G-quadruplex binders as cytostatic modulators of innate immune genes in cancer cells.

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.

MYCN Amplification, along with Wild-Type RB1 Expression, Enhances CDK4/6 Inhibitors' Efficacy in Neuroblastoma Cells.

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, MYCN 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 MYCN-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 MYCN-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 MYCN amplification and relatively high levels of RB1 expression.

Ligands stimulating antitumour immunity as the next G-quadruplex challenge.

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.

Topoisomerase I poison-triggered immune gene activation is markedly reduced in human small-cell lung cancers by impairment of the cGAS/STING pathway.

BACKGROUND: Current immunotherapy strategies have contrasting clinical results in human lung cancer patients as small-cell lung cancers (SCLC) often show features of immunological cold tumours. Topoisomerase 1 (TOP1) poisons are effective antitumor drugs with good efficacy against lung cancers. METHODS: We used molecular, genetic and bioinformatic approaches to determine the mechanism of micronuclei formation induced by two TOP1 poisons in different human cancer cells, including SCLC cell lines. RESULTS: TOP1 poisons stimulate similar levels of micronuclei in all tested cell lines but downstream effects can vary markedly. TOP1 poisons increase micronuclei levels with a mechanism involving R-loops as overexpression of RNaseH1 markedly reduces or abolishes both H2AX phosphorylation and micronuclei formation. TOP1 poison-induced micronuclei activate the cGAS/STING pathway leading to increased expression of immune genes in HeLa cells, but not in human SCLC cell lines, mainly due to lack of STING and/or cGAS expression. Moreover, the expression of STING and antigen-presenting machinery genes is generally downregulated in patient tumours of human lung cancer datasets. CONCLUSIONS: Altogether, our data reveal an immune signalling mechanism activated by TOP1 poisons, which is often impaired in human SCLC tumours.

G-quadruplex-R-loop interactions and the mechanism of anticancer G-quadruplex binders.

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.

DNA damage and genome instability by G-quadruplex ligands are mediated by R loops in human cancer cells.

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.

DROPA: DRIP-seq optimized peak annotator.

BACKGROUND: R-loops are three-stranded nucleic acid structures that usually form during transcription and that may lead to gene regulation or genome instability. DRIP (DNA:RNA Immunoprecipitation)-seq techniques are widely used to map R-loops genome-wide providing insights into R-loop biology. However, annotation of DRIP-seq peaks to genes can be a tricky step, due to the lack of strand information when using the common basic DRIP technique. RESULTS: Here, we introduce DRIP-seq Optimized Peak Annotator (DROPA), a new tool for gene annotation of R-loop peaks based on gene expression information. DROPA allows a full customization of annotation options, ranging from the choice of reference datasets to gene feature definitions. DROPA allows to assign R-loop peaks to the DNA template strand in gene body with a false positive rate of less than 7%. A comparison of DROPA performance with three widely used annotation tools show that it identifies less false positive annotations than the others. CONCLUSIONS: DROPA is a fully customizable peak-annotation tool optimized for co-transcriptional DRIP-seq peaks, which allows a finest gene annotation based on gene expression information. Its output can easily be integrated into pipelines to perform downstream analyses, while useful and informative summary plots and statistical enrichment tests can be produced.

DNA-PK triggers histone ubiquitination and signaling in response to DNA double-strand breaks produced during the repair of transcription-blocking topoisomerase I lesions.

Although defective repair of DNA double-strand breaks (DSBs) leads to neurodegenerative diseases, the processes underlying their production and signaling in non-replicating cells are largely unknown. Stabilized topoisomerase I cleavage complexes (Top1cc) by natural compounds or common DNA alterations are transcription-blocking lesions whose repair depends primarily on Top1 proteolysis and excision by tyrosyl-DNA phosphodiesterase-1 (TDP1). We previously reported that stabilized Top1cc produce transcription-dependent DSBs that activate ATM in neurons. Here, we use camptothecin (CPT)-treated serum-starved quiescent cells to induce transcription-blocking Top1cc and show that those DSBs are generated during Top1cc repair from Top1 peptide-linked DNA single-strand breaks generated after Top1 proteolysis and before excision by TDP1. Following DSB induction, ATM activates DNA-PK whose inhibition suppresses H2AX and H2A ubiquitination and the later assembly of activated ATM into nuclear foci. Inhibition of DNA-PK also reduces Top1 ubiquitination and proteolysis as well as resumption of RNA synthesis suggesting that DSB signaling further enhances Top1cc repair. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these new findings reveal that DSB production and signaling by transcription-blocking Top1 lesions impact on non-replicating cell fate and provide insights on the molecular pathogenesis of neurodegenerative diseases such as SCAN1 and AT syndromes, which are caused by TDP1 and ATM deficiency, respectively.

Anthracyclines as Topoisomerase II Poisons: From Early Studies to New Perspectives.

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.

Discovery of the first dual G-triplex/G-quadruplex stabilizing compound: a new opportunity in the targeting of G-rich DNA structures?

BACKGROUND: Guanine-rich DNA motifs can form non-canonical structures known as G-quadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex. METHODS: Virtual screening was employed to identify a small set of putative G-triplex ligands. The G-triplex stabilizing properties of these compounds were analyzed by CD melting assay. DSC, non-denaturing gel electrophoresis, NMR and molecular modeling studies were performed to investigate the interaction between the selected compound 1 and G-rich DNA structures. Cytotoxic activity of 1 was evaluated by MTT cell proliferation assay. RESULTS: The experiments led to the identification of a promising hit that was shown to bind preferentially to G-triplex and parallel-stranded G-quadruplexes over duplex and antiparallel G-quadruplexes. Molecular modeling results suggested a partial end-stacking of 1 to the external G-triad/G-tetrads as a binding mode. Biological assays showed that 1 is endowed with cytotoxic effect on human osteosarcoma cells. CONCLUSIONS: A tandem application of virtual screening along with the experimental investigation was employed to discover a G-triplex-targeting ligand. Experiments revealed that the selected compound actually acts as a dual G-triplex/G-quadruplex stabilizer, thus stimulating further studies aimed at its optimization. GENERAL SIGNIFICANCE: The discovery of molecules able to bind and stabilize G-triplex structures is highly appealing, but their transient state makes challenging their recognition. These findings suggest that the identification of ligands with dual G-triplex/G-quadruplex stabilizing properties may represent a new route for the design of anticancer agents targeting the G-rich DNA structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

Synthesis and Evaluation of New Naphthalene and Naphthoquinone Derivatives as Anticancer Agents.

DNA topoisomerase I inhibitors, both synthetic and of natural origin, are receiving increasing consideration primarily as drugs against refractory tumors. Alkannin and shikonin, two enantiomeric dyes from Alkanna tinctoria and Lithospermum erythrorhizon, have been known over many centuries as dyestuff, wound healing, anti-inflammatory, antibacterial and antitumor substances. Although multiple mechanisms appear to be implicated, their potency is associated with the inhibition of topoisomerase I and with the redox properties of the naphthazarin scaffold. Here, the synthesis of new naphthalene and naphthoquinone derivatives inspired by alkannin and shikonin is described and their structural and biological properties were examined. Different oxidation states of the naphthalene nucleus were examined to observe the effect of this parameter on cytotoxicity. Antiproliferative activities against a panel of human cancer cell lines were evaluated and the implication of topoisomerase I was assessed.

Antisense transcripts enhanced by camptothecin at divergent CpG-island promoters associated with bursts of topoisomerase I-DNA cleavage complex and R-loop formation.

DNA Topoisomerase I (Top1) is required to relax DNA supercoils generated by RNA polymerases (RNAPs). Top1 is inhibited with high specificity by camptothecin (CPT), an effective anticancer agent, and by oxidative base damage and ribonucleotides in DNA strands, resulting into Top1-DNA cleavage complexes (Top1ccs). To understand how Top1ccs affect genome stability, we have investigated the global transcriptional response to CPT-induced Top1ccs. Top1ccs trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication-independent and Top1-dependent manner. As CPT increases antisense transcript levels in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a transcription inhibitor, Top1ccs likely impair antisense RNA degradation. Time-course data showed a burst of Top1ccs increased by CPT at promoter sites and along transcribed regions, causing a transient block of RNAPII at the promoter. Moreover, cell immunofluorescence analyses showed that Top1ccs induce a transient increase of R-loops specifically at highly transcribed regions such as nucleoli in a Top1-dependent manner. Thus, a specific and highly dynamic transcriptional response to Top1ccs occurs at divergent active CpG-island promoters, which may include a transient stabilization of R-loops. The results clarify molecular features of a response pathway leading to transcription-dependent genome instability and altered transcription regulation.

Novel ametantrone-amsacrine related hybrids as topoisomerase IIß poisons and cytotoxic agents.

The precise definition of the structural requirements for effective topoisomerase II poisoning by drug molecules is still an elusive issue. In the attempt to better define a pharmacophoric pattern, we prepared several conjugates combining the chemical features of two well-known topoisomerase II poisons, amsacrine and ametantrone. Indeed, an appropriate fusion geometry, which entails the anthracenedione moiety of ametantrone appropriately connected to the methanesulfonamidoaniline side chain of amsacrine, elicits DNA-intercalating properties, the capacity to inhibit the human topoisomerase IIß isoform, and cytotoxic activity resembling that of the parent compounds. In addition, the properties of the lateral groups linked to the anthracenedione group play an important role in modulating DNA binding and cell cytotoxicity. Among the compounds tested, 10, 11, and 19 appear to be promising for further development.

Immunofluorescence microscopy of G-quadruplexes and R-loops.

A large variety of non-B secondary structures can be formed between DNA and RNA. In this chapter, we focus on G-quadruplexes (G4) and R-loops, which can have a close structural interplay. In recent years, increasing evidence pointed to the fact that they can strongly influence each other in vivo, both having physiological and pathological roles in normal and cancer cells. Here, we detail specific and accurate methods for purification of BG4 and S9.6 antibodies, and their subsequent use in immunofluorescence microscopy, enabling single-cell analysis of extent and localization of G4s and R-loops.

Human DNA topoisomerase I poisoning causes R loop-mediated genome instability attenuated by transcription factor IIS.

DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.

TDP1 mutation causing SCAN1 neurodegenerative syndrome hampers the repair of transcriptional DNA double-strand breaks.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

In hepatocellular carcinoma miR-519d is up-regulated by p53 and DNA hypomethylation and targets CDKN1A/p21, PTEN, AKT3 and TIMP2.

MiR-519d belongs to the chromosome 19 miRNA cluster (C19MC), the largest human miRNA cluster. One of its members, miR-519d, is over-expressed in hepatocellular carcinoma (HCC) and we characterized its contribution to hepatocarcinogenesis. In HCC cells, the over-expression of miR-519d promotes cell proliferation, invasion and impairs apoptosis following anticancer treatments. These functions are, at least in part, exerted through the direct targeting of CDKN1A/p21, PTEN, AKT3 and TIMP2. The mechanisms underlying miR-519d aberrant expression in HCC were assayed by genomic DNA amplification, methylation analysis and ChIP assay. The aberrant hypomethylation of C19MC and TP53 were respectively identified as an epigenetic change allowing the aberrant expression of miR-519d and one of the factors able to activate its transcription. In conclusion, we assessed the oncogenic role of miR-519d in HCC by characterizing its biological functions, including the modulation of response to anticancer treatments and by identifying CDKN1A/p21, PTEN, AKT3 and TIMP2 among its targets.

Expression of down-regulated ERV LTR elements associates with immune activation in human small-cell lung cancers.

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.