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

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 p45AUF1, p42AUF1, p40AUF1, p37AUF1 and Hsp27. Actin was used as a control.
© Copyright Policy
Related In: Results  -  Collection

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

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 p45AUF1, p42AUF1, p40AUF1, p37AUF1 and Hsp27. Actin was used as a control.

Mentions: Since a proteosome inhibitor only partially rescued PU-H71 depletion of Bcl6 (Fig. 2d), we wondered whether Hsp90 might also block degradation of Bcl6 mRNA. We exposed DLBCL cells to 0.5 μM of PU-H71 for 6, 12 and 24 h and measured Bcl6 mRNA levels by QPCR. Bcl6 mRNA was reduced in a time-dependent manner (up to 3 to 5.3 fold) at 24 h compared to untreated controls (Fig. 3a). Exposure of OCI-Ly7 cells to the transcriptional inhibitor Actinomycin D in the presence of PU-H71 reduced the half-life of Bcl6 mRNA to 59 min as compared to 87 min in vehicle-treated cells, representing a 32% increase in decay rate (Fig. 3b). Heat shock factors can protect certain mRNAs containing AU-rich elements (ARE) in their 3′ untranslated regions (UTR)26 and Hsp90 inhibitors can reduce the stability of certain ARE-containing cytokine transcripts27. Analysis of the BCL6 3′ UTR revealed the presence of a putative ARE sequence (AUUUA) between nucleotides 3162 and 3168 (not shown). The decay of ARE containing mRNAs can be induced by ARE binding proteins such as the heat shock protein 27 (Hsp27) and can be prevented by the p37AUF1, p40AUF and p45AUF1 isoforms of AUF1 (AU-rich element RNA-binding protein 1)26,28–30. Accordingly, PU-H71 increased the levels of Hsp27 and decreased the levels of p37AUF1, p40AUF1 and p45AUF1 in a dose-dependent manner (Fig. 3c).


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 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 p45AUF1, p42AUF1, p40AUF1, p37AUF1 and Hsp27. Actin was used as a control.
© Copyright Policy
Related In: Results  -  Collection

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

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 p45AUF1, p42AUF1, p40AUF1, p37AUF1 and Hsp27. Actin was used as a control.
Mentions: Since a proteosome inhibitor only partially rescued PU-H71 depletion of Bcl6 (Fig. 2d), we wondered whether Hsp90 might also block degradation of Bcl6 mRNA. We exposed DLBCL cells to 0.5 μM of PU-H71 for 6, 12 and 24 h and measured Bcl6 mRNA levels by QPCR. Bcl6 mRNA was reduced in a time-dependent manner (up to 3 to 5.3 fold) at 24 h compared to untreated controls (Fig. 3a). Exposure of OCI-Ly7 cells to the transcriptional inhibitor Actinomycin D in the presence of PU-H71 reduced the half-life of Bcl6 mRNA to 59 min as compared to 87 min in vehicle-treated cells, representing a 32% increase in decay rate (Fig. 3b). Heat shock factors can protect certain mRNAs containing AU-rich elements (ARE) in their 3′ untranslated regions (UTR)26 and Hsp90 inhibitors can reduce the stability of certain ARE-containing cytokine transcripts27. Analysis of the BCL6 3′ UTR revealed the presence of a putative ARE sequence (AUUUA) between nucleotides 3162 and 3168 (not shown). The decay of ARE containing mRNAs can be induced by ARE binding proteins such as the heat shock protein 27 (Hsp27) and can be prevented by the p37AUF1, p40AUF and p45AUF1 isoforms of AUF1 (AU-rich element RNA-binding protein 1)26,28–30. Accordingly, PU-H71 increased the levels of Hsp27 and decreased the levels of p37AUF1, p40AUF1 and p45AUF1 in a dose-dependent manner (Fig. 3c).

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