XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition

Abstract

Persistent R-loops (RNA-DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5′-3′-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition. Abbreviations BER Base excision repair Co-IP Co-immunoprecipitation DAVID Database for Annotation, Visualization and Integrated Discovery DSBs DNA double strand breaks FDR False discovery rate HR Homologous recombination IPA Ingenuity Pathway Analysis MS Mass spectrometry NHEJ Non-homologous end joining PAR Poly(ADP-ribose) PMSF Phenylmethylsulfonyl fluoride STRING Search Tool for the Retrieval of INteracting Genes/proteins TAP Tandem affinity purification Preserving genomic integrity is critical for ensuring survival and faithful transmission of genetic information. Compromised genomic integrity results in several debilitating diseases such as neurodegenerative disorders and cancer 1,2. The nucleic acid structures containing RNA-DNA hybrids with a displaced single-stranded DNA (R-loops) are generated in cells during transcription and their persistence leads to DNA damage and genomic

Molecular Basis of XRN2-Deficient Cancer Cell Sensitivity to Poly(ADP-ribose) Polymerase Inhibition

“…Additionally, XRN2 is involved in the general degradation of RNA, gene silencing, and rRNA maturation [17][18][19]. Overall, XRN2 ′ s role in RNA metabolism has been studied extensively; however, its function in other cellular processes remains poorly understood [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. Importantly, genetic alternations of XRN2, such as mutations, mRNA expression, and copy number variations, are common in a wide range of cancers; XRN2 polymorphisms contribute to an increased risk of spontaneous lung cancer [20][21][22][23].…”

“…Overall, XRN2 ′ s role in RNA metabolism has been studied extensively; however, its function in other cellular processes remains poorly understood [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. Importantly, genetic alternations of XRN2, such as mutations, mRNA expression, and copy number variations, are common in a wide range of cancers; XRN2 polymorphisms contribute to an increased risk of spontaneous lung cancer [20][21][22][23]. Finally, XRN2 was recently reported to play an important role in the invasiveness of certain cancers, and alteration in its expression correlates with poor survival of cancer patients [24].…”

PARP inhibitors combined with radiotherapy: are we ready?

“…The PARylation of PARP1 and histones facilitates the recruitment of repair proteins at the DNA damage site and chromatin relaxation, granting spatial access to these proteins. Repairing damaged DNA in the BER process involves proteins such as X-ray repair crosscomplementing gene 1 (XRCC1), DNA polymerase, and DNA ligase (Patidar et al, 2020). Among them, XRCC1 serves as a scaffold protein anchoring other repair components.…”

DNA damage induced by KP372-1 hyperactivates PARP1 and enhances lethality of pancreatic cancer cells with PARP inhibition

“…Afterwards, media were replaced with fresh media (without KP372-1). Next, standard immunofluorescence microscopy protocol was followed as described previously 25 . Briefly, cells were gently washed in 1× PBS, followed by fixation with ice-cold methanol:acetic acid (3: The slides were then electrophoresed at 20 V for 45 min at 4 °C in the neutral electrophoresis buffer.…”

“…Confocal immunofluorescence microscopy. The general procedure for confocal immunofluorescence microscopy is similar as described previously 25 . Briefly, cells were seeded on 6-well plates (~ 100,000 cells/well) containing glass slides and allowed to adhere overnight.…”