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.