Limits...
mTOR kinase inhibitors synergize with histone deacetylase inhibitors to kill B-cell acute lymphoblastic leukemia cells.

Beagle BR, Nguyen DM, Mallya S, Tang SS, Lu M, Zeng Z, Konopleva M, Vo TT, Fruman DA - Oncotarget (2015)

Bottom Line: However, emerging data indicate that mTOR inhibitors are most effective when combined with other target agents.We found that mTOR kinase inhibitors (TOR-KIs) synergize with HDAC inhibitors to cause apoptosis in B-ALL cells and the effect is greater when compared to rapamycin plus HDAC inhibitors.The combination of TOR-KIs with the clinically approved HDAC inhibitor vorinostat increased apoptosis in primary pediatric B-ALL cells in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology & Biochemistry, University of California, Irvine, CA.

ABSTRACT
High activity of the mechanistic target of rapamycin (mTOR) is associated with poor prognosis in pre-B-cell acute lymphoblastic leukemia (B-ALL), suggesting that inhibiting mTOR might be clinically useful. However, emerging data indicate that mTOR inhibitors are most effective when combined with other target agents. One strategy is to combine with histone deacetylase (HDAC) inhibitors, since B-ALL is often characterized by epigenetic changes that silence the expression of pro-apoptotic factors. Here we tested combinations of mTOR and pan-HDAC inhibitors on B-ALL cells, including both Philadelphia chromosome-positive (Ph+) and non-Ph cell lines. We found that mTOR kinase inhibitors (TOR-KIs) synergize with HDAC inhibitors to cause apoptosis in B-ALL cells and the effect is greater when compared to rapamycin plus HDAC inhibitors. The combination of TOR-KIs with the clinically approved HDAC inhibitor vorinostat increased apoptosis in primary pediatric B-ALL cells in vitro. Mechanistically, TOR-KI and HDAC inhibitor combinations increased expression of pro-death genes, including targets of the Forkhead Box O (FOXO) transcription factors, and increased sensitivity to apoptotic triggers at the mitochondria. These findings suggest that targeting epigenetic factors can unmask the cytotoxic potential of TOR-KIs towards B-ALL cells.

Show MeSH

Related in: MedlinePlus

B-ALL cell death is caspase-dependent(A) SUP-B15 cells were cultured with MLN0128 (100 nM), vorinostat (500 nM), or the combination (M + V) in the presence of increasing concentrations of pan-caspase inhibitor. The percentage of cell death was determined after 48 hr by 7-AAD staining (mean +/− SEM, n = 3-5). * p < 0.05; ** p < 0.01, two-way ANOVA. (B) PARP cleavage in BV173 cells was measured by western blotting of lysates at various times after addition of MLN0128 (100 nM) and vorinostat (500 nM). Cells treated with the BCL2/BCL-XL antagonist ABT-263 were used as a positive control. β-actin served as a loading control. Similar results were observed in SUP-B15 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4385838&req=5

Figure 4: B-ALL cell death is caspase-dependent(A) SUP-B15 cells were cultured with MLN0128 (100 nM), vorinostat (500 nM), or the combination (M + V) in the presence of increasing concentrations of pan-caspase inhibitor. The percentage of cell death was determined after 48 hr by 7-AAD staining (mean +/− SEM, n = 3-5). * p < 0.05; ** p < 0.01, two-way ANOVA. (B) PARP cleavage in BV173 cells was measured by western blotting of lysates at various times after addition of MLN0128 (100 nM) and vorinostat (500 nM). Cells treated with the BCL2/BCL-XL antagonist ABT-263 were used as a positive control. β-actin served as a loading control. Similar results were observed in SUP-B15 cells.

Mentions: B-ALL cell killing by combination treatment might induce cytotoxicity through necrosis or programmed cell death (i.e. apoptosis). The pan-caspase inhibitor QVD-OPH caused a concentration-dependent suppression of apoptosis in SUP-B15 cells treated with MLN0128/vorinostat (Fig. 4A), consistent with death by caspase-dependent apoptosis. The combination of MLN0128/vorinostat also increased PARP cleavage, a caspase-dependent event in apoptotic cells (Fig. 4B). We did not consistently detect cleaved caspases by immunoblot or flow cytometry (data not shown), perhaps owing to low levels of caspase expression in B-ALL cells [43, 44]. However, using enzyme assays we observed a transient increase in activity of both caspase-8 and caspase-3 in three B-ALL cell lines treated with MLN0128/vorinostat that was significantly higher than in cells treated with single agents (Fig. S4). Together these data support apoptosis as the cytotoxic mechanism of action for the MLN0128/vorinostat combination in B-ALL cells.


mTOR kinase inhibitors synergize with histone deacetylase inhibitors to kill B-cell acute lymphoblastic leukemia cells.

Beagle BR, Nguyen DM, Mallya S, Tang SS, Lu M, Zeng Z, Konopleva M, Vo TT, Fruman DA - Oncotarget (2015)

B-ALL cell death is caspase-dependent(A) SUP-B15 cells were cultured with MLN0128 (100 nM), vorinostat (500 nM), or the combination (M + V) in the presence of increasing concentrations of pan-caspase inhibitor. The percentage of cell death was determined after 48 hr by 7-AAD staining (mean +/− SEM, n = 3-5). * p < 0.05; ** p < 0.01, two-way ANOVA. (B) PARP cleavage in BV173 cells was measured by western blotting of lysates at various times after addition of MLN0128 (100 nM) and vorinostat (500 nM). Cells treated with the BCL2/BCL-XL antagonist ABT-263 were used as a positive control. β-actin served as a loading control. Similar results were observed in SUP-B15 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385838&req=5

Figure 4: B-ALL cell death is caspase-dependent(A) SUP-B15 cells were cultured with MLN0128 (100 nM), vorinostat (500 nM), or the combination (M + V) in the presence of increasing concentrations of pan-caspase inhibitor. The percentage of cell death was determined after 48 hr by 7-AAD staining (mean +/− SEM, n = 3-5). * p < 0.05; ** p < 0.01, two-way ANOVA. (B) PARP cleavage in BV173 cells was measured by western blotting of lysates at various times after addition of MLN0128 (100 nM) and vorinostat (500 nM). Cells treated with the BCL2/BCL-XL antagonist ABT-263 were used as a positive control. β-actin served as a loading control. Similar results were observed in SUP-B15 cells.
Mentions: B-ALL cell killing by combination treatment might induce cytotoxicity through necrosis or programmed cell death (i.e. apoptosis). The pan-caspase inhibitor QVD-OPH caused a concentration-dependent suppression of apoptosis in SUP-B15 cells treated with MLN0128/vorinostat (Fig. 4A), consistent with death by caspase-dependent apoptosis. The combination of MLN0128/vorinostat also increased PARP cleavage, a caspase-dependent event in apoptotic cells (Fig. 4B). We did not consistently detect cleaved caspases by immunoblot or flow cytometry (data not shown), perhaps owing to low levels of caspase expression in B-ALL cells [43, 44]. However, using enzyme assays we observed a transient increase in activity of both caspase-8 and caspase-3 in three B-ALL cell lines treated with MLN0128/vorinostat that was significantly higher than in cells treated with single agents (Fig. S4). Together these data support apoptosis as the cytotoxic mechanism of action for the MLN0128/vorinostat combination in B-ALL cells.

Bottom Line: However, emerging data indicate that mTOR inhibitors are most effective when combined with other target agents.We found that mTOR kinase inhibitors (TOR-KIs) synergize with HDAC inhibitors to cause apoptosis in B-ALL cells and the effect is greater when compared to rapamycin plus HDAC inhibitors.The combination of TOR-KIs with the clinically approved HDAC inhibitor vorinostat increased apoptosis in primary pediatric B-ALL cells in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology & Biochemistry, University of California, Irvine, CA.

ABSTRACT
High activity of the mechanistic target of rapamycin (mTOR) is associated with poor prognosis in pre-B-cell acute lymphoblastic leukemia (B-ALL), suggesting that inhibiting mTOR might be clinically useful. However, emerging data indicate that mTOR inhibitors are most effective when combined with other target agents. One strategy is to combine with histone deacetylase (HDAC) inhibitors, since B-ALL is often characterized by epigenetic changes that silence the expression of pro-apoptotic factors. Here we tested combinations of mTOR and pan-HDAC inhibitors on B-ALL cells, including both Philadelphia chromosome-positive (Ph+) and non-Ph cell lines. We found that mTOR kinase inhibitors (TOR-KIs) synergize with HDAC inhibitors to cause apoptosis in B-ALL cells and the effect is greater when compared to rapamycin plus HDAC inhibitors. The combination of TOR-KIs with the clinically approved HDAC inhibitor vorinostat increased apoptosis in primary pediatric B-ALL cells in vitro. Mechanistically, TOR-KI and HDAC inhibitor combinations increased expression of pro-death genes, including targets of the Forkhead Box O (FOXO) transcription factors, and increased sensitivity to apoptotic triggers at the mitochondria. These findings suggest that targeting epigenetic factors can unmask the cytotoxic potential of TOR-KIs towards B-ALL cells.

Show MeSH
Related in: MedlinePlus