A purine scaffold Hsp90 inhibitor destabilizes BCL-6 and has specific antitumor activity in BCL-6-dependent B cell lymphomas

Abstract

We report that heat shock protein 90 (Hsp90) inhibitors selectively kill diffuse large B cell lymphomas (DLBCLs) that depend on the BCL-6 transcriptional repressor. We found that endogenous Hsp90 interacts with BCL-6 in DLBCL cells and can stabilize BCL-6 mRNA and protein. Hsp90 formed a complex with BCL-6 at its target promoters, and Hsp90 inhibitors derepressed BCL-6 target genes. A stable mutant of BCL-6 rescued DLBCL cells from Hsp90 inhibitor-induced apoptosis. BCL-6 and Hsp90 were almost invariantly coexpressed in the nuclei of primary DLBCL cells, suggesting that their interaction is relevant in this disease. We examined the pharmacokinetics, toxicity and efficacy of PU-H71, a recently developed purine-derived Hsp90 inhibitor. PU-H71 preferentially accumulated in lymphomas compared to normal tissues and selectively suppressed BCL-6-dependent DLBCLs in vivo, inducing reactivation of key BCL-6 target genes and apoptosis. PU-H71 also induced cell death in primary human DLBCL specimens.

Figure 1

Hsp90 inhibition induces apoptosis preferentially in Bcl6-dependent DLBCL. (a) A panel of seven Bcl6-dependent (OCI-Ly7, SU-DHL6, OCI-Ly1, Farage, OCI-Ly3, SU-DHL4 and OCI-Ly10) and four Bcl6-independent (Pfeiffer, Toledo, Karpas422 and OCI-Ly4) DLBCL cell lines were exposed to PU-H71 (from 0.1 to 10 μM) or vehicle control (water) for 48 h and analyzed for viability. Dose-response curves were plotted. The X-axis shows the dose of PU-H71 in μM. The Y-axis shows the effect of PU-H71 as compared to control on cell viability. 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 to or higher than 0.90 for each curve. (b) A graphical heat map representation of PU-H71 and 17-DMAG GI₅₀ values. The color reference for each dose range (in μM) is shown on the right. Other cell features are shown in the successive rows. (c) Farage, OCI-Ly7, and SU-DHL4 cells treated for 24 h with control (first lane) or increasing concentrations of PU-H71 (0.1, 0.25, 0.5 and 1 μM) were examined by acridine orange/ethidium bromide staining to categorize the morphological aspect of dead cells. Percentages for each type of dead (apoptotic-like or necrotic in grey and black respectively) and viable cells (white) from triplicate experiments are shown. For each triplicate we categorized at least 300 cells per experimental condition. (d) Immunoblot showing the major fragment of PARP cleavage (89 kD) resulting from caspase activity in cells treated as in (c). (e) Caspase 7 and 3 activity (represented as percentage compared to control) was measured by the cleavage of a specific pro-fluorescent substrate in cells treated as in (c). The Y-axis indicates the caspase 7 and 3 activity over cell number determined by multiplexing with a metabolic assay. Results represent the mean of four biological replicates each of which was performed in experimental triplicates.

Figure 1

Hsp90 inhibition induces apoptosis preferentially in Bcl6-dependent DLBCL. (a) A panel of seven Bcl6-dependent (OCI-Ly7, SU-DHL6, OCI-Ly1, Farage, OCI-Ly3, SU-DHL4 and OCI-Ly10) and four Bcl6-independent (Pfeiffer, Toledo, Karpas422 and OCI-Ly4) DLBCL cell lines were exposed to PU-H71 (from 0.1 to 10 μM) or vehicle control (water) for 48 h and analyzed for viability. Dose-response curves were plotted. The X-axis shows the dose of PU-H71 in μM. The Y-axis shows the effect of PU-H71 as compared to control on cell viability. 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 to or higher than 0.90 for each curve. (b) A graphical heat map representation of PU-H71 and 17-DMAG GI₅₀ values. The color reference for each dose range (in μM) is shown on the right. Other cell features are shown in the successive rows. (c) Farage, OCI-Ly7, and SU-DHL4 cells treated for 24 h with control (first lane) or increasing concentrations of PU-H71 (0.1, 0.25, 0.5 and 1 μM) were examined by acridine orange/ethidium bromide staining to categorize the morphological aspect of dead cells. Percentages for each type of dead (apoptotic-like or necrotic in grey and black respectively) and viable cells (white) from triplicate experiments are shown. For each triplicate we categorized at least 300 cells per experimental condition. (d) Immunoblot showing the major fragment of PARP cleavage (89 kD) resulting from caspase activity in cells treated as in (c). (e) Caspase 7 and 3 activity (represented as percentage compared to control) was measured by the cleavage of a specific pro-fluorescent substrate in cells treated as in (c). The Y-axis indicates the caspase 7 and 3 activity over cell number determined by multiplexing with a metabolic assay. Results represent the mean of four biological replicates each of which was performed in experimental triplicates.

Figure 2

Bcl6 is an Hsp90 client protein. (a) Bcl6 and actin immunoblots performed in Farage, OCI-Ly7 and SU-DHL4 cell lines exposed for 24 h to increasing concentrations of PU-H71 (0.1, 0.25, 0.5 and 1 μM). (b) Western blots for Bcl6 were performed with two different antibodies (N3 and C19, against n –and c– terminal domains respectively) and for actin in OCI-Ly7 cells at the indicated time points after exposure to 0.5 μM of PU-H71. (c) Control or PU-H71-treated OCI-Ly7 cells were incubated with 5 μM Cycloheximide (CHX) for the indicated periods of time. Untreated cells were used as control. Immunoblots for Bcl6 (top) and actin (bottom) are shown for each time point and treatment condition (inset). The relative amount of total Bcl6 to actin was quantified by densitometry (Y-axis) and plotted with respect to time (X-axis). The Bcl6 to actin level in untreated cells (U) was defined as 100%. A similar result was obtained in SU-DHL4 and Farage cells (not shown). (d) PU-H71-treated (0.5 μM for 24 h) OCI-Ly7 cells were exposed to 1 μM of Bortezomib for 24 h and cell lysates analyzed by immunoblot for Bcl6 and actin. The relative amount of total Bcl6 to actin was quantified by densitometry (bottom). (e) Nuclear and cytoplasmic fractions from human centroblasts (CB), OCI-Ly7 and OCI-Ly4 were immunoblotted to determine the abundance of Hsp90 and Bcl6 proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Histone 3 were used as control for the cytoplasmic and nuclear compartments respectively. (f) (Left): OCI-Ly7 nuclear extracts were immunoprecipitated with antibodies for Bcl6, Hsp90 or IgG (Immunoglogublin G as control) and immunoblotted for Bcl6 (top) and Hsp90 (bottom). Nuclear extract lysates were used as input. (Right) OCI-Ly7 nuclear and cytoplasmic extracts were precipitated with control– or PU-H71– coated agarose beads followed by blotting for Bcl6 (top) or Hsp90 (bottom). (g) Quantitative chromatin immunoprecipitation assays performed with Bcl6 or Hsp90 specific antibodies v IgG control for six known Bcl6 target genes (ATR, TP53, ZNF443, CD74, CCNI and TNFAIP8). A TP53 upstream primer set and MS4A1 were used as negative controls. Results are expressed as fold enrichment calculated as the percentage of input for the specific antibody (Bcl6 or Hsp90) over IgG control. Experiments were performed in biological triplicates with triplicate QPCR measurements. (h) The transcript abundance of ATR, TP53 and CD69 was measured by QPCR in Farage, OCI-Ly7 and SU-DHL4 cells exposed to 0.5 μM of PU-H71. Results are expressed as fold change compared to baseline (time 0 h) and were normalized to GAPDH. Experiments were performed five times, each with duplicate QPCR measurements. (i) Viability of Farage and SU-DHL6 cells transfected with pcDNA3.1, BCL6^FL and BCL6^ΔPEST and treated with PU-H71 0.625 μM for 24 h. Results are expressed as percentage to control from triplicate experiments. (j) Caspase 7 and 3 activity (represented as percentage compared to control) was measured by the cleavage of a specific pro-fluorescent substrate in Farage cells transfected with pcDNA3.1, BCL6^FL and BCL6^ΔPEST and treated with control (white bars) or PU-H71 0.625 μM (black bars) for 24 h. The Y-axis indicates the caspase 7 and 3 activity over cell number determined by multiplexing with a metabolic assay to control.

Figure 2

Bcl6 is an Hsp90 client protein. (a) Bcl6 and actin immunoblots performed in Farage, OCI-Ly7 and SU-DHL4 cell lines exposed for 24 h to increasing concentrations of PU-H71 (0.1, 0.25, 0.5 and 1 μM). (b) Western blots for Bcl6 were performed with two different antibodies (N3 and C19, against n –and c– terminal domains respectively) and for actin in OCI-Ly7 cells at the indicated time points after exposure to 0.5 μM of PU-H71. (c) Control or PU-H71-treated OCI-Ly7 cells were incubated with 5 μM Cycloheximide (CHX) for the indicated periods of time. Untreated cells were used as control. Immunoblots for Bcl6 (top) and actin (bottom) are shown for each time point and treatment condition (inset). The relative amount of total Bcl6 to actin was quantified by densitometry (Y-axis) and plotted with respect to time (X-axis). The Bcl6 to actin level in untreated cells (U) was defined as 100%. A similar result was obtained in SU-DHL4 and Farage cells (not shown). (d) PU-H71-treated (0.5 μM for 24 h) OCI-Ly7 cells were exposed to 1 μM of Bortezomib for 24 h and cell lysates analyzed by immunoblot for Bcl6 and actin. The relative amount of total Bcl6 to actin was quantified by densitometry (bottom). (e) Nuclear and cytoplasmic fractions from human centroblasts (CB), OCI-Ly7 and OCI-Ly4 were immunoblotted to determine the abundance of Hsp90 and Bcl6 proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Histone 3 were used as control for the cytoplasmic and nuclear compartments respectively. (f) (Left): OCI-Ly7 nuclear extracts were immunoprecipitated with antibodies for Bcl6, Hsp90 or IgG (Immunoglogublin G as control) and immunoblotted for Bcl6 (top) and Hsp90 (bottom). Nuclear extract lysates were used as input. (Right) OCI-Ly7 nuclear and cytoplasmic extracts were precipitated with control– or PU-H71– coated agarose beads followed by blotting for Bcl6 (top) or Hsp90 (bottom). (g) Quantitative chromatin immunoprecipitation assays performed with Bcl6 or Hsp90 specific antibodies v IgG control for six known Bcl6 target genes (ATR, TP53, ZNF443, CD74, CCNI and TNFAIP8). A TP53 upstream primer set and MS4A1 were used as negative controls. Results are expressed as fold enrichment calculated as the percentage of input for the specific antibody (Bcl6 or Hsp90) over IgG control. Experiments were performed in biological triplicates with triplicate QPCR measurements. (h) The transcript abundance of ATR, TP53 and CD69 was measured by QPCR in Farage, OCI-Ly7 and SU-DHL4 cells exposed to 0.5 μM of PU-H71. Results are expressed as fold change compared to baseline (time 0 h) and were normalized to GAPDH. Experiments were performed five times, each with duplicate QPCR measurements. (i) Viability of Farage and SU-DHL6 cells transfected with pcDNA3.1, BCL6^FL and BCL6^ΔPEST and treated with PU-H71 0.625 μM for 24 h. Results are expressed as percentage to control from triplicate experiments. (j) Caspase 7 and 3 activity (represented as percentage compared to control) was measured by the cleavage of a specific pro-fluorescent substrate in Farage cells transfected with pcDNA3.1, BCL6^FL and BCL6^ΔPEST and treated with control (white bars) or PU-H71 0.625 μM (black bars) for 24 h. The Y-axis indicates the caspase 7 and 3 activity over cell number determined by multiplexing with a metabolic assay to control.

Figure 3

Hsp90 prevents BCL6 mRNA decay. (a) The relative abundance of BCL6 mRNA was determined by QPCR in Farage, OCI-Ly7 and SU-DHL4 cell lines at baseline and 6, 12 and 24 h after treatment with PU-H71 0.5 μM. Results are expressed as fold difference compared to baseline (time 0 h) and were normalized to GAPDH. Experiments were performed five times, each with duplicate QPCR measurements. (b) OCI-Ly7 cells were treated with PU-H71 0.5 μM or control for 2 h, followed by Actinomycin D (ActD) for the indicated times to block transcription. Bcl6 mRNA abundance was determined by QPCR using a standard curve. The plot averages three independent experiments for half-life derivation. Results are expressed as mRNA abundance (log ng) normalized to untreated cells. Bcl6 mRNA half-life in minutes is shown for both experimental conditions. (c) OCI-Ly7 cells were exposed to the indicated concentrations of PU-H71 (or control) and immunoblotted for the ARE-binding proteins p45^AUF1, p42^AUF1, p40^AUF1, p37^AUF1 and Hsp27. Actin was used as a control.

Figure 4

Hsp90 is expressed in the nuclear and cytoplasmatic compartments of DLBCL and primary cells respond to PU-H71. (a) The expression and cellular compartmentalization of Hsp90 was determined by immunohistochemistry in a panel of 70 DLBCLs. The heat map shows the percentage of expression of Hsp90-β for the nuclear and cytoplasmic compartments in the 61 Bcl6 positive DLBCL cases. Three representative images showing different percentages of nuclear and cytoplasmic localizations are shown on the left. The bars represent 25 μm. (b) Correlation between the normalized (to GAPDH) protein levels of Bcl6 and Hsp90 from single cell suspensions obtained from lymph node biopsies of 21 patients diagnosed with DLBCL. Cell lysates were immunoblotted with specific antibodies and analyzed by densitometry. (c) Cases in (b) were treated with either PU-H71 2.5 μM (bars) or control (line) for 48 h. The Y-axis represents the percent of viable cells compared to control (vehicle), which is represented by the line at 100%. Error bars represent the standard error of the mean for triplicates.

Figure 5

PU-H71 suppresses DLBCL xenografts. (a) Tumor growth plots in Farage, OCI-Ly7 and SU-DHL4 xenografted mice treated with control (blue circles) or PU-H71 at 75 mg per kg body weight per day (green circles) for 10 consecutive days. The Y-axis indicates tumor volume (in mm³) and X-axis days of treatment. The P values represent the comparison of tumor volumes at day nine by T-test. (b) Tumor burden (in mg) at day ten in control (blue bars) and PU-H71 at 75 mg per kg body weight per day (green bars) treated Farage, OCI-Ly7 and SU-DHL4 mice. (c) Serum levels of human β2-microglobulin (in μg ml^-1) at day 10 in control (blue bars) and PU-H71 at 75 mg per kg body weight per day (green bars) treated Farage, OCI-Ly7 and SU-DHL4 mice. (d) Kaplan-Meier survival curves for the pooled mice treated with control (blue line) and PU-H71 75 mg per kg body weight per day (green line) treated mice. (e) The relative abundance of Bcl6 protein (to actin) was determined by immunoblotting lysates from Farage, OCI-Ly7 and SU-DHL4 xenografts. Circles (blue for control, green for PU-H71) represent the densitometry values (in arbitrary units) of Bcl6 to actin. Bars represent the mean for each group. P values were obtained by T-test comparisons. (f) Representative images from Farage, OCI-Ly7 and SU-DHL4 mice tumors after being treated with control or PU-H71 75 mg per kg body weight per day and assayed for apoptosis by TUNEL. The number of apoptotic cells over total cells and the statistical significance are shown at the bottom. The bar represents 100 μm.

Figure 6

PU-H71 induces additional changes in protein levels and a specific gene expression signature in DLBCLs. (a) Relative abundance of the NEMO, c-Raf, Akt and Hsp70 proteins (to actin) was measured by immunoblotting in tumor lysates from Farage, OCI-Ly7 and SU-DHL4 mice. Circles represent the densitometry values (in AU) of the specific protein to actin ratios for each mouse xenograft. The bar represents the mean for each group. P values were obtained by T-test comparisons. (b) A graphical heat map representation of the transcript abundance of down-regulated and up-regulated genes measured after treatment of three pairs of Farage xenograft-bearing mice with 75 mg per kg body weight PU-H71 for 6 and 12 h v controls (0 h). The dendrogram on the top represents the unsupervised hierarchical clustering of the six samples. Selected gene ontology (GO) categories with enrichment scores (ES) for up- and down-regulated genes are shown on the right. (c) Selected transcripts from (b) were measured by Q-PCR in three cell lines (Farage, OCI-Ly7 and SU-DHL4) after 6 or 12 h in vitro exposure to 0.5 μM of PU-H71 v control (0 h). Results are expressed as fold change compared to baseline (time 0 h) and were normalized to the average of 3 housekeeping genes (GAPDH, B2M and HPRT, hypoxanthine phosphoribosyltransferase 1). Experiments were performed in triplicate with duplicate QPCR measurements.