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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

Integrity of eIF4F complex in normal B cells and lymphoma cells(A)In vitro cap affinity assay was performed in 4 MCL cell lines (as indicated in the figure) and CD19+ normal B cells and western blotting was performed using eIF4G, eIF4A and eIF4E antibodies. (B) Effect of eIF4E inhibition on eIF4F complex integrity was assessed in the lysates from stably transfected HEK293eIF4E/KO and HEK293control shRNA cells by the cap affinity assay. The experiment was performed in triplicate with similar results (C)In vitro cap affinity assay in HEK293 cells after transient overexpression of eIF4EWT and eIF4Ecap mutant. The experiment was performed in triplicate. (D) Colony-forming assay was performed in stably transfected HEK293eIF4E/KO and HEK293control shRNA cells. Bars represent mean ± SD from 3 replicates. The experiment was performed in triplicate.
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Figure 1: Integrity of eIF4F complex in normal B cells and lymphoma cells(A)In vitro cap affinity assay was performed in 4 MCL cell lines (as indicated in the figure) and CD19+ normal B cells and western blotting was performed using eIF4G, eIF4A and eIF4E antibodies. (B) Effect of eIF4E inhibition on eIF4F complex integrity was assessed in the lysates from stably transfected HEK293eIF4E/KO and HEK293control shRNA cells by the cap affinity assay. The experiment was performed in triplicate with similar results (C)In vitro cap affinity assay in HEK293 cells after transient overexpression of eIF4EWT and eIF4Ecap mutant. The experiment was performed in triplicate. (D) Colony-forming assay was performed in stably transfected HEK293eIF4E/KO and HEK293control shRNA cells. Bars represent mean ± SD from 3 replicates. The experiment was performed in triplicate.

Mentions: We assessed the formation of the active eIF4F (m7GTP-eIF4E-eIF4G) translation initiation complex by a pull down assay using an agarose-immobilized m7GTP cap analog to capture eIF4E and its binding partners eIF4G and eIF4A in MCL cell line cells. The relative amount of captured eIF4G or eIF4A serves as an indicator of the integrity of the eIF4F translation complex. Cell lysates from Jeko, Mino, Granta, JVM2 and CD19+ normal B cells were incubated with m7GTP and analyzed by immunoblotting for the level of eIF4A and eIF4G. Our data demonstrate that the cap-bound fraction from normal B cells contained very little eIF4G and eIF4A. However, all the MCL cell lysates were enriched with eIF4G, eIF4E and eIF4A (Figure 1A). In order to determine the association of eIF4E with eIF4G, we repeated this experiment by pulling down eIF4G from the cell lysates of MCL cell lines and demonstrated that the immunoprecipitates of eIF4G fraction in MCL cell lysates were indeed enriched compared to normal B cells and IgG control (Supplemental Figure 1A). A pull down assay using eIF4E antibody demonstrated that immunoprecipitates of the eIF4E fraction were enriched in malignant B cells, suggesting reciprocal binding between eIF4G and eIF4E in MCL cells (Supplemental Figure 1B). Overall, these data demonstrate that the cap bound fraction from lymphoma cells was enriched with eIF4G, eIF4E and eIF4A, demonstrating that aggressive lymphoma B cells exist in a translationally activated state.


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)

Integrity of eIF4F complex in normal B cells and lymphoma cells(A)In vitro cap affinity assay was performed in 4 MCL cell lines (as indicated in the figure) and CD19+ normal B cells and western blotting was performed using eIF4G, eIF4A and eIF4E antibodies. (B) Effect of eIF4E inhibition on eIF4F complex integrity was assessed in the lysates from stably transfected HEK293eIF4E/KO and HEK293control shRNA cells by the cap affinity assay. The experiment was performed in triplicate with similar results (C)In vitro cap affinity assay in HEK293 cells after transient overexpression of eIF4EWT and eIF4Ecap mutant. The experiment was performed in triplicate. (D) Colony-forming assay was performed in stably transfected HEK293eIF4E/KO and HEK293control shRNA cells. Bars represent mean ± SD from 3 replicates. The experiment was performed in triplicate.
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Figure 1: Integrity of eIF4F complex in normal B cells and lymphoma cells(A)In vitro cap affinity assay was performed in 4 MCL cell lines (as indicated in the figure) and CD19+ normal B cells and western blotting was performed using eIF4G, eIF4A and eIF4E antibodies. (B) Effect of eIF4E inhibition on eIF4F complex integrity was assessed in the lysates from stably transfected HEK293eIF4E/KO and HEK293control shRNA cells by the cap affinity assay. The experiment was performed in triplicate with similar results (C)In vitro cap affinity assay in HEK293 cells after transient overexpression of eIF4EWT and eIF4Ecap mutant. The experiment was performed in triplicate. (D) Colony-forming assay was performed in stably transfected HEK293eIF4E/KO and HEK293control shRNA cells. Bars represent mean ± SD from 3 replicates. The experiment was performed in triplicate.
Mentions: We assessed the formation of the active eIF4F (m7GTP-eIF4E-eIF4G) translation initiation complex by a pull down assay using an agarose-immobilized m7GTP cap analog to capture eIF4E and its binding partners eIF4G and eIF4A in MCL cell line cells. The relative amount of captured eIF4G or eIF4A serves as an indicator of the integrity of the eIF4F translation complex. Cell lysates from Jeko, Mino, Granta, JVM2 and CD19+ normal B cells were incubated with m7GTP and analyzed by immunoblotting for the level of eIF4A and eIF4G. Our data demonstrate that the cap-bound fraction from normal B cells contained very little eIF4G and eIF4A. However, all the MCL cell lysates were enriched with eIF4G, eIF4E and eIF4A (Figure 1A). In order to determine the association of eIF4E with eIF4G, we repeated this experiment by pulling down eIF4G from the cell lysates of MCL cell lines and demonstrated that the immunoprecipitates of eIF4G fraction in MCL cell lysates were indeed enriched compared to normal B cells and IgG control (Supplemental Figure 1A). A pull down assay using eIF4E antibody demonstrated that immunoprecipitates of the eIF4E fraction were enriched in malignant B cells, suggesting reciprocal binding between eIF4G and eIF4E in MCL cells (Supplemental Figure 1B). Overall, these data demonstrate that the cap bound fraction from lymphoma cells was enriched with eIF4G, eIF4E and eIF4A, demonstrating that aggressive lymphoma B cells exist in a translationally activated state.

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