Biology subjects

Larbret, F.

Publications and source records attributed to Larbret, F..

7 recordsLinked to original sources

bioRxiv2026

Spectral CytoFRET2 identifies lysine acetyltransferase inhibitors as modulators of vimentin assembly

Cytoskeletal plasticity is a defining feature of cancer progression, enabling tumor cells to adapt their morphology, mechanics, and migratory behavior during invasion and metastasis. Although actin filaments, microtubules, and intermediate filaments are known to cooperate in these processes, the molecular mechanisms coordinating their dynamics remain incompletely understood, particularly the role of post-translational modifications (PTMs). Here, we developed CytoFRET2, a multiparametric cytometry-based FRET platform that enables real-time and simultaneous monitoring of the dynamics of actin filaments, microtubules, and vimentin in living suspension cells. The system combines fluorescently tagged cytoskeletal reporters with spectral flow cytometry, allowing simultaneous high-content analysis of multiple cytoskeletal networks while overcoming autofluorescence and fluorescence interference from small molecules. Using spectral CytoFRET2, we screened a small library of epigenetic compounds targeting regulators of acetylation and methylation pathways. The screen revealed that inhibition of lysine deacetylases (KDACs) and sirtuins promoted stabilization of both microtubules and vimentin filaments, without impacting actin filament organization. In contrast, inhibition of lysine acetyltransferases (KATs), particularly with garcinol and anacardic acid, induced rapid vimentin disassembly. Mechanistically, the study reveals acetylation as a key post-translational modification regulating the dynamics of microtubules and vimentin filaments, with KAT inhibitors emerging as potent modulators of vimentin organization. Together, the findings establish spectral CytoFRET2 as a versatile platform for systematic investigation of cytoskeletal regulatory networks and therapeutic vulnerabilities in cancer.

cell biology↗

bioRxiv2026

PSMD14 drives melanoma cell survival and MAPK inhibitor resistance through histone H2A deubiquitination

Melanoma progression and resistance to targeted therapies remain major clinical challenges, driven in part by the remarkable phenotypic plasticity of melanoma cells. Identifying molecular mechanisms that couple tumor survival to adaptative drug responses is therefore essential. Here, we identify PSMD14, a proteasome-associated deubiquitinase, as an essential regulator of melanoma plasticity, growth, survival, and therapeutic resistance with strong prognostic significance in metastatic disease. An unbiased siRNA screen targeting the human deubiquitinase family revealed PSMD14 (proteasome 26S subunit, non-ATPase 14) as a top regulator of melanoma cell proliferation. Integrative analyses of DepMap, TCGA, and patient-derived datasets revealed that PSMD14 is frequently upregulated in melanoma, enriched in metastatic lesions, and significantly associated with poor patient outcome. Functional and pharmacological studies demonstrated that genetic depletion or inhibition of PSMD14 suppresses proliferation, clonogenic and long-term growth, and viability of melanoma cells across BRAF-, NRAS-, and NF1-driven genotypes, while inducing DNA damage and apoptosis. Consistently, PSMD14 inhibition markedly reduced tumor growth in Nras and Braf syngeneic mouse models. Mechanistically, we uncover a non-proteolytic role for PSMD14 as an epigenetic regulator of chromatin state. Proteomic and biochemical analyses identified histone H2A as a direct interactor and substrate of PSMD14. PSMD14 deubiquitinates H2A at lysine 119 independently of the proteasome, antagonizing the Polycomb E3 ligase RING1B. Loss of PSMD14 allows the increment of H2AK119 ubiquitination, transcriptional repression of pro-survival genes, including MCL1 and BCL2, and apoptotic cell death, effects rescued by RING1B depletion. Importantly, we demonstrate that the PSMD14-H2A axis governs melanoma adaptation to MAPK pathway inhibition. PSMD14 expression and H2AK119 ubiquitination dynamically correlate with therapeutic response, drug-tolerant persistence, and acquired resistance. Targeting PSMD14 genetically or pharmacologically enhances the efficacy of BRAF and MEK inhibitors, suppresses the emergence of drug-tolerant persister cells, and prevents tumor relapse in vivo. Together, this findings establish PSMD14 as a chromatin-rewiring enzyme that links proteostasis to epigenetic control of melanoma plasticity and therapy resistance, highlighting PSMD14 as a promising biomarker and therapeutic target in aggressive and drug-resistant melanoma.

cancer biology↗

bioRxiv2025

MELK controls tumor metabolism to promote resistance to melanoma therapy

Melanoma is the most aggressive form of skin cancer, and despite major advances in targeted and immune therapies, durable responses remain limited due to the emergence of resistance mechanisms. Metabolic reprogramming has emerged as a key driver of therapy resistance, allowing tumor cells to adapt to environmental and therapeutic pressures. Here, we identify the maternal embryonic leucine zipper kinase (MELK) as a critical mediator of resistance in melanoma. We demonstrate that MELK stimulates intracellular accumulation of amino acids, leading to activation of mTORC1 signaling and enhanced mitochondrial metabolism and biogenesis. This metabolic shift promotes resistance to immune checkpoint blockade. Importantly, preclinical MELK inhibition sensitizes resistant melanoma cells to immunotherapy, revealing a potential combinatorial strategy to overcome resistance. Our findings establish MELK as a central regulator of metabolic adaptation and therapeutic resistance in melanoma, highlighting its potential as a promising therapeutic target.

cancer biology↗

bioRxiv2024

Extracellular matrix stiffness determines the phenotypic behavior of dedifferentiated melanoma cells through a DDR1/2-dependent YAP mechanotransduction pathway

Extracellular matrix (ECM) stiffening, resulting from increased collagen deposition and cross-linking, is a key biophysical factor of the tumor microenvironment. Cutaneous melanoma is a deadly metastatic cancer. Its aggressiveness stems from high intratumoral heterogeneity, resulting from the plasticity of melanoma cells, which transit from a melanocytic state to dedifferentiated therapy-resistant and invasive phenotypes, characterized by mesenchymal and/or neural crest stem cell-like features. Phenotypic plasticity is regulated by stroma-derived soluble factors, but the functional impact of ECM stiffening on melanoma cell phenotypes remains ill defined. Here, we found that melanoma cell subpopulations display difference in mechanical responsiveness. Compared to melanocytic cells, mesenchymal dedifferentiated cells showed increased proliferation, migration and resistance to MAP kinase-targeted therapy when seeded on stiff collagen. By contrast, a soft ECM impaired their proliferation and migration and sensitized them to targeted therapy. In addition, extracellular mechanical signals are required to sustain melanoma cell identity and dedifferentiation features. Further analyses indicated that the mechanosensitivity nature of dedifferentiated cells relies on the expression and activation of collagen receptors DDR1 and DDR2 that control actomyosin cytoskeleton reorganization and YAP mechanotransduction pathway. Inhibiting both DDR in dedifferentiated melanoma cells abrogated their mechano-induced behavior and drug-resistant phenotype, while forcing their expression in melanocytic cells induced mechanical responsiveness and a less differentiated phenotype. Our results reveal that phenotypic reprogramming endows dedifferentiated melanoma cells with increased sensitivity and addiction to ECM stiffness. We propose that mechano-addiction mediated by DDR collagen receptors may represent a novel vulnerability for aggressive dedifferentiated cancer cells that can be exploited for therapeutic benefits.

cancer biology↗

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↗

bioRxiv2021

Blockade of pro-fibrotic response mediated by the miR-143/-145 cluster prevents targeted therapy-induced phenotypic plasticity and resistance in melanoma

Lineage dedifferentiation towards a mesenchymal-like state is a common mechanism of adaptive response and resistance to targeted therapy in melanoma. Yet, the transcriptional network driving this phenotypic plasticity remains elusive. Remarkably, this cellular state displays myofibroblast and fibrotic features and escapes MAPK inhibitors (MAPKi) through extracellular matrix (ECM) remodeling activities. Here we show that the anti-fibrotic drug Nintedanib/BIBF1120 is active to normalize the fibrous ECM network, enhance the efficacy of MAPK-targeted therapy and delay tumor relapse in a pre-clinical model of melanoma. We also uncovered the molecular networks that regulate the acquisition of this resistant phenotype and its reversion by Nintedanib, pointing the miR-143/-145 pro-fibrotic cluster as a driver of the therapy-resistant mesenchymal-like phenotype. Upregulation of the miR-143/-145 cluster under BRAFi/MAPKi therapy was observed in melanoma cells in vitro and in vivo and was associated with an invasive/undifferentiated profile of resistant cells. The 2 mature miRNAs generated from this cluster, miR-143-3p and miR-145-5p collaborated to mediate phenotypic transition towards a drug resistant undifferentiated mesenchymal-like state by targeting Fascin actin-bundling protein 1 (FSCN1), modulating the dynamic crosstalk between the actin cytoskeleton and the ECM through the regulation of focal adhesion dynamics as well as contributing to a fine-tuning of mechanotransduction pathways. Our study brings insights into a novel miRNA-mediated regulatory network that contributes to non-genetic adaptive drug resistance and provides proof-of-principle that preventing MAPKi-induced pro-fibrotic stromal response is a viable therapeutic opportunity for patients on targeted therapy.

cancer biology↗

bioRxiv2019

Targeting DDR1 and DDR2 overcomes matrix-mediated melanoma cell adaptation to BRAF-targeted therapy

Resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanomas remains a major obstacle that limits patient benefit. Microenvironment components including the extracellular matrix (ECM) can support tumor cell adaptation and tolerance to targeted therapies, however the underlying mechanisms remain poorly understood. Here, we investigated the process of matrix-mediated drug resistance (MM-DR) in response to BRAF inhibition in melanoma. We demonstrate that physical and structural cues from fibroblast-derived ECM abrogate anti-proliferative responses to BRAF/MEK inhibition. MM-DR is mediated by the drug-induced clustering of DDR1 and DDR2, two tyrosine kinase collagen receptors. Genetic depletion and pharmacological inhibition of DDR1 and DDR2 overcome ECM-mediated resistance to BRAF inhibition. In melanoma xenografts, targeting DDRs by Imatinib enhances BRAF inhibitor efficacy, counteracts drug-induced collagen remodeling and delays tumor relapse. Mechanistically, DDR-mediated MM-DR fosters a targetable pro-survival NIK/IKK/NF-{kappa}B2 pathway. Our study reveals a novel role of collagen-rich matrix and DDRs in tumor cell adaptation and therapy resistance, thus providing important insights into environment-mediated drug resistance and a pre-clinical rationale for targeting DDR1/2 signaling in combination with BRAF-targeted therapy in melanoma.

cancer biology↗