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M. Wroblewski et al.
and relapse.
In recent years, increasing evidence has linked epigenet-
ic dysregulation with aberrant gene expression ultimately leading to the development of cancer. It was shown that 50% of human cancers harbor mutations in enzymes that are important for proper chromatin organization.3 Members of the family of bromodomain and extra termi- nal domain (BET) proteins include BRD2, BRD3, BRD4 and BRDT which function as epigenetic readers and have been shown to facilitate transcription through interaction with acetylated histones.4 Chromatin binding of BET pro- teins is known to drive MYC expression5 which is an oncogenic driver in several hematologic malignancies such as acute myeloid leukemia (AML),6 lymphoma7 or multi- ple myeloma (MM).8
Consequently, epigenetic regulators have become attractive therapeutic targets and, despite their toxic and non-specific side effects, drugs interfering with epigenetic pathways have entered clinical practice.9
The BET inhibitor JQ1 binds with highest affinity to bromodomain 1 of BRD4 and prevents it binding to acety- lated histones at promoters and linage specific enhancers thereby decreasing transcription of some lineage specific genes.10 At a therapeutic level, it exerts potent anti-cancer effects in a broad range of human AML subtypes,5 acute lymphoblastic leukemia (ALL),11 and MM,12 and the struc- turally similar BET inhibitor (OTX015) recently showed promising clinical results in these patients.13,14
Although it has recently been shown that JQ1 decreases pluripotency of embryonic stem cells by suppression of Nanog and Lefty1,15 the consequences of BET inhibition for adult HSC have not yet been analyzed in detail.
The fact that BET inhibitors are already used in clinical studies today underlines the need for a systematic approach to understand the impact of pharmacological BET inhibition on healthy hematopoiesis.
We, therefore, addressed this issue and show for the first time that BET inhibition by JQ1 induces expansion of HSC thereby promoting recovery of the hematopoietic system after myelosuppression or stem cell transplantation.
Methods
More detailed information can be found in the Online Supplementary Appendix.
Animals
All experiments were carried out in accordance with the institu- tional guidelines for the welfare of animals and were approved by the local licensing authority (Behörde für Soziales, Gesundheit, Familie, Verbraucherschutz; Amt für Gesundheit und Verbraucherschutz, Hamburg, Germany, project n. G128/16).
Treatments and mixed bone marrow chimeras
Ly5.1 animals received daily intraperitoneal (i.p.) injections with 50 mg/kg JQ1 or control for 21 days. To generate bone marrow (BM) chimeras, BM of treated Ly5.1 mice was mixed with 2x105 untreated Ly5.2 supporter BM cells and injected intravenously (i.v.) into lethally irradiated Ly5.2 mice (9 Gy). Transplantations were performed with total numbers of BM cells per condition and not corrected for equal numbers of HSC. Detailed information about the normalization in secondary recipients and the calculation of repopulating units can be found in the Online Supplementary Appendix.
Hematopoietic recovery after sublethal myeloablation
Mice received daily i.p. injections with 50 mg/kg JQ1 or control for 21 days. Myeloablation was then induced via sublethal irradi- ation (5 Gy). Hematopoietic recovery was monitored in peripheral blood (PB) and BM.
Colony formation assays
Colony formation assays for quantification of hematopoietic stem and progenitor cells (GF M3434, H4034 Optimum) or megakaryocytes (MegaCult-C) were performed according to the manufacturer’s instructions.
ELISA and RT-qPCR
Transcript levels were analyzed using Power SYBR Green RT- qPCR and protein concentrations were determined using ELISA according to the manufacturer’s instruction.
Flow cytometry
Different cell populations in thymus, PB, BM and spleen were stained with antibodies and analyzed on a FACS Canto II flow cytometer.
Statistical analysis
Data represent mean±Standard Error of Mean (SEM) of repre- sentative experiments, which were carried out at least in dupli- cates, unless otherwise stated. Statistical significance was calculat- ed by Student's t-test or ANOVA. Statistical significance in Kaplan- Meier plots was calculated by log-rank test.
Results
JQ1 treatment alters normal hematopoiesis in a cell context-dependent manner
As a first step, we analyzed the mRNA expression pat- terns of Brd2-4 in all major hematopoietic subpopulations, because BET family members represent the main molecu- lar targets of JQ1.5 Expression of Brd4 mRNA was most abundant in megakaryocytes (MK), followed by B cells, T cells and HSC (Figure 1A). Brd2 expression showed a sim- ilar distribution whereas Brd3 expression was highest in MK and T cells (Online Supplementary Figure S1A and B).
To characterize the effect of BET inhibition by JQ1 on hematopoietic development in vivo, we analyzed 7-week old mice which were treated with 50 mg/kg JQ1 for three weeks, a dose level previously used in literature.5,16
As reported previously, JQ1 treatment resulted in delayed growth of mice (Online Supplementary Figure S2A). Moreover, JQ1-treated mice were leukopenic (Figure 1B) and showed reduced numbers of mononucleated cells (MNC) in the BM (Figure 1C). In contrast, the numbers of erythrocytes and platelets were not affected by JQ1 (Online Supplementary Figure S2B and C). Spleen and thy- mus weight were reduced upon treatment, indicating a JQ1-induced reduction of immune cells in secondary lym- phoid organs in agreement with the literature16 (Figure 1D and E). Indeed, flow-cytometric analysis of lymphoid and myeloid cell populations showed that absolute cell num- bers of both B220+ B cells and CD3+ T cells were lower after treatment (Figure 1F and G). In contrast, absolute cell numbers of myeloid cells remained constant (Online Supplementary Figure S2D) indicating a more lymphoid- specific and cell context-dependent role of BET inhibition by JQ1.
It has been shown that JQ1-mediated effects occur due
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