Ribonucleotide Reductase Inhibitor Hydroxyurea Research Articles

Synergistic Effect of a Combination of Proteasome and Ribonucleotide Reductase Inhibitors in a Biochemical Model of the Yeast Saccharomyces cerevisiae and a Glioblastoma Cell Line.

Proteasome inhibitors are used in the therapy of several cancers, and clinical trials are underway for their use in the treatment of glioblastoma (GBM). However, GBM becomes resistant to chemotherapy relatively rapidly. Recently, the overexpression of ribonucleotide reductase (RNR) genes was found to mediate therapy resistance in GBM. The use of combinations of chemotherapeutic agents is considered a promising direction in cancer therapy. The present work aimed to evaluate the efficacy of the combination of proteasome and RNR inhibitors in yeast and GBM cell models. We have shown that impaired proteasome function results in increased levels of RNR subunits and increased enzyme activity in yeast. Co-administration of the proteasome inhibitor bortezomib and the RNR inhibitor hydroxyurea was found to significantly reduce the growth rate of S. cerevisiae yeast. Accordingly, the combination of bortezomib and another RNR inhibitor gemcitabine reduced the survival of DBTRG-05MG compared to the HEK293 cell line. Thus, yeast can be used as a simple model to evaluate the efficacy of combinations of proteasome and RNR inhibitors.

The Rtf1/Prf1-dependent histone modification axis counteracts multi-drug resistance in fission yeast.

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

Excessive reactive oxygen species induce transcription-dependent replication stress

Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.

A missense mutation in the suc22 gene encoding the small subunit of ribonucleotide reductase significantly sensitizes fission yeast to chronic treatment with hydroxyurea.

Ribonucleotide reductase (RNR) is essential for the biosynthesis of dNTPs and a therapeutic target. We have identified a missense mutation in suc22 , which encodes the small subunit of RNR in fission yeast. The suc22-S239F mutation significantly sensitizes the cells to chronic but not acute treatment with the RNR inhibitor hydroxyurea. Preliminary data indicate that the drug sensitivity is likely due to decreased RNR activity. Since S239F is the first missense mutation reported for suc22 and the mutated residue is highly conserved, the results will be useful for future yeast genetic studies and potentially, the development of new therapeutics targeting RNR.

Class 1 Histone Deacetylases and Ataxia-Telangiectasia Mutated Kinase Control the Survival of Murine Pancreatic Cancer Cells upon dNTP Depletion.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with a dismal prognosis. Here, we show how an inhibition of de novo dNTP synthesis by the ribonucleotide reductase (RNR) inhibitor hydroxyurea and an inhibition of epigenetic modifiers of the histone deacetylase (HDAC) family affect short-term cultured primary murine PDAC cells. We used clinically relevant doses of hydroxyurea and the class 1 HDAC inhibitor entinostat. We analyzed the cells by flow cytometry and immunoblot. Regarding the induction of apoptosis and DNA replication stress, hydroxyurea and the novel RNR inhibitor COH29 are superior to the topoisomerase-1 inhibitor irinotecan which is used to treat PDAC. Entinostat promotes the induction of DNA replication stress by hydroxyurea. This is associated with an increase in the PP2A subunit PR130/PPP2R3A and a reduction of the ribonucleotide reductase subunit RRM2 and the DNA repair protein RAD51. We further show that class 1 HDAC activity promotes the hydroxyurea-induced activation of the checkpoint kinase ataxia-telangiectasia mutated (ATM). Unlike in other cell systems, ATM is pro-apoptotic in hydroxyurea-treated murine PDAC cells. These data reveal novel insights into a cytotoxic, ATM-regulated, and HDAC-dependent replication stress program in PDAC cells.

Oncogenic Kinase Cascades Induce Molecular Mechanisms That Protect Leukemic Cell Models from Lethal Effects of De Novo dNTP Synthesis Inhibition.

Simple SummaryLeukemic cells show differential sensitivity to apoptosis induction by the clinically relevant drug hydroxyurea. Since resistance to hydroxyurea can pose a therapeutic problem, we searched for mechanisms that protect such cells from the toxic effects of hydroxyurea. We used proteomics followed by mass spectrometry to accomplish this task and noted a loss of the RAF1 kinase in cells that are killed by hydroxyurea. Pharmacological inhibition of RAF1 and its target BCL-XL show that these proteins suppress apoptosis induction. Furthermore, inhibition of their upstream regulators BCR-ABL1 (in chronic myeloid leukemia cells) and FLT3-ITD (in acute myeloid leukemia cells) plus hydroxyurea produced favorable results. This approach may benefit patients that are not successfully treated with tyrosine kinase inhibitors. Taken together, we provide novel insights into strategies that eliminate chronic and acute myeloid leukemia cells with combinations of clinically established and currently tested pharmaceutical agents.The ribonucleotide reductase inhibitor hydroxyurea suppresses de novo dNTP synthesis and attenuates the hyperproliferation of leukemic blasts. Mechanisms that determine whether cells undergo apoptosis in response to hydroxyurea are ill-defined. We used unbiased proteomics to uncover which pathways control the transition of the hydroxyurea-induced replication stress into an apoptotic program in chronic and acute myeloid leukemia cells. We noted a decrease in the serine/threonine kinase RAF1/c-RAF in cells that undergo apoptosis in response to clinically relevant doses of hydroxyurea. Using the RAF inhibitor LY3009120, we show that RAF activity determines the sensitivity of leukemic cells toward hydroxyurea. We further disclose that pharmacological inhibition of the RAF downstream target BCL-XL with the drug navitoclax and RNAi combine favorably with hydroxyurea against leukemic cells. BCR-ABL1 and hyperactive FLT3 are tyrosine kinases that causally contribute to the development of leukemia and induce RAF1 and BCL-XL. Accordingly, the ABL inhibitor imatinib and the FLT3 inhibitor quizartinib sensitize leukemic cells to pro-apoptotic effects of hydroxyurea. Moreover, hydroxyurea and navitoclax kill leukemic cells with mutant FLT3 that are resistant to quizartinib. These data reveal cellular susceptibility factors toward hydroxyurea and how they can be exploited to eliminate difficult-to-treat leukemic cells with clinically relevant drug combinations.

Hydroxyurea Induces a Stress Response That Alters DNA Replication and Nucleotide Metabolism in Bacillus subtilis.

Hydroxyurea (HU) is classified as a ribonucleotide reductase (RNR) inhibitor and has been widely used to stall DNA replication by depleting deoxyribonucleoside triphosphate (dNTP) pools. Recent evidence in Escherichia coli shows that HU readily forms breakdown products that damage DNA directly, indicating that toxicity is a result of secondary effects. Because HU is so widely used in the laboratory and as a clinical therapeutic, it is important to understand its biological effects. To determine how Bacillus subtilis responds to HU-induced stress, we performed saturating transposon insertion mutagenesis followed by deep sequencing (Tn-seq), transcriptome sequencing (RNA-seq) analysis, and measurement of replication fork progression. Our data show that B. subtilis cells elongate, and replication fork progression is slowed, following HU challenge. The transcriptomic data show that B. subtilis cells initially mount a metabolic response likely caused by dNTP pool depletion before inducing the DNA damage response (SOS) after prolonged exposure. To compensate for reduced nucleotide pools, B. subtilis upregulates the purine and pyrimidine biosynthetic machinery and downregulates the enzymes producing ribose 5-phosphate. We show that overexpression of the RNR genes nrdEF suppresses the growth interference caused by HU, suggesting that RNR is an important target of HU in B. subtilis. Although genes involved in nucleotide and carbon metabolism showed considerable differential expression, we also find that genes of unknown function (y-genes) represent the largest class of differentially expressed genes. Deletion of individual y-genes caused moderate growth interference in the presence of HU, suggesting that cells have several ways of coping with HU-induced metabolic stress. IMPORTANCE Hydroxyurea (HU) has been widely used as a clinical therapeutic and an inhibitor of DNA replication. Some evidence suggests that HU inhibits ribonucleotide reductase, depleting dNTP pools, while other evidence shows that toxic HU breakdown products are responsible for growth inhibition and genotoxic stress. Here, we use multiple, complementary approaches to characterize the response of Bacillus subtilis to HU. B. subtilis responds by upregulating the expression of purine and pyrimidine biosynthesis. We show that HU challenge reduced DNA replication and that overexpression of the ribonucleotide reductase operon suppressed growth interference by HU. Our results demonstrate that HU targets RNR and several other metabolic enzymes contributing to toxicity in bacteria.

Use of trans‐3′5'4‐trihydroxystilbene (Resveratrol) as an adjunct in the treatment of Sickle Cell Disease

Sickle Cell Disease (SCD) and □-thalassemia are among the most common forms of hemoglobinopathy and life-threatening genetic diseases worldwide. So far, the only approved drug for these disorders is the ribonucleotide reductase inhibitor hydroxyurea (HU) falls short of being curative. The efficacy of HU in the management of thalassemia and SCD is generally attributed to its limited ability to boost the levels of fetal hemoglobin (Hb F,α2γ2), which decreases sickling by interfering with the polymerization of hemoglobin in patients with SCD and can partially correct the globin chain imbalance in □-thalassemia patients, while at the same time facilitating O2 transport to myoglobin in peripheral tissues. The naturally occurring dietary polyphenol resveratrol (trans-3′5'4-trihydroxystilbene, RV), has also been postulated to induce Hb F production via a biochemical mechanism like HU. Preclinical studies using the human erythroleukemic K562 cell line suggested RV can induce Hb F production in a more robust manner than HU. However, clinical studies on the efficacy and effectiveness of RV are limited and conflicting. This study sought to evaluate the clinical efficacy and safety of RV in managing SCD compared to HU. A systematic literature review was conducted in peer-reviewed, published full-text articles investigating RV in the management of SCD. Eligible studies were required to report explicitly, including participants diagnosed with β-thalassemia or SCD anemia or cultured cells with the SCD or thalassemia disease and where HU was included as a control. The analysis included thalassemia, RV type, RV dosage, duration of treatment, and outcome, Hb F induction and antioxidant activity. Additionally, a pilot study of RV in the management of adults with SCD who received treatment at National Hospital Abuja, Nigeria, was attempted but discontinued due to the COVID 19 pandemic. A case study of a single 23-year-old female SCD patient who had been taking RV [1000mg daily p.o.] for more than 6 years was included. Outcome variables evaluated include number of hospitalization episodes of pain crisis, need for blood transfusion, possible side effects and RBC indices. The early results of this metanalysis show that RV was as effective as HU in inducing production of Hb F in adult SCD patients and reducing hospital treatment days post RV treatment. Four primary treatment and curative therapies that have shown great potential and are currently used to prevent and treat acute and chronic complications in SCD, HU and blood transfusions, are the most well-established disease-modifying therapies used to prevent and treat complications associated with SCD. Other lesser used therapies include stem cell transplantation or the allogeneic hematopoietic stem cell transplant (HSCT), and the investigational gene therapy is the only known curative therapy that exists regarding SCD. The results of this study indicate that RV is an effective inducer of Hb F production in adults and shows promise as a cost effective and useful adjunct for the treatment of hemoglobinopathies including SCD and thalassemia.

Clb6-Cdc28 Promotes Ribonucleotide Reductase Subcellular Redistribution during S Phase.

A tightly controlled cellular deoxyribonucleotide (deoxynucleoside triphosphate [dNTP]) pool is critical for maintenance of genome integrity. One mode of dNTP pool regulation is through subcellular localization of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step of dNTP biosynthesis. In Saccharomyces cerevisiae, the RNR small subunit, Rnr2-Rnr4, is localized to the nucleus, whereas the large subunit, Rnr1, is cytoplasmic. As cells enter S phase or encounter DNA damage, Rnr2-Rnr4 relocalizes to the cytoplasm to form an active holoenzyme complex with Rnr1. Although the DNA damage-induced relocalization requires the checkpoint kinases Mec1-Rad53-Dun1, the S-phase-specific redistribution does not. Here, we report that the S-phase cyclin-cyclin-dependent kinase (CDK) complex Clb6-Cdc28 controls Rnr2-Rnr4 relocalization in S phase. Rnr2 contains a consensus CDK site and exhibits Clb6-dependent phosphorylation in S phase. Deletion of CLB6 or removal of the CDK site results in an increased association of Rnr2 with its nuclear anchor Wtm1, nuclear retention of Rnr2-Rnr4, and an enhanced sensitivity to the RNR inhibitor hydroxyurea. Thus, we propose that Rnr2-Rnr4 redistribution in S phase is triggered by Clb6-Cdc28-mediated phosphorylation of Rnr2, which disrupts the Rnr2-Wtm1 interaction and promotes the release of Rnr2-Rnr4 from the nucleus.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IgE-mediated mast cell activation through attenuation of NFκB and AP-1 transcription

Mast cell activation via the high-affinity IgE receptor (FcεRI) elicits production of inflammatory mediators central to allergic disease. As a synthetic antioxidant and a potent ribonucleotide reductase (RNR) inhibitor, Didox (3,4-dihyroxybenzohydroxamic acid) has been tested in clinical trials for cancer and is an attractive therapeutic for inflammatory disease. We found that Didox treatment of mouse bone marrow-derived mast cells (BMMC) reduced IgE-stimulated degranulation and cytokine production, including IL-6, IL-13, TNF and MIP-1a (CCL3). These effects were consistent using BMMC of different genetic backgrounds and peritoneal mast cells. While the RNR inhibitor hydroxyurea had little or no effect on IgE-mediated function, high concentrations of the antioxidant N-acetylcysteine mimicked Didox-mediated suppression. Furthermore, Didox increased expression of the antioxidant genes superoxide dismutase and catalase, and suppressed DCFH-DA fluorescence, indicating reduced reactive oxygen species production. Didox effects were not due to changes in FcεRI expression or cell viability, suggesting it inhibits signaling required for inflammatory cytokine production. In support of this, we found that Didox reduced FcεRI-mediated AP-1 and NFκB transcriptional activity. Finally, Didox suppressed mast cell-dependent, IgE-mediated passive systemic anaphylaxis in vivo. These data demonstrate the potential use for Didox asa means of antagonizing mast cell responses in allergic disease.

Bacterial Signaling Nucleotides Inhibit Yeast Cell Growth by Impacting Mitochondrial and Other Specifically Eukaryotic Functions.

ABSTRACTWe have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection.

Didox (3,4-dihydroxybenzohydroxamic acid) suppresses IL-33-induced cytokine production in primary mouse mast cells

While IgE is considered the primary mediator of mast cell activation, IL-33 contributes substantially in asthma, allergic rhinitis, and atopic dermatitis. To develop effective treatments for allergic disease, it is important to understand the role of therapeutic agents on IL-33 activation. We examined the effect of Didox (3,4-dihydroxybenzohydroxamic acid), an antioxidant and ribonucleotide reductase (RNR) inhibitor, on IL-33-mediated mast cell activation. Didox suppressed IL-6, IL-13, TNF, and MIP-1α (CCL3) production in bone marrow derived mast cells following IL-33 activation. This suppression was observed in different genetic backgrounds and extended to peritoneal mast cells. The antioxidant N-acetylcysteine mimicked the suppression of Didox, albeit at a much higher dose, while the RNR inhibitor hydroxyurea had no effect. Didox substantially suppressed IL-33-mediated NFκB and AP-1 transcriptional activities. These results suggest that Didox attenuates IL-33-induced mast cell activation and should be further studied as a potential therapeutic agent for inflammatory diseases involving IL-33.

Abstract B09: DNA damage inducible phosphorylation of ATR at threonine 1989 and quantitative analysis of the effect of ATR inhibition on DNA damage signaling using PTMScan

Abstract DNA repair pathways and checkpoint control are crucial in the maintenance of genome integrity. Agents that cause DNA damage, and agents that perturb DNA repair pathways, have been used successfully in the treatment of human cancer. The PI3K-like protein kinases ATR (ATM and Rad3-related) and ATM (ataxia telangiectasia mutated) are critical regulators of the DNA damage response (DDR), signaling to downstream effector molecules that in turn regulate cellular responses such as DNA repair, cell cycle arrest, and cell death. ATM is activated in response to DNA damage in part via autophosphorylation at serine 1981. ATR was long thought to exist in a constitutively active state in cells, with DNA damage-induced signaling occurring via recruitment of ATR to single stranded DNA and sites of replication stress. Recent work, however, has shown autophosphorylation of ATR at threonine 1989, a homologous site to ATM Ser1981. Like ATM Ser1981, phosphorylation of ATR Thr1989 occurs in response to DNA damage, indicating that phosphorylation at this site is important in ATR-mediated signaling. We have generated highly specific antibodies to both ATM Ser1981 and ATR Thr1989. Using these antibodies, we have identified conditions under which these sites are phosphorylated in cultured human cell lines. While phosphorylation of ATM was induced in response to a wide variety of DNA damage-inducing agents, phosphorylation of ATR was induced under a small subset of these conditions, most robustly with the alkylating agent mitomycin C, the ribonucleotide reductase inhibitor hydroxyurea and the topoisomerase II inhibitor etoposide. All of these agents inhibit DNA synthesis, albeit through different mechanisms. We then identified changes downstream of ATM and ATR under conditions of ATR Thr1989 phosphorylation. Checkpoint kinases Chk1 and Chk2 are phosphorylated and activated by ATR and ATM, respectively, though there is crosstalk between the two signaling pathways. Phosphorylation of ATR Thr1989, as well as phosphorylation of the ATR substrate Chk1 Ser317, but not phosphorylation of ATM Ser1981 or Chk2 Thr68, was abrogated by treatment with the ATR inhibitor VE-821. Using western blotting, we also detected a DNA damage-induced electrophoretic mobility shift of CtIP (CtBP-interacting protein), a protein involved in the generation of single stranded DNA. The change in CtIP was partially abrogated by the ATR inhibitor, indicating that posttranslational modification of CtIP may be related to ATR Thr1989 phosphorylation. Using cell lines with and without DNA damage, and in the presence or absence of kinase inhibitor, we performed PTMScan proteomic analysis to identify and quantify changes in phosphorylation levels of proteins and sites containing a consensus ATM/ATR substrate motif (S*Q/T*Q). Changes in phosphopeptide abundance between samples monitored by PTMScan provides insight into signaling components regulated by the ATM and ATR DNA damage response. Citation Format: Hayley J. Roberts, Matthew P. Stokes, Xiaoying Jia, Kimberly A. Lee, Susan M. Keezer. DNA damage inducible phosphorylation of ATR at threonine 1989 and quantitative analysis of the effect of ATR inhibition on DNA damage signaling using PTMScan [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B09.

Insufficient levels of the nrdAB-encoded ribonucleotide reductase underlie the severe growth defect of the Δhda E. coli strain.

The ATP-bound form of the Escherichia coli DnaA replication initiator protein remodels the chromosomal origin of replication, oriC, to load the replicative helicase. The primary mechanism for regulating the activity of DnaA involves the Hda and β clamp proteins, which act together to dramatically stimulate the intrinsic DNA-dependent ATPase activity of DnaA via a process termed Regulatory Inactivation of DnaA. In addition to hyperinitiation, strains lacking hda function also exhibit cold sensitive growth at 30°C. Strains impaired for the other regulators of initiation (i.e., ΔseqA or ΔdatA) fail to exhibit cold sensitivity. The goal of this study was to gain insight into why loss of hda function impedes growth. We used a genetic approach to isolate 9 suppressors of Δhda cold sensitivity, and characterized the mechanistic basis by which these suppressors alleviated Δhda cold sensitivity. Taken together, our results provide strong support for the view that the fundamental defect associated with Δhda is diminished levels of DNA precursors, particularly dGTP and dATP. We discuss possible mechanisms by which the suppressors identified here may regulate dNTP pool size, as well as similarities in phenotypes between the Δhda strain and hda+ strains exposed to the ribonucleotide reductase inhibitor hydroxyurea.

Lovastatin protects keratinocytes from DNA damage-related pro-apoptotic stress responses stimulated by anticancer therapeutics

BackgroundOral mucositis (OM) is a relevant adverse effect of anticancer therapy involving ionizing radiation (IR) and doxorubicin (Doxo). Because DNA damage of keratinocytes is causative for the pathogenesis of OM, we aim to identify pharmacological measures for geno- and cytoprotection of keratinocytes. MethodsWe investigated the influence of the lipid-lowering drug lovastatin on cell death, proliferation and DNA damage response (DDR) mechanisms of human keratinocytes following treatment with IR and Doxo. ResultsLovastatin protected keratinocytes from the cytotoxic and genotoxic effects of IR and Doxo as shown by a diminished induction of apoptosis as well as a reduced formation and slightly improved repair of DNA damage following Doxo and IR treatment, respectively. Lovastatin selectively blocked the activation of Chk1 and ATR kinases following treatment with IR, Doxo and the ribonucleotide reductase inhibitor hydroxyurea, indicating that the statin antagonizes ATR/Chk1-regulated replicative stress responses. Part of the cytoprotective activity of lovastatin seems to rest on a delayed entry of lovastatin treated cells into S-phase. Yet, because the statin also protected non-proliferating keratinocytes from IR- and Doxo-induced cytotoxicity, cell cycle independent protective mechanisms are involved, too. ConclusionsLovastatin attenuates pro-toxic DNA damage-related responses of keratinocytes stimulated by OM-inducing anticancer therapeutics. The data encourage forthcoming in vivo and clinical studies addressing the usefulness of statins in the prevention of OM.

Hydroxyurea Treatment Is Associated with Elevated Serum Erythropoietin Concentration but Suppressed Global Hypoxic Transcriptional Responses in Sickle Cell Disease

BackgroundThe level of distorted erythrocytes due to polymerization of hemoglobin S in sickle cell disease (SCD) (Science 1949;110:543) is a major determinant of the severity of hemolysis and microvascular occlusion (Lancet 2010;376:2018). Erythropoietin (EPO) is elevated in SCD due to hemolytic anemia and a related increase in hypoxia-inducible factors (HIFs) (Eur J Haematol 2007;78:183). Hydroxyurea (HU) is widely used in the treatment of SCD. HU inhibits ribonucleotide reductase (Semin Oncol 1992;19(3 Suppl 9):1-10) and promotes γ globin synthesis thereby increasing HbF-containing erythrocytes (F cells) while suppressing sickle β hemoglobin production (J Clin Invest 1984;74:652 and 2003;111:231). Increased level of F cells reduces hemolysis and ameliorates clinical complications in SCD. We and others have observed an increase in serum EPO level with HU treatment in SCD despite an increase in the hemoglobin concentration, and we hypothesized that this may be due to the known increased affinity of hemoglobin F for oxygen and related tissue hypoxia (Blood 2009;114:4639).MethodsMessenger RNA from peripheral blood mononuclear cells (PBMCs) was profiled using Affymetrix Human Exon 1.0 ST Array. Hypoxic transcriptional alteration was defined in 15 Chuvash polycythemia (CP) patients vs. 17 control individuals. CP leads to constitutive up-regulation of HIFs in the absence of anemia or hypoxia. Transcriptional alteration in SCD was determined in 13 HbSS subjects without HU treatment vs. 16 control individuals, and that induced by HU treatment was determined in 19 HbSS subjects with vs. 13 without HU treatment. For meta-analysis on serum EPO concentration, genomic DNA isolated from PBMCs was hybridized to the Illumina Human 610-Quad SNP array. Genotypes were imputed to 1000 genomes project phase 1 data. A linear regression model was applied adjusting for age, gender, hemoglobin concentration, and HU treatment.ResultsGene expression changes by HbSS highly correlated with those associated with homozygous VHLR200W (Pearson's r=0.79, Figure 1A). At 5% false discovery rate (FDR), expression levels of 377 genes were altered in both VHLR200W homozygotes and HbSS by >1.2 fold. For these hypoxic genes, the correlation of expression changes between HbSS and homozygous VHLR200W reached r=0.97 (Figure 1B). In contrast to our hypothesis, HU treatment in general suppressed expression changes induced by HbSS (r=-0.85, Figure 1C), especially for the hypoxic genes (r=-0.95, Figure 1D). In VHLR200W homozygotes, 62 of the hypoxic genes correlated with plasma EPO levels (adjusted P<0.05, n=42). These EPO-correlated genes were the most strongly up-regulated hypoxic genes in HbSS (red points in Figure 1B) and also the most strongly suppressed by HU treatment (red points in Figure 1D). Consistent with previous observations, we found that EPO was elevated by HU treatment in two SCD cohorts, and this persisted after adjusting for covariates including hemoglobin concentration which reflects hypoxic as well as inflammatory and hemolytic responses: Walk-PHaSST (β=0.49, P=2.5×10-15, n=586) and PUSH children (β=0.34, P=2.5×10-7, n=387). This observation suggests that biological signals independent of hypoxic regulation may contribute to EPO production under HU treatment. In a meta-analysis for the Walk-PHaSST and PUSH children cohorts, SNP rs60684937, located within the first intron of MAP2K6, an upstream regulator of HIF signaling (Mole Cell Biol 2005; 25:4853), was significantly associated with EPO levels at genome-wide significance (combined P=3.5×10-8). The C allele of the SNP decreased EPO levels in both Walk-PHaSST (β=-0.30, n=388) and PUSH children (β=-0.24, n=249) cohorts. This association was validated in an additional 89 SCD patients from the Howard cohort (β=-0.39, P=0.011). Further investigations are needed to determine whether the causal polymorphism affects protein function or gene regulation of the nearby genes.DiscussionOur study demonstrates a prominent release from hypoxic transcriptional responses by HU treatment in SCD despite an increase in serum EPO, a defining characteristic of an up-regulated hypoxic response. Our study hypothesizes that hypoxia-independent signals trigger EPO production in the setting of HU therapy and it identifies a potential genetic determinant in this alternative pathway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Checkpoint-dependent RNR induction promotes fork restart after replicative stress.

The checkpoint kinase Rad53 is crucial to regulate DNA replication in the presence of replicative stress. Under conditions that interfere with the progression of replication forks, Rad53 prevents Exo1-dependent fork degradation. However, although EXO1 deletion avoids fork degradation in rad53 mutants, it does not suppress their sensitivity to the ribonucleotide reductase (RNR) inhibitor hydroxyurea (HU). In this case, the inability to restart stalled forks is likely to account for the lethality of rad53 mutant cells after replication blocks. Here we show that Rad53 regulates replication restart through the checkpoint-dependent transcriptional response, and more specifically, through RNR induction. Thus, in addition to preventing fork degradation, Rad53 prevents cell death in the presence of HU by regulating RNR-expression and localization. When RNR is induced in the absence of Exo1 and RNR negative regulators, cell viability of rad53 mutants treated with HU is increased and the ability of replication forks to restart after replicative stress is restored.

WT1 Protects Leukemic Cells from Cytotoxic Replicative Stress

BACKGROUND:The transcription factor WT1 is a “pan-leukemic marker”. WT1 is overexpressed in a significant number of AML, CML, ALL, and MDS cells. Moreover, the persistence of WT1 expression after chemotherapy indicates residual leukemic stem cells. Hydroxyurea can decrease the white blood cell count in leukemic patients. Hydroxyurea inhibits ribonucleotide reductase and consequently depletes the cellular dNTP pool. This process induces replicative stress and activates the intra-S phase checkpoint. The prolonged arrest of DNA replication leads to double strand DNA breaks and apoptosis. The aim of our study was to investigate whether WT1 has an impact on the survival of AML and CML cells treated with hydroxyurea. Furthermore, we analyzed whether replicative stress affects WT1 expression. We also addressed whether clinically relevant drugs modulating WT1 influence apoptosis, autophagy, and DNA damage signaling in leukemic cells.METHODS:We used human AML and CML cells (MV4-11, RS4-11, NB4, HEL, K562) and primary AML cells with various genetic lesions (FLT3-ITD, MLL-AF4, PML-RARα, JAK2V617F, BCR-ABL; p53 mutant or null) or a complex karyotype. Cells were exposed to 0,5 mM hydroxyurea and analyzed by FACS, Western Blot and mRNA expression analyses. RNAi with morpholinos targeting the WT1 mRNA as well as the tyrosine kinase inhibitor imatinib (0,1-1 µM) and epigenetic modifiers of the histone deacetylase inhibitor family (HDACi: 1,5-5 µM entinostat and 10-100 nM panobinostat) were used to decrease WT1 levels.RESULTS:1.The stability of WT1 is causally linked to the survival of leukemic cells undergoing replicative stress. Hydroxyurea-treated leukemic cells retaining WT1 (K562 and RS4-11 cells) accumulate in S phase and survive. However, cells that lose WT1 (NB4, MV4-11, HEL, and primary AML cells) succumb to apoptosis (57-70% apoptosis after 24 h; p<0,001).2.The specific elimination of WT1 in K562 cells with morpholino antisense nucleotides (10 nM) significantly sensitizes the cells to hydroxyurea-induced apoptosis (26-30% increased apoptosis rates after 24 h; p<0,005).3.The hydroxyurea-induced loss of WT1 in sensitive cells is caspase-dependent but does not require the p53 tumor suppressor. The pan-caspase inhibitor ZVAD-FMK (20-50 µM) and the ectopic overexpression of mitochondrial anti-apoptotic proteins (BCL-XL/BCL2) prevents the hydroxyurea-induced depletion of WT1 and apoptosis in NB4 cells (from 62-69% to 9-14%; p<0,001).4.Moreover, our data reveal that hydroxyurea promotes the occurrence of previously unrecognized WT1 cleavage products. These are different from the known HTRA2-induced WT1 fragments, as they contain the WT1 zinc fingers required for DNA binding. These novel cleavage products also contain the domain for transcriptional activation, but lack the domain for transcriptional repression. We demonstrate that caspases cleave the full-length WT1 protein into such fragments, which accumulate in the nucleus of hydroxyurea-treated NB4 cells.5.Resistance of BCR-ABL-positive K562 CML cells to pro-apoptotic effects of hydroxyurea can be overcome with the tyrosine kinase inhibitor imatinib. This drug inhibits the WT1 promoter and sensitizes the cells to hydroxyurea (22-48% increased apoptosis rate after 24 h; p<0,005).6.The HDACi entinostat and panobinostat suppress the WT1 gene and accelerate the proteasomal degradation of WT1 by an increased expression of the E2 ubiquitin conjugase UBCH8. The depletion of WT1 by HDACi renders K562 cells sensitive towards hydroxyurea-induced killing and causes a synergistic activation of cell death (apoptosis increased by 43-55% after 24 h; p<0,001).7.In K562 cells hydroxyurea/entinostat combinations lead to a caspase-dependent cleavage of the DNA repair enzyme PARP-1 and to the accumulation of double-strand DNA breaks.8.We further show that entinostat significantly shifts the balance from a hydroxyurea-induced autophagy program to apoptosis (from 60% to 16% autophagy; p<0,001) in K562 cells. We reveal that entinostat depletes proteins critical for autophagy and autophagosome-lysosome fusion (Beclin-1 and HDAC6) in these cells.CONCLUSIONS:Our data illustrate that WT1 is a novel biological target of hydroxyurea, independent of a specific genetic lesion and irrespective of a functional p53 pathway. The inhibition of WT1 might be therapeutically exploitable for targeting approaches against leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Immunologic effects of hydroxyurea in sickle cell anemia.

Susceptibility to encapsulated bacteria is well known in sickle cell disease (SCD). Hydroxyurea use is common in adults and children with SCD, but little is known about hydroxyurea's effects on immune function in SCD. Because hydroxyurea inhibits ribonucleotide reductase, causing cell cycle arrest at the G1-S interface, we postulated that hydroxyurea might delay transition from naive to memory T cells, with inhibition of immunologic maturation and vaccine responses. T-cell subsets, naive and memory T cells, and antibody responses to pneumococcal and measles, mumps, and rubella vaccines were measured among participants in a multicenter, randomized, double-blind, placebo-controlled trial of hydroxyurea in infants and young children with SCD (BABY HUG). Compared with placebo, hydroxyurea treatment resulted in significantly lower total lymphocyte, CD4, and memory T-cell counts; however, these numbers were still within the range of historical healthy controls. Antibody responses to pneumococcal vaccination were not affected, but a delay in achieving protective measles antibody levels occurred in the hydroxyurea group. Antibody levels to measles, mumps, and rubella showed no differences between groups at exit, indicating that effective immunization can be achieved despite hydroxyurea use. Hydroxyurea does not appear to have significant deleterious effects on the immune function of infants and children with SCD. Additional assessments of lymphocyte parameters of hydroxyurea-treated children may be warranted. No changes in current immunization schedules are recommended; however, for endemic disease or epidemics, adherence to accelerated immunization schedules for the measles, mumps, and rubella vaccine should be reinforced.

Reciprocal regulation of autophagy and dNTP pools in human cancer cells

Ribonucleotide reductase (RNR) plays a critical role in catalyzing the biosynthesis and maintaining the intracellular concentration of 4 deoxyribonucleoside triphosphates (dNTPs). Unbalanced or deficient dNTP pools cause serious genotoxic consequences. Autophagy is the process by which cytoplasmic constituents are degraded in lysosomes to maintain cellular homeostasis and bioenergetics. However, the role of autophagy in regulating dNTP pools is not well understood. Herein, we reported that starvation- or rapamycin-induced autophagy was accompanied by a decrease in RNR activity and dNTP pools in human cancer cells. Furthermore, downregulation of the small subunit of RNR (RRM2) by siRNA or treatment with the RNR inhibitor hydroxyurea substantially induced autophagy. Conversely, cancer cells with abundant endogenous intracellular dNTPs or treated with dNTP precursors were less responsive to autophagy induction by rapamycin, suggesting that autophagy and dNTP pool levels are regulated through a negative feedback loop. Lastly, treatment with si-RRM2 caused an increase in MAP1LC3B, ATG5, BECN1, and ATG12 transcript abundance in xenografted Tu212 tumors in vivo. Together, our results revealed a previously unrecognized reciprocal regulation between dNTP pools and autophagy in cancer cells.