Abstract 1702: Loss of KEAP1 promotes R-loop formation independent of NRF2

Abstract This study investigates the mechanism of R-loop formation as a consequence of KEAP1 depletion. R-loops, described as three-stranded nucleic acid structures consisting of a DNA:RNA hybrid and a non-template single-stranded DNA, are assemblies often attributed with pathological consequences like genomic instability and replication stress. However, they have significant physiological roles, inter alia, regulation of transcription initiation, splicing and termination. Loss in R-loop resolution factors (like RNaseH nucleases, senataxin) can result in “unscheduled” or “pathological” R-loops, which can compromise genome stability. R-loops are commonly associated with an active transcription bubble, with nascent RNA trailing the RNA polymerase re-annealing to the template DNA strand. Increased transcriptional stress can lead to accumulation of R-loops. In a 2016 eLife study by Stork et al., estrogen induction resulted in concomitantly increased transcription and R-loop formation in the same subset of genes. Similar to estrogen induction, activation of the NRF2/KEAP1 pathway upon oxidative stress causes NRF2 to be released from KEAP1 and enter the nucleus to turn on transcription of its target genes. Kelch-like ECH associated protein 1, encoded by the KEAP1 gene, is a canonical binding partner of NRF2. Under basal conditions, KEAP1 binds NRF2 and targets it for degradation. Mutations in KEAP1 and epigenetic modifications have been reported in lung and breast cancer phenotypes. In lieu of chemical manipulation, NRF2 can be activated by knocking down KEAP1 via RNAi. We therefore asked whether transcription activation downstream of the NRF2/KEAP1 pathway can lead to accumulation of R-loops.We have observed the consequences of depleting KEAP1 (for NRF2 activation) with RNAi on global transcription using ethynyl uridine (EU) incorporation with click-iT chemistry and flow cytometry, on R-loop levels using S9.6 antibody immunocytochemistry and on transcriptional profile changes with RNA-seq. We observe that loss of KEAP1 leads to an increased accumulation of R-loops. Surprisingly, this is not accompanied by an expected increase in transcription as measured quantitatively with EU flow cytometry. This phenomenon was found to consistently occur in various cell lines across cancer types (breast, lung). Interestingly, depleting NRF2 cannot mitigate the effects of KEAP1 loss, suggesting that KEAP1 loss-associated R-loop formation is independent of NRF2 activation. Analysis of RNA-seq data shows that TOP2 is downregulated in KEAP1 deficient cells. As TOP2 has been previously known to regulate R-loop formation, we predict that KEAP1 loss can sensitize cells to topoisomerase inhibitors.In conclusion, interaction of KEAP1 with other players in its interactome outside of NRF2 may regulate R-loop formation and therefore R-loop-induced genome instability. Citation Format: Pramiti Mukhopadhyay, Nicklas Bassani, Alexander J. Bishop. Loss of KEAP1 promotes R-loop formation independent of NRF2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1702.