ABSTRACT
BACKGROUND: Evidences support social inequalities in cancer survival. Studies on hematological malignancies, and more specifically Acute Myeloid Leukemia (AML), are sparser. Our study assessed: 1/ the influence of patients' socioeconomic position on survival, 2/ the role of treatment in this relationship, and 3/ the influence of patients' socioeconomic position on treatment utilization. METHODS: This prospective multicenter study includes all patients aged 60 and older, newly diagnosed with AML, excluding promyelocytic subtypes, between 1st January 2009 to 31st December 2014 in the South-West of France. Data came from medical files. Patients' socioeconomic position was measured by an ecological deprivation index, the European Deprivation Index. We studied first, patients' socioeconomic position influence on overall survival (n = 592), second, on the use of intensive chemotherapy (n = 592), and third, on the use of low intensive treatment versus best supportive care among patients judged unfit for intensive chemotherapy (n = 405). RESULTS: We found an influence of patients' socioeconomic position on survival (highest versus lowest position HRQ5: 1.39 [1.05;1.87] that was downsized to become no more significant after adjustment for AML ontogeny (HRQ5: 1.31[0.97;1.76] and cytogenetic prognosis HRQ5: 1.30[0.97;1.75]). The treatment was strongly associated with survival. A lower proportion of intensive chemotherapy was observed among patients with lowest socioeconomic position (ORQ5: 0.41[0.19;0.90]) which did not persist after adjustment for AML ontogeny (ORQ5: 0.59[0.25;1.40]). No such influence of patients' socioeconomic position was found on the treatment allocation among patients judged unfit for intensive chemotherapy. CONCLUSIONS: Finally, these results suggest an indirect influence of patients' socioeconomic position on survival through AML initial presentation.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cancer Survivors , Healthcare Disparities , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/mortality , Patient Acceptance of Health Care , Socioeconomic Factors , Age Factors , Aged , Aged, 80 and over , Female , Follow-Up Studies , France , Humans , Longitudinal Studies , Male , Middle Aged , Prognosis , Prospective Studies , Survival Rate , Treatment OutcomeABSTRACT
Poisons of topoisomerase II (TOP2) kill cancer cells by preventing religation of intermediate DNA breaks during the enzymatic process and thus by accumulating enzyme-drug-DNA complexes called TOP2 cleavage-complex (TOP2cc). F14512 is a highly cytotoxic polyamine-vectorized TOP2 inhibitor derived from etoposide and currently in clinical trials. It was shown in vitro that F14512 has acquired DNA-binding properties and that the stability of TOP2cc was strongly increased. Paradoxically, at equitoxic concentrations in cells, F14512 induced less DNA breaks than etoposide. Here, we directly compared etoposide and F14512 for their rates of TOP2cc production and resolution in human cells. We report that targeting of TOP2α and not TOP2ß impacts cell killing by F14512, contrary to etoposide that kills cells through targeting both isoforms. Then, we show that despite being more cytotoxic, F14512 is less efficient than etoposide at producing TOP2α cleavage-complex (TOP2αcc) in cells. Finally, we report that compared with TOP2αcc mediated by etoposide, those generated by F14512 persist longer in the genome, are not dependent on TDP2 for cleaning break ends from TOP2α, are channeled to a larger extent to resection-based repair processes relying on CtIP and BRCA1 and promote RAD51 recruitment to damaged chromatin. In addition to the addressing of F14512 to the polyamine transport system, the properties uncovered here would be particularly valuable for a therapeutic usage of this new anticancer compound. More generally, the concept of increasing drug cytotoxicity by switching the repair mode of the induced DNA lesions via addition of a DNA-binding moiety deserves further developments. Mol Cancer Ther; 16(10); 2166-77. ©2017 AACR.
Subject(s)
DNA Breaks, Double-Stranded/drug effects , DNA Topoisomerases, Type II/genetics , Neoplasms/drug therapy , Topoisomerase II Inhibitors/administration & dosage , Apoptosis/drug effects , BRCA1 Protein/genetics , Chromatin/genetics , Genetic Vectors/drug effects , Humans , Neoplasms/genetics , Neoplasms/pathology , Podophyllotoxin/administration & dosage , Podophyllotoxin/analogs & derivatives , Polyamines/administration & dosage , Rad51 Recombinase/geneticsABSTRACT
The association of DNA Ligase IV (Lig4) with XRCC4 is essential for repair of DNA double-strand breaks (DSBs) by Non-homologous end-joining (NHEJ) in humans. DSBs cytotoxicity is largely exploited in anticancer therapy. Thus, NHEJ is an attractive target for strategies aimed at increasing the sensitivity of tumors to clastogenic anticancer treatments. However the high affinity of the XRCC4/Lig4 interaction and the extended protein-protein interface make drug screening on this target particularly challenging. Here, we conducted a pioneering study aimed at interfering with XRCC4/Lig4 assembly. By Molecular Dynamics simulation using the crystal structure of the complex, we first delineated the Lig4 clamp domain as a limited suitable target. Then, we performed in silico screening of ~95,000 filtered molecules on this Lig4 subdomain. Hits were evaluated by Differential Scanning Fluorimetry, Saturation Transfer Difference-NMR spectroscopy and interaction assays with purified recombinant proteins. In this way we identified the first molecule able to prevent Lig4 binding to XRCC4 in vitro. This compound has a unique tripartite interaction with the Lig4 clamp domain that suggests a starting chemotype for rational design of analogous molecules with improved affinity.
Subject(s)
DNA Ligase ATP/chemistry , DNA-Binding Proteins/chemistry , DNA/chemistry , Models, Molecular , Binding Sites , DNA/metabolism , DNA Breaks, Double-Stranded , DNA Ligase ATP/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Humans , Ligands , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Interaction Domains and Motifs , Reproducibility of Results , Structure-Activity RelationshipABSTRACT
With the aim of finding selective and biologically active G-quadruplex ligands, modified porphyrin with bulky cationic substituents, meso-5,10,15,20-tetrakis(4-guanidinophenyl)porphyrin tetrahydrochloride, referred to as guanidinium phenyl porphyrin, was prepared. The corresponding nickel(II) and cobalt(III) metallated porphyrins were also synthesized. Interaction with quadruplexes was examined by means of fluorescence resonance energy transfer melting and surface plasmon resonance-based assays: the three compounds proved to bind to G-quadruplex DNA in a similar and highly selective way. Guanidinium phenyl porphyrin and its nickel(II) metallated derivative exhibit moderate cytotoxicity toward cells in culture. Strikingly, the nickel porphyrin derivative was able to displace hPOT1 shelterin protein from telomeres in human cells. Nickel(II) guanidinium phenyl porphyrin, a cationic bulky porphyrin is a powerful specific G-quadruplex DNA ligand. It enters the cells and induces shelterin modification.
Subject(s)
G-Quadruplexes/drug effects , Guanidine/chemistry , Metalloporphyrins/pharmacology , Nickel/chemistry , Porphyrins/chemistry , Telomere-Binding Proteins/metabolism , Telomere/drug effects , Binding Sites/drug effects , Cell Proliferation/drug effects , Circular Dichroism , Cobalt/chemistry , Dose-Response Relationship, Drug , Fluorescence Resonance Energy Transfer , Humans , Ligands , Metalloporphyrins/chemical synthesis , Metalloporphyrins/chemistry , Molecular Structure , Protein Transport/drug effects , Shelterin Complex , Structure-Activity Relationship , Surface Plasmon Resonance , Tumor Cells, CulturedABSTRACT
Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)-DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)-X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4-Cer-XLF complexes on DNA ends.
Subject(s)
DNA End-Joining Repair/physiology , DNA Ligases/physiology , DNA-Binding Proteins/physiology , Cell-Free System/metabolism , Cells, Cultured , DNA Damage , DNA Helicases/metabolism , DNA Helicases/physiology , DNA Ligase ATP , DNA Ligases/metabolism , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/physiology , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Genomic Instability , Holoenzymes , Humans , Ku Autoantigen , PhosphorylationABSTRACT
In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs.
Subject(s)
Antigens, Nuclear/metabolism , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA-Binding Proteins/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Antigens, Nuclear/genetics , Cell Fractionation , Cell Line , Chromatin/chemistry , Chromatin/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Gene Knockdown Techniques , Humans , Ku Autoantigen , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase InhibitorsABSTRACT
G-quadruplex nucleic acids are emerging as therapeutic targets for small molecules referred to as small-molecule G-quadruplex ligands. The porphyrin H(2)-TMPyP4 was early reported to be a suitable motif for G-quadruplex DNA recognition. It probably binds to G-quadruplex nucleic acid through π-π stacking with the external G-quartets. We explored chemical modifications of this porphyrin such as insertion of various metal ions in the centre of the aromatic core and addition of bulky substituents that may improve the specificity of the compound toward G-quadruplex DNA. Porphyrin metallation, affording a G4-ligand with two symmetric faces, allowed the conclusion that the presence of an axial water molecule perpendicular to the aromatic plane lowered but did not hamper π-π stacking interactions between the aromatic parts of the ligand on the one hand and the external G-quartet on the other. The charge introduced in the centre of the porphyrin had little influence on binding. Thus, the ionic channel in the centre of G-quadruplex nucleic acids was not found to provide clear additional molecular clues for G-quadruplex nucleic acids targeting by porphyrins tested in the present study. Furthermore, we confirmed the unique G-quadruplex selectivity of a porphyrin modified with four bulky substituents at the meso positions and showed that although the compound is not "drug-like" it was capable of entering cells in culture and mediated some of the typical cellular effects of small-molecule G-quadruplex ligands.
Subject(s)
Enzyme Inhibitors/chemistry , G-Quadruplexes , Porphyrins/chemistry , Porphyrins/metabolism , Binding Sites , Cell Line , Cell Proliferation/drug effects , DNA Damage , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Fluorescence Resonance Energy Transfer , G-Quadruplexes/drug effects , Humans , Ligands , Manganese/chemistry , Manganese/metabolism , Structure-Activity Relationship , Telomerase/antagonists & inhibitors , Telomerase/metabolismABSTRACT
DNA-dependent protein kinase (DNA-PK) is a double-strand breaks repair complex, the subunits of which (KU and DNA-PKcs) are paradoxically present at mammalian telomeres. Telomere fusion has been reported in cells lacking these proteins, raising two questions: how is DNA-PK prevented from initiating classical ligase IV (LIG4)-dependent non-homologous end-joining (C-NHEJ) at telomeres and how is the backup end-joining (EJ) activity (B-NHEJ) that operates at telomeres under conditions of C-NHEJ deficiency controlled? To address these questions, we have investigated EJ using plasmid substrates bearing double-stranded telomeric tracks and human cell extracts with variable C-NHEJ or B-NHEJ activity. We found that (1) TRF2/RAP1 prevents C-NHEJ-mediated end fusion at the initial DNA-PK end binding and activation step and (2) DNA-PK counteracts a potent LIG4-independent EJ mechanism. Thus, telomeres are protected against EJ by a lock with two bolts. These results account for observations with mammalian models and underline the importance of alternative non-classical EJ pathways for telomere fusions in cells.