Biology subjects

Martin-Subero, J. I.

Publications and source records attributed to Martin-Subero, J. I..

12 recordsLinked to original sources

bioRxiv2026

Hierarchical classification of hematologic malignancies using epigenetic and genetic information

Molecular testing in hematology requires different assays for disease subgroup identification, risk stratification and selection of appropriate treatment regimens. Yet, molecular tests are not necessarily standardized between diagnostic laboratories, resulting in varying turnaround times and potentially divergent results. To resolve this issue and enable single-assay molecular testing, we have developed a hierarchical classification framework that combines epigenetic and genetic data from whole genome nanopore sequencing (WGNS) with machine learning to determine disease entities, epigenetic subgroups (epitypes) and genetic aberrations in hematopoietic neoplasms. We curated DNA methylation data from 5,420 samples and trained a classifier allowing entity-level diagnostics featuring 21 conditions, including healthy controls, acute and chronic myeloid and lymphoid neoplasms. This classifier was subsequently combined with entity-specific epitype classifiers predicting 44 therapeutically or prognostically relevant states, followed by integration of genetic data. Benchmarking of the combined (epi-)genetic testing strategy using WGNS confirmed high accuracy in the detection of diagnostic groups and risk stratification, and identified diagnosis-defining molecular alterations that were not reported by standard-of-care work-up.

cancer biology↗

bioRxiv2026

Mapping Disease Transitions from Premalignant, Asymptomatic to Advanced Myeloma through Integrative Epigenomic and Transcriptional Analyses

AbstractMultiple myeloma (MM) evolves from asymptomatic precursor conditions through progressive genetic and epigenetic remodeling, yet the regulatory mechanisms driving the development toward more active stages of the disease remain poorly understood. Here, we integrated bulk-based paired chromatin accessibility and activation, and transcriptomic profiling across disease stages to map regulatory remodeling during myeloma development. We identified a progressive increase in chromatin accessibility; furthermore, this epigenetic reconfiguration is accompanied by a stage-dependent shift from promoter-centered regulation in precursor states toward enhancer-dominated transcriptional control in active MM. Motif enrichment and regulatory network analyses identified both established and previously underappreciated transcription factors (TFs), including members of the IRF, MEF2, and FOX families, associated with disease-stage-specific transcriptional programs. Among these, MEF2D and FOXK2 emerged as candidate regulators of pathways involved in cell survival and chemotaxis. Functional perturbation demonstrated that MEF2D depletion markedly impaired MM cell viability, whereas inhibition of either MEF2D or FOXK2 reduced chemotactic migration. Together, these findings provide a stage-resolved framework of epigenetic and transcriptional remodeling across myeloma development, revealing regulatory programs established in precursor conditions and progressively reinforced during disease evolution, while identifying candidate transcriptional dependencies with potential biological and therapeutic relevance.

cancer biology↗

bioRxiv2025

Three-Dimensional Epigenome Roadmap of Human B-cell Differentiation Uncovers Mechanisms of Humoral Immunity and Oncogenesis

Human B-cell differentiation underlies humoral immunity, and its disruption leads to cancer, autoimmunity, and immunodeficiencies. However, the enhancer-based regulatory mechanisms governing this process remain poorly understood. To address this gap, we generated the first three-dimensional epigenomic roadmap of human B-cell differentiation in vivo. By profiling nine differentiation stages, we linked nearly half a million enhancers to their target genes through chromatin interactions, defined their activity states, and predicted bound transcription factors. We show that the acquisition of B-cell identity, distinct from common cellular processes, relies on large enhancer networks acting additively and synergistically to finely tune transcription. We further identify a 3D epigenetic priming mechanism underlying immune memory, whereby memory B cells retain primed DNA loops that enable faster responses to antigen re-exposure. Extending this roadmap to disease, we demonstrate that B-cell malignancies preserve enhancer signatures of their cell of origin while silencing distal tumor suppressor control. Finally, we uncover a previously unappreciated oncogenic mechanism in which intragenic deletions disrupt distal gene regulation via enhancer loss. Together, this resource provides a framework for understanding the genetic and epigenetic basis of humoral immunity and immune-related diseases.

genomics↗

bioRxiv2025

A transcriptional map of human tonsil architecture:beyond the sum of (single cell) parts

The tonsil is a highly compartmentalized organ in which different microanatomical structures orchestrate designated (immune) functions. We use this already well-studied tissue to survey spatial molecular imaging data (CosMx SMI) for studying immune responses in native tissue context; and, to demonstrate the advantages of SMI for faithfully recapitulating cellular composition in direct comparison with single-cell RNA sequencing. While SMI data still poses many analytical challenges and lacks standardization, we established a versatile analysis pipeline focused on the profitable particularities of these data: considering organization (microenvironment), interactions (signaling), and function (higher-order structures) across scales. Specifically, we resolve ~ 2M cells into 52 subpopulations across immune and, in particular, structural compartments. Various spatial niches partition tonsillar tissue into architecturally and functionally distinct regions, which we characterize through cell-cell colocalization and communication analyses, while performing various non-standard analyses at the level of spatial features. These topological readouts may help elucidate where certain immunological processes occur (e.g., class switch recombination); and, where signaling pathways are active (e.g., TNF and galectin, which have been implicated in diverse lymphomas). In all, we provide an analytical framework for Spatial Immunology, and showcase alternative views that such techniques and concomitant computational approaches can bring on tissue composition and architecture.

immunology↗

bioRxiv2023

Uncovering the cis-regulatory program of early human B-cell commitment and its implications in the pathogenesis of B-cell acute lymphoblastic leukemia

Dysregulation of the early stages of B-cell lymphopoiesis, orchestrating the development of cellular immunity, may induce malignant transformations. Therefore, it is essential to characterize the gene regulatory network (GRN) driving B-cell lymphopoiesis in healthy individuals to uncover malignancy mechanisms. To this end, we generated a dataset that included paired human data for chromatin accessibility and gene expression in eight B-cell precursor stages, providing the first deep characterization of early B-cell lymphopoiesis, including the identification of regulatory elements and the reconstruction of the GRN. Using this data, we recapitulated well-known regulatory elements and revealed new regulons, such as ELK3, enriched in pro-B cells with a putative role in cell cycle progression. Moreover, a single-cell multi-omics analysis validated and enhanced the resolution of the regulatory landscape recovered by bulk data, revealing MYBL2 and ZNF367 as specific regulons of cycling cell states, and CEBPA associated with lymphoid multipotent progenitors (LMPPs). Importantly, this dataset enabled us to uncover B-cell acute lymphoblastic leukemia (B-ALL) triggers. We identified different cellular origins of malignant transformation depending on the B-ALL subtype, including the association of the ETV6-RUNX1 with pro-B cells and the increased expression of ELK3 in this ALL subtype. Overall, our dataset provides the most comprehensive atlas to date of early human B-cell regulation (B-rex; https://translationalbio.shinyapps.io/brex/), facilitating further understanding of B-cell differentiation in health and disease.

immunology↗

bioRxiv2022

An Atlas of Cells in the Human Tonsil

Palatine tonsils are secondary lymphoid organs representing the first line of immunological defense against inhaled or ingested pathogens. Here, we present a comprehensive census of cell types forming the human tonsil by applying single-cell transcriptome, epigenome, proteome and adaptive immune repertoire sequencing as well as spatial transcriptomics, resulting in an atlas of >357,000 cells. We provide a glossary of 121 annotated cell types and states, and disentangle gene regulatory mechanisms that drive cells through specialized lineage trajectories. Exemplarily, we stratify multiple tonsil-resident myeloid slancyte subtypes, establish a distant BCL6 superenhancer as locally active in both follicle-associated T and B cells, and describe SIX5 as a potentially novel transcriptional regulator of plasma cell maturation. Further, our atlas is a reference map to understand alterations observed in disease. Here, we discover immune-phenotype plasticity in tumoral cells and microenvironment shifts of mantle cell lymphomas (MCL). To facilitate such reference-based analysis, we develop HCATonsilData and SLOcatoR, a computational framework that provides programmatic and modular access to our dataset; and allows the straightforward annotation of future single-cell profiles from secondary lymphoid organs.

immunology↗

bioRxiv2021

NOTCH1 drives immune-escape mechanisms in B cell malignancies

NOTCH1 is a recurrently mutated gene in Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL). Functional studies to investigate its role have been hampered by the inability to genetically manipulate primary human lymphoma cells, attributed to low transduction-efficacy and procedure-associated toxicity. To overcome these limitations, we have developed a novel method to retrovirally transfer genes into malignant human B cells. We generated isogenic human tumor cells from patients with CLL and MCL, differing only in their expression of NOTCH1. Our data demonstrate that NOTCH1 facilitates immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-{gamma} signaling. In addition, NOTCH1 causes silencing of the entire HLA-class II locus via suppression of the transcriptional co-activator CIITA. These NOTCH1-mediated immune escape mechanisms are associated with the expansion of CD4+ T cells in vivo, further contributing to the poor clinical outcome of NOTCH1-mutated CLL and MCL.

cancer biology↗

bioRxiv2020

The proliferative history shapes the DNA methylome of B-cell tumors and predicts clinical outcome

We report a systematic analysis of the biological and clinical implications of DNA methylation variability in five categories of B-cell tumors derived from B cells spanning the entire maturation spectrum. We used 2056 primary samples including training and validation series and show that 88% of the human DNA methylome is dynamically modulated under normal and neoplastic conditions. B-cell tumors display both epigenetic imprints of their cellular origin and de novo, disease-specific epigenetic alterations that in part are related to differential transcription factor binding. These differential methylation patterns were used by a machine-learning approach to create a diagnostic algorithm that accurately classifies 14 B-cell tumor entities and subtypes with different clinical management. Beyond this, we identified extensive patient-specific epigenetic variability targeting constitutively silenced chromatin regions, a phenomenon we could relate to the proliferative history of normal and neoplastic B cells. We observed that, depending on the maturation stage of the tumor cell of origin, mitotic activity leaves different imprints into the DNA methylome. Subsequently, we constructed a novel DNA methylation-based mitotic clock called epiCMIT (epigenetically-determined Cumulative MIToses), whose lapse magnitude represents a strong independent prognostic variable within specific B-cell tumor subtypes and is associated with particular driver genetic alterations. Our findings reveal DNA methylation as a holistic tracker of B-cell tumor developmental history, with implications in the differential diagnosis and prediction of the outcome of the patients.

cancer biology↗

bioRxiv2020

Sampling artifacts in single-cell genomics cohort studies

Robust protocols and automation now enable large-scale single-cell RNA and ATAC sequencing experiments and their application on biobank and clinical cohorts. However, technical biases introduced during sample acquisition can hinder solid, reproducible results and a systematic benchmarking is required before entering large-scale data production. Here, we report the existence and extent of gene expression and chromatin accessibility artifacts introduced during sampling and identify experimental and computational solutions for their prevention.

genomics↗

bioRxiv2019

Dynamics of genome architecture and chromatin function during human B cell differentiation and neoplastic transformation

Despite recent advances, the dynamics of genome architecture and chromatin function during human cell differentiation and its potential reorganization upon neoplastic transformation remains poorly characterized. Here, we integrate in situ Hi-C and nine additional omic layers to define and biologically characterize the dynamic changes in three-dimensional (3D) genome architecture across normal B cell differentiation and in neoplastic cells from different subtypes of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) patients. Beyond conventional active (A) and inactive (B) compartments, an integrative analysis of Hi-C data reveals the presence of a highly-dynamic intermediate compartment enriched in poised and polycomb-repressed chromatin. During B cell development, we detect that 28% of the compartments change at defined maturation stages and mostly involve the intermediate compartment. The transition from naive to germinal center B cells is associated with widespread chromatin activation, which mostly reverts into the naive state upon further maturation of germinal center cells into memory B cells. The analysis of CLL and MCL neoplastic cells points both to entity and subtype-specific alterations in chromosome organization. Remarkably, we observe that large chromatin blocks containing key disease-specific genes alter their 3D genome organization. These include the inactivation of a 2Mb region containing the EBF1 gene in CLL and the activation of a 6.1Mb region containing the SOX11 gene in clinically aggressive MCL. This study indicates that 3D genome interactions are extensively modulated during normal B cell differentiation and that the genome of B cell neoplasias acquires a tumor-specific 3D genome architecture.

genomics↗

bioRxiv2019

Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting co-regulated adjacent genes. Among target genes upregulated by this process, we found members of the NOTCH, NFkB, mTOR1 signaling and p53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status, and in addition identified PRDM5 as a novel essential gene for MM. Collectively our data indicates that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.

cancer biology↗

bioRxiv2019

Differential genomic and transcriptomic events associated with high-grade transformation of Chronic Lymphocytic Leukemia

The transformation of chronic lymphocytic leukemia (CLL) to high-grade diffuse large B-cell lymphoma (DLBCL), also called Richters Syndrome (RS), is a rare cancer with dismal prognosis. Drug discovery for RS is hampered by the lack of suitable experimental models, and effective therapies remain elusive rendering RS an area of high unmet clinical need. We performed whole genome sequencing (WGS) to interrogate paired CLL and RS samples from 17 patients enrolled in a prospective multicenter Phase 2 clinical trial (CHOP-OR) and we found that subclones affected by mutations in MAPK and PI3K pathways show a high expansion probability during transformation. We also demonstrate for the first time that non-coding mutation clusters in a PAX5 enhancer, situated 330kb upstream from the transcription initiation site, correlate with transformation. Finally, we confirm our findings by employing targeted DNA sequencing (TGS) and RNA expression profiling on an extended cohort of 38 patients.\n\nStatement of significanceThrough integrated analysis of WGS, TGS and RNA expression data, we identified drivers of transformation not previously implicated in RS, which can be targeted therapeutically and tested in the clinic. Our results have informed the design of a new clinical platform study, which is now open to recruitment in the UK.

genomics↗