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A mechanism for synergy with combined mTOR and PI3 kinase inhibitors.

Yang S, Xiao X, Meng X, Leslie KK - PLoS ONE (2011)

Bottom Line: Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone.Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27.While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics & Gynecology, The University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.

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Mechanism of synergistic effect for combination treatment of temsirolimus and BEZ235.A, Phosphorylation of 4E-BP1 (P-4E-BP1 T37/T46) was assessed after incubating cells with the indicated treatments for 24 hrs. Total 4E-BP1 expression serves as a loading control. B, Cells were treated with 1 nM or 10 nM temsirolimus or BEZ235 for 72 hrs then phosphorylation of rS6 (P-rS6 S235/S236) determined by Western blotting. Total rS6 serves as a loading control. C, Proposed mechanism for synergistic effect. Temsirolimus blocks one arm of mTORC1 signaling as evidenced by lack of phosphorylation of rS6. As a dual inhibitor of PI3K and mTOR, BEZ235 acts both upstream as well as downstream of mTORC1 and mTORC2 through inhibition of Akt (upstream) and 4E-BP1 (downstream) phosphorylation. BEZ235 has minimal effects on rS6 phosphorylation, but combination with temsirolimus blocks both arms of mTORC1 signaling. As a specific PI3K inhibitor, ZSTK474 acts upstream of the PI3K/Akt/mTOR signaling pathway, and in combination with temsirolimus, can block rS6 but not 4E-BP1 activation.
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pone-0026343-g007: Mechanism of synergistic effect for combination treatment of temsirolimus and BEZ235.A, Phosphorylation of 4E-BP1 (P-4E-BP1 T37/T46) was assessed after incubating cells with the indicated treatments for 24 hrs. Total 4E-BP1 expression serves as a loading control. B, Cells were treated with 1 nM or 10 nM temsirolimus or BEZ235 for 72 hrs then phosphorylation of rS6 (P-rS6 S235/S236) determined by Western blotting. Total rS6 serves as a loading control. C, Proposed mechanism for synergistic effect. Temsirolimus blocks one arm of mTORC1 signaling as evidenced by lack of phosphorylation of rS6. As a dual inhibitor of PI3K and mTOR, BEZ235 acts both upstream as well as downstream of mTORC1 and mTORC2 through inhibition of Akt (upstream) and 4E-BP1 (downstream) phosphorylation. BEZ235 has minimal effects on rS6 phosphorylation, but combination with temsirolimus blocks both arms of mTORC1 signaling. As a specific PI3K inhibitor, ZSTK474 acts upstream of the PI3K/Akt/mTOR signaling pathway, and in combination with temsirolimus, can block rS6 but not 4E-BP1 activation.

Mentions: Recently, several groups reported that 4E-BP1, not rS6, which are both downstream of mTORC1, is critical for controlling oncogenic Akt signaling [10], [11]. Additionally, it was demonstrated the BEZ235 is more effective at down-regulating 4E-BP1 phosphorylation as compared to rapamycin and second-generation mTOR inhibitors [11]. ZSTK474 targets PI3K, which is upstream of PI3K/Akt/mTOR signaling, while temsirolimus inhibits mTORC1, which is downstream of this pathway. This strategy, targeting both ends of the same pathway, clearly demonstrated synergy in cell proliferation assays (Fig. 4B). However, BEZ235 itself is a dual PI3K/mTOR inhibitor that functions both upstream and downstream of the PI3K/Akt/mTOR pathway and should be sufficient to completely block signaling; nevertheless, a synergistic effect was observed when BEZ235 was combined with another mTOR inhibitor, temsirolimus. This finding was unexpected, and we sought an explanation for why blocking mTORC1 with two different agents produced synergy. We addressed this question by examining the individual effect of BEZ235 and temsirolimus on downstream components of mTORC1 pathway signaling (Fig. 7A, B). The downstream components tested were 4E-BP1 and rS6 (Fig. 7C). Surprisingly, temsirolimus had little or no effect on inhibiting 4E-BP1 phosphorylation compared with control despite its ability to fully block phosphorylation of rS6 (Fig. 7B). On the other hand, BEZ235 completely blocked 4E-BP1 phosphorylation in all tested cell lines but had less effect on rS6 phosphorylation (Fig. 7A, B). When testing the phosphorylation of rS6, we discovered that temsirolimus inhibited it much more effectively (at 1 nM) than BEZ235 at the same concentration (Fig. 7B and Supporting Fig. S3). In summary, to fully block both of the important downstream effectors, both drugs were required. Figure 7C is a schematic model to summarize our findings on the specific mechanistic functions of PI3K/Akt/mTOR inhibitors in the treatment of cancers that are reliant on this pathway.


A mechanism for synergy with combined mTOR and PI3 kinase inhibitors.

Yang S, Xiao X, Meng X, Leslie KK - PLoS ONE (2011)

Mechanism of synergistic effect for combination treatment of temsirolimus and BEZ235.A, Phosphorylation of 4E-BP1 (P-4E-BP1 T37/T46) was assessed after incubating cells with the indicated treatments for 24 hrs. Total 4E-BP1 expression serves as a loading control. B, Cells were treated with 1 nM or 10 nM temsirolimus or BEZ235 for 72 hrs then phosphorylation of rS6 (P-rS6 S235/S236) determined by Western blotting. Total rS6 serves as a loading control. C, Proposed mechanism for synergistic effect. Temsirolimus blocks one arm of mTORC1 signaling as evidenced by lack of phosphorylation of rS6. As a dual inhibitor of PI3K and mTOR, BEZ235 acts both upstream as well as downstream of mTORC1 and mTORC2 through inhibition of Akt (upstream) and 4E-BP1 (downstream) phosphorylation. BEZ235 has minimal effects on rS6 phosphorylation, but combination with temsirolimus blocks both arms of mTORC1 signaling. As a specific PI3K inhibitor, ZSTK474 acts upstream of the PI3K/Akt/mTOR signaling pathway, and in combination with temsirolimus, can block rS6 but not 4E-BP1 activation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198385&req=5

pone-0026343-g007: Mechanism of synergistic effect for combination treatment of temsirolimus and BEZ235.A, Phosphorylation of 4E-BP1 (P-4E-BP1 T37/T46) was assessed after incubating cells with the indicated treatments for 24 hrs. Total 4E-BP1 expression serves as a loading control. B, Cells were treated with 1 nM or 10 nM temsirolimus or BEZ235 for 72 hrs then phosphorylation of rS6 (P-rS6 S235/S236) determined by Western blotting. Total rS6 serves as a loading control. C, Proposed mechanism for synergistic effect. Temsirolimus blocks one arm of mTORC1 signaling as evidenced by lack of phosphorylation of rS6. As a dual inhibitor of PI3K and mTOR, BEZ235 acts both upstream as well as downstream of mTORC1 and mTORC2 through inhibition of Akt (upstream) and 4E-BP1 (downstream) phosphorylation. BEZ235 has minimal effects on rS6 phosphorylation, but combination with temsirolimus blocks both arms of mTORC1 signaling. As a specific PI3K inhibitor, ZSTK474 acts upstream of the PI3K/Akt/mTOR signaling pathway, and in combination with temsirolimus, can block rS6 but not 4E-BP1 activation.
Mentions: Recently, several groups reported that 4E-BP1, not rS6, which are both downstream of mTORC1, is critical for controlling oncogenic Akt signaling [10], [11]. Additionally, it was demonstrated the BEZ235 is more effective at down-regulating 4E-BP1 phosphorylation as compared to rapamycin and second-generation mTOR inhibitors [11]. ZSTK474 targets PI3K, which is upstream of PI3K/Akt/mTOR signaling, while temsirolimus inhibits mTORC1, which is downstream of this pathway. This strategy, targeting both ends of the same pathway, clearly demonstrated synergy in cell proliferation assays (Fig. 4B). However, BEZ235 itself is a dual PI3K/mTOR inhibitor that functions both upstream and downstream of the PI3K/Akt/mTOR pathway and should be sufficient to completely block signaling; nevertheless, a synergistic effect was observed when BEZ235 was combined with another mTOR inhibitor, temsirolimus. This finding was unexpected, and we sought an explanation for why blocking mTORC1 with two different agents produced synergy. We addressed this question by examining the individual effect of BEZ235 and temsirolimus on downstream components of mTORC1 pathway signaling (Fig. 7A, B). The downstream components tested were 4E-BP1 and rS6 (Fig. 7C). Surprisingly, temsirolimus had little or no effect on inhibiting 4E-BP1 phosphorylation compared with control despite its ability to fully block phosphorylation of rS6 (Fig. 7B). On the other hand, BEZ235 completely blocked 4E-BP1 phosphorylation in all tested cell lines but had less effect on rS6 phosphorylation (Fig. 7A, B). When testing the phosphorylation of rS6, we discovered that temsirolimus inhibited it much more effectively (at 1 nM) than BEZ235 at the same concentration (Fig. 7B and Supporting Fig. S3). In summary, to fully block both of the important downstream effectors, both drugs were required. Figure 7C is a schematic model to summarize our findings on the specific mechanistic functions of PI3K/Akt/mTOR inhibitors in the treatment of cancers that are reliant on this pathway.

Bottom Line: Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone.Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27.While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics & Gynecology, The University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.

Show MeSH
Related in: MedlinePlus