Dataset Information

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

ABSTRACT: Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.

SUBMITTER: Hewitt G

PROVIDER: S-EPMC7895907 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Hewitt Graeme G Borel Valerie V Segura-Bayona Sandra S Takaki Tohru T Ruis Phil P Bellelli Roberto R Lehmann Laura C LC Sommerova Lucia L Vancevska Aleksandra A Tomas-Loba Antonia A Zhu Kang K Cooper Christopher C Fugger Kasper K Patel Harshil H Goldstone Robert R Schneider-Luftman Deborah D Herbert Ellie E Stamp Gordon G Brough Rachel R Pettitt Stephen S Lord Christopher J CJ West Stephen C SC Ahel Ivan I Ahel Dragana D Chapman J Ross JR Deindl Sebastian S Boulton Simon J SJ

Molecular cell 20201216 4

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR ...[more]

PMID: 33333017

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Project description:BackgroundPARP inhibitors have been the most promising target drugs with widely proven benefits among ovarian cancer patients. Although platinum-response, HR-related genes, or HRD genomic scar detection are acceptably used in assessment of Olaparib response, there are still evident limitations in the present approaches. Therefore, we aim to investigate more accurate approaches to predict Olaparib sensitivity and effective synergistic treatment strategies.MethodsWe probed two databases (TCGA and Qilu Hospital) in order to quest novel miRNAs associated with platinum-sensitivity or HR-related genes. Cellular experiments in vitro or in vivo and PDX models were utilized to validate their role in tumor suppression and Olaparib sensitizing. Furthermore, HR gene mutation was analyzed through WES to explore the relation between HR gene mutation and Olaparib response.ResultsHigh miR-509-3 expression indicated better response to platinum and longer progression-free and overall survival in two independent ovarian cancer patient cohorts (high vs. low miR-509-3 expression; PFS: TCGA P < 0.05, Qilu P < 0.05; OS: TCGA P < 0.05, Qilu P < 0.01). MiR-509-3 could impair the proliferation, migration, and invasion ability but enhance the sensitivity to Olaparib of ovarian cancer cell in vitro and in vivo by directly targeting HMGA2 and RAD51. In two PDX cases (PDX1 and PDX9), miR-509-3 could significantly increase the sensitivity to Olaparib along with the decrease of RAD51 positive rate (mean tumor weight NC + Olaparib vs. miR-509 + Olaparib; PDX1 P < 0.05, PDX9 P < 0.05). Additionally, in PDX8, miR-509-3 treatment dramatically reversed the Olaparib insensitivity (P < 0.05) by downregulating RAD51 expression. RAD51 functional detection revealed that all Olaparib sensitive cases exhibited low RAD51 positive rate (lesser than 50%) in treated groups. Furthermore, among the four HR gene mutation patients, three harbored HR core gene mutation and were sensitive to Olaparib while the remaining one with non-HR core gene mutation did not respond well to Olaparib.ConclusionsMiR-509-3 can sensitize ovarian cancer cells to Olaparib by impeding HR, which makes it a potential target in PARPi synergistic treatment. HR core gene analysis and RAD51 functional detection are prospectively feasible in prediction of PARPi response.

Project description:XL-MS mass spectrometry analysis Cross-linked peptides were resolved for mass spectrometry analysis using a Dionex UltiMate 3000 RSLnano liquid chromatography system. Peptides were initially loaded from the autosampler onto an Acclaim PepMap 100 C18 LC Trap Cartridge (0.3 mm inside diameter, 5 mm length) (Thermo Scientific, San Jose, CA) using a loading pump flow rate of 2 ul/minute. Peptides were subsequently resolved for mass spectrometry analysis using an analytical column (75 um inside diameter, 150 mm length) packed in-house with ReproSil-Pur C18-aQ 1.9 um resin (Dr. Masch GmbH, Germany). Chromatography was performed using combinations of buffer A (95% water, 5% acetonitrile, and 0.1% formic acid (v/v/v), pH 2.6), and buffer B (20% water, 80% acetonitrile, and 0.1% formic acid (v/v/v), pH 2.6). The following chromatography steps were performed using a flow rate of 180 nl/minute: (1) 2% B for 20 minutes (column equilibration); (2) a linear gradient from 2% to 10% B over 10 minutes; (3) a linear gradient from 10% to 40% B over either 120 minutes or 240 minutes; (4) a linear gradient from 40% to 95% B over 5 minutes; (5) 95% B for 14 minutes (column wash); (6) a linear gradient from 95% B to 2% B over 1 minute; (7) 2% B for 10 minutes (column re-equilibration). Eluted peptides were analyzed by mass spectrometry using an Orbitrap Fusio Lumo mass spectrometer (Thermo Scientific, San Jose, CA). An MS3 based method was used for identification of DSSO cross-linked peptides as follows: Full MS scans were performed using the Orbitrap mass analyzer (60,000 m/z resolution, 1.6 m/z isolation window, and 375-1500 m/z scan range); The top 3 peptides identified with charge state 4 to 8 were selected for MS2 fragmentation (25% CID energy) and subsequent detection with the Orbitrap mass analyzer (30,000 m/z resolution and a dynamic exclusion time of 40 s); Pairs of MS2 fragments with a mass difference of 31.9720 (20 ppm mass tolerance) were selected for MS3 fragmentation (CID energy 35%) and detection using the Linear Ion Trap mass analyzer; Each MS2 scan was followed by a maximum of 4 MS3 scans. Identification of DSSO Cross-Linked Peptides The m/z, charges from MS2 and corresponding m/z and intensities of fragment ion from MS3 spectra were extracted into ms2 file format using RawDistiller v. 1.0, in-house developed software with D(g, 6) settings to abstract parent ion profiles by Gaussian fitting and dynamic offline lock mass using six background polydimethylcyclosiloxane ions as internal calibrants. MS3 spectra were first searched using ProLuCID with a mass tolerance for parent ions and fragment ions set as 7 ppm and 800 ppm, respectively. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database combining 4540 proteins including 426 common contaminants. The methionine oxidation (O; 15.9949 Da) and remnants of the crosslinker alkene (C3H2O; +54.0106 Da) and thiol (C3H2SO; +85.9826 Da) were set as variable modifications. A static modification with a mass of 57.0125Da was added to cysteine residues. A script module was added into NSAF7 to search MSn data and identify putative crosslinkers based on their unique MS fragmentation patterns with the false positive rate was not more than 1% as illustrated in. The Xcorr of the peptides for a specified cross-linker was summed as scores S and use log2(10*log2(S+1)+1) to calculate the cross-linker scores. The non-cross-linking peptide/spectrum matches were sorted and selected using DTASelect/CONTRAST (v. 1.9) with an in-house software, swallow to filter spectra, peptides, and proteins at FDRs <1%. NSAF7 was used to create the final reports on all detected cross-linkers, peptides and non-redundant proteins identified across the different runs.

Dataset Information

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Publications

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

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