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PI3K/mTOR pathway inhibitors sensitize chronic myeloid leukemia stem cells to nilotinib and restore the response of progenitors to nilotinib in the presence of stem cell factor.

Airiau K, Mahon FX, Josselin M, Jeanneteau M, Belloc F - Cell Death Dis (2013)

Bottom Line: We previously demonstrated that nilotinib-induced apoptosis was reduced by stem cell factor (SCF) addition.However, this pathway seems not to be involved in the nilotinib-induced resistance of the CML stem cell population.Conversely, PI3K inhibition sensitized both CML progenitors and stem cells to nilotinib, suggesting that, downstream PI3K, two different kinase pathways are activated in CML progenitor and stem cell populations.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratoire d'Hématopoïèse Leucémique et Cibles Thérapeutiques, INSERM 1035, Université Bordeaux Segalen, 146 Rue Léo Saignat, Bordeaux Cedex 33076, France [2] CHU Bordeaux, Hôpital Haut-Lévêque, Laboratoire d'Hématologie, Avenue Magellan 33604 Pessac, France.

ABSTRACT
Nilotinib is a second-generation tyrosine kinase inhibitor, designed to specifically inhibit break-point cluster region (BCR)-Abelson (ABL) and developed to treat chronic myeloid leukemia (CML) in patients showing a resistance to imatinib. We previously demonstrated that nilotinib-induced apoptosis was reduced by stem cell factor (SCF) addition. Here, the SCF-activated survival pathway was investigated. BCR-ABL expression was accompanied by the activation of the SCF receptor: c-KIT. Nilotinib inhibited this activation that was restored by SCF binding. Parallel variations were observed for mammaliam target of rapamycin (mTOR) kinase and mTOR complex 1 substrate S6K. The inhibition of mTORC1 restored the response of BCR-ABL cell lines to nilotinib in the presence of SCF. PI3K inhibition restored nilotinib-induced apoptosis. On hematopoietic progenitors from CML patient's bone marrows, mTORC1 inhibition also restored nilotinib sensitivity in the presence of SCF, confirming its involvement in SCF-activated survival pathway. However, this pathway seems not to be involved in the nilotinib-induced resistance of the CML stem cell population. Conversely, PI3K inhibition sensitized both CML progenitors and stem cells to nilotinib, suggesting that, downstream PI3K, two different kinase pathways are activated in CML progenitor and stem cell populations.

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mTOR inhibition alleviates SFC-induced resistance to nilotinib and cooperates with BCR-ABL inhibition to induce apoptosis in SCF-treated cell lines. (a) K562 and LAMA-84 cells were seeded at 103 cells/well in 96-well plates. They were treated with increasing concentrations of nilotinib in the presence or absence of 200 nM RAD-001 and/or 100 ng/ml SCF alone or in combination as indicated. The number of viable cells was 2 evaluated after 3 days of culture on the basis of the ATP content. Results are displayed as the percentage of the untreated control value as a function of nilotinib concentration. (b) K562 and LAMA-84 cells were treated for 24 h in the presence or absence of 200 nM RAD-001Rad-001, 20 nM nilotinib, 100 ng/ml SCF alone or in combination as indicated. Apoptosis was measured by flow cytometry using DiOC6(3) as a probe. Drug-induced apoptosis was calculated as described in Materials and Methods and corrected for spontaneous apoptosis. Results are expressed as mean +/− S.D. of three experiments. (c and d) K562 (c) and LAMA-84 (d) cells were treated with 20 nM nilotinib alone or in the presence of 100 ng/ml SCF and/or 200 nM RAD-001 as indicated for 24 h. Cell lysates were then analyzed by western blot and probed with anti-phospho-c-kit, anti-phospho-mTOR and anti-phospho-S6K or anti-LC3b and anti-cleaved caspase 3 antibodies. Anti-tubulin antibody was used to verify the loading homogeneity
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fig3: mTOR inhibition alleviates SFC-induced resistance to nilotinib and cooperates with BCR-ABL inhibition to induce apoptosis in SCF-treated cell lines. (a) K562 and LAMA-84 cells were seeded at 103 cells/well in 96-well plates. They were treated with increasing concentrations of nilotinib in the presence or absence of 200 nM RAD-001 and/or 100 ng/ml SCF alone or in combination as indicated. The number of viable cells was 2 evaluated after 3 days of culture on the basis of the ATP content. Results are displayed as the percentage of the untreated control value as a function of nilotinib concentration. (b) K562 and LAMA-84 cells were treated for 24 h in the presence or absence of 200 nM RAD-001Rad-001, 20 nM nilotinib, 100 ng/ml SCF alone or in combination as indicated. Apoptosis was measured by flow cytometry using DiOC6(3) as a probe. Drug-induced apoptosis was calculated as described in Materials and Methods and corrected for spontaneous apoptosis. Results are expressed as mean +/− S.D. of three experiments. (c and d) K562 (c) and LAMA-84 (d) cells were treated with 20 nM nilotinib alone or in the presence of 100 ng/ml SCF and/or 200 nM RAD-001 as indicated for 24 h. Cell lysates were then analyzed by western blot and probed with anti-phospho-c-kit, anti-phospho-mTOR and anti-phospho-S6K or anti-LC3b and anti-cleaved caspase 3 antibodies. Anti-tubulin antibody was used to verify the loading homogeneity

Mentions: mTORC1 inhibition was performed using RAD-001. Figure 3a shows that, in the presence of SCF, the IC50 of nilotinib was increased by a fourfold in K562 and more than seven times in LAMA-84 cell lines (dotted lines). This differential effect could be due to the higher expression of c-KIT in LAMA-84 as compared with K562 cells (supplementary Figure S1). Although RAD-001 alone had no effect (data not shown), it restored in part the IC50 of nilotinib in the presence of SCF. Similarly, SCF inhibited nilotinib-induced apoptosis more efficiently in LAMA-84 than in K562 cells (Figure 3b) and, although this inhibition was completely abolished by the mTOR inhibitor in K562 cells, it was only reduced in LAMA-84 cells. Again, this result can be related to the relative expression of c-KIT in the two cell lines (Supplementary Figure S1). Thus, it seems that in the presence of SCF, the cell lines necessitate a further inhibition of the mTOR pathway to undergo apoptosis. To support this result, we used LAMA-84 cells transduced with vector coding for shBCR-ABL and GFP or control vector (GFP alone).9 SCF addition maintained a cell survival that was inhibited in part by RAD-001 (Supplementary Figure S2). However, it is not clear from these results whether SCF-induced c-KIT activation is directly responsible for mTOR activation or whether an alternative pathway is also activated.


PI3K/mTOR pathway inhibitors sensitize chronic myeloid leukemia stem cells to nilotinib and restore the response of progenitors to nilotinib in the presence of stem cell factor.

Airiau K, Mahon FX, Josselin M, Jeanneteau M, Belloc F - Cell Death Dis (2013)

mTOR inhibition alleviates SFC-induced resistance to nilotinib and cooperates with BCR-ABL inhibition to induce apoptosis in SCF-treated cell lines. (a) K562 and LAMA-84 cells were seeded at 103 cells/well in 96-well plates. They were treated with increasing concentrations of nilotinib in the presence or absence of 200 nM RAD-001 and/or 100 ng/ml SCF alone or in combination as indicated. The number of viable cells was 2 evaluated after 3 days of culture on the basis of the ATP content. Results are displayed as the percentage of the untreated control value as a function of nilotinib concentration. (b) K562 and LAMA-84 cells were treated for 24 h in the presence or absence of 200 nM RAD-001Rad-001, 20 nM nilotinib, 100 ng/ml SCF alone or in combination as indicated. Apoptosis was measured by flow cytometry using DiOC6(3) as a probe. Drug-induced apoptosis was calculated as described in Materials and Methods and corrected for spontaneous apoptosis. Results are expressed as mean +/− S.D. of three experiments. (c and d) K562 (c) and LAMA-84 (d) cells were treated with 20 nM nilotinib alone or in the presence of 100 ng/ml SCF and/or 200 nM RAD-001 as indicated for 24 h. Cell lysates were then analyzed by western blot and probed with anti-phospho-c-kit, anti-phospho-mTOR and anti-phospho-S6K or anti-LC3b and anti-cleaved caspase 3 antibodies. Anti-tubulin antibody was used to verify the loading homogeneity
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3824646&req=5

fig3: mTOR inhibition alleviates SFC-induced resistance to nilotinib and cooperates with BCR-ABL inhibition to induce apoptosis in SCF-treated cell lines. (a) K562 and LAMA-84 cells were seeded at 103 cells/well in 96-well plates. They were treated with increasing concentrations of nilotinib in the presence or absence of 200 nM RAD-001 and/or 100 ng/ml SCF alone or in combination as indicated. The number of viable cells was 2 evaluated after 3 days of culture on the basis of the ATP content. Results are displayed as the percentage of the untreated control value as a function of nilotinib concentration. (b) K562 and LAMA-84 cells were treated for 24 h in the presence or absence of 200 nM RAD-001Rad-001, 20 nM nilotinib, 100 ng/ml SCF alone or in combination as indicated. Apoptosis was measured by flow cytometry using DiOC6(3) as a probe. Drug-induced apoptosis was calculated as described in Materials and Methods and corrected for spontaneous apoptosis. Results are expressed as mean +/− S.D. of three experiments. (c and d) K562 (c) and LAMA-84 (d) cells were treated with 20 nM nilotinib alone or in the presence of 100 ng/ml SCF and/or 200 nM RAD-001 as indicated for 24 h. Cell lysates were then analyzed by western blot and probed with anti-phospho-c-kit, anti-phospho-mTOR and anti-phospho-S6K or anti-LC3b and anti-cleaved caspase 3 antibodies. Anti-tubulin antibody was used to verify the loading homogeneity
Mentions: mTORC1 inhibition was performed using RAD-001. Figure 3a shows that, in the presence of SCF, the IC50 of nilotinib was increased by a fourfold in K562 and more than seven times in LAMA-84 cell lines (dotted lines). This differential effect could be due to the higher expression of c-KIT in LAMA-84 as compared with K562 cells (supplementary Figure S1). Although RAD-001 alone had no effect (data not shown), it restored in part the IC50 of nilotinib in the presence of SCF. Similarly, SCF inhibited nilotinib-induced apoptosis more efficiently in LAMA-84 than in K562 cells (Figure 3b) and, although this inhibition was completely abolished by the mTOR inhibitor in K562 cells, it was only reduced in LAMA-84 cells. Again, this result can be related to the relative expression of c-KIT in the two cell lines (Supplementary Figure S1). Thus, it seems that in the presence of SCF, the cell lines necessitate a further inhibition of the mTOR pathway to undergo apoptosis. To support this result, we used LAMA-84 cells transduced with vector coding for shBCR-ABL and GFP or control vector (GFP alone).9 SCF addition maintained a cell survival that was inhibited in part by RAD-001 (Supplementary Figure S2). However, it is not clear from these results whether SCF-induced c-KIT activation is directly responsible for mTOR activation or whether an alternative pathway is also activated.

Bottom Line: We previously demonstrated that nilotinib-induced apoptosis was reduced by stem cell factor (SCF) addition.However, this pathway seems not to be involved in the nilotinib-induced resistance of the CML stem cell population.Conversely, PI3K inhibition sensitized both CML progenitors and stem cells to nilotinib, suggesting that, downstream PI3K, two different kinase pathways are activated in CML progenitor and stem cell populations.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratoire d'Hématopoïèse Leucémique et Cibles Thérapeutiques, INSERM 1035, Université Bordeaux Segalen, 146 Rue Léo Saignat, Bordeaux Cedex 33076, France [2] CHU Bordeaux, Hôpital Haut-Lévêque, Laboratoire d'Hématologie, Avenue Magellan 33604 Pessac, France.

ABSTRACT
Nilotinib is a second-generation tyrosine kinase inhibitor, designed to specifically inhibit break-point cluster region (BCR)-Abelson (ABL) and developed to treat chronic myeloid leukemia (CML) in patients showing a resistance to imatinib. We previously demonstrated that nilotinib-induced apoptosis was reduced by stem cell factor (SCF) addition. Here, the SCF-activated survival pathway was investigated. BCR-ABL expression was accompanied by the activation of the SCF receptor: c-KIT. Nilotinib inhibited this activation that was restored by SCF binding. Parallel variations were observed for mammaliam target of rapamycin (mTOR) kinase and mTOR complex 1 substrate S6K. The inhibition of mTORC1 restored the response of BCR-ABL cell lines to nilotinib in the presence of SCF. PI3K inhibition restored nilotinib-induced apoptosis. On hematopoietic progenitors from CML patient's bone marrows, mTORC1 inhibition also restored nilotinib sensitivity in the presence of SCF, confirming its involvement in SCF-activated survival pathway. However, this pathway seems not to be involved in the nilotinib-induced resistance of the CML stem cell population. Conversely, PI3K inhibition sensitized both CML progenitors and stem cells to nilotinib, suggesting that, downstream PI3K, two different kinase pathways are activated in CML progenitor and stem cell populations.

Show MeSH
Related in: MedlinePlus