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Research from Benoit Van den Eynde’s laboratory forms the basis of a Phase I/IIa clinical trial.
N-terminal cysteine acetylation and oxidation patterns may define protein stability
AbstractOxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.
Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.
Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.
Desmosome mutations impact the tumor microenvironment to promote melanoma proliferation.
Desmosomes are transmembrane protein complexes that contribute to cell-cell adhesion in epithelia and other tissues. Here, we report the discovery of frequent genetic alterations in the desmosome in human cancers, with the strongest signal seen in cutaneous melanoma, where desmosomes are mutated in more than 70% of cases. In primary but not metastatic melanoma biopsies, the burden of coding mutations in desmosome genes is associated with a strong reduction in desmosome gene expression. Analysis by spatial transcriptomics and protein immunofluorescence suggests that these decreases in expression occur in keratinocytes in the microenvironment rather than in primary melanoma cells. In further support of a microenvironmental origin, we find that desmosome gene knockdown in keratinocytes yields markedly increased proliferation of adjacent melanoma cells in keratinocyte and melanoma cocultures. Similar increases in melanoma proliferation are observed in media preconditioned with desmosome-deficient keratinocytes. Thus, gradual accumulation of desmosome mutations in neighboring cells may prime melanoma cells for neoplastic transformation.
MSP-tracker: A versatile vesicle tracking software tool used to reveal the spatial control of polarized secretion in Drosophila epithelial cells.
Understanding how specific secretory cargoes are targeted to distinct domains of the plasma membrane in epithelial cells requires analyzing the trafficking of post-Golgi vesicles to their sites of secretion. We used the RUSH (retention using selective hooks) system to synchronously release an apical cargo, Cadherin 99C (Cad99C), and a basolateral cargo, the ECM protein Nidogen, from the endoplasmic reticulum and follow their movements to the plasma membrane. We also developed an interactive vesicle tracking framework, MSP-tracker and viewer, that exploits developments in computer vision and deep learning to determine vesicle trajectories in a noisy environment without the need for extensive training data. MSP-tracker outperformed other tracking software in detecting and tracking post-Golgi vesicles, revealing that Cad99c vesicles predominantly move apically with a mean speed of 1.1µm/sec. This is reduced to 0.85 µm/sec by a dominant slow dynein mutant, demonstrating that dynein transports Cad99C vesicles to the apical cortex. Furthermore, both the dynein mutant and microtubule depolymerization cause lateral Cad99C secretion. Thus, microtubule organization plays a central role in targeting apical secretion, suggesting that Drosophila does not have distinct apical versus basolateral vesicle fusion machinery. Nidogen vesicles undergo planar-polarized transport to the leading edge of follicle cells as they migrate over the ECM, whereas most Collagen is secreted at trailing edges. The follicle cells therefore bias secretion of different ECM components to opposite sides of the cell, revealing that the secretory pathway is more spatially organized than previously thought.
Perturbing LSD1 and WNT rewires transcription to synergistically induce AML differentiation
Abstract Impaired differentiation is a hallmark of myeloid malignancies1,2. Therapies that enable cells to circumvent the differentiation block, such as all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), are by and large curative in acute promyelocytic leukaemia3, but whether ‘differentiation therapy’ is a generalizable therapeutic approach for acute myeloid leukaemia (AML) and beyond remains incompletely understood. Here we demonstrate that simultaneous inhibition of the histone demethylase LSD1 (LSD1i) and the WNT pathway antagonist GSK3 kinase4 (GSK3i) robustly promotes therapeutic differentiation of established AML cell lines and primary human AML cells, as well as reducing tumour burden and significantly extending survival in a patient-derived xenograft mouse model. Mechanistically, this combination promotes differentiation by activating genes in the type I interferon pathway via inducing expression of transcription factors such as IRF7 (LSD1i) and the co-activator β-catenin (GSK3i), and their selective co-occupancy at targets such as STAT1, which is necessary for combination-induced differentiation. Combination treatment also suppresses the canonical, pro-oncogenic WNT pathway and cell cycle genes. Analysis of datasets from patients with AML suggests a correlation between the combination-induced transcription signature and better prognosis, highlighting clinical potential of this strategy. Collectively, this combination strategy rewires transcriptional programs to suppress stemness and to promote differentiation, which may have important therapeutic implications for AML and WNT-driven cancers beyond AML.
Single-cell transcriptomic landscape deciphers intratumoral heterogeneity and subtypes of acral and mucosal melanoma.
PurposeTo identify the specific intratumoral and microenvironmental heterogeneity of acral (AM) and mucosal melanoma (MM), we aimed to delineate their distinct cellular compositions, evolutionary trajectories, and subtype-specific therapeutic strategies.Experimental designSingle-cell transcriptomic and genomic landscapes were analyzed across 42 melanoma samples (28 AM, 11 MM, and 3 non-acral cutaneous melanoma [CM]), supplemented by in vitro and in vivo validation. Tumor and stromal cells were profiled using scRNA-seq, whole-exome sequencing, and functional assays, including transwell migration, co-culture systems, and xenograft models.ResultsTumor cells exhibited divergent evolutionary routes, with MM dominated by MGP⁺/PCOLCE⁺ subpopulations showing high epithelial-to-mesenchymal transition (EMT) potential. MM displayed elevated neutrophil infiltration and CXCL3⁺ tumor-associated macrophages, while AM was enriched with PI16⁺ cancer-associated fibroblasts (CAFs) promoting tumor proliferation. Molecular classification revealed MM subtypes: an antigen-presenting subtype linked to favorable outcomes and a proliferative subtype associated with recurrence. TIGIT⁺ Treg cells were enriched in AM, suggesting targeted inhibition potential. Genomic analysis connected BRAF/NRAS mutations to ALDOA⁺ stem-like tumor cells and identified PTGDS as a therapeutic target in triple-WT melanomas.ConclusionsOur study provides a comprehensive comparison of AM and MM, uncovering subtype-specific stromal-immune interactions and molecular programs. The findings highlight actionable targets (e.g., TIGIT in AM, CXCL3⁺ macrophages in MM) and propose a framework for precision therapies, biomarker-driven trials, and risk stratification to improve outcomes in these aggressive melanomas.
Increased RhoA pathway activation downstream of αIIbβ3/SRC contributes to heterozygous Bernard Soulier syndrome.
Bernard Soulier Syndrome (BSS) is a severe bleeding disorder with moderate to severe thrombocytopenia, giant platelets, and platelet dysfunction, caused by biallelic mutations in GP1BA, GP1BB, or GP9 genes. We generated induced pluripotent stem cells (iPSC) from a BSS patient with a novel heterozygous GP1BA p.N103D mutation, resulting in moderate macrothrombocytopenia. The mutation does not affect megakaryocyte (MK) differentiation or GPIb-GPIX complex expression but reduces affinity to von Willebrand factor (VWF). It induces increased signaling independent of VWF and αIIbβ3-mediated outside-in signaling, causing a profound defect in proplatelet formation after adhesion on fibrinogen. Pre-activation of αIIbβ3 integrin and heightened stress fiber formation linked to RhoA pathway overactivation were observed, likely due to increased phosphorylation of SRC at Y419 downstream of GPIbα. Dasatinib, a SRC inhibitor, restored stress fiber formation. Using a 3D bone marrow model to mimic platelet release under flow, we demonstrated that the ROCK1/2 inhibitor Y27632 increased platelet number and restored platelet size in GPIbα N103D MKs, as well as in MKs from two other patients with heterozygous GP1BA mutations (p.L160P and p.N150S). However, Y27632 had no additional effect on platelet generation from MKs of two patients with biallelic BSS, suggesting a distinct molecular mechanism in biallelic cases.
LIBX-A401: A Novel Selective Inhibitor of Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) and Its Binding Mode.
Acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), a pivotal enzyme in lipid metabolism, has emerged as a therapeutic target for ferroptosis-related conditions and cancer. However, its reference inhibitor, rosiglitazone, has off-target activity on peroxisome proliferator-activated receptor gamma (PPARγ), a key regulator of lipid homeostasis. Here, the discovery of LIBX-A401, a potent ACSL4 inhibitor derived from rosiglitazone devoid of PPARγ activity, is reported. Its binding to ACSL4 is ATP-dependent, stabilizing the C-terminal domain and altering the fatty acid gate region, as shown by Hydrogen-Deuterium Exchange Mass Spectrometry. Photoaffinity labeling identified A329 within the fatty acid binding site, while molecular dynamics and mutagenesis highlighted Q302 as critical for LIBX-A401 binding. LIBX-A401 exhibits anti-ferroptotic properties in cells, supported by target engagement. These findings establish LIBX-A401 as a valuable tool to study ACSL4 in ferroptosis and cancer, while its elucidated binding mode paves the way for the rational design of improved inhibitors.
Fatty acid uptake activates an AXL-CAV1-β-catenin axis to drive melanoma progression.
Interaction between the tumor microenvironment and cancer cell plasticity drives intratumor phenotypic heterogeneity and underpins disease progression and nongenetic therapy resistance. Phenotype-specific expression of the AXL receptor tyrosine kinase is a pivotal player in dormancy, invasion, and resistance to treatment. However, although the AXL ligand GAS6 is present within tumors, how AXL is activated in metastasizing cells remains unclear. Here, using melanoma as a model, we reveal that AXL is activated by exposure to human adipocytes and to oleic acid, a monounsaturated fatty acid abundant in lymph and in adipocytes. AXL activation triggers SRC-dependent formation and nuclear translocation of a β-catenin-CAV1 complex required for melanoma invasiveness. Remarkably, only undifferentiated AXLHigh melanoma cells engage in symbiosis with human adipocytes, in part by triggering WNT5a-mediated lipolysis, leading to AXL-dependent, but FATP-independent, fatty acid uptake and nuclear localization of the β-catenin-CAV1 complex. Significantly, human melanomas in the vicinity of adipocytes exhibit high levels of nuclear CAV1. The results unveil an AXL- and CAV1-dependent mechanism through which a nutritional input drives phenotype-specific activation of a prometastasis program. Given the key role of AXL in a broad range of cancers, the results offer major insights into the mechanisms of cancer cell dormancy and therapy resistance.