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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

CLL apoptosis corresponds with eIF4G cleavage and 4E-BP1 dephosphorylationA. Dose-dependent treatment analysis of Ibrutinib, Idelalisib, and NVP-BEZ235 in the MEC1 CLL cell line to compare the IC50 for each inhibitor. B. Annexin V/DAPI apoptotic assay comparing 10uM Ibrutinib, 10uM Idelalisib, and 10uM NVP-BEZ235 treatment in CLL cells co-cultured with Huh7.5 cells (n = 15). Based on the results, NVP-BEZ235 was able to cause more apoptotic death during the 48hr period. C. At 6 hours, 10uM NVP-BEZ235 causes greater dephosphorylation of 4E-BP1 as well as greater cleavage of eIF4G in HS-5 co-cultured CLL cells compared to 10 uM Ibrutinib and 10uM Idelalisib. D. Decreasing 4E-BP1 phosphorylation and increasing eIF4G cleavage correspond with higher apoptotic death in primary CLL samples (errors bars = S.E.M.).
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Figure 4: CLL apoptosis corresponds with eIF4G cleavage and 4E-BP1 dephosphorylationA. Dose-dependent treatment analysis of Ibrutinib, Idelalisib, and NVP-BEZ235 in the MEC1 CLL cell line to compare the IC50 for each inhibitor. B. Annexin V/DAPI apoptotic assay comparing 10uM Ibrutinib, 10uM Idelalisib, and 10uM NVP-BEZ235 treatment in CLL cells co-cultured with Huh7.5 cells (n = 15). Based on the results, NVP-BEZ235 was able to cause more apoptotic death during the 48hr period. C. At 6 hours, 10uM NVP-BEZ235 causes greater dephosphorylation of 4E-BP1 as well as greater cleavage of eIF4G in HS-5 co-cultured CLL cells compared to 10 uM Ibrutinib and 10uM Idelalisib. D. Decreasing 4E-BP1 phosphorylation and increasing eIF4G cleavage correspond with higher apoptotic death in primary CLL samples (errors bars = S.E.M.).

Mentions: To compare these three inhibitors, we first performed an IC50 proliferation assay for each of the three inhibitors in the MEC1 CLL cell line. From this experiment, we determined NVP-BEZ235 to have a lower IC50 value (Figure 4A). We then co-cultured 15 CLL patient B-cells with Huh7.5 cells, an adenocarcinoma cell line that provides a proper microenvironment for survival and viability of CLL cells [64], and treated the primary cells with each inhibitor for 48 hours at their maximum effective doses of 10uM [23, 70, 72]. Based on Annexin V/DAPI measurements, NVP-BEZ235 was able to significantly lower cell viability compared to Ibrutinib and Idelalisib. This lowered viability correlated with a potent induction of apoptosis in CLL cells as NVP-BEZ235 caused a significant increase in Annexin V+/DAPI+ cell death compared with side-by-side treatment of Ibrutinib and Idelalisib (Figure 4B).


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)

CLL apoptosis corresponds with eIF4G cleavage and 4E-BP1 dephosphorylationA. Dose-dependent treatment analysis of Ibrutinib, Idelalisib, and NVP-BEZ235 in the MEC1 CLL cell line to compare the IC50 for each inhibitor. B. Annexin V/DAPI apoptotic assay comparing 10uM Ibrutinib, 10uM Idelalisib, and 10uM NVP-BEZ235 treatment in CLL cells co-cultured with Huh7.5 cells (n = 15). Based on the results, NVP-BEZ235 was able to cause more apoptotic death during the 48hr period. C. At 6 hours, 10uM NVP-BEZ235 causes greater dephosphorylation of 4E-BP1 as well as greater cleavage of eIF4G in HS-5 co-cultured CLL cells compared to 10 uM Ibrutinib and 10uM Idelalisib. D. Decreasing 4E-BP1 phosphorylation and increasing eIF4G cleavage correspond with higher apoptotic death in primary CLL samples (errors bars = S.E.M.).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: CLL apoptosis corresponds with eIF4G cleavage and 4E-BP1 dephosphorylationA. Dose-dependent treatment analysis of Ibrutinib, Idelalisib, and NVP-BEZ235 in the MEC1 CLL cell line to compare the IC50 for each inhibitor. B. Annexin V/DAPI apoptotic assay comparing 10uM Ibrutinib, 10uM Idelalisib, and 10uM NVP-BEZ235 treatment in CLL cells co-cultured with Huh7.5 cells (n = 15). Based on the results, NVP-BEZ235 was able to cause more apoptotic death during the 48hr period. C. At 6 hours, 10uM NVP-BEZ235 causes greater dephosphorylation of 4E-BP1 as well as greater cleavage of eIF4G in HS-5 co-cultured CLL cells compared to 10 uM Ibrutinib and 10uM Idelalisib. D. Decreasing 4E-BP1 phosphorylation and increasing eIF4G cleavage correspond with higher apoptotic death in primary CLL samples (errors bars = S.E.M.).
Mentions: To compare these three inhibitors, we first performed an IC50 proliferation assay for each of the three inhibitors in the MEC1 CLL cell line. From this experiment, we determined NVP-BEZ235 to have a lower IC50 value (Figure 4A). We then co-cultured 15 CLL patient B-cells with Huh7.5 cells, an adenocarcinoma cell line that provides a proper microenvironment for survival and viability of CLL cells [64], and treated the primary cells with each inhibitor for 48 hours at their maximum effective doses of 10uM [23, 70, 72]. Based on Annexin V/DAPI measurements, NVP-BEZ235 was able to significantly lower cell viability compared to Ibrutinib and Idelalisib. This lowered viability correlated with a potent induction of apoptosis in CLL cells as NVP-BEZ235 caused a significant increase in Annexin V+/DAPI+ cell death compared with side-by-side treatment of Ibrutinib and Idelalisib (Figure 4B).

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