Molecular Recognition of BRDT by JQ1
To assess competitive binding to BRDT(1), we devised homogeneous, luminescence proximity assays that are capable of quantifying binding of a synthetic, biotinylated tetra-acetylated histone 4 peptide (H4Kac4, residues 1–20) to recombinant epitope-tagged murine or human BRDT(1). Dose-ranging studies of (+)-JQ1 demonstrated potent inhibition of H4Kac4 binding with a half-maximum inhibitory concentration (IC50
) value of 10 nM for murine BRDT(1) and 11 nM for human BRDT(1) (D). In contrast, the (−)-JQ1 enantiomer was inactive for either ortholog, establishing stereospecific, ligand-competitive binding. To confirm competitive inhibition of the acetyl-lysine binding module, isothermal titration calorimetry (ITC) was performed on human BRDT(1), employing a synthetic H4Kac4 peptide in the presence and absence of (+)-JQ1 (E). This peptide was found to bind two protein modules concomitantly with a Kd
of 25.5 μM, in good agreement with published data (Morinière et al., 2009
). This interaction was directly inhibited in the presence of (+)-JQ1 that partially saturated the acetyl-lysine binding (ratio 1:0.8) due to solubility limits of the inhibitor. The remaining unoccupied sites bound the H4Kac4 peptide with identical affinity and expected stoichiometry (Table S1
). Binding free energies and dissociation constants (KD
) determined by ITC for each bromodomain of human and murine BRDT confirm potent, specific (ratio 1:1) interactions with all domains (KD
= 44–190 nM; F). Notably, (+)-JQ1 binds murine BRDT(1) with high ligand efficiency (LE = 0.30), compared to reported inhibitors of protein-protein interactions (Wells and McClendon, 2007
The mode of binding of (+)-JQ1 to BRDT(1) was established by X-ray crystallography at high-resolution ( and Table S2
). Similar to the structure of murine BRDT(1) (Morinière et al., 2009
), human BRDT(1) exhibits the canonical structural features of a bromodomain, namely a left-handed bundle of four antiparallel α helices (αZ, αA, αB, and αC) and a hydrophobic acetyl-lysine binding pocket defined by interhelical ZA and BC loops (A–2C) (Dhalluin et al., 1999
). The observed, extraordinary shape complementarity between (+)-JQ1 and BRDT(1) explains the increased potency of (+)-JQ1 compared to the physiologic H4Kac4 ligand. In addition, (+)-JQ1 engages all surface residues analogous to those on murine BRDT(1) mediating molecular recognition of H4K5ac8ac (D and 2E) (Morinière et al., 2009
). Notable examples include a hydrogen bond linking the triazole ring and the evolutionarily conserved asparagine (Asn109), as well as extensive hydrophobic contacts with the ZA and BC loop regions (B). These biochemical and structural studies confirm and explain potent, acetyl-lysine competitive inhibition of BRDT(1) by (+)-JQ1 (hereafter referred to as JQ1).
Molecular Recognition of BRDT by JQ1
Testis Bioavailability of JQ1
To test the ability of JQ1 to reach its contraceptive target cells, the testicular bioavailability of JQ1 was assessed by serum and tissue pharmacokinetic analysis. Following a single intraperitoneal (i.p.) dose of JQ1 (50 mg/kg) in male mice, measurements of JQ1 concentration were made in serum, testis, and brain (A and Table S3
). Overall, JQ1 exhibits excellent testicular bioavailability (AUCtestis
= 259%), suggesting preferential distribution into this tissue compartment with rapid (Tmax
= 0.25 hr) and pronounced exposure (Cmax
= 34 μg/ml). Corroborating barrier permeability by JQ1, nearly uniform blood-brain barrier permeability was observed after single-dose pharmacokinetic studies (AUCbrain
BRDT Inhibition with a Testis-Permeable Small Molecule Reduces Testis Size and Sperm Counts and Motility
JQ1 Impairs Sperm Count and Motility
To determine the possible consequences of blocking BRDT function in vivo, we tested the spermatogenic effects of JQ1 administered to male mice. First, juvenile or adult male mice were administered daily i.p. injections of JQ1 (50 mg/kg once daily [QD]) or vehicle control over a 3 or 6 week period. After drug or vehicle treatment, mice were either sacrificed or mated to females while continuing to receive JQ1. All JQ1-treated males had a significant reduction in testis volume (B and 3C). With daily injections of 50 mg/kg JQ1, males treated from 3–6 weeks of age showed a reduction to 75.4% of control, males treated from 6–9 weeks of age showed a reduction to 54.7%, and males treated for 6 weeks with JQ1 (6–12 weeks of age) showed a reduction to 40.6% (C). Thus, longer treatment with JQ1 has the most prominent effect on testicular volume.
To examine the effect on sperm production, sperm counts were determined after JQ1 or vehicle treatment. Epididymal sperm number was reduced to 28% of control after 3 weeks of 50 mg/kg daily exposure (3–6 weeks of age) and 11% of control after 6 weeks of treatment (6–12 weeks of age) (D). Analysis of sperm motility after 3 weeks of JQ1 (50 mg/kg QD) revealed a significant, 4.5-fold reduction in sperm motility (16.0% for the JQ1-treated versus 72.4% for the control; E). Following 6 weeks of daily JQ1, only 5% of the sperm demonstrated progressive motility compared to 85% of sperm from controls (). Thus, daily exposure to JQ1 quantitatively reduced sperm number and motility.
Motility and Histological Effect of JQ1 Treatment on the Epididymides of Male Mice, Related to
Recently, the tolerability of twice-daily administration of JQ1 was reported in a confirmatory study of JQ1 in hematologic malignancies (Mertz et al., 2011
). Pharmacokinetic study of twice-daily (BID) JQ1 (50 mg/kg) confirmed excellent penetration of the blood:testis barrier and demonstrated a marked increase in drug exposure in the testis (AUC0–24hr
= 96,640 hr
ng/ml; F and Table S4
), accompanied by apparent partitioning into this compartment (AUCtestis
= 236%). With BID injections of JQ1 (JQ1 50 mg/kg), adult males treated for 3 weeks (9–12 weeks) showed reduction in testis weight to 63.3% of controls (G). Paralleling the reduction in testis weights, BID treatment resulted in reduction in sperm in the cauda epididymis to 35.6% of the control (H), a dramatic 5.8-fold reduction in sperm motility (11.2% for JQ1-treated versus 64.5% for controls; I), firmly establishing the tolerability and antispermatogenic effect of BID JQ1 in mice. Altogether, these findings are similar to defects observed in mice deficient in BRDT(1) (Shang et al., 2007
JQ1 Selectively Targets Male Germ Cells without Affecting Hormone Levels
Based on the observed inhibition of spermatogenesis, we performed further mechanistic studies of BRDT inhibition. Consistent with the reduction in testis volume, the cross-sectional area of seminiferous tubules from testes of males treated daily from 3–6 weeks with JQ1 were 84.9% of controls, males treated from 6–12 weeks with JQ1 were 56.4%, and males treated from 9–12 weeks with JQ1 BID were 65.0% of controls (J). Histological examination established a JQ1-dependent decrease in the amount and number of tubules that had obvious and abundant round spermatids and spermatozoa in the lumen (A–4H). This was particularly apparent in stage VII tubules, in which a marked reduction in round spermatids was observed in JQ1-treated mice. Of note, mice treated with JQ1 BID for 3 weeks had a 79% reduction in round spermatids in stage VII tubules (112.7 ± 7.4 [controls] versus 23.9 ± 3.6 [JQ1-treated]; p < 0.0001). Analysis of epididymides of JQ1-treated males also showed a similar finding in which fewer sperm were observed in the epididymal lumen compared to their abundance in controls (). Detailed microscopic analysis of the testes of mice treated with JQ1 revealed seminiferous tubule degeneration, sloughing of germ cells, and multinucleated symplasts (F and 4H; black arrows), findings also described in cyclin A1 (Ccna1
) knockout mice, which are infertile due to a spermatocyte block (Liu et al., 1998
). Thus, JQ1 treatment causes a block in spermatogenesis and a resultant reduction in testicular production of spermatozoa.
Histological Analysis of the Antispermatogenic Effects of JQ1
Because absence of glycoprotein hormone and abrogation of androgen signaling pathways cause male infertility (Matzuk and Lamb, 2008
), we measured the serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone after treatment with JQ1. Differences in the levels of FSH, LH, and testosterone were not statistically significant between control and JQ1-treated males (K–3M). Likewise, weights of the seminal vesicles, a major androgen-responsive tissue, were similar (). These results are consistent with the presence of histologically normal testosterone-producing Leydig cells in JQ1-treated males (A and 4B; red arrows). Thus, JQ1 effects are specific to germ cells and do not alter hormone-dependent processes.
Molecular Analysis of the Germ Cell Defects in JQ1-Treated Testes
To molecularly define the consequences of BRDT inhibition by JQ1, we performed genome-wide expression analysis of males treated with 50 mg/kg JQ1 daily for 6 weeks (6–12 weeks of age). Hierarchical clustering separated samples by treatment assignment and identified 1,685 unique genes enriched or upregulated and 675 unique genes depleted or downregulated (2-fold or greater; Q value < 0.05) with JQ1 exposure (A). The broad transcriptional events observed—more than an order of magnitude greater than prior studies of JQ1 (Delmore et al., 2011
) —suggested a pronounced effect on multistage differentiation more than selective effects on discrete transcriptional programs. Multiple germ-cell-expressed genes were suppressed upon treatment with JQ1, including Ccna1
(expressed in pachytene spermatocytes), Msy2
(pachytene spermatocytes and postmeiotic germ cells), polo-like kinase 1 (Plk1
; diplotene and diakinesis stage spermatocytes, secondary spermatocytes, and round spermatids), aurora kinase C (Aurkc
; chromocenter clusters in diplotene spermatocytes), and A-kinase anchoring protein 4 (Akap4
; postmeiotic germ cells). These results are significant because knockout mice lacking cyclin A1 have a spermatocyte block (Liu et al., 1998
knockout mice are sterile due to postmeiotic germ cell block and multinucleated spermatids (Yang et al., 2005
); absence of AKAP4 prevents progressive motility (Miki et al., 2002
); humans with aurora kinase C mutations demonstrate polyploid nuclei secondary to meiosis I arrest (Dieterich et al., 2007
); and the mammalian PLK1 protein, similar to fruit fly polo kinase, is believed to function in the completion of meiotic division.
Selective Depletion of Germ Cell Transcripts by JQ1
To determine the stages of spermatogenesis at which JQ1 functions, we then queried these data with functionally defined gene sets reflecting early (pachytene spermatocyte) or late (spermatid) transcriptional signatures (Schultz et al., 2003
) and observed coordinate depletion (normalized enrichment score [NES] = −2.5 to −2.1; p < 0.001) in three independent modules by gene set enrichment analysis (GSEA).
Our expression profiling results were confirmed and extended by gene-specific RT-PCR (D and Table S6
). Genes expressed early in spermatogenesis such as Plzf
, a spermatogonial marker, and Stra8
, expressed in differentiating spermatogonia and preleptotene spermatocytes, are 2.0- and 1.3-fold enriched, respectively, in testes of JQ1-treated mice compared to controls. In addition to Ccna1
, and Akap4
, other key genes expressed during meiosis or spermiogenesis, including Brdt
(mid- to late-spermatocytes), Papolb
(step 1–7 round spermatids), Klf17
(step 4–7 spermatids), and Prm1
(step 7–16 spermatids), are 2.1- to 7.3-fold lower in testes of mice treated with JQ1 versus control. Unlike the Brdt
mutant mouse (Shang et al., 2007
), in which pachytene spermatocyte-expressed gene Hist1h1t
is upregulated, JQ1 treatment leads to a 2.6-fold downregulation of this gene, in line with suppression of Brdt
, and Plk1
. This difference is likely secondary to enhanced blockade of BRDT function by JQ1 in spermatocytes compared to the Brdt
To further characterize the consequences of these molecular changes, we performed histological analysis of zona pellucida binding protein 1 (ZPBP1) and transition protein 2 (TNP2). ZPBP1 is an acrosomal matrix protein first detected in round spermatids as the acrosome forms (Lin et al., 2007
), and TNP2 is expressed in the nuclei of step 10–15 spermatids during histone-to-protamine transition (Zhao et al., 2004
). Consistent with transcriptional analysis, JQ1 treatment for 3 or 6 weeks reduced the number of round spermatids, elongating spermatids, elongated spermatids, and spermatozoa that are positive for ZPBP1 (A–6D and ) and TNP2 (E and 6F and ). Depending on the severity of the cellular loss in any specific tubule, JQ1 treatment caused variable losses of spermatocytes positive for GASZ (), a piRNA pathway protein expressed in pachytene spermatocytes in adults (Ma et al., 2009
). The significant effect of JQ1 on the seminiferous tubule compartment confirms the transfer of JQ1 across the blood:testis boundary to alter spermatogenesis.
Histological Evidence of Antispermatogenic Effects of JQ1
Histological Characterization of the Testes of Mice Treated with JQ1 or Control, Related to
The Bradner laboratory has demonstrated a pronounced antimitotic effect of JQ1 on dividing cancer cells (Delmore et al., 2011
; Filippakopoulos et al., 2010
). In BRD4-dependent cancers, the antitumor efficacy of JQ1 is associated with G1 cell-cycle arrest. Toxicity to proliferative compartments such as bone marrow or bowel has not been observed. To firmly exclude a BRD4-mediated, nonspecific antimitotic effect of JQ1 in testes, we stained testis sections for phosphorylated serine 10 of histone H3 (pH3Ser10), which accumulates in the nuclei of mitotic spermatogonia during chromatin condensation. Changes in pH3Ser10 are not seen, supporting a selective effect of JQ1 on spermatocytes and during spermiogenesis (G and 6H). Further supporting a nontoxic effect of JQ1 on spermatogonia, quantitative microscopic analysis failed to identify any statistically significant decrease in the abundance of cyclin-D1-positive nuclei or any increase in TUNEL-positive cells ().
BRDT Inhibition by JQ1 Does Not Affect Proliferating Gonocytes and Spermatogonia, Related to
In translational models of solid and hematologic malignancies, the cancer-specific antiproliferative activity of JQ1 has been mechanistically linked to addiction to the BRD4
proto-oncogene (Filippakopoulos et al., 2010
) or oncogenic MYC
(a BRD4 target gene) (Delmore et al., 2011
; Zuber et al., 2011
). In cancer models, BET bromodomain inhibition by JQ1 results in the coordinated downregulation of the Myc transcriptional program (Delmore et al., 2011
; Zuber et al., 2011
). Though Myc
is expressed in type B spermatogonia and early prophase spermatocytes (Wolfes et al., 1989
), JQ1 exposure did not suppress the expression of MYC target genes, as assessed by GSEA using the functionally defined MYC signature validated by Dang and colleagues (C) (Zeller et al., 2003
). Paradoxically, MYC-dependent genes trended toward increased expression (NES = 1.8; p < 0.001), possibly due to enrichment of spermatogonial RNA amidst depletion of spermatids and spermatozoa. Together, these orthogonal measurements of spermatogonial transcription, appearance, and function rule out a nonspecific antiproliferative effect of JQ1 on BET bromodomains within mitotic germ cells.
BRDT Inhibition Confers a Reversible Contraceptive Effect in Males
To determine the effects of alternative JQ1 treatment regimens on the fertility of male mice, JQ1 (50 mg/kg/day) and vehicle control were each delivered to 10 male mice for 6 weeks. To ensure that JQ1 and vehicle were having proper effects, three males from each group were sacrificed and were shown to have reduced testis size (control: 107.4 ± 8.8; JQ1: 62.8 ± 7.5 mg; p < 0.05), sperm counts (control: 12.3 ± 0.6 × 106; JQ1: 3.8 ± 0.3 × 106; p < 0.0001), and sperm motility (control: 72.4 ± 2.5%; JQ1: 16.0 ± 0.1%; p < 0.0001). After this pretreatment period, the remaining seven JQ1-treated and seven control males were mated continuously to two adult females per male while continuing to receive JQ1 or vehicle. After the first month of breeding, the seven control males had sired 14 litters of offspring. However, only four of the seven JQ1-treated males sired offspring (only six litters of offspring and a reduced number of pups). For the three JQ1-treated males that had not yet sired offspring, these mice demonstrated normal mating behavior as evident by the presence of copulation plugs. Thus, even at a low dose of JQ1, there is a partial contraceptive effect.
As the mice appeared to be healthy at 50 mg/kg/day of JQ1, we escalated the dosage of JQ1 in the second mating month to 75 mg/kg/day. The male mice appeared to fall into two groups: those that show a contraceptive effect with 50 mg/kg/day followed by 75 mg/kg/day (A) and those that still produced at least one litter under this lower-dose regimen (B). For the three mice that were most responsive to the low-dose regimen, only one was able to sire a litter of two offspring (born on the first day of month 2). For the latter group, the four males that sired offspring were treated in mating month 3 with 50 mg/kg twice per day. These males were not observed to sire any offspring in month 3 (B). Thus, depending on individual variability of each of the males, total daily dosages between 50 and 100 mg/kg can produce a complete contraceptive effect in male mice.
BRDT Inhibition with JQ1 Causes a Reversible Contraceptive Effect in Male Mice
To assess the reversibility of the JQ1-induced contraceptive effect, we examined whether fertility returned after JQ1 treatment was halted. Among the three infertile mice treated with the low-dose regimen, infertility remained complete at 1 month of recovery (mating month 3), indicating a durable effect of JQ1 on spermatogenesis. However, in mating month 4, all three males sired offspring with a statistically similar number of pups per female as observed for the controls (A). The average days to effective copulation was estimated to be 31.7 ± 6.0 days (range: 26–38 days) based on the birth of the first litters after treatment was stopped and the 19 day gestation period of mice. After a total mating period of 7 months (i.e., 4 months after halting the low-dose regimen), testis volume, seminiferous tubule area, testis histology, sperm motility, and sperm counts were statistically similar to the control group (C–7E and ), consistent with fully recovered fertility. Thus, mice treated with JQ1 at 50 mg/kg/day and then 75 mg/kg/day recover fertility within 6 weeks of withholding the drug and show no long-term effects of JQ1 treatment. For the mice that required escalation to the high-dose regimen (50 mg/kg BID), JQ1 was halted after the first month of this treatment (i.e., treatment month 3). In months 4 and 5, these males failed to sire offspring (B). However, all JQ1-treated males sired offspring in month 6 and sired similar litter sizes as controls in months 6–8. The average days to effective copulation for this cohort was estimated to be 65.7 ± 7.7 days (range: 58–81 days). Thus, longer JQ1 treatment and/or the increased dosage of JQ1 resulted in an extended (~1 month) period of infertility.
Reversal of Testicular Phenotypes following Withdrawal of JQ1, Related to
To more precisely determine the integrity and function of the testes and spermatozoa following withdrawal of JQ1 therapy, two additional independent experiments were performed. In the first experiment, JQ1-treated adult male mice were examined at the end of 6 weeks of treatment (50 mg/kg/day) or after 2 or 4 months following cessation of therapy. After 2 months, the testis weight and sperm counts and motility were increased, and after 4 months, the weights and sperm parameters approached normal levels (). Histologically, seminiferous tubules of JQ1-treated mice are indistinguishable from controls (I and 7J). In the second experiment, a cohort of mice was treated with vehicle or 3 weeks of high-dose JQ1 (50 mg/kg BID). Mice were sacrificed at 3 weeks of treatment and 10, 30, and 60 days after JQ1 withdrawal. Males treated with high-dose JQ1 continue to show defects in testicular parameters 30 days after halting treatment but exhibit complete recovery of all parameters within 60 days (F–7H), consistent with the documented recovery of fertility. Thus, depending on duration and overall dosage of JQ1, testicular and semen parameters normalize within 1–3 months to allow productive copulation and return of fertility.
Lastly, offspring that were born from JQ1-treated males showed normal size, activity, and behavior as offspring born from controls (K and data not shown). When offspring from vehicle and JQ1-treated fathers were allowed to mate with adult females (2 females/cage) at 6 weeks of age, both sets of males (n = 6 for each group) displayed normal mating behavior, sired offspring from each female, and produced statistically similar litter sizes from matings over the first month (control: 7.73 ± 0.70 pups/litter; JQ1: 7.50 ± 0.34 pups/litter). At 11 weeks of age, the testis weights (control: 98.7 ± 2.8 mg; JQ1: 95.8 ± 3.2 mg), sperm counts (control: 11.8 ± 0.3 × 106; JQ1: 11.2 ± 0.6 × 106), and sperm motility (control: 69.5 ± 1.7%; JQ1: 69.8 ± 1.3%) of the offspring were statistically similar. Together, these findings indicate that JQ1 did not have any long-term transgenerational effects on testis physiology or reproductive capacity.