Methylation of ESCRT-III components regulates the timing of cytokinetic abscission.

Abscission is the final stage of cytokinesis, which cleaves the intercellular bridge (ICB) connecting two daughter cells. Abscission requires tight control of the recruitment and polymerization of the Endosomal Protein Complex Required for Transport-III (ESCRT-III) components. We explore the role of post-translational modifications in regulating ESCRT dynamics. We discover that SMYD2 methylates the lysine 6 residue of human CHMP2B, a key ESCRT-III component, at the ICB, impacting the dynamic relocation of CHMP2B to sites of abscission. SMYD2 loss-of-function (genetically or pharmacologically) causes CHMP2B hypomethylation, delayed CHMP2B polymerization and delayed abscission. This is phenocopied by CHMP2B lysine 6 mutants that cannot be methylated. Conversely, SMYD2 gain-of-function causes CHMP2B hypermethylation and accelerated abscission, specifically in cells undergoing cytokinetic challenges, thereby bypassing the abscission checkpoint. Additional experiments highlight the importance of CHMP2B methylation beyond cytokinesis, namely during ESCRT-III-mediated HIV-1 budding. We propose that lysine methylation signaling fine-tunes the ESCRT-III machinery to regulate the timing of cytokinetic abscission and other ESCRT-III dependent functions.

Identification of Cellular Protein Targets of a Half-Sandwich Iridium(III) Complex Reveals Its Dual Mechanism of Action via Both Electrophilic and Oxidative Stresses.

Identification of intracellular targets of anticancer drug candidates provides key information on their mechanism of action. Exploiting the ability of the anticancer (C∧N)-chelated half-sandwich iridium(III) complexes to covalently bind proteins, click chemistry with a bioorthogonal azido probe was used to localize a phenyloxazoline-chelated iridium complex within cells and profile its interactome at the proteome-wide scale. Proteins involved in protein folding and actin cytoskeleton regulation were identified as high-affinity targets. Upon iridium complex treatment, the folding activity of Heat Shock Protein HSP90 was inhibited in vitro and major cytoskeleton disorganization was observed. A wide array of imaging and biochemical methods validated selected targets and provided a multiscale overview of the effects of this complex on live human cells. We demonstrate that it behaves as a dual agent, inducing both electrophilic and oxidative stresses in cells that account for its cytotoxicity. The proposed methodological workflow can open innovative avenues in metallodrug discovery.

VC-resist glioblastoma cell state: vessel co-option as a key driver of chemoradiation resistance.

Glioblastoma (GBM) is a highly lethal type of cancer. GBM recurrence following chemoradiation is typically attributed to the regrowth of invasive and resistant cells. Therefore, there is a pressing need to gain a deeper understanding of the mechanisms underlying GBM resistance to chemoradiation and its ability to infiltrate. Using a combination of transcriptomic, proteomic, and phosphoproteomic analyses, longitudinal imaging, organotypic cultures, functional assays, animal studies, and clinical data analyses, we demonstrate that chemoradiation and brain vasculature induce cell transition to a functional state named VC-Resist (vessel co-opting and resistant cell state). This cell state is midway along the transcriptomic axis between proneural and mesenchymal GBM cells and is closer to the AC/MES1-like state. VC-Resist GBM cells are highly vessel co-opting, allowing significant infiltration into the surrounding brain tissue and homing to the perivascular niche, which in turn induces even more VC-Resist transition. The molecular and functional characteristics of this FGFR1-YAP1-dependent GBM cell state, including resistance to DNA damage, enrichment in the G2M phase, and induction of senescence/stemness pathways, contribute to its enhanced resistance to chemoradiation. These findings demonstrate how vessel co-option, perivascular niche, and GBM cell plasticity jointly drive resistance to therapy during GBM recurrence.

Human dental pulp stem cells derived extracellular matrix promotes mineralization via Hippo and Wnt pathways.

Extracellular matrix (ECM) is an intricate structure providing the microenvironment niche that influences stem cell differentiation. This study aimed to investigate the efficacy of decellularized ECM derived from human dental pulp stem cells (dECM_DPSCs) and gingival-derived mesenchymal stem cells (dECM_GSCs) as an inductive scaffold for osteogenic differentiation of GSCs. The proteomic analysis demonstrated that common and signature matrisome proteins from dECM_DPSCs and dECM_GSCs were related to osteogenesis/osteogenic differentiation. RNA sequencing data from GSCs reseeded on dECM_DPSCs revealed that dECM_DPSCs upregulated genes related to the Hippo and Wnt signaling pathways in GSCs. In the inhibitor experiments, results revealed that dECM_DPSCs superiorly promoted GSCs osteogenic differentiation, mainly mediated through Hippo and Wnt signaling. The present study emphasizes the promising translational application of dECM_DPSCs as a bio-scaffold rich in favorable regenerative microenvironment for tissue engineering.

Proteogenomic profiling uncovers differential therapeutic vulnerabilities between TCF3::PBX1 and TCF3::HLF translocated B-cell acute lymphoblastic leukemia.

Not available.

The distinct localization of CDC42 isoforms is responsible for their specific functions during migration.

The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins: the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), which only differ in their carboxy-terminal sequence, including the CAAX motif essential for their association with membranes. We show that these divergent sequences do not directly affect the range of CDC42's potential binding partners but indirectly influence CDC42-driven signaling by controlling the subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u associates with the leading-edge plasma membrane of migrating cells, where it recruits the Par6-PKCζ complex to fulfill its polarity function. In contrast, CDC42b mainly localizes to intracellular membrane compartments, where it regulates N-WASP-mediated endocytosis. Both CDC42 isoforms contribute their specific functions to promote the chemotaxis of neural precursors, demonstrating that their expression pattern is decisive for tissue-specific cell behavior.

Proteogenomic Characterization of Bladder Cancer Reveals Sensitivity to Apoptosis Induced by Tumor Necrosis Factor-related Apoptosis-inducing Ligand in FGFR3-mutated Tumors.

BACKGROUND: Molecular understanding of muscle-invasive (MIBC) and non-muscle-invasive (NMIBC) bladder cancer is currently based primarily on transcriptomic and genomic analyses. OBJECTIVE: To conduct proteogenomic analyses to gain insights into bladder cancer (BC) heterogeneity and identify underlying processes specific to tumor subgroups and therapeutic outcomes. DESIGN, SETTING, AND PARTICIPANTS: Proteomic data were obtained for 40 MIBC and 23 NMIBC cases for which transcriptomic and genomic data were already available. Four BC-derived cell lines harboring FGFR3 alterations were tested with interventions. INTERVENTION: Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), second mitochondrial-derived activator of caspases mimetic (birinapant), pan-FGFR inhibitor (erdafitinib), and FGFR3 knockdown. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Proteomic groups from unsupervised analyses (uPGs) were characterized using clinicopathological, proteomic, genomic, transcriptomic, and pathway enrichment analyses. Additional enrichment analyses were performed for FGFR3-mutated tumors. Treatment effects on cell viability for FGFR3-altered cell lines were evaluated. Synergistic treatment effects were evaluated using the zero interaction potency model. RESULTS AND LIMITATIONS: Five uPGs, covering both NMIBC and MIBC, were identified and bore coarse-grained similarity to transcriptomic subtypes underlying common features of these different entities; uPG-E was associated with the Ta pathway and enriched in FGFR3 mutations. Our analyses also highlighted enrichment of proteins involved in apoptosis in FGFR3-mutated tumors, not captured through transcriptomics. Genetic and pharmacological inhibition demonstrated that FGFR3 activation regulates TRAIL receptor expression and sensitizes cells to TRAIL-mediated apoptosis, further increased by combination with birinapant. CONCLUSIONS: This proteogenomic study provides a comprehensive resource for investigating NMIBC and MIBC heterogeneity and highlights the potential of TRAIL-induced apoptosis as a treatment option for FGFR3-mutated bladder tumors, warranting a clinical investigation. PATIENT SUMMARY: We integrated proteomics, genomics, and transcriptomics to refine molecular classification of bladder cancer, which, combined with clinical and pathological classification, should lead to more appropriate management of patients. Moreover, we identified new biological processes altered in FGFR3-mutated tumors and showed that inducing apoptosis represents a new potential therapeutic option.

Extracellular vesicles and co-isolated endogenous retroviruses from murine cancer cells differentially affect dendritic cells.

Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression.

DNA damage induces nuclear envelope rupture through ATR-mediated phosphorylation of lamin A/C.

The integrity of the nuclear envelope (NE) is essential for maintaining the structural stability of the nucleus. Rupture of the NE has been frequently observed in cancer cells, especially in the context of mechanical challenges, such as physical confinement and migration. However, spontaneous NE rupture events, without any obvious physical challenges to the cell, have also been described. The molecular mechanism(s) of these spontaneous NE rupture events remain to be explored. Here, we show that DNA damage and subsequent ATR activation leads to NE rupture. Upon DNA damage, lamin A/C is phosphorylated in an ATR-dependent manner, leading to changes in lamina assembly and, ultimately, NE rupture. In addition, we show that cancer cells with intrinsic DNA repair defects undergo frequent events of DNA-damage-induced NE rupture, which renders them extremely sensitive to further NE perturbations. Exploiting this NE vulnerability could provide a new angle to complement traditional, DNA-damage-based chemotherapy.

Codependencies of mTORC1 signaling and endolysosomal actin structures.

The mechanistic target of rapamycin complex 1 (mTORC1) is part of the amino acid sensing machinery that becomes activated on the endolysosomal surface in response to nutrient cues. Branched actin generated by WASH and Arp2/3 complexes defines endolysosomal microdomains. Here, we find mTORC1 components in close proximity to endolysosomal actin microdomains. We investigated for interactors of the mTORC1 lysosomal tether, RAGC, by proteomics and identified multiple actin filament capping proteins and their modulators. Perturbation of RAGC function affected the size of endolysosomal actin, consistent with a regulation of actin filament capping by RAGC. Reciprocally, the pharmacological inhibition of actin polymerization or alteration of endolysosomal actin obtained upon silencing of WASH or Arp2/3 complexes impaired mTORC1 activity. Mechanistically, we show that actin is required for proper association of RAGC and mTOR with endolysosomes. This study reveals an unprecedented interplay between actin and mTORC1 signaling on the endolysosomal system.

Global proteomic identifies multiple cancer-related signaling pathways altered by a gut pathobiont associated with colorectal cancer.

In this work, we investigated the oncogenic role of Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut bacterium associated with colorectal cancer (CRC). We showed that SGG UCN34 accelerates colon tumor development in a chemically induced CRC murine model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 revealed that 164 proteins and 725 phosphorylation sites were differentially regulated. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced by SGG UCN34, as ~ 90% of proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed up-regulation of several cancer hallmark pathways such as MAPK, mTOR and integrin/ILK/actin, affecting epithelial and stromal colonic cells. Importantly, an independent analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGG's capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology. Taken together, our results demonstrate the oncogenic role of SGG UCN34 in a murine model of CRC associated with activation of multiple cancer-related signaling pathways.

Polθ is phosphorylated by PLK1 to repair double-strand breaks in mitosis.

DNA double-strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle1. In interphase, DSBs are mainly repaired by non-homologous end joining and homologous recombination2. However, these pathways are completely inhibited in mitosis3-5, leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta6 (Polθ) repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Polθ function is activated in mitosis upon phosphorylation by Polo-like kinase 1 (PLK1). Phosphorylated Polθ is recruited by a direct interaction with the BRCA1 C-terminal domains of TOPBP1 to mitotic DSBs, where it mediates joining of broken DNA ends. Loss of Polθ leads to defective repair of mitotic DSBs, resulting in a loss of genome integrity. This is further exacerbated in cells that are deficient in homologous recombination, where loss of mitotic DSB repair by Polθ results in cell death. Our results identify mitotic DSB repair as the underlying cause of synthetic lethality between Polθ and homologous recombination. Together, our findings reveal the critical importance of mitotic DSB repair in the maintenance of genome integrity.

Phenotypic and proteomic analysis of plasma extracellular vesicles highlights them as potential biomarkers of primary Sjögren syndrome.

Sjögren syndrome (SjS) is an autoimmune disease characterized by the destruction of the exocrine gland epithelia, causing a dryness of mucosa called sicca symptoms, and whose main life-threatening complication is lymphoma. There is a need for new biomarkers in this disease, notably diagnostic biomarkers for patients with genuine sicca symptoms that do not meet current criteria, and prognostic biomarkers for patients at risk of lymphoma. Plasma extracellular vesicles (EVs) are promising biomarker candidates in several diseases, but their potential has not yet been explored in SjS. In this proof-of-concept study, we characterized EVs from primary SjS patients (pSS, n=12) at the phenotypic and proteomic levels, compared to EVs from healthy donor (HD, n=8) and systemic lupus erythematosus patients (SLE, n=12). Specific plasma EVs subpopulations, derived from neutrophils, endothelial, and epithelial cells, were found increased in pSS. We also identified a pSS proteomic signature in plasma EVs, including neutrophil-, epithelial-, and endothelial-related proteins, such as integrin alpha M (ITGAM), olfactomedin-4 (OLFM4), Ras-related protein RAB10, and CD36. Overall, our results support the relevance of plasma EVs as biomarkers in SjS.

Fibroblasts generate topographical cues that steer cancer cell migration.

Fibroblasts play a fundamental role in tumor development. Among other functions, they regulate cancer cells' migration through rearranging the extracellular matrix, secreting soluble factors, and establishing direct physical contacts with cancer cells. Here, we report that migrating fibroblasts deposit on the substrate a network of tubular structures that serves as a guidance cue for cancer cell migration. Such membranous tubular network, hereafter called tracks, is stably anchored to the substrate in a ß5-integrin-dependent manner. We found that cancer cells specifically adhere to tracks by using clathrin-coated structures that pinch and engulf tracks. Tracks thus represent a spatial memory of fibroblast migration paths that is read and erased by cancer cells directionally migrating along them. We propose that fibroblast tracks represent a topography-based intercellular communication system capable of steering cancer cell migration.

Therapeutic potential of extracellular vesicles derived from cardiac progenitor cells in rodent models of chemotherapy-induced cardiomyopathy.

Background: Current treatments of chemotherapy-induced cardiomyopathy (CCM) are of limited efficacy. We assessed whether repeated intravenous injections of human extracellular vesicles from cardiac progenitor cells (EV-CPC) could represent a new therapeutic option and whether EV manufacturing according to a Good Manufacturing Practices (GMP)-compatible process did not impair their bioactivity. Methods: Immuno-competent mice received intra-peritoneal injections (IP) of doxorubicin (DOX) (4 mg/kg each; cumulative dose: 12 mg/kg) and were then intravenously (IV) injected three times with EV-CPC (total dose: 30 billion). Cardiac function was assessed 9-11 weeks later by cardiac magnetic resonance imaging (CMR) using strain as the primary end point. Then, immuno-competent rats received 5 IP injections of DOX (3 mg/kg each; cumulative dose 15 mg/kg) followed by 3 equal IV injections of GMP-EV (total dose: 100 billion). Cardiac function was assessed by two dimensional-echocardiography. Results: In the chronic mouse model of CCM, DOX + placebo-injected hearts incurred a significant decline in basal (global, epi- and endocardial) circumferential strain compared with sham DOX-untreated mice (p = 0.043, p = 0.042, p = 0.048 respectively) while EV-CPC preserved these indices. Global longitudinal strain followed a similar pattern. In the rat model, IV injections of GMP-EV also preserved left ventricular end-systolic and end-diastolic volumes compared with untreated controls. Conclusions: Intravenously-injected extracellular vesicles derived from CPC have cardio-protective effects which may make them an attractive user-friendly option for the treatment of CCM.

Longitudinal characterization of primary osteosarcoma and derived subcutaneous and orthotopic relapsed patient-derived xenograft models.

Osteosarcoma is a rare bone cancer in adolescents and young adults with a dismal prognosis because of metastatic disease and chemoresistance. Despite multiple clinical trials, no improvement in outcome has occurred in decades. There is an urgent need to better understand resistant and metastatic disease and to generate in vivo models from relapsed tumors. We developed eight new patient-derived xenograft (PDX) subcutaneous and orthotopic/paratibial models derived from patients with recurrent osteosarcoma and compared the genetic and transcriptomic landscapes of the disease progression at diagnosis and relapse with the matching PDX. Whole exome sequencing showed that driver and copy-number alterations are conserved from diagnosis to relapse, with the emergence of somatic alterations of genes mostly involved in DNA repair, cell cycle checkpoints, and chromosome organization. All PDX patients conserve most of the genetic alterations identified at relapse. At the transcriptomic level, tumor cells maintain their ossification, chondrocytic, and trans-differentiation programs during progression and implantation in PDX models, as identified at the radiological and histological levels. A more complex phenotype, like the interaction with immune cells and osteoclasts or cancer testis antigen expression, seemed conserved and was hardly identifiable by histology. Despite NSG mouse immunodeficiency, four of the PDX models partially reconstructed the vascular and immune-microenvironment observed in patients, among which the macrophagic TREM2/TYROBP axis expression, recently linked to immunosuppression. Our multimodal analysis of osteosarcoma progression and PDX models is a valuable resource to understand resistance and metastatic spread mechanisms, as well as for the exploration of novel therapeutic strategies for advanced osteosarcoma.

Medium-throughput image-based phenotypic siRNA screen to unveil the molecular basis of B cell polarization.

Cell polarity is an essential and highly conserved process governing cell function. Cell polarization is generally triggered by an external signal that induces the relocation of the centrosome, thus defining the polarity axis of the cell. Here, we took advantage of B cells as a model to study cell polarity and perform a medium-throughput siRNA-based imaging screen to identify new molecular regulators of polarization. We first identified candidates based on a quantitative proteomic analysis of proteins differentially associated with the centrosome of resting non-polarized and stimulated polarized B cells. We then targeted 233 candidates in a siRNA screen and identified hits regulating the polarization of the centrosome and/or lysosomes in B cells upon stimulation. Our dataset of proteomics, images, and polarity indexes provides a valuable source of information for a broad community of scientists interested in the molecular mechanisms regulating cell polarity.

The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity.

The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomics screen for polysome-associated proteins in astrocytes and focused on ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially binds to a number of mRNAs, including Kcnj10, encoding the inward-rectifying potassium (K+) channel Kir4.1. By developing an astrocyte-specific, conditional RACK1 knockout mouse model, we show that RACK1 represses production of Kir4.1 in hippocampal astrocytes and PAPs. Upregulation of Kir4.1 in the absence of RACK1 increases astrocytic Kir4.1-mediated K+ currents and volume. It also modifies neuronal activity attenuating burst frequency and duration. Reporter-based assays reveal that RACK1 controls Kcnj10 translation through the transcript's 5' untranslated region. Hence, translational regulation by RACK1 in astrocytes represses Kir4.1 expression and influences neuronal activity.