Biology subjects

Lupien, M.

Publications and source records attributed to Lupien, M..

At least 19 recordsLinked to original sources

bioRxiv2026

KDM6B interacts with nucleo-adhesome components CSRP2 and TGFB1I1 to regulate EMT

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.

cell biology↗

bioRxiv2025

Genetic and Epigenetic Reprogramming of Transposable Elements Drives ecDNA-Mediated Metastatic Prostate Cancer

Extrachromosomal DNAs (ecDNAs), which replicate and segregate in a non-Mendelian manner, serve as vectors for accelerated tumor evolution. By integrating chromatin accessibility, whole-genome sequencing, and Hi-C-based genome topology data from a cohort of metastatic Castration-Resistant Prostate Cancer (mCRPC) cases, we show that epigenetically activated repeat DNA, amplified in ecDNAs, drive oncogene overexpression. Specifically, we identify a subgroup of mCRPCs (20%) characterized by clusters of accessible LINE1 repeat DNA elements flanking the androgen receptor (AR) gene. These LINE1 elements are co-amplified with AR and provide binding sites for prostate-lineage transcription factors, including AR, FOXA1 and HOXB13. Accessible LINE1 elements establish novel 3D chromatin interactions with the AR gene, forging a new regulatory plexus driving AR overexpression and confers resistance to androgen signaling inhibitors. Our findings indicate how tumor evolution is driven by the convergence of genetic and epigenetic alterations on repeat DNA, activating and amplifying them to allow oncogene overexpression. Statement of significanceWe show how tumor evolution is driven by the convergence of genetic and epigenetic alterations on repeat DNA elements, resulting in their activation as regulatory elements and co-amplification in ecDNAs with oncogenes in mCRPC.

cancer biology↗

bioRxiv2025

iSHARC: Integrating scMultiome data for heterogeneity and regulatory analysis in cancer

SummaryThe 10x Genomics single cell Multiome (scMultiome) assay enables the simultaneous profiling of chromatin accessibility and gene expression from the same nucleus, and has increasingly been utilized in revealing cellular heterogeneity and gene regulation in cancers. However, a dedicated bioinformatics pipeline specifically designed for this type of data is still lacking. Here we present iSHARC, a streamlined pipeline for quality control, modality integration, clustering, cell type annotation, and regulatory mechanism analysis of individual scMultiome data, as well as for integrating multiple samples. The main advantages of iSHARC are: 1) easy implementation, execution and extension through a modular Snakemake workflow management system; 2) flexible analysis and parameters customization via a single configuration file; and 3) comprehensive accessibility by providing different access points to results and detailed summary reports from a single run. Availability and implementationThis pipeline is an open-source software under the MIT license and it is freely available at https://github.com/yzeng-lol/iSHARC. Contactyong.zeng@uhn.ca or hansen.he@uhn.ca or mathieu.lupien@uhn.ca Supplementary informationSupplementary data are appended.

bioinformatics↗

bioRxiv2024

Distinct gene regulatory networks govern hematopoietic and leukemia stem cells

The underlying gene regulatory networks (GRN) that govern leukemia stem cells (LSC) in acute myeloid leukemia (AML) and hematopoietic stem cells (HSC) are not well understood. Here, we identified GRNs by integrating gene expression (GE) and chromatin accessibility data derived from functionally defined cell populations enriched for HSC and LSC. We analyzed n=32 LSC+ and n=32 LSC-cell fractions from n=22 AML patients, along with n=7 stem and n=10 progenitor enriched cell populations sorted from human umbilical cord blood (hUCB), producing a database of n{approx}17,000 transcription factor (TF) regulatory interactions for hUCB-HSPC and AML. We developed an iterative algorithm that associates the degree of chromatin openness with TF binding preferences, and the GE of candidate TF and target genes within 100kb upstream of transcription start sites. A putative regulatory structure was found to be enriched in HSC-enriched cell populations, comprising TF-target gene interactions between ETS1, EGR1, RUNX2, and ZNF683 oriented in a self-reinforcing configuration. A regulatory loop comprising FOXK1 and MEIS1, rather than the 4-factor HSC subnetwork, was detected in the LSC-specific GRN. The core HSC and LSC TF networks were extended using protein-protein interaction (PPI) data to determine connectivity with interacting genes whose expression strongly associated with LSC/HSC frequency estimates, producing a database of n=103,516 PPI target pathways. The effect of perturbing genes along the identified pathways on functional HSC and LSC frequency was predicted based on statistical regression analyses. To validate GRN predictions, we used pharmacologic and CRISPR targeting, in addition to re-examining published functional data associated with several network nodes that were predicted to impact stemness. Notably, we found that inhibition of CDK6 in AML samples markedly reduced LSC numbers as assessed in de novo serial xenotransplantation studies (fold change {approx} 10), as predicted by the LSC GRN model. Additionally, in-house CRISPR-based knockdown of ETS1 resulted in a significant decrease in HSC quiescence-associated microRNA-126 expression, and increased HSC frequency. Taken together, our models provide a comprehensive view of the underlying regulatory structures governing functional human HSC and LSC. This approach has translational potential as it can be used as a high-throughput in-silico screening tool for the systematic identification of gene targets for LSC elimination and HSC expansion.

bioinformatics↗

bioRxiv2024

Identification of leukemia stem cell subsets with distinct transcriptional, epigenetic and functional properties

The leukemia stem cell (LSC) compartment is a complex reservoir fueling disease progression in acute myeloid leukemia (AML). The existence of heterogeneity within this compartment is well documented but prior studies have focused on genetic heterogeneity without being able to address functional heterogeneity. Understanding this heterogeneity is critical for the informed design of therapies targeting LSC, but has been hampered by LSC scarcity and the lack of reliable cell surface markers for viable LSC isolation. To overcome these challenges, we turned to the patient-derived OCI-AML22 cell model. This model includes functionally, transcriptionally and epigenetically characterized LSC broadly representative of LSC found in primary AML samples. Focusing on the pool of LSC, we used an integrated approach combining xenograft assays with single-cell analysis to identify two LSC subtypes with distinct transcriptional, epigenetic and functional properties. These LSC subtypes differed in depth of quiescence, differentiation potential and repopulation capacity and could be isolated based on CD112 expression. A majority of AML patient samples had transcriptional signatures reflective of either LSC subtype, and some even showed coexistence within an individual sample. This work provides a framework for further investigation of the LSC compartment in AML.

cancer biology↗

bioRxiv2023

Simulating cell-free chromatin using preclinical models for cancer-specific biomarker discovery

Cell-free chromatin (cf-chromatin) is a rich source of biomarkers across various conditions, including cancer. Tumor-derived circulating cf-chromatin can be profiled for epigenetic features, including nucleosome positioning and histone modifications that govern cell type-specific chromatin conformations. However, the low fractional abundance of tumor-derived cf-chromatin in blood and constrained access to plasma samples pose challenges for epigenetic biomarker discovery. Conditioned media from preclinical tissue culture models could provide an unencumbered source of pure tumor-derived cf-chromatin, but large cf-chromatin complexes from such models do not resemble the nucleosomal structures found predominantly in plasma, thereby limiting the applicability of many analysis techniques. Here, we developed a robust and generalizable framework for simulating cf-chromatin with physiologic nucleosomal distributions using an optimized nuclease treatment. We profiled the resulting nucleosomes by whole genome sequencing and confirmed that inferred nucleosome positioning reflected gene expression and chromatin accessibility patterns specific to the cell type. Compared with plasma, simulated cf-chromatin displayed stronger nucleosome positioning patterns at genomic locations of accessible chromatin from patient tissue. We then utilized simulated cf-chromatin to develop methods for genome-wide profiling of histone post-translational modifications associated with heterochromatin states. Cell-free chromatin immunoprecipitation and sequencing (cf-ChIP-Seq) of H3K27me3 identified heterochromatin domains associated with repressed gene expression, and when combined with H3K4me3 cfChIP-Seq revealed bivalent domains consistent with an intermediate state of transcriptional activity. Combining cfChIP-Seq of both modifications provided more accurate predictions of transcriptional activity from the cell of origin. Altogether, our results demonstrate the broad applicability of preclinical simulated cf-chromatin for epigenetic liquid biopsy biomarker discovery.

genomics↗

bioRxiv2022

A primary patient-derived model for investigating functional heterogeneity within the human Leukemic Stem Cell Compartment

The ability of leukemic stem cells (LSC) to evade therapy and fuel leukemic progression causing relapse impedes therapeutic success in acute myeloid leukemia (AML). The LSC pool within a patient sample is not homogenous but comprises distinct LSC subsets that vary in self-renewal and propagation properties. The stemness programs that underlie LSC types are poorly understood since human LSC studies require primary patient samples where LSC numbers are low and isolation methods impure. To overcome these challenges, we developed a patient-derived AML model system (OCI-AML22) displaying a functionally, transcriptionally and epigenetically defined cellular hierarchy driven by functional LSCs that can be immunophenotypically identified and isolated. Through single cell and functional approaches, the OCI-AML22 LSC fraction was found to contain distinct LSCs that vary in proliferative and differentiation properties. OCI-AML22 represents a valuable resource to decipher mechanisms driving stemness and the multiple layers of heterogeneity within LSCs.

cancer biology↗

bioRxiv2022

PRMT5 regulates ATF4 transcript splicing and oxidative stress response

Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.

cancer biology↗

bioRxiv2021

Non-coding mutations reveal cancer driver cistromes in luminal breast cancer

Whole genome sequencing of primary breast tumors enabled the identification of cancer driver genes 1,2 and non-coding cancer driver plexuses from somatic mutations 3-6. However, differentiating driver and passenger events among non-coding genetic variants remains a challenge to understand the etiology of cancer and inform delivery of personalized cancer medicine. Herein, we reveal an enrichment of non-coding mutations in cis-regulatory elements that cover a subset of transcription factors linked to tumor progression in luminal breast cancers. Using a cohort of 26 primary luminal ER+PR+ breast tumors, we compiled a catalogue of [~]100,000 unique cis-regulatory elements from ATAC-seq data. Integrating this catalogue with somatic mutations from 350 publicly available breast tumor whole genomes, we identified four recurrently mutated individual cis-regulatory elements. By then partitioning the non-coding genome into cistromes, defined as the sum of binding sites for a transcription factor, we uncovered cancer driver cistromes for ten transcription factors in luminal breast cancer, namely CTCF, ELF1, ESR1, FOSL2, FOXA1, FOXM1 GATA3, JUND, TFAP2A, and TFAP2C in luminal breast cancer. Nine of these ten transcription factors were shown to be essential for growth in breast cancer, with four exclusive to the luminal subtype. Collectively, we present a strategy to find cancer driver cistromes relying on quantifying the enrichment of non-coding mutations over cis-regulatory elements concatenated into a functional unit drawn from an accessible chromatin catalogue derived from primary cancer tissues.

cancer biology↗

bioRxiv2021

Dichotomous regulation of lysosomes by MYC and TFEB controls hematopoietic stem cell fate

It is critical to understand how quiescent long-term hematopoietic stem cells (LT-HSC) sense demand from daily and stress-mediated cues and transition into bioenergetically active progeny to differentiate and meet these cellular needs. Here, we show that lysosomes, which are sophisticated nutrient sensing and signaling centers, are dichotomously regulated by the Transcription Factor EB (TFEB) and MYC to balance catabolic and anabolic processes required for activating LT-HSC and guiding their lineage fate. TFEB-mediated induction of the endolysosomal pathway causes membrane receptor degradation, limiting LT-HSC metabolic and mitogenic activation, which promotes quiescence, self-renewal and governs erythroid-myeloid commitment. By contrast, MYC engages biosynthetic processes while repressing lysosomal catabolism to drive LT-HSC activation. Collectively, our study identifies lysosomes as a central regulatory hub for proper and coordinated stem cell fate determination.

cell biology↗

bioRxiv2021

Accessibility Over Transposable Elements Reveals Genetic Determinants of Stemness Properties in Normal and Leukemic Hematopoiesis

Despite most acute myeloid leukemia (AML) patients achieving complete remission after induction chemotherapy, two-thirds will relapse with fatal disease within five years. AML is organized as a cellular hierarchy sustained by leukemia stem cells (LSC) at the apex, with LSC properties directly linked to tumor progression, therapy failure, and disease relapse 1-5. Despite the central role of LSC in poor patient outcomes, little is known about the genetic determinants driving their stemness properties. As LSCs share many functional and molecular properties with normal hematopoietic stem cells (HSC) 6, we investigated accessible chromatin unique across normal hematopoietic and cancer cell states and identified transposable elements (TEs) as genetic determinants of both primitive populations in comparison with their downstream mature progeny. A clinically-relevant TE chromatin accessibility-based LSCTE121 signature was developed that enabled patient classification based on survival outcomes. Through functional assays, primitive cell specific-TE subfamilies were found to serve as docking sites for stem cell-associated regulators of genome topology or lineage-specific transcription factors, including LYL1 in LSCs. Finally, using chromatin editing tools, we establish that chromatin accessibility at LTR12C elements in LSCs are necessary to maintain stemness properties. Our work identifies TEs as genetic drivers of primitive versus mature cell states, where distinct TE subfamilies account for stemness properties in normal versus leukemic hematopoietic stem cells.

cancer biology↗

bioRxiv2021

Cis-Regulatory Element Hijacking by Structural Variants Overshadows Topological Changes in Primary Prostate Cancer

Prostate cancer is a heterogeneous disease whose progression is linked to genome instability. However the impact of this instability on the three-dimensional chromatin organization and how this drives progression is unclear. Using primary benign and tumour tissue, we find a high concordance in the higher-order three-dimensional genome organization across normal and prostate cancer cells. This concordance argues for constraints to the topology of prostate tumour genomes. Nonetheless, we identify changes to focal chromatin interactions and show how structural variants can induce these changes to guide cis-regulatory element hijacking. Such events result in opposing differential expression on genes found at antipodes of rearrangements. Collectively, our results argue that cis-regulatory element hijacking from structural variant-induced altered focal chromatin interactions overshadows higher-order topological changes in the development of primary prostate cancer.

cancer biology↗

bioRxiv2020

Viral integration transforms chromatin to drive oncogenesis

Human papillomavirus (HPV) drives almost all cervical cancers and up to [~]70% of head and neck cancers. Frequent integration into the host genome occurs only for tumourigenic strains of HPV. We hypothesized that changes in the epigenome and transcriptome contribute to the tumourigenicity of HPV. We found that viral integration events often occurred along with changes in chromatin state and expression of genes near the integration site. We investigated whether introduction of new transcription factor binding sites due to HPV integration could invoke these changes. Some regions within the HPV genome, particularly the position of a conserved CTCF binding site, showed enriched chromatin accessibility signal. ChIP-seq revealed that the conserved CTCF binding site within the HPV genome bound CTCF in 4 HPV+ cancer cell lines. Significant changes in CTCF binding pattern and increases in chromatin accessibility occurred exclusively within 100 kbp of HPV integration sites. The chromatin changes co-occurred with out-sized changes in transcription and alternative splicing of local genes. We analyzed the essentiality of genes upregulated around HPV integration sites of The Cancer Genome Atlas (TCGA) HPV+ tumours. HPV integration upregulated genes which had significantly higher essentiality scores compared to randomly selected upregulated genes from the same tumours. Our results suggest that introduction of a new CTCF binding site due to HPV integration reorganizes chromatin and upregulates genes essential for tumour viability in some HPV+ tumours. These findings emphasize a newly recognized role of HPV integration in oncogenesis.

cancer biology↗

bioRxiv2019

GLUT1 inhibition blocks growth of RB1-positive Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data implicates E2F Targets pathway activity as a surrogate of OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) are strongly correlated with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.

cancer biology↗

bioRxiv2018

High-resolution structural genomics reveals new therapeutic vulnerabilities in glioblastoma

We investigated the role of 3D genome architecture in instructing functional properties of glioblastoma stem cells (GSCs) by generating the highest-resolution 3D genome maps to-date for this cancer. Integration of DNA contact maps with chromatin and transcriptional profiles identified specific mechanisms of gene regulation, including individual physical interactions between regulatory regions and their target genes. Residing in structurally conserved regions in GSCs was CD276, a gene known to play a role in immuno-modulation. We show that, unexpectedly, CD276 is part of a stemness network in GSCs and can be targeted with an antibody-drug conjugate to curb self-renewal, a key stemness property. Our results demonstrate that integrated structural genomics datasets can be employed to rationally identify therapeutic vulnerabilities in self-renewing cells.\n\nSIGNIFICANCEIn adult GBM, GSCs act as therapy-resistant reservoirs to nucleate tumor recurrence. New therapeutic approaches that target these cell populations hold the potential of significantly improving patient care and overall prognosis for this always-lethal cancer. Our work describes new links between 3D genome architecture and stemness properties in GSCs. In particular, through integration of multiple genomics and structural genomics datasets, we found an unexpected connection between immune-related genes and self-renewal programs in GBM. Among these, we show that targeting CD276 with knockdown strategies or specific antibody-drug conjugates achieve suppression of self-renewal. Strategies to target CD276+ cells are currently in clinical trials for solid tumors. Our results indicate that CD276-targeting agents could be deployed in GBM to specifically target GSC populations.\n\nHIGHLIGHTSO_LIWe generated high (sub-5 kb) resolution Hi-C maps for stem-like cells from GBM patients.\nC_LIO_LIIntegration of Hi-C and genomics datasets dissects mechanisms of gene regulation.\nC_LIO_LI3D genomes poise immune-related genes, including CD276, for expression.\nC_LIO_LITargeting CD276 curbs self-renewal properties of GBM cells.\nC_LI

cancer biology↗

bioRxiv2018

A Chemical Toolbox for the Study of Bromodomains and Epigenetic Signaling

Bromodomains (BRDs) are evolutionary conserved epigenetic protein interaction modules which recognize (\"read\") acetyl-lysine, however their role(s) in regulating cellular states and their potential as targets for the development of targeted treatment strategies is poorly understood. Here we present a set of 25 chemical probes, selective tool small molecule inhibitors, covering 29 human bromodomain targets. We comprehensively evaluate the selectivity of this probe-set using BROMOscan(R) and demonstrate the utility of the set using studies of muscle cell differentiation and triple negative breast cancer (TNBC). We identified cross talk between histone acetylation and the glycolytic pathway resulting in a vulnerability of TNBC cell lines to inhibition of BRPF2/3 BRDs under conditions of glucose deprivation or GLUT1 inhibition. This chemical probe set will serve as a resource for future applications in the discovery of new physiological roles of bromodomain proteins in normal and disease states, and as a toolset for bromodomain target validation.

cell biology↗

bioRxiv2018

Chromatin Blueprint Of Glioblastoma Stem Cells Reveals Common Drug Candidates For Distinct Subtypes

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as Glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.

cancer biology↗

bioRxiv2018

Metabolic adaptations underlie epigenetic vulnerabilities in chemoresistant breast cancer.

Cancer cell survival upon cytotoxic drug exposure leads to changes in cell identity, dictated by the epigenome. Several metabolites serve as substrates or co-factors to chromatin-modifying enzymes, suggesting that metabolic changes can underlie change in cell fate. Here, we show that progression of triple-negative breast cancer (TNBC) to taxane-resistance is characterized by altered methionine metabolism and S-adenosylmethionine (SAM) availability, giving rise to DNA hypomethylation in regions enriched for transposable elements (TE). Compensatory redistribution of H3K27me3 forming Large Organized Chromatin domains of lysine (K) modification (LOCK) prevents expression of TE in taxane-resistant cells. Pharmacological inhibition of EZH2, the H3K27me3 methyltransferase, alleviates TE repression, leading to the accumulation of dsRNA and activation of the interferon viral mimicry-response, specifically inhibiting the growth of taxane-resistant TNBC. Together, our work delineates a role for metabolic adaptations in redefining the epigenome of taxane-resistant TNBC cells and underlies an epigenetic vulnerability toward pharmacological inhibition of EZH2.

genomics↗