Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen ...hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.
Mammalian DNA topoisomerases II are targets of anticancer anthracyclines that act by stabilizing enzyme-DNA complexes wherein DNA strands are cut and covalently linked to the protein. This molecular ...mechanism is the molecular basis of anthracycline anticancer activity as well as the toxic effects such as cardiomyopathy and induction of secondary cancers. Even though anthracyclines have been used in the clinic for more than 50 years for solid and blood cancers, the search of breakthrough analogs has substantially failed. The recent developments of personalized medicine, availability of individual genomic information, and immune therapy are expected to change significantly human cancer therapy. Here, we discuss the knowledge of anthracyclines as Topoisomerase II poisons, their molecular and cellular effects and toxicity along with current efforts to improve the therapeutic index. Then, we discuss the contribution of the immune system in the anticancer activity of anthracyclines, and the need to increase our knowledge of molecular mechanisms connecting the drug targets to the immune stimulatory pathways in cancer cells. We propose that the complete definition of the molecular interaction of anthracyclines with the immune system may open up more effective and safer ways to treat patients with these drugs.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
G quadruplexes (G4s) and R loops are noncanonical DNA structures that can regulate basic nuclear processes and trigger DNA damage, genome instability, and cell killing. By different technical ...approaches, we here establish that specific G4 ligands stabilize G4s and simultaneously increase R-loop levels within minutes in human cancer cells. Genome-wide mapping of R loops showed that the studied G4 ligands likely cause the spreading of R loops to adjacent regions containing G4 structures, preferentially at 3′-end regions of expressed genes, which are partially ligand-specific. Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4 ligands in BRCA2-proficient and BRCA2-silenced cancer cells. Moreover, even if the studied G4 ligands increased noncanonical DNA structures at similar levels in nuclear chromatin, their cellular effects were different in relation to cell-killing activity and stimulation of micronuclei, a hallmark of genome instability. Our findings therefore establish that G4 ligands can induce DNA damage by an R loop-dependent mechanism that can eventually lead to different cellular consequences depending on the chemical nature of the ligands.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
G-quadruplex (G4) binders have been investigated to discover new anticancer drugs worldwide in past decades. As these ligands are generally not highly cytotoxic, the discovery rational was mainly ...based on increasing the cell-killing potency. Nevertheless, no G4 binder has been shown yet to be effective in cancer patients. Here, G4 binder activity at low dosages will be discussed as a critical feature to discover ligands with therapeutic effects in cancer patients. Specific effects of G4 binders al low doses have been reported to occur in cancer and normal cells. Among them, genome instability and the stimulation of cytoplasmic processes related to autophagy and innate immune response open to the use of G4 binders as immune-stimulating agents. Thus, we propose a new rational of drug discovery, which is not based on cytotoxic potency but rather on immune gene activation at non-cytotoxic dosage.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Co-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both ...positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown.
Here, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.
Our findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation.
Although accumulation of DNA damage and genomic instability in resting cells can cause neurodegenerative disorders, our understanding of how transcription produces DNA double-strand breaks (DSBs) ...is limited. Transcription-blocking topoisomerase I cleavage complexes (TOP1ccs) are frequent events that prime DSB production in non-replicating cells. Here, we report a mechanism of their formation by showing that they arise from two nearby single-strand breaks (SSBs) on opposing DNA strands: one SSB from the removal of transcription-blocking TOP1ccs by the TDP1 pathway and the other from the cleavage of R-loops by endonucleases, including XPF, XPG, and FEN1. Genetic defects in TOP1cc removal (TDP1, PNKP, and XRCC1) or in the resolution of R-loops (SETX) enhance DSB formation and prevent their repair. Such deficiencies cause neurological disorders. Owing to the high frequency of TOP1cc trapping and the widespread distribution of R-loops, these persistent transcriptional DSBs could accumulate over time in neuronal cells, contributing to the neurodegenerative diseases.
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•Persistent TOP1ccs induce transcription block and R-loop formation•Removal of transcription-blocking TOP1ccs by the TDP1 pathway generates SSBs•Dual incision of R-loops by the nucleases XPF, XPG, and FEN1 generates SSBs•Transcriptional DSBs are formed by these two nearby SSBs on opposing DNA strands
Cristini et al. identify a mechanism of DSB formation in non-replicating cells, which strictly depends on transcription. They are formed by two single-strand breaks on opposing DNA strands resulting from the processing of both R-loops and topoisomerase I, and genetic defects increasing these transcriptional DSBs cause neurological disorders.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Targeting G-quadruplex structures is currently viewed as a promising anticancer strategy. Searching for potent and selective G-quadruplex binders, here we describe a small series of new monohydrazone ...derivatives designed as analogues of a lead which was proved to stabilize G-quadruplex structures and increase R loop levels in human cancer cells. To investigate the G-quadruplex binding properties of the new molecules, in vitro biophysical studies were performed employing both telomeric and oncogene promoter G-quadruplex-forming sequences. The obtained results allowed the identification of a highly selective G-quadruplex ligand that, when studied in human cancer cells, proved to be able to stabilize both G-quadruplexes and R loops and showed a potent cell killing activity associated with the formation of micronuclei, a clear sign of genome instability.
Abstract
G-quadruplexes (G4s) are non-canonical nucleic acid structures involved in fundamental biological processes. As G4s are promising anticancer targets, in past decades the search for effective ...anticancer G4 binders aimed at the discovery of more cytotoxic ligands interfering with specific G4 structures at oncogenes or telomeres. Here, we have instead observed a significant activation of innate immune genes by two unrelated ligands at non-cytotoxic concentrations. The studied G4 binders (pyridostatin and PhenDC3) can induce an increase of micronuclei triggering the activation of the cytoplasmic STING (stimulator of interferon response cGAMP interactor 1) signaling pathway in human and murine cancer cells. Ligand activity can then lead to type I interferon production and innate immune gene activation. Moreover, specific gene expression patterns mediated by a G4 binder in cancer cells correlate with immunological hot features and better survival in human TCGA (The Cancer Genome Atlas) breast tumors. The findings open to the development of cytostatic G4 binders as effective immunomodulators for combination immunotherapies in unresponsive tumors.
Small-cell lung cancer (SCLC) is an aggressive cancer characterized by immunosuppressive features leading to poor responses to current immunotherapies. Activation of transposable elements (TE) can ...trigger an innate immune response, which can synergize with immunotherapeutic protocols in patients. However, TE activity in relation to immune gene response is not fully known in human SCLC. Here, we compared TE expression in 104 human SCLC and 24 normal tissues and established their involvement in innate immune responses. We observed that different intergenic TEs, mainly endogenous retroviral (ERV) families, are deregulated in SCLC. Similarly to other cancers, we detected a subset of LTRs that correlate with innate immune gene signatures and cytosolic RNA sensors, such as RIG-I. These LTRs are downregulated in SCLC tumors vs. normal tissues, and are mainly located at transcriptional repressed regions, marked with H3K4me2 in different cell lines. Analyses of different genomic datasets show that chromatin repression is likely due to de-methylase LSD1 activity. Moreover, high expression levels of ERV LTRs predict a better survival upon chemotherapy of SCLC patients. The findings reveal a specific pattern of TE-mediated activation of innate immune genes in SCLC, which can be exploited to establish more effective immunotherapeutic combinations.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. ...In the context of genomic alterations,
amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two
-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating
-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying
amplification and relatively high levels of RB1 expression.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK