Biology subjects

Verhoeyen, E.

Publications and source records attributed to Verhoeyen, E..

9 recordsLinked to original sources

bioRxiv2025

IKAROS Gene Regulatory Network Reveal ERG as a Vulnerability in B-cell Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is driven by transcriptional dysregulation that impairs B-cell differentiation and sustains leukemic growth. A defining feature of high-risk B-ALL is mutations in IKZF1, which encodes the tumor suppressor IKAROS. Here, we map IKAROS gene regulatory networks in IKZF1-mutated Ph B-ALL using an inducible IKAROS system and multi-omic profiling. IKAROS restoration reprograms chromatin accessibility and transcriptional control, shifting regulation from an ETS-dominated state to one enriched for B-cell lineage factors. Among repressed transcription factors, we identify ERG as a key regulatory node directly bound and antagonized by IKAROS. IKAROS binds regulatory elements near ERG and other progenitor-associated genes, coinciding with reduced ERG expression and repression of transcriptional programs linked to early B-cell developmental stages. Analysis of single-cell multiome data from human B-cell progenitors shows that ERG and IKAROS have opposing stage-specific activities and identifies a developmental stage-specific regulatory region in ERG intron 3 which is bound by IKAROS, and functionally important for ERG gene expression. Functional assays using CRISPRi and ETS inhibitors, along with gene dependency data from DepMap, confirm ERG dependency in IKZF1-deficient B-ALL. Our findings identify ERG as a context-specific dependency in IKZF1-deficient B-ALL, providing a mechanistic basis for the observed mitigation of poor prognosis for IKZF1-mutation in patients with co-occurring ERG deletions.

cancer biology↗

bioRxiv2025

CAR T cells targeting Nectin-4 safely overcome resistance to anti-Nectin-4 antibody-drug conjugate in solid tumors

Chimeric antigen receptor T (CAR T) cells represent a promising therapeutic option for a variety of cancers, including solid tumors. Nectin-4 is a cell adhesion molecule expressed at different levels in many solid tumors, including breast and urothelial carcinoma (UC). Enfortumab vedotin (EV), an antibody-drug conjugate (ADC) against Nectin-4, has significantly improved survival in patients with metastatic UC. Skin adverse reactions are frequently observed due to Nectin-4 expression in the epidermis. Here, we developed second-generation CAR T cells against Nectin-4 (N4CART) with a scFv that does not recognize human Nectin-4 expressed in skin keratinocytes. To study the effects of N4CART cell therapy, we used preclinical models of cell-derived xenografts (CDX) and patient-derived xenograft (PDX) of breast cancer expressing moderate to high levels of Nectin-4. We showed a marked efficacy with induction of remissions. Interestingly, N4CART cells kill Nectin-4-positive breast, urothelial and colon tumor cells, which are resistant to EV. Thus, N4CART cells represent a valuable and safe therapy for the treatment of patients with Nectin-4 expressing tumors, including those that are resistant to EV. Finally, baboon-envelope pseudotyped lentiviral vectors (LV) outperformed VSVG-LVs for Nectin-4 CAR expressing in {beta} T cells resulting in an efficient anti-tumoral response in PDX mice.

immunology↗

bioRxiv2025

Development of CRISPR/Cas13-based analytical tools to study RNA-Protein Interactions

RNA-protein interactions (RPIs) are as important as protein-protein interactions (PPIs) for the formation of membraneless organelles (MLOs) and play a vital role in various biological processes. Despite remarkable advances in PPI analysis technologies in recent years, the development of RPI analysis tools has lagged behind. To advance RPI analysis, we integrated three established PPI tools--bimolecular fluorescence complementation (BiFC), NanoBiT, and split-TurboID--with the RNA-targeting CRISPR/Cas13. We applied these tools to analyze paraspeckles, one of the best known MLOs formed by interactions between the long non-coding RNA NEAT1 and the RNA-binding protein NONO. The optimized BiFC-dCas13 allows live cell imaging and quantitative detection of the NEAT1-NONO interaction. The NanoBiT-dCas13 detects dynamic changes in the NEAT1-NONO interaction in an immediate and reversible manner. As a proximity labeling tool, the Split-TurboID-dCas13 induces biotinylation of proteins surrounding paraspeckles, leading to the identification of the N6-methyladenosine reader protein YTHDC1 as a novel paraspeckles-associated protein. The BiFC-dCas13, NanoBiT-dCas13, and Split-TurboID-dCas13 systems have a broad utility for the analysis of RPIs and MLOs.

bioengineering↗

bioRxiv2023

ATP citrate lyase is an essential player of the metabolic rewiring induced by PTEN loss during T-ALL development.

Alterations inactivating the tumor suppressor gene PTEN drive the development of solid and hematological cancers, such as T-cell acute lymphoblastic leukemia (T-ALL), whereby PTEN loss defines poor-prognosis patients. We investigated the metabolic rewiring induced by PTEN loss in T-ALL, aiming at identifying novel metabolic vulnerabilities. We showed that the enzyme ATP citrate lyase (ACLY) is strictly required for the transformation of thymic immature progenitors and for the growth of human T-ALL, which remain dependent on ACLY activity even upon transformation. Whereas PTEN mutant mice all died within 17 weeks, the concomitant ACLY deletion prevented disease initiation in 70% of the animals, where it prevented the apoptosis of pre-malignant DP thymocytes by up-regulating BCL-2. Transcriptomic and metabolic analysis of primary T-ALL cells next translated our findings to the human pathology, showing that PTEN-altered T-ALL cells selectively activate ACLY and are specifically sensitive to its genetic targeting. ACLY activation thus represents a metabolic vulnerability with therapeutic potential for high-risk T-ALL patients.

cancer biology↗

bioRxiv2022

Distinct Subsets of Multi-Lymphoid Progenitors Support Ontogeny-Related Changes in Human Lymphopoiesis

Changes in lymphocyte production patterns occurring across human ontogeny remain poorly defined. In this study, we demonstrate that human lymphopoiesis is supported by three waves of embryonic, fetal, and postnatal multi-lymphoid progenitors (MLPs) differing in CD7 and CD10 expression and their output of CD127-/+ early lymphoid progenitors (ELP). Our results reveal that, like the fetal-to-adult switch in erythropoiesis, transition to postnatal life coincides with a shift from multilineage to B lineage-biased lymphopoiesis and an increase in production of CD127+ ELPs which persists until puberty. A further developmental transition is observed in elderly individuals where B-cell differentiation bypasses the CD127+ compartment and branches directly from CD10+ MLPs. Functional analyses indicate that these changes are determined at the level of the hematopoietic stem cell. Besides reconciling controversies about the identity and function of human MLPs, these results may shed light on the causes of age-related differences in the incidence of lymphoblastic leukemia.

immunology↗

bioRxiv2022

Multimodal Mapping of Human Lymphopoiesis Reveals B and T/NK/ILC Lineages are Subjected to Cell-Intrinsic versus Flt3L-Dependent Regulation

The developmental cartography of human lymphopoiesis remains incompletely understood. Here, we establish a multimodal map that extends the current view of lymphoid development. Our results demonstrate that lymphoid specification follows independent direct or stepwise differentiation pathways converging toward the emergence of CD117lo multi-lymphoid progenitors (MLPs) that undergo a proliferation arrest before entering the CD127- (T/NK/ILC) or CD127+ (B) lymphoid pathways. While the emergence of CD127- early lymphoid progenitors is driven by Flt3 signaling, differentiation of their CD127+ counterparts is regulated cell-intrinsically and depends exclusively on the divisional history of their precursors. Single-cell mapping of lymphoid differentiation trajectories reveals that a dissociation between proliferation and differentiation phases allows amplification of the precursor pools prior to the onset of antigen receptor rearrangement. Besides demonstrating that B and T/NK/ILC lineages are subjected to differential cell-autonomous versus Flt3-inducible regulation, our results go a long way to reconciling human and mouse models of lymphoid architecture.

immunology↗

bioRxiv2022

Nanoblades allow high-level genome editing in organoids

Genome engineering has become more accessible thanks to the RNA programmable endonucleases such as the CRISPR/Cas9 system. However, using this editing technology in synthetic organs called organoids is still very inefficient. This is due to the delivery methods used for the CRISPR-Cas9 machinery, which include electroporation of CRISPR/Cas9 DNA, mRNA or ribonucleoproteins (RNPs) containing the CAS9-gRNA complex. However, these procedures are toxic to some extent for the organoids. Here we describe the use of the Nanoblade technology, which outperformed by far knock-out (KO) levels achieved to date by gene editing in murine and human tissue derived organoids. We reached up to 80% of gene KO in organoids after treatment with nanoblades. Indeed, high-level nanoblade-mediated KO for the androgen receptor (AR) encoding gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene was achieved with single gRNA or dual gRNA containing nanoblades in murine prostate and colon organoids. Likewise, nanoblades achieved high levels of gene editing in human organoids ranging between 20% and 50%. Most importantly, in contrast to other gene editing methods, this was obtained without toxicity for the organoids. Only four weeks are required to obtain stable gene KO in organoids and nanoblades simplify and allow rapid genome editing in organoids with little to no side-effects such as possible unwanted INDELS in off-target sites.

bioengineering↗

bioRxiv2019

Efficient and robust NK-Cell transduction with Baboon Envelope pseudotyped lentivector: a major tool for immunotherapy

NK-cell resistance to transduction is a major technical hurdle for developing NK-cell immunotherapy. By using Baboon envelope pseudotyped lentiviral vectors (BaEV-LVs) encoding eGFP, we obtained a transduction rate of 23.0{+/-}6.6% in freshly-isolated NK-cells (FI-NK) and 83.4{+/-}10.1% in NK-cells obtained from the NK-cell Activation and Expansion System (NKAES), even at low MOI, with a sustained transgene expression for at least 21 days. BaEV-LVs outperformed Vesicular Stomatitis Virus type-G (VSV-G)-, RD114-and Measles Virus (MV)-pseudotyped LVs (p<0.001). mRNA expression of both BaEV receptors, ASCT1 and ASCT2, was detected in FI-NK and NKAES, with much higher expression in NKAES. Transduction with BaEV-LVs encoding for CAR-CD22 resulted in robust CAR-expression on 44.2%{+/-}14.2% of NKAES cells, which allowed the specific killing of the NK-resistant pre-B-ALL-RS4;11 cell line. Using a larger vector, encoding a dual CD19/CD22-CAR separated by T2A, we were able to transduce and re-expand dual-CAR-expressing NKAES, even with low viral titer. These dual-CAR-NK efficiently and specifically killed both CD19KO-and CD22KO-RS4;11 cells, which may overcome antigen-loss escape in the clinical setting. Our results suggest that BaEV-LVs may efficiently enable NK-cell biological studies and translation of NK-cell-based immunotherapy to the clinic.

immunology↗

bioRxiv2017

Efficient genome editing in primary cells and in vivo using viral-derived "Nanoblades" loaded with Cas9/sgRNA ribonucleoproteins

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Using engineered murine leukemia virus-like particles loaded with Cas9/sgRNA ribonucleoproteins (\"Nanoblades\"), we were able to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades were also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for \"all-in-one\" homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

cell biology↗