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Before irradiation, BM of JQ1-treated mice contained 1.6- fold higher numbers of HSC when compared to placebo- treated mice. Interestingly, irradiation led to a similar reduc- tion in HSC in both groups two weeks after treatment, indi- cated by an unchanged HSC ratio of 1.6-fold (Figure 5A). Similar observations were made for MPP (Figure 5B). This indicates that, despite increasing cell cycle activity, JQ1 does not sensitize HSC to irradiation at a dose level of 5 Gy.
In response to irradiation, cell counts of all major hematopoietic lineages decreased by 75-99% in both groups, with B and T cells being most affected one week after irradiation (Online Supplementary Figure S5A). Interestingly, and in line with the increased numbers of HSC and MPP, JQ1-treated mice showed a significantly faster recovery of all lineages when compared to animals before irradiation.
Within the whole post-irradiation period, the relative recovery of leukocytes was significantly faster in JQ1-treat-
ed mice than in the control cohort (Figure 5C). Six weeks after irradiation, total leukocyte counts of JQ1-treated mice fully recovered to levels before irradiation (94±7%; n=5; *P<0.05), whereas recovery was slower in the control group (47±5%; n=5; *P<0.05). Strikingly, recovery of most hematopoietic lineages such as B cells, T cells, monocytes, thrombocytes or granulocytes was accelerated when ani- mals received JQ1 treatment before irradiation (Figure 5D- H). In contrast, the recovery of erythrocytes was delayed upon JQ1 treatment; however, levels never dropped to crit- ical values and fully recovered after irradiation (Online Supplementary Figure S5B).
Although JQ1 induces cycling of HSC, this mild increase does not seem to be sufficient to render HSC more suscep- tible to irradiation with 5 Gy. Still harboring increased num- bers of HSC two weeks after irradiation, JQ1-pre-treated animals recover faster from sublethal myelosuppression than their placebo-treated littermates.
ABC
DEFG
Figure 3. JQ1 treatment results in expansion and mobilization of hematopoietic stem cells (HSC). Animals received daily intraperi- toneal (i.p.) injections of 50 mg/kg JQ1 for 21 days after which the dif- ferent parameters were analyzed. (A) Different FACS-sorted hematopoietic stem and progeni- tor cells from bone marrow (BM) of mice were analyzed for baseline
HIJ mRNA expression of Brd4 via qRT- PCR (n=2-3; *P<0.05). (B-D) Flowcytometric quantification of phenotypic lineage-ckit+sca1+ cells (LSK) (B), ST-HSC and LT-HSC (C), as well as MPP (D) in BM (n=5-9; *P<0.05). (E-G) Colony formation assays to quantify hematopoietic progenitors in BM (E), spleen (F), and peripheral blood (G) (n=3-8; *P<0.05). (H) Replating of CFU from BM (n=3; *P<0.05). (I) Flowcytometric quantification of BrdU incorporation in LT-HSC in BM (n=5; *P<0.05). (J) Flowcytometric quantification of apoptosis in LT-HSC in BM using
Annexin V (n=5; *P<0.05).
haematologica | 2018; 103(6)