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after lethal irradiation is impaired when the transplanted hematopoietic stem and progenitor cells harbor an shRNA-mediated knockdown of Brd4 (Brd4 KD).26 The reduced repopulative capacity of Brd4 KD HSCP at the LSK level initially seems to contradict our findings. One obvious reason for this discrepancy might be that the shRNA strategy only focuses on the inhibition of Brd4, whereas JQ1 targets the entire BET family. In addition, BRD4 contains other protein domains such as an extra-ter- minal domain, the function of which is not influenced by JQ1.27
Although the effect of JQ1 described in our study most likely results from the increased number of HSC, other data link it to increased HSC fitness. In colony formation assays, the increased CFU numbers arise from the expand- ed pool of stem and progenitor cells. However, their increased regenerative potential seen in replating assays may also result from increased HSC fitness. Similarly, the increased contribution of JQ1-treated HSC to hematopoiesis in secondary recipients results from the increased HSC numbers in the graft. However, the decline of the relative contribution to hematopoiesis was slower in the animals that received JQ1-treated HSC, which might indicate enhanced fitness. As in our study it is diffi- cult to discriminate between the effects of JQ1 on HSC quantity and quality, future experiments transplanting equal numbers of JQ1- or control-treated HSC are war- ranted.
It is worthy of note that the supporter BM cells from the first transplantation outcompeted the DMSO-treated HSC in secondary recipients starting at 12 months after trans- plantation. Although it is well known that the fitness of HSC dramatically decreases in secondary recipients,22 we also observed an unusually reduced median survival, par- ticularly in the DMSO arm, although this effect was not statistically significant; this has to be considered when interpreting the data. Therefore, future studies are needed to identify the underlying factors for this effect.
Although allogeneic or autologous HSCT is an impor- tant option for the treatment of leukemia, severe autoim- munity or myelosuppression,28 due to different limitations, not all patients benefit. It has been shown that the quan- tity and quality of HSCs are critical factors determining patient survival and time required for engraftment after HSCT.29
Between 2-5% of stem cell donors do not adequately respond to G-CSF treatment and the number of CD34+
HSC mobilized is not sufficient; these donors are called ‘poor mobilizers’.30-33 As a donor’s response to G-CSF varies,34 and HSCT programs have to, therefore, deal with these obvious uncertainties, alternative approaches are needed to obtain sufficient numbers of HSC for transplan- tation. We were able to show that, besides HSC prolifera- tion, BET inhibition by JQ1 also increases the mobiliza- tion of HSC into the PB. Our data, therefore, indicate that BET inhibition might represent an alternative approach to yield increased numbers of HSC. However, future studies are necessary to verify whether this approach is safe or applicable for the G-CSF-independent mobilization of stem cells in humans.
In this context, it should be considered that JQ1 treat- ment has opposing effects on some mature hematopoietic cell populations and HSC. This leads to the question as to whether the reduction of B and T cells might compromise the beneficial effects on HSC during repopulation of the hematopoietic system. In cases in which the detrimental effect on B and T cells persists even after discontinuation of BET inhibitor treatment in a transplantation setting, this would make HSCT recipients more prone to infections, thereby counteracting the beneficial effects of increased HSC numbers in the grafts. Therefore, further experi- ments are warranted to clarify the consequences of (tran- sient) BET inhibition on B and T cells and the outcome of HSCT.
Acknowledgments
The authors would like to thank the FACS Core Facility (UKE, Hamburg, Germany) for helping with flow cytometry.
Funding
SL was supported by the German Research Council (DFG LO1863/3-1), by the Margarethe Clemens Stiftung, by a Starting Grant of the European Research Council and is the recipient of a Heisenberg professorhip (DFG, LO1863/4-1); MWr was supported by the Medical Faculty of the University of Hamburg (FFM program); RB is an Erwin-Schrödinger fellow of the Austrian Science Fund (FWF); SK is grateful for support by the Structural Genomics Consortium (SGC); MWa is supported by the Research Training Group Translational Research Innovation - Pharma (TRIP), supported by the Else Kröner- Fresenius Foundation (EKFS). SAJ is supported by the German Research Council (DFG, JO 815/3-2) and the German Cancer Aid-funded PiPAC consortium (70112505). JQ and LW are supported by the NIH/NCI P01 CA066996.
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