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Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma.

Demosthenous C, Han JJ, Stenson MJ, Maurer MJ, Wellik LE, Link B, Hege K, Dogan A, Sotomayor E, Witzig T, Gupta M - Oncotarget (2015)

Bottom Line: Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1.Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation.These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA.

ABSTRACT
Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

No MeSH data available.


Related in: MedlinePlus

Effect of the dual mTOR kinase inhibitor CC214-1 on eIF4F complex(A) Effect of CC214-1 on eIF4F complex integrity by in vitro cap affinity assay in Jeko and Mino cells. (B) Cap dependent and independent translation was assessed by luciferase reporter assay by the use of bicistronic plasmid in the presence of CC214-1. Bars represent mean ± SD from 3 replicates. (C) Survival was evaluated in Mino, JVM2, Granta and Jeko cells treated with CC214-1. (D) Effect of CC214-1 on survival inhibition in malignant cells from fresh MCL patient was evaluated using annexin V/PI staining and flow cytometry.
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Figure 3: Effect of the dual mTOR kinase inhibitor CC214-1 on eIF4F complex(A) Effect of CC214-1 on eIF4F complex integrity by in vitro cap affinity assay in Jeko and Mino cells. (B) Cap dependent and independent translation was assessed by luciferase reporter assay by the use of bicistronic plasmid in the presence of CC214-1. Bars represent mean ± SD from 3 replicates. (C) Survival was evaluated in Mino, JVM2, Granta and Jeko cells treated with CC214-1. (D) Effect of CC214-1 on survival inhibition in malignant cells from fresh MCL patient was evaluated using annexin V/PI staining and flow cytometry.

Mentions: Since there is currently no drug that can directly inhibit the deregulated eIF4F complex in cancer cells, we assessed the effect of the next generation dual mTOR inhibitor CC214-1 on the translation initiation complex downstream of mTOR. We performed a cap affinity assay in Jeko and Mino MCL cells treated with CC214-1. CC214-1 caused a dose dependent inhibition in the association of eIF4G and eIF4A with eIF4E in both Jeko and Mino cells without much effect on the global quantity of these proteins (Figure 3A). Further evidence that CC214-1 was inhibiting the eIF4F complex formation was the increase in eIF4E bound 4E-BP1 after CC214-1 treatment of Jeko and Mino cells. Total levels of 4E-BP1 in the 10% input of CC214-1 treated immunoprecipitates were decreased (Figure 3A). Rapamycin, an mTORC1 inhibitor failed to alter the binding of eIF4G and 4E-BP1 to cap in Mino cells (Supplemental Figure 3A). We, then, evaluated the consequences of CC214-1 treatment on the 4E-BP1 phosphorylation in MCL cell lines. We observed a dose dependent decrease in the hyper-phosphorylated form of 4E-BP1 in response to CC214-1 but not with mTORC1 inhibitor at the given concentration. This effect was observed in multiple 4E-BP1 phosphorylation sites including 4E-BP1 at serine 65 in Jeko cells (Supplemental Figure 3B). However, both CC214-1 and mTORC1 inhibitor inhibited the S6 ribosomal protein phosphorylation. CC214-1 inhibited global protein translation in both Jeko and Mino cells (Supplemental Figure 4).


Translation initiation complex eIF4F is a therapeutic target for dual mTOR kinase inhibitors in non-Hodgkin lymphoma.

Demosthenous C, Han JJ, Stenson MJ, Maurer MJ, Wellik LE, Link B, Hege K, Dogan A, Sotomayor E, Witzig T, Gupta M - Oncotarget (2015)

Effect of the dual mTOR kinase inhibitor CC214-1 on eIF4F complex(A) Effect of CC214-1 on eIF4F complex integrity by in vitro cap affinity assay in Jeko and Mino cells. (B) Cap dependent and independent translation was assessed by luciferase reporter assay by the use of bicistronic plasmid in the presence of CC214-1. Bars represent mean ± SD from 3 replicates. (C) Survival was evaluated in Mino, JVM2, Granta and Jeko cells treated with CC214-1. (D) Effect of CC214-1 on survival inhibition in malignant cells from fresh MCL patient was evaluated using annexin V/PI staining and flow cytometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496233&req=5

Figure 3: Effect of the dual mTOR kinase inhibitor CC214-1 on eIF4F complex(A) Effect of CC214-1 on eIF4F complex integrity by in vitro cap affinity assay in Jeko and Mino cells. (B) Cap dependent and independent translation was assessed by luciferase reporter assay by the use of bicistronic plasmid in the presence of CC214-1. Bars represent mean ± SD from 3 replicates. (C) Survival was evaluated in Mino, JVM2, Granta and Jeko cells treated with CC214-1. (D) Effect of CC214-1 on survival inhibition in malignant cells from fresh MCL patient was evaluated using annexin V/PI staining and flow cytometry.
Mentions: Since there is currently no drug that can directly inhibit the deregulated eIF4F complex in cancer cells, we assessed the effect of the next generation dual mTOR inhibitor CC214-1 on the translation initiation complex downstream of mTOR. We performed a cap affinity assay in Jeko and Mino MCL cells treated with CC214-1. CC214-1 caused a dose dependent inhibition in the association of eIF4G and eIF4A with eIF4E in both Jeko and Mino cells without much effect on the global quantity of these proteins (Figure 3A). Further evidence that CC214-1 was inhibiting the eIF4F complex formation was the increase in eIF4E bound 4E-BP1 after CC214-1 treatment of Jeko and Mino cells. Total levels of 4E-BP1 in the 10% input of CC214-1 treated immunoprecipitates were decreased (Figure 3A). Rapamycin, an mTORC1 inhibitor failed to alter the binding of eIF4G and 4E-BP1 to cap in Mino cells (Supplemental Figure 3A). We, then, evaluated the consequences of CC214-1 treatment on the 4E-BP1 phosphorylation in MCL cell lines. We observed a dose dependent decrease in the hyper-phosphorylated form of 4E-BP1 in response to CC214-1 but not with mTORC1 inhibitor at the given concentration. This effect was observed in multiple 4E-BP1 phosphorylation sites including 4E-BP1 at serine 65 in Jeko cells (Supplemental Figure 3B). However, both CC214-1 and mTORC1 inhibitor inhibited the S6 ribosomal protein phosphorylation. CC214-1 inhibited global protein translation in both Jeko and Mino cells (Supplemental Figure 4).

Bottom Line: Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1.Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation.These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA.

ABSTRACT
Deregulated mRNA translation has been implicated in disease development and in part is controlled by a eukaryotic initiation complex eIF4F (composed of eIF4E, eIF4G and eIF4A). We demonstrate here that the cap bound fraction from lymphoma cells was enriched with eIF4G and eIF4E indicating that lymphoma cells exist in an activated translational state. Moreover, 77% (110/142) of diffuse large B cell lymphoma tumors expressed eIF4E and this was associated with an inferior event free survival. Over-expression of wild-type eIF4E (eIF4E(WT)) but not cap-mutant eIF4E (eIF4E(cap mutant)) increased the activation of the eIF4F complex. Treatment with the active-site dual mTOR inhibitor CC214-1 reduced the level of the eIF4F complex by decreasing the cap bound fraction of eIF4G and increasing the levels of 4E-BP1. CC214-1 inhibited both the cap dependent and global protein translation. CC214-1 inhibited c-Myc, and cyclin D3 translation by decreasing polysomal fractions from lymphoma cells. Inhibition of eIF4E with shRNA further decreased the CC214-1 induced inhibition of the eIF4F complex, c-Myc, cyclin D3 translation, and colony formation. These studies demonstrate that the eIF4F complex is deregulated in aggressive lymphoma and that dual mTOR therapy has therapeutic potential in these patients.

No MeSH data available.


Related in: MedlinePlus