Limits...
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.

Show MeSH

Related in: MedlinePlus

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, BCL6FL and BCL6ΔPEST 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, BCL6FL and BCL6ΔPEST 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2805915&req=5

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, BCL6FL and BCL6ΔPEST 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, BCL6FL and BCL6ΔPEST 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.

Mentions: Because the Bcl6-dependent DLBCL subtype was particularly susceptible to Hsp90 inhibition we wondered whether Bcl6 might be an Hsp90 client. Along these lines we found that a 24 h exposure to PU-H71 induced a dose-dependent reduction in Bcl6 protein abundance in Farage, OCI-Ly7 and SU-DHL4 Bcl6-dependent DLBCL cells (Fig. 2a). PU-H71 also induced the depletion of the known Hsp90 client proteins c-Raf22, Akt23 and NEMO (NF-kappa-B essential modifier)24 as well as a compensatory increase in Hsp70 (Heat shock protein 70) (Supplementary Fig. 3a). In addition, PU-H71 induced a similar pattern of dose-dependent changes in protein abundance, including Bcl6 depletion, in the Bcl6-independent DLBCL cell lines (Toledo, Pfeiffer and Karpas422) (Supplementary Fig. 3b). To determine the kinetics of PU-H71-induced Bcl6 protein depletion, we treated OCI-Ly7 cells with 0.5 μM PU-H71 for 4, 6, 12, 18, 24 and 48 h. After 18 h of treatment, Bcl6 was barely detectable (Fig. 2b) using two different antibodies for Bcl6 (N3 and C19). PU-H71 accelerated Bcl6 protein decay after protein translation blockade by Cycloheximide. Relative Bcl6 protein abundance decreased from 100% to 50% in 5 h (± 1 h) in the presence of PU-H71 compared to 24 h (± 2 h) in vehicle-treated cells (Fig. 2c). PU-H71-induced downregulation of Bcl6 was also partially blocked by treatment with the proteasome inhibitor Bortezomib (Fig. 2d).


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)

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, BCL6FL and BCL6ΔPEST 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, BCL6FL and BCL6ΔPEST 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805915&req=5

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, BCL6FL and BCL6ΔPEST 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, BCL6FL and BCL6ΔPEST 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.
Mentions: Because the Bcl6-dependent DLBCL subtype was particularly susceptible to Hsp90 inhibition we wondered whether Bcl6 might be an Hsp90 client. Along these lines we found that a 24 h exposure to PU-H71 induced a dose-dependent reduction in Bcl6 protein abundance in Farage, OCI-Ly7 and SU-DHL4 Bcl6-dependent DLBCL cells (Fig. 2a). PU-H71 also induced the depletion of the known Hsp90 client proteins c-Raf22, Akt23 and NEMO (NF-kappa-B essential modifier)24 as well as a compensatory increase in Hsp70 (Heat shock protein 70) (Supplementary Fig. 3a). In addition, PU-H71 induced a similar pattern of dose-dependent changes in protein abundance, including Bcl6 depletion, in the Bcl6-independent DLBCL cell lines (Toledo, Pfeiffer and Karpas422) (Supplementary Fig. 3b). To determine the kinetics of PU-H71-induced Bcl6 protein depletion, we treated OCI-Ly7 cells with 0.5 μM PU-H71 for 4, 6, 12, 18, 24 and 48 h. After 18 h of treatment, Bcl6 was barely detectable (Fig. 2b) using two different antibodies for Bcl6 (N3 and C19). PU-H71 accelerated Bcl6 protein decay after protein translation blockade by Cycloheximide. Relative Bcl6 protein abundance decreased from 100% to 50% in 5 h (± 1 h) in the presence of PU-H71 compared to 24 h (± 2 h) in vehicle-treated cells (Fig. 2c). PU-H71-induced downregulation of Bcl6 was also partially blocked by treatment with the proteasome inhibitor Bortezomib (Fig. 2d).

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