XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition

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

XRN2 Links Transcription Termination to DNA Damage and Replication Stress

We thank the Proteomics Core Facility. We thank Dr. Robert J. Crouch for providing us with GFP- and GFP-RNase H expression plasmids. We also thank Dr. Stephen H. Leppla for providing us with antibodies directed against RNA:DNA hybrids (R loops) (S9.6). We thank Novus Biologicals for generously providing XRN2 and Rrp45 antibodies. We also thank the members of the Boothman lab for critical reading of this manuscript.Author Summary Genomic instability is one of the primary causes of disease states, in particular cancer. One major cause of genomic instability is the formation of DNA double strand breaks (DSBs), which are one of the most dangerous types of DNA lesions the cell can encounter. If not repaired in a timely manner, one DSB can lead not only to cell death. If misrepaired, one DSB can lead to a hazardous chromosomal aberration, such as a translocation, that can eventually lead to cancer. The cell encounters and repairs DSBs that arise from naturally occurring cellular processes on a daily basis. A number of studies have demonstrated that aberrant structures that form during transcription under certain circumstances, in particular RNA:DNA hybrids (R loops), can lead to DSB formation and genomic instability, especially during DNA synthesis. Thus, it is important to understand how the cell responds and repairs transcription-mediated DNA damage in general and R loop-related DNA damage in particular. This paper both demonstrates that the XRN transcription termination factor links transcription and DNA damage, but also provides a better understanding of how the cell prevents transcription-related DNA damage.Yeshttp://www.plosgenetics.org/static/editorial#pee

Cystargolide-based amide and ester Pz analogues as proteasome inhibitors and anti-cancer agents

A series of cystargolide-based β-lactone analogues containing nitrogen atoms at the Pz portion of the scaffold were prepared and evaluated as proteasome inhibitors, and for their cytotoxicity profile toward several cancer cell lines. Inclusion of one, two or even three nitrogen atoms at the Pz portion of the cystargolide scaffold is well tolerated, producing analogues with low nanomolar proteasome inhibition activity, in many cases superior to carfilzomib. Additionally, analogue 8g, containing an ester and pyrazine group at Pz, was shown to possess significant activity toward RPMI 8226 cells (IC50 = 21 nM) and to be less cytotoxic toward the normal tissue model MCF10A cells than carfilzomib

XRN2 is a DNA damage-responsive factor.

<p>(<b>A</b>) To interrogate potential <i>in vivo</i> XRN2-interacting partners, exponentially growing HeLa cells were collected and lysed for FPLC analyses. Individual fractions were separated by SDS-PAGE and indicated proteins visualized by immunoblot (IB). (<b>B</b>) Interactions between XRN2 and the DNA damage regulators Ku80, 53BP1, and BRCA1 were interrogated by immunoprecipitation (IP). (<b>C</b>) XRN2 foci were quantitated in human fibroblasts stably expressing an shRNA control (shScr) or a Kub5-Hera shRNA (shk-h) in mock (unt)-, IR (1 Gy)-, and UV (20 J/m²)-treated cells. (<b>D</b>) Sub-cellular XRN2 localization and S9.6 foci were visualized in mock-, UV (20 J/m²)-, or UV (20 J/m²)- + α amanitin (α-aman)-treated shScr fibroblast cells by immunofluorescence. (*p<0.05).</p

Loss of XRN2 leads to increase amounts of replication stress.

<p>(<b>A</b>) Basal levels of 53BP1 were monitored in PCNA positive cells, an S-phase indicator, in shXRN2 and shScr cells by immunofluorescence (IF). (<b>B, C</b>) Basal levels of phosphorylated RPA, were monitored by IF in shXRN2, shScr, shk-h fibroblasts and MCF-7 cells transfected with control or XRN2 specific siRNAs. (<b>D</b>) Basal levels of phosphorylated ATR were monitored by IF in MCF-7 cells transfected with a siRNA control and a siXRN2. (<b>E</b>) Basal levels of phosphorylated Chk1 were monitored in.shScr, shXRN2, and shk-h fibroblasts by IF. (<b>F)</b> Basal levels of phospho-Chk1-pS-317 and RPA32-pS-(4/8), replication stress indicators, in shXRN2 (-) versus shScr (+) cells were monitored by western blotting. (<b>G</b>) DNA replication elongation was monitored in log-phase shScr, shXRN2, or shk-h fibroblasts by DNA fiber analyses. (**p<0.01).</p

XRN2-deficient cells are hypersensitive to various chemotherapeutic agents.

<p>(<b>A-D</b>) shScr and shXRN2 fibroblasts and MDA-MB-231 cells transfected with a siRNA control or targeting XRN2 were either mock-treated or exposed to: (<b>A, B</b>) ionizing radiation (IR); (<b>C, D</b>) ultraviolet light (UV). (<b>E-F</b>) shScr and shXRN2 fibroblasts were either mock-treated or exposed to: (<b>E</b>) H₂O₂ or (<b>F</b>) Aphidicolin (APH). Cells were then monitored for survival using colony forming assays. Colonies of >50 normal-appearing cells were quantified for mock- versus agent-exposed cells. (**p<0.01).</p

Loss of XRN2 leads to increased DSB formation and genomic instability.

<p>(<b>A</b>) Steady state levels of XRN2 protein in shScr (+) compared to shXRN2 (-) fibroblast cells were monitored by western blotting and immunofluorescence. (<b>B, C</b>) Basal levels of 53BP1, pATM^ser1981, γ-H2AX, and BRCA1 foci/nuclei were quantitated in shScr, shXRN2, and MCF-7 cells treated with control and XRN2 specific siRNA. (<b>D, E</b>) Genomic aberrations were quantified in shScr, shXRN2, and shk-h cells using derived metaphase spreads. (**p<0.01).</p

DDR regulators accumulate at the 3’ end of genes after XRN2 loss.

<p>(<b>A-E</b>) Accumulation of the DNA damage regulators, ATM, BRCA1, γ-H2AX, 53BP1, and CtIP, was monitored at the 3’ pause sites of the (<b>A</b>) <i>ENSA</i>, (<b>B</b>) <i>β-actin</i>, (<b>C</b>) <i>Akirin</i>,<i>1</i> (<b>D</b>) <i>Gemin7</i> genes and (<b>E</b>) an intronic region of <i>Gemin7</i> by chromatin immunoprecipitation. n = 3, S.E. is indicated. (<b>F</b>) Model for XRN2 functions in DNA repair pathway choice. In normal conditions, XRN2 binds to the NHEJ factor 53BP1 promoting DSB repair via the NHEJ pathway. In the absence of XRN2, NHEJ is inhibited downstream of 53BP1, allowing DSB repair via the HR pathway.</p

R-loop formation and transcription contribute to delayed DSB repair kinetics in XRN2-deficient cells.

<p><b>(A)</b> Levels of R loops were monitored in mock- or IR (1 Gy)-exposed shXRN2 compared to shScr fibroblast cells by immunofluorescence. <b>(B-C)</b> Regression of 53BP1 foci/nucleus was monitored by IF in IR (1 Gy)-treated shXRN2 and shScr cells that were exposed to α-amanitin (α-aman) or transfected with GFP-RNaseH. (*p<0.05).</p