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RPPA-based protein profiling reveals eIF4G overexpression and 4E-BP1 serine 65 phosphorylation as molecular events that correspond with a pro-survival phenotype in chronic lymphocytic leukemia.

Shull AY, Noonepalle SK, Awan FT, Liu J, Pei L, Bollag RJ, Salman H, Ding Z, Shi H - Oncotarget (2015)

Bottom Line: Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib.We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells.Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Georgia Regents University, Augusta, Georgia, USA.

ABSTRACT
Chronic lymphocytic leukemia (CLL), the most common adult leukemia, remains incurable despite advancements in treatment regimens over the past decade. Several expression profile studies have been pursued to better understand CLL pathogenesis. However, these large-scale studies only provide information at the transcriptional level. To better comprehend the differential protein changes that take place in CLL, we performed a reverse-phase protein array (RPPA) analysis using 167 different antibodies on B-cell lysates from 18 CLL patients and 6 normal donors. From our analysis, we discovered an enrichment of protein alterations involved with mRNA translation, specifically upregulation of the translation initiator eIF4G and phosphorylation of the cap-dependent translation inhibitor 4E-BP1 at serine 65. Interestingly, 4E-BP1 phosphorylation occurred independently of AKT phosphorylation, suggesting a disconnect between PI3K/AKT pathway activation and 4E-BP1 phosphorylation. Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib. We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells. Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival.

No MeSH data available.


Related in: MedlinePlus

4E-BP1 serine 65 phosphorylation is independent of AKT/mTOR activation in CLLA. Normalized phosphorylation fold changes of AKT/mTOR substrates in CLL samples from RPPA (error bars = 95% CI). B. Paired comparison of CLL samples demonstrating the disconnection between AKT T308 phosphorylation and 4E-BP1 serine 65 phosphorylation. C. IgM activation of CLL sample 8816 and normal donor SC30. 4E-BP1 seems to be constitutively phosphorylated in CLL regardless of AKT pT308 activation, whereas the phosphorylation of 4E-BP1 in normal B-cells is dependent upon IgM-mediated AKT activation.
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Figure 3: 4E-BP1 serine 65 phosphorylation is independent of AKT/mTOR activation in CLLA. Normalized phosphorylation fold changes of AKT/mTOR substrates in CLL samples from RPPA (error bars = 95% CI). B. Paired comparison of CLL samples demonstrating the disconnection between AKT T308 phosphorylation and 4E-BP1 serine 65 phosphorylation. C. IgM activation of CLL sample 8816 and normal donor SC30. 4E-BP1 seems to be constitutively phosphorylated in CLL regardless of AKT pT308 activation, whereas the phosphorylation of 4E-BP1 in normal B-cells is dependent upon IgM-mediated AKT activation.

Mentions: We then compared the distinct phosphorylation events of the AKT/mTOR pathway to reveal the overall phosphorylation patterns of AKT/mTOR substrates in CLL. By normalizing the phosphoprotein levels to the internal levels of the corresponding total protein, we observed 4E-BP1 at serine 65 had higher phosphorylation levels in CLL compared to healthy donors. However, the high phosphorylation at this site did not correspond with other AKT/mTOR related phosphorylation sites, including AKT threonine 308, AKT serine 473, p70S6K threonine 389, and mTOR serine 2448, which were all relatively hypophosphorylated (Figure 3A). These phosphorylation patterns are quite interesting due to the fact that 4E-BP1 phosphorylation typically is dependent upon AKT phosphorylation [68, 69].


RPPA-based protein profiling reveals eIF4G overexpression and 4E-BP1 serine 65 phosphorylation as molecular events that correspond with a pro-survival phenotype in chronic lymphocytic leukemia.

Shull AY, Noonepalle SK, Awan FT, Liu J, Pei L, Bollag RJ, Salman H, Ding Z, Shi H - Oncotarget (2015)

4E-BP1 serine 65 phosphorylation is independent of AKT/mTOR activation in CLLA. Normalized phosphorylation fold changes of AKT/mTOR substrates in CLL samples from RPPA (error bars = 95% CI). B. Paired comparison of CLL samples demonstrating the disconnection between AKT T308 phosphorylation and 4E-BP1 serine 65 phosphorylation. C. IgM activation of CLL sample 8816 and normal donor SC30. 4E-BP1 seems to be constitutively phosphorylated in CLL regardless of AKT pT308 activation, whereas the phosphorylation of 4E-BP1 in normal B-cells is dependent upon IgM-mediated AKT activation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4546493&req=5

Figure 3: 4E-BP1 serine 65 phosphorylation is independent of AKT/mTOR activation in CLLA. Normalized phosphorylation fold changes of AKT/mTOR substrates in CLL samples from RPPA (error bars = 95% CI). B. Paired comparison of CLL samples demonstrating the disconnection between AKT T308 phosphorylation and 4E-BP1 serine 65 phosphorylation. C. IgM activation of CLL sample 8816 and normal donor SC30. 4E-BP1 seems to be constitutively phosphorylated in CLL regardless of AKT pT308 activation, whereas the phosphorylation of 4E-BP1 in normal B-cells is dependent upon IgM-mediated AKT activation.
Mentions: We then compared the distinct phosphorylation events of the AKT/mTOR pathway to reveal the overall phosphorylation patterns of AKT/mTOR substrates in CLL. By normalizing the phosphoprotein levels to the internal levels of the corresponding total protein, we observed 4E-BP1 at serine 65 had higher phosphorylation levels in CLL compared to healthy donors. However, the high phosphorylation at this site did not correspond with other AKT/mTOR related phosphorylation sites, including AKT threonine 308, AKT serine 473, p70S6K threonine 389, and mTOR serine 2448, which were all relatively hypophosphorylated (Figure 3A). These phosphorylation patterns are quite interesting due to the fact that 4E-BP1 phosphorylation typically is dependent upon AKT phosphorylation [68, 69].

Bottom Line: Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib.We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells.Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, Georgia Regents University, Augusta, Georgia, USA.

ABSTRACT
Chronic lymphocytic leukemia (CLL), the most common adult leukemia, remains incurable despite advancements in treatment regimens over the past decade. Several expression profile studies have been pursued to better understand CLL pathogenesis. However, these large-scale studies only provide information at the transcriptional level. To better comprehend the differential protein changes that take place in CLL, we performed a reverse-phase protein array (RPPA) analysis using 167 different antibodies on B-cell lysates from 18 CLL patients and 6 normal donors. From our analysis, we discovered an enrichment of protein alterations involved with mRNA translation, specifically upregulation of the translation initiator eIF4G and phosphorylation of the cap-dependent translation inhibitor 4E-BP1 at serine 65. Interestingly, 4E-BP1 phosphorylation occurred independently of AKT phosphorylation, suggesting a disconnect between PI3K/AKT pathway activation and 4E-BP1 phosphorylation. Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib. We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells. Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival.

No MeSH data available.


Related in: MedlinePlus