Apoptosis signaling in hematopoietic stem and progenitor cells
Homeostasis within the hematopoietic stem and progenitor cell (HSPC) pool is provided by an orchestrated interplay of proliferation, differentiation and cell death. While the former features and in particular self-renewal are well described, knowledge on HSPC survival and cell death still is limited. In their physiological home, the so called “stem cell niche”, HSPCs are provided continuously with a plethora of survival signals, consisting of cytokines (SCF, Flt3L), cell-cell contact (Notch signaling), and cell-matrix contact signals (integrins). Although long-term stem cells (LT-HSC) are thought to be relatively cell death-resistant, different genetic models give evidence that apoptosis actively contributes to HSPCs pool homeostasis. Two anti-apoptotic Bcl-2 family members, Bcl-xL and Mcl-1, have earlier been shown to be essential for murine HSPC survival during development and under homeostatic conditions indicating that especially the intrinsic apoptosis pathway is important for HSPC homeostasis.
Apoptosis gets even more relevant when the HSPC pool is exposed to stress. On one hand, cell death can be induced directly by stress signals (i.e. chemo- or radiotherapy) which transiently restrict hematopoiesis and lead to BM aplasia with anemia, thrombocytopenia and neutropenia. One the other hand, apoptosis is required to counteract excessive proliferation after termination of hematopoietic regeneration. Another therapeutically relevant form of stress leading to HSPC apoptosis occurs, when these cells are used for hematopoietic stem cell transplantation (HSCT). If apoptosis is inhibited during HSCT, transplantation efficacy can be robustly increased and reconstitution can be accelerated.
Pro-survival Bcl-2 proteins (Bcl-2 itself, Bcl-xL, Mcl-1 and A1) act by inhibiting Bax and Bak activation and thus mitochondrial outer membrane permeabilization (MOMP). Following to cell stress it comes to the activation of one or more pro-apoptotic BH3-only proteins, which then bind to their anti-apoptotic antagonists. Consequently, Bax and Bak are released and activated. BH3-only proteins thus act upstream of the mitochondrial apoptosis pathway as “cell stress sensors”. They are activated in a very cell stress but also cell type specific manner.
In HSPCs, we previously identified the BH3-only protein Puma to be important for DNA damage-induced apoptosis downstream of p53 (i.e. following to irradiation) (Labi/Erlacher et al, Genes and Development, 2010). In contrast, two other BH3-only proteins, Bim and Bmf, are main players during early and long-term reconstitution of the hematopoietic system following HSCT. Whereas Bmf-induced apoptosis seems to be more relevant during early stages of engraftment, HSPCs seem to die in a Bim-dependent manner even beyond the time of hematopoietic regeneration (Labi/Bertele/Erlacher, EMBO Mol. Med., 2013).
In summary, survival and apoptosis are tightly regulated to maintain homeostasis within the HSPC pool. Importantly, deregulation of apoptosis signaling results in disease: Insufficient cell death contributes to the formation of leukemia and lymphoma, whereas excessive apoptosis of HSPCs is a main feature of bone marrow failures and myelodysplastic syndromes. For many congenital bone marrow failures, excessive p53 activation has been shown to be causative for HSPC apoptosis and BM exhaustion (i.e. Dyskeratosis congenita, Fanconi Anemia, Diamond-Blackfan Anemia).
Currently, we focus on the BH3-only protein Puma, the main pro-apoptotic target of p53, and analyze its function during hematopoietic regeneration, aging and premature bone marrow exhaustion.
In a second project, we try to inhibit HSPC apoptosis in a transient manner: as described above, the BH3-only proteins Bim and Bmf limit early engraftment and long-term reconstitution of murine and human HSPCs, and their inhibition results in increased transplantation efficiency. We thus provide evidence that inhibition of the intrinsic apoptosis pathway could serve as a suitable therapeutic option to reduce HSCT-related morbidity and mortality. However, since permanent apoptosis inhibition in lymphocytes and/or their progenitors can trigger lymphomagenesis, such inhibition needs to be transient when used therapeutically. For this purpose we are currently using adenoviral constructs as well as a protein transduction system.
A further project aims to identify proteins which are essentially required for survival of human stem cells and for hematopoietic regeneration, and upstream signaling pathways which act in a protective manner.
For our projects, we established various in vitro and in vivo models including lentiviral RNAi systems suitable for human HSPCs, colony forming assays as well as deifferent transplantation models (murine and xenotransplantation models) which now will be available for our future projects.