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E.-M. Demmerath et al.
Minor in vivo effects of the anti-apoptotic cytokines FLT3L and thrombopoietin
We next wondered whether substances known to inhib- it apoptosis by interfering with the BCL-2 protein family would be able to protect human hematopoiesis from irra- diation-induced injury in vivo. We focused on the cytokines FLT3L and TPO that suppress activation of the BH3-only proteins BIM and BMF in both murine and human HSPCs but also have proliferative effects.18 Sublethally irradiated mice were treated daily with FLT3L, TPO or their combination. While treatment of either FLT3L or TPO alone did not have any effects on regenera- tion of human hematopoiesis (data not shown), combined treatment for seven days resulted in a relative increase of human cells in BM and a mildly increased number of human cells in the spleen (Figure 5A-D and Online Supplementary Figure S5A). Both effects were transient, and no increase in human cell numbers was observed when cytokine combination was administered for 14 days (Figure 5E-H). To investigate the frequency of human HSPCs giving rise to colonies, we isolated 150,000 human CD45+ cells from BM of untreated and treated mice and seeded them into methylcellulose medium. There was no significant difference in colony numbers between mice treated with cytokines and control mice (Online Supplementary Figure S5B). Finally, we started TPO/FLT3L treatment 24 h prior to irradiation, but again did not see any protective effects on human hematopoiesis in vivo (data not shown).
Discussion
Although there is a strong clinical need to reduce hema- tologic side effects in cancer patients treated with chemo- or radiotherapy, to date, only few therapeutic options are available.11,12 This is in contrast to the many chemical com- pounds and endogenous substances that were described to be radioprotective in vitro or in mouse models. A major limitation in translating preclinical findings to routine clin- ical practice is the lack of suitable model systems. While research on primates is laborious, expensive and not feasi- ble everywhere, xenograft models that allow studies on human cells in vivo can be a good alternative.
The first aim of this study was to generate a xenograft mouse model of human hematopoiesis that can be used to analyze hematopoietic regeneration following sublethal stresses. Upon successful engraftment of human hematopoietic cells, recipient mice were subjected to total body irradiation (TBI) or daily etoposide treatment. Immediately after irradiation, or in parallel to etoposide administration, treatment with possibly protective sub- stances was initiated and continued for one week before the human hematopoiesis was analyzed in detail. Although the substances tested in this work did not per- form convincingly, we successfully demonstrated that our model can be used to investigate the presumed radiopro- tective effects of given molecules or chemical compounds on the human hematopoietic system treated with chemo- or radiotherapy in vivo. In addition, our model can be used to test novel cytotoxic drugs for their hematotoxicity by substituting TBI with repeated drug treatment. One short- coming of our model is that the murine microenvironment does not provide optimal support to human hematopoiet-
ic cells. This should, however, be no major issue since radio- and intensive chemotherapy also harm the human hematopoietic niche, thereby imposing an additional layer of stress to HSPCs. A more relevant shortcoming is that irradiation and cytotoxic drugs result in depletion of both human and murine cells, implicating that human cells have to compete against the more dominant murine cells during the stage of subsequent regeneration. Relative increase in human cells within a murine tissue, as observed after TBI and 7-day treatment with TPO/FLT3, or when we applied etoposide together with dmPGE2, might thus indicate that human cells are favored even when absolute human cell numbers do not increase.
The substances used here were carefully selected on the basis of published data and own earlier work. However, despite the very promising data obtained in mouse mod- els,9,10 neither EGF nor PGE2 were able to promote hematopoietic regeneration in our model system. In the case of EGF, non-conserved pathways seem to underlie this inconsistency. As for PGE2, both its antiproliferative and toxic effects might contribute to its lack of in vivo effi- cacy. Anyhow, the severe gastrointestinal side effects pro- voked by the combination of dmPGE2 and TBI/etoposide would render such a treatment unfeasible. FLT3L and TPO are both known to increase viability and promote prolifer- ation of human HSPCs in vitro. Along that line, we observed some beneficial effects on human hematopoietic regeneration in vivo, even though these were marginal and transient.
One could speculate that the maximal beneficial effect on human hematopoietic regeneration can be achieved by the use of substances that foster both survival and prolif- eration of HSPCs. Certainly, short-term complications such as febrile neutropenia or transfusion-dependent ane- mia and thrombocytopenia could be reduced by increas- ing the proliferative capacity of HSPCs following chemo- or radiotherapy. Repeated cycles of forced proliferation, however, could result in premature stem cell exhaustion and BM failure in the long term; this has already been shown when mice were repeatedly treated with SCF.19 Forced proliferation of DNA-damaged HSPCs, as caused by chemo- or radiotherapy, together with selection pres- sure could also increase the risk of genetic instability and clonal evolution eventually leading to t-MDS and second- ary AML.
Ideally, radioprotective substances should not stimulate proliferation but only inhibit apoptosis of healthy BM cells. Apoptosis resistance reduces therapy-induced myelosuppression, and at the same time lowers the risk of secondary leukemia by prolonging the time available for DNA repair and reducing compensatory proliferation and selection pressure.2,15,20,21 ROS scavengers, for example, are radioprotective by preventing both DNA damage and apoptosis.11
Direct inhibition of the pro-apoptotic effector proteins, BAX and BAK, could also keep hematopoietic cells alive without affecting proliferation. We recently showed that such transient apoptosis resistance can be achieved by short-term overexpression or protein transduction of the anti-apoptotic protein BCL-XL.22 Translation to clinical use will probably be pushed ahead by the development of specific BAX/BAK inhibitors.
Similar approaches will be useful to reduce risk of graft failure and shorten the time to full hematopoietic regener- ation in the case of autologous or allogeneic hematopoiet-
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haematologica | 2019; 104(4)