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Triptolide is a bioactive ingredient in traditional Chinese medicine that exhibits diverse biologic properties, including anticancer properties. Among its many putative targets, this compound has been reported to bind to XPB, the largest subunit of general transcription factor TFIIH, and to cause degradation of the largest subunit Rpb1 of RNA polymerase II (RNAPII). In this study, we clarify multiple important questions concerning the significance and basis for triptolide action at this core target. Triptolide decreased Rpb1 levels in cancer cells in a manner that was correlated tightly with its cytotoxic activity. Compound exposure blocked RNAPII at promoters and decreased chromatin-bound RNAPII, both upstream and within all genes that were examined, also leading to Ser-5 hyperphosphorylation and increased ubiqutination within the Rbp1 carboxy-terminal domain. Notably, cotreatment with inhibitors of the proteasome or the cyclin-dependent kinase CDK7 inhibitors abolished the ability of triptolide to ablate Rpb1. Together, our results show that triptolide triggers a CDK7-mediated degradation of RNAPII that may offer an explanation to many of its therapeutic properties, including its robust and promising anticancer properties. Cancer Res; 72(20); 5363–73. ©2012 AACR.

G-quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate basic processes such as transcription and replication. Unscheduled formation of R-loops is regarded as highly deleterious to cells, as R loops can induce replicative stress and DNA damage leading to genome instability. G4s are formed by four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by monovalent cations. R loops are triple-stranded structures that contain an RNA-DNA hybrid duplex and a displaced single-stranded DNA. We have recently shown that Topoisomerase I (Top1) can affect genome-wide levels of R loops in human cancer cells consistently with the knowledge that DNA superhelical tension is a main driving force allowing non-B DNA structure formation. As G4 ligands were suggested to synergize with Top1 inhibitors, we asked the question of whether G4 ligands can affect R loops. We have thus investigated by immunofluorescence microscopy the effects of two different G4 binders, Pyridostatin (PDS) and FG (compound 1 in Amato et al J Med Chem 2016), on R-loops in human cancer cells. These G4 binders can increase both G4s and R loops while an inactive derivative of FG cannot induce them. Interestingly, the induction of G4s and R loops well correlate to each other in time and intensity with PDS being more effective than FG. After 24 hours, G4 binders induce a cell cycle arrest at the G2/M phase associated to double-stranded DNA cleavage as detected by γH2AX and 53BP1 foci formation and to activation of checkpoint response as shown by ATM phosphorylation. Interestingly, overexpression of RNaseH1 reduces both R loops and γH2AX foci induced by G4 binders showing that the ligands induce DNA cleavage via an R loop-mediated mechanism. Then, we silenced the BRCA2 gene by RNAi in U2OS cells and the results show that BRCA2 depletion increased γH2AX foci induced by PDS while overexpression of RNaseH1 rescue DNA cleavage induction. We also determined genomic R loop maps by DRIP-seq, and bioinformatic analyses of the specific location of G4-stabilized R loops provided information consistent with a model in which a G4 opposite to a DNA:RNA hybrid can stabilize R loops. Our study establishes for the first time that G4 binders stabilize either G4s and R loops in human cancer cells, and that they induce genome instability with a mechanism dependent on R loop formation. Partially supported by AIRC, Milan. Citation Format: Alessio De Magis, Stefano Giustino Manzo, Marco Russo, Olivier Sordet, Rita Morigi, Giovanni Capranico. R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4838.

DNA Topoisomerase I (Top1) is specifically inhibited by camptothecin (CPT), a natural product with effective anticancer activity. CPT can stabilize a DNA-Top1 DNA cleavage complex that can lead to irreversible DNA breakage at replication forks, and transcription-dependent genome instability. The bulk of cellular Top1 activity is required at transcribing regions to modulate DNA supercoils generated by elongating RNA polymerases. However, the molecular effects of CPT inhibition of Top1 at transcriptional levels are not fully established. By next generation sequencing analyses of bisulfite-treated RNAs extracted from human HCT116 cancer cells, we here show that CPT promoted high levels of antisense tags specifically at active promoters containing CpG islands (CGI). We found several not annotated transcripts close to Refseq genes, the majority of which were promoter-associated antisense transcripts increased by CPT. We identified 256 and 84 of such transcripts in HCT116 cells and Top1-silenced HCT116-siRNATop1 cells, respectively. We validated CPT-stimulated antisense transcripts by PCR in both cell lines, and found that several of them could form R-loops at corresponding genomic regions by affinity purification with a recombinant inactive mutant RNaseH1. Drug effects were independent from replication, and required both Top1 and ongoing transcription. In addition, CPT induced an immediate burst and a subsequent rapid reduction of Top1-DNA cleavage complexes at active, but not inactive, promoters indicating a partial, time-dependent removal of Top1 from chromatin. Our findings demonstrate that Top1 activity prevents accumulation of antisense R-loops and RNAs at human active CGI promoters by relaxing negative DNA supercoils. The transcriptional CPT effects can contribute to drug therapeutic activity in cancer and other diseases. We will discuss our findings in relation to transcription regulation and genome instability caused by interference with Top1 activity. Citation Format: Jessica Marinello, Giovanni Chillemi, Susana Bueno, Stefano G. Manzo, Giovanni Capranico. Antisense transcripts and R-loops caused by DNA topoisomerase I inhibition by camptothecin at human active CpG island promoters. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 637. doi:10.1158/1538-7445.AM2013-637

Transgenic mice have been generated bearing three fusion genes consisting of: (a) a 900-base pair rat thyroglobulin promoter followed by a gene coding for a chloramphenicol acetyl transferase activity; (b) the same promoter followed by the complementary DNA of the human activated Ki-ras oncogene; (c) a 2000-base pair rat thyroglobulin promoter followed by the complementary DNA of the human activated Ki-ras. We have shown that the 900-base pair rat thyroglobulin promoter is able to direct the expression of the reporter gene specifically in the thyroid gland of transgenic mice. The mice bearing the two Ki-ras constructs, which express the transgene in thyroid glands, show thyroid abnormalities, although at very low incidence. These lesions appear after a long latency and with a benign aspect, thus suggest that, in agreement with literature data on naturally occurring human thyroid tumors, the action of an activated ras gene is not sufficient to attain a complete malignant conversion of thyroid glands in vivo. However, ras expression in thyroid follicular cells represents a favorable ground for tumor development, as shown by the fact that goitrogen stimulation experiments increase the occurrence of tumors.