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1.
Nat Commun ; 14(1): 3620, 2023 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-37365178

RESUMO

Metastasis is the major cause of cancer-related deaths. Neuroblastoma (NB), a childhood tumor has been molecularly defined at the primary cancer site, however, the bone marrow (BM) as the metastatic niche of NB is poorly characterized. Here we perform single-cell transcriptomic and epigenomic profiling of BM aspirates from 11 subjects spanning three major NB subtypes and compare these to five age-matched and metastasis-free BM, followed by in-depth single cell analyses of tissue diversity and cell-cell interactions, as well as functional validation. We show that cellular plasticity of NB tumor cells is conserved upon metastasis and tumor cell type composition is NB subtype-dependent. NB cells signal to the BM microenvironment, rewiring via macrophage mgration inhibitory factor and midkine signaling specifically monocytes, which exhibit M1 and M2 features, are marked by activation of pro- and anti-inflammatory programs, and express tumor-promoting factors, reminiscent of tumor-associated macrophages. The interactions and pathways characterized in our study provide the basis for therapeutic approaches that target tumor-to-microenvironment interactions.


Assuntos
Neoplasias da Medula Óssea , Neuroblastoma , Humanos , Criança , Medula Óssea/patologia , Monócitos/metabolismo , Transcriptoma , Epigenômica , Neoplasias da Medula Óssea/genética , Neoplasias da Medula Óssea/metabolismo , Neoplasias da Medula Óssea/patologia , Neuroblastoma/metabolismo , Microambiente Tumoral/genética
2.
Cancer Lett ; 554: 216028, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36462556

RESUMO

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.


Assuntos
Sarcoma de Ewing , Humanos , Animais , Camundongos , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Peixe-Zebra/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Avaliação Pré-Clínica de Medicamentos , Xenoenxertos , Apoptose , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , Linhagem Celular Tumoral
3.
Nat Chem Biol ; 19(3): 301-310, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36302897

RESUMO

Velcrin compounds kill cancer cells expressing high levels of phosphodiesterase 3A (PDE3A) and Schlafen family member 12 (SLFN12) by inducing complex formation between these two proteins, but the mechanism of cancer cell killing by the PDE3A-SLFN12 complex is not fully understood. Here, we report that the physiological substrate of SLFN12 RNase is tRNALeu(TAA). SLFN12 selectively digests tRNALeu(TAA), and velcrin treatment promotes the cleavage of tRNALeu(TAA) by inducing PDE3A-SLFN12 complex formation in vitro. We found that distinct sequences in the variable loop and acceptor stem of tRNALeu(TAA) are required for substrate digestion. Velcrin treatment of sensitive cells results in downregulation of tRNALeu(TAA), ribosome pausing at Leu-TTA codons and global inhibition of protein synthesis. Velcrin-induced cleavage of tRNALeu(TAA) by SLFN12 and the concomitant global inhibition of protein synthesis thus define a new mechanism of apoptosis initiation.


Assuntos
Neoplasias , RNA de Transferência de Leucina , Linhagem Celular Tumoral , Morte Celular , Apoptose , Biossíntese de Proteínas
4.
Nat Commun ; 12(1): 3230, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050156

RESUMO

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Ósseas/diagnóstico , DNA Tumoral Circulante/sangue , Sarcoma de Ewing/diagnóstico , Adolescente , Adulto , Biomarcadores Tumorais/genética , Neoplasias Ósseas/sangue , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Estudos de Casos e Controles , Criança , Pré-Escolar , DNA Tumoral Circulante/genética , Análise Mutacional de DNA , Feminino , Humanos , Lactente , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Mutação , Sarcoma de Ewing/sangue , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Sequenciamento Completo do Genoma , Adulto Jovem
5.
mBio ; 10(4)2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311879

RESUMO

Nosocomial infections with Acinetobacter baumannii are a global problem in intensive care units with high mortality rates. Increasing resistance to first- and second-line antibiotics has forced the use of colistin as last-resort treatment, and increasing development of colistin resistance in A. baumannii has been reported. We evaluated the transcriptional regulator PmrA as potential drug target to restore colistin efficacy in A. baumannii Deletion of pmrA restored colistin susceptibility in 10 of the 12 extensively drug-resistant A. baumannii clinical isolates studied, indicating the importance of PmrA in the drug resistance phenotype. However, two strains remained highly resistant, indicating that PmrA-mediated overexpression of the phosphoethanolamine (PetN) transferase PmrC is not the exclusive colistin resistance mechanism in A. baumannii A detailed genetic characterization revealed a new colistin resistance mechanism mediated by genetic integration of the insertion element ISAbaI upstream of the PmrC homolog EptA (93% identity), leading to its overexpression. We found that eptA was ubiquitously present in clinical strains belonging to the international clone 2, and ISAbaI integration upstream of eptA was required to mediate the colistin-resistant phenotype. In addition, we found a duplicated ISAbaI-eptA cassette in one isolate, indicating that this colistin resistance determinant may be embedded in a mobile genetic element. Our data disprove PmrA as a drug target for adjuvant therapy but highlight the importance of PetN transferase-mediated colistin resistance in clinical strains. We suggest that direct targeting of the homologous PetN transferases PmrC/EptA may have the potential to overcome colistin resistance in A. baumanniiIMPORTANCE The discovery of antibiotics revolutionized modern medicine and enabled us to cure previously deadly bacterial infections. However, a progressive increase in antibiotic resistance rates is a major and global threat for our health care system. Colistin represents one of our last-resort antibiotics that is still active against most Gram-negative bacterial pathogens, but increasing resistance is reported worldwide, in particular due to the plasmid-encoded protein MCR-1 present in pathogens such as Escherichia coli and Klebsiella pneumoniae Here, we showed that colistin resistance in A. baumannii, a top-priority pathogen causing deadly nosocomial infections, is mediated through different avenues that result in increased activity of homologous phosphoethanolamine (PetN) transferases. Considering that MCR-1 is also a PetN transferase, our findings indicate that PetN transferases might be the Achilles heel of superbugs and that direct targeting of them may have the potential to preserve the activity of polymyxin antibiotics.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana , Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ordem dos Genes , Humanos , Mutação
6.
Antimicrob Agents Chemother ; 60(12): 7263-7271, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27671072

RESUMO

Infections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug-resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug-resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study, we developed a highly efficient and versatile genome-editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profiles. We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline-resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC90 of tigecycline from 25 µg/ml in the parental strains to 3.1 µg/ml in the ΔadeR mutants, indicating its importance in the drug resistance phenotype. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole-genome sequencing revealed loss-of-function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline-resistant clinical isolates. This study highlights the development and application of a powerful genome-editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinically relevant strains.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Edição de Genes/métodos , Minociclina/análogos & derivados , Transportadores de Cassetes de Ligação de ATP/metabolismo , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Sequência de Bases , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genoma Bacteriano/genética , Testes de Sensibilidade Microbiana , Minociclina/farmacologia , Análise de Sequência de DNA , Tigeciclina
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