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Dual PI3K/mTOR inhibition is required to effectively impair microenvironment survival signals in mantle cell lymphoma.

Rosich L, Montraveta A, Xargay-Torrent S, López-Guerra M, Roldán J, Aymerich M, Salaverria I, Beà S, Campo E, Pérez-Galán P, Roué G, Colomer D - Oncotarget (2014)

Bottom Line: Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage.We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases.NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL.

View Article: PubMed Central - PubMed

Affiliation: Experimental Therapeutics in Lymphoid Malignancies Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

ABSTRACT
Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis and drug resistance. Antitumor activity has been observed with mTOR inhibitors. However, they have shown limited clinical efficacy in relation to drug activation of feedback loops. Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage. Here, we have performed a comparative analysis of the mTOR inhibitor everolimus, the pan-PI3K inhibitor NVP-BKM120 and the dual PI3K/mTOR inhibitor NVP-BEZ235 in primary MCL cells. We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases. Among the three drugs, NVP-BEZ235 induced the highest change in gene expression profile. Functional validation demonstrated that NVP-BEZ235 inhibited angiogenesis, migration and tumor invasiveness in MCL cells. NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL. Our findings support the use of the dual PI3K/mTOR inhibitor NVP-BEZ235 as a promising approach to interfere with the microenvironment-related processes in MCL.

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Cytotoxic effect of everolimus, NVP-BEZ235 and NVP-BKM120 and PI3K/Akt/mTOR signaling inhibition in primary MCL cellsA, Primary MCL cells were treated with 5 μM everolimus, 1 μM NVP-BEZ235 or 1 μM NVP-BKM120 for 48 hours and cytotoxicity was measured by Annexin V labeling. Mean ± SEM of all the samples analyzed (n=11). B, Primary MCL cells (n=9) were cocultured with or without HS-5 and incubated with the corresponding drugs as above. Cell viability was assessed by Annexin V labeling at 48 hours and calculated relative to the respective untreated control, with or without stroma. Mean ± SEM of the cases analyzed. C, MCL cells were exposed for 8 hours to the corresponding drugs as previously. Analysis of phosphorylated and total levels of Akt, RPS6, 4EBP1 and EIF4E were determined by Western blot. Ratio between phosphorylated and total protein levels was calculated and relative protein quantification in treated versus control extracts was conducted with Image Gauge software (Fujifilm). α-tubulin was probed as a loading control. Two representative cases are shown (MCL nº.1 and nº.7). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
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Figure 1: Cytotoxic effect of everolimus, NVP-BEZ235 and NVP-BKM120 and PI3K/Akt/mTOR signaling inhibition in primary MCL cellsA, Primary MCL cells were treated with 5 μM everolimus, 1 μM NVP-BEZ235 or 1 μM NVP-BKM120 for 48 hours and cytotoxicity was measured by Annexin V labeling. Mean ± SEM of all the samples analyzed (n=11). B, Primary MCL cells (n=9) were cocultured with or without HS-5 and incubated with the corresponding drugs as above. Cell viability was assessed by Annexin V labeling at 48 hours and calculated relative to the respective untreated control, with or without stroma. Mean ± SEM of the cases analyzed. C, MCL cells were exposed for 8 hours to the corresponding drugs as previously. Analysis of phosphorylated and total levels of Akt, RPS6, 4EBP1 and EIF4E were determined by Western blot. Ratio between phosphorylated and total protein levels was calculated and relative protein quantification in treated versus control extracts was conducted with Image Gauge software (Fujifilm). α-tubulin was probed as a loading control. Two representative cases are shown (MCL nº.1 and nº.7). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

Mentions: Cells from 11 primary MCL cases were exposed to everolimus (5 μM), NVP-BEZ235 (1 μM) or NVP-BKM120 (1 μM) for 48 hours and cytotoxicity was measured by Annexin V labeling (Table 1). Drug optimal doses were selected based on previous studies.[10;18;24] These doses showed a low cytotoxic effect in healthy B and T lymphocytes (data not shown). Figure 1A the three compounds induced significant cytotoxicity compared to control (**, P < 0.01; ***, P < 0.001). Among them, NVP-BEZ235 induced a high cytotoxic effect with a mean response of 40.80 ± 21.30 % which was significantly higher than that observed with everolimus (mean response of 22.74 ± 17.63 %; **, P < 0.01). The antitumor effect of the pan-PI3K inhibitor NVP-BKM120 reached 31.93 ± 17.31 %. The sensitivity to these drugs was not related to genomic alterations of PI3K/Akt/mTOR (PTEN deletion, PIK3CA and AKT1 amplifications) or TP53 alterations (Table 1).


Dual PI3K/mTOR inhibition is required to effectively impair microenvironment survival signals in mantle cell lymphoma.

Rosich L, Montraveta A, Xargay-Torrent S, López-Guerra M, Roldán J, Aymerich M, Salaverria I, Beà S, Campo E, Pérez-Galán P, Roué G, Colomer D - Oncotarget (2014)

Cytotoxic effect of everolimus, NVP-BEZ235 and NVP-BKM120 and PI3K/Akt/mTOR signaling inhibition in primary MCL cellsA, Primary MCL cells were treated with 5 μM everolimus, 1 μM NVP-BEZ235 or 1 μM NVP-BKM120 for 48 hours and cytotoxicity was measured by Annexin V labeling. Mean ± SEM of all the samples analyzed (n=11). B, Primary MCL cells (n=9) were cocultured with or without HS-5 and incubated with the corresponding drugs as above. Cell viability was assessed by Annexin V labeling at 48 hours and calculated relative to the respective untreated control, with or without stroma. Mean ± SEM of the cases analyzed. C, MCL cells were exposed for 8 hours to the corresponding drugs as previously. Analysis of phosphorylated and total levels of Akt, RPS6, 4EBP1 and EIF4E were determined by Western blot. Ratio between phosphorylated and total protein levels was calculated and relative protein quantification in treated versus control extracts was conducted with Image Gauge software (Fujifilm). α-tubulin was probed as a loading control. Two representative cases are shown (MCL nº.1 and nº.7). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
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Figure 1: Cytotoxic effect of everolimus, NVP-BEZ235 and NVP-BKM120 and PI3K/Akt/mTOR signaling inhibition in primary MCL cellsA, Primary MCL cells were treated with 5 μM everolimus, 1 μM NVP-BEZ235 or 1 μM NVP-BKM120 for 48 hours and cytotoxicity was measured by Annexin V labeling. Mean ± SEM of all the samples analyzed (n=11). B, Primary MCL cells (n=9) were cocultured with or without HS-5 and incubated with the corresponding drugs as above. Cell viability was assessed by Annexin V labeling at 48 hours and calculated relative to the respective untreated control, with or without stroma. Mean ± SEM of the cases analyzed. C, MCL cells were exposed for 8 hours to the corresponding drugs as previously. Analysis of phosphorylated and total levels of Akt, RPS6, 4EBP1 and EIF4E were determined by Western blot. Ratio between phosphorylated and total protein levels was calculated and relative protein quantification in treated versus control extracts was conducted with Image Gauge software (Fujifilm). α-tubulin was probed as a loading control. Two representative cases are shown (MCL nº.1 and nº.7). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
Mentions: Cells from 11 primary MCL cases were exposed to everolimus (5 μM), NVP-BEZ235 (1 μM) or NVP-BKM120 (1 μM) for 48 hours and cytotoxicity was measured by Annexin V labeling (Table 1). Drug optimal doses were selected based on previous studies.[10;18;24] These doses showed a low cytotoxic effect in healthy B and T lymphocytes (data not shown). Figure 1A the three compounds induced significant cytotoxicity compared to control (**, P < 0.01; ***, P < 0.001). Among them, NVP-BEZ235 induced a high cytotoxic effect with a mean response of 40.80 ± 21.30 % which was significantly higher than that observed with everolimus (mean response of 22.74 ± 17.63 %; **, P < 0.01). The antitumor effect of the pan-PI3K inhibitor NVP-BKM120 reached 31.93 ± 17.31 %. The sensitivity to these drugs was not related to genomic alterations of PI3K/Akt/mTOR (PTEN deletion, PIK3CA and AKT1 amplifications) or TP53 alterations (Table 1).

Bottom Line: Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage.We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases.NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL.

View Article: PubMed Central - PubMed

Affiliation: Experimental Therapeutics in Lymphoid Malignancies Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.

ABSTRACT
Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis and drug resistance. Antitumor activity has been observed with mTOR inhibitors. However, they have shown limited clinical efficacy in relation to drug activation of feedback loops. Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage. Here, we have performed a comparative analysis of the mTOR inhibitor everolimus, the pan-PI3K inhibitor NVP-BKM120 and the dual PI3K/mTOR inhibitor NVP-BEZ235 in primary MCL cells. We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases. Among the three drugs, NVP-BEZ235 induced the highest change in gene expression profile. Functional validation demonstrated that NVP-BEZ235 inhibited angiogenesis, migration and tumor invasiveness in MCL cells. NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL. Our findings support the use of the dual PI3K/mTOR inhibitor NVP-BEZ235 as a promising approach to interfere with the microenvironment-related processes in MCL.

Show MeSH
Related in: MedlinePlus