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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.

We investigated the mechanism of how the papillomavirus E2 transcription factor can activate promoters through activator protein (AP)1 binding sites. Using an unbiased approach with an inducible cell line expressing the viral transcription factor E2 and transcriptome analysis, we found that E2 induces the expression of the two AP1 components c-Fos and FosB in a Brd4-dependent manner. In vitro RNA interference confirmed that c-Fos is one of the AP1 members driving the expression of viral oncogenes E6/E7. Mutation analysis and in vivo RNA interference identified an essential role for c-Fos/AP1 and also for the bromodomain protein Brd4 for papillomavirus-induced tumorigenesis. Lastly, chromatin immunoprecipitation analysis demonstrated that E2 binds together with Brd4 to a canonical E2 binding site (E2BS) in the promoter of c-Fos, thus activating c-Fos expression. Thus, we identified a novel way how E2 activates the viral oncogene promoter and show that E2 may act as a viral oncogene by direct activation of c-Fos involved in skin tumorigenesis., Author Summary Human Papillomaviruses (HPV) are the etiological agents of cervical cancer and of skin cancer in individuals with the inherited disease epidermodysplasia verruciformis (EV). While the role of the viral oncogenes E6/E7 as drivers of tumorigenesis in cervical cancer has been firmly established, the contribution of the early viral genes in skin cancer is less clear. For EV-associated HPV8 and for the skin cancer model system using cottontail rabbit PV, an important role of the viral E2 protein in tumorigenesis was suggested earlier and regulation of cellular genes by E2 through different mechanisms was demonstrated. We show now that the viral E2 and cellular Brd4 act together to induce the cellular gene c-Fos, which as a member of the AP-1 complex, is involved in the regulation of cellular genes and the viral promoter driving the expression of viral oncogenes. As c-Fos has also been shown to be essential for skin cancer, E2 contributes to tumorigenesis via expression of E6/E7 as well as by increasing c-Fos.

DNA topoisomerases have an important function in preventing replication/transcription (R/T) conflicts caused by RNA polymerases elongating in the opposite (or same) direction of advancing replication fork. Published data demonstrated that such persistent R/T conflicts can be rescued by overexpression of RNase H1, which specifically degrades the RNA of DNA:RNA hybrids present in R-loop structures. R loops are a common non-B DNA structure of cellular genomes that mainly occur during transcription. R loops can lead to genome instability; however, they also play a role in important cellular functions. Here, we have determined the dynamics of cellular R loops generated by chemical poisoning of DNA topoisomerases (TOP1 and TOP2) and the role of the proteasome in DNA damage induction by performing detailed kinetics analyses of cellular R loops by immunofluorescence microscopy. Treatment of HeLa and U2OS cells with clinically used topoisomerase inhibitors (camptothecin, LMP-776, MJ-III-65, doxorubicin, sabarubicin) triggers an immediate increase of nucleoplasmic R loops from 2 to 10-20 minutes of treatment, followed by a dramatic reduction of R-loop levels at 1 hour. Concomitantly with the reduction of R-loop structures, DNA damage was detected as phosphorylation of histone H2AX, a marker of DNA double-stranded breaks (DSB). Pretreating cells with MG132 (a proteasome inhibitor) stabilized the presence of R loops at longer treatment times and concomitantly reduced DSB formation, suggesting that the degradation of poisoned topoisomerases is necessary to trigger the pathway resulting in DSB formation. Overexpression of RNaseH1 in U2OS cells abolishes the transient increase of R loops and the induction of DNA damage. Topoisomerase poisoning is able to effectively kill the studied human cancer cells. However, overexpression of RNaseH1 or MG132 markedly reduced cell killing induced by R/T conflicts generated by topoisomerase inhibitors. The results are overall consistent with a necessary function of nuclear proteasome in generating a free DNA break from abortive topoisomerase cleavage complexes that will eventually lead to DSBs. Citation Format: Jessica Marinello, Maria Delcuratolo, Yves Pommier, Monica Binaschi, Andrea Pellacani, Giovanni Capranico. DNA topoisomerase-mediated transcription-replication conflicts cause DNA damage by a transient increase of R loops and proteasome activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B188.

DNA topoisomerases (Top) are important players in maintaining the genome stability of cells by removing negative and positive DNA supercoils during transcription, replication and other DNA transactions. Recent evidence demonstrated that Top 1 silencing and poisoning by camptothecin affect the formation of R-loops, which are RNA/DNA hybrid structures involved in genome instability and are favored by negative supercoils of the DNA. The findings overall demonstrate that Top 1 reduces R loop levels during transcription whereas it favors R loop formation at early origins of DNA replication. Here, we address the question of whether DNA topoisomerase IIβ can also contribute to steady-state levels of R loop structures in the genome of U2OS cancer cells. We have then investigated the effects of Top2β silencing and Top2 poisons, doxorubicin and etoposide, on R loops and DNA damage in human U2OS cancer cells. Similarly to published data with camptothecin (Marinello et al., Nucleic Acids Research, 2013), Top2 poisons increase cellular R loops by IF after short treatment times and reduce them after 1 hour of treatment. The bi-phasic effect of poisons is mainly dependent on transcription. Moreover, we have determined the changes of R-loop levels by IF after Top2β silencing and the findings demonstrate that the enzyme can strongly modulate the formation of R loops as a full Top2β depletion increases nuclear R-loop levels. In addition, Top2β depletion leads to a slight increase of phosphorylation of H2AX histone along with cell cycle delay and eventually cell death. Thus, Top2β depletion can trigger genomic DNA breakage through alterations of R-loops. In order to establish which are the genomic regions of altered R loop levels, we have mapped Top2β-dependent R loop alterations by the DRIP method showing that specific genomic sites are affected by Top2β. Bioinformatic analyses of R loop maps in U2OS cells will be presented and discussed at the meeting. Altogether the findings demonstrate a critical role of Top2β in governing R loop structures in human cancer cells indicating that DNA torsional tension is a main driving factor of R loop formation and hence genome instability in cancer cells. Citation Format: Maria Delcuratolo, Jessica Marinello, Giovanni Capranico. Topoisomerase IIβ silencing increases R loops at specific genomic loci associated with an increase of γH2AX and cell cycle progression delay in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 510. doi:10.1158/1538-7445.AM2017-510

In this study, simultaneous positron emission tomography (PET)/magnetic resonance (MR) imaging was employed to evaluate the feasibility of the PET tracers 2-deoxy-2- 18 F-fluoro-D-glucose ( 18 F-FDG), 11 C-choline, and 18 F-fluorothymidine ( 18 F-FLT) to detect papillomavirus-induced tumors in an established rabbit model system. The combined PET/MR allowed the analysis of tracer uptake of the tumors using the morphologic information acquired by MR. New Zealand White rabbits were infected with cottontail rabbit papillomavirus genomes and were imaged for up to 10 months with a simultaneous PET/MR system during the course of infection. The uptake characteristics of the PET tracers 11 C-choline and 18 F-FLT of tumors and reference tissues were examined relative to the clinical standard, 18 F-FDG. Tracer biodistribution of various organs was measured by gamma-counting after the last PET scan and compared to the in vivo PET/MR 18 F-FDG uptake. Increased tracer uptake was found 2 months postinfection in primary tumors with 18 F-FDG and 11 C-choline, whereas 18 F-FLT failed to detect the tumors at all measured time points. Our data show that the PET tracer 18 F-FDG is superior for imaging papillomavirus-induced tumors in rabbits compared to 11 C-choline and 18 F-FLT. However, 11 C-choline imaging, which has previously been applied to detect various tumor entities in patients, appears to be an alternative to 18 F-FDG.