Metabolic regulation of homologous recombination repair by MRE11 lactylation

Abstract

Lactylation is a lactate-induced post-translational modification best known for its roles in epigenetic regulation. Herein, we demonstrate that MRE11, a crucial homologous recombination (HR) protein, is lactylated at K673 by the CBP acetyltransferase in response to DNA damage and dependent on ATM phosphorylation of the latter. MRE11 lactylation promotes its binding to DNA, facilitating DNA end resection and HR. Inhibition of CBP or LDH downregulated MRE11 lactylation, impaired HR, and enhanced chemosensitivity of tumor cells in patient-derived xenograft and organoid models. A cell-penetrating peptide that specifically blocks MRE11 lactylation inhibited HR and sensitized cancer cells to cisplatin and PARPi. These findings unveil lactylation as a key regulator of HR, providing fresh insights into the ways in which cellular metabolism is linked to DSB repair. They also imply that the Warburg effect can confer chemoresistance through enhancing HR and suggest a potential therapeutic strategy of targeting MRE11 lactylation to mitigate the effects.

Keywords: CPP; MRE11; cell-penetrating peptide; chemoresistance; homologous recombination repair; lactylation; the Warburg effect.

Figure 1.. Lactylation plays a key role in DNA repair

(A) Analysis of HRD score in basal-like breast cancer samples. Statistical significance was tested using two-sided Mann-Whitney-Wilcoxon tests. (B) Western blots of HEK293T cells as indicated. (C) The HR levels of the indicated HEK293T cells were detected by fluorescence-activated cell sorting (FACS). (D) Representative picture of γ-H2AX foci in U2OS cells treated with as indicated. Scale bars, 10 μm. (E) Quantification of the data in (D). (F) Western blots of samples as indicated. (G) The HR levels of the indicated HEK293T cells were detected by FACS. (H) Representative picture of γ-H2AX foci in U2OS cells treated with as indicated. Scale bars, 10 μm. (I) Quantification of the data in (H). (J) Western blots of OVCAR-8 cells treated with sodium lactate (NALA) for 24 h. (K and L) OVCAR-8 (K) or MDA-MB-231 cells (L) were utilized for colony formation assays as indicated. (M) Western blots of OVCAR-8 cells treated with LDHi (10 or 20 mM) for 24 h. (N–P) OVCAR-8 (N and O) or MDA-MB-231 cells (P) were utilized for colony formation assays as indicated. (Q) Western blots of MDA-MB-231 cells as indicated. (R) Cells from (Q) were utilized to perform colony formation as indicated. Statistical analyses were performed with the Student’s t test. *p < 0.05; **p < 0.01, NS stands for no significant change. See also Figure S1.

Figure 2.. MRE11 K673 is lactylated by CBP

(A) Detecting lactylation of several proteins by western blots using co-immunoprecipitation (coIP) sample as indicated. (B–D) Detecting lactylation of MRE11 in cells treated with NALA or LDHi (B), depleted LDHA/B (C), or treated with several inducing DNA damage chemicals (D) as indicated by western blot. (E) Screening the “writer(s)” of MRE11 lactylation by transfecting combined MRE11 and acetyltransferase as indicated. (F and G) Detecting lactylation of MRE11 in cells depleted CBP (F) or treated with chemicals as indicated by western blot. Plko.1 is a lentivirus plasmid empty vector. (H and I) CoIP showing interactions between CBP and MRE11. (J) Detecting MRE11 lactylation using an in vitro lactylation assay on the indicated samples. (K) Schematic depicting MRE11 key domains and several lysine sites which were identified to be possibly lactylated by MS were pointed out. (L) Illustration of MRE11 K673 lactylation identified by MS. (M–O) Detecting lactylation of MRE11 by western blots as indicated. (P) Detecting MRE11 lactylation using as indicated an in vitro lactylation assay on the indicated. Quantifications of western blots in (B), (D), and (I) using Image J software. See also Figure S2.

Figure 3.. Lactylation of MRE11 enhances its DNA binding

(A) Western blots of as indicated coIP and input samples. (B) MRE11 proteins were lactylated in vitro. (C and D) Lactylated MRE11 proteins or unlactylated MRE11 proteins from (B) were used to perform an EMSA as indicated. (E and F) MRE11 WT and MRE11 K673R proteins were used to perform an EMSA as indicated. (G and H) Representative picture of MRE11(G) or MRE11-K673la (H) foci in U2OS cells as indicated treatment in upper panel. Scale bars, 10 μm. The lower panel is quantification. (I and J) Western blots of chromatin fraction from the indicated cells. (K) Representative picture of MRE11 foci in U2OS cells as indicated treatment in upper panel. Scale bars, 10 μm. The lower panel is quantification. (L) Western blots of chromatin fraction from the indicated cells. (M) Representative pictures of MRE11 foci were shown in left panel. Scale bars, 10 μm. The right panel is quantification. Quantifications of western blot or gels in (C)–(F), (I), (J), and (L) by Image J software. Statistical analyses were performed with the Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; NS stands for no significant change. See also Figure S3.

Figure 4.. MRE11 lactylation promotes DNA resection

(A and B) Western blots of samples from cells treated with NALA, LDHi, and/or cisplatin for 24 h (A) or, depleted LDHA/B and/or cisplatin for 24 h (B) as indicated. (C–E) Representative pictures of RPA2 (C), BrdU (D), and RAD51 (E) foci in U2OS cells with the indicated treatment are shown in upper panel. Scale bars, 10 μm. The lower panel is quantification. (F) Western blots of the carried MRE11 WT or MRE11 K673R OVCAR-8 cells treated with NALA and/or bleomycin for 24 h as indicated. (G–I) Representative picture of RPA2 (G), BrdU (H), and RAD51 (I) foci in as indicated U2OS cells in upper panel. Scale bars, 10 μm. The lower panel is quantification. (J) The diagram of AsiSI-ER U2OS system for detecting single-strand DNA generated by DNA end resection. (K) Detecting DNA end resection by qPCR using AsiSI-ER U2OS system. Statistical analyses were performed with the Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; NS stands for no significant change. See also Figures S3 and S4.

Figure 5.. Inhibition of MRE11 lactylation impairs DNA damage repair

(A) The HR levels of the carried MRE11 WT or MRE11 K673R HEK293T cells treated with/without NALA for 24 h as indicated were detected by FACS. (B) Western blots of the carried MRE11 WT or MRE11 K673R HEK293T cells treated with as indicated. (C) Representative pictures of comet assays as indicated. Scale bars, 10 μm. (D) Quantification of the data in (C). (E) Genomic instability was examined by metaphase spread assays as indicated. (F and G) Representative pictures of γ-H2AX staining in as indicated lung sections of mice (5 mice/group). Scale bars, 100 μm. (H) Representative pictures of intestinal regenerating villi of the indicated mice. Scale bars, 50 μm. (I) Representative pictures of γ-H2AX staining in as indicated lung sections of the indicated mice (5 mice/group). Scale bars, 100 μm. (J) Representative pictures of intestinal regenerating villi of as indicated mice. Scale bars, 50 μm. (K and L) Western blots of samples the indicated. Statistical analyses were performed with the Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; NS stands for no significant change. See also Figure S5.

Figure 6.. Inhibition of MRE11 lactylation sensitizes cancer cell to chemotherapy

(A) Analysis of HRD score in basal-like breast cancer samples. n = 140 samples. Statistical significance was tested using two-sided Mann-Whitney-Wilcoxon tests. (B–H) Colony formation assays were performed as indicated. (I) Western blots of four colon cancer patient-derived organoids (PDOs) as indicated. (J and K) As indicated, two PDOs with high lactylation of MRE11 K673 were employed to perform growth assays with CCK-8 (cell counting kit-8) assays. (L) PDO 223 with low lactylation of MRE11 K673 was employed to perform growth assays with CCK-8. (M) Western blots of three colon cancer patients derived xenografts (PDXs), as indicated. (N–P) Nu/Nu mice were transplanted subcutaneously with PDX from (M) and treated as indicated. Tumor images were acquired as shown in (N); tumor volumes were calculated (O); and weights (P) were measured. n = 5; data points in (O) and (P) represent mean tumor weight ± SD. Statistical analyses were performed with the Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; NS stands for no significant change. See also Figure S6.

Figure 7.. Targeting lactylation of MRE11 K673 via a peptidic inhibitor enhances chemotherapy sensitivity

(A) Schematic illustration of designed peptides. Green: cell-penetrating peptide (CPP). Red: site for MRE11 lactylation. (B–E) Western blots of samples as indicated. (F) Representative picture of MRE11 K673la, RPA2, and RAD51 foci in U2OS cells with the indicated treatment. Scale bars, 10 μm. (G–I) Quantification of K673la (G), RPA2 (H), and RAD51 (I) foci from the data in (F). (J) The HR levels of as indicated HEK293T cells were detected by FACS. (K) HCT116 cells were utilized to perform CCK-8 assays as indicated. (L) MDA-MB-231 cells were utilized to perform CCK-8 assays as indicated. (M) As indicated MDA-MB-231 cells were utilized to perform CCK-8 assays. (N and O) Nu/Nu mice were transplanted subcutaneously with PDX from Figure 6M and treated as indicated. Tumor images were acquired as shown in (N) and weights (O) were measured. n = 5; data points in (O) represent mean tumor weight ± SD. Statistical analyses were performed with the Student’s t test. *p < 0.05;**p < 0.01; ***p < 0.001; NS stands for no significant change. See also Figure S7.