mTOR regulates DNA damage response through NF-κB-mediated FANCD2 pathway in hematopoietic cells

Abstract

Hematopoietic stem/progenitor cells (HSPCs) function to give rise to mature blood cells. Effective DNA damage response (DDR) and maintenance of genomic stability are crucial for normal functioning of HSPCs. Mammalian target of rapamycin (mTOR) integrates signals from nutrients and growth factors to control protein synthesis, cell growth, survival and metabolism, and has been shown to regulate DDR in yeast and human cancer cells through the p53/p21 signaling cascade. Here, we show that gene targeting of mTOR in HSPCs causes a defective DDR due to a variety of DNA damage agents, mimicking that caused by deficient FANCD2, a key component of the Fanconi anemia (FA) DDR machinery. Mechanistically, mTOR(-/-) HSPCs express drastically reduced FANCD2. Consistent with these genetic findings, inactivation of mTOR in human lymphoblast cells by pp242 or Torin 1, mTOR kinase inhibitors, suppresses FANCD2 expression and causes a defective DDR that can be rescued by reconstitution of exogenous FANCD2. Further mechanistic studies show that mTOR deficiency or inactivation increases phosphorylation and nuclear translocation of nuclear factor (NF)-κB, which results in an enhanced NF-κB binding to FANCD2 promoter to suppress FANCD2 expression. Thus, mTOR regulates DDR and genomic stability in hematopoietic cells through a noncanonical pathway involving NF-κB-mediated FANCD2 expression.

Figure 1

mTOR deficiency leads to defective DDR. (a) Western blotting of mTOR protein in bone marrow cells. (b–d) DDR of mTOR ^−/− and mTOR^+/+ Lin⁻ cells in vitro. Lin⁻ cells were stimulated with or without melphalan (0.3 mg/ml). After 16 h, gH2AX foci formation was visualized by immunofluorescence and quantified (c). The images are representative of at least 30 cells (c). The percentage of positive cells (X6 gH2AX foci) was assessed from 30–50 nuclei (c). Damaged DNA was visualized by comet assay, and olive tail moment in at least 50 cells was calculated (d). After 48 h, the apoptotic cells were measured by FACS analysis of annexin V⁺ cells (b). (e) DDR of mTOR ^−/− and mTOR^+/+ Lin⁻ cells in vivo. Mice were injected i.p. with MMC (1 mg/kg body weight) and killed after 72 h. Lin⁻ cells were isolated and analyzed for damaged DNA by comet assay. (f) p53 activity in mTOR ^−/− and mTOR^+/+ Lin⁻ cells. Phospho- and total p53 in Lin⁻ cells was detected by Western blot. Data are represented as mean±_s.d. **P<0.01.

Figure 2

FANCD2 deficiency recapitulated DDR phenotypes of mTOR deficiency. (a) Quantitative RT-PCR analysis of FANCD2 expression in LSK cells. (b) Quantitative RT-PCR analysis of FANCA, FANCC, ATM and ATR expression in LSK cells. (c, d) DDR of FNACD2 ^−/− and FANCD2^+/+ Lin cells in vitro. Lin cells were stimulated with or without melphalan (0.3 μg/ml). After 16 h, damaged DNA was visualized by comet assay, and olive tail moment in at least 50 cells was calculated (c). After 48 h, the apoptotic cells were measured by FACS analysis of annexin V⁺ cells (d). (e) DDR of FNACD2 ^−/− and FANCD2^+/+ Lin cells in vivo. Mice were injected i.p. with MMC (1 mg/kg body weight) and killed after 72 h. Lin cells were isolated and analyzed for damaged DNA by comet assay. Data are represented as mean±_s.d. *P<0.05; **pP<0.01.

Figure 3

mTOR inhibition mimics FANCD2 deficiency with defective DDR in human cells. (a, b) FANCD2 expression in human cells upon mTOR inhibition. (a) Human JY B lymphoblasts (Hu-FANCD2^+/+) were treated overnight with or without mTOR kinase inhibitor pp242 (2.4 μ_M) (left) or Torin 1 (100 n_M) (right) and analyzed by quantitative RT-PCR for FANCD2 expression. (b) Hu-FANCD2^+/+ cells, human Fanconi anemia patient-derived PD20 cells deficient for FANCD2 (Hu-FANCD2 ^−/− ), and FANCD2-reconstituted Hu-FANCD2 ^−/− cells (Hu-FANCD2 ^−/− / FANCD2) were preincubated with or without pp242 for 1 h and then cultured overnight in the presence or absence of MMC (100 ng/ml). The cells were subjected to western blotting for FANCD2 and phopho-S6K. Topo1 was blotted as a loading control. Ub-FANCD2: ubiquitinized FANCD2. (c, d) DDR of Hu-FANCD2^+/+ cells after a treatment with pp242 and/or MMC. (e, f) Chromosome breaks and radial formation of Hu-FANCD2^+/+ cells after a treatment with pp242 and/or MMC. Hu-FANCD2^+/+ cells were preincubated with or without pp242 for 1 h and then cultured for 2 days in the presence or absence of MMC. Chromosomes were then subjected to cytogenetic analysis. A total of 50 cells from each sample were examined and scored. Arrowheads point to chromosome breaks in the Hu-FANCD2^+/+ + MMC cells or to radial structures in the Hu-FANCD2^+/+ + MMC + pp242 cells. As too many breaks were observed in Hu-FANCD2^+/+ + MMC + pp242 cells, the number of chromosome breaks per cell in these cells was artificially set to 10. (g) DDR of Hu-FANCD2 ^−/− and Hu-FANCD2^−/− /FANCD2 cells after a treatment with pp242 and/or MMC. Data are represented as mean±_s.d. *P<0.05; **P<0.01.

Figure 4

NF-κB mediates mTOR-dependent FANCD2 expression. (a) Western blotting of phospho- and total NF-κB p65 and p50 in Lin⁻ cells. (b) Western blotting of phospho- and total NF-κB p65 in whole-cell extracts (WCE), cytosolic and nuclear fractions of Hu-FANCD2^+/+ cells treated overnight with or without pp242 (2.4 μ_M) (left) or Torin 1 (100 n_M) (right). (c) Electrophoretic mobility shift assay (EMSA) on DNA binding activity of NF-κB to canonical, as well as four FANCD2-specific NF-κB consensus sites by using pp242-treated Hu-FANCD2^+/+ cells (upper). Supershift assays were performed to determine the binding specificity of NF-κB by using NF-κB-specific antibodies against p65 or p50 (lower). NF-1 DNA binding activity was assayed as a control. (d) Left hand panel: western blotting of phospho- and total NF-κB p65 in Lin⁻ cells treated overnight with or without NF-κB inhibitor JSH-23 (10 μ_M). Right hand panel: quantitative RT-PCR analysis of FANCD2 expression in JSH-23-treated Lin⁻ cells. (e) Left hand panel: western blotting of phospho-NF-κB p65 in Lin⁻ cells treated overnight with or without NF-κB inhibitory peptide NEMO (15 μ_M) or control peptide (15 μ_M). Right hand panel: quantitative RT-PCR analysis of FANCD2 expression in NEMO-treated Lin cells. (f) Left hand panel: western blotting of phospho- and total NF-κB p65 in NF-κB p65^+/+ (WT) and NF-κB p65 ^−/− MEFs treated for 12 h with or without pp242. Middle panel: western blotting of phospho- and total IKKα in IKKα^+/+ (WT) and IKKα ^−/− MEFs treated for 12 h with or without pp242. Right hand panel: quantitative RT-PCR analysis of FANCD2 expression in pp242-treated MEFs. Data are represented as mean±_s.d. **P<0.01.