Selectively targeting BCL6 using a small molecule inhibitor is a potential therapeutic strategy for ovarian cancer

Ovarian cancer is one of the tumors with the highest fatality rate among gynecological tumors. The current 5-year survival rate of ovarian cancer is <35%. Therefore, more novel alternative strategies and drugs are needed to treat ovarian cancer. The transcription factor B-cell lymphoma 6 (BCL6) is critically associated with poor prognosis and cisplatin resistance in ovarian cancer treatment. Therefore, BCL6 may be an attractive therapeutic target for ovarian cancer. However, the role of targeting BCL6 in ovarian cancer remains elusive. Here, we developed a novel BCL6 small molecule inhibitor, WK369, which exhibits excellent anti-ovarian cancer bioactivity, induces cell cycle arrest and causes apoptosis. WK369 effectively inhibits the growth and metastasis of ovarian cancer without obvious toxicity in vitro and in vivo. meanwhile, WK369 can prolong the survival of ovarian cancer-bearing mice. It is worth noting that WK369 also has significant anti-tumor effects on cisplatin-resistant ovarian cancer cell lines. Mechanistic studies have shown that WK369 can directly bind to the BCL6-BTB domain and block the interaction between BCL6 and SMRT, leading to the reactivation of p53, ATR and CDKN1A. BCL6-AKT, BCL6-MEK/ERK crosstalk is suppressed. As a first attempt, our study demonstrates that targeting BCL6 may be an effective approach to treat ovarian cancer and that WK369 has the potential to be used as a candidate therapeutic agent for ovarian cancer.


Molecular Docking
A molecular docking study was performed by employing the crystal structures of the BCL6-BTB domain co-crystallized with the ligand pyrazolo-pyrimidine macrocyclic (PDB ID code: 5N1Z) by searching the Protein Data Bank 1 Docking assays were performed by using AutoDock4.2.6 4 , and the parameter files and the binding poses were obtained by using AutoDockTool 1.5.6 and the Lamarckian genetic algorithm, respectively.The co-crystallized ligand was used to define the active sites for docking, and the size of the box was 60*60*60.The number of conformational docking simulations was 200, and the lowest binding energy was selected for analysis.

Homogenous Time Resolved Fluorescence (HTRF) Assay
A total volume of 8 L of BCL6-GST protein and SMRT-6His peptide were added to each well in 384-well plates (Greiner Bio-one, 784045), and then 2 L of diluent buffer with the compounds to be tested was added.After 1 h, a total volume of 10 L of anti-6His-XL665 (Cisbio) and anti-GST-Tb (Cisbio) was added to reach a 20  L final volume.After overnight incubation, the plate was read on a microplate reader (BioTek Cytation5) at 665 nm and 620 nm.

Surface plasmon resonance assay
Surface plasmon resonance (SPR) analysis was conducted with a Biacore T200 instrument (GE Healthcare) with CM5 sensor chip (GE Healthcare).To test WK369 binding of BCL6-BTB protein, serially diluted concentrations of WK369 were injected into the flow system.Experiments were conducted using phosphate buffered saline (PBS) and the analyte was injected at the flow rate of 30  L/min.The association time was 60 s and the dissociation time was 30 s.Since WK369 was dissolved in PBS containing 5% dimethyl sulfoxide and a solvent correction assay was performed to adjust the results.BCL6-BTB protein was immobilized on the sensor chip (CM5) using the amine-coupling method according to standard protocols.

Animal immunity
The eight-week-old male C57BL/6 mice were immunized with antigen NP18-CGG and each mouse was injected intraperitoneally with 100 g.Two days later, the mice were treated with 50 mg/kg/d of FX1 or WK369 by ip injection.The mice were euthanized after 12 days of continuous administration, spleen and serum were collected.Flow detection of the proportion of GC-B cells in mice, serum for enzyme linked immunosorbent assay.

Flow Cytometric Analysis
Single-cell suspensions were prepared and red blood cells were lysed by ACK.

Hematoxylin and Eosin (H&E) staining
The paraffin-embedded paraffin tissue was cut into 4 μm thin sections, immersed in xylene I solution, xylene II solution, xylene and ethanol 1:1 mixture for 10 min, then immersed in 100%, 95%, 85%, 75% ethanol solution and pure water for 3 min, immersed in hematoxylin dyeing solution for about 10 min.The color was separated with 1% hydrochloric acid ethanol for 10 sec, and then immersed in 75%, 85% and 95% ethanol for 3 min in turn after 30 min in tap water.The tissue sections were stained with eosin for 5 sec, then immersed in 95% ethanol, 100% ethanol, xylene and ethanol 1:1 mixture, xylene I solution, xylene II solution for 5 min, and finally sealed with neutral resin.

Immunohistochemistry (IHC)
The paraffin-embedded paraffin tissue was cut into 4 μm thin sections.After dewaxing, sections were washed with antigen repair solution for 20 min and PBS for 5 min.Tissue sections were soaked in 3% H2O2-methanol solution for 15 min in the dark to remove catalase, and then washed with PBST for three times.Then rinse with distilled water 3 times, 2 min each time.Anti-ki67 rabbit antibody (1:50) was added and incubated overnight at 4 °C.The sections were rinsed again and incubated with the corresponding secondary antibody for 60 min at room temperature, and 3,3 '-diaminbenzidine (DAB) for 5-10 min.Hematoxylin staining was performed.The sections were then rinsed with ddH2O for 10 min, washed with alcohol (75% to 100%), and cleared in xylene.Finally, the sheet is sealed with resin.

Statistical Analyses
Statistical analysis was carried out using Prism 8 (GraphPad Software).Data are represented as the mean ± sd.Statistical comparisons were calculated by One-way ANOVA followed by Tukey's test.P < 0.05 was considered statistical significance.

Supplemental references
WK369 at various concentrations were injected into the flow system.The kinetics and affinity assay were examined at 25 ℃ at a flow rate of 30  L/min.The KD values were calculated with the kinetics and affinity analysis option of Biacore 8K evaluation software (version 3.0).Quantitative Real-Time PCR PCR primers are listed as follows: p53: CCCTTCCCAGAAAACCTACC and AATCAACCCACAGCTGCAC, CDKN1A: CTGAAGGGTCCCCAGGTG and TAGGGCTTCCTCTTGGAGAA, ATR: GGAGATTTCCTGAGCATGTTCGG and GGCTTCTTTACTCCAGACCA, CD69: CTGGTCACCCATGGAAGTG and CATGCTGCTGACCTCTGTGT, GAPDH: GAAGGTGAAGGTCGGAGTC and GAAGATGGTGATGGGATTTC model 8-week-old female BALB/c nude mice were obtained from the Animal Center of East China Normal University.All animal experimental protocols were approved by the Animal Investigation Committee of the Institute of Biomedical Sciences, East China Normal University.ES-2 cells that stably expressed luciferase (ES-2-luc) were embedded in Matrigel (BD Biosciences) and intrasplenically injected into mice.Tumors were allowed to grow for 1 week and treatment of the mice with either compound or control was started after grouping of the mice according to average bioluminescence.The IVIS Imaging System (Xenogen Corporation) was used to monitor pancreatic tumor growth and metastasis.Images and measurements of bioluminescent signals were acquired and analyzed using Living Image and Xenogen software.

Figure S1 .
Figure S1.BCL6 is overexpressed in Ovarian Cancer and Promotes Ovarian

Figure S2 .
Figure S2.WK369 is a Novel and Potent of BCL6 Small Molecular Inhibitor.

Figure S4 .
Figure S4.The high-resolution mass spectrum (HR MS) of WK369.High-resolution

Figure S7 .
Figure S7.WK369 inhibits ovarian cancer cell and DLBCL cancer cell

Figure S9 .
Figure S9.WK369 prevents ovarian cancer growth in vivo and no cases of toxic

Figure S11 .
Figure S11.Effect of WK369 in combination with Cisplatin on ovarian cancer

Figure S13 .
Figure S13.Main pharmacokinetic parameters of WK369 after oral administration or