title: Disruption of SLX4-MUS81 Function Increases the Relative Biological Effectiveness of Proton Radiation.
abstract: OBJECTIVE
  Clinical proton beam therapy has been based on the use of a generic relative biological effectiveness (RBE) of ∼1.1. However, emerging data have suggested that Fanconi anemia (FA) and homologous recombination pathway defects can lead to a variable RBE, at least in vitro. We investigated the role of SLX4 (FANCP), which acts as a docking platform for the assembly of multiple structure-specific endonucleases, in the response to proton irradiation.
  
  
  METHODS
  Isogenic cell pairs for the study of SLX4, XPF/ERCC1, MUS81, and SLX1 were irradiated at the mid-spread-out Bragg peak of a clinical proton beam (linear energy transfer 2.5 keV/μm) or with 250 kVp x-rays, and the clonogenic survival fractions were determined. To estimate the RBE of the protons relative to cobalt-60 photons (Co60Eq), we assigned a RBE(Co60Eq) of 1.1 to x-rays to correct the physical dose measured. Standard DNA repair foci assays were used to monitor the damage responses, and the cell cycle distributions were assessed by flow cytometry. The poly(ADP-ribose) polymerase inhibitor olaparib was used for comparison.
  
  
  RESULTS
  Loss of SLX4 function resulted in an enhanced proton RBE(Co60Eq) of 1.42 compared with 1.11 for wild-type cells (at a survival fraction of 0.1; P<.05), which correlated with increased persistent DNA double-strand breaks in cells in the S/G2 phase. Genetic analysis identified the SLX4-binding partner MUS81 as a mediator of resistance to proton radiation. Both proton irradiation and olaparib treatment resulted in a similar prolonged accumulation of RAD51 foci in SLX4/MUS81-deficient cells, suggesting a common defect in the repair of DNA replication fork-associated damage.
  
  
  CONCLUSIONS
  A defect in the FA pathway at the level of SLX4 results in hypersensitivity to proton radiation, which is, at least in part, due to impaired MUS81-mediated processing of replication forks that stall at clustered DNA damage. In vivo and clinical studies are needed to confirm these findings in human cancers.
authors.affiliation: Laboratory of Cellular and Molecular Radiation Oncology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Division of Radiation Physics, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Tracy S A
authors.initials: TSA
authors.lastname: Underwood
authors.affiliation: Division of Radiation Physics, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Jong
authors.initials: J
authors.lastname: Kung
authors.affiliation: Laboratory of Cellular and Molecular Radiation Oncology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Meng
authors.initials: M
authors.lastname: Wang
authors.affiliation: Division of Radiation Physics, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Hsiao-Ming
authors.initials: HM
authors.lastname: Lu
authors.affiliation: Division of Radiation Physics, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Harald
authors.initials: H
authors.lastname: Paganetti
authors.affiliation: Laboratory of Cellular and Molecular Radiation Oncology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Kathryn D
authors.initials: KD
authors.lastname: Held
authors.affiliation: Laboratory of Cellular and Molecular Radiation Oncology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Hong
authors.affiliation: Laboratory of Cellular and Molecular Radiation Oncology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Efstathiou
authors.affiliation: Laboratory of Cellular and Molecular Radiation Oncology, Department of Radiation Oncology, Massachusetts General Hospital, Boston, Massachusetts. Electronic address: hwillers@mgh.harvard.edu.
authors.forename: Henning
authors.initials: H
authors.lastname: Willers
mesh_terms: D000818:Animals; D002588:Cesium Radioisotopes; D003037:Cobalt Radioisotopes; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000378:metabolism*; D004706:Endodeoxyribonucleases; D004720:Endonucleases / Q000172:deficiency / Q000378:metabolism*; D005199:Fanconi Anemia / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D016014:Linear Models; D051379:Mice; D010793:Phthalazines / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology; D061766:Proton Therapy; D011522:Protons; D051135:Rad51 Recombinase / Q000378:metabolism; D011836:Radiation Tolerance; D045522:Recombinases / Q000172:deficiency / Q000378:metabolism*; D012062:Relative Biological Effectiveness
chemical_list: D002588:Cesium Radioisotopes; D003037:Cobalt Radioisotopes; D004268:DNA-Binding Proteins; D010793:Phthalazines; D010879:Piperazines; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011522:Protons; D045522:Recombinases; C095888:xeroderma pigmentosum group F protein; D051135:Rad51 Recombinase; C071447:ERCC1 protein, human; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C498772:MUS81 protein, human; C542320:SLX1 protein, human; C542319:SLX4 protein, human; C531550:olaparib