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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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PI3K inhibition cooperates with BCR-ABL inhibition to induce apoptosis in BCR-ABL-expressing cells. (a) K562 and LAMA-84 cells were treated or not with 20 nM nilotinib in the presence or absence of 100 ng/ml SCF for 24 h, and AKT phosphorylation was analyzed by western blot. (b and c) K562 and LAMA-84 cells were treated with nilotinib 20 nM in the presence or absence of 100 ng/ml SCF and 10 μM LY294002 as indicated. Induced apoptosis was measured and calculated as described in Figure 1a (b), and mTOR and S6K phosphorylation was analyzed by western blot (c). (d) K562 and LAMA-84 cells were treated with nilotinib 20 nM in the presence or absence of 100 ng/ml SCF and with 1 μM CAL-101 (dark grey bars) or 1 μM GDC-0491 (light grey bars) or 1 μM BEZ-235 (middle grey bars) or 10 μM LY294002 (white bars) as indicated. Induced apoptosis was measured and calculated as described in Figure 1a
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fig4: PI3K inhibition cooperates with BCR-ABL inhibition to induce apoptosis in BCR-ABL-expressing cells. (a) K562 and LAMA-84 cells were treated or not with 20 nM nilotinib in the presence or absence of 100 ng/ml SCF for 24 h, and AKT phosphorylation was analyzed by western blot. (b and c) K562 and LAMA-84 cells were treated with nilotinib 20 nM in the presence or absence of 100 ng/ml SCF and 10 μM LY294002 as indicated. Induced apoptosis was measured and calculated as described in Figure 1a (b), and mTOR and S6K phosphorylation was analyzed by western blot (c). (d) K562 and LAMA-84 cells were treated with nilotinib 20 nM in the presence or absence of 100 ng/ml SCF and with 1 μM CAL-101 (dark grey bars) or 1 μM GDC-0491 (light grey bars) or 1 μM BEZ-235 (middle grey bars) or 10 μM LY294002 (white bars) as indicated. Induced apoptosis was measured and calculated as described in Figure 1a

Mentions: In order to identify the mechanism leading to mTOR activity, we analyzed the PI3K pathway described as the canonical pathway responsible for both AKT and mTOR activation.19 Conversely to mTOR, AKT was not dephosphorylated after BCR-ABL inhibition or phosphorylated by SCF addition (Figure 4a). However, the upstream inhibition of the PI3K using LY294002 cooperated with BCR-ABL inhibition to restore the apoptosis in the cell lines (Figure 4b). When BCR-ABL expression is inhibited by RNA interference, LY294002 addition blocked the cell rescue by SCF (Supplementary Figure S2). Moreover, PI3K and BCR-ABL inhibitors cooperated to inhibit mTOR phosphorylation and mTORC1 activity (Figure 4c), suggesting that both these pathways participated to mTOR activation. As LY294002 is not a clinically used PI3K inhibitor, we tested other PI3K inhibitors, currently involved in clinical trials. CAL-101 is a specific inhibitor of the PI3Kγ, GDC-0491 is able to inhibit the four PI3K isoforms and BEZ-235 is a dual PI3K/mTOR inhibitor. CAL-101 was not able to restore CML cell sensitivity to nilotinib in the presence of SCF (Figure 4d), conversely to GDC-0491 and BEZ-235 which induced apoptosis in a dose-dependent effect (Supplementary Figure S23).


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)

PI3K inhibition cooperates with BCR-ABL inhibition to induce apoptosis in BCR-ABL-expressing cells. (a) K562 and LAMA-84 cells were treated or not with 20 nM nilotinib in the presence or absence of 100 ng/ml SCF for 24 h, and AKT phosphorylation was analyzed by western blot. (b and c) K562 and LAMA-84 cells were treated with nilotinib 20 nM in the presence or absence of 100 ng/ml SCF and 10 μM LY294002 as indicated. Induced apoptosis was measured and calculated as described in Figure 1a (b), and mTOR and S6K phosphorylation was analyzed by western blot (c). (d) K562 and LAMA-84 cells were treated with nilotinib 20 nM in the presence or absence of 100 ng/ml SCF and with 1 μM CAL-101 (dark grey bars) or 1 μM GDC-0491 (light grey bars) or 1 μM BEZ-235 (middle grey bars) or 10 μM LY294002 (white bars) as indicated. Induced apoptosis was measured and calculated as described in Figure 1a
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fig4: PI3K inhibition cooperates with BCR-ABL inhibition to induce apoptosis in BCR-ABL-expressing cells. (a) K562 and LAMA-84 cells were treated or not with 20 nM nilotinib in the presence or absence of 100 ng/ml SCF for 24 h, and AKT phosphorylation was analyzed by western blot. (b and c) K562 and LAMA-84 cells were treated with nilotinib 20 nM in the presence or absence of 100 ng/ml SCF and 10 μM LY294002 as indicated. Induced apoptosis was measured and calculated as described in Figure 1a (b), and mTOR and S6K phosphorylation was analyzed by western blot (c). (d) K562 and LAMA-84 cells were treated with nilotinib 20 nM in the presence or absence of 100 ng/ml SCF and with 1 μM CAL-101 (dark grey bars) or 1 μM GDC-0491 (light grey bars) or 1 μM BEZ-235 (middle grey bars) or 10 μM LY294002 (white bars) as indicated. Induced apoptosis was measured and calculated as described in Figure 1a
Mentions: In order to identify the mechanism leading to mTOR activity, we analyzed the PI3K pathway described as the canonical pathway responsible for both AKT and mTOR activation.19 Conversely to mTOR, AKT was not dephosphorylated after BCR-ABL inhibition or phosphorylated by SCF addition (Figure 4a). However, the upstream inhibition of the PI3K using LY294002 cooperated with BCR-ABL inhibition to restore the apoptosis in the cell lines (Figure 4b). When BCR-ABL expression is inhibited by RNA interference, LY294002 addition blocked the cell rescue by SCF (Supplementary Figure S2). Moreover, PI3K and BCR-ABL inhibitors cooperated to inhibit mTOR phosphorylation and mTORC1 activity (Figure 4c), suggesting that both these pathways participated to mTOR activation. As LY294002 is not a clinically used PI3K inhibitor, we tested other PI3K inhibitors, currently involved in clinical trials. CAL-101 is a specific inhibitor of the PI3Kγ, GDC-0491 is able to inhibit the four PI3K isoforms and BEZ-235 is a dual PI3K/mTOR inhibitor. CAL-101 was not able to restore CML cell sensitivity to nilotinib in the presence of SCF (Figure 4d), conversely to GDC-0491 and BEZ-235 which induced apoptosis in a dose-dependent effect (Supplementary Figure S23).

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