DNA intercalating drugs: Mechanisms of action in cancer treatment.

In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.

The anthracycline doxorubicin (Doxo) and its analogs daunorubicin (Daun), epirubicin (Epi), and idarubicin (Ida) have been cornerstones of anticancer therapy for nearly five decades. However, their clinical application is limited by severe side effects, especially dose-dependent irreversible cardiotoxicity. Other detrimental side effects of anthracyclines include therapy-related malignancies and infertility. It is unclear whether these side effects are coupled to the chemotherapeutic efficacy. Doxo, Daun, Epi, and Ida execute two cellular activities: DNA damage, causing double-strand breaks (DSBs) following poisoning of topoisomerase II (Topo II), and chromatin damage, mediated through histone eviction at selected sites in the genome. Here we report that anthracycline-induced cardiotoxicity requires the combination of both cellular activities. Topo II poisons with either one of the activities fail to induce cardiotoxicity in mice and human cardiac microtissues, as observed for aclarubicin (Acla) and etoposide (Etop). Further, we show that Doxo can be detoxified by chemically separating these two activities. Anthracycline variants that induce chromatin damage without causing DSBs maintain similar anticancer potency in cell lines, mice, and human acute myeloid leukemia patients, implying that chromatin damage constitutes a major cytotoxic mechanism of anthracyclines. With these anthracyclines abstained from cardiotoxicity and therapy-related tumors, we thus uncoupled the side effects from anticancer efficacy. These results suggest that anthracycline variants acting primarily via chromatin damage may allow prolonged treatment of cancer patients and will improve the quality of life of cancer survivors.

Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.

Regulation of RNA polymerase subunit synthesis in Escherichia coli: Utilization of DNA-intercalating drugs as a probe

DNA-targeting drugs may damage DNA or chromatin. Many anti-cancer drugs damage both, making it difficult to understand their mechanisms of action. Using molecules causing DNA breaks without altering nucleosome structure (bleomycin) or destabilizing nucleosomes without damaging DNA (curaxin), we investigated the consequences of DNA or chromatin damage in normal and tumor cells. As expected, DNA damage caused p53-dependent growth arrest followed by senescence. Chromatin damage caused higher p53 accumulation than DNA damage; however, growth arrest was p53-independent and did not result in senescence. Chromatin damage activated the transcription of multiple genes, including classical p53 targets, in a p53-independent manner. Although these genes were not highly expressed in basal conditions, they had chromatin organization around the transcription start sites (TSS) characteristic of most highly expressed genes and the highest level of paused RNA polymerase. We hypothesized that nucleosomes around the TSS of these genes were the most sensitive to chromatin damage. Therefore, nucleosome loss upon curaxin treatment would enable transcription without the assistance of sequence-specific transcription factors. We confirmed this hypothesis by showing greater nucleosome loss around the TSS of these genes upon curaxin treatment and activation of a p53-specific reporter in p53-null cells by chromatin-damaging agents but not DNA-damaging agents.

The purpose of this overview was to make a broad inventory of investigational drugs for medicinal cancer treatment and, specifically, to indicate the evidence of clinical efficacy. Information was retrieved from electronic database searches in Medline and CANCERLIT and relevant published reviews. As the most recent findings are first reported as conference abstracts, an important basis for identification of new drugs and clinical results was a hand search of 13,392 abstracts from five major recent cancer conferences. A total of 209 investigational approaches or drugs were identified and classified into one of eight groups according to proposed mechanism of action. For 28 drugs/approaches survival data were available from randomized controlled trials. Statistically significant benefit was observed for only 12. In earlier phases no or modest anticancer activity was reported. It is speculated that the expanding knowledge in tumour biology might not easily translate into new substantially better anticancer drugs.

A small, international, closed-door conference on Novel Agents in the Treatment of Lung Cancer, held in Cambridge, Massachusetts, October 17–18, 2003, was convened to present and discuss findings from recent and ongoing trials of investigational drugs for the treatment of lung cancer. Invited

We have studied the role of sequence context upon RNA polymerase II arrest by a cyclobutane pyrimidine dimer using an in vitro transcription system consisting of templates containing a specifically located cyclobutane pyrimidine dimer (CPD) and purified RNA polymerase II (RNAP II) and initiation factors. We selected a model sequence containing a well characterized site for RNAP II arrest in vitro, the human histone H3.3 gene arrest site. The 13-base pair core of the arrest sequence contains two runs of T in the nontranscribed strand that impose a bend in the DNA. We hypothesized that arrest of RNAP II might be affected by the presence of a CPD, based upon the observation that a CPD located at the center of a dA6.dT6 tract eliminates bending (Wang, C.-I., and Taylor, J.-S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9072-9076). We examined the normal H3.3 sequence and a mutant sequence containing a T --> G transversion, which reduces bending and efficiency of arrest. We show that a CPD in the transcribed strand at either of two locations in the arrest site is a potent block to transcription. However, a CPD in the nontranscribed strand only transiently pauses RNAP II. The CPD in concert with a mutation in the arrest site can reduce the extent of bending of the DNA and improve readthrough efficiency. These results demonstrate the potential importance of sequence context for the effect of CPDs within transcribed sequences.

Molecules that inhibit DNA dependent processes are the most commonly used agents for the treatment of cancer. The genotoxicity associated with their mechanisms of action, unfortunately, make them extremely toxic to the patient and cancer cells alike. The work presented in this thesis outlines the development of Py-Im polyamides as non-genotoxic DNA-targeted antitumor molecules that interfere with RNA polymerase II elongation. We initially characterized the pharmacokinetic profiles of two hairpin polyamides to establish their bioavailability in the serum and tissues after a single administration. We next determined the molecular mechanism that contributes to toxicity of a hairpin polyamide in human prostate cancer cells in cell culture and we demonstrated antitumor effects of the compound against LNCaP xenografts in mice. Finally, we conducted animal toxicity experiments on 4 polyamides that vary on the gamma-turn with respect to the substitution of amino and acetamide groups at the alpha and beta positions. From this study we identified a second generation compound that retains antitumor activity with significantly reduce animal toxicity. This work sets the foundation for the development of Py-Im polyamides as DNA targeted therapeutics for the treatment of advanced prostate cancer.

DNA sequences rich in runs of guanine have the potential to form G4 DNA, a four-stranded non-canonical DNA structure stabilized by formation and stacking of G quartets, planar arrays of four hydrogen-bonded guanines. It was reported recently that G4 DNA can be generated in Escherichia coli during transcription of plasmids containing G-rich sequences in the non-transcribed strand. In addition, a stable RNA/DNA hybrid is formed with the transcribed strand. These novel structures, termed G loops, are suppressed in recQ(+) strains, suggesting that their persistence may generate genomic instability and that the RecQ helicase may be involved in their dissolution. However, little is known about how such non-canonical DNA structures are processed when encountered by an elongating polymerase. To assess whether G4-forming sequences interfere with transcription, we studied their effect on transcription elongation by T7 RNA polymerase and mammalian RNA polymerase II. We used a reconstituted transcription system in vitro with purified polymerase and initiation factors and with substrates containing G-rich sequences in either the transcribed or non-transcribed strand downstream of the T7 promoter or the adenovirus major late promoter. We report that G-rich sequences located in the transcribed strand do not affect transcription by either polymerase, but when the sequences are located in the non-transcribed strand, they partially arrest both polymerases. The efficiency of arrest increases with negative supercoiling and also with multiple rounds of transcription compared with single events.

CBL0137 is a clinical-stage anti-cancer drug candidate that is active against multiple preclinical cancer models. It is a carbazole-based DNA intercalator, discovered by cell-based screening for p53 activation in cells lacking DNA damage. Binding of curaxin to DNA results in alterations in DNA length, charge, and flexibility, which together impair the ability of DNA to wrap around the histone core. Treatment of cells with CBL0137 leads to decondensation of chromatin accompanied by histone eviction. This causes the binding of histone chaperone FACT to epitopes normally hidden within nucleosomes and the activation of p53 by FACT-bound CK2. p53 activation occurs in the absence of detectable DNA damage. However, CBL0137 is also toxic to p53-deficient cells, suggesting that its toxicity is mediated by events upstream of p53 activation. Trying to understand the mechanisms of CBL0137 toxicity, we found that FACT is essential for the viability of tumor but not normal cells. FACT was discovered as a transcription elongation factor performing disassembly and reassembly of nucleosomes in cell-free conditions. However, no reduction of transcription is observed with FACT depletion in cells. Moreover, measurement of FACT chromatin binding and gene expression upon FACT loss suggest that FACT negatively regulates gene expression. mammalian FACT does not disassemble nucleosomes during transcription but binds “open” nucleosomes, with partially unwrapped DNA, appearing during transcription, and tethers nucleosome components together to prevent histone loss from chromatin. In cells treated with CBL0137 nucleosomes are mostly lost in regions enriched with tandem AC/TG dinucleotide repeats, known as microsatellites. These regions accumulate FACT in treated cells. To understand the effects of FACT loss from other chromatin regions, we measured nucleosome distribution in cells with genetically-depleted FACT. Regions normally enriched with FACT lose nucleosomes in a transcription-dependent manner. Thus, curaxin induces chromatin disassembly via a direct effect on nucleosomal DNA and inhibition of FACT-mediated nucleosome restoration at transcribed regions. To explain the reasons for the higher toxicity of curaxin in tumor versus normal cells, we hypothesized that chromatin is, in general, less stable in tumor than in normal cells. Thus, less drug is needed to cause unrepairable nucleosome disassembly in tumor cells. We compared chromatin organization in syngeneic normal, immortalized, and transformed cells and found that during tumor progression, there is loss of histones, reduction of condensed chromatin and increase of DNA accessibility to the transcriptional machinery. The same signs are observed in human tumors. In summary, curaxin kills cells via the destabilization of nucleosomes and disassembly of chromatin, a phenomenon that we have named “chromatin damage.” Tumor cells are more sensitive to chromatin damage due to their preexisting, less stable chromatin. Citation Format: Alfiya Safina, Poorva Sandlesh, Jianmin Wang, Katerina V. Gurova. Induction of nucleosome disassembly with small molecules as a novel anti-cancer approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1747.

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain a robust spindle assembly checkpoint (SAC) response. Here, we used live-cell imaging of human cells, RNA-seq and qPCR to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC signaling and cell fate. However, we were unable to detect significant changes in mitotic transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on Aurora B centromeric localization, SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, mitotic progression, arrest, exit or death is determined by centromere structural integrity, rather than de novo transcription.

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain a robust spindle assembly checkpoint (SAC) response. Here, we used live-cell imaging of human cells, RNA-seq and qPCR to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC signaling and cell fate. However, we were unable to detect significant changes in mitotic transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on Aurora B centromeric localization, SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, mitotic progression, arrest, exit or death is determined by centromere structural integrity, rather than de novo transcription.

Molecules that bind DNA by intercalating its bases remain among the most potent cancer therapies and antimicrobials due to their interference with DNA-processing proteins. To accelerate the discovery of novel intercalating drugs, we designed a fluorescence resonance energy transfer (FRET)-based probe that reports on DNA intercalation, allowing rapid and sensitive screening of chemical libraries in a high-throughput format. We demonstrate that the method correctly identifies known DNA intercalators in approved drug libraries and discover previously unreported intercalating compounds. When introduced in cells, the oligonucleotide-based probe rapidly distributes in the nucleus, allowing direct imaging of the dynamics of drug entry and its interaction with DNA in its native environment. This enabled us to directly correlate the potency of intercalators in killing cultured cancer cells with the ability of the drug to penetrate the cell membrane. The combined capability of the single probe to identify intercalators in vitro and follow their function in vivo can play a valuable role in accelerating the discovery of novel DNA-intercalating drugs or repurposing approved ones.

<h3>Abstract</h3> Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain the spindle assembly checkpoint (SAC) for several hours in response to unattached kinetochores. Here we used live-cell imaging of human cells in culture, combined with RNA-seq and qPCR, to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC robustness and cell fate. However, we were unable to detect significant changes in transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, the capacity of human cells to progress, sustain, exit or die in mitosis relies on centromere integrity, rather than de novo transcription.