Page 40 - Haematologica June

942
M. Wroblewski et al.
B-cell progenitors (Lin-B220+CD43+IgM–) were present in normal numbers after treatment (Figure 2A). However, numbers of late pre-B (Lin–B220+CD43–IgM–) and imma- ture B cells (Lin–B220+IgM+) were reduced by 2- and 5-fold, respectively (Figure 2B and C).
Moreover, JQ1-mediated BET inhibition caused deple- tion of absolute numbers of T cells (Figure 1G). Analysis of T-cell development in the thymus showed that, despite the reduction of T-cell numbers in the BM (Figure 1G), there were no developmental differences in double nega- tive (DN) or double positive (DP) thymocyte subpopula- tions (Figure 2D and E). Within the developmental stages of DN thymocytes, JQ1 did not influence the proportions of DN1, DN2, DN3 and DN4 progenitor subpopulations (Figure 2F). Notably, all differentiation stages of T-cell development showed increased apoptosis rates, whereas apoptosis of B-cell subsets was unchanged (Figure 2G and H).
On a transcriptional level, JQ1 treatment decreased the expression of anti-apoptotic mediator Bcl2 in the majority of sorted T-cell subsets (Figure 2I). In contrast, treatment did not affect the expression of pro-apoptotic Bak1 in these cell types (Online Supplementary Figure S1E). Further analyses revealed no changes in differential expression of other BH3 genes important for apoptosis during particular steps of the development of DN (Mcl1, Bcl2a1a, Bcl2l11), DP (Mcl1, Bclxl, Bcl2l11) or single positive CD4+ or CD8+ (Bclxl, Mcl1, Bcl2l11) T cells18-21 (Online Supplementary Figure S1F-I). This suggests that JQ1 induces apoptosis in T cells by reducing Bcl2 expression.
JQ1 treatment results in expansion and mobilization of HSC
Prompted by the high expression of BET family mem- bers in HSCs, we further dissected their expression pat- tern in different stages during HSC differentiation, such as LT-HSC, ST-HSC, GMP and MEP (Figure 3A). Interestingly, only Brd4 expression was highest in LT-HSC and this decreased with further differentiation via GMP to MEP, whereas no differential expression of Brd2 and 3 was detectable (Online Supplementary Figure S1C and D). Therefore, we examined the impact of JQ1 treatment on hematopoietic stem and progenitor cells (HSPC) in vivo.
Despite reduced cellularity in BM, the absolute numbers of phenotypic lineage-ckit+sca1+ cells (LSK) were increased 3-fold upon JQ1 treatment (Figure 3B). To rule out the pos- sibility that JQ1 treatment only affects developing hematopoiesis in young mice, we validated the JQ1- induced increase in LSK cells in adult mice (Online Supplementary Figure S2I). Interestingly, an increased cell count was only present in LT-HSC (CD34–CD135–LSK), ST-HSC (CD34+CD135–LSK) and MPP (CD34+CD135+ LSK) (Figure 3C and D), but not in more differentiated lin- eages such as GMP (lin–sca1–ckit+CD34+FcgRII+) or MEP (lin–sca1–ckit+ CD34–FcgRII–) (Online Supplementary Figure S2E and F).
To assess the functionality of hematopoietic progeni- tors, we evaluated the colony forming capacity of JQ1- treated MNC from BM and spleen in vitro. We found a 2- to 3-fold increase in total numbers of colony forming units (CFU) in BM and spleen in mice that had received JQ1 treatment (Figure 3E and F). Moreover, JQ1 treatment mobilized early hematopoietic progenitors into the PB, where we could detect a 2.5-fold increase in CFU per mL of blood (Figure 3G).
To further characterize their proliferation and ability to preserve stemness, we serially replated the CFU multiple times (Figure 3H). Intriguingly, and in contrast to control, JQ1-treated BM gave rise to constant CFU numbers after each replating, whereas CFU counts decreased rapidly in the control group. This indicates that, in addition to increasing proliferation, JQ1 treatment helps maintain the regenerative potential of hematopoietic progenitors, indi- cating a preservation of stemness.
To investigate potential species-to-species variations, we treated purified human CD34+ cells from BM with JQ1 in vitro and assed the colony-forming capacity via colony formation assays. Interestingly, JQ1 induced an increase in CFU numbers when compared to placebo treatment (Online Supplementary Figure S2J), indicating that JQ1 has a similar effect on both murine and human HSC.
To determine whether the accumulation of HSC results from increased proliferation or reduced differentiation, we analyzed BrdU incorporation into HSC in vivo. We found that LT-HSC from JQ1-treated mice incorporated more BrdU than those from control-treated littermates (Figure 3I), suggesting a JQ1-induced increase in HSC-cycling. Interestingly, more differentiated ST-HSC or the pool of LSK cells did not show increased cell cycle activity, indi- cating specificity of the JQ1-induced effect in LT-HSC (Online Supplementary Figure S2G and H). In addition, HSC proliferation was accompanied by a marginal increase in apoptosis of HSC (Figure 3J). These data strongly suggest that JQ1 promotes HSC proliferation, ultimately leading to an increase in the size of the HSC pool.
JQ1 treatment increases hematopoietic reconstitution after HSC transplantation
To measure HSC numbers and multipotency at a func- tional level, we carried out limiting dilution competitive repopulation transplantations. Therefore, we treated CD45.1 mice for three weeks with JQ1 or control before sacrifice. Subsequently, a mix of supporter BM from CD45.2 mice and JQ1- or control-treated BM from CD45.1 mice was transplanted into lethally irradiated CD45.2 recipients. Engraftment of HSC was evaluated by analyzing recipient PB contribution over four months.
Consistent with our in vitro studies, mice transplanted with JQ1-treated BM had higher levels of donor-derived chimerism during the whole observation period, indicat- ing an increased fraction of HSPC in the transplants (Figure 4A and B). Relative calculation of repopulating units (RU) revealed 2.5-fold more HSC in transplants when donors received JQ1 treatment [RUD (DMSO) = 2.15; RUD (JQ1) = 4.9]. In addition, extreme limiting dilu- tion analysis (ELDA) showed a 3-fold increase of HSC fre- quency in BM upon JQ1 treatment (DMSO: 1/19480 vs. JQ1: 1/6284; n=8-12; *P<0.05) (Figure 4C). Importantly, JQ1 and DMSO-treated HSC reconstituted all major hematopoietic lineages with similar frequencies (Online Supplementary Figure S3A-C). We, therefore, conclude that JQ1 induces HSC pool expansion in donors without induction of lineage priming after transplantation.
After six months, primary recipients were sacrificed and their BM was transplanted into secondary recipients. As it is well known that HSC engraftment is less effective in secondary recipients, we normalized the measured fre- quencies of CD45.1+ cells in secondary recipients (Online Supplementary Figure S4) to the known frequencies of their respective donor animals; this allowed us to analyze
haematologica | 2018; 103(6)