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

Cerchietti LC, Lopes EC, Yang SN, Hatzi K, Bunting KL, Tsikitas LA, Mallik A, Robles AI, Walling J, Varticovski L, Shaknovich R, Bhalla KN, Chiosis G, Melnick A - Nat. Med. (2009)

Bottom Line: Hsp90 formed a complex with BCL-6 at its target promoters, and Hsp90 inhibitors derepressed BCL-6 target genes.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Oncology, Weill Cornell Medical College of Cornell University, New York, New York, USA.

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.

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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.
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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.

Mentions: In order to determine the anti-lymphoma activity of Hsp90 inhibitors, a panel of DLBCL cell lines was exposed to increasing concentrations of PU-H71. DLBCLs can be divided into subtypes with distinct gene expression signatures and response to drugs and biological agents. One system for dividing DLBCLs classifies them according to their expression of B-cell receptor (BCR) or oxidative phosphorylation genes20. The BCR DLBCLs display coordinated repression of Bcl6 target genes, depend on Bcl6 for their survival20 and are preferentially sensitive to Bcl6 targeting by specific peptides17,21 and small-interfering RNA (Supplementary Fig. 1). In response to PU-H71, Bcl6-dependent DLBCL cell lines showed decreased growth compared to Bcl6-independent DLBCL cell lines (Fig. 1). The concentration of PU-H71 that inhibited the growth of the cell lines by 50% compared to control (GI50) in BCcl6-dependent DLBCLs was 1.39 μM (± 1.00 μM) compared to a GI50 of 71 μM (± 41 μM) in the Bcl6-independent group (P = 0.001, T test) (Fig. 1a). Other features such as abundance of Hsp90-α or Hsp90-β, BCL2 translocation, TP53 mutation status or the activated B-cell (ABC) or germinal center B-cell (GCB) type gene expression signatures were not associated with the differential response of these cell lines to Hsp90 inhibition (Fig. 1b, Supplementary Fig. 2 and Supplementary Table 1). An identical effect was shown with the Hsp90 inhibitor 17-DMAG (Fig. 1b and Supplementary Table 1). PU-H71 killed DLBCL cells in a dose-dependent manner, preferentially through induction of apoptosis, as shown by nuclear fragmentation observed in ethidium bromide/acridine orange staining, PARP (poly (ADP-ribose) polymerase) cleavage and induction of caspase 7 and 3 activity (Fig. 1c–e).


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

Cerchietti LC, Lopes EC, Yang SN, Hatzi K, Bunting KL, Tsikitas LA, Mallik A, Robles AI, Walling J, Varticovski L, Shaknovich R, Bhalla KN, Chiosis G, Melnick A - Nat. Med. (2009)

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.
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Related In: Results  -  Collection

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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.
Mentions: In order to determine the anti-lymphoma activity of Hsp90 inhibitors, a panel of DLBCL cell lines was exposed to increasing concentrations of PU-H71. DLBCLs can be divided into subtypes with distinct gene expression signatures and response to drugs and biological agents. One system for dividing DLBCLs classifies them according to their expression of B-cell receptor (BCR) or oxidative phosphorylation genes20. The BCR DLBCLs display coordinated repression of Bcl6 target genes, depend on Bcl6 for their survival20 and are preferentially sensitive to Bcl6 targeting by specific peptides17,21 and small-interfering RNA (Supplementary Fig. 1). In response to PU-H71, Bcl6-dependent DLBCL cell lines showed decreased growth compared to Bcl6-independent DLBCL cell lines (Fig. 1). The concentration of PU-H71 that inhibited the growth of the cell lines by 50% compared to control (GI50) in BCcl6-dependent DLBCLs was 1.39 μM (± 1.00 μM) compared to a GI50 of 71 μM (± 41 μM) in the Bcl6-independent group (P = 0.001, T test) (Fig. 1a). Other features such as abundance of Hsp90-α or Hsp90-β, BCL2 translocation, TP53 mutation status or the activated B-cell (ABC) or germinal center B-cell (GCB) type gene expression signatures were not associated with the differential response of these cell lines to Hsp90 inhibition (Fig. 1b, Supplementary Fig. 2 and Supplementary Table 1). An identical effect was shown with the Hsp90 inhibitor 17-DMAG (Fig. 1b and Supplementary Table 1). PU-H71 killed DLBCL cells in a dose-dependent manner, preferentially through induction of apoptosis, as shown by nuclear fragmentation observed in ethidium bromide/acridine orange staining, PARP (poly (ADP-ribose) polymerase) cleavage and induction of caspase 7 and 3 activity (Fig. 1c–e).

Bottom Line: Hsp90 formed a complex with BCL-6 at its target promoters, and Hsp90 inhibitors derepressed BCL-6 target genes.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology and Oncology, Weill Cornell Medical College of Cornell University, New York, New York, USA.

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.

Show MeSH
Related in: MedlinePlus