mTOR Regulates DNA Damage Response of Hematopoietic Stem and Progenitor Cells Through Modulation of Fanconi Anemia Core Complex

Abstract 864

The mammalian target of rapamycin (mTOR) integrates signals from nutrients, growth factors, and cellular energy status to control protein synthesis, cell growth, proliferation, survival and metabolism in various cancer cells, but its physiological function in the hematopoiesis process and signaling role in hematopoietic stem cell (HSC) regulation remain unknown. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate megakaryocyte and dendritic cell proliferation and differentiation. Hyperactivation of mTOR by deletion of the negative regulators of mTOR, TSC1/TSC2 or PTEN, causes a loss of quiescence and long-term exhaustion of HSCs. Since conventional gene targeting of mTOR leads to early embryonic lethality, a conditional mTOR knockout mouse model has recently been generated. We have produced mTOR^flox/flox; Mx-Cre compound mice that allow interferon-induced mTOR deletion in bone marrow (BM) following a transplantation and polyI:C induction protocol. We found that depletion of mTOR drastically affected hematopoiesis: the mTOR^flox/flox;Mx-Cre BM recipient mice showed a marked reduction in total BM cellularity and in the numbers and frequency of myeloid and lymphoid cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, after induced mTOR deletion, resulting in bone marrow failure and lethality. Interestingly, the numbers of hematopoietic stem and progenitor cells (HSPCs; Lin⁻Sca-1⁺c-Kit⁺) and HSCs (CD150⁺ CD41⁻CD48⁻ Lin⁻Sca-1⁺c-Kit⁺) in bone marrow increased transiently by approximately 5- and 8-fold, respectively, while the numbers of early progenitors (CMP, GMP, MEP, CLP) were mildly affected in the mutant mice 7–14 days after polyI:C treatment. While the more mature lineage committed mTOR^−/− blood cells showed a cell cycle blockage and significantly increased apoptosis, mTOR^−/− HSPCs and HSCs displayed a loss of quiescence and increased proliferation, as assessed by 5-bromodeoxyuridine incorporation assays, and a normal survival index. Transplantation of mTOR^−/− BM cells into immunodeficient or syngeneic mice demonstrated that the mTOR^−/− HSPCs failed to engraft and repopulate in the recipients. At the molecular level, mRNA microarray, quantitative real-time PCR and immunoblotting analyses of mTOR^−/− HSPCs or Lin⁻ cells revealed that the cell cycle inhibitor Rb was downregulated while the positive regulator of cell cycle E2F5 and pro-survival regulators MCL1 and BCL-xL were upregulated. mTOR deficiency abolished the activation of translational regulators S6K and 4E-BP but led to an increased activation of Akt. In addition, mTOR deficiency sensitized Lin⁻ cells to DNA damage induced in vitro or in vivo by melphalan or mitomycin C (MMC), evidenced by a marked increase in γH2AX foci as well as DNA double-strand breaks (comet-tailed value of 30.2 ± 7.6 in mTOR^−/− cells treated in vitro with melphalan and 37.6 ± 3.4 in mTOR^−/− cells treated in vivo with MMC compared to 7.6 ± 2.1 in melphalan-treated WT cells and 17.3 ± 6.7 in MMC-treated WT cells, respectively). The increased DNA damage response can be attributed to an ∼300-fold reduction of the expression of FANCD2, a key component of the Fanconi DNA damage repair complex. Significantly, the effect of mTOR deficiency on Fanconi gene expression was specific to FANCD2, because the expression of other Fanconi proteins such as FANCA and FANCC was not affected in mTOR^−/− Lin⁻ cells. Intriguingly, the mTOR^−/− Lin⁻ cells phenocopied the DNA damage response of FANCD2^−/− Lin⁻ cells in vitro and in vivo. Similar effects of reduced FANCD2 expression and dampened DNA damage response were observed in human lymphoblasts treated with pp242, a mTOR kinase inhibitor. FANCD2-deficient human Fanconi anemia patient cells recapitulated the pp242-induced DNA damage phenotypes that could be rescued by FANCD2 reconstitution. Taken together, these results demonstrate that mTOR is a critical regulator of HSC quiescence and engraftment through the regulation of cell cycle machinery and is essential in multiple stages of hematopoiesis. Moreover, mTOR is required for maintaining genomic stability of HSPCs through modulation of the Fanconi anemia DNA damage response pathway.