Epigenetic alterations in glioblastomas: Diagnostic, prognostic and therapeutic relevance.

Glioblastoma, the most common and heterogeneous tumor affecting brain parenchyma, is dismally characterized by a very poor prognosis. Thus, the search of new, more effective treatments is a vital need. Here, we will review the druggable epigenetic features of glioblastomas that are, indeed, currently explored in preclinical studies and in clinical trials for the development of more effective, personalized treatments. In detail, we will review the studies that have led to the identification of epigenetic signatures, IDH mutations, MGMT gene methylation, histone modification alterations, H3K27 mutations and epitranscriptome landscapes of glioblastomas, in each case discussing the corresponding targeted therapies and their potential efficacy. Finally, we will emphasize how recent technological improvements permit to routinely investigate many glioblastoma epigenetic biomarkers in clinical practice, further enforcing the hope that personalized drugs, targeting specific epigenetic features, could be in future a therapeutic option for selected patients.

Subclones with variants of uncertain clinical significance might contribute to ineffective hemopoiesis and leukemia predisposition.

BACKGROUND: Splicing modifications, genomic instability, and hypomethylation are central mechanisms promoting myelodysplasia and acute myeloid leukemia (AML). In this real-life retrospective study, to elucidate pathophysiology of clonal hemopoiesis in hematological malignancies, we investigated clinical significance of mutations in leukemia-related genes of known pathogenetic significance and of variants of uncertain clinical significance (VUS) in a cohort of patients with MDS and AML. METHODS: A total of 59 consecutive subjects diagnosed with MDS, 48 with AML, and 17 with clonal cytopenia with unknown significance were screened for somatic mutations in AML-related genes by next-generation sequencing. RESULTS: We showed that TET2, SETBP1, ASXL1, EZH2, RUNX1, SRSF2, DNMT3A, and IDH1/2 were commonly mutated. MDS patients also showed a high genetic complexity, especially for SETBP1. Moreover, the presence of SETBP1 wild-type or two or more simultaneous VUS variants identified a subgroup of AML and MDS patients with better outcome, while the presence of single SETBP1 VUS variant was related to a worse prognosis, regardless TET2 mutational status. CONCLUSIONS: In conclusions, we linked both pathogenic and VUS variants in AML-related genes to clonal hematopoiesis; therefore, we proposed to consider those variants as prognostic markers in leukemia and myelodysplasia. However, further studies in larger prospective cohorts are required to validate our results.

MGMT and Whole-Genome DNA Methylation Impacts on Diagnosis, Prognosis and Therapy of Glioblastoma Multiforme.

Epigenetic changes in DNA methylation contribute to the development of many diseases, including cancer. In glioblastoma multiforme, the most prevalent primary brain cancer and an incurable tumor with a median survival time of 15 months, a single epigenetic modification, the methylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene, is a valid biomarker for predicting response to therapy with alkylating agents and also, independently, prognosis. More recently, the progress from single gene to whole-genome analysis of DNA methylation has allowed a better subclassification of glioblastomas. Here, we review the clinically relevant information that can be obtained by studying MGMT gene and whole-genome DNA methylation changes in glioblastomas, also highlighting benefits, including those of liquid biopsy, and pitfalls of the different detection methods. Finally, we discuss how changes in DNA methylation, especially in glioblastomas bearing mutations in the Isocitrate Dehydrogenase (IDH) 1 and 2 genes, can be exploited as targets for tailoring therapy.

Methylome Profiling in Fabry Disease in Clinical Practice: A Proof of Concept.

Anderson−Fabry disease (FD) is an X-linked disease caused by a functional deficit of the α-galactosidase A enzyme. FD diagnosis relies on the clinical manifestations and research of GLA gene mutations. However, because of the lack of a clear genotype/phenotype correlation, FD diagnosis can be challenging. Recently, several studies have highlighted the importance of investigating DNA methylation patterns for confirming the correct diagnosis of different rare Mendelian diseases, but to date, no such studies have been reported for FD. Thus, in the present investigation, we analyzed for the first time the genome-wide methylation profile of a well-characterized cohort of patients with Fabry disease. We profiled the methylation status of about 850,000 CpG sites in 5 FD patients, all carrying the same mutation in the GLA gene (exon 6 c.901C>G) and presenting comparable low levels of α-Gal A activity. We found that, although the whole methylome profile did not discriminate the FD group from the unaffected one, several genes were significantly differentially methylated in Fabry patients. Thus, we provide here a proof of concept, to be tested in patients with different mutations and in a larger cohort, that the methylation state of specific genes can potentially identify Fabry patients and possibly predict organ involvement and disease evolution.

The rationale for druggability of CCDC6-tyrosine kinase fusions in lung cancer.

Gene fusions occur in up to 17% of solid tumours. Oncogenic kinases are often involved in such fusions. In lung cancer, almost 30% of patients carrying an activated oncogene show the fusion of a tyrosine kinase to an heterologous gene. Several genes are partner in the fusion with the three kinases ALK, ROS1 and RET in lung. The impaired function of the partner gene, in combination with the activation of the kinase, may alter the cell signaling and promote the cancer cell addiction to the oncogene. Moreover, the gene that is partner in the fusion to the kinase may affect the response to therapeutics and/or promote resistance in the cancer cells. Few genes are recurrent partners in tyrosine kinase fusions in lung cancer, including CCDC6, a recurrent partner in ROS1 and RET fusions, that can be selected as possible target for new strategies of combined therapy including TKi.

The between Now and Then of Lung Cancer Chemotherapy and Immunotherapy.

Lung cancer is the most common cancer worldwide. Disappointingly, despite great effort in encouraging screening or, at least, a close surveillance of high-risk individuals, most of lung cancers are diagnosed when already surgically unresectable because of local advancement or metastasis. In these cases, the treatment of choice is chemotherapy, alone or in combination with radiotherapy. Here, we will briefly review the most successful and recent advances in the identification of novel lung cancer genetic lesions and in the development of new drugs specifically targeting them. However, lung cancer is still the leading cause of cancer-related mortality also because, despite impressive initial responses, the patients often develop resistance to novel target therapies after a few months of treatment. Thus, it is literally vital to continue the search for new therapeutic options. So, here, on the basis of our recent findings on the role of the tumor suppressor CCDC6 protein in lung tumorigenesis, we will also discuss novel therapeutic approaches we envision for lung cancer.

New therapeutic perspectives in CCDC6 deficient lung cancer cells.

Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC.

A Rare Adult Primary Intracranial Sarcoma, DICER1-Mutant Identified by Epigenomic Profiling: A Case Report.

Diagnoses of primary malignant mesenchymal brain tumors are a challenge for pathologists. Here, we report the case of a 52-year-old man with a primary brain tumor, histologically diagnosed as a high-grade glioma, not otherwise specified (NOS). The patient underwent two neurosurgeries in several months, followed by radiotherapy and chemotherapy. We re-examined the tumor samples by methylome profiling. Methylome analysis revealed an epi-signature typical of a primary intracranial sarcoma, DICER1-mutant, an extremely rare tumor. The diagnosis was confirmed by DNA sequencing that revealed a mutation in DICER1 exon 25. DICER1 mutations were not found in the patient's blood cells, thus excluding an inherited DICER1 syndrome. The methylome profile of the DICER1 mutant sarcoma was then compared with that of a high-grade glioma, a morphologically similar tumor type. We found that several relevant regions were differentially methylated. Taken together, we report the morphological, epigenetic, and genetic characterization of the sixth described case of an adult primary intracranial sarcoma, DICER1-mutant to-date. Furthermore, this case report underscores the importance of methylome analysis to refine primary brain tumor diagnosis and to avoid misdiagnosis among morphologically similar subtypes.

Host fecal DNA specific methylation signatures mark gut dysbiosis and inflammation in children affected by autism spectrum disorder.

The gut-brain axis involves several bidirectional pathway communications including microbiome, bacterial metabolites, neurotransmitters as well as immune system and is perturbed both in brain and in gastrointestinal disorders. Consistently, microbiota-gut-brain axis has been found altered in autism spectrum disorder (ASD). We reasoned that such alterations occurring in ASD may impact both on methylation signatures of human host fecal DNA (HFD) and possibly on the types of human cells shed in the stools from intestinal tract giving origin to HFD. To test this hypothesis, we have performed whole genome methylation analysis of HFD from an age-restricted cohort of young children with ASD (N = 8) and healthy controls (N = 7). In the same cohort we have previously investigated the fecal microbiota composition and here we refined such analysis and searched for eventual associations with data derived from HFD methylome analysis. Our results showed that specific epigenetic signatures in human fecal DNA, especially at genes related to inflammation, associated with the disease. By applying methylation-based deconvolution algorithm, we found that the HFD derived mainly from immune cells and the relative abundance of those differed between patients and controls. Consistently, most of differentially methylated regions fitted with genes involved in inflammatory response. Interestingly, using Horvath epigenetic clock, we found that ASD affected children showed both epigenetic and microbiota age accelerated. We believe that the present unprecedented approach may be useful for the identification of the ASD associated HFD epigenetic signatures and may be potentially extended to other brain disorders and intestinal inflammatory diseases.

Targeted inhibition of the methyltransferase SETD8 synergizes with the Wee1 inhibitor adavosertib in restraining glioblastoma growth.

Despite intense research efforts, glioblastoma remains an incurable brain tumor with a dismal median survival time of 15 months. Thus, identifying new therapeutic targets is an urgent need. Here, we show that the lysine methyltransferase SETD8 is overexpressed in 50% of high-grade gliomas. The small molecule SETD8 inhibitor UNC0379, as well as siRNA-mediated inhibition of SETD8, blocked glioblastoma cell proliferation, by inducing DNA damage and activating cell cycle checkpoints. Specifically, in p53-proficient glioblastoma cells, SETD8 inhibition and DNA damage induced p21 accumulation and G1/S arrest whereas, in p53-deficient glioblastoma cells, DNA damage induced by SETD8 inhibition resulted in G2/M arrest mediated by Chk1 activation. Checkpoint abrogation, by the Wee1 kinase inhibitor adavosertib, induced glioblastoma cell lines and primary cells, DNA-damaged by UNC0379, to progress to mitosis where they died by mitotic catastrophe. Finally, UNC0379 and adavosertib synergized in restraining glioblastoma growth in a murine xenograft model, providing a strong rationale to further explore this novel pharmacological approach for adjuvant glioblastoma treatment.

Epigenetic remodelling of Fxyd1 promoters in developing heart and brain tissues.

FXYD1 is a key protein controlling ion channel transport. FXYD1 exerts its function by regulating Na+/K+-ATPase activity, mainly in brain and cardiac tissues. Alterations of the expression level of the FXYD1 protein cause diastolic dysfunction and arrhythmias in heart and decreased neuronal dendritic tree and spine formation in brain. Moreover, FXYD1, a target of MeCP2, plays a crucial role in the pathogenesis of the Rett syndrome, a neurodevelopmental disorder. Thus, the amount of FXYD1 must be strictly controlled in a tissue specific manner and, likely, during development. Epigenetic modifications, particularly DNA methylation, represent the major candidate mechanism that may regulate Fxyd1 expression. In the present study, we performed a comprehensive DNA methylation analysis and mRNA expression level measurement of the two Fxyd1 transcripts, Fxyd1a and Fxyd1b, in brain and heart tissues during mouse development. We found that DNA methylation at Fxyd1a increased during brain development and decreased during heart development along with coherent changes in mRNA expression levels. We also applied ultra-deep methylation analysis to detect cell to cell methylation differences and to identify possible distinct methylation profile (epialleles) distribution between heart and brain and in different developmental stages. Our data indicate that the expression of Fxyd1 transcript isoforms inversely correlates with DNA methylation in developing brain and cardiac tissues suggesting the existence of a temporal-specific epigenetic program. Moreover, we identified a clear remodeling of epiallele profiles which were distinctive for single developmental stage both in brain and heart tissues.

Looking Beyond the Glioblastoma Mask: Is Genomics the Right Path?

Glioblastomas are the most frequent and malignant brain tumor hallmarked by an invariably poor prognosis. They have been classically differentiated into primary isocitrate dehydrogenase 1 or 2 (IDH1 -2) wild-type (wt) glioblastoma (GBM) and secondary IDH mutant GBM, with IDH wt GBMs being commonly associated with older age and poor prognosis. Recently, genetic analyses have been integrated with epigenetic investigations, strongly implementing typing and subtyping of brain tumors, including GBMs, and leading to the new WHO 2021 classification. GBM genomic and epigenomic profile influences evolution, resistance, and therapeutic responses. However, differently from other tumors, there is a wide gap between the refined GBM profiling and the limited therapeutic opportunities. In addition, the different oncogenes and tumor suppressor genes involved in glial cell transformation, the heterogeneous nature of cancer, and the restricted access of drugs due to the blood-brain barrier have limited clinical advancements. This review will summarize the more relevant genetic alterations found in GBMs and highlight their potential role as potential therapeutic targets.

Evaluation of MGMT Gene Methylation in Neuroendocrine Neoplasms.

Unresectable neuroendocrine neoplasms (NENs) often poorly respond to standard therapeutic approaches. Alkylating agents, in particular temozolomide, commonly used to treat high-grade brain tumors including glioblastomas, have recently been tested in advanced or metastatic NENs, where they showed promising response rates. In glioblastomas, prediction of response to temozolomide is based on the assessment of the methylation status of the MGMT gene, as its product, O 6-methylguanine-DNA methyltransferase, may counteract the damaging effects of the alkylating agent. However, in NENs, such a biomarker has not been validated yet. Thus, we have investigated MGMT methylation in 42 NENs of different grades and from various sites of origin by two different approaches: in contrast to methylation-specific PCR (MSP), which is commonly used in glioblastoma management, amplicon bisulfite sequencing (ABS) is based on high-resolution, next-generation sequencing and interrogates several additional CpG sites compared to those covered by MSP. Overall, we found MGMT methylation in 74% (31/42) of the NENs investigated. A higher methylation degree was observed in well-differentiated tumors and in tumors originating in the gastrointestinal tract. Comparing MSP and ABS results, we demonstrate that the region analyzed by the MSP test is sufficiently informative of the MGMT methylation status in NENs, suggesting that this predictive parameter could routinely be interrogated also in NENs.

Exploiting immune-dependent effects of microtubule-targeting agents to improve efficacy and tolerability of cancer treatment.

Microtubule-targeting agents (MTAs), like taxanes and vinca alkaloids, are tubulin-binding drugs that are very effective in the treatment of various types of cancers. In cell cultures, these drugs appear to affect assembly of the mitotic spindle and to delay progression through mitosis and this correlates with their ability to induce cell death. Their clinical efficacy is, however, limited by resistance and toxicity. For these reasons, other spindle-targeting drugs, affecting proteins such as certain kinesins like Eg5 and CENP-E, or kinases like Plk1, Aurora A and B, have been developed as an alternative to MTAs. However, these attempts have disappointed in the clinic since these drugs show poor anticancer activity and toxicity ahead of positive effects. In addition, whether efficacy of MTAs in cancer treatment is solely due to their ability to delay mitosis progression remains controversial. Here we discuss recent findings indicating that the taxane paclitaxel can promote a proinflammatory response by activation of innate immunity. We further describe how this can help adaptive antitumor immune response and suggest, on this basis and on the recent success of immune checkpoint inhibitors in cancer treatment, that a combination therapy based on low doses of taxanes and immune checkpoint inhibitors may be of high clinical advantage in terms of wide applicability, reduced toxicity, and increased antitumor response.

New combinatorial strategies to improve the PARP inhibitors efficacy in the urothelial bladder Cancer treatment.

BACKGROUND: Novel therapeutic strategies are urgently needed for the treatment of metastatic Urothelial Bladder Cancer. DNA damaging repair (DDR) targeting has been introduced in cinical trials for bladder cancer patients that carry alterations in homologous DNA repair genes, letting to envisage susceptibility to the Poly (adenosine diphosphate [ADP]) ribose polymerase (PARP) inhibitors. MAIN BODY: PARP inhibition, by amplifying the DNA damage, augments the mutational burden and promotes the immune priming of the tumor by increasing the neoantigen exposure and determining upregulation of programmed death ligand 1 (PD-L1) expression. Thus, the combination of PARP-inhibition and the PD/PD-L1 targeting may represent a compelling strategy to treat bladder cancer and has been introduced in recent clinical trials. The targeting of DDR has been also used in combination with epigenetic drugs able to modulate the expression of genes involved in DDR, and also able to act as immunomodulator agents suggesting their use in combination with immune-checkpoint inhibitors. CONCLUSION: In conclusion, it may be envisaged the combination of three classes of drugs to treat bladder cancer, by targeting the DDR process in a tumor context of DDR defect, together with epigenetic agents and immune-checkpoint inhibitors, whose association may amplify the effects and reduce the doses and the toxicity of each single drug.

CCDC6 and USP7 expression levels suggest novel treatment options in high-grade urothelial bladder cancer.

BACKGROUND: The muscle invasive form of urothelial bladder cancer (UBC) is a deadly disease. Currently, the therapeutic approach of UBC is mostly based on surgery and standard chemotherapy. Biomarkers to establish appropriate drugs usage are missing. Deficiency of the tumor suppressor CCDC6 determines PARP-inhibitor sensitivity. The CCDC6 levels are modulated by the deubiquitinase USP7. In this work we scored CCDC6 and USP7 expression levels in primary UBC and we evaluated the expression levels of CCDC6 in correlation with the effects of the PARP-inhibitors combined with the USP7 inhibitor, P5091, in vitro. Since PARP-inhibitors could be enhanced by conventional chemotherapy or DNA damage inducers, we tested the new agent RRx-001, able to induce DNA damage, to prove the benefit of combined treatments in bladder cancer cells. METHODS: The J82, T24, 5637 and KU-19-19 bladder cancer cells were exposed to USP7 inhibitor P5091 in presence of cycloheximide to analyse the CCDC6 stability. Upon the CCDC6 degradation induced by P5091, the cells sensitivity to PARP-inhibitor was evaluated by cell viability assays. The ability of the DNA damage inducer RRx-001 to modulate CCDC6 protein levels and H2AX phosphorylation was detected at immunoblot. The combination of USP7 inhibitor plus RRx-001 enhanced the PARP-inhibitor sensitivity, as evaluated by cell viability assays. The results of the scores and correlation of CCDC6 and USP7 expression levels obtained by UBC primary biopsies staining were used to cluster patients by a K-mean cluster analysis. RESULTS: P5091 determining CCDC6 degradation promoted bladder cancer cells sensitivity to PARP-inhibitor drugs. RRx-001, by inducing DNA damage, enhanced the effects of the combined treatment. The immunohistochemical staining of both CCDC6 and USP7 proteins allowed to cluster the high grade (G3) UBC patients, on the basis of CCDC6 expression levels. CONCLUSIONS: In high grade UBC the identification of two clusters of patients based on CCDC6 and USP7 expession can possibly indicate the use of PARP-inhibitor drugs, in combination with USP7 inhibitor in addition to the DNA damage inducer RRx-001, that also acts as an immunomodulatory agent, offering novel therapeutic strategy for personalized medicine in bladder cancer patients.

Wee1 Rather Than Plk1 Is Inhibited by AZD1775 at Therapeutically Relevant Concentrations.

Wee1 kinase is an inhibitor of cyclin-dependent kinase (cdk)s, crucial cell cycle progression drivers. By phosphorylating cdk1 at tyrosine 15, Wee1 inhibits activation of cyclin B-cdk1 (Cdk1), preventing cells from entering mitosis with incompletely replicated or damaged DNA. Thus, inhibiting Wee1, alone or in combination with DNA damaging agents, can kill cancer cells by mitotic catastrophe, a tumor suppressive response that follows mitosis onset in the presence of under-replicated or damaged DNA. AZD1775, an orally available Wee1 inhibitor, has entered clinical trials for cancer treatment following this strategy, with promising results. Recently, however, AZD1775 has been shown to inhibit also the polo-like kinase homolog Plk1 in vitro, casting doubts on its mechanism of action. Here we asked whether, in the clinically relevant concentration range, AZD1775 inhibited Wee1 or Plk1 in transformed and non-transformed human cells. We found that in the clinically relevant, nanomolar, concentration range AZD1775 inhibited Wee1 rather than Plk1. In addition, AZD1775 treatment accelerated mitosis onset overriding the DNA replication checkpoint and hastened Plk1-dependent phosphorylation. On the contrary selective Plk1 inhibition exerted opposite effects. Thus, at therapeutic concentrations, AZD1775 inhibited Wee1 rather than Plk1. This information will help to better interpret results obtained by using AZD1775 both in the clinical and experimental settings and provide a stronger rationale for combination therapies.

Analysis of CCDC6 as a novel biomarker for the clinical use of PARP1 inhibitors in malignant pleural mesothelioma.

OBJECTIVES: CCDC6 (coiled-coil domain containing 6) is a player of the HR response to DNA damage and has been predicted to interact with BAP1, another HR-DNA repair gene highly mutated in Malignant Pleural Mesothelioma (MPM), an aggressive cancer with poor prognosis. CCDC6 levels are modulated by the deubiquitinase USP7, and CCDC6 defects have been reported in several tumors determining PARP-inhibitors sensitivity. Our aim was to investigate the functional role of CCDC6 in MPM carcinogenesis and response to PARP-inhibitors. MATERIALS AND METHODS: The interaction between CCDC6 and BAP1 was confirmed in MPM cells, by co-immunoprecipitation. Upon USP7 inhibition, that induces CCDC6 degradation, the ability to repair the DSBs and the sensitivity to PARP inhibitors, was explored by HR reporter and by cells viability assays, respectively. A TMA including 34 MPM cores was immunostained for CCDC6, USP7 and BAP1 and the results correlated by statistical analysis. RESULTS: MPM cells depleted of CCDC6 showed defects in DSBs repair and sensitivity to PARP inhibitors. The silencing of CCDC6 when combined with the overexpression of BAP1-mutant (Δ221-238) enhanced the HR-DNA repair defects and the PARP inhibitors sensitivity. In the TMA of MPM primary samples, the staining of CCDC6 and of its de-ubiquitinase USP7 showed a significant correlation in the tested primary samples (p = 0.01). CCDC6 was barely detected in 30% of the tumors that also carried BAP1 defects. CONCLUSION: The combination of CCDC6 and BAP1 staining may indicate therapeutic options for DDR targeting, acting in synergism with cisplatinum.

Retrospective analysis of coagulation factor II receptor (F2R) sequence variation and coronary heart disease in hypertensive patients.

OBJECTIVES: The purpose of this study was to evaluate the role of genetic variants within the coagulation factor II receptor (F2R) in the occurrence of coronary heart disease (CHD). METHODS AND RESULTS: Four SNPs (-1738 G/A, 2860 G/A, 2930 T/C, and 9113 C/A) and an ins/del polymorphism -506-/GGCCGCGGGAAGC (D/I), replicating a consensus sequence for Ets-1 transcription factor, and their related haplotypes were tested for association to CHD in 1600 hypertensive patients divided in 2 groups according to presence (cases, n=559) and absence (controls, n=1041) of CHD. Allele I at -506 locus was associated with increased risk of CHD under additive, dominant, and recessive models of inheritance (all P<0.01). Three haplotypes carrying I allele were consistently associated with an increased risk of CHD (all P<0.05). Patients homozygous for the C allele at the 2930 locus also showed an increased risk of CHD (P<0.05). To test the functionality of -506 locus, nuclear extracts were incubated with -506D and -506I sequences by EMSA and F2R promoter activity (F2R-A) were assessed in HUVECs transfected with vectors carrying -506D and -506I sequences and exposed to hypoxia. Presence of the -506I sequence was associated with a 26% reduction of affinity binding to nuclear proteins and to blunted F2R-A in response to hypoxia as compared with the -506D sequence (all P<0.05). CONCLUSIONS: F2R genetic variants may influence the natural history of CHD in patients at high risk of cardiovascular events.

Evidence that PP2A activity is dispensable for spindle assembly checkpoint-dependent control of Cdk1.

Progression through mitosis, the cell cycle phase deputed to segregate replicated chromosomes, is granted by a protein phosphorylation wave that follows an activation-inactivation cycle of cyclin B-dependent kinase (Cdk) 1, the major mitosis-promoting enzyme. To ensure correct chromosome segregation, the safeguard mechanism spindle assembly checkpoint (SAC) delays Cdk1 inactivation by preventing cyclin B degradation until mitotic spindle assembly. At the end of mitosis, reversal of bulk mitotic protein phosphorylation, downstream Cdk1 inactivation, is required to complete mitosis and crucially relies on the activity of major protein phosphatases like PP2A. A role for PP2A, however, has also been suggested in spindle assembly and SAC-dependent control of Cdk1. Indeed, PP2A was found in complex with SAC proteins while small interfering RNAs (siRNAs)-mediated downregulation of PP2A holoenzyme components affected mitosis completion in mammalian cells. However, whether the SAC-dependent control of Cdk1 required the catalytic activity of PP2A has never been directly assessed. Here, using two PP2A inhibitors, okadaic acid and LB-100, we provide evidence that PP2A activity is dispensable for SAC control of Cdk1 in human cells.