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Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma.

Cloughesy TF, Yoshimoto K, Nghiemphu P, Brown K, Dang J, Zhu S, Hsueh T, Chen Y, Wang W, Youngkin D, Liau L, Martin N, Becker D, Bergsneider M, Lai A, Green R, Oglesby T, Koleto M, Trent J, Horvath S, Mischel PS, Mellinghoff IK, Sawyers CL - PLoS Med. (2008)

Bottom Line: Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration.Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT

Background: There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.

Methods and findings: Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).

Conclusions: Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.

Trial registration: http://www.ClinicalTrials.gov (#NCT00047073).

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Induction of Akt Signaling in a Subset of Rapamycin-Treated Tumors(A) Determination of Akt activation in tumor tissue during mTOR inhibitor therapy. The left panel is a cartoon illustrating the mTOR/S6K1 dependent feedback loop of the PI3k-Akt pathway. The right panel shows changes in phosphorylation of the Akt-substrate PRAS40 (threonine 246) during rapamycin therapy correspond to changes in Akt phosphorylation (serine 473). Tumors from patients 2 and 5 show an increase in pAkt and pPRAS40 immunostaining during rapamycin treatment, whereas the tumor from patient 11 shows a decrease in the same markers on rapamycin.(B) Changes in PRAS40 phosphorylation between S1 and S2 for 14/15 rapamycin patients. The horizontal line inside the box indicates the median value. The lower and upper border of the box corresponds to the 25th and 75th percentile, respectively. The whiskers extend to the 95% range. Paraffin blocks from patient 14 were not sufficient for quantification. N.S. indicates that the difference in pPRAS40 staining intensity not statistically significant at the 0.05 level according to the Wilcoxon test.(C) Kaplan Meier analysis illustrating the relationship between pPRAS40 induction and time-to-tumor progression.
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pmed-0050008-g004: Induction of Akt Signaling in a Subset of Rapamycin-Treated Tumors(A) Determination of Akt activation in tumor tissue during mTOR inhibitor therapy. The left panel is a cartoon illustrating the mTOR/S6K1 dependent feedback loop of the PI3k-Akt pathway. The right panel shows changes in phosphorylation of the Akt-substrate PRAS40 (threonine 246) during rapamycin therapy correspond to changes in Akt phosphorylation (serine 473). Tumors from patients 2 and 5 show an increase in pAkt and pPRAS40 immunostaining during rapamycin treatment, whereas the tumor from patient 11 shows a decrease in the same markers on rapamycin.(B) Changes in PRAS40 phosphorylation between S1 and S2 for 14/15 rapamycin patients. The horizontal line inside the box indicates the median value. The lower and upper border of the box corresponds to the 25th and 75th percentile, respectively. The whiskers extend to the 95% range. Paraffin blocks from patient 14 were not sufficient for quantification. N.S. indicates that the difference in pPRAS40 staining intensity not statistically significant at the 0.05 level according to the Wilcoxon test.(C) Kaplan Meier analysis illustrating the relationship between pPRAS40 induction and time-to-tumor progression.

Mentions: Physiologic activation of the Akt pathway is regulated, in part, by a negative feedback loop involving phosphorylation of insulin receptor substrate 1 (IRS1) by the mTOR effector molecule S6 kinase 1 (Figure 4A) [23–26]. mTOR inhibition by rapamycin can cancel this negative feedback and activate Akt in some cancer cell lines and tumor samples, but the potential clinical impact is unknown [8,27,28]. We assessed Akt activity in S1 and S2 samples in the rapamycin-treated patients using phosphosite-specific antibodies against the serine/threonine kinase Akt (serine 473) and its downstream substrate PRAS 40 (threonine 246), which serves as a biomarker for Akt activity (Figure 4A). PRAS40 has also been recently shown to inhibit mTOR, and this inhibition is relieved by Akt phosphorylation [29–31]. Seven of 14 (50%) patients had a statistically significant (p < 0.05, Wilcoxon test) increase in PRAS40 phosphorylation in their S2 sample (Figure 4B). Of note, one patient (11) had a significant decrease in PRAS40 phosphorylation (and pS473 Akt) at S2 (Figure 4B), which could reflect the potential inhibition of the TORC2 mTOR complex (implicated as the pS473 Akt kinase) by rapamycin after prolonged exposure [32,33].


Antitumor activity of rapamycin in a Phase I trial for patients with recurrent PTEN-deficient glioblastoma.

Cloughesy TF, Yoshimoto K, Nghiemphu P, Brown K, Dang J, Zhu S, Hsueh T, Chen Y, Wang W, Youngkin D, Liau L, Martin N, Becker D, Bergsneider M, Lai A, Green R, Oglesby T, Koleto M, Trent J, Horvath S, Mischel PS, Mellinghoff IK, Sawyers CL - PLoS Med. (2008)

Induction of Akt Signaling in a Subset of Rapamycin-Treated Tumors(A) Determination of Akt activation in tumor tissue during mTOR inhibitor therapy. The left panel is a cartoon illustrating the mTOR/S6K1 dependent feedback loop of the PI3k-Akt pathway. The right panel shows changes in phosphorylation of the Akt-substrate PRAS40 (threonine 246) during rapamycin therapy correspond to changes in Akt phosphorylation (serine 473). Tumors from patients 2 and 5 show an increase in pAkt and pPRAS40 immunostaining during rapamycin treatment, whereas the tumor from patient 11 shows a decrease in the same markers on rapamycin.(B) Changes in PRAS40 phosphorylation between S1 and S2 for 14/15 rapamycin patients. The horizontal line inside the box indicates the median value. The lower and upper border of the box corresponds to the 25th and 75th percentile, respectively. The whiskers extend to the 95% range. Paraffin blocks from patient 14 were not sufficient for quantification. N.S. indicates that the difference in pPRAS40 staining intensity not statistically significant at the 0.05 level according to the Wilcoxon test.(C) Kaplan Meier analysis illustrating the relationship between pPRAS40 induction and time-to-tumor progression.
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pmed-0050008-g004: Induction of Akt Signaling in a Subset of Rapamycin-Treated Tumors(A) Determination of Akt activation in tumor tissue during mTOR inhibitor therapy. The left panel is a cartoon illustrating the mTOR/S6K1 dependent feedback loop of the PI3k-Akt pathway. The right panel shows changes in phosphorylation of the Akt-substrate PRAS40 (threonine 246) during rapamycin therapy correspond to changes in Akt phosphorylation (serine 473). Tumors from patients 2 and 5 show an increase in pAkt and pPRAS40 immunostaining during rapamycin treatment, whereas the tumor from patient 11 shows a decrease in the same markers on rapamycin.(B) Changes in PRAS40 phosphorylation between S1 and S2 for 14/15 rapamycin patients. The horizontal line inside the box indicates the median value. The lower and upper border of the box corresponds to the 25th and 75th percentile, respectively. The whiskers extend to the 95% range. Paraffin blocks from patient 14 were not sufficient for quantification. N.S. indicates that the difference in pPRAS40 staining intensity not statistically significant at the 0.05 level according to the Wilcoxon test.(C) Kaplan Meier analysis illustrating the relationship between pPRAS40 induction and time-to-tumor progression.
Mentions: Physiologic activation of the Akt pathway is regulated, in part, by a negative feedback loop involving phosphorylation of insulin receptor substrate 1 (IRS1) by the mTOR effector molecule S6 kinase 1 (Figure 4A) [23–26]. mTOR inhibition by rapamycin can cancel this negative feedback and activate Akt in some cancer cell lines and tumor samples, but the potential clinical impact is unknown [8,27,28]. We assessed Akt activity in S1 and S2 samples in the rapamycin-treated patients using phosphosite-specific antibodies against the serine/threonine kinase Akt (serine 473) and its downstream substrate PRAS 40 (threonine 246), which serves as a biomarker for Akt activity (Figure 4A). PRAS40 has also been recently shown to inhibit mTOR, and this inhibition is relieved by Akt phosphorylation [29–31]. Seven of 14 (50%) patients had a statistically significant (p < 0.05, Wilcoxon test) increase in PRAS40 phosphorylation in their S2 sample (Figure 4B). Of note, one patient (11) had a significant decrease in PRAS40 phosphorylation (and pS473 Akt) at S2 (Figure 4B), which could reflect the potential inhibition of the TORC2 mTOR complex (implicated as the pS473 Akt kinase) by rapamycin after prolonged exposure [32,33].

Bottom Line: Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration.Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT

Background: There is much discussion in the cancer drug development community about how to incorporate molecular tools into early-stage clinical trials to assess target modulation, measure anti-tumor activity, and enrich the clinical trial population for patients who are more likely to benefit. Small, molecularly focused clinical studies offer the promise of the early definition of optimal biologic dose and patient population.

Methods and findings: Based on preclinical evidence that phosphatase and tensin homolog deleted on Chromosome 10 (PTEN) loss sensitizes tumors to the inhibition of mammalian target of rapamycin (mTOR), we conducted a proof-of-concept Phase I neoadjuvant trial of rapamycin in patients with recurrent glioblastoma, whose tumors lacked expression of the tumor suppressor PTEN. We aimed to assess the safety profile of daily rapamycin in patients with glioma, define the dose of rapamycin required for mTOR inhibition in tumor tissue, and evaluate the antiproliferative activity of rapamycin in PTEN-deficient glioblastoma. Although intratumoral rapamycin concentrations that were sufficient to inhibit mTOR in vitro were achieved in all patients, the magnitude of mTOR inhibition in tumor cells (measured by reduced ribosomal S6 protein phosphorylation) varied substantially. Tumor cell proliferation (measured by Ki-67 staining) was dramatically reduced in seven of 14 patients after 1 wk of rapamycin treatment and was associated with the magnitude of mTOR inhibition (p = 0.0047, Fisher exact test) but not the intratumoral rapamycin concentration. Tumor cells harvested from the Ki-67 nonresponders retained sensitivity to rapamycin ex vivo, indicating that clinical resistance to biochemical mTOR inhibition was not cell-intrinsic. Rapamycin treatment led to Akt activation in seven patients, presumably due to loss of negative feedback, and this activation was associated with shorter time-to-progression during post-surgical maintenance rapamycin therapy (p < 0.05, Logrank test).

Conclusions: Rapamycin has anticancer activity in PTEN-deficient glioblastoma and warrants further clinical study alone or in combination with PI3K pathway inhibitors. The short-term treatment endpoints used in this neoadjuvant trial design identified the importance of monitoring target inhibition and negative feedback to guide future clinical development.

Trial registration: http://www.ClinicalTrials.gov (#NCT00047073).

Show MeSH
Related in: MedlinePlus