Polo-like kinase 1 (PLK1) is a mitotic kinase that is frequently overexpressed across many cancer types and strongly associated with poor prognosis and resistance to treatment. Comprehensive genomic, genetic, and pharmacological screens consistently pinpoint PLK1 as a therapeutic target in oncology. Despite promising preclinical anti-tumor efficacy of PLK1 inhibitors, clinical outcome as a monotherapy has been modest, necessitating the development of next-generation PLK1 inhibitors and combination strategies. The role of PLK1 extends beyond cell cycle regulation to tumor progression, immune evasion, and the tumor microenvironment. Emerging evidence supports synergistic potential of new-generation PLK1 inhibitors, such as Onvansertib, with chemo- and immune-therapies. This mini-review and perspective present current insights on PLK1 overexpression and its mechanistic impact on cancer aggressiveness and therapy resistance. We highlight the need for refined patient stratification and innovative combination regimens to exploit PLK1 inhibition in cancer treatment.

Death-associated protein kinase-Related Apoptosis-inducing protein Kinase 1 (DRAK1/STK17A) is a serine/threonine kinase of the Death Associated Protein Kinase (DAPK) family. STK17A is widely expressed and enriched in immune tissues, and is primarily localized in the nucleus, though it can translocate to the cytoplasm in response to specific stimuli. STK17A stimulates apoptosis and cytoskeletal dynamics, but its physiological roles remain incompletely defined, in part due to limited availability of potent/selective chemical probes and the absence of STK17A in commonly used rodent models. In this review, we summarize current knowledge on STK17A, including its structure, evolution, expression patterns, molecular interactions, and roles in cancer as well as in autoimmune, cardiovascular, infectious, and neurological disorders. We also compare STK17A with its closest homolog, STK17B, highlighting both shared features and functional distinctions. The review further examines recent medicinal chemistry efforts that have yielded the first small-molecule modulators of STK17A (DRAK1) and STK17B (DRAK2), including dual inhibitors and emerging selective scaffolds. These compounds can serve as valuable chemical probes and hold promising therapeutic potential. Nonetheless, challenges of selectivity and functional validation remain, emphasizing the need for continued medicinal chemistry efforts to unlock the full potential of STK17A as a therapeutic target across cancer, autoimmune, and neurodegenerative diseases.

Duchenne muscular dystrophy (DMD) is a severe X-linked disease caused by pathogenic variants in the DMD gene, resulting in the absence of functional dystrophin. Antisense oligonucleotide (ASO)-based therapies aim to restore the open reading frame and produce a truncated but functional dystrophin protein. Although several ASOs are approved in the United States and Japan via accelerated approval procedures, dystrophin restoration in patient biopsies remains low, underlining the need to improve ASO potency. One major limitation is poor ASO biodistribution to skeletal muscle, influenced by both ASO chemistry and pathological features of dystrophic tissue. In DMD patients and mdx mice, microvascular abnormalities and impaired angiogenesis likely restrict ASO delivery. Here, we hypothesized that enhancing muscle vascularization could improve ASO biodistribution and therapeutic outcomes. Mdx mice were treated with a pro-angiogenic treatment prior to ASO administration targeting exon 23 of dystrophin pre-mRNA. Angiogenic stimulation increased capillary density and improved ASO delivery to muscles (3.8-fold), exon skipping (1.8-fold), and dystrophin expression (1.5-fold) compared to ASO alone. These molecular improvements were associated with increased myofiber size, larger mean cross-sectional area, and decreased serum myomesin levels, without signs of toxicity. This study provides proof of concept that promoting angiogenesis can enhance the potency of ASO-based treatments, offering a complementary strategy to improve therapeutic outcomes in DMD.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are central regulators of cardiovascular development and stress responses and have been implicated in the cardiovascular toxicity of tyrosine kinase inhibitors. While ERK signaling in cardiomyocytes has been extensively investigated, the isoform- and cell-specific contribution of ERK2 in vascular smooth muscle cells (VSMCs) to cardiac dysfunction remains incompletely understood. To address this issue, we generated mice with systemic deletion of Erk1 (Erk1-/-), SM22α-Cre-mediated deletion of Erk2 (SM22α-E2KO; targeting VSMCs and cardiomyocytes), and α-myosin heavy chain-Cre-mediated deletion of Erk2 in cardiomyocytes alone (MHCα-E2KO). SM22α-E2KO mice developed progressive eccentric left ventricular hypertrophy, systolic dysfunction, impaired exercise capacity, and markedly reduced survival, whereas MHCα-E2KO and Erk1-/- mice exhibited preserved cardiac structure and function under basal conditions. Vascular functional studies revealed enhanced phenylephrine-induced vasoconstriction and impaired acetylcholine-mediated relaxation in SM22α-E2KO mice, despite preserved ERK2 expression in aortic endothelial cells. Aortic RhoA/Rho-kinase activity was significantly increased in SM22α-E2KO mice, and pharmacological inhibition of Rho-kinase normalized vascular hypercontractility. Transcriptomic analyses of the heart demonstrated downregulation of excitation-contraction coupling and metabolic pathways, accompanied by activation of inflammatory and fibrotic signaling. Notably, partial loss of Erk1 further exacerbated mortality and cardiac dysfunction in SM22α-E2KO mice, indicating a compensatory role of ERK1 in the setting of ERK2 deficiency. Collectively, these findings identify ERK2 signaling in VSMCs as a critical determinant of vascular tone and cardiac systolic function and demonstrate that combined vascular and myocardial ERK2 deficiency promotes heart failure through Rho-kinase-dependent vascular dysfunction.

We read with great interest the recent study by Karaulic et al [...].

Background: Clear cell renal cell carcinoma (ccRCC) is predominantly treated with anti-angiogenic therapies (AATs), such as sunitinib and axitinib. While these therapies initially improve outcomes, resistance frequently emerges, limiting long-term efficacy. Understanding the molecular mechanisms underlying AAT resistance is essential to optimize treatment strategies. Methods: To identify factors involved in AAT resistance, we performed integrated transcriptomic and proteomic analyses on ccRCC cell lines subjected to either transient AAT treatment or with established acquired resistance. Functional validation was performed using in vitro assays (proliferation, migration, invasion) and in vivo zebrafish models. Plasma levels of candidate proteins were also measured in ccRCC patients and correlated with clinical outcomes. Results: Connective Tissue Growth Factor (CTGF) was consistently upregulated following treatment and in resistant cell lines. CTGF, a secreted protein regulated by Yes-associated protein (YAP) in the Hippo pathway, is known to promote angiogenesis, fibrosis, and tumor progression. Functionally, CTGF enhanced tumor cell aggressiveness in vitro and in vivo. Patient-derived samples also exhibited elevated CTGF levels in resistant tumors. Crucially, higher plasma CTGF levels were associated with shorter progression-free survival in ccRCC patients receiving AATs. Conclusion: CTGF is a key mediator of resistance to AATs in ccRCC, by promoting tumor progression and remodeling the tumor microenvironment. CTGF may thus serve as both a predictive biomarker and a therapeutic target. These findings support further investigation of CTGF inhibition as a strategy to overcome AAT resistance and improve treatment outcomes in ccRCC patients.

Breast cancer is the most prevalent cancer among women worldwide, with various subtypes that require distinct treatment approaches. Among these, Triple-Negative Breast Bancer (TNBC) is recognized as the most aggressive form, often associated with poor prognosis due to its lack of targeted therapeutic options. This review specifically focuses on Radiotherapy (RT) as a treatment modality for TNBC, evaluating recent advancements and ongoing challenges, particularly the issue of radioresistance. RT remains an essential part in the management of breast cancer, including TNBC. Over the years, multiple improvements have been made to enhance RT effectiveness and minimize resistance. The introduction of advanced techniques such as Stereotactic Body Radiation Therapy (SBRT) and Stereotactic Radiosurgery (SRS) has significantly improved precision and reduced toxicity. More recently, proton radiation therapy, a novel RT modality, has been introduced, offering enhanced dose distribution and reducing damage to surrounding healthy tissues. Despite these technological advancements, a subset of TNBC patients continues to exhibit resistance to RT, leading to recurrence and poor treatment outcomes. To overcome radioresistance, there is an increasing interest in combining RT with targeted therapeutic agents that sensitize cancer cells to radiation. Radiosensitizing drugs have been explored to enhance the efficacy of RT by making cancer cells more susceptible to radiation-induced damage. Potential candidates include DNA damage repair inhibitors, immune checkpoint inhibitors, and small-molecule targeted therapies that interfere with key survival pathways in TNBC cells. In conclusion, while RT remains a crucial modality for TNBC treatment, radioresistance remains a significant challenge. Future research should focus on optimizing RT techniques while integrating radiosensitizing agents to improve treatment efficacy. By combining RT with targeted drug therapy, a more effective and personalized treatment approach can be developed, ultimately improving patient outcomes and reducing recurrence rates in TNBC.

Sunitinib is an oral tyrosine kinase inhibitor used in treating advanced renal cell carcinoma (RCC) that exhibits significant efficacy but faces resistance in 30% of patients. Identifying the molecular mechanisms underlying resistance could enable the development of strategies to enhance sunitinib sensitivity. In this study, we showed that sunitinib induces a metabolic shift leading to increased serine synthesis in RCC cells. Activation of the GCN2-ATF4 stress response pathway was identified as the mechanistic link between sunitinib treatment and elevated serine production. The increased serine biosynthesis supported nucleotide synthesis and sustained cell proliferation, migration, and invasion following sunitinib treatment. Inhibiting key enzymes in the serine synthesis pathway, such as phosphoglycerate dehydrogenase and phosphoserine aminotransferase 1, enhanced the sensitivity of resistant cells to sunitinib. Beyond RCC, similar activation of serine synthesis following sunitinib treatment occurred in a variety of other cancer types, suggesting a shared adaptive response to sunitinib therapy. Together, this study identifies the de novo serine synthesis pathway as a potential target to overcome sunitinib resistance, offering insights into therapeutic strategies applicable across diverse cancer contexts. Significance: Sunitinib treatment induces metabolic reprogramming to provide essential metabolite building blocks for tumor survival, resistance, and progression by upregulating serine biosynthesis, which represents a targetable dependency to enhance therapeutic efficacy.

Immunofluorescence-based detection of proteins in fixed cells is a powerful tool for research in cell and developmental biology. While a variety of immunofluorescence protocols exist, they can be time consuming or require expensive equipment which may not be accessible to all laboratories. A common challenge in these protocols is the numerous washing steps, particularly in experiments with numerous conditions. To address this, here we introduce the IF-CRIB device, a 3D-printable wash rack specifically designed for applications involving a high number of round coverslips with adherent cultured cells. We detail its design and the 3D printing process which can be easily used by any laboratory and we highlight that it facilitates the numerous washing steps. In addition, we present the IF-Express protocol, an optimized and effective method that enables fast and consistent immunofluorescence results. As an example of the utility of the IF-CRIB device and the IF-Express protocol, we describe their application in the screening and characterization of several NIH3T3 cell clones expressing a degradable form of ERK2 kinase (ERK2-dTAG) after treatment with the dTAG-13 compound. The generation of ERK2-dTAG clones involves a knock-in strategy. We provide a detailed methodology for clone selection, immunofluorescence screening, and characterization of ERK2-dTAG, including degradation kinetics, dose-response analysis, and nuclear translocation assays to assess ERK2-dTAG functionality. The IF-CRIB device and IF-Express protocol has been proven to be efficient for the obtention and characterization of ERK2dTAG-expressing clones thereby offering a powerful framework for studying ERK2 dynamics in cell biology and disease models.

PURPOSE:To monitor intraocular mediator dynamics in treatment-naïve neovascular age-related macular degeneration (nAMD) patients treated with anti-VEGF intravitreal injections (IVIs) to identify individual mediator patterns correlating with treatment response. DESIGN:Interventional, monocentric, prospective, clinical study. PARTICIPANTS:Treatment-naïve nAMD patients. METHODS:Aqueous humor samples (100-200 μL) were collected by clear cornea paracentesis at baseline (before the first anti-VEGF IVI) and before the second and third anti-VEGF IVIs. The levels of 13 intraocular mediators were measured (VEGF-A, VEGF-C, PlGF, IL-1β, IL-6, IL-10, IL-18, CXCL1, CXCL5, CXCL7, CXCL8, MIP-1α and TNFα) using multiplex arrays. MAIN OUTCOMES MEASURES:The primary endpoint was the changes in intraocular inflammatory mediator levels between baseline and month 3. Secondary endpoints were the changes in best-corrected visual acuity (BCVA) and Central Retinal Thickness (CRT) between baseline and month 4. RESULTS:Fifteen eyes were included in the study. BCVA remained stable throughout the study (p = 0.07). CRT, foveal thickness, and the presence of intraretinal and subretinal fluid significantly decreased after anti-VEGF IVIs (p < 0.0001, p < 0.0001, p < 0.001 and p < 0.001, respectively). After anti-VEGF IVIs, VEGF-A levels significantly decreased (p < 0.0001). No significant differences in all other mediator levels were observed. Three patients had baseline VEGF-A levels ≤50 pg/mL: they showed higher baseline IL-6 levels (p = 0.05), and elevated IL-6 (p = 0.03), PlGF (p = 0.02), VEGF-C (p = 0.005), IL-8 (p = 0.04), and TNFα (p = 0.013) levels after the first IVI. Good clinical responders had significantly higher baseline VEGF-A levels (p = 0.007). Patients who required a fourth IVI within 8 weeks of the loading dose had higher baseline TNFα levels (p = 0.05); higher MIP-1α levels after the first IVI (p = 0.045); and elevated TNFα (p = 0.026) and IL-8 (p = 0.029) levels after the second IVI. CONCLUSIONS:The aqueous humor levels of the studied mediators remained stable after anti-VEGF IVIs, except for a significant decrease in VEGF-A levels in all patients. Patients with low baseline intraocular VEGF-A levels (i.e., ≤50 pg/mL) showed an intraocular inflammatory profile with elevated IL-6, PlGF, VEGF-C, IL-8 and TNFα levels. Treatment response correlated with high baseline VEGF-A levels. An interval > 8 weeks between the third and fourth anti-VEGF IVIs was associated with a pro-angiogenic/pro-inflammatory environment.

BACKGROUND/OBJECTIVES:The advent of tyrosine kinase inhibitors (TKI), therapeutic antibodies and inducers of apoptosis has revolutionized cancer treatment, yet their application in pediatric tumors, particularly medulloblastoma, remains understudied. Understanding the expression of these targets in specific genetic subgroups could unveil potential repositioning opportunities for already approved drugs. METHODS:We analyzed RNA-sequencing data from the R2 Genomics Analysis and Visualization Platform (N = 763 patients, multiple cohorts) and the TCGA database (six individual cohorts 828 patients) to assess the expression of 73 potential targets of TKIs and antibodies targeting immune checkpoint inhibitors (ICI) or membrane receptors and inducers of apoptosis. These treatments, FDA-approved or in phase II clinical trials for solid or hematologic cancers, and their targets were evaluated in both non-metastatic and metastatic patients when data was available. Additionally, we examined treatments tailored to mutated targets crucial for tumorigenesis or resistance to conventional therapies. RESULTS:Overexpression of certain targets beyond predefined cutoff values in Kaplan-Meier analyses correlated with either prolonged or shortened overall survival. Targets associated with shorter survival suggested potentially relevant treatments, thereby highlighting the importance of defining specific treatments for distinct genetic subgroups. Notably, certain immune checkpoint inhibitors showed relevance for specific subgroups but detriment for others. As a positive control, our analysis confirmed the use of axitinib, an anti-angiogenic treatment, as demonstrated by our recent publication. Surprisingly, a treatment developed for hematological tumors, venetoclax, demonstrated potential efficacy in medulloblastoma. CONCLUSIONS:Medulloblastoma displays subtype-specific expressions of FDA-approved TKI, ICI and pro-apoptotic drug targets, impacting overall survival. Clinical trials investigating these approved treatments in medulloblastoma are therefore warranted.

This review explores treating metastatic clear cell renal cell carcinoma (ccRCC) through current therapeutic modalities—anti-angiogenic therapies and immunotherapies. While these approaches represent the forefront, their limitations and variable patient responses highlight the need to comprehend underlying resistance mechanisms. We specifically investigate the role of fibrosis, prevalent in chronic kidney disease, influencing tumour growth and treatment resistance. Our focus extends to unravelling the intricate interplay between fibrosis, immunotherapy resistance, and the tumour microenvironment for effective therapy development. The analysis centres on connective tissue growth factor (CTGF), revealing its multifaceted role in ccRCC—promoting fibrosis, angiogenesis, and cancer progression. We discuss the potential of targeting CTGF to address the problem of fibrosis in ccRCC. Emphasising the crucial relationship between fibrosis and the immune system in ccRCC, we propose that targeting CTGF holds promise for overcoming obstacles to cancer treatment. However, we recognise that an in-depth understanding of the mechanisms and potential limitations is imperative and, therefore, advocate for further research. This is an essential prerequisite for the successful integration of CTGF-targeted therapies into the clinical landscape.

Metastatic triple-negative breast cancers (TNBC) are among the most aggressive types of breast cancer and are often treated with adjuvant radiotherapy and chemotherapy. Despite initial efficacy, relapses are common, leading to poor prognosis. Understanding the response of tumor microenvironment to radiotherapy is crucial, particularly comparing photon (X) and proton (P) radiotherapy due to proton radiation's reduced side effects. Methods: We investigated the effects of single and multiple X and P irradiations on various cell types within the tumor microenvironment, including vascular and lymphatic endothelial cells, fibroblasts, and TNBC tumor cells. VEGFC, a key factor in lymphatic vessel formation and metastasis, was a primary focus. We used protein arrays to evaluate the effects of irradiation and examined the impact of VEGFC inactivation on the sensitivity to X and P radiation. Additionally, we tested tumor-forming capabilities of irradiated cells and assessed the impact of genetic or therapeutic VEGFC inhibition on TNBC growth. Transcriptomic and proteomic analyses further characterized the differences between X and P tumors, providing deeper insights into their distinct molecular profiles. Results: Both X and P irradiations caused a transient increase in VEGFC levels, along with other pro-angiogenic, pro-lymphangiogenic, and pro-fibrotic factors, such as angiopoietin 2, artemin, endostatin, IGFBP2, serpinE1, PDGFA, and DPPIV. Endothelial cells exposed to multiple rounds of radiation showed enhanced proliferation but lost the ability to form pseudo vessels, indicating an endothelial-mesenchymal transition. Tumor cells lacking VEGFC were more sensitive to radiation, and anti-VEGFC antibodies significantly suppressed TNBC cells proliferation, both naive and multi-irradiated. Tumor xenografts formed by multi-irradiated cells grew larger in nude mice, particularly following proton irradiation, while X-irradiated tumors exhibited a more pro-lymphangiogenic phenotype compared to P-irradiated tumors. Conclusions: Our findings show that while P multi-irradiated TNBC cells form larger tumors, X multi-irradiated tumors are more aggressive, with elevated expression of genes linked to angiogenesis, lymphangiogenesis, and endothelial-mesenchymal transition. Targeting VEGFC during photon or proton radiotherapy could reduce metastasis and improve TNBC prognosis. ### 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.

Background In clear cell renal cell carcinoma (ccRCC), first-line treatment combines nivolumab (anti-PD-1) and ipilimumab (anti-CTLA4), yielding long-term remissions but with only a 40% success rate. Our study explored the potential of enhancing ccRCC treatment by concurrently using CXCR2 inhibitors alongside immunotherapies. Methods We analyzed ELR + CXCL levels and their correlation with patient survival during immunotherapy. RCT001, a unique CXCR2 inhibitor, was examined for its mechanism of action, particularly its effects on human primary macrophages. We tested the synergistic impact of RCT001 in combination with immunotherapies in both mouse models of ccRCC and human ccRCC in the presence of human PBMC. Resuts Elevated ELR + CXCL cytokine levels were found to correlate with reduced overall survival during immunotherapy. RCT001, our optimized compound, acted as an inverse agonist, effectively inhibiting angiogenesis and reducing viability of primary ccRCC cells. It redirected M2-like macrophages without affecting M1-like macrophage polarization directed against the tumor. In mouse models, RCT001 enhanced the efficacy of anti-CTLA4 + anti-PD1 by inhibiting tumor-associated M2 macrophages and tumor-associated neutrophils. It also impacted the activation of CD4 T lymphocytes, reducing immune-tolerant lymphocytes while increasing activated natural killer and dendritic cells. Similar effectiveness was observed in human RCC tumors when RCT001 was combined with anti-PD-1 treatment. Conclusions RCT001, by inhibiting CXCR2 through its unique mechanism, effectively suppresses ccRCC cell proliferation, angiogenesis, and M2 macrophage polarization. This optimization potentiates the efficacy of immunotherapy and holds promise for significantly improving the survival prospects of metastatic ccRCC patients.

CXCR1/2 biomolecules play vital roles in cancer cell proliferation, tumor inflammation, and angiogenesis, making them attractive drug targets. In clear cell renal cell carcinoma (RCC) and head and neck squamous cell carcinoma (HNSCC), where CXCR1/2 is overexpressed, inhibition studies are limited. Building upon previous research efforts, we investigated new N,N'-diarylurea analogues as ELR+CXCL-CXCR1/2 inhibitors. Evaluations on RCC and HNSCC cell lines and 3D spheroid cultures identified compound 10 as a lead molecule, exhibiting significant inhibition of invasion, migration, and neo-angiogenesis. It demonstrated strong interference with the signaling pathway, with high selectivity toward kinases. In vivo studies on zebrafish embryos and RCC xenografted mice showed notable anticancer, antimetastatic, and antiangiogenic effects after oral administration and minimal toxicity. Compound 10 emerges as a promising candidate for further preclinical development as an oral anticancer and antiangiogenic drug targeting the ELR+CXCL-CXCR1/2 pathway.

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

Background and objectives Age influences the development and progression of clear cell renal cell carcinoma (ccRCC). Management of non-metastatic (M0) ccRCC involves surgery, adjuvant immune checkpoint inhibition (e.g., pembrolizumab) and surveillance. For metastatic (M1) ccRCC, treatment combines immune checkpoint inhibitors with anti-angiogenic therapies, achieving long-term remission in approximately 30% of patients. Despite these advancements, a definitive cure remains elusive for most individuals. Furthermore, predictive criteria for relapse that incorporate age-specific variations are lacking, highlighting the critical need for prognostic models to optimize outcomes across diverse age groups. This study aimed to validate tumor aggressiveness markers in ccRCC, focusing on age as a determinant factor. Methods Data from The Cancer Genome Atlas (TCGA) were analyzed to identify age-related gene expression differences, which were then validated using an independent cohort and ccRCC cell lines. Random Survival Forest models were employed to predict overall survival (OS) and disease-free survival (DFS) post nephrectomy and progression-free survival upon tyrosine kinase inhibitors and immunotherapies. Key findings and limitations Of 110 age-differentiated genes identified, 31 were significantly correlated with OS and DFS. Poor prognostic markers included CIAO1, EIF2B1, NARF, UBE2G2, and SART3, while TMEM167B emerged as a good prognostic marker, and a potential predictive biomarker for immunotherapy efficacy. Limitations include the need for larger independent cohort validation and deeper exploration of gene mechanisms. Conclusions and clinical implications The study presents age-informed prognostic models that integrate gene expression profiles, offering a foundation for personalized treatment strategies. TMEM167B stands out as a novel candidate for improving age-specific ccRCC management. Advancing Practice What does the study add? This study explores age-dependent differentially expressed genes in clear cell renal cell carcinoma (ccRCC) and proposes a potential age-specific prognostic gene signature that could be applicable to both non-metastatic and metastatic patients. This signature may assist in identifying individuals at higher risk of relapse. Among the identified genes, TMEM167B is highlighted as a candidate associated with favorable prognosis and shows promise as a potential predictive biomarker for immunotherapy efficacy. Patient Summary Aggressiveness in ccRCC varies with age and gene expression. Tailored treatments based on these factors may enhance outcomes and minimize side effects, particularly for patients over 75 years of age. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the French ministry of research, the CNRS, The League against cancer, the French association for cancer research (ARC, labelled team and contract ARCAGEING2023020006332), the foundation for medical research (FRM). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: TCGA data base and publication of Beuselinck et al I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript