Extracellular matrix (ECM) stiffening is a biophysical hallmark of solid tumors. Cutaneous melanoma is an aggressive malignancy characterized by high heterogeneity and phenotypic plasticity in which melanoma cells switch from a proliferative and differentiated phenotype to an invasive, dedifferentiated and therapy-resistant state. However, the impact of ECM stiffness on the diverse cellular phenotypes of melanoma remains poorly defined. Here, we show that melanoma cell subpopulations differ in their responses to mechanical signals. Compared to melanocytic/transitory cells, dedifferentiated cells exhibited heightened sensitivity to stiff collagen matrices, characterized by increased cell spreading, focal adhesion maturation, YAP nuclear translocation and contractility. ECM stiffening enhanced proliferation, migration and invasion in dedifferentiated cells, whereas highly proliferative and poorly migratory melanocytic/transitory cells were less affected by collagen stiffness. Importantly, a soft ECM sensitized dedifferentiated cells, but not melanocytic/transitory cells, to BRAF/MEK inhibition. Mechanistically, the mechanosensitivity of dedifferentiated cells relies on collagen receptors DDR1 and DDR2, which control cytoskeleton reorganization and YAP mechanosignaling. Genetic or pharmacological inhibition of DDR, actomyosin contractility, or YAP suppressed stiffness-induced proliferation and migration, reduced traction forces, and restored sensitivity to targeted therapy in dedifferentiated cells. Conversely, ectopic DDR1/DDR2 expression confers mechanosensitive properties to melanocytic cells. Our results thus reveal that phenotypic plasticity endows dedifferentiated melanoma cells with increased addiction to mechanical cues and implicate DDR1/2-YAP-dependent signaling in this aggressive behavior.

ABSTRACT 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.

Cherubism is a rare genetic disorder caused by SH3BP2 mutations. This sterile autoinflammatory disease is characterized by jaw osteolysis, in which bone tissue is replaced by multinucleated giant cells containing fibrous tissue. The cherubism mouse model (Sh3bp2 KI) is characterized by systemic bone loss as well as inflammatory phenotypes induced and maintained by TNFα. IL-1β, produced by the NRLP3 inflammasome through recruitment of Caspase-1, is involved in the development of sterile autoinflammatory disease. We previously reported a cherubism patient with elevated serum IL-1β, and cherubism mice also have elevated serum IL-1β levels. Thus, we wanted to disentangle the role of IL-1β in cherubism. To that end, we deleted Caspase-1 in Sh3bp2 KI mice to tamp down IL-1β production. However, deleting Caspase-1 did not rescue the systemic bone and inflammatory phenotypes.

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.

Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm. Diagnosis of CEL is often challenging, notably because of the lack of recurrent and specific molecular event. We report here a case of CEL occurring in a 49-year-old man who presented a persistent hypereosinophilia (HE) associated with anemia and thrombopenia. Karyotyping showed a translocation t(5;12)(q31;p13). Targeted RNA Sequencing identified a novel ETV6::RAPGEF6 fusion gene, confirmed by RT-PCR. Despite several lines of treatment, the patient died after 16 months of duration with transformation to acute myeloid leukemia (AML).Contrary to myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK), where fusion genes are both class defining and involve tyrosine kinase genes as the 3' partner, fusion genes are exceedingly rare in non-MLN-TK myeloid malignancies with HE. The most reported fusion gene is ETV6::ACSL6, in rare cases. Previously, overexpression of IL3 (interleukin 3) has been described in myeloid neoplasms with ETV6::ACSL6 (previously named ACS2). In our case of CEL with the ETV6::RAPGEF6 fusion, we also demonstrated overexpression of IL3, which could potentially result from the proximity of the IL3 gene to RAPGEF6, similar to what is observed with ACSL6 and IL3. The use of RNA sequencing in routine diagnosis of CEL could provide evidence for clonal event such as gene fusion, improving diagnosis as well as prognosis and therapeutic approaches.

Transcription factor EB (TFEB) is a key transcription factor that orchestrates the cellular response to stress. Dysregulation of TFEB is associated with a range of human diseases, and understanding the regulatory mechanisms of TFEB is crucial for identifying potential drug targets. In this study, we used Caenorhabditis elegans to screen for E3 ubiquitin ligases regulating the activity of TFEB's homolog, HLH-30, upon pathogenic infection. We identified WWP-1 as a regulator of HLH-30-dependent immune response controlling HLH-30 stability to mediate host defense in vivo. We found that HLH-30 interacts with WWP-1, supporting a model of WWP-1 directly regulating HLH-30. Furthermore, we found that WWP-1's human homolog WWP2 binds TFEB, directly induces TFEB ubiquitination and stabilizes TFEB. Finally, we found that WWP2 is required for TFEB-dependent host response in human monocytes-derived macrophages upon infection. Overall, our work has identified an evolutionarily conserved regulation of TFEB by WWP2 and highlighted its role in modulating stress response.

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. ### Competing Interest Statement The authors have declared no competing interest.

BackgroundMultiple Myeloma (MM) is the second most common hematological malignancy, characterized by the accumulation of monoclonal plasmocytes in the bone marrow. Despite advancements with proteasome inhibitors, immunomodulatory agents, and CD38-targeting antibodies, MM remains largely incurable due to resistant clones and frequent relapses. The success of the proteasome inhibitor bortezomib (BTZ) in MM treatment highlights the critical role of the ubiquitin-proteasome system (UPS) in this disease. Deubiquitinases (DUBs), which regulate protein stability, interactions, and localization by removing ubiquitin modifications, have emerged as promising therapeutic targets in various cancers, including MM.MethodsThrough a comprehensive loss-of-function screen, we identified USP39 as a critical survival factor for MM cells. Gene Set Enrichment Analysis (GSEA) was employed to correlate USP39 mRNA levels with clinical outcomes in MM patients. USP39 protein expression was evaluated via immunohistochemistry (IHC) on bone marrow samples from MM patients and healthy controls. The impact of USP39 knockdown via SiRNA was assessed through in vitro assays measuring cellular metabolism, clonogenic capacity, cell cycle progression, apoptosis, and sensitivity to BTZ. Co-immunoprecipitation and deubiquitination assays were conducted to elucidate the interaction and regulation of ZEB1 by USP39. Finally, in vitro and in vivo zebrafish experiments were used to characterize the biological consequences of ZEB1 regulation by USP39.ResultsOur study found that elevated USP39 mRNA levels are directly associated with shorter survival in MM patients. USP39 protein expression is significantly higher in MM patient plasmocytes compared to healthy individuals. USP39 knockdown inhibits clonogenic capacity, induces cell cycle arrest, triggers apoptosis, and overcomes BTZ resistance. Gain-of-function assays revealed that USP39 stabilizes the transcription factor ZEB1, enhancing the proliferation and the trans-migratory potential of MM cells.ConclusionsOur findings highlight the critical role of the deubiquitinase USP39, suggesting that the USP39/ZEB1 axis could serve as a potential diagnostic marker and therapeutic target in MM.

BackgroundLarge language models (LLMs) have raised both interest and concern in the academic community. They offer the potential for automating literature search and synthesis for systematic reviews but raise concerns regarding their reliability, as the tendency to generate unsupported (hallucinated) content persist. ObjectiveThe aim of the study is to assess the performance of LLMs such as ChatGPT and Bard (subsequently rebranded Gemini) to produce references in the context of scientific writing. MethodsThe performance of ChatGPT and Bard in replicating the results of human-conducted systematic reviews was assessed. Using systematic reviews pertaining to shoulder rotator cuff pathology, these LLMs were tested by providing the same inclusion criteria and comparing the results with original systematic review references, serving as gold standards. The study used 3 key performance metrics: recall, precision, and F1-score, alongside the hallucination rate. Papers were considered “hallucinated” if any 2 of the following information were wrong: title, first author, or year of publication. ResultsIn total, 11 systematic reviews across 4 fields yielded 33 prompts to LLMs (3 LLMs×11 reviews), with 471 references analyzed. Precision rates for GPT-3.5, GPT-4, and Bard were 9.4% (13/139), 13.4% (16/119), and 0% (0/104) respectively (P<.001). Recall rates were 11.9% (13/109) for GPT-3.5 and 13.7% (15/109) for GPT-4, with Bard failing to retrieve any relevant papers (P<.001). Hallucination rates stood at 39.6% (55/139) for GPT-3.5, 28.6% (34/119) for GPT-4, and 91.4% (95/104) for Bard (P<.001). Further analysis of nonhallucinated papers retrieved by GPT models revealed significant differences in identifying various criteria, such as randomized studies, participant criteria, and intervention criteria. The study also noted the geographical and open-access biases in the papers retrieved by the LLMs. ConclusionsGiven their current performance, it is not recommended for LLMs to be deployed as the primary or exclusive tool for conducting systematic reviews. Any references generated by such models warrant thorough validation by researchers. The high occurrence of hallucinations in LLMs highlights the necessity for refining their training and functionality before confidently using them for rigorous academic purposes.

Rationale Multiple Myeloma (MM) stands as the second most common hematological malignancy characterized by the accumulation of monoclonal plasmocytes within the bone marrow. Despite the introduction of proteasome inhibitors, immunomodulatory agents and CD38-targeting antibodies which have extended survival rates, the disease remains incurable for most patients due to the emergence of resistant clones and frequent relapses. The efficacy of the proteasome inhibitor bortezomib (BTZ) in MM treatment underscores the critical role of the ubiquitin proteasome system (UPS) in this cancer. Deubiquitinases (DUBs), a class of enzymes governing the stability, interactions or localization of cellular proteins by removing ubiquitin modifications, have emerged as promising therapeutic targets across various cancers, including MM. Methods Through an exhaustive loss-of-function approach, we have identified for the first time USP39 DUB as a pivotal survival determinant for MM cells. Results Our analysis reveals a direct correlation between heightened USP39 mRNA levels and shorter survival in MM patients. Additionally, robust USP39 protein expression is observed in MM patient plasmocytes compared to healthy counterparts. Knockdown of Usp39 not only impedes clonogenic capabilities, but also induces apoptosis, triggers cell cycle arrest and overcomes BTZ resistance. Complementary gain-of-function assays, further elucidate how USP39, by stabilizing the transcription factor ZEB1, enhances the trans-migratory potential of MM cells. Conclusions In summary, our findings underscores the pivotal role of the deubiquitinase USP39, suggesting that targeting the USP39/ZEB1 axis hold promise as a prospective diagnostic marker and therapeutic target in MM. ### Competing Interest Statement The authors have declared no competing interest. * ### ABBREVATION ASCT : stem cell auto transplantation BM : bone marrow BTZ : bortezomib DUB : deubiquitinase EMT : epithelial-to-mesenchymal transition GFP : green fluorescent protein GSEA : Gene Set enrichment analysis HCC : hepatocellular carcinoma IMiDs : immunomodulatory drugs JAAMs : JAB1/PAB1/MPN-domain containing metallo-enzyme MGUS : monoclonal gammopathy of undetermined significance MJDs : Machado-Joseph disease protein domain proteases MM : multiple myeloma Monoclonal antibodies : mAbs NR : non relevant OTUs : ovarian tumor-related proteases PIs : proteasome inhibitors siRNA : small interfering RNA Ub : ubiquitin Ubis : Ubiquitinases UCHs : ubiquitin carboxy-terminal hydrolases UPS : ubiquitin proteasome system USP39 : ubiquitin-specific peptidase 39 USPs : ubiquitin-specific proteases WT : wild type ZEB1 : Zinc-finger E-box-binding homeobox 1

Supplementary Figure 1. b-AP15 and its derivative VLX1570 inhibit USP14 activity in melanoma cells. Supplementary Figure 2. USP14 inhibition irreversibly induces growth inhibition and cytotoxicity in melanoma cells. Supplementary Figure 3. USP14 overexpression reduces the cytotoxic effect of b-AP15 on melanoma cell. Supplementary Figure 4. siRNA-mediated depletion of USP14 reduces melanoma cell growth and focus formation. Supplementary Figure 5. USP14 targeting induces caspase-independent and ROS-dependent cell death. Supplementary Figure 6. USP14 inhibition induces a rapid accumulation of polyubiquitinated proteins and HSP70 in melanoma cells

Supplementary Figure 1 from Targeting Cancer Cell Metabolism: The Combination of Metformin and 2-Deoxyglucose Induces p53-Dependent Apoptosis in Prostate Cancer Cells

Supplementary Figures 5-6 from Spleen Tyrosine Kinase Functions as a Tumor Suppressor in Melanoma Cells by Inducing Senescence-like Growth Arrest

Supplementary Figure 2 from Spleen Tyrosine Kinase Functions as a Tumor Suppressor in Melanoma Cells by Inducing Senescence-like Growth Arrest

Supplementary Figure from Secretion of IL1 by Dedifferentiated Melanoma Cells Inhibits JAK1-STAT3–Driven Actomyosin Contractility of Lymph Node Fibroblastic Reticular Cells

<p>All significant (p {less than or equal to} 0.05) categories resulting from each list produced the over-represented biological themes shown in supplementary Table 1.</p>

<p>Legend to supplemental Figure 1: Ingenuity Pathway Analysis identifies a network of genes upregulated in both resistant clones compared to parental cells. The network is displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). All grey nodes correspond to up-regulated genes in both IM-R and PD-R resistant cells. As described in the legend provided, nodes are displayed using various shapes that represent the functional class of the gene product. Edges are displayed with various labels that describe the nature of the relationship between the nodes (A, activation; B, binding; E, expression; I, inhibition; P, phosphorylation; T, transcription). Edges without a label represent binding only. White nodes were identified by the pathway analysis as part of the network.</p>

Supplementary Methods, Legends for Table, Figures 1-6 from Spleen Tyrosine Kinase Functions as a Tumor Suppressor in Melanoma Cells by Inducing Senescence-like Growth Arrest

Supplementary Table 1 from Spleen Tyrosine Kinase Functions as a Tumor Suppressor in Melanoma Cells by Inducing Senescence-like Growth Arrest

Supplementary Figure legends and methods