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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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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.
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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.

Mentions: In order to determine the efficacy of PU-H71 in vivo, Farage, OCI-Ly7 and SU-DHL4 DLBCL xenografts were established in ten SCID mice each (total n = 30). Once palpable tumors were detected, pairs of mice were randomized to receive either PU-H71 75 mg per kg body weight per day (n = 5 per cell line) or vehicle (n = 5 per cell line). Animals were sacrificed when two or more controls reached the maximum tumor burden permitted in our animal protocol. Compared to control, PU-H71 potently reduced DLBCL size and weight (Fig. 5a,b). The serum levels of human β2-microglobulin, a marker of tumor burden, were also reduced by PU-H71 (Fig. 5c). Using tumor growth to critical mass as a surrogate for survival, PU-H71 (n = 15) significantly prolonged the survival v controls (n = 15) (Cox’s F test, P < 0.0001; Fig. 5d). In contrast, PU-H71 had no effect on the Bcl6-independent DLBCL Toledo xenografts at a similar dose (Supplementary Fig. 8). PU-H71 induced depletion of Bcl6 protein in the tumor xenografts (Fig. 5e and Supplementary Fig. 9a) and increased the fraction of cells undergoing apoptosis as detected by TUNEL assay and PARP cleavage (Fig. 5f and Supplementary Fig. 9a). Similar to its effects in vitro, PU-H71 decreased the abundance of c-Raf, Akt and NEMO and induced Hsp70 in the DLBCL xenografts (Fig. 6a and Supplementary Fig. 9b). There was no evidence of toxicity from behavioral, macroscopic or microscopic standpoints (Supplementary Fig. 10a and data not shown). Long-term exposure (20 days) to PU-H71 75 mg per kg body weight per day in OCI-Ly7 xenografts caused significant retardation of tumor growth followed by a steady phase in which the tumors maintained a relatively constant volume of 500 mm3 (5 to 6 times the initial size) (Supplementary Fig. 11a). A similar effect was evident with the slow-growing BCL6-dependent SU-DHL6 xenograft, although these tumors were more completely suppressed and did not grow beyond 100 mm3 (Supplementary Fig. 11a). PU-H71 at 75 mg per kg body weight per day was able to decelerate tumor growth in these models even when the tumors were allowed to reach 500 mm3 prior to initiating treatment (Supplementary Fig. 11b). Again, there was no evidence of toxicity in these animals (data not shown). A more extensive toxicity study performed in normal Balb/c mice treated with either 50 mg per kg body weight per day (n = 5) or 75 mg per kg body weight per day (n = 5) of PU-H71 for 10 days (compared to vehicle; n = 5) showed no evidence of macroscopic or microscopic toxicity (Supplementary Fig. 10b and Supplementary Fig. 12). There was also no evidence of hematologic, renal or hepatic toxicity as determined by complete blood counts, blood chemistry, liver function tests and thyroid hormone testing (since PU-H71 is iodinated) (Supplementary Fig. 10c).


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)

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.
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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.
Mentions: In order to determine the efficacy of PU-H71 in vivo, Farage, OCI-Ly7 and SU-DHL4 DLBCL xenografts were established in ten SCID mice each (total n = 30). Once palpable tumors were detected, pairs of mice were randomized to receive either PU-H71 75 mg per kg body weight per day (n = 5 per cell line) or vehicle (n = 5 per cell line). Animals were sacrificed when two or more controls reached the maximum tumor burden permitted in our animal protocol. Compared to control, PU-H71 potently reduced DLBCL size and weight (Fig. 5a,b). The serum levels of human β2-microglobulin, a marker of tumor burden, were also reduced by PU-H71 (Fig. 5c). Using tumor growth to critical mass as a surrogate for survival, PU-H71 (n = 15) significantly prolonged the survival v controls (n = 15) (Cox’s F test, P < 0.0001; Fig. 5d). In contrast, PU-H71 had no effect on the Bcl6-independent DLBCL Toledo xenografts at a similar dose (Supplementary Fig. 8). PU-H71 induced depletion of Bcl6 protein in the tumor xenografts (Fig. 5e and Supplementary Fig. 9a) and increased the fraction of cells undergoing apoptosis as detected by TUNEL assay and PARP cleavage (Fig. 5f and Supplementary Fig. 9a). Similar to its effects in vitro, PU-H71 decreased the abundance of c-Raf, Akt and NEMO and induced Hsp70 in the DLBCL xenografts (Fig. 6a and Supplementary Fig. 9b). There was no evidence of toxicity from behavioral, macroscopic or microscopic standpoints (Supplementary Fig. 10a and data not shown). Long-term exposure (20 days) to PU-H71 75 mg per kg body weight per day in OCI-Ly7 xenografts caused significant retardation of tumor growth followed by a steady phase in which the tumors maintained a relatively constant volume of 500 mm3 (5 to 6 times the initial size) (Supplementary Fig. 11a). A similar effect was evident with the slow-growing BCL6-dependent SU-DHL6 xenograft, although these tumors were more completely suppressed and did not grow beyond 100 mm3 (Supplementary Fig. 11a). PU-H71 at 75 mg per kg body weight per day was able to decelerate tumor growth in these models even when the tumors were allowed to reach 500 mm3 prior to initiating treatment (Supplementary Fig. 11b). Again, there was no evidence of toxicity in these animals (data not shown). A more extensive toxicity study performed in normal Balb/c mice treated with either 50 mg per kg body weight per day (n = 5) or 75 mg per kg body weight per day (n = 5) of PU-H71 for 10 days (compared to vehicle; n = 5) showed no evidence of macroscopic or microscopic toxicity (Supplementary Fig. 10b and Supplementary Fig. 12). There was also no evidence of hematologic, renal or hepatic toxicity as determined by complete blood counts, blood chemistry, liver function tests and thyroid hormone testing (since PU-H71 is iodinated) (Supplementary Fig. 10c).

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