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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

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.

Supplementary Figure 2. p53 phosphorylation.

Legends for supplementary figures

Supplementary Figure 1.p53 expression and phosphorylation.

Supplementary Figure 3. BTK and apoptosis.

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.

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 14-3-3o at an isoform-unique cysteine residue, Cys38. This modification leads to isoform-specific stabilisation of two 14-3-3o PPIs in a manner that is cooperative with a well characterised molecular glue, fusicoccin A. Our findings reveal a novel stabilisation mechanism for 14-3-3o, 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.

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.

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.

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.

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.

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.

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.

SScience allows us to understand the world we live in. Centuries of research have helped us learn more about our place in the universe, and we have discovered a perhaps even more fascinating mystery: how the organism we inhabit works and the reasons why it stops doing so. In this field, as in many others, we have gone from just admiring nature’s wonders to being able to control them.

Background: Endometrial cancer (EC) is the commonest gynaecologic malignancy in many countries and its incidence is rising. Although excellent prognosis is associated with early stage disease, response to systemic treatment for metastatic or recurrent EC is often low and treatment options are limited.Aims-Methods: The aim of the study was to propose improved targeted drug treatments suitable for subsequent testing in pre-clinical models of EC. Cell proliferation assay (MTS) was used to assess viability of EC cell lines following treatment with drug inhibitors and Western blotting to explore the effect of inhibitors in molecular pathways. Results: We identified that CUDC-907, a PI3K and HDAC inhibitor, was the most effective monotherapy treatment of a panel of drugs screened in EC cells. Moreover, several combination treatments showed synergism in EC cell lines, with the most efficacious being CUDC-907 combined with the MEK inhibitor PD0325901. This indicates that simultaneous inhibition of two main oncogenic pathways, PI3K and MEK, could improve drug sensitivity in EC.Conclusions: In summary, we propose a range of targeted inhibitory drugs, alone or in combination, showing in vitro efficacy in endometrial cancer cells, which could provide novel therapeutic strategies for advanced EC.

Proteomic mapping of cell surfaces is an invaluable tool for drug development and clinical diagnostics. This work describes a new ‘snapshot imprinting’ method designed to obtain proteomic maps of cell surfaces, with the aim of identifying cell surface markers and epitopes for diagnostic and therapeutic applications. The analysis of two cancer cell lines, HN5 and MDA-MB-468, is described herein as a proof of concept, along with the selective targeting of three identified epitopes of epidermal growth factor receptor using molecularly imprinted polymer nanoparticles. 438 proteins were identified using this technique, with 283 considered to be transmembrane or extracellular proteins. The major advantage of the molecular imprinting approach developed here is the ability to analyse cell surface proteins without tedious fractionation, affinity separation or labelling. We believe that this system of protein analysis may provide a basic molecular diagnostics toolbox for precise, personalised treatment of cancer and other diseases.

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-XL 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-XL with A1331852 was associated with a displacement of both BAX and BAK from BCL-XL 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.

The progressive accumulation of senescent cells in tissues in response to damage importantly contributes to pathophysiological conditions such as fibrosis, diabetes, cancer, Alzheimer's and ageing. Consistent with this, eliminating senescent cells prolongs the lifespan and healthspan in animals and ameliorates certain diseases. Detecting and clearing senescent cells from human tissues could therefore have a significant diagnostic and prognostic impact. However, identifying senescent cells in vivo has proven to be complex. To address this, we characterized and validated a panel of novel membrane markers of senescence. Here, we show the application of molecularly imprinted nanoparticles (nanoMIPs) against an extracellular epitope of one of these markers, B2M, to detect senescent cells in vitro and in vivo. We show that nanoMIPs do not elicit toxic responses in the cells or in mice and successfully recognize old animals, which have a higher proportion of senescent cells in their organs. Importantly, nanoMIPs loaded with drugs can specifically kill senescent cells. Our results provide a proof-of-principle assessment of specific and safe nanotechnology-based approaches for senescent cell detection and clearance with potential clinical relevance.

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., The aminothiol WR-1065 covalently modifies 14-3-3σ to stabilse its interactions with p53 and ERα. It enhances the effect of fusicoccin A via a cooperative mechanism that leads to 14-3-3 partner-protein specific activty against cancer cells.

BackgroundThe 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.MethodsHeLa 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.ResultsIn 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.ConclusionsThe 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.

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.

The clearance of senescent cells has the potential to become a therapeutic strategy that could be used in many pathologies, ranging from pulmonary fibrosis and diabetes to ageing itself. The initial genetic experiments performed in mouse models are now being recapitulated using chemical compounds that preferentially kill senescent cells, known as senolytics. Senolytic drugs hold an immense clinical potential but their lack of specificity could lead to side effects that would limit their use, especially when treating otherwise healthy aged individuals. Thus, it would be convenient to develop more targeted approaches to the elimination of senescent cells. Here, we discuss the ongoing efforts to design targeted senolytics, with special focus on the utilization of the extracellular epitopes displayed by the senescent surfaceome, and we summarize the avenues of research that have shown the most promising results so far: nanotechnology, cellular therapies and antibody-based drug delivery.

Radiotherapy is still a treatment of choice for many malignancies, often in combination with other strategies. However, its efficacy is limited by the dose that can be safely administered without eliciting serious side-effects, as well as the fact that recurrence is common, particularly in large tumours. Combining radiotherapy with drugs that could sensitise cells to radiation and/or reduce the factors that promote the recovery of the surviving cancer cells is a promising approach. Ionising radiation has been shown to induce senescence and the accumulation of senescent cells creates a microenvironment that facilitates neoplastic growth. This provides a rationale to test the addition of anti-senescent drugs, some of which are already available in the clinic, to radiotherapy protocols. Here, we discuss the relevance of radiotherapy-induced senescent cell accumulation and the potential interventions to minimise its negative effects.

Ageing is a multifactorial process that affects almost every living organism. The fact that we have already uncovered many of the molecular pathways that determine the ageing phenotype in humans has allowed us to start studying strategies to slow down or prevent the deleterious effects of time in tissue physiology. Several ongoing lines of research are aimed at defining new therapies that could be used in regenerative medicine, based on the knowledge accumulated on the molecular physiology of ageing. Among those, the most promising focus on preventing the accumulation of old cells, also known as senescent cells. These have been shown to disrupt tissue physiology and to be responsible, at least in part, for many of the symptoms associated with ageing. Consistent with this, clearance of senescent cells results in prolonged lifespan and health span in mammals and thus could be a potent anti-ageing therapy. In this chapter, we will discuss these and other novel approaches for regenerative medicine being developed in the context of the current understanding of cellular ageing.

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.

ObjectivesTo identify differences in the mutational profile of endometrial tumours between British White (BW) and South Asian (BSA) women.MethodsWe 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.ResultsIn 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).ConclusionWe 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.

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

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.

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.

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.

ISSN 2045-1393 Q What exactly is meant by the term ‘precision medicine’ Although the empirical chemotherapy of the last century allowed some spectacular advances, the outlook for many patients with cancer remains unaltered. Moreover, the toxicities associated with chemotherapy are often severe, particularly in the elderly. The aim of the multibillion dollar cancer industry is to develop medicines that will allow targeted therapy of malignancy, in much the same manner as we use antibiotics to treat bacterial infections. ‘Precision medicines’ are the means by which we will be able to deliver effective ‘personalized cancer therapy’ without significant systemic toxicities. Precision medicines come in various shapes and sizes. Monoclonal antibodies, owing to their specificity for cell surface epitopes, have been used for many years. Novel antibody drug conjugates with enhanced efficacy and antibodies that are able to redirect the immune response are two notable recent advances. However, advances in structural biology, with the resolution of the structure of many key kinases and apoptotic inhibitors have allowed the development of a plethora of highly specific

The Nobel Prize in Physiology or Medicine for 1906 was shared by two scientists that set the basis for understanding how the brain works: Camillo Golgi and Santiago Ramón y Cajal were awarded the honour “in recognition of their work on the structure of the nervous system”. Yet, contrary to what usually happens in these situations, one of them was wrong and tried to sabotage the theories of the other one, refusing to admit his mistakes even when he gave his acceptance speech. How did Santiago Ramón y Cajal, a humble Spanish doctor, manage to upstage the legendary Italian pathologist and change forever the way we see the brain?

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.

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.

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.

p53 is a critical tumor suppressor in humans. It functions mostly as a transcriptional factor and its activity is regulated by numerous post-translational modifications. Among different covalent modifications found on p53 the most controversial one is lysine methylation. We found that human G9a (hG9a) unlike its mouse orthologue (mG9a) potently stimulated p53 transcriptional activity. Both ectopic and endogenous hG9a augmented p53-dependent transcription of pro-apoptotic genes, including Bax and Puma, resulting in enhanced apoptosis and reduced colony formation. Significantly, shRNA-mediated knockdown of hG9a attenuated p53-dependent activation of Puma. On the molecular level, hG9a interacted with histone acetyltransferase, p300/CBP, resulting in increased histone acetylation at the promoter of Puma. The bioinformatics data substantiated our findings showing that positive correlation between G9a and p53 expression is associated with better survival of lung cancer patients. Collectively, this study demonstrates that depending on the cellular and organismal context, orthologous proteins may exert both overlapping and opposing functions. Furthermore, this finding has important ramifications on the use of G9a inhibitors in combination with genotoxic drugs to treat p53-positive tumors.

The authors report a case of purine analogue–refractory hairy-cell leukemia with biallelic BRAF V600E mutations and a high leukemic burden that was treated successfully with vemurafenib, a BRAF inhibitor.

Oxygen availability has important effects on cell physiology. Although hyperoxic and hypoxic stresses have been well characterized, little is known about cellular functions in the oxygen levels commonly found in vivo. Here, we show that p53-dependent apoptosis in response to different DNA-damaging agents was reduced when normal and cancer cells were cultured at physiological oxygen tensions instead of the usual atmospheric levels. Different from what has been described in hypoxia, this was neither determined by decreases in p53 induction or its transactivation activity, nor by differences in the intracellular accumulation of reactive oxygen species. At these physiological oxygen levels, we found a constitutive activation of the ERK1/2 MAPK in all the models studied. Inhibition of this signaling pathway reversed the protective effect in some but not all cell lines. We conclude that a stress-independent constitutive activation of prosurvival pathways, including but probably not limited to MAPK, can protect cells in physiological oxygen tensions against genotoxic stress. Our results underscore the need of considering the impact of oxygen levels present in the tissue microenvironment when studying cell sensitivity to treatments such as chemotherapy and radiotherapy.

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

The mechanisms of action of polycystin-1 (PC1) have been difficult to dissect because of its interaction with multiple factors, the heterogeneity of the genetic mutations, and the complexity of the experimental animal models. Here, stable knockdown of PC1 in MDCK epithelial cells was achieved by lentiviral-mediated delivery of a specific small interfering RNA for PKD1. The reduction of PC1 expression prevented tubulogenesis in three-dimensional collagen type I culture in response to hepatocyte growth factor and induced formation of cysts. PC1 knockdown created a condition of haploinsufficiency that led to hyperproliferation, increased adhesion to collagen type I, and increased apoptosis. It was shown that the suppression of PC1 was associated with the increased expression of integrin-alpha2beta1 and reduced apoptosis in cells grown on collagen type I. The engagement of integrin-alpha2beta1 seemed to be essential for the survival because PC1 knockdown cells were significantly less susceptible to anoikis by a mechanism that was reversible by anti-integrin-alpha2beta1 blocking antibodies. Overall, these data link integrin-alpha2beta1 to some of the biologic functions that are ascribed to PC1 and establish the potential of this approach for the direct study of PC1 functions in a genetically defined background. Furthermore, these findings indicate that reduction of PC1 expression levels, rather than the loss of heterozygosity, may be sufficient to induce cystogenesis.

The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) was identified initially as a gene induced in senescent cells and itself has been shown to cause permanent growth arrest/senescence. Reactive oxygen species (ROS), a byproduct of oxidative processes, can also induce an irreversible growth arrest similar to senescence. Here we show that p21 increased intracellular levels of ROS both in normal fibroblasts and in p53-negative cancer cells. N-acetyl-L-cysteine, an ROS inhibitor, rescued p21-induced senescence, showing that ROS elevation is necessary for induction of the permanent growth arrest phenotype. p16(Ink4a), a CDK4- and CDK6-specific inhibitor, failed to increase ROS levels, and cell cycle arrest induced by p16 was reversible following its down-regulation, demonstrating the specificity of this p21 effect. A p21 mutant that lacked the ability to bind proliferating cell nuclear antigen (PCNA) retained the ability to induce both ROS and permanent growth arrest. All of these findings establish that p21 mediates senescence by a mechanism involving ROS accumulation which does not require either its PCNA binding or the CDK inhibitory functions shared with p16.

Stra6 is the retinoic acid (RA)-inducible gene encoding the cellular receptor for holo-retinol binding protein. This transmembrane protein mediates the internalization of retinol, which then upregulates RA-responsive genes in target cells. Here, we show that Stra6 can be upregulated by DNA damage in a p53-dependent manner, and it has an important role in cell death responses. Stra6 expression induced significant amounts of apoptosis in normal and cancer cells, and it was also able to influence p53-mediated cell fate decisions by turning an initial arrest response into cell death. Moreover, inhibition of Stra6 severely compromised p53-induced apoptosis. We also found that Stra6 induced mitochondria depolarization and accumulation of reactive oxygen species, and that it was present not only at the cellular membrane but also in the cytosol. Finally, we show that these novel functions of Stra6 did not require downstream activation of RA signalling. Our results present a previously unknown link between the RA and p53 pathways and provide a rationale to use retinoids to upregulate Stra6, and thus enhance the tumour suppressor functions of p53. This may have implications for the role of vitamin A metabolites in cancer prevention and treatment.

p21Waf1/Cip1/Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs) and one of the best characterized p53 direct target genes. Moreover, it is one of the principal inducers of senescence, a permanent arrest phenotype related to ageing and the prevention of cell transformation. It was initially thought that p21 had tumour suppressor properties, due to its ability to stop cell cycle at different stages, either transitorily or permanently. However, recent evidence points to a much more complex picture. It is now well established that p21 itself can trigger apoptosis in certain situations, even independently of p53. On the other hand, p21-mediated cell arrest can actually limit the sensitivity to apoptotic stimuli. To complicate matters even further, p21 has been shown to have other direct pro-survival functions and could even be contributing to tumourigenesis through the secretion of growth factors by arrested cells. At the core of these antagonistic functions of p21 is the generation of Reactive Oxygen Species (ROS), which have been shown to be important both in the induction of apoptosis and the establishment of senescence, and could also participate in the negative effects on tissue homeostasis of the senescent cell secretome. p21 has the ability to stop cancer cell growth and is not mutated in cancer. However, without a better understanding of its pleiotropic functions we will not be able to harness its clinical potential. It is therefore important to investigate the mechanisms by which p21 affects cell physiology and its use of ROS as messengers and effectors.