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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.

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Related in: MedlinePlus

MLN0128 induces dephosphorylation and nuclear translocation of FOXO factors (A) Lysates from SUP-B15 cells treated for the indicated times with vehicle alone, MLN0128 (100 nM), vorinostat (500 nM), or the combination (M + V)Blots were probed for the proteins and phosphosites shown on the right. (B) SUP-B15 cells were treated for the indicated times with MLN0128 (100 nM) before cell fractionation into nuclear and cytoplasmic extracts. Fractions were subjected to western blotting with anti-FOXO1 antibody. Antibodies to β-actin and histone H3 were used to confirm the purity of cytoplasmic and nuclear fractions. (C) SUP-B15 cells were treated for 4 hr with vehicle or 100 nM MLN0128. mRNA was obtained and expression of the indicated gene products was determined by Q-PCR. Graph depicts the fold increase in mRNA in cells treated with MLN0128 versus vehicle (mean +/− SEM, n = 3). *** p < 0.001, one-way ANOVA. BIM and TRAIL mRNA was not increased at 6 or 8 hr post-treatment (not shown). (D) SUP-B15 cells were infected with retroviruses expressing a human CD4 marker gene lacking the cytoplasmic tail and magnetically sorted to enrich hCD4+ cells. The vectors contained no insert (EV) or cDNAs encoding triple-alanine mutants of FOXO1 or FOXO3a fused to the hormone binding domain of the estrogen receptor (ER). Cells were then treated with vehicle or 4OHT for 48hr in the absence or presence of vorinostat (500 nM). The percentage of cell death was determined after 48 hr by 7-AAD staining (mean +/− SEM, n = 3). * p < 0.05; paired two-tailed t-test.
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Figure 5: MLN0128 induces dephosphorylation and nuclear translocation of FOXO factors (A) Lysates from SUP-B15 cells treated for the indicated times with vehicle alone, MLN0128 (100 nM), vorinostat (500 nM), or the combination (M + V)Blots were probed for the proteins and phosphosites shown on the right. (B) SUP-B15 cells were treated for the indicated times with MLN0128 (100 nM) before cell fractionation into nuclear and cytoplasmic extracts. Fractions were subjected to western blotting with anti-FOXO1 antibody. Antibodies to β-actin and histone H3 were used to confirm the purity of cytoplasmic and nuclear fractions. (C) SUP-B15 cells were treated for 4 hr with vehicle or 100 nM MLN0128. mRNA was obtained and expression of the indicated gene products was determined by Q-PCR. Graph depicts the fold increase in mRNA in cells treated with MLN0128 versus vehicle (mean +/− SEM, n = 3). *** p < 0.001, one-way ANOVA. BIM and TRAIL mRNA was not increased at 6 or 8 hr post-treatment (not shown). (D) SUP-B15 cells were infected with retroviruses expressing a human CD4 marker gene lacking the cytoplasmic tail and magnetically sorted to enrich hCD4+ cells. The vectors contained no insert (EV) or cDNAs encoding triple-alanine mutants of FOXO1 or FOXO3a fused to the hormone binding domain of the estrogen receptor (ER). Cells were then treated with vehicle or 4OHT for 48hr in the absence or presence of vorinostat (500 nM). The percentage of cell death was determined after 48 hr by 7-AAD staining (mean +/− SEM, n = 3). * p < 0.05; paired two-tailed t-test.

Mentions: Broad spectrum HDACi like vorinostat have the potential to alter cellular signal transduction through altered transcription or non-histone target proteins. To investigate the mechanism of synergistic killing of B-ALL cells by MLN0128/vorinostat, we first assessed phosphorylation of signaling proteins in the PI3K/AKT/mTOR pathway. As expected, vorinostat increased global histone acetylation in SUP-B15 cells; however, vorinostat did not affect phosphorylation of proteins in the PI3K/AKT/mTOR pathway (Fig. 5A). In contrast, MLN0128 treatment reduced phosphorylation of the mTORC1 substrate 4EBP1 (T37/46) and the mTORC2 substrate AKT (S473), with only a slight effect on histone acetylation (Fig. 5A). MLN0128 also blocked phosphorylation of ribosomal protein S6 (S240/244) downstream of mTORC1 (Fig. 5A). Similar results were observed in the Blin-1 and Nalm-6 cell lines (data not shown). MLN0128 and vorinostat did not influence ERK phosphorylation in SUP-B15 cells (Fig. 5A). These data suggest that vorinostat does not alter the activity of major oncogenic signaling pathways in B-ALL cell lines, nor does vorinostat modulate the signaling impact of TOR-KIs.


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)

MLN0128 induces dephosphorylation and nuclear translocation of FOXO factors (A) Lysates from SUP-B15 cells treated for the indicated times with vehicle alone, MLN0128 (100 nM), vorinostat (500 nM), or the combination (M + V)Blots were probed for the proteins and phosphosites shown on the right. (B) SUP-B15 cells were treated for the indicated times with MLN0128 (100 nM) before cell fractionation into nuclear and cytoplasmic extracts. Fractions were subjected to western blotting with anti-FOXO1 antibody. Antibodies to β-actin and histone H3 were used to confirm the purity of cytoplasmic and nuclear fractions. (C) SUP-B15 cells were treated for 4 hr with vehicle or 100 nM MLN0128. mRNA was obtained and expression of the indicated gene products was determined by Q-PCR. Graph depicts the fold increase in mRNA in cells treated with MLN0128 versus vehicle (mean +/− SEM, n = 3). *** p < 0.001, one-way ANOVA. BIM and TRAIL mRNA was not increased at 6 or 8 hr post-treatment (not shown). (D) SUP-B15 cells were infected with retroviruses expressing a human CD4 marker gene lacking the cytoplasmic tail and magnetically sorted to enrich hCD4+ cells. The vectors contained no insert (EV) or cDNAs encoding triple-alanine mutants of FOXO1 or FOXO3a fused to the hormone binding domain of the estrogen receptor (ER). Cells were then treated with vehicle or 4OHT for 48hr in the absence or presence of vorinostat (500 nM). The percentage of cell death was determined after 48 hr by 7-AAD staining (mean +/− SEM, n = 3). * p < 0.05; paired two-tailed t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: MLN0128 induces dephosphorylation and nuclear translocation of FOXO factors (A) Lysates from SUP-B15 cells treated for the indicated times with vehicle alone, MLN0128 (100 nM), vorinostat (500 nM), or the combination (M + V)Blots were probed for the proteins and phosphosites shown on the right. (B) SUP-B15 cells were treated for the indicated times with MLN0128 (100 nM) before cell fractionation into nuclear and cytoplasmic extracts. Fractions were subjected to western blotting with anti-FOXO1 antibody. Antibodies to β-actin and histone H3 were used to confirm the purity of cytoplasmic and nuclear fractions. (C) SUP-B15 cells were treated for 4 hr with vehicle or 100 nM MLN0128. mRNA was obtained and expression of the indicated gene products was determined by Q-PCR. Graph depicts the fold increase in mRNA in cells treated with MLN0128 versus vehicle (mean +/− SEM, n = 3). *** p < 0.001, one-way ANOVA. BIM and TRAIL mRNA was not increased at 6 or 8 hr post-treatment (not shown). (D) SUP-B15 cells were infected with retroviruses expressing a human CD4 marker gene lacking the cytoplasmic tail and magnetically sorted to enrich hCD4+ cells. The vectors contained no insert (EV) or cDNAs encoding triple-alanine mutants of FOXO1 or FOXO3a fused to the hormone binding domain of the estrogen receptor (ER). Cells were then treated with vehicle or 4OHT for 48hr in the absence or presence of vorinostat (500 nM). The percentage of cell death was determined after 48 hr by 7-AAD staining (mean +/− SEM, n = 3). * p < 0.05; paired two-tailed t-test.
Mentions: Broad spectrum HDACi like vorinostat have the potential to alter cellular signal transduction through altered transcription or non-histone target proteins. To investigate the mechanism of synergistic killing of B-ALL cells by MLN0128/vorinostat, we first assessed phosphorylation of signaling proteins in the PI3K/AKT/mTOR pathway. As expected, vorinostat increased global histone acetylation in SUP-B15 cells; however, vorinostat did not affect phosphorylation of proteins in the PI3K/AKT/mTOR pathway (Fig. 5A). In contrast, MLN0128 treatment reduced phosphorylation of the mTORC1 substrate 4EBP1 (T37/46) and the mTORC2 substrate AKT (S473), with only a slight effect on histone acetylation (Fig. 5A). MLN0128 also blocked phosphorylation of ribosomal protein S6 (S240/244) downstream of mTORC1 (Fig. 5A). Similar results were observed in the Blin-1 and Nalm-6 cell lines (data not shown). MLN0128 and vorinostat did not influence ERK phosphorylation in SUP-B15 cells (Fig. 5A). These data suggest that vorinostat does not alter the activity of major oncogenic signaling pathways in B-ALL cell lines, nor does vorinostat modulate the signaling impact of TOR-KIs.

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