RRM1 single nucleotide polymorphism -37C→A correlates with progression-free survival in NSCLC patients after gemcitabine-based chemotherapy

<p>Abstract</p> <p>Background</p> <p>The ribonucleotide reductase M1 (RRM1) gene encodes the regulatory subunit of ribonucleotide reductase, the molecular target of gemcitabine. The overexpression of RRM1 mRNA in tumor tissues is reported to be associated with gemcitabine resistance. Thus, single nucleotide polymorphisms (SNPs) of the RRM1 gene are potential biomarkers of the response to gemcitabine chemotherapy. We investigated whether RRM1 expression in peripheral blood mononuclear cells (PBMCs) or SNPs were associated with clinical outcome after gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.</p> <p>Methods</p> <p>PBMC samples were obtained from 62 stage IIIB and IV patients treated with gemcitabine-based chemotherapy. RRM1 mRNA expression levels were assessed by real-time PCR. Three RRM1 SNPs, -37C→A, 2455A→G and 2464G→A, were assessed by direct sequencing.</p> <p>Results</p> <p>RRM1 expression was detectable in 57 PBMC samples, and SNPs were sequenced in 56 samples. The overall response rate to gemcitabine was 18%; there was no significant association between RRM1 mRNA expression and response rate (<it>P </it>= 0.560). The median progression-free survival (PFS) was 23.3 weeks in the lower expression group and 26.9 weeks in the higher expression group (<it>P </it>= 0.659). For the -37C→A polymorphism, the median PFS was 30.7 weeks in the C(-)37A group, 24.7 weeks in the A(-)37A group, and 23.3 weeks in the C(-)37C group (<it>P </it>= 0.043). No significant difference in PFS was observed for the SNP 2455A→G or 2464G→A.</p> <p>Conclusions</p> <p>The RRM1 polymorphism -37C→A correlated with PFS in NSCLC patients treated with gemcitabine-based chemotherapy. No significant correlation was found between PBMC RRM1 mRNA expression and the efficacy of gemcitabine.</p

Development of a capillary electrophoretic method for simultaneous determination of gemcitabine and its impurity

Gemcitabin, 2',2'-difluoro-2'-deoksicitidin, antimetabolit pirimidina, je antineoplastični lijek koji se koristi u liječenju brojnih uznapredovalih ili metastatskih karcinoma kao monoterapija ili u kombinaciji s drugim citostaticima, ovisno o indikaciji. Cilj je ovog istraživanja bio razviti novu, brzu, jednostavnu i ekološki prihvatljivu kapilarnoelektroforetsku metodu za istovremenu analizu gemcitabina i njegovog onečišćenja cizotina. Kao prikladna tehnika odabrana je kapilarna elektroforeza kao "zelena" alternativa već razvijenoj HPLC metodi za određivanje sadržaja gemcitabina i određivanje onečišćenja uvrštenoj u 8. izdanje Europske farmakopeje. Analiza je vršena u kapilari duljine 35 cm (27 cm) pri temperaturi od 25 °C, na valnoj duljini detekcije od 275 i 237 nm. Tijekom razvoja nove metode ispitane su različite vrste radnih pufera: boratni (pH 9,3), fosfatni (pH 7,0) i acetatni (pH 3,0). Na temelju rezultata eksperimenata najbolji puferom pokazao se fosfatni pufer (pH 7,0), dok je bolje razdvajanje i oblik pikova postignut dodatkom veće koncentracije SDS-a. Optimalnim se pokazala 50 mM koncentracija SDS-a. Ispitan je i utjecaj napona na vrijeme analize te razlučivanje između analita. Kod napona od 10 kV postignuto je bolje razdvajanje gemcitabina i citozina. Međutim, primjenom nižeg napona produljuje se vrijeme putovanja analita kroz kapilaru pa je potrebno voditi računa o tome da je razlučivanje između analita što bolje, no istovremeno uz što kraće vrijeme analize. Predlaže se daljnja optimizacija metode kako bi se postiglo prikladno razlučivanje pikova te njihov odgovarajući oblik i simetrija uz što kraće vrijeme analize.Gemcitabine, 2', 2'-difluoro-2'-deoxycytidine, pyrimidine antimetabolite, is an antineoplastic drug used to treat many advanced or metastatic cancers as monotherapy or in combination with other cytostatics, depending on the indication. The aim of this study was to develop a new, fast, simple and environmentally friendly capillary electrophoretic method for simultaneous identification and determination of gemcitabine and its impurity cytosine. Capillary electrophoresis was chosen as a suitable "green" alternative to an already developed HPLC method for the determination of gemcitabine and its impurities included in the 8th edition of European Pharmacopoeia. The analysis was carried out in 35 cm (27 cm) capillary at a temperature of 25 ° C and detection was at 275 i 237 nm. Three different buffers were studied while developing the method: borate (pH 9.3), phosphate (pH 7.0) and acetate (pH 3.0). Based on the experimental results, best buffer was shown to be the phosphate buffer (pH 7.0), while a better separation and peak shape were achieved by adding a higher concentration of SDS. 50 mM SDS was chosen as the optimal concentration. The effect of voltage on the analysis time and resolution between the analytes was investigated. It has been shown that a better separation of gemcitabine and cytosine was achieved at 10 kV. However, applying a lower voltage is prolonging the migration time of the analytes through the capillary. Therefore, it is necessary to take into account that a good resolution is achieved but that the analysis time is not too long. Further method optimization is required to achieve the appropriate peak separation, shape and symmetry with as short analysis time as possible

Whole genome doubling confers unique genetic vulnerabilities on tumor cells

Whole genome doubling (WGD) generates genetically unstable tetraploid cells that fuel tumorigenesis. Cells that undergo WGD must acquire adaptive characteristics to accommodate their tetraploid state, and these adaptations may confer unique vulnerabilities that can be exploited therapeutically. We analyzed the genomes of ~9,700 primary human cancer samples to uncover genetic alterations that are specifically enriched in WGD+ cancer cells. Through integrating our genetic analysis with gene essentiality data acquired from Project Achilles, we identified gene dependencies in WGD+ cells. Moreover, we identified genes that are essential for the viability of WGD+ cancer cells, but non-essential to non-transformed diploid cells. We demonstrated that the gene encoding for the mitotic kinesin KIF18A is dispensable for mitosis in diploid cells, but becomes critical for accurate chromosome segregation and viability in WGD+ cells, making it an attractive drug target. Collectively, this work revealed new strategies to specifically target WGD+ cancer cells, namely targeting the gene KIF18A, while sparing the normal diploid cells from which they arise.2022-06-04T00:00:00

RRM2 enhances MYCN-driven neuroblastoma formation and acts as a synergistic target with CHK1 inhibition

High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential

Síntese e design racional de novos análogos de nucleósidos e de nucleótidos como potenciais inibidores de cinases com interesse terapêutico

Tese de mestrado em Química, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018Os nucleósidos e nucleótidos são moléculas cruciais para a vida, desempenhando funções chave em inúmeros processos biológicos fundamentais como a síntese do DNA e RNA, divisão celular e metabolismo. O desenvolvimento de análogos sintéticos destes compostos tem sido explorado ao longo dos anos, tendo conduzido a vários fármacos que são utilizados no tratamento do cancro e de doenças virais. Para além disso, existem evidências do potencial de compostos deste tipo para inibir colinesterases e para exibir atividade antimicrobiana. Apesar do seu enorme potencial terapêutico, no que se refere às suas propriedades anticancerígenas e antivirais, existem alguns problemas associados ao seu usual mecanismo de ação. Existem vários mecanismos de resistência que as células exibem face à ação destes compostos, como por exemplo, a diminuição de proteínas transportadoras de nucleósidos, o que impossibilita a passagem destas moléculas para dentro das células, devido à sua polaridade. Deste modo, o desenvolvimento de novos análogos contendo substituintes estáveis e apolares e exibindo novos mecanismos de ação ou atuando em alvos biológicos pouco explorados, poderá ultrapassar estes problemas. Neste trabalho foram sintetizados novos análogos de nucleósidos, contendo o grupo guanidina, devido ao facto de ser um potencial mimético de uma base azotada, partindo de derivados de xilofuranose contendo diferentes O-substituintes na posição 3. Foram abordados dois diferentes métodos de redução/guanidinilação e os compostos desejados foram obtidos com sucesso. A partir desses derivados também foi possível sintetizar estruturas inovadoras de isonucleótidos, contendo um grupo fosfato ou um grupo fosforamidato na posição 5 e uma unidade benziltriazole numa O-ramificação da posição 3 como um sistema mimético de uma nucleobase. Foram abordadas as reações de fosforilação e Staudinger-fosfito para a síntese destes compostos, respetivamente. Por fim, foram também obtidos novos análogos de nucleótidos contendo o grupo uracilo na posição anomérica e um grupo (metil)triazolil fosfonato como um possível mimético estável e neutro do grupo difosfato. Para a síntese destes compostos as reações de ciclização térmica e N-glicosilação foram exploradas. A afinidade destes compostos para as enzimas CDK2 e GSK3β foi estudada por docking molecular, uma vez que estas enzimas são potenciais alvos terapêuticos para o cancro e para a doença de Alzheimer/diabetes, respetivamente. De um modo geral, as moléculas ou características estruturais que permitiram obter melhores resultados em termos de afinidade de ligação às enzimas foram: guanidino furanoses contendo o grupo benziltriazole, derivados de metil triazoles contendo o substituinte O-benzilo na posição 3 e grupo fosfato O-substituído por grupos fenilos. Deste modo, os resultados foram bastante positivos e motivadores para a realização de futuros testes biológicos.Nucleosides and nucleotides are crucial molecules for life that play a key role in fundamental biological processes such as a synthesis of DNA and RNA, cell division and metabolism. The development of synthetic analogues of these molecules has been explored throughout the years and has led to a number of drugs that are used nowadays for the treatment of cancer and viral diseases. Furthermore, some reports showed the potential of these types of compounds to display cholinesterase inhibitory properties and antimicrobial activity. On the other hand, despite their significant therapeutic profile, considering their anticancer and antiviral properties, there are some problems related to their mechanisms of action. Various mechanisms of resistance are exhibited by cells against the action of these compounds, such as the decrease on the level of nucleoside transporters, which perturbs their passage to the intracellular medium due to their polarity. Thus, the development of new analogues containing stable and less polar moieties and exhibiting new mechanism of action or focussing rather less exploited biological targets, are required to overcome these problems. In this work, new nucleoside analogues comprising a guanidine group as a potential mimetic of a nucleobase, were synthetized. These compounds were obtained successfully from bis-functionalized xylofuranose derivatives containing different O-substituents in position 3, using two different reduction/guanidinylation methods. Innovative isonucleotides were obtained from these precursors, comprising a phosphate or a phosphoramidate group and a triazole moiety in the position 3, as a mimetic of a nucleobase. For the access of these compounds, the synthetic strategies involved phosphorylation and Staudinger-phosphite reactions, respectively. Moreover, new nucleotide analogues containing a uracil moiety in the anomeric position and comprising a (triazolyl)methyl phosphonate system as a stable and neutral mimetic of a diphosphate group, were synthetized. These compounds were obtained by N-glycosylation and thermal cyclization reactions. The affinity of these compounds for CDK2 and GSK3β was studied by molecular docking, since these enzymes are potential therapeutic targets for cancer and Alzheimer’s disease/diabetes, respectively. The molecules/structural features that enabled obtaining better results in terms of binding affinities to the enzymes are the following: guanidino sugars comprising a benzyl triazole system, methyl triazole derivatives having an O-benzyl group in the position 3 and phosphate moieties containing O-phenyl groups. These results are very promising and encourage further biological testing of these molecules

Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007.Vita.Includes bibliographical references.Efforts towards the Synthesis of Fully N-Differentiated Heparin-like Glycosaminoglycans. Heparin-like glycosaminoglycans (HLGAGs) are complex information-carrying biopolymers and are an important component of the coagulation cascade. They have also been implicated in interactions with growth factors, cytokines, virus entry, and other functions. Currently, no general synthesis of arbitrary HLGAG sequences has been demonstrated. The modular synthesis of glycosaminoglycans requires straightforward methods for the production of large quantities of protected uronic acid building blocks. An efficient route to methyl 3-0- benzyl-1,2-O-isopropylidene-a-L-idopyranosiduronate from diacetone glucose in nine steps and 36% overall yield is described. Additionally, a general method for the conversion of glycals to the corresponding 1,2-cis-isopropylidene-a-glycosides is reported. Epoxidation of glycals with dimethyldioxirane followed by ZnC12-catalyzed addition of acetone converted a variety of protected glycals into 1,2-cis-isopropylidene-a-glycosides in good yield. The reaction is compatible with a range of protecting groups, as well as free hydroxyl groups. This method has been applied to develop a synthesis of 3-O-benzyl-1,2-O-isopropylidene-P-D-glucopyranosiduronate in seven steps and 32% overall yield.(cont.) These compounds are useful as glycosyl acceptors and as intermediates that may be further elaborated into uronic acid trichloroacetimidate glycosyl donors for the assembly of glycosaminoglycan structures. The glucosamine residues in HLGAGs have been found to exist as amines, acetamides, and N-sulfonates. In order to develop a completely general, modular synthesis of heparin, three degrees of orthogonal nitrogen protection are required. Reported is a strategy for the synthesis of fully N-differentiated heparin oligosaccharides in the context of target octasaccharide 3-1, which contains an N-acetate, N-sulfonates, and a free amine. The protecting group scheme used in the synthesis blocked the N-acetate as a N-diacetate, the N-sulfonates as azido groups, and the amine as a N-CBz; free hydroxyls were masked as benzyl ethers and O-sulfonates as acetate esters. Disaccharide and tetrasaccharide modules were synthesized using this strategy; however, the union of tetrasaccharide trichloroacetimidate 3-4 with disaccharide acceptor 3-5 unexpectedly formed the undesired P-linked glycoside in addition to the a-linkage anticipated for iduronic acid nucleophiles, resulting in an inseparable 6:1 a: p mixture of products. Detailed studies into the basis for this unexpected result were conducted and are also reported.(cont.) Investigations into the Mechanism of Inactivation of RTPR by Gemcitabine Triphosphate. Ribonucleoside triphosphate reductase (RTPR) is an adenosylcobalamin (AdoCbl) dependant enzyme that catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates via controlled radical chemistry. The antitumor agent 2',2'-difluoro-2'- deoxycytidine (gemcitabine, F2C) has been shown to owe some of its in vivo activity to inhibition of human RNR by the 5'-diphosphate (F2CDP). Previous studies have shown that RTPR is rapidly inactivated by one equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). This inactivation is associated with the release of two equivalents of fluoride and modification of RTPR by a Co-S bond between C419 and the cobalamin cofactor. In order to further characterize this inactivation, isotopically labeled derivatives of F2CTP were synthesized: radiolabeled 1'-[3H]-F2C and mass labeled 1'-[2H]-F2C and 3'-[2H]-F2C. These compounds were converted to F2CTP through a set of enzymatic phosphorylation steps which overcome difficulties found using traditional, chemical methods. Biochemical investigations were performed using these labeled derivatives to track the fate of the base and sugar during RTPR inactivation by F2CTP.(cont.) The release of cytosine base, previously overlooked in this system, was detected utilizing 5-[3H]-F2CTP: 0.7 equiv. of cytosine were released, with 0.15-0.2 equiv. of unreacted F2CTP remaining. Size exclusion chromatography (SEC) was used to quantify covalent labeling of RTPR by F2CTP: 0.15 equiv. were detected using 5-[3H]-F2CTP, 0.45 equiv. were detected using 1'-[3H]-F2CTP. A small molecule nucleotide product was identified in inactivation mixtures quenched with NaBH4 and identified as an isomer of cytidine, indicating the loss of both fluorides and the addition of an oxygen at the 2' carbon. RTPR inactivated with 1'-[3H]-F2CTP was digested with trypsin and peptides containing radioactivity purified. Identical peptides were prepared using partially deuterated F2CTP, allowing identification by MALDI-MS. Post source decay (PSD) MS/MS methods were used to further characterize these peptides, identifying the site of label as the C-terminal tryptic peptide of RTPR at C731 and C736. The cysteines were labeled through conjugate addition with a furanone-like precursor that had lost cytosine, triphosphate, and both fluorines. The results of these studies have allowed for the first time the proposal of a mechanistic hypothesis for RTPR inactivation by F2CTP.by Gregory J.S. Lohman.Ph.D

Etudes fonctionnelles et biophysiques de Hug1 ; une protéine intrinsèquement désordonnée impliquée dans le métabolisme des nucléotides

Face aux agressions constantes que subit l ADN, les cellules ont développé des mécanismes de protection, nommés checkpoints pour maintenir l intégrité de leur génome. Chez Saccharomyces cerevisiae, la kinase Rad53 joue un rôle central dans ces voies et son activation conduit à de nombreux effets cellulaires tels que le ralentissement du cycle cellulaire, le ralentissement de la réplication, l activation de la transcription de certains gènes, l activation de la réparation Lors d un crible transcriptomique, utilisant une souche exprimant une forme hyperactive de Rad53, nous avons identifié le gène HUG1 comme l un des gènes les plus transcrits suite à l activation de la voie RAD53. Cependant les fonctions de Hug1 demeurent énigmatiques.Pour mieux comprendre les fonctions de Hug1 dans la réponse aux dommages de l ADN, nous avons recherché ses partenaires physiques et avons identifié les protéines Rnr2 et Rnr4, les deux composants de la petite sous-unité de la Ribonucléotide Réductase (RNR). La RNR est un complexe enzymatique qui catalyse l étape limitante de synthèse des nucléotides. Nous avons alors cherché à caractériser cette interaction par diverses méthodes. Nous avons ainsi montré que Hug1 est une protéine intrinsèquement désordonnée capable d interagir physiquement avec la petite sous-unité de la RNR et qu au moins onze acides aminés de Hug1 sont impliqués dans son interaction avec la RNR. Lors de nos investigations, nous avons observé que le fait d étiqueter Rnr2 en position C-terminale sensibilisait les souches aux stress génotoxiques et que cette sensibilité était supprimée si on abrogeait la fonction de HUG1, faisant de Hug1 un nouvel inhibiteur de la RNR. Ainsi nous sommes parvenus à proposer un modèle de régulation de la RNR par Hug1.To maintain genome integrity, cells have developed protection mechanisms, called checkpoints, in response to DNA damage insults. In Saccharomyces cerevisiae, Rad53 protein kinase is one of the major actors in these mechanisms, and its activation triggers several cellular responses such as cell cycle delay, replication delay, transcription modifications, activation of DNA repair pathways Using an hyperactivative allele of RAD53, we identified HUG1, as one of the most induced gene in a transcriptomic analysis upon RAD53 pathway activation. However Hug1 s functions remains elusive.To better understand Hug1 s functions in DNA damage response, we searched for physical partners and identified Rnr2 and Rnr4 proteins, which are the two small subunits of Ribonucleotide Reductase (RNR). The RNR is an enzymatic complex that catalyses nucleotide reduction, a step limiting for dNTPs synthesis. We next experimentally tackled the Hug1-RNR interaction using various methods. We showed so that Hug1 is a small intrinsically disordered protein able to interact physically with the small RNR subunit and that at least eleven amino acids in Hug1 are involved in this interaction. During our investigations, we observed that C-terminal tagging of Rnr2 sensitizes strains to genotoxics stress and that this sensitivity was suppressed when HUG1 s function is abrogated. Hence, we showed that Hug1 is a negative RNR regulator and propose a model for Hug1 s function.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Investigations of the inhibition mechanisms of human ribonucleotide reductase by gemcitabine-5'-diphosphate and saccharomyces cerevisiae ribonucleotide reductase by Sml1

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.Vita. Cataloged from PDF version of thesis.Includes bibliographical references.Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits ([alpha] and [beta]) for activity. The [alpha] subunit binds the substrates and the allosteric effectors that govern specificity and turnover. The 32 subunit houses the diferric Y* cofactor required to initiate nucleotide reduction. Human cells possess two type of P subunits of RNR: one ([beta]) is involved in DNA replication and the second (p53[beta]') is required for mitochondrial DNA replication and likely plays some role in DNA repair. Gemcitabine (2',2'-difluoro-2'-deoxycytidine, F2C) is used clinically in a variety of cancer treatments and the phosphorylated F2C targets many enzymes involved in nucleotide metabolism, including RNR. The studies presented here with [1 '-3H]- and [5- 3H]-F 2CDP have established that F2CDP is a sub-stoichiometric mechanism based inhibitor (0.5 equivalents F2CDP/[alpha]) of both the E. coli and the human RNRs in the presence of a reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 equivalents/[alpha]) and is accompanied by the release of 0.5 equivalent cytosine/[alpha]. Studies using size exclusion chromatography reveal that in the E. coli RNR, an u212 tight complex is generated subsequent to enzyme inactivation by F2CDP, while in the human RNR, an [alpha]6[beta]6 or [alpha]6[beta]'6 tight complex is generated. The second part of this thesis focuses on the Sml inhibition mechanism in S. cerevisiae. Smll is a 12 kDa small protein RNR inhibitor.(cont.) It regulates RNR activity by binding directly to a to repress RNR activity. The binding of Smll to a has been proposed to block the reduction of the active site disulfide formed concomitantly with dNTP production, leaving a in the oxidized form. A fluorescence titration method was employed to measure the Kd of Smll with different forms of c. Our data suggest that Smll binds to a by a mechanism that involves its C-terminal helix (likely the hydrophobic face) and a region of a that includes W688. The kinetics studies suggest that Smll behaves as an uncompetitive inhibitor relative to the substrate, and binds to the oxidized form of [alpha] in preference to the reduced form.by Jun Wang.Ph.D