Targeting STAT5 in Hematologic Malignancies through Inhibition of the Bromodomain and Extra-Terminal (BET) Bromodomain Protein BRD2

Bromodomain inhibitors block STAT5-dependent gene expression

To determine whether BET bromodomain-containing proteins are critical to STAT5 transcriptional function, we first examined the effect of the bromodomain inhibitor JQ1 on reporter cell systems in which luciferase is regulated by individual transcription factors. JQ1 caused a dose-dependent inhibition of STAT5-dependent luciferase expression (Fig. 1A). In contrast, JQ1 had no effect on transcription mediated by the highly related transcription factor STAT3. Interestingly, at maximally effective doses, JQ1 led to approximately a 60% decrease in reporter gene expression, suggesting that BET bromodomain proteins are not essential for basal STAT5 transcriptional activity, but may be required for maximal activity. In contrast, Jak inhibitor 1, which completely abolishes the activating STAT tyrosine phosphorylation, decreased STAT5-dependent transcription in this assay by approximately 80%.

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Figure 1.

JQ1 decreases STAT5 activity. A, reporter cell lines were treated with the indicated doses of JQ1 or Jak inhibitor 1 (JAKi; 1 μmol/L) for 1 hour, after which IL-6 or prolactin was added to activate STAT3 or STAT5, respectively. Six hours later, luciferase activity was quantitated by luminometry and normalized to cell viability. B, HEL erythroleukemia cells were transfected with one of two luciferase constructs regulated by a STAT5-dependent promoter (NCAM-luc or B-luc). Cells were then treated with JQ1 at the indicated concentration, and normalized luciferase activity was determined. Jak inhibitor 1 (JAKi; 1 μmol/L) was used as a positive control. C, expression of STAT5 target genes BCL-x, CIS, and PIM1 was measured by quantitative RT-PCR (normalized to 18S RNA) and presented relative to vehicle treatment control, in the indicated leukemia cell lines. Cells were treated for 6 hours with vehicle control, 0.5 μmol/L JQ1, or 1 μmol/L TKI (Jak inhibitor 1 for HEL and DND41 and imatinib for ALL-SIL and K562). D, HEL erythroleukemia cells and ALL-SIL T-ALL cells were treated with vehicle (DMSO), JQ1 (0.25 μmol/L), or the indicated TKIs [Jak inhibitor 1 (0.5 μmol/L) or imatinib (0.0625 μmol/L)] for 30 hours, after which cells were harvested and immunoblot analyses were performed with the indicated proteins. Total STAT5 level was used as a loading control. Densitometric quantification of two separate experiments is shown on the right. E, TALL-1 cells were pretreated with vehicle, JQ1 (1 μmol/L), or Jak inhibitor 1 (1 μmol/L) for 1 hour, after which IL-2 (50 U/mL) was added to stimulate STAT5 activation. RNA was analyzed 90 minutes after IL-2 stimulation.

We next determined the effect of JQ1 in leukemia cell lines in which STAT5 is constitutively activated through a variety of mechanisms. We used two reporter constructs in which luciferase is regulated by distinct STAT5-dependent promoter sequences derived from the NCAM2 gene (NCAM-luc) or the BCL6 gene (B-luc). JQ1 treatment led to a dose-dependent reduction of STAT5-dependent luciferase activity mediated by both of these promoters in multiple lymphoid and myeloid leukemia cell types (Fig. 1B and Supplementary Fig. S1).

Constitutively activated STAT5 drives cancer pathogenesis by increasing expression of genes regulating cell-cycle progression and promoting survival. Thus, we determined the effect of JQ1 on the expression levels of well-characterized endogenous STAT5 targets genes (Supplementary Fig. S2), including BCL-x (21, 22), CIS (20), and PIM1 (23). JQ1 inhibited the expression of STAT5 target genes in leukemia cell lines with constitutively activated STAT5 driven by Jak2 (HEL and DND41) or Abl (ALL-SIL and K562; Fig. 1C). Protein expression of STAT5 target genes was also reduced by JQ1, as was the previously described target of JQ1, Myc (ref. 15; Fig. 1D). As these endogenous genes may also be regulated by other transcription factors, the response to JQ1 (and kinase inhibitors) was, as expected, more variable than that seen with the reporter systems. However, these results also suggest that JQ1 does not cause nonspecific inhibition of transcription. Because autocrine or paracrine production of cytokines is an important mechanism of STAT5 activation, we next evaluated systems in which STAT5 phosphorylation is cytokine induced. JQ1 inhibited interleukin (IL)-2–induced STAT5 target gene expression in T-lymphocytic leukemia cells (Fig. 1E). Taken together, these data demonstrate that JQ1 inhibits STAT5-dependent transcriptional function, and this inhibition is independent of the mechanism driving STAT5 activation.

To further evaluate whether bromodomain inhibition blocks STAT5 transcriptional function, we tested whether a second BET bromodomain inhibitor I-BET, which is structurally distinct from JQ1, also inhibits STAT5 transcriptional activity. We also evaluated an inactive (-)-JQ1 enantiomer, which is structurally incapable of inhibiting BET bromodomains (15). We found that I-BET was as effective as JQ1 in inhibiting STAT5-dependent transcription using luciferase reporter cells (Fig. 2A). As expected, the (-)-JQ1 enantiomer had no activity in this assay (Fig. 2A). Furthermore, both JQ1 and I-BET reduced expression of endogenous STAT5 target genes in ALL cells (Fig. 2B). These results indicate that structurally unrelated bromodomain inhibitors can inhibit STAT5 transcriptional function.

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Figure 2.

Inhibition of Brd2 reduces STAT5 transcriptional function. A, ALL-SIL cells were transfected with a STAT5-luciferase construct (NCAM-luc) and treated with vehicle, JQ1 (1 μmol/L), iBET (1 μmol/L), or the enantiomer (-)-JQ1 (1 μmol/L), after which STAT5-dependent gene expression was determined by dual luciferase assay. B, DU528 cells were treated with vehicle, JQ1 (1 μmol/L), or iBET (1 μmol/L) for 6 hours, after which expression of the indicated STAT5 target genes was determined by quantitative RT-PCR. HEL cells (C and E) or ALL-SIL cells (D) were treated with the indicated shRNA constructs targeting BRD2, after which expression of the indicated genes was determined by quantitative RT-PCR.

JQ1 inhibits STAT5 function by blocking BRD2

We next focused on determining which BET bromodomain proteins are necessary for STAT5 transcriptional function. In particular, we examined BRD2, BRD3, and BRD4, as BRDT is only expressed in testis and ovary. To do this, we used lentiviral vector-mediated shRNAs to knock down each individual BET protein in leukemia cells, and determined the effect on expression of STAT5 target genes. The efficacy and specificity of shRNAs targeting BRD2, BRD3, and BRD4 were validated by RT-PCR analysis (Supplementary Fig. S3). Despite 80% knockdown of BRD3 or BRD4 by shRNAs, no reproducible decrease was seen in the expression of the STAT5 target genes Bcl-x, CIS, and PIM1 (Supplementary Fig. S4). In contrast, knockdown of BRD2 led to a prominent reduction in expression of STAT5 target genes in multiple leukemia cell lines (Fig. 2C–E). These experiments confirm a specific association between depletion of BRD2 and reduction of STAT5 target gene expression in all cell types examined, further supporting the hypothesis that reduction of STAT5 transcriptional function by JQ1 is mediated through inhibition of BRD2.

Because BRD2 is associated with transcription factor-dependent gene expression in the setting of hyperacetylated chromatin (24), we considered whether BRD2 regulates transcription of Bcl-x, CIS, and PIM1 independent of STAT5. To examine this hypothesis, we knocked down BRD2 in the T-lymphoblastic leukemia cell lines TALL-1, which lacks constitutive STAT5 activation. Knockdown of BRD2 did not decrease expression of BCL-x, CIS, or PIM1 in these cell lines, in contrast with what was seen in cells having constitutively activated STAT5 (Supplementary Fig. S5). These results provide further evidence that downregulation of BCL-x, CIS, and PIM1 expression after BRD2 depletion is due to inhibition of STAT5 transcriptional function.

BRD2 inhibition by JQ1 can inhibit STAT5 function without affecting STAT5 phosphorylation

Although JQ1 has been found to be able to decrease STAT5 phosphorylation in a subset of acute B-lymphocytic leukemia cells through downregulation of the IL-7 receptor (IL-7R; ref. 25), we found that JQ1 can inhibit STAT5 transcription in a wide variety of cell types in which IL-7R is not involved in STAT5 activation, including breast cancer cells (Fig. 1A). Therefore, we considered alternative hypotheses for how BRD2 inhibition by JQ1 reduces STAT5 function. First, we evaluated the effect of this drug on STAT5 protein expression and its activating tyrosine phosphorylation in systems not dependent on the IL-7R. In both HEL myeloid leukemia cells (in which STAT5 is activated by the constitutively activated kinase JAK2V617F) and ALL-SIL T-ALL cells (in which STAT5 is activated by the fusion tyrosine kinase NUP214-ABL), JQ1 had no effect on STAT5 protein levels or tyrosine phosphorylation (Fig. 3A). In contrast, the TKIs Jak inhibitor 1 and imatinib completely abrogated STAT5 phosphorylation, respectively. Similarly, knocking down BRD2 in ALL-SIL with multiple distinct constructs did not influence STAT5 expression or phosphorylation (Fig. 3B).

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Figure 3.

Brd2 inhibition by JQ1 or shRNA knockdown does not affect STAT5 phosphorylation or DNA binding. A, HEL erythroleukemia cells and ALL-SIL T-ALL cells were treated with vehicle (DMSO), JQ1 (0.5 μmol/L), or the indicated TKIs (1 μmol/L) for 6 hours, after which cells were harvested and immunoblot analyses were performed with STAT5 and phosphorylated STAT5. B, ALL-SIL cells were infected with one of three shRNA constructs targeting BRD2 or GFP (as a control). Three days after puromycin selection, cells were harvested and immunoblot analyses were performed with the indicated antibodies. C, cells were treated with vehicle, JQ1 (0.5 μmol/L), or imatinib (1 μmol/L) for 6 hours, after which cells were harvested and ChIP was performed. STAT5 and RNA polymerase II binding were analyzed relative to a known STAT5-negative binding region. D, comparison of CIS transcripts close to the transcriptional start site (designated as pre-CIS) and the mature form of CIS RNA level by quantitative RT-PCR (normalized to 18S RNA).

We next considered the possibility that JQ1 is inhibiting the interaction of STAT5 with its cognate genomic regulatory sequences. To examine this, we performed ChIP to evaluate whether JQ1 inhibits STAT5 binding to the well-characterized regulatory sites for CIS. Despite reduction of CIS expression after 6 hours of treatment with JQ1, JQ1 did not significantly reduce STAT5 binding to its genomic-binding site (Fig. 3C). In contrast, the TKI imatinib, which inhibited STAT5 phosphorylation, significantly reduced DNA binding by STAT5. Given that STAT5 could still bind to the CIS promoter in the presence of JQ1, we evaluated whether the binding of RNA polymerase II was affected by this treatment. Kinase inhibitors, which blocked STAT5 phosphorylation and DNA binding, inhibited the recruitment of RNA polymerase II to the CIS promoter (Fig. 3C). IN contrast, JQ1 had little effect on RNA polymerase recruitment to this site. Thus, we investigated whether JQ1 reduces transcriptional initiation or elongation by measuring the abundance of sequences close to the transcriptional start site and comparing them with the mature form of CIS RNA. We found equivalent decreases in sequences located close to the transcriptional start sites as with mature RNA transcripts of CIS (Fig. 3D), suggesting that although transcriptional initiation was inhibited, elongation seemed to be unaffected. Taken together, these results show that JQ1 can decrease STAT5 transcriptional function without affecting STAT5 phosphorylation, DNA binding, or recruitment of RNA polymerase II, and support the notion that JQ1 likely reduces STAT5 transcriptional initiation by targeting essential cofactors for STAT5, likely BRD2.

Although JQ1 had no effect on the phosphorylation of STAT5 in most cell lines examined, we did observe that JQ1 decreased STAT5 phosphorylation in lymphoid malignancies such as Hodgkin lymphoma and acute T-lymphoblastic leukemia, in which STAT5 activation is mediated by signaling through Jak kinases (Fig. 4A). This is consistent with what has been observed in a subset of B-ALL in which STAT5 activation is Jak dependent (25). To exclude the possibility that JQ1 was acting directly as a kinase inhibitor in this setting, we tested it for activity against a range of kinases using in vitro kinase assays. At a concentration of 500 nmol/L, JQ1 had no effect on the kinase activity of JAK1, JAK2, JAK3, or TYK2, or the unrelated tyrosine kinase SRC (Fig. 4B).

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Figure 4.

JQ1 is not a direct JAK kinase inhibitor. A, the indicated cell line was treated with JQ1 (0.5 μmol/L) and harvested at the indicated time points, after which immunoblot analyses were performed for phosphorylated and total STAT5. B, the effect of JQ1 (0.5 μmol/L) on the activity of selected tyrosine kinases was analyzed using SelectScreen Kinase Profiling (Invitrogen). Jak inhibitor 1 (0.5 μmol/L) was used as positive control for inhibition of Jak family kinases.

BRD2 inhibition strongly synergizes with TKIs to decrease leukemia cell survival

Leukemogenesis driven by a variety of oncogenic tyrosine kinases is dependent on STAT5 activation. Because BRD2 is essential for maximal STAT5 function, we next considered whether inhibition of BRD2 has an effect on leukemia cell survival. Reduction of BRD2 by itself does not significantly decrease the viability of leukemia cells (data not shown). However, when HEL cells, which are driven by a mutated form of the kinase Jak2, are cotreated with BRD2 knockdown and Jak inhibitor 1, there is a dramatic cooperative effect, with a decrease in the IC₅₀ concentration of 50% to 75% (Fig. 5A). Similarly, reduction of BRD2 in ALL-SIL cells, which express NUP-214-ABL, reduces the IC₅₀ of the kinase inhibitor imatinib by 50% (Fig. 5B). Consistent with this finding, induction of apoptosis of leukemia cells by kinase inhibitors is enhanced by knockdown of BRD2 proportional to the decrease in BRD2 expression (Fig. 5C). These results suggest that dual inhibition of STAT5 function by blocking phosphorylation and inhibiting an important transcriptional coregulator leads to maximal inhibition of STAT5 function and reduction of leukemia cell survival.

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Figure 5.

Inhibition of Brd2 increases sensitivity of leukemia cells to TKIs. HEL (A) and ALL-SIL (B) cells were infected with lentiviral vectors containing distinct shRNAs targeting BRD2. After 4 days of selection in puromycin, cells were treated with TKIs as indicated. Cell viability was measured 72 hours after drug treatment. C, parental ALL-SIL cells or cells with Brd2 knockdown were treated with imatinib for 48 hours, after which apoptosis was quantitated by annexin V and propidium iodide staining followed by flow cytometry. D, the indicated leukemia cell lines were treated for 72 hours with JQ1 or the indicated TKI, alone or in combination. Isobologram analysis was performed on the basis of change in viable cell number after treatment. Data points below the line indicate synergy between the two agents. E, ALL-SIL cells were treated with JQ1 (0.5 μmol/L), imatinib (0.125 μmol/L), or both for 48 hours, after which apoptosis was quantified by annexin V staining and flow cytometry. F, ALL-SIL or DND41 T lymphocytic leukemia cells were infected with a vector expressing either GFP alone or GFP and constitutively activated STAT5a (caSTAT5a). Cells were then treated with JQ1 (0.25 μmol/L), Jak inhibitor 1 (1 μmol/L), or imatinib (0.125 μmol/L), or the indicated combinations for 72 hours, and the fraction of GFP⁺ cells was quantified by flow cytometry and normalized to the vehicle (DMSO) control.

Having shown that a reduction of BRD2 decreases STAT5 transcriptional function in several leukemia cell types and that there is a BRD2-dependent mechanism of STAT5 inhibition by JQ1, we next explored the therapeutic implications of JQ1 in leukemia, particularly in aggressive T-ALL in which STAT5 is driven by constitutively activated tyrosine kinases. We found JQ1 used as a single agent only modestly decreased the viability of T-ALL cell lines with constitutive STAT5 activation, and required relatively high concentrations (Supplementary Fig. S6A). This low activity occurred despite a prominent decrease in Myc expression (Supplementary Fig. S6B), as has been reported for JQ1. This suggested that reduction in Myc expression alone is not sufficient for the therapeutic effect of JQ1 in T-ALL cells. In contrast, we found strong consistent synergy between JQ1 and TKIs, including imatinib and Jak inhibitor 1, in reducing leukemia cell viability across a broad range of doses in multiple cell lines (Fig. 5D). Consistent with this finding, combination treatment of JQ1 and TKIs led to a synergistic induction of apoptosis (Fig. 5E).

To determine whether STAT5 was the key target in leukemia cells treated with TKIs and JQ1, we examined the effect of a constitutively activated form of STAT5, STAT5a1*6. ALL-SIL or DND41 cells were transfected with a construct expressing GFP alone or with STAT5a1*6. Cells were then treated with vehicle, JQ1, and/or a TKI, and the relative proportion of GFP-expressing cells was determined by flow cytometry. In the presence of constitutively activated STAT5, treatment with TKIs (imatinib for ALL-SIL or Jak inhibitor 1 for DND41) or JQ1 alone led to a slight enrichment of GFP⁺ cells. However, treatment with TKIs and JQ1 in combination led to a significant increase in the proportion of GFP⁺ cells (Fig. 5F), suggesting that constitutively activated STAT5 rescues these leukemia cells from cell death induced by TKIs and JQ1. These results indicate that the effects of JQ1 and TKIs are, at least partially, mediated through STAT5 inhibition, supporting the importance of this transcription factor as a therapeutic target.

Finally, we wished to determine whether primary T-ALL cells were also sensitive to JQ1 and TKIs. We obtained bone marrow mononuclear cells from four patients with untreated T-ALL in which greater than 90% of the cells were lymphoblasts. As the number of cells available was insufficient to assess STAT5 phosphorylation, we used sensitivity to kinase inhibitors as a surrogate marker for STAT5 activation. Of the four, none was sensitive to imatinib and one (patient 1) was sensitive to JAK inhibition (data not shown). This is consistent with reports that the NUP214–ABL1 translocation is a rare event in T-ALL (26). We found that these primary T-ALL cells showed variable sensitivity to JQ1 treatment, though again cells from patient 1 showed the greatest sensitivity to JQ1 (Fig. 6A). Consistent with our observations in T-ALL cell lines, we observed synergistic reduction of viability of leukemia cells from patient 1 when JQ1 and JAK inhibitor I were combined (Fig. 6B and C).

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Figure 6.

The combination of JQ1 and TKIs shows toxicity to primary T-ALL cells but not normal hematopoietic cells. A, bone marrow mononuclear cells harvested from four untreated patients with T-ALL were treated with JQ1 at the indicated concentrations for 72 hours, at which point viable cell number was determined by ATP-dependent bioluminescence. B, bone marrow mononuclear cells harvested from patient 1 with patients with T-ALL were treated with JQ1 (0.125 μmol/L), JAK inhibitor 1 (1 μmol/L), or the combination for 72 hours, after which viable cell number was quantitated by ATP-dependent bioluminescence. C, isobologram analysis was performed on the basis of loss of cell viability for 14 different combinations of JQ1 and JAKi. Data points below the line indicate synergistic effects. D, mononuclear cells from umbilical cord blood (n = 4), or ALL-SIL or DU528 T-lymphocytic leukemia cells were treated for 72 hours with JQ1 (0.25 μmol/L) or TKIs (imatinib 0.125 μmol/L or JAKi 0.5 μmol/L), alone or in combination, and viable cell number was quantitated by ATP-dependent bioluminescence.

To exclude the possibility that the combination of JQ1 with a kinase inhibitor is nonspecifically cytotoxic to immature hematopoietic cells, we obtained mononuclear cells from umbilical cord blood, which is highly enriched in progenitor cells. In contrast with what is seen in leukemia cells, JQ1 and TKIs, either alone or in combination, had minimal effects on the viability of normal hematopoietic cells (Fig. 6D and Supplementary Fig. S7). Together, these results suggest that the use of JQ1 combined with TKIs in patients with ALL would not be associated with toxicity to normal hematopoietic cells.