Abstract
Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Mutation accumulation in HSCs precedes the development of leukemia and lymphoma, and these “pre-leukemic HSCs” can survive after chemotherapy, contributing to the relapse of the disease. Thus, understanding for the DNA damage response at a HSC level is a matter of critical importance for lifelong hematopoiesis, yet the protection mechanism for HSCs from DNA damage accumulation remains to be elucidated. During our study on the response of HSCs to ionizing radiation (IR), we have detected higher responsiveness of HSCs to DNA damage compared with committed progenitor cells: higher p53 activation was observed in HSC-enriched LSK (Lin-Sca1+cKit+) cells and LT-HSCs (CD150+CD41-CD48-LSK) than in myeloid progenitor-enriched LKS- cells. Of note, when treated with 4 Gy IR, LSK cells exhibited stronger upregulation of pro-apoptotic genes Bax, Noxa and Puma compared with LKS- cells, whereas upregulation of survival-contributing p21 and Mdm2 genes was comparable between the two populations. Corresponding to such characteristic behavior, we have identified apoptosis-stimulating protein of p53 1 (Aspp1) as a novel specific regulator of HSCs that provides HSCs with high sensitivity to apoptosis. We found that mRNA and protein of Aspp1 were specifically detected in LSK cells and LT-HSCs. To uncover the roles of Aspp1 in the regulation of HSCs, we evaluated HSCs of adult Aspp1 knockout (KO) mice. These mutant mice exhibited a major increase in the absolute number of LSK cells (1.5-fold; P<0.05) and LT-HSCs (2-fold; P<0.0005). Furthermore, self-renewal capacity of Aspp1-null HSCs was significantly enhanced as measured by serial competitive bone marrow (BM) transplantation assays (P<0.01). To assess the cause of enhanced self-renewal of Aspp1-null HSCs, we examined gene expression profile of Aspp1-null LSK cells before and after BM transplantation using multiplex quantitative RT-PCR array. Aspp1-null LSK cells showed higher expression of multiple quiescence-related genes including Tek, Mpl and Ndn. In line with this, Ki67 staining revealed that Aspp1-null LSK cells showed resistance to the loss of quiescence after serial BM transplantation (P<0.01), and Aspp1 KO mice showed accelerated recovery of peripheral blood and BM when treated with a single dose of 5-FU (P<0.05). Moreover, when serially transplanted or subjected to 4 Gy IR in vivo, Aspp1-null LSK cells exhibited higher resistance to apoptosis which was detected as decreased proportion of Annexin V-positive cells (P<0.05). Gene expression analysis consistently revealed that the induction of pro-apoptotic genes Bax, Noxa and Puma was impaired in irradiated Aspp1-null LSK cells. As a result of the reduced apoptosis, Aspp1-null LSK cells exhibited the tendency to retain persistent DNA damage after genotoxic stress as assessed by γH2AX and 53BP1 foci (chi-square test, P<0.05). Importantly, by breeding Aspp1 KO mice with Mx1-Cre mice and p53flox/flox mice, we verified that Aspp1 synergized with p53 to regulate self-renewal and genomic integrity of HSCs beyond its canonical p53-dependent function. Aspp1 loss further enhanced self-renewal capacity of HSCs in a p53-null background when assayed by serial BM transplantation (P<0.05). Likewise, Aspp1 deficiency further accentuated the accumulation of DNA damage after IR exposure in the absence of p53 (P<0.05). Consequently, whereas approximately half of the recipients receiving p53-null LSK cells died of thymic lymphoma, the recipient mice transplanted with LSK cells deficient for both Aspp1 and p53 were 100% lethal within 6 months after BM transplantation (log-rank test, P<0.01). These mice succumbed to hematological malignancies, mostly T-cell acute lymphoblastic lymphoma and leukemia (ALL) (88%) but also B-cell (6%) and myeloid (6%) malignancies. Taken together, our study demonstrates that Aspp1 attenuates HSC quiescence and induces apoptosis in damaged HSCs, in both p53-dependent and -independent manners, thereby inhibiting the development of leukemia and lymphoma in conjunction with p53 in HSCs. As loss of Aspp1 expression due to aberrant methylation of its promoter has already been proven to be an independent poor prognosis factor in ALL patients, Aspp1 may be a potential target for stem cell-directed therapy of leukemia and lymphoma.
Disclosures
No relevant conflicts of interest to declare.
Combined mild hypoxia and bone marrow mesenchymal stem cells improve expansion and HOXB4 gene expression of human cord blood CD34+ stem cells