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Reactive oxygen species (ROS), the principal mediators of oxidative stress, induce responses such as apoptosis or permanent growth arrest/senescence in normal cells. Moreover, p53 activation itself contributes to ROS accumulation. Here we show that treatment of p53-null cancer cells with sublethal concentrations of ROS triggered an arrest with some morphological similarities to cellular senescence. Different from a classical senescent arrest in G1, the ROS-induced arrest was predominantly in the G2 phase of the cell cycle, and its establishment depended at least in part on an intact Chk1-dependent checkpoint. Chk1 remained phosphorylated only during the repair of double strand DNA breaks, after which Chk1 was inactivated, the G2 arrest was suppressed, and some cells recovered their ability to proliferate. Inhibition of Chk1 by an RNAi approach resulted in an increase in cell death in p53-null cells, showing that the Chk1-dependent G2 checkpoint protected cells that lacked a functional p53 pathway from oxidative stress. It has been proposed that the induction of a senescent-like phenotype by antineoplastic agents can contribute therapeutic efficacy. Our results indicate that oxidative stress-induced growth arrest of p53-null tumor cells cannot be equated with effective therapy owing to its reversibility and supports the concept that targeting Chk1 may enhance the effects of DNA-damaging agents on cancer progression in such tumors.Oncogene (2006) 25, 6037–6047. doi:10.1038/sj.onc.1209629; published online 1 May 2006 [ABSTRACT FROM AUTHOR]

Cellular senescence, a form of terminal cell cycle arrest, is as a key driver of organismal ageing and an important factor in age-related diseases. Insights into the senescent phenotype have led to the development of novel therapeutic strategies, collectively known as senotherapies, that aim to ameliorate the detrimental effects of senescent cell accumulation in tissues. The senotherapeutic field has rapidly evolved over the past decade, with clinical translation of the first drugs discovered currently underway. What began as the straightforward removal of senescent cells using repurposed compounds, which were given the name of senolytics, has grown into an expanding field that uses different state of the art approaches to achieve the goal of preventing the build-up of senescent cells in the body. Here, we summarize the emergence of a new generation of senotherapies, based on improving the efficacy and safety of the original senolytics by making them targeted, but also branching out into drugs that prevent senescence (senoblockers) or revert it (senoreversers).The use of nanotechnology, specific antibodies, cell-based approaches and restored immunosurveillance is likely to revolutionize the field of senotherapies in the near future, hopefully allowing it to realize its full clinical potential., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Fasting can affect the body's inflammatory response, and this has been linked to potential health benefits, including improvements for people with rheumatic diseases. In this work, we evaluated, in vitro, how changes in nutrient availability alter the inflammatory response of macrophages. Macrophage-differentiated THP1 cells were cultured, deprived of FCS or subjected to cycles of FCS deprivation and restoration to mimic intermittent fasting. Changes in the macrophage phenotype, the cells' response to inflammatory stimuli and the level of mitochondrial alteration were assessed. The results indicate that while periods of serum starvation are associated with a decrease in IL1β and TNFα expression, consistent with an anti-inflammatory response, intermittent serum starvation cycles promote a pro-inflammatory phenotype. Rapid changes in reducing capacity and mitochondrial response were also observed. Of note, while some changes, such as the production of oxygen free radicals, were reversed with refeeding, others, such as a decrease in reducing capacity, were maintained and even increased. This study shows that different fasting protocols can have diverging effects and highlights that time-limited nutrient changes can significantly affect macrophage functions in cell cultures. These findings help elucidate some of the mechanisms by which specific fasting dietary interventions could help control inflammatory diseases.

Intermittent fasting (IF) has proven to be a feasible dietary intervention for the wider population. The recent increase in IF clinical trials highlights its potential effects on health, including changes in body composition, cardiometabolic status, and aging. Although IF may have clinical applications in different populations, studies suggest there may be sex-specific responses in parameters such as body composition or glucose and lipid metabolism. Here, the existing literature on IF clinical trials is summarized, the application of IF in both disease prevention and management is discussed, and potential disparities in response to this type of diet between men and women are assessed. Moreover, the potential mechanisms that may be contributing to the sexually dimorphic response, such as age, body composition, tissue distribution, or sex hormones are investigated. This review underscores the need to further study these sex-specific responses to IF to define the most effective time frames and length of fasting periods for men and women. Tailoring IF to specific populations with a personalized approach may help achieve its full potential as a lifestyle intervention with clinical benefits., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mouse Double Minute 2 (MDM2) is a key negative regulator of the tumor suppressor protein p53. MDM2 overexpression occurs in many types of cancer and results in the suppression of WT p53. The 14-3-3 family of adaptor proteins are known to bind MDM2 and the 14-3-3σ isoform controls MDM2 cellular localization and stability to inhibit its activity. Therefore, small molecule stabilization of the 14-3-3σ/MDM2 protein-protein interaction (PPI) is a potential therapeutic strategy for the treatment of cancer. Here, we provide a detailed biophysical and structural characterization of the phosphorylation-dependent interaction between 14-3-3σ and peptides that mimic the 14-3-3 binding motifs within MDM2. The data show that di-phosphorylation of MDM2 at S166 and S186 is essential for high affinity 14-3-3 binding and that the binary complex formed involves one MDM2 di-phosphorylated peptide bound to a dimer of 14-3-3σ. However, the two phosphorylation sites do not simultaneously interact so as to bridge the 14-3-3 dimer in a 'multivalent' fashion. Instead, the two phosphorylated MDM2 motifs 'rock' between the two binding grooves of the dimer, which is unusual in the context of 14-3-3 proteins. In addition, we show that the 14-3-3σ-MDM2 interaction is amenable to small molecule stabilization. The natural product fusicoccin A forms a ternary complex with a 14-3-3σ dimer and an MDM2 di-phosphorylated peptide resulting in the stabilization of the 14-3-3σ/MDM2 PPI. This work serves as a proof-of-concept of the drugability of the 14-3-3/MDM2 PPI and paves the way toward the development of more selective and efficacious small molecule stabilizers., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: It is known that the microenvironmental cytokine interferon gamma (IFN-γ) provides a survival advantage for chronic lymphocytic leukemia (CLL) cells. However, the mechanisms involved in this effect have not been properly investigated., Methods: Herein, we conducted a comprehensive screening of the effects of IFN-γ on signaling pathways and gene expression profiles in CLL cells by using western blotting, real-time quantitative reverse transcription (RT-qPCR) and high-throughput RNA sequencing (RNA-seq)., Results: We found that IFN-γ not only activated the pro-survival signal transducer and activator of transcription 3 (STAT3), but also activated the protein kinase B and extracellular signal-regulated kinase signaling pathways. RNA-seq analysis showed that IFN-γ stimulation changed the expression profiles of more than 500 genes, with 391 being up-regulated and 123 down-regulated. These genes are involved in numerous biological processes, including anti-apoptosis, cell migration, and proliferation. IFN-γ significantly up-regulated the expression of CD38, BCL6, CXCL9, BCL2A1, SCOS3, IL-10, HGF, EGFR, THBS-1, FN1, and MUC1, which encode proteins potentially associated with disease progression, worse prognosis or poor response to treatment. Blocking janus kinases1/2 (JAK1/2) or STAT3 signal by specific inhibitors affected the expression of most genes, suggesting a pivotal role of the JAK1/2-STAT3 pathway in IFN-γ pro-survival effects in CLL., Conclusions: Our data demonstrate that IFN-γ regulates a complex pro-survival signal network in CLL through JAK1/2-STAT3, which provides a rational explanation for IFN-γ promoting CLL cells survival and drug resistance., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The accumulation of senescent cells has an important role in the phenotypical changes observed in ageing and in many age-related pathologies. Thus, the strategies designed to prevent these effects, collectively known as senotherapies, have a strong clinical potential. Senolytics are a type of senotherapy aimed at specifically eliminating senescent cells from tissues. Several small molecule compounds with senolytic properties have already been identified, but their specificity and range of action are variable. Because of this, potential novel senolytics are being actively investigated. Given the involvement of HDACs and the PI3K pathway in senescence, we hypothesized that the dual inhibitor CUDC-907, a drug already in clinical trials for its antineoplastic effects, could have senolytic effects. Here, we show that CUDC-907 was indeed able to selectively induce apoptosis in cells driven to senesce by p53 expression, but not when senescence happened in the absence of p53. Consistent with this, CUDC-907 showed senolytic properties in different models of stress-induced senescence. Our results also indicate that the senolytic functions of CUDC-907 depend on the inhibitory effects of both HDACs and PI3K, which leads to an increase in p53 and a reduction in BH3 pro-survival proteins. Taken together, our results show that CUDC-907 has the potential to be a clinically relevant senolytic in pathological conditions in which stress-induced senescence is involved.

Although clinical outcomes of CLL have improved with the use of BCL-2 inhibitor, ABT-199, acquired resistance eventually occurs in many cases, which leads to CLL disease progression. Thus, understanding the mechanisms that mediate this relapse is important to design improved therapies. Herein, we report that cytokine IFN-γ, secreted by dysfunctional T cells, enhanced CLL cells resistance to ABT-199. IFN-γ stimulation significantly increased the expression of BCL-2, MCL-1 and BCL-xL. Blocking JAK1/2-STAT3 signaling pathway impaired the expression of these anti-apoptotic proteins after IFN-γ stimulation. The combination of ABT-199 with JAK1/2 inhibitor Ruxolitinib or STAT3 inhibitors Stattic and C188-9 increased malignant B cell death. In summary, we show that IFN-γ enhanced CLL cells resistance to ABT-199 at least in part by up-regulating BCL-2, MCL-1 and BCL-xL expression via JAK1/2-STAT3 pathway, and thus blocking this pathway with inhibitors increased ABT-199 efficiency to induce CLL cell apoptosis, suggesting a potential therapeutically relevant combination to overcome ABT-199 resistance.

Background: This study attempts to provide a picture of the hesitancy to vaccination against COVID-19 in Spain during the 2021 spring-autumn vaccination campaign, both in the general population and in healthcare professionals., Methods: The participants were recruited using social media such as Facebook and Twitter, in addition to the cooperation of health personnel contacted with the collaboration of medical scientific societies. A cross-sectional study was carried out that included the response of an online questionnaire. The data were collected from April 30 to September 26, 2021. To assess the different associations between variables to be measured, we fit Poisson regression models with robust variance., Results: Responses were obtained from 3,850 adults from the general population group and 502 health professionals. Of the overall sample, 48.6% of participants from the general population were vaccinated against COVID-19, whereas in the healthcare professionals, 94.8% were vaccinated. The prevalence of general population vaccination increased with age, and was higher in women than men. Most participants did not show a preference for any vaccine itself. However, the prevalence of people vaccinated with their preferred vaccine was higher for the ones vaccinated with Pfizer's vaccine. 6.5% of the general population reported being reticent to be vaccinated. People from younger age groups, people with lower educational levels and those who were not from a risk group showed greater reluctance to be vaccinated. No gender differences in reluctancy were found., Conclusions: Health professionals were significantly less likely to refuse vaccination even though they had more doubts about the safety and efficacy of vaccines. On the other hand, younger people, those with a lower level of education and those who were not from a risk group were the most hesitant., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Saigí-Rubió et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cellular senescence has proved to be a strong contributor to ageing and age-related diseases, such as cancer and atherosclerosis. Therefore, the protein content of senescent cells is highly relevant to drug discovery, diagnostics and therapeutic applications. However, current technologies for the analysis of proteins are based on a combination of separation techniques and mass spectrometry, which require handling large sample sizes and a large volume of data and are time-consuming. This limits their application in personalised medicine. An easy, quick and inexpensive procedure is needed for qualitative and quantitative analysis of proteins expressed by a cell or tissue. Here, we describe the use of the "snapshot imprinting" approach for the identification of proteins differentially expressed by senescent cells. Molecularly imprinted polymer nanoparticles (MIPs) were formed in the presence of whole cells. Following trypsinolysis, protein epitopes protected by complex with MIPs were eluted from the nanoparticles and analysed by LC-MS/MS. In this work, "snapshot imprinting" was performed parallel to a standard proteomic "shaving approach", showing similar results. The analysis by "snapshot imprinting" identified three senescent-specific proteins: cell division cycle 7-related protein kinase, partitioning defective three homolog B and putative ATP-dependent RNA helicase DHX57, the abundance of which could potentially make them specific markers of senescence. Identifying biomarkers for the future elimination of senescent cells grants the potential for developing therapeutics for age-related diseases., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Determining which cancer patients will be sensitive to a given therapy is essential for personalised medicine. Thus, it is important to develop new tools that will allow us to stratify patients according to their predicted response to treatment. The aim of work presented here was to use molecular imprinting for determining the sensitivity of lung cancer cell lines to ionising radiation based on cell surface proteomic differences. Molecularly imprinted polymer nanoparticles (nanoMIPs) were formed in the presence of whole cells. Following trypsinolysis, protein epitopes protected by complexing with MIPs were eluted from the nanoparticles and analysed by LC-MS/MS. The analysis identified two membrane proteins, neutral amino acid transporter B (0) and 4F2 cell-surface antigen heavy chain, the abundance of which in the lung cancer cells could indicate resistance of these cells to radiotherapy. This proof-of-principle experiments shows that this technology can be used in the discovery of new biomarkers and in development of novel diagnostic and therapeutic tools for a personalised medicine approach to treating cancer., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

The COVID-19 pandemic has been a phenomenal challenge to global health care and will continue to be so in the upcoming months. Beyond its medical toll, COVID-19 has also exacerbated pre-existing social issues and created new inequalities. This has generated a series of ethical problems that will need to be carefully analyzed to avoid repeating similar mistakes in the context of other crises. Among those, we discuss here the bioethical implications of preserving individual freedom in the context of the early response to a pandemic and propose a global approach to the issue that could be applied in future health challenges., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Macip and Yuguero.)

When a cell is damaged, it must decide how to respond. As a consequence of a variety of stresses, cells can induce well-regulated programmes such as senescence, a persistent proliferative arrest that limits their replication. Alternatively, regulated programmed cell death can be induced to remove the irreversibly damaged cells in a controlled manner. These programmes are mainly triggered and controlled by the tumour suppressor protein p53 and its complex network of effectors, but how it decides between these wildly different responses is not fully understood. This review focuses on the key proteins involved both in the regulation and induction of apoptosis and senescence to examine the key events that determine cell fate following damage. Furthermore, we examine how the regulation and activity of these proteins are altered during the progression of many chronic diseases, including cancer.

Cervical cancer is one of the most common malignancies in women, with a poor survival rate. Thus, there is a need to define effective combination strategies to improve therapy. In this study, we report that dsRNA poly(I:C) up-regulated the expression of IFNβ and apoptosis-associated genes in cervical cancer cells, activating both intrinsic and extrinsic apoptotic pathways, and eventually inducing cell death. Similarly, proteasome inhibitors also effectively induced cervical cancer cell apoptosis, probably through prevention of p53 degradation, inhibiting NF-κB signal activation and decreasing BCL-2 expression. Importantly, the combination of poly(I:C) with proteasome inhibitors enhanced caspase-8 and caspase-9 activation, and synergistically induced cervical cancer cell apoptosis. Both activated p38 signals and increased ROS levels, and their combination extended these effects. Collectively, we show that the activation of multiple pro-apoptotic pathways by poly(I:C) and proteasome inhibitors underpin a synergistic effect on inducing cervical cancer cell death, suggesting a potential therapeutic combination with clinical relevance., (Copyright © 2022. Published by Elsevier Inc.)

Retinoids are a group of vitamin A-related chemicals that are essential to chordate mammals. They regulate a number of basic processes, including embryogenesis and vision. From ingestion to metabolism and the subsequent cellular effects, retinoid levels are tightly regulated in the organism to prevent toxicity. One component of this network, the membrane receptor STRA6, has been shown to be essential in facilitating the cellular entry and exit of retinol. However, recent data suggests that STRA6 may not function merely as a retinoid transporter but also act as a complex signalling hub in its own right, being able to affect cell fate through the integration of retinoid signalling with other key pathways, such as those involving p53, JAK/STAT, Wnt/β catenin and calcium. This may open new therapeutic strategies in diseases like cancer, where these pathways are often compromised. Here, we look at the growing evidence regarding the novel roles of STRA6 beyond its well characterized classic functions., (© 2021. The Author(s).)

A wide range of diseases have been shown to be influenced by the accumulation of senescent cells, from fibrosis to diabetes, cancer, Alzheimer's and other age-related pathologies. Consistent with this, clearance of senescent cells can prolong healthspan and lifespan in in vivo models. This provided a rationale for developing a new class of drugs, called senolytics, designed to selectively eliminate senescent cells in human tissues. The senolytics tested so far lack specificity and have significant off-target effects, suggesting that a targeted approach could be more clinically relevant. Here, we propose to use an extracellular epitope of B2M, a recently identified membrane marker of senescence, as a target for the specific delivery of toxic drugs into senescent cells. We show that an antibody-drug conjugate (ADC) against B2M clears senescent cells by releasing duocarmycin into them, while an isotype control ADC was not toxic for these cells. This effect was dependent on p53 expression and therefore more evident in stress-induced senescence. Non-senescent cells were not affected by either antibody, confirming the specificity of the treatment. Our results provide a proof-of-principle assessment of a novel approach for the specific elimination of senescent cells using a second generation targeted senolytic against proteins of their surfaceome, which could have clinical applications in pathological ageing and associated diseases., (© 2021. The Author(s).)

14-3-3 proteins are an important family of hub proteins that play important roles in many cellular processes via a large network of interactions with partner proteins. Many of these protein-protein interactions (PPI) are implicated in human diseases such as cancer and neurodegeneration. The stabilisation of selected 14-3-3 PPIs using drug-like 'molecular glues' is a novel therapeutic strategy with high potential. However, the examples reported to date have a number of drawbacks in terms of selectivity and potency. Here, we report that WR-1065, the active species of the approved drug amifostine, covalently modifies 14-3-3σ at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3σ PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3σ, an isoform with particular involvement in cancer pathways. This mechanism can be exploited to harness the enhanced potency conveyed by covalent drug molecules and dual ligand cooperativity. This is demonstrated in two cancer cell lines whereby the cooperative behaviour of fusicoccin A and WR-1065 leads to enhanced efficacy for inducing cell death and attenuating cell growth., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: The requirement of promyelocytic leukaemia protein (PML) in interferon (IFN)-induced cell apoptosis is well-established. However, the exact mechanisms by which the multiple isoforms of PML protein participate in this process remain not well-understood. We previously demonstrated that PML isoform II (PML-II) positively regulates induced gene expression during a type I IFN response and evaluate here how PML-II contributes to IFNα-induced cell death., Methods: HeLa cells were transiently depleted of PML-II by siRNA treatment and the response of these cells to treatment with IFNα assessed by molecular assays of mRNA and proteins associated with IFN and apoptosis responses., Results: In HeLa cells, death during IFNα stimulation was reduced by prior PML-II depletion. PML-II removal also considerably decreased the induced expression of pro-apoptotic ISGs such as ISG54 (IFIT2), and substantially impaired or prevented expression of PUMA and TRAIL, proteins that are associated with the intrinsic and extrinsic apoptotic pathways respectively. Thirdly, PML-II depletion enhanced ERK and AKT pro-survival signaling activation suggesting that PML-II normally suppresses signaling via these pathways, and that lack of PML-II hence led to greater than normal activation of AKT signaling upon IFNα stimulation and consequently increased resistance to IFNα-induced apoptosis., Conclusions: The positive contribution of PML-II to the expression of various IFNα-induced pro-apoptotic proteins and its inhibition of pro-survival signaling together provide a mechanistic explanation for reduced apoptosis under conditions of PML deficiency and may account for at least part of the role of PML as a tumor suppressor gene. Video Abstract.

The outbreak of the novel coronavirus disease 2019 (COVID-19) has emerged as one of the biggest global health threats worldwide. As of October 2020, more than 44 million confirmed cases and more than 1,160,000 deaths have been reported globally, and the toll is likely to be much higher before the pandemic is over. There are currently little therapeutic options available and new potential targets are intensively investigated. Recently, Bruton tyrosine kinase (BTK) has emerged as an interesting candidate. Elevated levels of BTK activity have been reported in blood monocytes from patients with severe COVID-19, compared with those from healthy volunteers. Importantly, various studies confirmed empirically that administration of BTK inhibitors (acalabrutinib and ibrutinib) decreased the duration of mechanical ventilation and mortality rate for hospitalized patients with severe COVID-19. Herein, we review the current information regarding the role of BTK in severe acute respiratory syndrome coronavirus 2 infections and the suitability of its inhibitors as drugs to treat COVID-19. The use of BTK inhibitors in the management of COVID-19 shows promise in reducing the severity of the immune response to the infection and thus mortality. However, BTK inhibition may be contributing in other ways to inhibit the effects of the virus and this will need to be carefully studied., (©2020 American Association for Cancer Research.)

Most cancers evolve to disable the p53 pathway, a key tumour suppressor mechanism that prevents transformation and malignant cell growth. However, only ~50% exhibit inactivating mutations of p53, while in the rest its activity is suppressed by changes in the proteins that modulate the pathway. Therefore, restoring p53 activity in cells in which it is still wild type is a highly attractive therapeutic strategy that could be effective in many different cancer types. To this end, drugs can be used to stabilise p53 levels by modulating its regulatory pathways. However, despite the emergence of promising strategies, drug development has stalled in clinical trials. The need for alternative approaches has shifted the spotlight to the 14-3-3 family of proteins, which strongly influence p53 stability and transcriptional activity through direct and indirect interactions. Here, we present the first detailed review of how 14-3-3 proteins regulate p53, with special emphasis on the mechanisms involved in their binding to different members of the pathway. This information will be important to design new compounds that can reactivate p53 in cancer cells by influencing protein-protein interactions. The intricate relationship between the 14-3-3 isoforms and the p53 pathway suggests that many potential drug targets for p53 reactivation could be identified and exploited to design novel antineoplastic therapies with a wide range of applications.

The BCL-2-specific inhibitor, ABT-199 (venetoclax) has exhibited remarkable clinical activity in nearly all cases of chronic lymphocytic leukemia. In contrast, responses are usually much less in diffuse large B-cell lymphoma (DLBCL), despite high level expression of BCL-2 in over 40% of cases, indicating that co-expression of related anti-apoptotic BCL-2 family proteins may limit the activity of ABT-199. We have investigated the roles of BCL-2 proteins in DLBCL cells using a panel of specific BCL-2 homology 3 (BH3)-mimetics and identified subgroups of these cells that exhibited marked and specific dependency on either BCL-2, BCL-X L or MCL-1 for survival. Dependency was associated with selective sequestration of the pro-apoptotic proteins BIM, BAX and BAK by the specific anti-apoptotic BCL-2 protein which was important for cellular survival. Sensitivity to BH3-mimetics was independent of genetic alterations involving the BCL-2 family and only partially correlated with protein expression levels. Treatment with ABT-199 displaced BAX and BIM from BCL-2, subsequently leading to BAK activation and apoptosis. In contrast, apoptosis induced by inhibiting BCL-X L with A1331852 was associated with a displacement of both BAX and BAK from BCL-X L and occurred independently of BIM. Finally, the MCL-1 inhibitor S63845 induced mainly BAX-dependent apoptosis mediated by a displacement of BAK, BIM and NOXA from MCL-1. In conclusion, our study indicates that in DLBCL, the heterogeneous response to BH3-mimetics is mediated by selective interactions between BAX, BAK and anti-apoptotic BCL-2 proteins., (Copyright© 2020 Ferrata Storti Foundation.)

Objectives: To identify differences in the mutational profile of endometrial tumours between British White (BW) and South Asian (BSA) women., Methods: We analysed primary tumours from matched cohorts of British White (BW) and British South Asian (BSA) women resident in Leicestershire diagnosed with EC. Next Generation Sequencing was performed to investigate mutational differences in a panel of 10 genes previously identified as being commonly mutated in EC. The presence of somatic Mismatch Repair (MMR) gene deficiencies was determined by immunohistochemistry., Results: In total, 57 tumours (27 BSA and 30 BW) were sequenced. There was no significant difference in the overall mutation frequency of the 10 genes analysed; however, numerous differences were observed between the groups. There was a positive association between PIK3CA and PTEN mutations in the BSA group, with 78% of PIK3CA-mutant tumours harbouring a PTEN mutation, whereas only 11% of PIK3CA wild-type (wt) tumours were PTEN mutant positive (p = 0.0012). In BW women, 90% of ARID1A mutant tumours had co-existent PI3K pathway mutations versus 50% of wild-type (wt) ARID1A patients (p = 0.0485). This trend was not significant in the BSA group (p = 0.66). The age at diagnosis was significantly higher in the BW group with a somatic MMR gene deficiency compared to those with no deficiency (72.8 years versus 59.6 years, p = 0.007), whereas this difference was not seen in the BSA group (64 years versus 60 years, p = 0.37)., Conclusion: We have identified differences in the mutational profile of primary EC tumours from BW and BSA women. Further research is needed to confirm these findings and to explore their potential implications for early detection, treatment response and prognosis., Competing Interests: The authors have declared that no competing interests exist.

The average human life expectancy has increased globally, and continues to rise, owing to the substantive progress made in healthcare, medicine, sanitation, housing and education. This ultimately enriches society with a greater proportion of elderly people. Sustaining a healthy aged population is key to diminish the societal and economic impact of age-related infirmities. This is especially challenging because tissue function, and thus wellbeing, naturally progressively decline as humans age. With age increasing the risk of developing diseases, one of the therapeutic options is to interfere with the molecular and cellular pathways involved in age-related tissue dysfunction, which is in part caused by the accumulation of senescent cells. One strategy to prevent this could be using drugs that selectively kill these cells (senolytics). In parallel, some compounds have been identified that prevent or slow down the progression of senescence or some of its features (senostatics). Senolytic and senostatic therapies have been shown to be efficient in vivo , but they also have unwanted dose-dependent side effects, including toxicity. Important advances might be made using bioactive compounds from plants and foods (nutraceuticals) if, as is proposed, they offer similar effectiveness with fewer side effects. The focus of this review is on the use of nutraceuticals in interfering with cellular senescence., (Copyright © 2020 Kaur, Macip and Stover.)

One of the hallmarks of aging is the progressive accumulation of senescent cells in organisms, which has been proposed to be a contributing factor to age-dependent organ dysfunction. We recently reported that Bruton's tyrosine kinase (BTK) is an upstream component of the p53 responses to DNA damage. BTK binds to and phosphorylates p53 and MDM2, which results in increased p53 activity. Consistent with this, blocking BTK impairs p53-induced senescence. This suggests that sustained BTK inhibition could have an effect on organismal aging by reducing the presence of senescent cells in tissues. Here, we show that ibrutinib, a clinically approved covalent inhibitor of BTK, prolonged the maximum lifespan of a Zmpste24 -/- progeroid mice, which also showed a reduction in general age-related fitness loss. Importantly, we found that certain brain functions were preserved, as seen by reduced anxiety-like behaviour and better long-term spatial memory. This was concomitant to a decrease in the expression of specific markers of senescence in the brain, which confirms a lower accumulation of senescent cells after BTK inhibition. Our data show that blocking BTK has a modest increase in lifespan in Zmpste24 -/- mice and protects them from a decline in brain performance. This suggests that specific inhibitors could be used in humans to treat progeroid syndromes and prevent the age-related degeneration of organs such as the brain., (© 2019 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

CUDC-907, a dual PI3K/HDAC inhibitor, has been proposed to have therapeutic potential in hematopoietic malignancies. However, the molecular mechanisms of its effects in chronic lymphocytic leukaemia (CLL) remain elusive. We show that CLL cells are sensitive to CUDC-907, even under conditions similar to the protective microenvironment of proliferation centres. CUDC-907 inhibited PI3K/AKT and HDAC activity, as expected, but also suppressed RAF/MEK/ERK and STAT3 signalling and reduced the expression of anti-apoptotic BCL-2 family proteins BCL-2, BCL-xL, and MCL-1. Moreover, CUDC-907 downregulated cytokines BAFF and APRIL and their receptors BAFFR, TACI, and BCMA, thus blocking BAFF-induced NF-κB signalling. T cell chemokines CCL3/4/17/22 and phosphorylation of CXCR4 were also reduced by CUDC-907. These data indicated that CUDC-907 abrogates different protective signals and suggested that it might sensitize CLL cells to other drugs. Indeed, combinations of low concentrations of CUDC-907 with inhibitors of BCL2, BTK, or the NF-κB pathway showed a potent synergistic effect. Our data indicate that, apart from its known functions, CUDC-907 blocks multiple pro-survival pathways to overcome microenvironment protection in CLL cells. This provides a rationale to evaluate the clinical relevance of CUDC-907 in combination therapies with other targeted inhibitors., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Many genes of the human genome display pleiotropic activity, playing an important role in two or more unrelated pathways. Surprisingly, some of these functions can even be antagonistic, often letting to divergent functional outcomes depending on microenviromental cues and tissue/cell type-dependent parameters. Lately, the Bruton's tyrosine kinase (BTK) has emerged as one of such pleiotropic genes, with opposing effects in cancer pathways. While it has long been considered oncogenic in the context of B cell malignancies, recent data shows that BTK can also act as a tumour suppressor in other cells, as an essential member of the p53 and p73 responses to damage. Since BTK inhibitors are already being used clinically, it is important to carefully review these new findings in order to fully understand the consequences of blocking BTK activity in all the cells of the organism.

Bruton's tyrosine kinase (BTK) is a key component of B cell receptor signalling. Because of this, BTK plays an important role in cell proliferation and survival in various B cell malignancies. However, in certain contexts, BTK can also have tumour suppressor functions. We have previously shown that BTK activates the p53 transcriptional activity by binding to and phosphorylating p53, as well as acting on MDM2 to reduce its inhibitory effects. This results in increased p53 functions, including enhanced cell death. Here, we report that BTK can also induce cell death and increase responses to DNA damage independently of p53. This is concomitant to the induction of p21, PUMA and MDM2, which are classic target genes of the p53 family of proteins. Our results show that these p53-independent effects of BTK are mediated through p73. Similar to what we observed in the p53 pathway, BTK can upregulate p73 after DNA damage and induce expression of its target genes, suggesting that BTK is a modulator of p73 functions and in the absence of p53. This effect allows BTK to have pro-apoptotic functions independently of its effects on the p53 pathway and thus play an important role in the DNA damage-related induction of apoptosis in the absence of p53. This provides a novel role of BTK in tumour suppression and contributes to the understanding of its complex pleiotropic functions., Competing Interests: The authors declare no conflict of interest.

Endometrial cancer (EC) is now the most prevalent gynaecological malignancy in the Western world. Black or African American women (BoAA) have double the mortality of Caucasian women, and their tumours tend to be of higher grade. Despite these disparities, little is known regarding the mutational landscape of EC between races. Hence, we wished to investigate the molecular features of ECs within The Cancer Genome Atlas (TCGA) dataset by racial groupings. In total 374 Caucasian, 109 BoAA and 20 Asian patients were included in the analysis. Asian women were diagnosed at younger age, 54.2 years versus 64.5 years for Caucasian and 64.9 years for BoAA women (OR 3.432; p=0.011); BoAA women were more likely to have serous type tumors (OR 2.061; p=0.008). No difference in overall survival was evident. The most frequently mutated gene in Caucasian and Asian tumours was PTEN (63% and 85%), unlike BoAA cases where it was TP53 (49%). Mutation and somatic copy number alteration (SCNA) analysis revealed an enrichment of TP53 mutations in BoAAs; whereas POLE and RPL22 mutations were more frequent in Caucasians. Major recurrent SCNA racial differences were observed at chromosomes 3p, 8, 10, and 16, which clustered BoAA tumors into 4 distinct groups and Caucasian tumors into 5 groups. There was a significantly higher frequency of somatic mutations in DNA mismatch repair genes in Asian tumours, in particular PMS2 (p=0.0036). In conclusion, inherent racial disparities appear to be present in the molecular profile of EC, which could have potential implications on clinical management., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

p53 is a tumour suppressor that is activated in response to various types of stress. It is regulated by a complex pattern of over 50 different post-translational modifications, including ubiquitination by the E3 ligase MDM2, which leads to its proteasomal degradation. We have previously reported that expression of Bruton's Tyrosine Kinase (BTK) induces phosphorylation of p53 at the N-terminus, including Serine 15, and increases its protein levels and activity. The mechanisms involved in this process are not completely understood. Here, we show that BTK also increases MDM2 and is necessary for MDM2 upregulation after DNA damage, consistent with what we have shown for other p53 target genes. Moreover, we found that BTK binds to MDM2 on its PH domain and induces its phosphorylation. This suggested a negative regulation of MDM2 functions by BTK, supported by the fact BTK expression rescued the inhibitory effects of MDM2 on p53 transcriptional activity. Indeed, we observed that BTK mediated the loss of the ubiquitination activity of MDM2, a process that was dependent on the phosphorylation functions of BTK. Our data together shows that the kinase activity of BTK plays an important role in disrupting the MDM2-p53 negative feedback loop by acting at different levels, including binding to and inactivation of MDM2. This study provides a potential mechanism to explain how BTK modulates p53 functions., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Senescence is a cellular process that is thought to have prognostic and therapeutic relevance in conditions such as cancer, aging, and fibrosis. However, current protocols for identifying senescent cells in vitro and in vivo have several drawbacks. Most markers used lack sufficient specificity and false positives and negatives in common. In addition, classical staining techniques often require lengthy protocols and do not offer objective quantification. Recently, several novel markers of senescence associated with the plasma membrane have been identified. Here, we propose to take advantage of these markers to define a customizable FACS-based protocol to detect senescent cells using antibodies tagged with fluorescence dyes. This method has the advantage of being fast and allowing quantitation. Furthermore, its specificity is increased using several markers simultaneously.

Chronic lymphocytic leukemia (CLL) cells multiply and become more resistant to immunochemotherapy in "proliferation centers" within tissues, whereas apoptosis occurs in the periphery. Various models recapitulate these microenvironments in vitro, such as stimulation with CD154 and IL4. Using this system, we observed a 30- to 40-fold induction of wild-type p53 protein in 50 distinct human CLL specimens tested, without the induction of either cell-cycle arrest or apoptosis. In contrast, the mRNA levels for p53 did not increase, indicating that its elevation occurred posttranscriptionally. Mechanistic investigations revealed that under the conditions studied, p53 was phosphorylated on residues associated with p53 activation and increased half-life. However, p53 protein induced in this manner could transcriptionally activate only a subset of target genes. The addition of a DNA-damaging agent further upregulated p53 protein levels, which led to apoptosis. p53 induction relied on the increase in intracellular reactive oxygen species observed after CD154 and IL4 stimulation. We propose that chronic oxidative stress is a characteristic of the microenvironment in B-cell "proliferation centers" in CLL that are capable of elevating the basal expression of p53, but to levels below the threshold needed to induce arrest or apoptosis. Our findings suggest that reactivation of the full transcriptional activities of p53 in proliferating CLL cells may offer a possible therapeutic strategy. Cancer Res; 76(21); 6311-9. ©2016 AACR., (©2016 American Association for Cancer Research.)

Dinaciclib is a cyclin-dependent kinase inhibitor with clinical potential in different cancers, including chronic lymphocytic leukaemia (CLL). In order to better understand its cytotoxic action, we characterized its effects on signalling pathways important for the survival of CLL cells. We found that dinaciclib induced apoptosis through the activation of caspases 8 and 9, which was independent of the presence of cytokines to mimic the environment of proliferation centres or IGVH mutation status. Moreover, treatment with dinaciclib led to the inhibition of oncogenic pathways normally activated in stimulated CLL cells, such as STAT3, NF-κB, p38, PI3K/AKT and RAF/MEK/ERK. Dinaciclib was also able to block the expression of anti-apoptotic proteins of the BCL2 family such as MCL1 and BCL-xL (also termed BCL2L1). Finally, we showed that low concentrations of dinaciclib enhanced cell sensitivity to ibrutinib and the BCL2 inhibitor ABT-199, two drugs with known effects on CLL. Taken together, our data show that dinaciclib targets multiple pro-survival signalling pathways in CLL, which provides a mechanistic explanation for its potent induction of apoptosis. They also support a therapeutic application of cyclin-dependent kinase inhibitors in CLL in combination with other relevant targeted therapies., (© 2016 John Wiley & Sons Ltd.)

p53 is a tumor suppressor that prevents the emergence of transformed cells by inducing apoptosis or senescence, among other responses. Its functions are regulated tightly by posttranslational modifications. Here we show that Bruton's tyrosine kinase (BTK) is a novel modulator of p53. We found that BTK is induced in response to DNA damage and p53 activation. BTK induction leads to p53 phosphorylation, which constitutes a positive feedback loop that increases p53 protein levels and enhances the transactivation of its target genes in response to stress. Inhibiting BTK reduced both p53-dependent senescence and apoptosis. Further, BTK expression also upregulated DNA damage signals and apoptosis. We conclude that despite being involved in oncogenic signals in blood malignancies, BTK has antineoplastic properties in other contexts, such as the enhancement of p53's tumor suppressor responses. Along with evidence that BTK expression correlates with good prognosis in some epithelial tumors, our findings may encourage a reevaluation of the clinical uses of BTK inhibitors in cancer therapy. Cancer Res; 76(18); 5405-14. ©2016 AACR., (©2016 American Association for Cancer Research.)

HIF-1 is a transcription factor that mediates the cellular responses to low oxygen environments, mainly as a result of having an oxygen-labile subunit, HIF-1α. HIF-1α has been carefully studied in the context of severe hypoxic stresses (<1% O2), but it is also known to be present at oxygen tensions commonly found in normal tissues in vivo (∼1-13% O2), albeit at much lower levels. Its role under these physiological conditions is not fully understood. Here, we show that a transcriptionally active HIF-1α was up-regulated at 5% O2, both in normal and cancer cells, but only some of its target genes were elevated as a result. HIF-1α induction was in part dependent on the activation of the ERK1/2 MAPK signalling pathway, which we have previously shown is active at 5% O2. We also found that HIF-1α does not contribute to the protection against DNA damage that can be observed in low oxygen environments, and that there are certain DNA damaging agents, such as doxorubicin and actinomycin D, that prevent HIF-1α induction independently of p53. Moreover, absence of HIF-1α significantly reduced the growth advantage of cells cultured at 5% O2. In view of these data, we conclude that HIF-1α can be induced and activated at physiological oxygen tensions in a MAPK-dependent manner and that, although this does not lead to pro-survival responses to stress, it determines the increased cell proliferation rates that are common under these conditions.