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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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SCF inhibits nilotinib-induced apoptosis independently of BCL-2 family proteins. (a) Apoptosis was measured by flow cytometry using DiOC6(3) as a probe for K562 and LAMA-84 cell lines and FITC-annexin V for CML bone marrow CD34+ cells. Cells were incubated for 24 h in the presence or absence of 100 ng/ml SCF and 20 nM nilotinib. 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 for the cell lines and seven experiments for the CML CD34+ cells. (b and c) K562 and LAMA-84 cells were treated with 20 nM nilotinib in the presence or absence of SCF, and the expression of BIM, BCL-xL and cleaved caspase 3 (b) or phospho-ERK1/2 and ERK (c) were analyzed by western blot. Anti-tubulin antibody was used to verify the loading homogeneity. The figure shows one representative experiment of three performed
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fig1: SCF inhibits nilotinib-induced apoptosis independently of BCL-2 family proteins. (a) Apoptosis was measured by flow cytometry using DiOC6(3) as a probe for K562 and LAMA-84 cell lines and FITC-annexin V for CML bone marrow CD34+ cells. Cells were incubated for 24 h in the presence or absence of 100 ng/ml SCF and 20 nM nilotinib. 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 for the cell lines and seven experiments for the CML CD34+ cells. (b and c) K562 and LAMA-84 cells were treated with 20 nM nilotinib in the presence or absence of SCF, and the expression of BIM, BCL-xL and cleaved caspase 3 (b) or phospho-ERK1/2 and ERK (c) were analyzed by western blot. Anti-tubulin antibody was used to verify the loading homogeneity. The figure shows one representative experiment of three performed

Mentions: We previously demonstrated that SCF was able to inhibit nilotinib-induced apoptosis on BCR-ABL-expressing cells when nilotinib was used at concentrations targeting the BCR-ABL tyrosine kinase but was unable to inhibit the c-KIT tyrosine kinase.9 These results were confirmed on Figure 1a, where apoptosis induced in 24 h by 20 nM nilotinib was reduced by at least 50% in two BCR-ABL-positive cell lines and fresh CD34+cells from CML patient's bone marrows. Moreover, the nilotinib-induced BIM accumulation and BCL-xL downregulation were not modified by the addition of SCF, whereas the cleavage of caspase 3, specific of apoptosis, was partly inhibited (Figure 1b). Similarly, ERK1/2 (extracellular signal-regulated kinases) phosphorylation, responsible for BIM degradation, was not completely restored in the presence of SCF, explaining the sustained accumulation of BIM (Figure 1c). Thus, although TKI-induced imbalance between the BCL-2 family proteins was necessary for apoptosis,16 it was not sufficient for the completion of this cell death, suggesting the inhibition of other antiapoptotic signals activated by BCR-ABL.


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)

SCF inhibits nilotinib-induced apoptosis independently of BCL-2 family proteins. (a) Apoptosis was measured by flow cytometry using DiOC6(3) as a probe for K562 and LAMA-84 cell lines and FITC-annexin V for CML bone marrow CD34+ cells. Cells were incubated for 24 h in the presence or absence of 100 ng/ml SCF and 20 nM nilotinib. 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 for the cell lines and seven experiments for the CML CD34+ cells. (b and c) K562 and LAMA-84 cells were treated with 20 nM nilotinib in the presence or absence of SCF, and the expression of BIM, BCL-xL and cleaved caspase 3 (b) or phospho-ERK1/2 and ERK (c) were analyzed by western blot. Anti-tubulin antibody was used to verify the loading homogeneity. The figure shows one representative experiment of three performed
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: SCF inhibits nilotinib-induced apoptosis independently of BCL-2 family proteins. (a) Apoptosis was measured by flow cytometry using DiOC6(3) as a probe for K562 and LAMA-84 cell lines and FITC-annexin V for CML bone marrow CD34+ cells. Cells were incubated for 24 h in the presence or absence of 100 ng/ml SCF and 20 nM nilotinib. 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 for the cell lines and seven experiments for the CML CD34+ cells. (b and c) K562 and LAMA-84 cells were treated with 20 nM nilotinib in the presence or absence of SCF, and the expression of BIM, BCL-xL and cleaved caspase 3 (b) or phospho-ERK1/2 and ERK (c) were analyzed by western blot. Anti-tubulin antibody was used to verify the loading homogeneity. The figure shows one representative experiment of three performed
Mentions: We previously demonstrated that SCF was able to inhibit nilotinib-induced apoptosis on BCR-ABL-expressing cells when nilotinib was used at concentrations targeting the BCR-ABL tyrosine kinase but was unable to inhibit the c-KIT tyrosine kinase.9 These results were confirmed on Figure 1a, where apoptosis induced in 24 h by 20 nM nilotinib was reduced by at least 50% in two BCR-ABL-positive cell lines and fresh CD34+cells from CML patient's bone marrows. Moreover, the nilotinib-induced BIM accumulation and BCL-xL downregulation were not modified by the addition of SCF, whereas the cleavage of caspase 3, specific of apoptosis, was partly inhibited (Figure 1b). Similarly, ERK1/2 (extracellular signal-regulated kinases) phosphorylation, responsible for BIM degradation, was not completely restored in the presence of SCF, explaining the sustained accumulation of BIM (Figure 1c). Thus, although TKI-induced imbalance between the BCL-2 family proteins was necessary for apoptosis,16 it was not sufficient for the completion of this cell death, suggesting the inhibition of other antiapoptotic signals activated by BCR-ABL.

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