BACKGROUND:Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous breast cancer subtype with limited treatment options. Predicting patient response to chemo-immunotherapy remains challenging, highlighting the need for robust stratification strategies. METHODS:We performed a multi-parametric analysis combining histological, genomic, transcriptomic, proteomic, and immune profiling in the immunocompetent MMTV-R26Met TNBC mouse model and compared outcomes with patient data from human TNBC cohorts and TNBC tumor microarray. To enable therapeutic testing and functional validation, we established syngeneic grafts from primary tumors and used them to evaluate combined chemotherapy (epirubicin) and anti-PD-1 immunotherapy. RESULTS:Multi-parametric analysis of TNBC heterogeneity modeled by the MMTV-R26Met mice identified four distinct TNBC clusters, defined by unique intrinsic (molecular/genomic) and extrinsic (immune) features, which closely parallel patient subtypes, including rare metaplastic forms, and correlate with clinical outcomes. Both intrinsic and immune hallmarks of primary tumors were conserved across serial syngeneic transplantations, confirming the translational value of this preclinical platform. Treatment assessments indicated cluster-specific therapeutic vulnerabilities associated with molecular and immune traits. Specifically, whereas chemo-immunotherapy is beneficial to neutrophil-enriched tumors, immunotherapy alone appears to be more effective in macrophage-enriched tumors. Our findings indicate that TNBC treatment response is shaped by the interplay between tumor-intrinsic and immune features. CONCLUSION:Our study provides a robust preclinical platform for precision immuno-oncology, enabling stratification of TNBC patients for tailored onco-immunotherapies.

The primary cilium is a sensory organelle present in all eukaryotic cells that acts as a signaling hub that receives, integrates and transmits signals carried out by extracellular stimuli, playing a fundamental role in cell behavior, development and tissue homeostasis. Genetic mutations in genes encoding for structural components and regulators of primary cilium underlie severe developmental defects, also known as ciliopathies. Understanding cilium biology may, thus, provide novel therapeutic strategies for ciliopathies. Here, we report that A-Kinase Anchor protein 2(AKAP2) dynamically localizes within the primary cilium and forms complexes with kinesin motor proteins and components of the intra-flagellar transport system. Genetic deletion experiments and functional analysis demonstrate that AKAP2/PKA complex controls cilium biogenesis and intraciliary trafficking of cargo receptors, acting as a positive regulator of Shh pathway. In the medaka fish model, depletion of AKAP2 affects ciliogenesis and ciliary trafficking, delays embryonic development and significantly increases the embryonic death rate. Our findings identify AKAP2 as a ciliary scaffold protein that finely controls ciliogenesis, intraciliary trafficking and Hedgehog signaling cascade. Manipulating ciliary AKAP2 signaling, thus, provides a new therapeutic window for ciliopathy disorders.

Lung cancers frequently increase iron demand to sustain growth, which makes them vulnerable to ferroptosis. While Cullin 2-RING ubiquitin ligases (CRL2s) are critical regulators of stress responses and redox balance, their role in ferroptosis mechanisms remains largely unknown. Here, we identify the E3 ligase CRL2FEM1B as a key regulator of the ferroptotic response. CRL2FEM1B recruits BTB and CNC homolog 1 (BACH1), a transcriptional regulator of ferroptosis, for degradation by recognizing a degron that is directly formed by the redox-sensing molecule heme. By degrading BACH1 in response to heme, CRL2FEM1B acts as a switch that dynamically modulates the transcriptional activation of ferroptosis-protective genes, particularly solute carrier family 7 member 11 (SLC7A11). Loss of CRL2FEM1B stabilizes BACH1 and suppresses SLC7A11, thereby sensitizing lung tumor cells to ferroptosis inducers in vitro and in preclinical models. Our findings identify CRL2FEM1B as a target to increase the efficacy of ferroptosis inducers in lung cancer treatment and define a broader principle whereby endogenous metabolites regulate protein degradation by enabling substrate-E3 ligase interactions.

Pancreatic cancer remains a devastating disease with limited therapeutic options. Accumulating evidence shows that cancer-associated fibroblasts (CAFs) and tumor-associated macrophages, the predominant cells in the pancreatic cancer (PDAC) tumor microenvironment, hinder antitumor immunity. However, the role of extracellular vesicles (EVs) in such a process is poorly understood. In this study, using human bone marrow-derived monocytes and PDAC tumor cells, we showed that tumor cell-derived EVs (TC-EVs) induced monocyte differentiation toward M2-like, immunosuppressive, CD200R+PD-L1+HLA-DRlo macrophages that express ALOX15B, that we identify as an independent PDAC poor-prognosis biomarker using a human PDAC metacohort. We also demonstrated that TC-EVs reprogrammed human primary PDAC CAFs, causing a fibronectin network reorganization associated with changes in extracellular matrix (ECM) composition, including alterations of WNT pathway elements such as secreted frizzled related protein-1 (SFRP1) enrichment. We also revealed that monocytes cultured on SFRP1-enriched ECM differentiated into M2-like, immunosuppressive macrophages. Last, we demonstrated that both directly and indirectly TC-EV- or SFRP1-enriched ECM-driven differentiated macrophages hindered T cell activation and subsequent antitumor activity. Our findings highlight potentially novel dual mechanisms of TC-EV-mediated crosstalk, involving ALOX15B+ macrophages and SFRP1+ CAFs, that simultaneously contribute to foster the immunosuppressive ecosystem of PDAC.

Multiciliated cells (MCCs) are essential for generating directional fluid flow across specialized epithelia in various vertebrate organs. MCC differentiation involves a tightly regulated program characterized by massive centriole amplification. Although transcriptional control of MCC development is well characterized, insights into proteome dynamics have been limited due to the lack of suitable models. Here, we report the generation of a stable inducible MCC line, derived from Xenopus A6 kidney epithelial cells. Upon induction of the master regulator multicilin (MCI), most A6-MCI cells synchronously differentiate into mature MCCs in 48 h. Using this resource, custom antibodies, and super-resolution imaging, we characterized Xenopus deuterosomes, the platforms that allow massive centriole synthesis in vertebrate MCCs. We performed detailed proteomic profiling throughout differentiation, uncovered previously uncharacterized regulators and highlighted a critical role for CDK7 in Xenopus and human MCC differentiation. Our work provides a valuable resource for mechanistic studies of MCC biology and opens avenues to identify novel therapeutic targets for motile ciliopathies.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with poor prognosis and limited therapeutic options. Early biomarkers for the detection and prediction of treatment response are sorely lacking. The tumour secretome, the set of proteins released by cancer cells, represents a promising source of biomarkers and provides insights into tumour biology, as these factors may be detectable in blood and suitable for non-invasive monitoring. However, most secretome studies have relied on established cell lines or mouse models, poorly reflecting human tumour heterogeneity. To address this gap, we generated a comprehensive proteomic dataset of secretomes from 48 low-passage, treatment-naïve, patient-derived primary PDAC cultures, which retain the molecular and phenotypic features of their tumours of origin. Across samples, we identified 4,204 proteins, including 793 shared by all cultures. Annotation showed that most of these proteins matched extracellular vesicle contents and canonical secreted proteins that may reach the circulation. Consequently, this dataset provides a valuable resource for the identification of circulating biomarkers and for comparative analyses of PDAC secretomes.

Castration-resistant prostate cancer (CRPC) is an advanced and ultimately incurable stage of the disease that arises despite androgen deprivation and remains challenging to treat due to the limited and short-lived efficacy of current therapies. Heat shock protein 27 (HSP27), a molecular chaperone, has been implicated in prostate cancer (PC) progression and therapy resistance; however, its mechanistic roles remain incompletely understood. This study aimed to delineate the HSP27 interactome in PC cells during PC progression and to explore its functional significance, particularly in relation to the mTOR signaling pathway, which is activated in most cases, primarily due to PTEN loss. We performed affinity purification-mass spectrometry (AP-MS) to identify HSP27-interacting proteins in a panel of prostate cell lines with increasing aggressiveness: PNT1A (non-malignant), LNCaP (androgen-sensitive), DU-145, and PC-3 (both androgen-independent CRPC models). Functional enrichment and network analyses were conducted to uncover pathways associated with HSP27 interactors. Validation experiments included Western blotting, co-immunoprecipitation, and pharmacological inhibition using OGX-427 and mTOR inhibitors (Everolimus, Sapanisertib) in prostate cancer cell lines, patient-derived organoids (PDOs), and xenograft models. HSP27 exhibited a progressively expanded interactome in CRPC models, with enrichment of proteins involved in stress adaptation, proteostasis, and mTOR signaling. These insights highlight HSP27 not only as a molecular chaperone but also as a dynamic network hub that may promotes survival in stress-adapted tumor states. In PC-3 cells, HSP27 stabilized key mTORC1 components, including RAPTOR, S6K1, and 4E-BP1 via its chaperone function, thereby enhancing mTORC1 activation. Combined inhibition of HSP27 using the antisense oligonucleotide OGX-427 (Apatorsen) and mTOR blockade via Sapanisertib induced a robust synergistic anti-tumor effect across diverse preclinical models, including advanced PC Patient-derived organoid (PDOs) and CRPC xenografts. Our findings reveal novel insights into HSP27’s role in PC progression and its modulation of the mTOR signaling pathway in CRPC, highlighting dual HSP27/mTOR inhibition as a promising therapeutic approach for advanced, treatment-resistant disease.

Set1 is the catalytic subunit of SET1C or COMPASS, which methylates histone H3K4 and serves as a scaffold for the association of seven tightly bound polypeptides. We have employed yeast two-hybrid screenings to determine the interactome of Set1 and each subunit, providing a unique resource for exploring known and novel roles of the complex. Our screenings identified a multitude of potential interactors involved in chromatin regulation, DNA replication, meiotic breaks, and Ty transposition, processes previously associated with SET1C. Consistent with Set1 being an RNA-binding protein, the screens link SET1C to multiple aspects of RNA biogenesis, including pre-mRNA splicing and polyadenylation. The results reveal that several importins are candidate interactors of Set1, along with RGG motif-containing proteins, providing insights into the mechanisms by which Set1 moves between cytoplasmic and nuclear compartments. We further reveal that reconstituted SET1C interacts with the AT hook domain of the chromatin remodeler Snf2 and methylates multiple arginines within this domain. In vivo, we report that the ARTSTRGR AT-hook motif is methylated in a Set1-dependent manner revealing new interplay between lysine and arginine methylation.

Staphylococcus aureus is a Gram-positive opportunistic pathogen and a top priority bacterium in the fight against antimicrobial resistance. Its high propensity to develop resistance, its high virulence, and its ability to form biofilms and persist intracellularly result in difficult-to-treat infections against, which new chemical classes are urgently needed. Here, we investigated the antibacterial activity of oxadiazolone-core derivatives (OX) against planktonic, intracellular, and biofilm-associated S. aureus. Among the tested compounds, MpPPOX exhibited a bactericidal effect on extracellular bacteria with an MIC similar to that of vancomycin; iBPOX mainly inhibited intracellular replication, while HPOX strongly impaired initial biofilm formation. These results prompted us to identify the potential target enzymes of the three OXs via activity-based protein profiling, combined with mass spectrometry. The antibiofilm HPOX compound was indeed found to primarily react with enzymes involved in biofilm formation and associated virulence, while iBPOX and the most active MpPPOX inhibitor targeted multiple (Ser/Cys)-based enzymes. Among these, the FabH protein has been confirmed as a vulnerable target of MpPPOX. Overall, this study underscores the multitarget nature of the OXs, which covalently bind to several (Ser/Cys)-based enzymes of interest. Such property makes them highly versatile chemotypes that could be used as broad-spectrum antimicrobial agents, notably by improving the antibiofilm activity of ineffective or poorly active drugs.

Abstract Circulating tumor cells (CTCs) are the potential seeds of distant metastases; however, little is known about how they survive in the bloodstream. Using a large cohort of colorectal cancer (CRC) patients, we found that the pseudokinase receptor PTK7 is highly expressed in primary tumors and metastatic lesions. Consistent with previous reports, high PTK7 expression is associated with reduced disease-free survival and increased metastatic dissemination. Surprisingly, PTK7 is absent from most CTCs and undergoes a cell-autonomous ON tumor /OFF CTC /ON metastasis switch that can be recapitulated in a xenografted mouse model, in in vitro systems, and a fluidic platform. PTK7-negative cancer cells exhibit increased expression of YAP1-driven genes, senescence-like features, and enhanced resistance to hemodynamic stress following loss of cell-cell and cell-matrix adhesion. This adaptive phenotype depends on metalloproteases, notably ADAM17, whose cleavage activity remodels the CTCs surfaceome. Functionally, the PTK7 OFF CTC state confers enhanced metastatic potential in vivo , and can be pharmacologically suppressed using metalloprotease inhibitors. Collectively, our findings identify a reversible, cell-autonomous, protease-driven surfaceome remodeling program that enables metastatic adaptation during hematogenous dissemination. Highlights / statement of significance By investigating potential markers for circulating colorectal tumor cells with strong metastatic potential, we describe a reversible and cell-autonomous remodeling of the circulating tumor cell surfaceome in patients that confers resistance to anoikis and stress induced by entry into the bloodstream. One Sentence Summary The dynamic regulation of PTK7 serves as a surrogate marker for tumor cell plasticity, aggressiveness, survival in the bloodstream, and efficiency in forming metastases. Trial registration CTC colon Cohort: registered on https://ClinicalTrials.gov identifier NCT03256084 ; date of registration 2017-07-17 B-Org cohort: registered on https://ClinicalTrials.gov NCT05384184 ; date of registration 2019-06-06 Ethics statement for animal experiments Studies on animals were conducted in accordance with the current ethical standards of the European Community (Directive 2010/63/EU), the Ethics Committee for Animal Experimentation (CEEA#14) and the French Ministry of Higher Education and Research, which approved and authorized the entire procedure described in this paper (project number APAFIS #35294).

Background and aims Obesity drives adipose tissue (AT) expansion and chronic inflammation, leading to metabolic dysfunction through adipocyte hypertrophy, impaired ASPC differentiation, immune infiltration, and fibrosis. Stromal vascular remodeling prominently features expansion of Gdf15- and Trem2-expressing lipid-associated macrophages (LAMs), which respond to adipocyte stress and act as lipid scavengers to buffer excess lipids released by adipocytes. Here, we examined macrophage reprogramming in p21+/Tert and p21+/TertCi mice, which express active TERT or its catalytically inactive TERTCi mutant from the endogenous Cdkn1a promoter. Results Following HFD exposure, conditional expression of TERT or TERTCi resulted in a pronounced downregulation of p21 in macrophage subsets, accompanied by a reduction in AT inflammation. Notably, TERT and TERTCi expression reshaped the adipose-tissue macrophage (ATM) landscape depleting Trem2+ and Gdf15+ LAMs while preserving resident macrophages. This shift was accompanied by marked suppression of the Trem2 transcriptional program and down-regulation of PPAR-γ and NR1H3 in LAMs and by impaired ASPC-LAM signaling pathways that normally drive LAM recruitment and activation. Proteomic profiling further showed that p21+/Tert ASPCs secreted markedly higher levels of proteins associated with non-conventional secretion, while extracellular matrix–associated factors and cytokines/chemokines including key mediators of ASPC–LAM communication such as Ccl2, C3, and Csf1 were substantially reduced. However, only p21+/Tert mice, and not p21+/TertCi mice, exhibited significant metabolic improvements, indicating that macrophage remodeling alone is insufficient to restore systemic metabolic function. Consistent with this, enhanced ASPC expansion and differentiation, supporting improved adipose-tissue remodeling, was observed exclusively in p21+/Tert obese mice. Conclusions TERT remodels adipose tissue immunity independently of its enzymatic activity, and TERT-driven reprogramming of the ASPC secretome may emerge as a promising strategy to combat obesity-related metabolic dysfunction. Highlights ### Competing Interest Statement J.G. has acted as a consultant for Unity Biotechnology, Geras Bio, Myricx Pharma Ltd., and Merck KgaA, owns equity in Geras Bio and share options in Myricx Pharma Ltd. And is a named inventor in MRC and imperial College patents related to senolytic therapies (unrelated to the work described here). J.G.s lab received Pfizer and Unity Biotechnology unrelated to the work described here. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, Thalatel Inserm, https://ror.org/02vjkv261, AGEMED Institut National du Cancer, PLBIO 2019 MRC Clinical Sciences Centre, https://ror.org/00kts8c89, MC_U120085810, MC_UP1605/7 Cancéropôle PACA, https://ror.org/01mwvah42, Emergence

Resistance to anticancer therapies remains a major obstacle to improving survival rates in cancer; it is often driven by epigenetic alterations. Recently, we discovered a new mechanism of therapy resistance that is not fully driven by epigenetic remodeling but involves a switch in the activity of an epigenetic enzyme, EZH2. Despite its well-established activity as part of PRC2 for mediating gene repression by H3K27me3 deposition, new evidence suggests the importance of other, so-called noncanonical activities mediated by variation of PRC2 composition and posttranslational modifications (PTMs) of EZH2. Interestingly, we showed that noncanonical EZH2 was associated with resistance to retinoic acid (RA) in acute promyelocytic leukemia (APL) and that depletion of pan-EZH2 activities was beneficial in killing relapse-initiating cells. However, the precise noncanonical roles of EZH2 remain unclear, especially in non-APL acute myeloid leukemia (AML). Using public transcriptomic data, we explored these functions in non-APL AML. We showed that patients with high EZH2 levels are enriched in metabolic profiles associated with chemotherapy resistance (HighOXPHOS) and that noncanonical EZH2 is linked to relapse after AraC treatment. Notably, AraC-resistant AML cells responded well to the EZH2 degrader MS177 but not to the enzymatic inhibitor tazemetostat. Although MS177 combined with AraC had limited effect, strong synergy was observed when MS177 was combined with RA, venetoclax, or azacytidine. This suggests that degrading EZH2 sensitizes resistant AML cells to specific therapies. Finally, omics analyses revealed specific PTMs and partner variations in EZH2 that may mediate resistance. These findings highlight the contribution of noncanonical EZH2 to AraC resistance in non-APL AML and support the therapeutic potential of targeting these activities to overcome treatment resistance.

Colorectal cancer (CRC) remains a significant public health challenge, with an urgent need of novel prognostic biomarkers and therapeutic targets. PTK7, a membrane tyrosine pseudokinase and Wnt receptor, is overexpressed in CRC and linked to poor prognosis. In CRC, PTK7 expression correlates with metastatic progression and decreased survival in non-metastatic patients, positioning PTK7 as both a potential biomarker and a promising drug target. Notably, a targeted antibody-drug conjugate (ADC) against PTK7 has recently shown efficacy in inducing tumor regression in patient-derived xenograft (PDX) models of solid tumors and has progressed to a Phase I clinical trial with promising results. Other PTK7-targeting ADCs and anti-PTK7 CAR-T cells recently entered into preclinical development. At the plasma membrane, PTK7 can interact with a range of membrane receptors, including VEGFR, EGFR, ROR2, and Plexins, enhancing their signaling capabilities. Using biotin proximity labeling in HCT116 CRC cells, we identified a novel network of PTK7-associated membrane proteins. Among these, we focused on EPHA2, an active tyrosine kinase receptor also implicated in cancer. Our findings indicate that PTK7 and EPHA2 interact via their extracellular domains (ECDs), and that PTK7 depletion significantly upregulates EPHA2 expression and signaling in response to its ligand EFNA-1 in HCT116 cells. Additionally, PTK7 regulates EPHA2’s endosomal sorting through a Rab11-dependent mechanism, as well as its K63-linked ubiquitination and subsequent lysosomal degradation. Mechanistically, PTK7 does not appear to participate directly in EFNA-1 binding but instead promotes multimerization of active EPHA2, a crucial step in amplifying EPHA2 signaling. This study aims to elucidate the functional role of the interaction between PTK7 and EPHA2 in colorectal cancer progression. By mapping the PTK7:EPHA2 interface using AlphaFold3 predictions and introducing targeted deletions and mutations within the ECDs of both receptors, we seek to understand how disrupting this interaction influences tumorigenic and metastatic processes. Our findings indicate that PTK7 depletion triggers cell rounding and detachment, with a marked increase in total and activated EPHA2 levels, which may drive pro-metastatic activity in vivo. Outcomes may significantly impact the use of anti-PTK7-ADCs or CAR-T cells and orient new therapeutic strategies to design agents interfering with PTK7 and EPHA2 interaction and function. Charlotte Dessaux, Constantin Semenchenko, Luc Camoin, Stéphane Audebert, Emilie Baudelet, Avais Daulat, Flavio Maina, Jean-Paul Borg. Exploring a novel crosstalk between PTK7 and EPHA2 tyrosine kinase receptors in metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2006.

Despite extensive proof for the tumour-supporting function of cancer-derived small extracellular vesicles (sEVs), attributions of pathological effects to specific sEV subpopulations are poorly described. In this study, we aimed to characterise a distinct sEV species under the control of Syntenin, a key regulator of endosomal sEV biogenesis, regarding its proteomic cargo and pro-tumourigenic functions. Using mass spectrometry (MS), we detected 178 down- and 236 up-regulated proteins on sEVs from breast cancer cells upon Syntenin knockout (KO). Pathway enrichment analysis suggested that Syntenin depletion was particularly associated with adhesion-related processes. Accordingly, sEVs from Syntenin-deficient 4T1 and MCF-7 breast cancer cells showed a reduced expression of several focal adhesion and cell-cell junction proteins. Syntenin silencing reduced the Fibronectin-binding capacity of sEVs from both cell lines, which was mediated by sEV-associated Integrin alpha-V/beta-3 (αVβ3). Compared to sEVs from wildtype cells, Syntenin KO sEVs showed decreased tropism towards the Fibronectin-rich liver microenvironment in vivo, provided less adhesive support for 4T1 cells and thereby failed to induce cancer cell migration, which appeared to be independent of EV uptake. In summary, this study revealed that Syntenin has a large-scale effect on the proteomic cargo of sEVs and regulates their adhesive, organotropic and pro-migratory properties in breast cancer.

The appearance of hybrid epithelial-mesenchymal (E/M) cells expressing E-cadherin is favourable for the establishment of pro-invasive function. Although the potential role of E-cadherin in cancer invasion is now accepted, the molecular mechanisms involved in this process are not completely elucidated. To gain further insight, we focused our analysis on invadopodia formation, an early event in the invasion process. We used models of E/M hybrid cell lines, tissue sections and patient-derived xenografts from a multi-centre clinical trial. E-cadherin involvement in invadopodia formation was assessed using a gelatin-FITC degradation assay. Mechanistic studies were performed by using proteomic analysis, siRNA strategy and proximity ligation assay. We showed that E-cadherin is a critical component of invadopodia. This unexpected localization results from a synergistic trafficking of E-cadherin and MT1-MMP through a Rab vesicle-dependent pathway. Modulation of E-cadherin expression or activation impacted invadopodia formation. Moreover, colocalization of E-cadherin and Actin in "ring structures" as precursors of invadopodia reveals that E-cadherin is required for invadopodia structuration. E-cadherin, initially localised in the adherens junctions, could be recycled to nascent invadopodia where it will interact with several components enriched in invadopodia, such as Arp2/3, Cortactin or MT1-MMP. The trans-adhesive properties of E-cadherin are therefore essential for structuring invadopodia. This new localisation of E-cadherin and its unexpected role in cell invasion shine a new light on hybrid E/M transition features in tumoral invasion.

Ubiquitin removal by deubiquitinases (DUBs) is crucial for protein activity and homeostasis. While tumor cells adapt to treatment and environmental stress, the role of DUBs in sensing mechanical signals from the extracellular matrix (ECM) remains an unexplored area. Using melanoma cells cultured on collagen matrices of varying stiffness and activity-based ubiquitin probe profiling combined with quantitative proteomics, we identify ubiquitin specific peptidase 9 X-linked (USP9X) as a stiffness-sensitive DUB acting through the discoidin domain receptor (DDR)/actomyosin signaling pathway. USP9X regulates levels of the mechanosensor YAP by preventing its proteasomal degradation via deubiquitination. Inhibition or knockdown of USP9X reduced YAP expression, impaired tumor cell migration, invasion, and ECM contraction, and decreased metastatic potential in vivo. Targeting USP9X also enhanced the effectiveness of BRAF-targeted therapies by limiting YAP-mediated mechanosensing, drug resistance, and tumor relapse. These findings establish USP9X as a mechanoresponsive DUB essential for cancer cell adaptation to mechanical cues, proposing it as a targetable mechanosensitive therapeutic target in cancer.

The constant emergence of drug-resistant mycobacteria, together with the lack of new antibiotics entering the market, has become a global public health problem that threatens the effective treatment of infectious diseases. The development of single molecules targeting different proteins should significantly reduce the emergence of resistant strains, and therefore represent a promising strategy to overcome such an issue. In this challenging context, a new series of 30 lipophilic compounds based on the β-lactone-core has been synthesized by varying the nature of the substituents on the lactone ring. The evaluation of their antibacterial activity against M. tuberculosis and M. abscessus, two major pathogenic mycobacteria, highlighted potential candidates. The VM038, VM040 and VM045 were active only against M. tuberculosis, while VM025, VM026 and VM043 inhibited the growth of both M. tuberculosis and the S and R variants of M. abscessus. Competitive click chemistry activity-based protein profiling revealed several potential M. abscessus target enzymes of VM043, the best extracellular growth inhibitor. Finally, when tested against intracellular bacteria, although VM043 was found inactive, VM025 & VM026 proved to be potent and promising inhibitors of intramacrophagic M. abscessus growth with minimal inhibitory concentrations (MIC50Raw) comparable to the standard antibiotic imipenem. Overall, these results strengthen the added value of our VM β-lactone derivatives not only in the fight against pathogenic mycobacteria, leading to the arrest of M. abscessus and/or M. tuberculosis growth through multitarget enzyme inhibition, but also as efficient probes to identify novel potential therapeutic targets using chemoproteomics approaches.