockade. CD8+ T cells, the main effectors of anticancer responses, recognize MHC I-associated peptides (MAPs). Our work aimed to characterize the repertoire of MAPs presented by HR+ and TNBC tumors. Using mass spectrometry, we identified 57,094 unique MAPs in 26 primary breast cancer samples. MAP source genes highly overlapped between both subtypes. We identified 25 tumor-specific antigens (TSAs) mainly deriving from aberrantly expressed regions. TSAs were most frequently identified in TNBC samples and were more shared among The Cancer Genome Atlas (TCGA) database TNBC than HR+ samples. In the TNBC cohort, the predicted number of TSAs positively correlated with leukocytic infiltration and overall survival, supporting their immunogenicity in vivo. We detected 49 tumor-associated antigens (TAAs), some of which derived from cancer-associated fibroblasts. Functional expansion of specific T cell assays confirmed the in vitro immunogenicity of several TSAs and TAAs. Our study identified attractive targets for cancer immunotherapy in both breast cancer subtypes. The higher prevalence of TSAs in TNBC tumors provides a rationale for their responsiveness to checkpoint blockade.


Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/patologia , Antígenos de Neoplasias/genética , Imunoterapia/métodos , Linfócitos T CD8-Positivos/patologia
4.
Genome Biol ; 24(1): 188, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37582761

RESUMO

MHC-I-associated peptides deriving from non-coding genomic regions and mutations can generate tumor-specific antigens, including neoantigens. Quantifying tumor-specific antigens' RNA expression in malignant and benign tissues is critical for discriminating actionable targets. We present BamQuery, a tool attributing an exhaustive RNA expression to MHC-I-associated peptides of any origin from bulk and single-cell RNA-sequencing data. We show that many cryptic and mutated tumor-specific antigens can derive from multiple discrete genomic regions, abundantly expressed in normal tissues. BamQuery can also be used to predict MHC-I-associated peptides immunogenicity and identify actionable tumor-specific antigens de novo.


Assuntos
Neoplasias , Proteogenômica , Humanos , Antígenos de Neoplasias/genética , Antígenos de Histocompatibilidade Classe I , Neoplasias/genética , Peptídeos/genética , RNA
5.
EMBO J ; 42(7): e110496, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36843541

RESUMO

Aberrant splicing is typically attributed to splice-factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation-independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice-factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice-site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high-eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre-mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.


Assuntos
Processamento Alternativo , Leucemia Mieloide Aguda , Humanos , Fatores de Processamento de RNA/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Splicing de RNA , Fatores de Iniciação em Eucariotos/genética , Leucemia Mieloide Aguda/genética , Mutação
6.
Front Immunol ; 13: 867443, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35401501

RESUMO

Early T-cell development is precisely controlled by E proteins, that indistinguishably include HEB/TCF12 and E2A/TCF3 transcription factors, together with NOTCH1 and pre-T cell receptor (TCR) signalling. Importantly, perturbations of early T-cell regulatory networks are implicated in leukemogenesis. NOTCH1 gain of function mutations invariably lead to T-cell acute lymphoblastic leukemia (T-ALL), whereas inhibition of E proteins accelerates leukemogenesis. Thus, NOTCH1, pre-TCR, E2A and HEB functions are intertwined, but how these pathways contribute individually or synergistically to leukemogenesis remain to be documented. To directly address these questions, we leveraged Cd3e-deficient mice in which pre-TCR signaling and progression through ß-selection is abrogated to dissect and decouple the roles of pre-TCR, NOTCH1, E2A and HEB in SCL/TAL1-induced T-ALL, via the use of Notch1 gain of function transgenic (Notch1ICtg) and Tcf12+/- or Tcf3+/- heterozygote mice. As a result, we now provide evidence that both HEB and E2A restrain cell proliferation at the ß-selection checkpoint while the clonal expansion of SCL-LMO1-induced pre-leukemic stem cells in T-ALL is uniquely dependent on Tcf12 gene dosage. At the molecular level, HEB protein levels are decreased via proteasomal degradation at the leukemic stage, pointing to a reversible loss of function mechanism. Moreover, in SCL-LMO1-induced T-ALL, loss of one Tcf12 allele is sufficient to bypass pre-TCR signaling which is required for Notch1 gain of function mutations and for progression to T-ALL. In contrast, Tcf12 monoallelic deletion does not accelerate Notch1IC-induced T-ALL, indicating that Tcf12 and Notch1 operate in the same pathway. Finally, we identify a tumor suppressor gene set downstream of HEB, exhibiting significantly lower expression levels in pediatric T-ALL compared to B-ALL and brain cancer samples, the three most frequent pediatric cancers. In summary, our results indicate a tumor suppressor function of HEB/TCF12 in T-ALL to mitigate cell proliferation controlled by NOTCH1 in pre-leukemic stem cells and prevent NOTCH1-driven progression to T-ALL.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo
7.
Blood Adv ; 6(2): 509-514, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34731885

RESUMO

Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.


Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Leucemia Mieloide Aguda , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética
8.
Cell Rep ; 31(7): 107660, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32433969

RESUMO

In human cells, the expression of ∼1,000 genes is modulated throughout the cell cycle. Although some of these genes are controlled by specific transcriptional programs, very little is known about their post-transcriptional regulation. Here, we analyze the expression signature associated with all 687 RNA-binding proteins (RBPs) and identify 39 that significantly correlate with cell cycle mRNAs. We find that NF45 and NF90 play essential roles in mitosis, and transcriptome analysis reveals that they are necessary for the expression of a subset of mitotic mRNAs. Using proteomics, we identify protein clusters associated with the NF45-NF90 complex, including components of Staufen-mediated mRNA decay (SMD). We show that depletion of SMD components increases the binding of mitotic mRNAs to the NF45-NF90 complex and rescues cells from mitotic defects. Together, our results indicate that the NF45-NF90 complex plays essential roles in mitosis by competing with the SMD machinery for a common set of mRNAs.


Assuntos
Mitose/fisiologia , Proteína do Fator Nuclear 45/metabolismo , Proteínas do Fator Nuclear 90/metabolismo , Estabilidade de RNA/fisiologia , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Mitose/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteína do Fator Nuclear 45/genética , Proteínas do Fator Nuclear 90/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
9.
Genome Med ; 12(1): 40, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345368

RESUMO

BACKGROUND: Endogenous retroelements (EREs) constitute about 42% of the human genome and have been implicated in common human diseases such as autoimmunity and cancer. The dominant paradigm holds that EREs are expressed in embryonic stem cells (ESCs) and germline cells but are repressed in differentiated somatic cells. Despite evidence that some EREs can be expressed at the RNA and protein levels in specific contexts, a system-level evaluation of their expression in human tissues is lacking. METHODS: Using RNA sequencing data, we analyzed ERE expression in 32 human tissues and cell types, including medullary thymic epithelial cells (mTECs). A tissue specificity index was computed to identify tissue-restricted ERE families. We also analyzed the transcriptome of mTECs in wild-type and autoimmune regulator (AIRE)-deficient mice. Finally, we developed a proteogenomic workflow combining RNA sequencing and mass spectrometry (MS) in order to evaluate whether EREs might be translated and generate MHC I-associated peptides (MAP) in B-lymphoblastoid cell lines (B-LCL) from 16 individuals. RESULTS: We report that all human tissues express EREs, but the breadth and magnitude of ERE expression are very heterogeneous from one tissue to another. ERE expression was particularly high in two MHC I-deficient tissues (ESCs and testis) and one MHC I-expressing tissue, mTECs. In mutant mice, we report that the exceptional expression of EREs in mTECs was AIRE-independent. MS analyses identified 103 non-redundant ERE-derived MAPs (ereMAPs) in B-LCLs. These ereMAPs preferentially derived from sense translation of intronic EREs. Notably, detailed analyses of their amino acid composition revealed that ERE-derived MAPs presented homology to viral MAPs. CONCLUSIONS: This study shows that ERE expression in somatic tissues is more pervasive and heterogeneous than anticipated. The high and diversified expression of EREs in mTECs and their ability to generate MAPs suggest that EREs may play an important role in the establishment of self-tolerance. The viral-like properties of ERE-derived MAPs suggest that those not expressed in mTECs can be highly immunogenic.


Assuntos
Retroelementos , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Citocinas/farmacologia , Células Dendríticas , Células Epiteliais/metabolismo , Humanos , Espectrometria de Massas , Camundongos Knockout , Análise de Sequência de RNA , Timo/citologia , Fatores de Transcrição/genética , Proteína AIRE
10.
Cancer Immunol Res ; 8(4): 544-555, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32047025

RESUMO

High-grade serous ovarian cancer (HGSC), the principal cause of death from gynecologic malignancies in the world, has not significantly benefited from advances in cancer immunotherapy. Although HGSC infiltration by lymphocytes correlates with superior survival, the nature of antigens that can elicit anti-HGSC immune responses is unknown. The goal of this study was to establish the global landscape of HGSC tumor-specific antigens (TSA) using a mass spectrometry pipeline that interrogated all reading frames of all genomic regions. In 23 HGSC tumors, we identified 103 TSAs. Classic TSA discovery approaches focusing only on mutated exonic sequences would have uncovered only three of these TSAs. Other mutated TSAs resulted from out-of-frame exonic translation (n = 2) or from noncoding sequences (n = 7). One group of TSAs (n = 91) derived from aberrantly expressed unmutated genomic sequences, which were not expressed in normal tissues. These aberrantly expressed TSAs (aeTSA) originated primarily from nonexonic sequences, in particular intronic (29%) and intergenic (22%) sequences. Their expression was regulated at the transcriptional level by variations in gene copy number and DNA methylation. Although mutated TSAs were unique to individual tumors, aeTSAs were shared by a large proportion of HGSCs. Taking into account the frequency of aeTSA expression and HLA allele frequencies, we calculated that, in Caucasians, the median number of aeTSAs per tumor would be five. We conclude that, in view of their number and the fact that they are shared by many tumors, aeTSAs may be the most attractive targets for HGSC immunotherapy.


Assuntos
Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Cistadenocarcinoma Seroso/patologia , Imunoterapia/métodos , Mutação , Neoplasias Ovarianas/patologia , Proteogenômica/métodos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo
11.
Genes Chromosomes Cancer ; 59(2): 125-130, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31515871

RESUMO

Infant acute lymphoblastic leukemias (ALL) are rare hematological malignancies occurring in children younger than 1 year of age, most frequently associated with KMT2A rearrangements (KMT2A-r). The smaller subset without KMT2A-r, which represents 20% of infant ALL cases, is poorly characterized. Here we report two cases of chemotherapy-sensitive non-KMT2A-r infant ALL. Transcriptome analyses revealed identical ACIN1-NUTM1 gene fusions in both cases, derived from cryptic chromosomal rearrangements undetected by standard cytogenetic approaches. Two isoforms of the gene fusion, joining exons 3 or 4 of ACIN1 to exon 3 of NUTM1, were identified. Both fusion transcripts contained the functional DNA-binding SAP (SAF-A/B, Acinus, and PIAS) domain of ACIN1 and most of NUTM1. The detection of the ACIN1-NUTM1 fusion by RT-PCR allowed the molecular monitoring of minimal residual disease in a clinical setting. Based on publicly available genomic datasets and literature review, we predict that NUTM1 gene fusions are recurrent events in infant ALL. As such, we propose two clinically relevant assays to screen for NUTM1 rearrangements in bone marrow cells, independent of the fusion partner: NUMT1 immunohistochemistry and NUTM1 RNA expression. In sum, our study identifies ACIN1-NUTM1 as a recurrent and possibly cryptic fusion in non-KMT2A-r infant ALL, provides clinical tools to screen for NUTM1-rearranged leukemia and contributes to the refinement of this new subgroup.


Assuntos
Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Aberrações Cromossômicas , Citogenética , Fusão Gênica , Rearranjo Gênico/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imuno-Histoquímica , Recém-Nascido , Leucemia Mieloide Aguda/genética , Masculino , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo
12.
Blood Adv ; 3(21): 3307-3321, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31698461

RESUMO

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (<3 years of age). It remains plagued with extremely poor treatment outcomes (<40% cure rates), mostly due to primary chemotherapy refractory disease and/or early relapse. Recurrent and mutually exclusive chimeric fusion oncogenes have been detected in 60% to 70% of cases and include nucleoporin 98 (NUP98) gene rearrangements, most commonly NUP98-KDM5A. Human models of NUP98-KDM5A-driven AMKL capable of faithfully recapitulating the disease have been lacking, and patient samples are rare, further limiting biomarkers and drug discovery. To overcome these impediments, we overexpressed NUP98-KDM5A in human cord blood hematopoietic stem and progenitor cells using a lentiviral-based approach to create physiopathologically relevant disease models. The NUP98-KDM5A fusion oncogene was a potent inducer of maturation arrest, sustaining long-term proliferative and progenitor capacities of engineered cells in optimized culture conditions. Adoptive transfer of NUP98-KDM5A-transformed cells into immunodeficient mice led to multiple subtypes of leukemia, including AMKL, that phenocopy human disease phenotypically and molecularly. The integrative molecular characterization of synthetic and patient NUP98-KDM5A AMKL samples revealed SELP, MPIG6B, and NEO1 as distinctive and novel disease biomarkers. Transcriptomic and proteomic analyses pointed to upregulation of the JAK-STAT signaling pathway in the model AMKL. Both synthetic models and patient-derived xenografts of NUP98-rearranged AMKL showed in vitro therapeutic vulnerability to ruxolitinib, a clinically approved JAK2 inhibitor. Overall, synthetic human AMKL models contribute to defining functional dependencies of rare genotypes of high-fatality pediatric leukemia, which lack effective and rationally designed treatments.


Assuntos
Biomarcadores , Modelos Animais de Doenças , Leucemia Megacarioblástica Aguda/etiologia , Leucemia Megacarioblástica Aguda/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Proteína 2 de Ligação ao Retinoblastoma/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biologia Computacional/métodos , Suscetibilidade a Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Leucemia Megacarioblástica Aguda/terapia , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Front Oncol ; 9: 772, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31475115

RESUMO

Shwachman-Diamond syndrome (SDS) is a rare and systemic disease mostly caused by mutations in the SBDS gene and characterized by pancreatic insufficiency, skeletal abnormalities, and a bone marrow dysfunction. In addition, SDS patients are predisposed to develop myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), typically during adulthood and associated with TP53 mutations. Although most SDS diagnoses are established in childhood, the nature and frequency of serial bone marrow cell investigations during the patients' lifetime remain a debatable topic. The precise molecular mechanisms leading to AML progression in SDS patients have not been fully elucidated because the patient cohorts are small and most disease monitoring is conducted using standard histological and cytogenetic approaches. Here we report a rare case of a patient with SDS who was diagnosed with AML at 5 years of age and survived. Intermittent neutropenia preceded the AML diagnostic but serial bone marrow monitoring according to the standard of care revealed no cytogenetic anomalies nor signs of clonal hematopoiesis. Using next generation sequencing approaches to find cytogenetically cryptic pathogenic mutations, we identified the cancer hotspot mutation c.394C>T/p.Arg132Cys in IDH1 with high variant allelic frequency in bone marrow cells, suggesting clonal expansion of a major leukemic clone karyotypically normal, in the SDS-associated AML. The mutation was somatic and likely occurred at the leukemic transformation stage, as it was not detected in a matched normal tissue nor in bone marrow smear prior to AML diagnosis. Gain-of-function mutations in IDH1, such as c.394C>T/p.Arg132Cys, create a neo-activity of isocitrate dehydrogenase 1 converting α-ketoglutarate into the oncometabolite D-2-hydroxyglutarate, inhibiting α-ketoglutarate-dependent enzymes, such as histone and DNA demethylases. Overall, our results suggest that along with previously described abnormalities such as TP53 mutations or monosomy7, 7q-, which are all absent in this patient, additional mechanisms including IDH1 mutations drive SDS-related AML and are likely associated with variable outcomes. Sensitive techniques complementary to standard cytogenetics, such as unbiased or targeted panel-based next generation sequencing approaches, warrant testing for monitoring of myelodysplasia, clonal hematopoiesis, and leukemia in the context SDS. Such analyses would also assist treatment decisions and allow to gain insight into the disease biology.

14.
Life Sci Alliance ; 2(4)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31427380

RESUMO

Mutations identified in acute myeloid leukemia patients are useful for prognosis and for selecting targeted therapies. Detection of such mutations using next-generation sequencing data requires a computationally intensive read mapping step followed by several variant calling methods. Targeted mutation identification drastically shifts the usual tradeoff between accuracy and performance by concentrating all computations over a small portion of sequence space. Here, we present km, an efficient approach leveraging k-mer decomposition of reads to identify targeted mutations. Our approach is versatile, as it can detect single-base mutations, several types of insertions and deletions, as well as fusions. We used two independent cohorts (The Cancer Genome Atlas and Leucegene) to show that mutation detection by km is fast, accurate, and mainly limited by sequencing depth. Therefore, km allows the establishment of fast diagnostics from next-generation sequencing data and could be suitable for clinical applications.


Assuntos
Leucemia Mieloide Aguda/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Algoritmos , Biologia Computacional/métodos , Bases de Dados Genéticas , Genoma , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias , RNA-Seq , Software , Sequenciamento do Exoma
15.
Blood ; 134(3): 263-276, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31076446

RESUMO

FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56 highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Biomarcadores , Ciclo Celular/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , DNA Metiltransferase 3A , Modelos Animais de Doenças , Duplicação Gênica , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos Transgênicos , Mutação , Nucleofosmina , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sequências de Repetição em Tandem , Sítio de Iniciação de Transcrição , Transcriptoma
16.
Sci Rep ; 9(1): 7203, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31076589

RESUMO

Endothelial cells have multifaceted interactions with the immune system, both as initiators and targets of immune responses. In vivo, apoptotic endothelial cells release two types of extracellular vesicles upon caspase-3 activation: apoptotic bodies and exosome-like nanovesicles (ApoExos). Only ApoExos are immunogenic: their injection causes inflammation and autoimmunity in mice. Based on deep sequencing of total RNA, we report that apoptotic bodies and ApoExos are loaded with divergent RNA cargos that are not released by healthy endothelial cells. Apoptotic bodies, like endothelial cells, contain mainly ribosomal RNA whereas ApoExos essentially contain non-ribosomal non-coding RNAs. Endogenous retroelements, bearing viral-like features, represented half of total ApoExos RNA content. ApoExos also contained several copies of unedited Alu repeats and large amounts of non-coding RNAs with a demonstrated role in autoimmunity such as U1 RNA and Y RNA. Moreover, ApoExos RNAs had a unique nucleotide composition and secondary structure characterized by strong enrichment in U-rich motifs and unstably folded RNAs. Globally, ApoExos were therefore loaded with RNAs that can stimulate a variety of RIG-I-like receptors and endosomal TLRs. Hence, apoptotic endothelial cells selectively sort in ApoExos a diversified repertoire of immunostimulatory "self RNAs" that are tailor-made for initiation of innate immune responses and autoimmunity.


Assuntos
Vesículas Extracelulares/genética , Perfilação da Expressão Gênica/métodos , Células Endoteliais da Veia Umbilical Humana/citologia , RNA/imunologia , Apoptose , Proteína DEAD-box 58/metabolismo , Células Endoteliais da Veia Umbilical Humana/química , Humanos , RNA/genética , Edição de RNA , Receptores Imunológicos , Análise de Sequência de RNA , Receptores Toll-Like/metabolismo
17.
Sci Transl Med ; 10(470)2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518613

RESUMO

Tumor-specific antigens (TSAs) represent ideal targets for cancer immunotherapy, but few have been identified thus far. We therefore developed a proteogenomic approach to enable the high-throughput discovery of TSAs coded by potentially all genomic regions. In two murine cancer cell lines and seven human primary tumors, we identified a total of 40 TSAs, about 90% of which derived from allegedly noncoding regions and would have been missed by standard exome-based approaches. Moreover, most of these TSAs derived from nonmutated yet aberrantly expressed transcripts (such as endogenous retroelements) that could be shared by multiple tumor types. Last, we demonstrated that, in mice, the strength of antitumor responses after TSA vaccination was influenced by two parameters that can be estimated in humans and could serve for TSA prioritization in clinical studies: TSA expression and the frequency of TSA-responsive T cells in the preimmune repertoire. In conclusion, the strategy reported herein could considerably facilitate the identification and prioritization of actionable human TSAs.


Assuntos
Antígenos de Neoplasias/metabolismo , DNA Intergênico/genética , Neoplasias/genética , Neoplasias/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Imunização , Interferon gama/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peptídeos/química , Biossíntese de Proteínas , Proteogenômica , Linfócitos T/imunologia
18.
Genes Chromosomes Cancer ; 57(6): 311-319, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29427526

RESUMO

The advent of large scale genomic sequencing technologies significantly improved the molecular classification of acute megakaryoblastic leukaemia (AMKL). AMKL represents a subset (∼10%) of high fatality pediatric acute myeloid leukemia (AML). Recurrent and mutually exclusive chimeric gene fusions associated with pediatric AMKL are found in 60%-70% of cases and include RBM15-MKL1, CBFA2T3-GLIS2, NUP98-KDM5A and MLL rearrangements. In addition, another 4% of AMKL harbor NUP98 rearrangements (NUP98r), with yet undetermined fusion partners. We report a novel NUP98-BPTF fusion in an infant presenting with primary refractory AMKL. In this NUP98r, the C-terminal chromatin recognition modules of BPTF, a core subunit of the NURF (nucleosome remodeling factor) ATP-dependent chromatin-remodeling complex, are fused to the N-terminal moiety of NUP98, creating an in frame NUP98-BPTF fusion, with structural homology to NUP98-KDM5A. The leukemic blasts expressed two NUP98-BPTF splicing variants, containing one or two tandemly spaced PHD chromatin reader domains. Our study also identified an unreported wild type BPTF splicing variant encoding for 2 PHD domains, detected both in normal cord blood CD34+ cells and in leukemic blasts, as with the fly BPTF homolog, Nurf301. Disease course was marked by rapid progression and primary chemoresistance, with ultimately significant tumor burden reduction following treatment with a clofarabine containing regimen. In sum, we report 2 novel NUP98-BPTF fusion isoforms that contribute to refine the NUP98r subgroup of pediatric AMKL. Multicenter clinical trials are critically required to determine the frequency of this fusion in AMKL patients and explore innovative treatment strategies for a disease still plagued with poor outcomes.


Assuntos
Antígenos Nucleares/genética , Leucemia Megacarioblástica Aguda/genética , Proteínas do Tecido Nervoso/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fatores de Transcrição/genética , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Humanos , Lactente , Cariotipagem , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Masculino , Splicing de RNA
20.
FASEB J ; 31(11): 5012-5018, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28754713

RESUMO

The ubiquitin-associated protein 2-like (UBAP2L) gene remains poorly studied in human and mouse development. UBAP2L interacts with the Polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and determines the activity of mouse hematopoietic stem cells in vivo Here we show that loss of Ubap2l leads to disorganized respiratory epithelium of mutant neonates, which die of respiratory failure. We also show that UBAP2L overexpression leads to epithelial-mesenchymal transition-like phenotype in a non-small cell lung carcinoma (NSCLC) cell line. UBAP2L is amplified in 15% of human primary lung adenocarcinoma specimens. Such patients express higher levels of UBAP2L and show a reduction in survival when compared with those who do not have this gene amplification. Supporting a possible role for UBAP2L in lung tumor progression, NSCLC cells engineered to express low levels of this gene produce much smaller tumors in vivo than wild-type control cells. Together, these results suggest that UBAP2L contributes to epithelial lung cell identity in mice and that it plays an important role in human lung adenocarcinoma.-Aucagne, R., Girard, S., Mayotte, N., Lehnertz, B., Lopes-Paciencia, S., Gendron, P., Boucher, G., Chagraoui, J., Sauvageau, G. UBAP2L is amplified in a large subset of human lung adenocarcinoma and is critical for epithelial lung cell identity and tumor metastasis.


Assuntos
Adenocarcinoma/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Transporte/biossíntese , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biossíntese , Mucosa Respiratória/metabolismo , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/genética , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Mucosa Respiratória/patologia
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