Figure 7.
Figure 7. PRMT5 recruits BCL6 to its target genes to induce gene repression. (A) Enrichment of BCL6, SMRT, HA-tagged PRMT5, and IgG at BCL6 targets in OCI-LY1 cells treated with vehicle or 200 nM GSK591 for 72 hours. *P < .05; **P < .01. (B) Messenger RNA abundance of BCL6 target genes in OCI-LY1, OCI-LY7, SUDHL4, and SUDHL6 cells treated with vehicle or 200 nM GSK591 for 96 hours. *P < .05; **P < .01. (C) Immunoblot demonstrating inhibition of SDMA using the SYM10 antibody at 72 hours after treatment with 200 nM GSK591. IP demonstrating decrease in symmetric dimethylation of BCL6 shown in supplemental Figure 7A. (D) Pathway analysis of gene expression changes in SUDHL6 cells treated with vehicle or 200 nM GSK591 for 24 or 96 hours. The Fisher exact test was used to calculate enrichment P values for each gene set, and the Benjamini-Hochberg method was used for false discovery rate control. (E) GSK591 concentration that results in 50% growth inhibition (GI50) of BCL6-dependent and BCL6-independent DLBCL cell lines treated with vehicle or increasing concentrations of GSK591 for 6 days. Raw growth inhibition curves of GSK591 alone are shown in supplemental Figure 8A. (F) Messenger RNA abundance of BCL6 targets in OCI-LY1 cells with combined treatment of 25 μM FX1 and 200 nM GSK591 for 48 hours. *P < .05; **P < .01 relative to vehicle. †P < 0.05; ††P < .01 relative to each drug alone. (G) Combination indexes of the BCL6 inhibitor FX1 with GSK591 after treating cells with increasing concentrations of GSK591 for 6 days and FX1 for 2 days. Data are representative of 3 triplicates ± standard error of the mean (SEM). Raw growth inhibition curves of each drug alone and in combination are shown in supplemental Figure 8C. (H) Mean fluorescence intensity of carboxyfluorescein diacetate succinimidyl ester of live CD19+ (GhostDye− or DAPI−) human DLBCL samples on day 6 after cells were exposed to GSK591 on day 0 then treated with FX1 3 days later. Data representative of 3 triplicates ± SEM. *P < .05; **P < .01 relative to VEH. †P < 0.05; ††P < .01 relative to each drug alone. (I) Cell viability (GhostDye− or DAPI−) of CD19+ human DLBCL from panel H. Data are representative of 3 triplicates ± SEM. *P < .05; **P < .01 relative to vehicle. †P < .05; ††P < .01 relative to each drug alone. FACS, fluorescence-activated cell sorting; FL, follicular lymphoma; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; mRNA, messenger RNA; VEH, vehicle.

PRMT5 recruits BCL6 to its target genes to induce gene repression. (A) Enrichment of BCL6, SMRT, HA-tagged PRMT5, and IgG at BCL6 targets in OCI-LY1 cells treated with vehicle or 200 nM GSK591 for 72 hours. *P < .05; **P < .01. (B) Messenger RNA abundance of BCL6 target genes in OCI-LY1, OCI-LY7, SUDHL4, and SUDHL6 cells treated with vehicle or 200 nM GSK591 for 96 hours. *P < .05; **P < .01. (C) Immunoblot demonstrating inhibition of SDMA using the SYM10 antibody at 72 hours after treatment with 200 nM GSK591. IP demonstrating decrease in symmetric dimethylation of BCL6 shown in supplemental Figure 7A. (D) Pathway analysis of gene expression changes in SUDHL6 cells treated with vehicle or 200 nM GSK591 for 24 or 96 hours. The Fisher exact test was used to calculate enrichment P values for each gene set, and the Benjamini-Hochberg method was used for false discovery rate control. (E) GSK591 concentration that results in 50% growth inhibition (GI50) of BCL6-dependent and BCL6-independent DLBCL cell lines treated with vehicle or increasing concentrations of GSK591 for 6 days. Raw growth inhibition curves of GSK591 alone are shown in supplemental Figure 8A. (F) Messenger RNA abundance of BCL6 targets in OCI-LY1 cells with combined treatment of 25 μM FX1 and 200 nM GSK591 for 48 hours. *P < .05; **P < .01 relative to vehicle. †P < 0.05; ††P < .01 relative to each drug alone. (G) Combination indexes of the BCL6 inhibitor FX1 with GSK591 after treating cells with increasing concentrations of GSK591 for 6 days and FX1 for 2 days. Data are representative of 3 triplicates ± standard error of the mean (SEM). Raw growth inhibition curves of each drug alone and in combination are shown in supplemental Figure 8C. (H) Mean fluorescence intensity of carboxyfluorescein diacetate succinimidyl ester of live CD19+ (GhostDye− or DAPI−) human DLBCL samples on day 6 after cells were exposed to GSK591 on day 0 then treated with FX1 3 days later. Data representative of 3 triplicates ± SEM. *P < .05; **P < .01 relative to VEH. †P < 0.05; ††P < .01 relative to each drug alone. (I) Cell viability (GhostDye− or DAPI−) of CD19+ human DLBCL from panel H. Data are representative of 3 triplicates ± SEM. *P < .05; **P < .01 relative to vehicle. †P < .05; ††P < .01 relative to each drug alone. FACS, fluorescence-activated cell sorting; FL, follicular lymphoma; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; mRNA, messenger RNA; VEH, vehicle.

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