2.17. Statistical analysis
The results are expressed as the mean ± SD. The significance of the
difference between two groups was analysed by Student’s t test. For
animal experiments, data were analysed by two-way ANOVA. All experiments
were performed at least three times, except for the animal
experiments. All statistical analyses were carried out using GraphPad
Prism 5.0 software. The significant differences in the means were
determined at the level of *P < 0.05, ** P < 0.01
and ***P < 0.001.
Results
Identification of WK500B as a novel BCL6 BTB inhibitor.
To identify novel BCL6BTB inhibitors with improved
activities both in vitro and in
vivo ,
a preliminary screen was performed
of a library of approximately 500
compounds using a luciferase reporter assay, which allowed us to assess
BCL6-mediated transcriptional repression activity, and 2 hits were
identified at a concentration of 10 μM among the investigated ones.
Intriguingly,
these
effective compounds shared the same structural skeleton of
diaminopyrimidine (Supplementary Fig. 1A).
Therefore,
more analogs were further synthesized and modified based on the
structural of diaminopyrimidine. Subsequently, those derivatives were
then subjected to secondary screen to evaluate BCL6-mediated
transcriptional repression activity and the disruption of
BCL6BTB/SMRT complex formation by a Homogenous
Time-Resolved Fluorescence (HTRF) assay
(Supplementary Fig. 1A).
FX1,
which was previously reported as a BCL6 inhibitor (Cardenas et al.,
2016), was included as the positive control.
Among
the investigated derivatives, 7 compounds exhibited strong repression to
BCL6 in luciferase reporter assay and inhibited BCL6 and SMRT
interaction in HTRF assay (Fig. 1A).
WK500B
was identified as the most active
analogues among the investigated compounds (Fig. 1B). WK500B almost
completely disrupted the transcriptional repression function of the BCL6
BTB domain at 10 μM, which abrogated the repression of the
(GAL4)5-TK-LUC reporter by the
GAL4-DBD-BCL6BTB fusion protein, whereas FX1 had
little effect on the BCL6 BTB domain at the same concentration (Fig.
1C). Consistent with its greater inhibitory activity against
BCL6BTB versus that of FX1, WK500B blocked the
interaction of the corepressor SMRT with BCL6 BTB with
an IC50 of 1.37 ± 0.12
μM, and the IC50of FX1 was 29.78 ± 2.45 μM (Fig.
1D). To study whether WK500B could bind to the
BCL6BTB, a surface plasmon resonance (SPR) assay was
performed (Homola, 2003). Surprisingly, WK500B directly bound to the
BCL6 BTB domain with a dissociation constant (KD) value
of 1.61 μM (Fig. 1E). In addition, we also performed a molecular docking
assay to predict the binding mode of the WK500B and BCL6 BTB domain. As
shown in Fig. 1F, the carbonyl oxygen of the acyl residues and the N
atom of the pyridinyl on the left part of WK500B were bound to the
relevant amino acid residue R24. In addition, two hydrogen bonds were
formed between the N-H of the piperazine ring of WK500B and the N atom
of the imidazolyl group of His14. In short, these results suggest that
WK500B could bind to the BCL6 BTB domain.
WK500B disruptsBCL6BTB/SMRT
complex formation and reactivates the expression of BCL6 target genes.
The above results indicated that WK500B bound to the BCL6 BTB domain
with high affinity and disrupted BCL6 BTB/SMRT complex formation in the
extracellular
space.
To assess the impact of WK500B on the BCL6 BTB/SMRT interaction inside
cells, an immunofluorescence assay was performed in SUDHL4 cells. As we
predicted, the colocalization of BCL6 with SMRT was disrupted by WK500B
(Fig. 2A), and endogenous BCL6 expression was not affected by WK500B
(Fig. 2A, Supplementary Fig. S2A). Targeting the BCL6 BTB domain could
avoid the adverse effects resulting from the complete abrogation of BCL6
functions (Huang, Hatzi & Melnick, 2013; Valls et al., 2017), and
WK500B had no effect on inflammatory responses (Supplementary Fig. S2B).
These findings established that WK500B bound to the BCL6 BTB domain and
blocked recruitment of SMRT by BCL6.
BCL6 is a transcriptional repressor that plays a key role in GC
formation and the pathogenesis of DLBCLs by repressing the expression of
downstream target genes through binding with corepressors at gene
promoters (Huang & Melnick, 2015; Polo et al.,
2007). To determine the effect of
WK500B on BCL6 transcriptional repression activity, we examined the
expression of BCL6 target genes in DLBCL cell lines (SUDHL4 and Farage)
that were exposed to WK500B. As shown in Fig. 2B, WK500B significantly
increased the reactivation of the expression of known BCL6 target genes
(p53, CDKN1A, CXCR4 and CD69) (Cardenas et al., 2016; Phan &
Dalla-Favera, 2004; Ranuncolo et al., 2007; Ranuncolo, Polo & Melnick,
2008) compared with the vehicle, while the positive control compound FX1
showed little effect at the same concentration. To further explore the
selectivity of WK500B, Toledo cell line, which lacks BCL6 expression was
then exposed to WK500B at 10 μM for 24 h. In contrast to SUDHL4 and
Farage, WK500B had no effect on any of these genes in the
BCL6-independent Toledo cell line (Figure 2C). Together, these data show
that WK500B disrupts BCL6 corepressor recruitment and specifically
reactivates BCL6 target genes in BCL6-dependent DLBCLs.
WK500B significantly inhibits DLBCL growth and induces cell
cycle arrest and apoptosis in
vitro.
Most previous studies have shown that BCL6 target genes are essential
for DLBCL survival and reactivating these target genes can cause DLBCL
cell lethality (Cardenas et al., 2016; Cerchietti & Melnick, 2013;
Valls et al., 2017). Given that WK500B significantly reactivated BCL6
target genes (Fig. 2B),
five
DLBCL cell lines (SUDHL4, SUDHL6, OCI-LY7, Farage and DOHH2) were
exposed to WK500B for 72 h, and the viabilities of these cells were
detected through an MTS assay. As expected,
WK500B
effectively inhibited DLBCL proliferation at low concentrations, and the
IC50 values of different DLBCL cell lines were equal to
1 μM (Fig. 3A, B). In addition,
four normal cell lines, including the human normal liver cell line LO2,
human skin fibroblast cell line HAF, human normal colon epithelium cell
line NCM460 and human prostatic cell line PNT1A, were also exposed to
WK500B to further explore its selectivity toward normal cells.
Excitingly,
WK500B
had little killing effect on the normal cells even at 10 μM
(IC50 > 10 μM), indicating that WK500B
selectively inhibited DLBCL proliferation with low toxicity toward
normal cells (Fig. 3A, B). Moreover, WK500B had an even more significant
effect on inhibiting DLBCL proliferation, and its IC50values were 30-fold lower than those of FX1 (Fig. 3B). Subsequently,
SUDHL4 cells with lentivirus expressing BCL6 shRNA were treated with
WK500B. The results indicated that WK500B could effectively inhibit
control vector cells in the investigated range of 1.25 − 10.0 μM in a
dose-dependent manner, while it showed little effect on BCL6 knockdown
cells in the same concentration range, supporting the notion that WK500B
inhibits DLBCL cell growth by targeting BCL6 (Fig. 3C).
BCL6 mediates the survival and cell cycle progression of B-cell lymphoma
cells (Polo et al., 2004). We determined whether WK500B affected DLBCL
cell cycle progression and cell apoptosis by using flow cytometry. The
results (Fig. 3D, E) indicated that
WK500B induced significant cell cycle arrest at the S phase and caused
the dose-dependent induction of apoptosis at doses of 2.5, 5.0 and 10
µM. The positive control FX1 did not induce apoptosis or cell cycle
arrest at the same concentration (data not shown). Taken together, these
results demonstrated that WK500B induced the death of DLBCLs in
vitro .
WK500B exhibits
favourable pharmacokinetics and abrogates germinal centre formation in
vivo.
To determine whether WK500B could serve as the prototype of BCL6
inhibitor for further clinical development,
a
human liver microsome study was primarily carried out to evaluatein vitro metabolism of WK500B. Intriguingly, WK500B had good
metabolic stability in vitro (Supplementary Table 1). Encouraged
by this result, pharmacokinetic analyses were subsequently performed
after oral administration (p.o., 10 mg/kg) and intravenous injection
(i.p., 5.0 mg/kg) of WK500B in mice to further gain insights into the
preclinical pharmacokinetics. As shown in Supplementary Table 2, the
plasma half-life (T1/2) values were 7.93 ± 0.81 h for
p.o. administration and 6.41 ± 0.87 h for i.p. injection, respectively,
and relatively low plasma clearance (CL ) was observed, which
indicated that WK500B has improved metabolic stability in vivo .
The mean values of the apparent volume of distribution
(Vd) were very high for both p.o. and i.p.
administration, which suggested that WK500B was widely distributed in
tissues. In addition, the area under the curve (AUC0−∞)
was high for both p.o. and i.p. administration, indicating that WK500B
was absorbed easily. Moreover, WK500B has excellent oral bioavailability
(F = 82.49%). These results demonstrated that WK500B is a bioavailable
candidate for oral administration and has improved drug-likeness.
BCL6
is important for the formation of GCs for humoural immunity, and mice
engineered with a mutation of BCL6 BTB have normal B cell development
but fail to form GCs and show
disruption of the affinity maturation of immunoglobulins (Huang, Hatzi
& Melnick, 2013). To examine the impact of WK500B on GCs, C57/BL6 mice
were immunized with
NP18CGG and treated
by gavage with daily doses of WK500B and FX1 (positive control) at 50
mg/kg two days later.
After
12 days of dosing, the mice were sacrificed.
As
expected, the WK500B treatment group showed significantly abolished GC
formation
(GL7+FAS+B220+)
compared with the vehicle group (Fig. 4A). Moreover, the WK500B
treatment group exhibited better inhibitory activity, with a frequency
of B cells of 0.4%, than the FX1 group, in which the frequency of GC B
cells was 0.9% (Fig. 4A). In
addition, spleens were also subjected to immunofluorescent staining to
assess the number of GCs. Ig D staining revealed
normal B cell follicular structures,
and staining with peanut agglutinin, a GC B cell-specific marker, showed
a profound loss of GCs (Fig. 4B), similar to the results of Flow
cytometric. Notably, WK500B had a more significant GC inhibitory effect
than FX1.
Follicular helper T (Tfh) cells contribute to the development of GC B
cells, and BCL6 also acts a pivotal part in Tfh cells (Huang, Hatzi &
Melnick, 2013; Kitano et al., 2011). In mice with
BCL6BTB mutations, the frequency of Tfh cells was
depleted compared with that in BCL6+/+ mice
(Huang, Hatzi & Melnick, 2013).
Similar
to that observed in mice with conditional deletion of BCL6 in GC B
cells, the proportion of Tfh cells
(CXCR5+PD1+CD4+)
was much lower in the WK500B treatment group than that in the untreated
group (Fig. 4C).
Impaired immunoglobulin affinity maturation is found in mice with BCL6
BTB mutations (Huang, Hatzi & Melnick, 2013), and we next examined
whether WK500B can recapitulate this phenotype by enzyme-linked
immunosorbent assay (ELISA). As
expected, WK500B treatment group brought about antibody-deficient mice
whose titres of high-affinity immunoglobulin G1 specific to NP5-BSA as
well as the total IgG1 specific to NP23-BSA were dramatically lower than
the blank control group (Fig. 4D). Collectively, these results
demonstrated that WK500B could effectively inhibit BCL6 biological
functions in vivo .
WK500B effectively suppresses the growth of lymphoma without
toxic effects in
vivo.
To further evaluate whether WK500B could protect against DLBCL
proliferation in vivo , SCID mouse xenograft models were
established by subcutaneous injection of SUDHL4 DLBCL cells.
When
visible tumours reached approximately 100 mm3 in size,
mice were injected with 12.5 mg/kg/day and 25 mg/kg/day WK500B or 25
mg/kg/day FX1 (positive control) or vehicle by oral administration. The
tumour volume and mouse body weight were detected every three days. As
expected, WK500B significantly inhibited tumour growth
and had a more potent
tumour-inhibitory effect than FX1 (Fig. 5A, B and C). Importantly, the
expression of BCL6 target genes in tumours was more strongly reactivated
in the WK500B-treated groups than in the control group, which was
consistent with the in vitro observations
(Fig. 2B), providing
pharmacodynamic evidence of WK500B engaging with BCL6 in tumour tissue
(Fig. 5E). In addition, the tumour tissues were analysed by
immunohistochemistry (IHC). Compared with control group, the
proliferation marker Ki67 was dramatically decreased in the
WK500B-treated groups, which confirmed the anticancer effects of WK500Bin vivo ( Fig. 5F). Furthermore, we tested whether WK500B
bring about toxic effects in mice. No obvious toxicity was observed
according to the measurement of the mouse body weight (Fig. 5D), and the
mice had no abnormal behaviour or side effects during the treatment
period. Moreover, no obvious organ damage was observed based on H&E
staining (Supplementary Fig. 3). To further evaluate the toxicity of
WK500B in mice,
C57BL/6
mice received WK500B orally at 50 mg/kg/day for 14 days, and the
biochemical parameters and complete blood counts from C57BL/6 mice were
examined. Intriguingly, WK500B had negligible effects on mice treated
with compound (Supplementary Tables 3 and 4). Hence, these data
demonstrated that WK500B is an
effective anti-lymphoma agent in vivo and
is nontoxic to animals.
WK500B synergizes with EZH2 and PRMT5 inhibitors.
Since lymphomas are typically highly heterogeneous, BCL6 inhibitors in
combination with other targeted agents are required. BCL6 has been found
to cooperate with EZH2 and PRMT5 to mediate GC formation (Beguelin et
al., 2016; Lu et al., 2018). Then, to evaluate whether WK500B has
synergistic effects with EZH2 inhibitor or PRMT5 inhibitor, we exposed
SUDHL4 cells to a combination of the PRMT5 inhibitor GSK591 or the EZH2
inhibitor GSK343 with WK500B, and we observed increased de-repression of
BCL6 target genes compared to that induced by either compound alone
(Fig. 6A). Next, the combinatorial activity was determined by
administering increasing doses of WK500B with GSK591 or GSK343 and
measuring cell viability. As expected, the combination of GSK591 with
WK500B more significantly suppressed DLBCL cell growth than either
GSK591 or WK500B alone, resulting in a synergistic combination effect
(Fig. 6B). Consistent with the findings for GSK591, synergistic killing
activity in SUDHL4 cells was also observed when combining GSK343 with
WK500B (Fig. 6B). Collectively, the results indicate that WK500B
synergizes with the EZH2 and PRMT5 inhibitors.
Discussion
B cells transiting the GC reaction
manifest many hallmarks of cancer cells, and the deregulation of BCL6
expression leads to B cell malignant transformation. BCL6 has been
documented a convincing therapeutic target in DLBCL and other GC-derived
lymphomas (Leeman-Neill & Bhagat, 2018).
Inhibitors that disrupt
interactions between BCL6BTB and its corepressors show
anticancer activity and lack toxicity (Cerchietti et al., 2009; Polo et
al., 2004). Although a series of
BCL6 inhibitors have been reported (Fig. 7A), the IC50values of BCL6 inhibitors are high and molecules with a higher affinity
for BCL6 have not been studied in vivo (Fig. 7B), which is
perhaps due to their poor pharmacokinetic properties or other reasons
(Kamada et al., 2017; Kerres et al., 2017; McCoull et al., 2017). Hence,
the development of clinical-grade BCL6 inhibitors with improved
drug-likeness is an urgent mission.
Here, we conducted a screen by using a luciferase reporter assay and an
HTRF assay to identify a novel
potent BCL6 inhibitor, WK500B, which directly bound to
BCL6BTB, significantly reactivated the expression of
BCL6 target genes, impacted the growth of DLBCLs in vitro and
induced apoptosis and cell cycle arrest. BCL6BTBmutant mice had an impaired GC response after immunization with a T
cell–dependent antigen (Huang, Hatzi & Melnick, 2013). WK500B
disturbed the biological functions of BCL6 and phenocopied the
BCL6BTB phenotype in vivo , which significantly
inhibited GC formation, reduced the proportion of Tfh cells and impaired
immunoglobulin affinity maturation. Moreover, WK500B suppressed the
growth of DLBCL cells free of eliciting detectable off-target effects
against normal tissues. No inhibitors that directly target BCL6 have
been approved by the FDA, which may be due to their poor
pharmacokinetic/pharmacodynamic properties (Cheng et al., 2018; Kamada
et al., 2017; Kerres et al., 2017; McCoull et al., 2017; Yasui et al.,
2017). WK500B displayed favourable pharmacokinetics with a long plasma
half-life (T1/2) and low plasma clearance (CL ) as
well as a high apparent volume of distribution (Vd) and
area under the curve (AUC0−∞). Extraordinarily, WK500B
also has excellent oral bioavailability, which is not possessed by other
BCL6 inhibitors. Taken together, WK500B is orally available and shows
superior druggability compared with other reported BCL6 inhibitors (Fig.
7A, B).
In addition, other aspects of the BCL6 mechanism have been harnessed to
design combined targeted therapy regimens (Cardenas, Oswald, Yu, Xue,
MacKerell & Melnick, 2017). Combinations of WK500B with an EZH2
inhibitor and a PRMT5 inhibitor showed enhanced anti-lymphoma activity
and synergistic effects on the de-repression of BCL6 target genes.
Future clinical trials will be important to investigate the efficacy of
targeting BCL6 in combination with other therapies. Recent reports have
implicated BCL6 as an important therapeutic target in solid tumours,
including glioblastoma (Xu et al., 2017), breast cancer (Walker et al.,
2015), ovarian cancer (Wang, Xu, Weng, Wei, Yang & Du, 2015) and
non-small cell lung cancer (NSCLC) (Deb et al., 2017). Moreover, an
increasing number of studies have shown that targeting the BCL6 BTB
domain can overcome drug tolerance in cancer cells, (Duy et al., 2011;
Fernando et al., 2019; Madapura et al., 2017; Song et al., 2018; Wu, Lv,
Wang, Li & Guo, 2018). We conjectured that WK500B may kills other
cancer cells and overcome drug tolerance, which need more experiments to
verify our hypothesis. This is of great significance to expand the scope
of BCL6 inhibitors.
In summary, WK500B not only disrupted BCL6 biological functions in
vitro and in vivo but also presented superior druggability, and
combination therapy with WK500B and other targeted chemotherapeutic
agents yielded dramatic antitumour effects. Therefore, WK500B could
potentially be used as a novel effective and orally available anticancer
agent for treatment of DLBCL.