Figure legends
Fig. 1 WK500B is identified as a BCL6BTBinhibitor. (A) The repression to BCL6 activity versus inhibition of interaction between BCL6 and SMRT of FX1 and 7 derivative analogs. (B) Chemical structure of WK500B. (C) WK500B attenuated BCL6BTB-mediated transcriptional repression in luciferase reporter assays. (D) WK500B was tested by a HTRF assay for inhibition of the BCL6-SMRT interaction. (E) KD of WK500B binding to BCL6 protein determined by an SPR assay. (F) The binding model of WK500B with BTB of BCL6. (*, P < 0.05; **, P < 0.01 versus control)
Fig. 2 WK500B blocks BCL6BTB-mediated corepressor recruitment and induces de-repression of BCL6 target genes.(A) The colocalization of BCL6 and SMRT was disrupted by WK500B. Scale bar, 10 μm. (B) WK500B reactivated the de-repression of BCL6 target genes. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control). (C) mRNA abundance of the BCL6 target genes was measured in SUDHL4, Farage (both BCL6 dependent) and Toledo (BCL6 independent) cell lines exposed to WK500B.
Fig. 3 WK500B inhibits DLBCL cell growth and induces apoptosisin vitro. (A) Anti-proliferative effects of WK500B on DLBCL cell lines and normal cell lines after 72 h of treatment. (B) IC50 values of WK500B and FX1 in DLBCL cell lines and normal cell lines after 72 h of treatment. (C) SUDHL4 cells were transfected with BCL6 specific shRNA (shBCL6) or Scramble shRNA (shScr) and then seeded in 96-well plates and treated with WK500B. After 72 h, cell growth was measured by MTS assay. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control). (D) Cell cycle profile of SUDHL4 cells treated with WK500B for 24 h. (E) The effect on apoptosis of SUDHL4 cells that were treated with WK500B for 24 h.
Fig. 4 WK500B inhibits GC formation in vivo. C57BL/6 mice were immunized intraperitoneally with NP-CGG and by gavage with WK500B at a dosage of 50 mg/kg/d for 12 days. (A) Representative flow cytometry plot of splenic GC B cells (B220+GL7+FAS+) (left) and quantification (right). (B) Immunofluorescence histology of spleens stained for peanut agglutinin (red) and Ig D (green). Scale bars represent 100 μm. Quantification of the area of GCs was performed by ImageJ software. (C) Flow cytometry detection of Tfh cells (CD4+CXCR5+PD1+) (left) and quantification (right). (D) Titres of the NP-specific immunoglobulin G1 in serum measured with NP5-BSA and NP23-BSA and presented in relative units (RU) as serial dilutions of serum relative to the antibody end-point titres. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control).
Fig. 5 WK500B suppresses BCL6-driven DLBCL growth in vivo. SUDHL4 cells were injected subcutaneously into SCID mice. After 18 days of treatment, the mice were sacrificed, and the tumours were photographed (A) and weighed (B). Scale bars, 1 cm. Tumour volumes (C) and body weights (D) of mice treated with different compounds were measured every 3 days. (E) mRNA expression of the BCL6 target genes from tumours was measured by RT-qPCR assays. (F) Images of IHC staining of Ki67 are shown. Scale bars, 50 μm. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control).
Fig. 6 WK500B synergizes with EZH2 and PRMT5 inhibitors. (A) mRNA abundances of BCL6 target genes in SUDHL4 cells treated with 2 μM GSK591 or GSK343 for 72 h, 2 μM WK500B for 24 h, or a combination. (B) WK500B and PRMT5 inhibitor or EZH2 inhibitor cooperate to kill SUDHL4 cells, and the combination indexes (CIs) are shown at the indicated concentrations. (*, P < 0.05; **, P < 0.01; ***, P < 0.001 versus control).
Fig. 7 Comparison of reported small molecular BCL6 inhibitors and WK500B. (A) Chemical structures of reported small molecular BCL6 inhibitors and WK500B. (B) Comparison of reported small molecular BCL6 inhibitors and WK500B. Y, indicates that the title requirement is met. N, indicates that the datum was not reported. Otherwise, the violation is listed in detail. cLogP and tPSA were calculated using Chemdraw.