Biology subjects

Lazarevic, V.

Publications and source records attributed to Lazarevic, V..

8 recordsLinked to original sources

bioRxiv2026

Engineering autologous ossicles for the personalized modeling of acute myeloid leukemia

Acute Myeloid Leukemia (AML) is the most lethal hematological malignancy in adults and is characterized by significant genetic and cytogenetic heterogeneity. This diversity drives substantial patient-to-patient variability in disease progression and therapeutic response. Increasing evidence indicates that bone marrow mesenchymal stem/stromal cells (MSCs) play a critical role in AML emergence, evolution and treatment resistance. However, progress in the field is challenged by the lack of experimental models supporting sustained AML survival while faithfully recapitulating the human bone marrow niche. Humanized ossicles (hOss) have recently emerged as ectopic human bone/bone marrow microenvironments composed of MSCs and hematopoietic elements, enabling improved AML engraftment in vivo. Nonetheless, those models typically exploit healthy MSCs largely because primary patient-derived cells exhibit diminished ossification potential. Here, we introduce OssiGel, a novel engineered human cartilage extracellular matrix produced by a mesenchymal cell line. We hypothesized that combining OssiGel with primary MSCs would enable the robust formation of hOss in healthy and malignant settings. We report that OssiGel supports fast and reproducible formation of hOss through endochondral ossification using MSCs isolated from both healthy and AML bone marrow samples. Within hOss, AML-MSCs were shown to persist long-term and to generate a human stromal microenvironment supporting the establishment of autologous AML hematopoietic cells, enabling the generation of autologous (autologOss). Single-cell RNA sequencing (scRNA-seq) revealed that autologOss recapitulate the majority of the AML blood populations observed in the corresponding patient bone marrow samples. Moreover, proof-of-concept sequencing of AML-MSCs from autologOss allowed predictive interrogation of interactions with leukemic populations and revealed the acquisition of an inflammatory-like phenotype, characteristic of AML. Taken together, our study establishes OssiGel as a novel platform for the robust engineering of autologous ossicles from AML patient samples. This provides a relevant tool to decipher patient-specific cellular interactions and drug responses, with potential translational value to broad hematological malignancies.

bioengineering↗

bioRxiv2026

Chromatin landscape and epigenetic heterogeneity of acute myeloid leukemia

Acute myeloid leukemia (AML) is an aggressive hematologic cancer characterized by proliferation of immature myeloblasts. It shows profound molecular heterogeneity, which has been primarily studied through genetic abnormalities, providing the basis for disease classification, prognostication, and therapeutic choice. However, genetic factors alone may not fully explain AML pathogenesis and diversity, while leaving the role of abnormal epigenome, particularly chromatin state, largely unexplored in a large cohort of patients. Here we show that AML is classified into 16 subgroups with distinct chromatin accessibility profiles based on ATAC-seq in 1,563 AML cases, including novel AML subgroups not previously recognized in conventional genomic classifications. By integrating multi-omics analyses of genome, transcriptome, and major histone marks, we show that these epigenetic subgroups exhibit unique features in clinical presentation, gene mutations, differentiation states, gene expression, and super-enhancer profiles, which are validated across independent cohorts. Single-cell sequencing demonstrates the presence of subgroup-specific ATAC signatures that are shared by all leukemic cells, confirming the definitive role of the epigenome in the ATAC-based classification. Mechanistically, each subgroup is associated with a distinct gene regulatory network centered on key transcription factors, where subgroup-specific super-enhancers play a pivotal role. These ATAC subgroups also have prognostic significance independent of genomic classification, and help reveal unexpected drug sensitivities. In summary, ATAC-based chromatin profiling in this large sample set, combined with multi-omics data, provides new insights into AML pathogenesis beyond genomic profiling and also serves as an invaluable resource for AML research.

cancer biology↗

bioRxiv2025

The cellular state space of AML unveils novel NPM1 subtypes with distinct clinical outcomes and immune evasion properties

Acute myeloid leukemia (AML) is a genetically and cellularly heterogeneous disease. We characterized 120 AMLs using genomic and transcriptomic analyses, including single-cell RNA sequencing. Our results reveal an extensive cellular heterogeneity that distorts the bulk transcriptomic profiles. Selective examination of the transcriptional signatures of >90,000 immature AML cells identified four main clusters, thereby extending current genomic classification of AML. Notably, NPM1 mutated AML could be stratified into two novel, clinically relevant classes, with NPM1class I associated with downregulation of MHC class II and excellent survival following hematopoietic stem cell transplantation (HSCT). NPM1class II was instead associated with resistance to allogeneic T cells in an ex vivo co culture assay, and importantly, dismal survival following HSCT. These findings provide new insights into the cellular state space of AML, define new diagnostic entities, and highlight potential therapeutic intervention points. Key PointsO_LIThe bulk transcriptional profiles of AML are mainly driven by a diverse set of cellular signatures. C_LIO_LISingle cell RNA-sequencing of the most common AML subtypes reveals marked heterogeneity extending beyond current genomic classification schemes. C_LIO_LINPM1-mutated AML can be divided into two new classes, with distinct immune evasion mechanisms and survival after transplantation. C_LI

cancer biology↗

bioRxiv2021

Bassoon controls synaptic vesicle pools via regulation of presynaptic phosphorylation and cAMP homeostasis

Neuronal presynaptic terminals contain hundreds of neurotransmitter-filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon featured a decreased SV release competence and increased resting pool of SV as observed by imaging of SV release in cultured neurons. Further analyses in vitro and in vivo revealed a dysregulation of CDK5/calcineurin and cAMP/PKA presynaptic signalling resulting in an aberrant phosphorylation of their downstream effectors synapsin 1 and SNAP25, which are well-known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalised the SV pools in neurons lacking bassoon. Finally, we demonstrated that CDK5-dependent regulation of PDE4 activity controls SV release competence by interaction with cAMP/PKA signalling. These data reveal that bassoon organises SV pools via regulation of presynaptic phosphorylation and indicate an involvement of PDE4 in the control of neurotransmitter release.

neuroscience↗

bioRxiv2020

Clinical metagenomics bioinformatics pipeline for the identification of hospital-acquired pneumonia pathogens antibiotic resistance genes from bronchoalveolar lavage samples

BackgroundShortening the time-to-result for pathogen detection and identification and antibiotic susceptibility testing for patients with Hospital-Acquired and Ventilator-Associated pneumonia (HAP-VAP) is of great interest. For this purpose, clinical metagenomics is a promising non-hypothesis driven alternative to traditional culture-based solutions: when mature, it would allow direct sequencing all microbial genomes present in a BronchoAlveolar Lavage (BAL) sample with the purpose of simultaneously identifying pathogens and Antibiotic Resistance Genes (ARG). In this study, we describe a new bioinformatics method to detect pathogens and their ARG with good accuracy, both in mono- and polymicrobial samples. MethodsThe standard approach (hereafter called TBo), that consists in taxonomic binning of metagenomic reads followed by an assembly step, suffers from lack of sensitivity for ARG detection. Thus, we propose a new bioinformatics approach (called TBwDM) with both models and databases optimized for HAP-VAP, that performs reads mapping against ARG reference database in parallel to taxonomic binning, and joint reads assembly. ResultsIn in-silico simulated monomicrobial samples, the recall for ARG detection increased from 51% with TBo to 97.3% with TBwDM; in simulated polymicrobial infections, it increased from 41.8% to 82%. In real sequenced BAL samples (mono and polymicrobial), detected pathogens were also confirmed by traditional culture approaches. Moreover, both recall and precision for ARG detection were higher with TBwDM than with TBo (35 points difference for recall, and 7 points difference for precision). ConclusionsWe present a new bioinformatics pipeline to identify pathogens and ARG in BAL samples from patients with HAP-VAP, with higher sensitivity for ARG recovery than standard approaches and the ability to link ARG to their host pathogens.

bioinformatics↗

bioRxiv2018

From genotype to antibiotic susceptibility phenotype in Enterobacteriaceae: a clinical perspective

Predicting the antibiotic susceptibility phenotype from genomic data is challenging, especially for some specific antibiotics in Enterobacteriaceae. Here we aimed to assess the performance of whole genomic sequencing (WGS) for predicting the antibiotic susceptibility in various Enterobacteriaceae species using the detection of antibiotic resistance genes (ARGs), specific mutations and a knowledge-based decision algorithm. We sequenced (Illumina MiSeq 2x250b) 187 clinical isolates from species possessing (n=98) or not (n=89) an intrinsic AmpC-type cephalosporinase. Antibiotic susceptibility was performed by the disc diffusion method. Reads were assembled by A5-miseq and ARGs were identified from the ResFinder database using Diamond. Mutations on GyrA and ParC topoisomerases were studied. We assessed the prediction rates for amoxicillin, co-amoxiclav, piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, meropenem, amikacin, gentamicin and fluoroquinolones. A total of 1,870 isolate/antibiotic combinations were considered. In 295 cases (15.8%) no attempt of prediction was made. Among the 1,575 attempts, 1,537 (97.6%) were correct (1,011 for predicting susceptibility and 526 for predicting resistance), 15 (0.9%) were major errors (MEs) and 23 (1.5%) were very major errors (VMEs). The concordance rates were similar between non-AmpC and AmpC-producing Enterobacteriaceae (907/935 [97.0%] vs 630/640 [98.4%], Chi2 test p=0.07), but more VMEs were observed in non-AmpC producing strains than in those producing an AmpC (20/935 [2.1%] vs 3/640 [0.5%], Chi2 test p=0.007). The majority of VMEs were putatively due to the overexpression of chromosomal genes. In conclusion, the inference of antibiotic susceptibility from genomic data showed good performances for non-AmpC and AmpC-producing Enterobacteriaceae species.

microbiology↗

bioRxiv2018

The intestinal microbiota predisposes to traveller’s diarrhoea and to the carriage of multidrug-resistant Enterobacteriaceae after travelling to tropical regions

The risk of acquisition of multidrug-resistant Enterobacteriaceae (MRE) and of occurrence of diarrhoea is high when travelling to tropical regions. The relationships between these phenomena and the composition of human gut microbiota have not yet been assessed. Here, we investigated the dynamics of changes of metabolically active microbiota by sequencing total RNA from faecal samples taken before and after travel to tropical regions. We found that the occurrence of diarrhoea during the travel was associated with a higher relative abundance of Prevotella copri before departure and after return. The composition of microbiota, before travel as well as at return, was not correlated with the acquisition of MRE. However, the clearance of MRE one month after return was linked to a specific pattern of bacterial species that was also found before and after return.

microbiology↗

bioRxiv2016

Clinical metagenomics of bone and joint infections: a proof of concept study

BackgroundBone and joint infections (BJI) are severe infections that require a tailored and protracted antibiotic treatment. The diagnostic of BJI relies on the culture of surgical specimens, yet some bacteria would not grow because of extreme oxygen sensitivity or fastidious growth. Hence, metagenomic sequencing could potentially address those limitations. In this study, we assessed the performances of metagenomic sequencing of BJI samples for the identification of pathogens and the prediction of antibiotic susceptibility.\n\nMethodsA total of 179 samples were considered. The DNA was extracted with a kit aiming to decrease the amount of human DNA (Molzym), and sequenced on an Illumina HiSeq2500 in 2x250 paired-end reads. The taxonomy was obtained by MetaPhlAn2, the bacterial reads assembled with MetaSPAdes and the antibiotic resistance determinants (ARDs) identified using a database made of Resfinder+ARDs from functional metagenomic studies.\n\nResultsWe could sequence the DNA from 24 out of 179 samples. For monomicrobial samples (n=8), the presence of the pathogen was confirmed by metagenomics in all cases. For polymicrobial samples (n=16), 32/55 bacteria (58.2%) were found at the species level (41/55 [74.5%] at the genus level). Conversely, a total of 273 bacteria not found in culture were identified, 182 being possible pathogens undetected in culture and 91 contaminants. A correct antibiotic susceptibility could be inferred in 94.1% cases for monomicrobial samples and in 76.5% cases in polymicrobial samples.\n\nConclusionsWhen sufficient amounts of DNA can be extracted from samples, we found that clinical metagenomics is a potential tool to support conventional culture.

microbiology↗