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METTL16 antagonizes MRE11-mediated DNA end resection and confers synthetic lethality to PARP inhibition in pancreatic ductal adenocarcinoma

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. Characterization of genetic alterations will improve our understanding and therapies for this disease. Here, we report that PDAC with elevated expression of METTL16, one of the ‘writers’ of RNA N6-methyladenosine modification, may benefit from poly-(ADP-ribose)-polymerase inhibitor (PARPi) treatment. Mechanistically, METTL16 interacts with MRE11 through RNA and this interaction inhibits MRE11’s exonuclease activity in a methyltransferase-independent manner, thereby repressing DNA end resection. Upon DNA damage, ATM phosphorylates METTL16 resulting in a conformational change and autoinhibition of its RNA binding. This dissociates the METTL16–RNA–MRE11 complex and releases inhibition of MRE11. Concordantly, PDAC cells with high METTL16 expression show increased sensitivity to PARPi, especially when combined with gemcitabine. Thus, our findings reveal a role for METTL16 in homologous recombination repair and suggest that a combination of PARPi with gemcitabine could be an effective treatment strategy for PDAC with elevated METTL16 expression.

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Fig. 1: METTL16 suppresses HR repair.
Fig. 2: METTL16’s inhibition of HR is reversed by ATM phosphorylation.
Fig. 3: METTL16 represses DNA end resection in an MRE11-dependent manner.
Fig. 4: RNA orchestrates METTL16-mediated MRE11 inactivation.
Fig. 5: METTL16 phosphorylation at Ser419 induces conformational change and autoinhibition of RNA binding.
Fig. 6: High METTL16 expression is correlated with increased sensitivity to PARPi and other chemotherapy reagents.
Fig. 7: Elevated METTL16 expression indicates a better response to PARPi and survival in patients.
Fig. 8: PARPi and gemcitabine in combination effectively kill tumor cell expression METTL16 in vivo.

Data availability

The human pancreatic ductal adenocarcinoma genomic data were derived from the TCGA Research Network: http://cancergenome.nih.gov/. The coordinates and structure factors for METTL16 N-terminal domain or METTL16 catalytic domain in complex with an RNA molecule were deposited in the PDB under accession nos. 6B92 and 6DU4, respectively. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

This research was supported by funding from the National Natural Science Foundation of China (grant nos. 81874184 and 82072736 to K.T., grant nos. 32090032 and 32070713 to J.Y., grant nos. 82172994 and 82002985 to Y. Chen), Key Research and Development Projects of Hubei Province (grant no. 2021BCA116 to K.T.) and the Mayo Foundation to Z.L. X.Z. is supported by the China Scholar Council (grant no. 201806160023). J.A.K. is supported by NIH grant no. T32 GM65841. We thank J. Wang for MRE11 WT and mutant vectors. We thank justicon, Freepik, max.icons, Good Ware, Flat Icons and Vitaly Gorbachev for icons from www.flaticon.com.

Author information

Authors and Affiliations

Authors

Contributions

Z.L., K.T. and J.Y. conceived and designed the study. X.Z. and F.Z. performed most of the experiments and wrote the manuscript. G.C. and G.M. performed the molecular dynamics simulations. Y. Chen helped with the GST, GST-MRE11 WT and GST-MRE11 MD5 protein purification. M.D. and J.H. helped with the nuclease reaction assay. W.K., Q.Z. and Y. Cao helped to analyze the ionizing radiation-induced foci data. J.A.K., K.L. and K.T. reviewed and edited the manuscript. R.A.D. and T.T.P. provided support for generating protein purification. H.G., C.Z. and Q.Z. helped with flow cytometric analysis. Y.S., Z.W., S.Z. and P.Y. carried out xenograft experiments. Y.Y., Z.X. and L.C. helped with immunohistochemical staining in specimen microarray. Y.Z. and X.T. provided technical expertise with the RNA pull-down assay. J.H., G.G. and J.L. helped with vectors construction.

Corresponding authors

Correspondence to Jian Yuan, Kaixiong Tao or Zhenkun Lou.

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Competing interests

The authors declare no competing interests.

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Peer review information

Nature Cancer thanks Jiri Bartek, Jianjun Chen, Alexander Kleger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 METTL16 does not affect NHEJ.

a, Gating strategy for HR/NHEJ, MMEJ reporter assays, repair efficiency = Q2/(Q1 + Q2). b, Gating strategy to determine the percentage of cells in each cell cycle. c, Cell cycle analysis of HEK293T cells with or without METTL16 knockdown. d,e, Left, schematic of the compatible or incompatible DNA ends NHEJ reporter system. Right, relative NHEJ repair efficiency in HEK293T cells with or without METTL16 knockdown. f, Relative MMEJ repair efficiency in HEK293T cells with or without METTL16 knockdown. g, Immunoblot of METTL16 in U2OS cells after being infected with the indicated lentiviruses. β-Actin was used as a loading control. h-k, Representative micrographs and quantification data for 53BP1 (h and i respectively) or RIF1 (j and k respectively) foci formation in the indicated U2OS cell lines at 0 h or 2 h following 5 Gy X-ray irradiation treatment. Scale bars are displayed (h,j). Data are represented as the mean ± SD from three independent experiments (c-f). Data are represented as the mean ± SEM; each point represents a cell; the cells used for analysis in each experiment were from a single replicate; n indicates the cell number used for quantification in each group (i,k). P-values are indicated (c-f,i,k); ns, not significant. Statistical significance was determined by two-tailed unpaired t-tests (c-f,i,k). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (g).

Source data

Extended Data Fig. 2 METTL16 suppresses HR and is regulated by ATM phosphorylation at Ser419.

a, Immunoblot of METTL16 in the indicated U2OS cells. β-Actin was used as a loading control. b, Sanger sequencing results of METTL16 locus in U2OS cells with METTL16 WT or METTL16 knockout. Red arrows indicate the mutation sites. c, Immunoblot of METTL16 in the indicated U2OS cells. β-Actin was used as a loading control. d,e, Representative micrographs (d) and quantification data (e) for γ-H2AX foci formation in the indicated U2OS cell lines without treatment or at 1 h and 8 h after 2 Gy X-ray irradiation treatment. f, The indicated U2OS cells were collected without treatment, or at 1 h, 4 h, 8 h, and 12 h following 10 Gy X-ray irradiation treatment. Cell lysates were immunoblotted with the indicated antibodies. g, Quantitative analysis of the relative levels of γ-H2AX normalized to H2AX loading control in (f). Data are represented as the mean ± SD from three independent experiments. h,i, Representative micrographs (h) and quantification data (i) of neutral comet assay in the indicated U2OS cells without treatment or at 0.5 h and 4 h after 5 Gy X-ray irradiation treatment. Scale bars are displayed (d,h). Data are represented as the mean ± SEM; each point represents a cell; the cells used for analysis in each experiment were from a single replicate; n indicates the cell number used for quantification in each group (e,i). P-values are indicated (e,g,i). Statistical significance was determined by two-tailed unpaired t-tests (e,i), or two-way ANOVA (g). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a,c,f).

Source data

Extended Data Fig. 3 METTL16 inhibits DNA end resection through MRE11.

a-f, Representative micrographs and quantification data for BRCA1 (a and b respectively), CtIP (c and d respectively), or PALB2 (e and f respectively) foci formation in the indicated U2OS cell lines. g, The indicated U2OS cells were collected and cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as a loading control. h, Total or chromatin-enriched extracts of indicated U2OS cells were collected and immunoblotted with the indicated antibodies. i, Representative micrographs for RPA32 (upper) or BrdU (lower) foci formation in the indicated U2OS cells. j, Immunoblots of MRE11 and METTL16 in the indicated U2OS cells with or without 10 Gy X-ray irradiation treatment. β-Actin was used as a loading control. k,l, Representative micrographs (k) and quantification data (l) for MRE11 foci formation in the indicated U2OS cell lines. m, Endogenous IP between MRE11 and RAD50 or NBS1 with or without METTL16 overexpression. n,o, Representative micrographs (n) and quantification data (o) for NBS1 foci formation in the indicated U2OS cell lines. p, The indicated U2OS cells treated with 10 Gy X-ray irradiation were collected at the indicated time point and cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as a loading control. q, Co-IP of the indicated METTL16 vectors with MRE11. Scale bars are displayed (a,c,e,i,k,n). Data are represented as the mean ± SEM; each point represents a cell; the cells used for analysis in each experiment were from a single replicate; n indicates the cell number used for quantification in each group (b,d,f,l,o). P-values are indicated (b,d,f,l,o). Statistical significance was determined by two-tailed unpaired t-tests (b,d,f,l,o). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (g,h,j,m,p,q).

Source data

Extended Data Fig. 4 METTL16 inhibits MRE11 exonuclease activity by forming METTL16-RNA-MRE11 complex.

a, Schematic structure and sequence of U6 snRNA. b, Schematic of the interaction between METTL16 and MAT2A_hp1. c, Schematic structure and sequence of MAT2A_hp1. d,e, RNA pull-down of MAT2A_hp1 RNA with the indicated METTL16 or MRE11 vectors with or without 10 Gy X-ray irradiation treatment. f, Expression and purification of MRE11/RAD50 (MR), NBS1, and METTL16 proteins in vitro. Flag-tagged MRE11 and His-tagged RAD50 were co-expressed. g,h, Purified MR (MRE11 and RAD50), NBS1 and control RNA or U6 snRNA were incubated with 5′ 32P labeled blunt (g) or overhang (h) DNA substrate in vitro. The cleavage products were shown below by autoradiography. i, Immunoblot of METTL16 in the indicated U2OS cells. β-Actin was used as a loading control. j,k, Representative micrographs (j) and quantification data (k) for γ-H2AX foci formation in the indicated U2OS cell lines without treatment or at 1 h and 8 h after 2 Gy X-ray irradiation treatment. l,m, Representative micrographs (l) and quantification data (m) of neutral comet assay in the indicated U2OS cells without treatment or at 0.5 h and 4 h after 5 Gy X-ray irradiation treatment. n, Representative micrographs for RPA32 (upper) or BrdU (lower) foci formation in the indicated U2OS cells. Scale bars are displayed (j,l,n). Data are represented as the mean ± SEM; each point represents a cell; the cells used for analysis in each experiment were from a single replicate; n indicates the cell number used for quantification in each group (k,m). P-values are indicated (k,m). Statistical significance was determined by two-tailed unpaired t-tests (k,m). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (d-i).

Source data

Extended Data Fig. 5 METTL16 functions in DNA end resection independently of its MTase activity.

a, Co-IP of the indicated METTL16 vectors with MRE11 with or without 10 Gy X-ray irradiation treatment. b-e, RNA pull-down of the indicated RNAs with METTL16 or MRE11. UM, unmodified. f, Relative HR repair efficiency in the indicated HEK293T cells. Data are presented as mean ± SD from three independent experiments. g, Immunoblot of METTL16 in the indicated U2OS cells. β-Actin was used as a loading control. h,i, Representative micrographs (h) and quantification data (i) for γ-H2AX foci formation in the indicated U2OS cell lines without treatment or at 1 h and 8 h after 2 Gy X-ray irradiation treatment. j,k, Representative micrographs (j) and quantification data (k) of neutral comet assay in the indicated U2OS cells without treatment or at 0.5 h and 4 h after 5 Gy X-ray irradiation treatment. l-n, Representative micrographs (l) and quantification data (m,n) for RPA32 or BrdU foci formation in the indicated U2OS cells. o, Total or chromatin-enriched extracts of the indicated U2OS cells were collected and immunoblotted with the indicated antibodies. Scale bars are displayed (h,j,l). Data are represented as the mean ± SEM; each point represents a cell; the cells used for analysis in each experiment were from a single replicate; n indicates the cell number used for quantification in each group (i,k,m,n). P-values are indicated (f,i,k,m,n). Statistical significance was determined by two-tailed unpaired t-tests (f,i,k,m,n). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a-e,g,o).

Source data

Extended Data Fig. 6 METTL16 undergoes a conformational change and auto-inhibits RNA binding upon DNA damage-mediated phosphorylation at Ser419.

a,b, RNA pull-down of MAT2A_hp1 RNA with the indicated METTL16 vectors with or without 10 Gy X-ray irradiation treatment. c, Unmodified METTL16 peptide dissociates from the METTL16 N-terminal domain in the molecular dynamics simulation. d,e, RMS fluctuation for each of the residues of the phosphopeptide (pS419) (d) or unmodified METTL16 peptide (e). f,g, RMS deviation in METTL16 N-terminal domain in the presence of a phosphopeptide (pS419) (f) peptide or unmodified Ser419-containing METTL16 (g). h, HEK293T cells transfected with GFP-METTL16 (C-terminus) were pretreated as indicated. Harvested cells were immunoprecipitated with GFP beads and immunoblotted with the indicated antibodies. i, Co-IP of the indicated METTL16 vectors. j, RNA pull-down of MAT2A_hp1 RNA with Flag-METTL16 after transfected with the indicated GFP-METTL16 vectors. k,l, Co-IP of the indicated METTL16 vectors. m, METTL16 null U2OS cells were transfected with the indicated METTL16 vectors and cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as a loading control. n,o, Representative images (n) and quantification (o) of Duo-link in situ in the indicated U2OS cells without X-ray irradiation treatment. Scale bar is displayed (n). Data are presented as mean ± SD from three independent experiments (o). P-value is indicated and statistical significance was determined by two-tailed unpaired t-tests (o). p, HEK293T cells were transfected with Flag-METTL16 vector following 10 Gy X-ray irradiation treatment. Cell lysates were subjected to native-polyacrylamide gel electrophoresis (Native-PAGE) and immunoblotted with Flag antibody. q, Co-IP of the indicated METTL16 vectors. Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a,b,h-n,p,q).

Source data

Extended Data Fig. 7 Phosphorylation of METTL16 leads to dissociation with MRE11 by DNA damage-inducing reagents.

a, Immunoblots of METTL3 and METTL14 in different PDAC cell lines. β-Actin was used as a loading control. b,c, Relative METTL16 mRNA expression level (b) and the correlation of METTL16 mRNA and protein level (c) in the panel of PDAC cell lines. Data are presented as mean ± SD from three independent experiments (b). d, Quantification data for RPA32 foci formation in the indicated PDAC cell lines with or without 5 Gy X-ray irradiation treatment. Data are presented as mean ± SEM; each point represents a cell; the cells used for analysis in each experiment were from a single replicate; n indicates the cell number used for quantification in each group. e,f, HEK293T cells transfected with Flag-METTL16 vector were treated with the indicated DNA damage-induced agents. Cell lysates were subjected to immunoprecipitation with Flag beads and immunoblotted with the indicated antibodies. g,h, HEK293T cells transfected with Flag-METTL16 vector were treated with olaparib for the indicated time period or 10 Gy X-ray irradiation. Cell lysates were subjected to immunoprecipitation with Flag beads and immunoblotted with the indicated antibodies. i,j, Sanger sequencing results of METTL16 locus in SW1990 or PANC-1 cells with METTL16 WT or METTL16 knockout. Red arrows indicate the mutation sites. P-values are indicated (c,d). Statistical significance was determined by linear regression (c), or two-tailed unpaired t-tests (d). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a,e-h).

Source data

Extended Data Fig. 8 METTL16’s regulation of PARPi sensitivity is dependent on its phosphorylation and RNA binding capacity.

a,b, Immunoblot of METTL16 in the indicated SW1990 cell lines. β-Actin was used as a loading control. c, Cell survival of the indicated SW1990 cells for 2-D colony formation in response to olaparib. d, Relative colony number of the indicated SW1990 cells for soft agar colony formation in response to olaparib. e, Immunoblot of METTL16 in the indicated SW1990 cell lines. β-Actin was used as a loading control. f, Cell survival of the indicated SW1990 cells for 2-D colony formation in response to olaparib. g, Relative colony number of the indicated SW1990 cells for soft agar colony formation in response to olaparib. Data are presented as mean ± SD from three independent experiments (c,d,f,g). P-values are indicated (c,d,f,g). Statistical significance was determined by two-way ANOVA (c,f), or two-tailed unpaired t-tests (d,g). Experiments were repeated three times independently with similar results; data of one representative experiment are shown (a,b,e).

Source data

Extended Data Fig. 9 PARPi synergizes with gemcitabine in killing high METTL16 tumor cells in vitro.

a, Cell survival of the indicated SW1990 cells for 2-D colony formation treated with olaparib or gemcitabine or their combo. b, Relative colony number of the indicated SW1990 cells for soft agar colony formation treated with olaparib or gemcitabine or their combo. c, Cell survival of the indicated PANC-1 cells for 2-D colony formation treated with olaparib or gemcitabine or their combo. d, Relative colony number of the indicated PANC-1 cells for soft agar colony formation treated with olaparib or gemcitabine or their combo. Data are presented as mean ± SD from three independent experiments (a-d). P-values are indicated (a-d). Statistical significance was determined by two-tailed unpaired t-tests (a-d).

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Supplementary information

Reporting Summary

Supplementary Tables

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Source Data Extended Data Fig. 6

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Unprocessed western blots and/or gels.

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Zeng, X., Zhao, F., Cui, G. et al. METTL16 antagonizes MRE11-mediated DNA end resection and confers synthetic lethality to PARP inhibition in pancreatic ductal adenocarcinoma. Nat Cancer 3, 1088–1104 (2022). https://doi.org/10.1038/s43018-022-00429-3

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