FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells

Abstract

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy.

The Role of γH2AX in Replication Stress-induced Carcinogenesis: Possible Links and Recent Developments

“…In addition, reactive oxygen species (ROS) were recently shown to induce replicative stress by enhancing replicationtranscription conflicts and R-loop formation (30). Moreover, the group of Valeria Naim identified Senataxin (SETX), an RNA:DNA helicase involved in the resolution of R-loops, as a factor protecting cells from transcription-associated replication stress (31). Collectively, the aforementioned studies confirmed the existing causes of replicative stress and enabled us to comprehend their underlying mechanisms.…”

Replication stress as a driver of cellular senescence and aging

“…Genomic Instability . Replicative stress incurs DNA damage in the form of chromosomal aberrations and DSBs that attenuate replicative lifespan of cells and induce aging-associated tissue dysfunction 9 , 121 , 123 . Telomere Attrition .…”

“…DSB formation is also incurred by replication fork blockage imposed by transcription-replication conflicts, and this DNA damage induction is enhanced in the presence of co-occurring three-stranded DNA-RNA hybrids with the associated non-template ssDNA (R-loops) that exacerbate replication fork stalling 120 . R-loop persistence in mitosis resulting from depletion of the R-loop resolving helicase senataxin (SETX) has also been shown to induce chromosomal fragility and DNA under-replication 121 . Deficiency in the SETX-interacting factor senataxin-associated exonuclease 1 (San1) induces early onset murine cardiomyopathy associated with elevated cardiomyocyte R-loop and DSB levels, evidencing the role of replication stress-induced DNA damage accrual in premature induction of age-related pathologies 122 .…”

RNA biogenesis and RNA metabolism factors as R-loop suppressors: a hidden role in genome integrity

“…R-loop accumulation upon Sen1/SETX deficiency was subsequently confirmed genome-wide in yeast and human cells ( Chan et al 2014 ; Costantino and Koshland 2018 ; San Martin-Alonso et al 2021 ). As in yeast, the loss of SETX in human cells causes replication stress, making the Fanconi anemia pathway essential for viability ( Said et al 2022 ).…”