DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells

Abstract

Limited DNA end resection is the key to impaired homologous recombination in BRCA1-mutant cancer cells. Here, using a loss-of-function CRISPR screen, we identify DYNLL1 as an inhibitor of DNA end resection. The loss of DYNLL1 enables DNA end resection and restores homologous recombination in BRCA1-mutant cells, thereby inducing resistance to platinum drugs and inhibitors of poly(ADP-ribose) polymerase. Low BRCA1 expression correlates with increased chromosomal aberrations in primary ovarian carcinomas, and the junction sequences of somatic structural variants indicate diminished homologous recombination. Concurrent decreases in DYNLL1 expression in carcinomas with low BRCA1 expression reduced genomic alterations and increased homology at lesions. In cells, DYNLL1 limits nucleolytic degradation of DNA ends by associating with the DNA end-resection machinery (MRN complex, BLM helicase and DNA2 endonuclease). In vitro, DYNLL1 binds directly to MRE11 to limit its end-resection activity. Therefore, we infer that DYNLL1 is an important anti-resection factor that influences genomic stability and responses to DNA-damaging chemotherapy.

Extended Data Figure 1, Related to Fig. 1.

a, Comparison of ATMIN and BRCA1 alterations in Ovarian Cancer according to TCGA data (316 samples, Nature, 2011) from the cBioPortal. Red bar, Amplification. Blue bar, Deep deletion. Red square, mRNA Upregulation. Blue square, mRNA Downregulation. Gray bar, no mutation. Green square, missense mutation. Black square, truncating mutation. b, Survival assay of RPE1 with siCtrl and siDYNLL1. c-e, Survival assay of BRCA1-mutant cells UWB1.289, MDA-MB-236 and L56Br-C1 treated with olaparib and cisplatin, transfected with siCtrl and siDYNLL1. f, g Survival assay of BRCA1 siRNA transfected RPE1 and Hela cells treated with olaparib and cisplatin, co-transfected with siCtrl and siDYNLL1. h, Immuno-blots showing depletion of DYNLL1 and BRCA1. Experiments were repeated independently with similar results for 3 times. i, KURAMUCHI with siCtrl and siDYNLL1, treated with Olaparib or Cisplatin. For all panels, data is represented as mean SEM from three different experiments. j, Progression Free Survival (PFS) of ovarian carcinoma patients with BRCA2 mutation based on above or below median expression values of DYNLL1 (DYNLL1 high n=14, DYNLL1 low n=18, Source: Ovarian cancer, TCGA Dataset Nature 2011). Statistical significance was assessed by the one-sided Mantel–Cox test with P-value as indicated.

Extended Data Figure 2, Related to Fig. 2.

Immunofluorescence and quantification of RAD51 foci (a and b) and RPA32 foci (c) in Cov362 cells and Cov362 DYNLL1−/− cells exposed to 5Gy IR. Staining for Rad51 is 6 h and RPA32 is 4 h after IR. For panel a, N=105, unpaired t test two tailed for Ctrl and sgDYNLL1#1 p-value < 0.0001. for Ctrl and sgDYNLL1#2 p=0.0003, for sgDYNLL1#1 and sgDYNLL1#2 p=0.5679. In (b) Cov362 cells and Cov362 DYNLL1−/− cells were also transfected with control siRNA and siBRCA1, immunoblot (left panel). Data is represented as mean SEM from three different experiments (n=100). For panel c, N=100, unpaired t test two tailed for Ctrl and sgDYNLL1#1 p-value = 0.0002. for Ctrl and sgDYNLL1#2 p<0.0001, for sgDYNLL1#1 and sgDYNLL1#2 p=0.5679. d, immunoblot of Cov362 and Cov362 DYNLL1−/− cells after 10 uM Olaparib treatment for 48 h with indicated antibodies. Experiments were repeated independently for 3 times with similar results. e, Analysis of 53BP1 foci as shown in Fig.2b from Cov362 cells and Cov362 DYNLL1−/− cells treated with 10 uM Olaparib for 24 h. Data is represented as mean SEM from three different experiments (n=102 cells of each genotype). f-h, Immunofluorescence and quantification of 53BP1 foci (f) n=95 p=0.1019 unpaired t test two sided, RPA32 foci (g) n=100 p=0.0238 unpaired t test two sided and RAD51 foci (h) n=94 p=0.3161 unpaired t test two sided in RPE1 cells transfected with siCtrl and siDYNLL1 exposed to 5Gy IR. Staining for 53BP1 is 1 h, Rad51 is 6 h and RPA32 is 4 h after IR. For all panels, data is represented as mean SEM from three different experiments.

Extended Data Figure 3, Related to Fig. 3.

a, Samples from the Pancreatic Cancer Endocrine neoplasms (PAEN-AU) were grouped into 4 categories based on combinatorial high (above median) and low (below median) expression of BRCA1 and DYNLL1, frequency of somatic structural variations (SV) (left panel) and frequency of intra-chromosomal SVs (deletion/Duplication/ Insertion/ Intra-Chromosomal Translocation) (right panel) were plotted. b, Samples from the PAEN-AU (32 samples) cohort were grouped into 4 categories based on combinatorial high (above median) and low (below median) expression of BRCA1 and DYNLL1 (left panel) or expression of BRCA1 and 53BP1 (right panel). Frequency of homologous sequence discovered in the junction of Deletion + Duplication/ Insertion/ Intra-Chromosomal Translocation were plotted. For a and b panel, statistical differences (calculated using one sided Wilcoxon Rank Sum test) are indicated. In the boxplots, upper whisker is defined to be 1.5×IQR more than the third quartile and the lower whisker is defined to be 1.5×IQR lower than the first quartile respectively, where IQR is the difference between the third quartile and the first quartile, i.e. the box length. c, Quantification of mRNA level of indicated genes in Cov362 (ctrl) and Cov362 ATMIN^−/−cells (sgATMIN). N=4, for all panels expression was normalized to actin, data is represented as mean SEM from 4 different experiments. Unpaired t test two-sided Ctrl vs #1 and Ctrl vs #2 for MRE11 are P=0.0335 and P=0.0038, for NBN are P=0.0152 and P=0.0257, for BLM are P=0.0130 and P=0.0203, for DNA2 are P<0.0001 and P=0.0179. d, Quantification of mRNA levels of indicated genes in Cov362 (ctrl) and Cov362 DYNLL1−/− (sgDYNLL1). N=6, for all panels expression was normalized to actin, data is represented as mean SEM from 6 different experiments. e, Quantification of subcellular fraction of indicated proteins (n=3) in Cov362 cells and Cov362 DYNLL1−/− cells. Total protein and chromatin bound protein was normalized to H2AX levels, and levels of indicated proteins in Cov362 DYNLL1−/− cells graphically represented relative to the control Cov362 cells. Data is represented as mean SEM. Unpaired t test two sided Ctrl vs #1 and Ctrl vs #2 for MRE11 are P<0.0001 and P<0.0001, for Rad50 are P=0.0035 and P=0.0007, for NBN are P<0.0001 and P<0.0001, for BLM are P<0.0001 and P<0.0001, for DNA2 are P=0.0002 and P<0.0001. f, Flag immunoprecipitation of FLAG-DYNLL1 and immunoblot with indicated antibodies. g, Immunofluorescence and quantification of DYNLL1 and -H2AX foci in RPE1 cells transfected with siCtrl and siDYNLL1, 1h after 5Gy IR. Experiments were repeated independently for 3 times with similar results. h, Immunoblot of DYNLL1 in RPE1 cells exposed to 5 Gy IR and sub-cellular fractionation at indicated times. Experiments were repeated independently for 3 times with similar results.

Extended Data Figure 4, Related to Fig. 4.

a, Structure of DYNLL1 dimer with potentially relevant residues indicated. b, Immunoprecipitation of indicated DYNLL1 mutants with 53BP1 and MRE11. Experiments were repeated independently for 3 times with similar results. c, Resection products of independent experiments WT or mutant recombinant DYNLL1 (purified proteins inner panel) with MRN-RPA-BLM-DNA2 and a [32] P-labeled linear 2.7kb dsDNA substrate. Experiments were repeated independently for 3 times with similar results. d, GST pull-down of GST-tagged DYNLL1-S88D incubated with purified human MRE11 or human DNA, EXO1, BLM, or the human RPA trimer (RPA70-RPA32-RPA14). Experiments were repeated independently for 3 times with similar results. e, Recombinant DYNLL1 WT protein was incubated with RPA-BLM and with a [32P]-labeled linear 2.7 kb dsDNA substrate to monitor DNA unwinding. Experiments were repeated independently for 3 times with similar results.

Figure 1.. Genome wide CRISPR screen reveals DYNLL1 loss causes PARPi and platinum resistance in BRCA1-mutant HGSOCs.

a, Schematic of CRISPR-based screen for PARPi or cisplatin resistance. b, Relative guide abundance before and after Olaparib and cisplatin treatment in Cov362 cells (data provided in Supplemental Table 1 and 2). c, Progression free survival (PFS) of ovarian carcinoma patients with BRCA1 mutant (top DYNLL1 high expression n=16, DYNLL1 low expression n=20) and BRCA1 wild type (bottom DYNLL1 high n=135, DYNLL1 low n=111) based on above or below median expression values of DYNLL1 (Source: Ovarian cancer, TCGA Dataset Nature 2011). Statistical significance was assessed by the one-sided Mantel–Cox test with P-value as indicated. d, Immunoblot of ATMIN and DYNLL1 from Cov362 cells with deletions of ATMIN or DYNLL1. #1 and #2 are independent stable clones. Survival assay of the indicated Cov362 clones treated with e, PARP inhibitors: Olaparib or Niraparib and f, Platinum drugs: Cisplatin or Carboplatin. g, Immunoblot of tagged DynLL1 in Cov362 cells with deletions of ATMIN or DYNLL1. d-f, experiments were repeated 3 times independently with similar results. Survival assay of h) Cov362 DYNLL1 −/− clone and i) Cov362 ATMIN −/− clone expressing tagged DYNLL1, treated with Olaparib or Cisplatin. Data is represented as mean SEM from 3 different experiments.

Figure 2.. DYNLL1 loss leads to restoration of DNA end resection and HR.

Immunofluorescence and quantification of Cov362 cells and Cov362 DYNLL1−/− cells treated with 10 uM Olaparib for 24 h stained with antibodies against a) RAD51, b) 53BP1, and c) RPA32 (for a, 143 cells b, 102 cells and c, 146 cells were analyzed, unpaired t test two tailed p<0.0001). -H2AX staining was done for all panels (data not shown). d, Schematic of AsiSI based DNA end resection assay (top) in U2OS cells and PCR-based quantification of ssDNA formation at 335bp or 1664bp downstream of the AsiSI induced break site (bottom) in cells transfected with indicated siRNAs. N=3 unpaired t test two tailed p<0.0001. e, Change in ssDNA length in Cov362 control and Cov362 DYNLL1−/− cells after 10 uM Olaparib treatment for 48 h, representative images (left) and quantification (right). 150 DNA fibers are analyzed, unpaired t test two tailed p-value between Ctrl and sgDYNLL1< 0.0001, p-value between sgDYNLL1 #1 and #2 =0.0848. Data is represented as mean SEM from 3 independent experiments.

Figure 3.. Impact of DYNLL1 on chromosomal aberrations in HGSOC samples and interaction with the DNA end resection machinery.

a, Samples from the Australian Ovarian Cancer Study cohort (OV-AU) were grouped into 4 categories based on combinatorial high (above median) and low (below median) expression of BRCA1 and DYNLL1, frequency of somatic structural variations (SV) (left panel) and frequency of intra-chromosomal SVs (Deletion + Duplication/ Insertion/ Intra-Chromosomal Translocation) (right panel) were plotted. b, Samples from the OV-AU cohort were grouped into 4 categories based on combinatorial high (above median) and low (below median) expression of BRCA1 and DYNLL1 (left panel) or expression of BRCA1 and 53BP1 (right panel). Frequency of homologous sequence discovered in the junction of Deletion + Duplication/ Insertion/ Intra-Chromosomal Translocation were plotted. Statistical differences (Mann Whitney U test, p values) are indicated. c, Representative immunoblot from 3 experiments of indicated proteins in Cov362 cells and Cov362 DYNLL1−/− cells after sub-cellular fractionation. Quantification (n=3) of chromatin enrichment shown in Extended Data 3e. d, Flag immunoprecipitation of GFP-DYNLL1 with FLAG-tagged DNA end resection proteins. Experiments were repeated 4 times independently with similar results. e, U2OS stable clone (U2OS19) containing 256 lac-operator and 96 tetracycline response element copies were transiently transfected with mCherry-LacI fused to DYNLL1 and either GFP- empty or fused to indicated proteins. Quantification of co-localization of mCherry-DYNLL1 with indicated GFP-tagged proteins on the LacO array is indicated (mean ± s.d., n=3).

Figure 4.. Identification and characterization of DYNLL1 mutants that impact genome stability in cells and DNA end resection in vitro.

a, Immunoprecipitation of DYNLL1 mutants with end resection complex enzymes. Experiments were repeated 3 times independently with similar results. b, Immunoblot of tagged- DYNLL1 (wildtype and mutants) in Cov362 DYNLL1−/− cells (bottom) and survival assay following olaparib treatment (top) Data is represented as mean SEM from 3 different experiments. c, Resection products of WT or mutant recombinant DYNLL1 (purified proteins top panel) with MRN-RPA-BLM-DNA2 and a [32] P-labeled linear 2.7kb dsDNA substrate (middle panel) and quantification of resection efficiency (bottom panel, n=3). d, GST pull down of GST-tagged DYNLL1- WT and S88D mutant and MRE11 isolated from insect cells. e, Resection product of a 5’ end labelled 100 bp dsDNA incubated with indicated concentration of purified recombinant human DYNLL1 and the MRE11-RAD50-NBS1 complex. f, Immunofluorescence and quantification of MRE11 foci in Cov362 cells and Cov362 DYNLL1−/− cells treated with 10 uM Olaparib for 24 h. 198 cells were analyzed, unpaired t test two tailed p-value between Ctrl and sgDYNLL1< 0.0001. Data is represented as mean SEM from three different experiments.