BCL6 repression of EP300 in human diffuse large B cell lymphoma cells provides a basis for rational combinatorial therapy

Abstract

B cell lymphoma 6 (BCL6), which encodes a transcriptional repressor, is a critical oncogene in diffuse large B cell lymphomas (DLBCLs). Although a retro-inverted BCL6 peptide inhibitor (RI-BPI) was recently shown to potently kill DLBCL cells, the underlying mechanisms remain unclear. Here, we show that RI-BPI induces a particular gene expression signature in human DLBCL cell lines that included genes associated with the actions of histone deacetylase (HDAC) and Hsp90 inhibitors. BCL6 directly repressed the expression of p300 lysine acetyltransferase (EP300) and its cofactor HLA-B-associated transcript 3 (BAT3). RI-BPI induced expression of p300 and BAT3, resulting in acetylation of p300 targets including p53 and Hsp90. Induction of p300 and BAT3 was required for the antilymphoma effects of RI-BPI, since specific blockade of either protein rescued human DLBCL cell lines from the BCL6 inhibitor. Consistent with this, combination of RI-BPI with either an HDAC inhibitor (HDI) or an Hsp90 inhibitor potently suppressed or even eradicated established human DLBCL xenografts in mice. Furthermore, HDAC and Hsp90 inhibitors independently enhanced RI-BPI killing of primary human DLBCL cells in vitro. We also show that p300-inactivating mutations occur naturally in human DLBCL patients and may confer resistance to BCL6 inhibitors. Thus, BCL6 repression of EP300 provides a basis for rational targeted combinatorial therapy for patients with DLBCL.

Figure 1. EP300 and BAT3 are BCL6 target genes.

(A) Graphical representation from the connectivity map (C-map) analysis of BPI revealing a potential functional relationship with Hsp90 inhibitors and HDAC inhibitors (left) and of our working hypothesis that these drugs are linked through BCL6 repression of EP300 (right). (B) SUDHL-6, Farage, and OCI-Ly7 cells treated for 6 and 12 hours with either BPI (10 μM) or control (CP) were analyzed for EP300 and BAT3 mRNA abundance. Results are shown as fold induction versus baseline (0 hours) and normalized to HPRT. (C) SUDHL-6, Farage, and OCI-Ly7 nuclear extracts from cells treated for 18 hours with either BPI (10 μM) or control (CP) were analyzed for p300 and BAT3 protein abundance. EP300 was detected by immunoprecipitation followed by immunoblotting and normalized to IgG (left panel, densitometry analysis at the bottom). BAT3 nuclear abundance was determined by immunoblotting and normalized to GAPDH (right panel, densitometry analysis at the bottom). (D) QChIP was performed with BCL6 antibody versus actin antibody as control at the EP300 and BAT3 loci. Specific primers were designed in regions with the presence of at least 1 BCL6 consensus binding sequence (as shown on the right) and compared with the upstream regions in the same genes (negative controls). Results are expressed as fold enrichment calculated as percentage of the input for BCL6/actin antibodies (y axis). On the right, graphical representation of the primer amplification site in the EP300 5′ UTR and the promoter of BAT3, together with the respective BCL6 consensus binding sequences. Data are presented as mean with 95% CI.

Figure 2. RI-BPI increases the lysine-acetyltransferase activity of p300.

(A) p300-HAT activity was measured in OCI-Ly7, OCI-Ly10, and SU-DHL6 cells before (white bars) and after (black bars) treatment with BPI (10 μM) for 24 hours normalized to control-treated cells (CP). The HAT activity associated with p300 was determined by p300 immunoprecipitation versus IgG control followed by incubation of the immunoprecipitates with specific HAT substrates and cofactors. The resulting acetylated product was measured by spectrophotometry (OD_440nm). Results are expressed as fold-specific p300-HAT activity in RI-BPI– versus CP-treated cells. (B) Immunoblotting was performed for acetyl-p53 (Lys382) and p53 in the cytosol and nuclear fractions of OCI-Ly7 cells before and after treatment with RI-BPI (10 μM) for 24 hours. (C) Immunoprecipitation was performed for Hsp90 following by immunoblotting with anti–acetyl-lysine (upper panel) or anti-Hsp90 (lower panel) as control in nuclear extracts of OCI-Ly7 cells treated with RI-BPI (10 μM) for 24 or 48 hours versus CP. (D) OCI-Ly7 cells were treated for 24 hours with BPI (10 μM) or control (CP). Hsp70, AKT1, and RAF1 protein abundance were determined by immunoblotting of whole cell lysates. Actin was used as loading control. Densitometry analysis is shown on the right. Data are presented as mean with 95% CI.

Figure 3. RI-BPI–induced cell death is rescued by p300 and BAT3 inhibition.

(A) A panel of 7 BCL6-dependent DLBCL cell lines (OCI-Ly7, SU-DHL6, OCI-Ly1, Farage, OCI-Ly3, SU-DHL4, and OCI-Ly10) was exposed in triplicate to RI-BPI (10 μM) (dark gray bars), the p300-HAT inhibitor Lys-CoA (light gray bars), and the combination of both (black bars) for 48 hours (versus respective CPs). Cell viability (as percentage of CP-treated cells) is shown on the y axis. The experiment was carried out in triplicate with biological duplicates. (B) The BCL6-dependent SU-DHL4, SU-DHL6, and OCI-Ly3 cell lines were transfected with siRNA for BAT3 (siBAT3) or nontargeting sequence (siNT) and treated with RI-BPI (10 μM) or control. After 48 hours, viability was determined. Results are expressed as percentage of viable cells normalized to control (siNT). The experiment was carried out in 4 replicates with biological triplicates. Immunoblotting for BAT3 corresponding to the transfected cells is shown in Supplemental Figure 6. Data are presented as mean with 95% CI.

Figure 4. RI-BPI synergizes with HDAC inhibitors.

(A) 7 BCL6-dependent DLBCL cell lines were exposed to 6 concentrations of SAHA (from 0.5 to 10 μM), TSA (from 25 to 400 nM), or vehicle control (DMSO, 0.1% in water) for 48 hours and analyzed for viability. Dose-effect (percentage of dead cells) curves were plotted. Data points represent experimental data for a particular dose effect. The curves were derived using these data points and the Compusyn software. (B) The same cells were treated with 6 concentrations of SAHA or TSA and the combination of these drugs with 6 concentrations of RI-BPI at a constant ratio (concurrent administration). Conservative GI₅₀ isobolograms for the combination of SAHA or TSA with BPI for each drug were plotted. Data points falling on the line indicate an additive effect, points below the line indicate synergy, and points above the line indicate infra-additive effect. The dose values for each GI₅₀ for each cell line are shown in Supplemental Table 1. (C) For the cell lines that were resistant (i.e., the GI₅₀ was higher than the upper dose limit) to 1 or more drugs, a potentiation effect with BPI was calculated. Cells were treated with BPI (10 μM) or SAHA (1 μM) or TSA (100 nM) or the combination of these for 48 hours (sequential schedule BPI→drug). Cell viability was determined and compared with control-treated cells. (D) The 7 DLBCL cell lines were exposed to RI-BPI (10 μM) (white bars), SAHA (1 μM) (light gray bars), RI-BPI plus SAHA (black bars), or vehicle control (dark gray bars, not visible) for 48 hours and analyzed for caspase 7/3 activity. y axis represents caspase 7/3 activity (fold). Data are presented as mean with 95% CI.

Figure 5. RI-BPI synergizes with Hsp90 inhibitors.

(A) 7 BCL6-dependent DLBCL cell lines (OCI-Ly7, SU-DHL6, OCI-Ly1, Farage, OCI-Ly3, SU-DHL4, and OCI-Ly10) were exposed to 6 concentrations of PU-H71 (from 0.5 to 5 μM), 17-DMAG (from 0.5 to 5 μM), or vehicle control (water or DMSO, respectively) for 48 hours and analyzed for viability. Dose-effect (percentage of dead cells) curves were plotted. The x axis shows the dose of the Hsp90 inhibitor. The y axis shows the fractional effect of the drug as compared with control on cell viability. The experiment was done in 4 replicates. The goodness of fit for the experimental data to the median-effect equation (linear correlation coefficient) obtained from the logarithmic form of this equation was equal or higher than 0.90 for each curve. (B) The same panel of cells was treated with 6 concentrations of PU-H71 or 17-DMAG and the combination of these drugs with 6 concentrations of RI-BPI at a constant ratio. Conservative GI₅₀ isobolograms for the combination of PU-H71 or 17-DMAG with RI-BPI for each drug were plotted. The dose values for each GI₅₀ for each cell line are shown in Supplemental Table 1. (C) The 7 DLBCL cell lines were exposed in triplicate to RI-BPI (10 μM) (white bars), PU-H71 (1 μM) (light gray bars), RI-BPI plus PU-H71 (black bars), or vehicle control (water, dark gray bars, not visible) for 48 hours and analyzed for caspase 7/3 activity. The y axis represents the caspase 7/3 activity as compared with each cell line control (fold). Data are presented as mean with 95% CI.

Figure 6. SAHA enhances RI-BPI antilymphoma effect in vivo.

(A and B) Left panels: tumor growth plots in Farage (A) and OCI-Ly7 (B) xenografted mice treated with control (DMSO, 10% in saline, n = 10, black squares), RI-BPI (25 mg/kg/d) (n = 5, white squares), SAHA (20 mg/kg/d) (n = 5, gray squares), or the combination of RI-BPI and SAHA (n = 5, black circles) for 10 consecutive days. The y axis indicates tumor volume (in mm³) and the x axis days of treatment. P values represent the comparison of tumor volumes in treated to control mice at day 9 by t test. Right panels, top: Serum levels of human β2-microglobulin (in μg/ml) at day 10 in Farage (A) and OCI-Ly7 (B) mice treated with control (C), RI-BPI (B), SAHA (S), and a combination (B+S). Right panels, bottom: tumor burden (in grams) at day 10 in Farage (A) and OCI-Ly7 (B) mice treated with control, RI-BPI, SAHA, and a combination. In all the cases, the P values were obtained by t test comparisons of treated versus control mice. (C) Representative immunohistochemistry images from Farage and OCI-Ly7 mouse tumors after treatment with control, SAHA, RI-BPI, or the combination of RI-BPI and SAHA assayed for apoptosis by TUNEL staining. Scale bar: 200 μm. Data are presented as mean with 95% CI.

Figure 7. PU-H71 enhances RI-BPI antilymphoma effects in vivo.

(A and B) Left panels: tumor growth plots in Farage (A) and OCI-Ly7 (B) xenografted mice treated with control (water, n = 10, black squares), RI-BPI (25 mg/kg/d) (n = 5, white squares), PU-H71 (75 mg/kg/d) (n = 5, gray squares), or a combination of RI-BPI and PU-H71 (n = 5, black circles) for 10 consecutive days. The y axis indicates tumor volume (in mm³) and the x axis days of treatment. P values represent the comparison of tumor volumes in treated to control mice at day 9 by t test. Right panels, top: serum levels of human β2-microglobulin (in μg/ml) at day 10 in Farage (A) and OCI-Ly7 (B) mice treated with control (C), RI-BPI (R), PU-H71 (P), and a combination (R+P). Right panels, bottom: tumor burden (in grams) at day 10 in Farage (A) and OCI-Ly7 (B) mice treated with control, RI-BPI, PU-H71, and a combination. In all cases, P values were obtained by t test comparisons of treated versus control mice. (C) Representative immunohistochemistry images from Farage and OCI-Ly7 mouse tumors after treatment with control, PU-H71, RI-BPI, or the combination of RI-BPI and PU-H71 and assayed for apoptosis by TUNEL staining. Scale bar: 200 μm. Data are presented as mean with 95% CI.

Figure 8. EP300 genetic lesions and the impact of combinatorial therapy on primary DLBCL specimens.

(A) Representation of the EP300 mutations found in DLBCL cell lines (n = 10) and patient samples (n = 93). The EP300 protein (N¹ to C²⁴¹⁴ terminus) is represented as a white bar with the KAT domain (1224 to 1669) in gray. Synonymous mutations are italicized. For nonsynonymous mutations, the substituted amino acid is indicated. Truncated proteins are indicated with an asterisk. The data correspondent to the cell line RC-K8 is from the literature (23). Patient samples are categorized accordingly to their DLBCL subtype as ABC, GCB, and unclassifiable (UC). (B) CD19⁺ single-cell suspensions from lymph node biopsies of 8 confirmed BCL6⁺ DLBCL specimens were cocultured with HK dendritic cells in a dual chamber separated by a 0.4-μm porous membrane. CD19⁺ cells were exposed to vehicle (white bars), 10 μM of RI-BPI (light gray bars), 1 μM of SAHA (dark gray bars), or the combination of RI-BPI and SAHA (black bars) for 48 hours. Cell viability (represented as percentage of control-treated cells) is shown on the y axis. Individual cases as well as the average for all the cases are shown on the x axis. The experiment was carried out in 4 replicates. (C) Similar experimental conditions as in B but for cells treated with vehicle (white bars), 10 μM of RI-BPI (light gray bars), 1 μM of PU-H71 (dark gray bars), or the combination of RI-BPI and PU-H71 (black bars) for 48 hours. Data are presented as mean with 95% CI.