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Resistance to Dasatinib in primary chronic lymphocytic leukemia lymphocytes involves AMPK-mediated energetic re-programming.

Martinez Marignac VL, Smith S, Toban N, Bazile M, Aloyz R - Oncotarget (2013)

Bottom Line: The contrasting metabolic features revealed by our strategy could be used to metabolically target CLL lymphocyte subsets creating new therapeutic windows for this disease for mTORC1 or AMPK inhibitors.Indeed, we report that Metformin, a drug used to treat diabetes was selectively cytotoxic to Dasatinib sensitive samples.Ultimately, we suggest that a similar strategy could be applied to other cancer types by using Dasatinib and/or relevant tyrosine kinase inhibitors.

View Article: PubMed Central - PubMed

Affiliation: McGill University, Lady Davis Institute and Segal Cancer Center, Jewish General Hospital, Montreal, Canada.

ABSTRACT
Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. Although promising new therapies for this incurable disease are being tested in clinical trials, the therapeutic relevance of metabolic rewiring in chronic lymphocytic leukemia (CLL) is poorly understood. The aim of this study was to identify targetable metabolic differences in primary CLL lymphocytes by the use of Dasatinib. Dasatinib is a multi-tyrosine kinase inhibitor used to treat chronic myelogenous leukemia (CML) and is being tested in clinical trials for several cancers including CLL. This drug has been shown to be beneficial to CML patients suffering from diabetes by reducing their glucose plasma levels. In keeping with this previous observation, we report that Dasatinib induced glucose use while reducing lactate production, suggesting that this tyrosine kinase inhibitor decreases aerobic glycolysis and shifts glucose use in primary CLL lymphocytes. Our results suggest that primary CLL lymphocytes (independently of traditional prognostic factors) can be stratified in two subsets by their sensitivity to Dasatinib in vitro. Increased glucose use induced by Dasatinib or by inhibition of mitochondrial respiration was not sufficient to sustain survival and ATP levels in CLL samples sensitive to Dasatinib. The two subsets of primary CLL lymphocytes are characterized as well by a differential dependency on mitochondrial respiration and the use of anabolic or catabolic processes to cope with induced metabolic/energetic stress. Differential metabolic reprogramming between subsets is supported by the contrasting effect on the survival of Dasatinib treated CLL lymphocytes with pharmacological inhibition of two master metabolic regulators (mTorc1 and AMPK) as well as induced autophagy. Alternative metabolic organization between subsets is further supported by the differential basal expression (freshly purified lymphocytes) of active AMPK, regulators of glucose metabolism and modulators of AKT signaling. The contrasting metabolic features revealed by our strategy could be used to metabolically target CLL lymphocyte subsets creating new therapeutic windows for this disease for mTORC1 or AMPK inhibitors. Indeed, we report that Metformin, a drug used to treat diabetes was selectively cytotoxic to Dasatinib sensitive samples. Ultimately, we suggest that a similar strategy could be applied to other cancer types by using Dasatinib and/or relevant tyrosine kinase inhibitors.

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Complex I and downstream mTor pathway in Dasatinib sensitizationA.Western blots showing that non toxic doses of Metformin (1mM), CGP-57,380 (1μM), Rapamycin (100nM) and 4EGI-1(10μM) increased AMPK (Thr172) in sensitive samples while they did not affecte 4EBP-1 (Thr37/46) phosphorylation, an mTor downstream target. B.Annexin V/PI staining and FSC-SSC assay showing 5mM Metformin effect after 48h treatment. The graphs are for representative samples and show that Metformin at 5mM induced cell death and apoptosis which were selectively in sensitive samples (t-Test p=0.035). C.Western blots showing 5mM Metformin effect on mTor and AMPK pathway. A higher concentration of 5mM Metformin treatments at 12h increased significantly (*) AMPK (Thr172) in resistant and sensitive samples together with a decrease in mTor-2448 in both sets however the inhibition of mTOR (Ser2448) as well as 4EBP-1 (Thr37/46) phosphorylation was significantly decreased (*) in sensitive samples compared to resistant samples by 2 folds for mTor (Paired t-Test p=0.029) and more than 3 folds in the case of 4EBP-1 (Paired t-Test p=0.011).
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Figure 4: Complex I and downstream mTor pathway in Dasatinib sensitizationA.Western blots showing that non toxic doses of Metformin (1mM), CGP-57,380 (1μM), Rapamycin (100nM) and 4EGI-1(10μM) increased AMPK (Thr172) in sensitive samples while they did not affecte 4EBP-1 (Thr37/46) phosphorylation, an mTor downstream target. B.Annexin V/PI staining and FSC-SSC assay showing 5mM Metformin effect after 48h treatment. The graphs are for representative samples and show that Metformin at 5mM induced cell death and apoptosis which were selectively in sensitive samples (t-Test p=0.035). C.Western blots showing 5mM Metformin effect on mTor and AMPK pathway. A higher concentration of 5mM Metformin treatments at 12h increased significantly (*) AMPK (Thr172) in resistant and sensitive samples together with a decrease in mTor-2448 in both sets however the inhibition of mTOR (Ser2448) as well as 4EBP-1 (Thr37/46) phosphorylation was significantly decreased (*) in sensitive samples compared to resistant samples by 2 folds for mTor (Paired t-Test p=0.029) and more than 3 folds in the case of 4EBP-1 (Paired t-Test p=0.011).

Mentions: We next assessed the effect of Dasatinib on significant targets twelve hours after Dasatinib treatment with equally cytotoxic concentrations meaning that each sample was treated with its own IC50. In this condition, in vitro treatment with the drug resulted in decreased mTor phosphorylation (Ser2448) and UCP2 protein levels by 5 fold (p=0.004) and 2 fold (p=0.008), respectively in all samples tested (FIGURE 3B, left panel). The decreased levels of UCP2 suggest a drug-induced increase in mitochondrial membrane potential. This was supported by Dasatinib induction of increased mitochondrial biomass staining with Mitotracker green in all the tested samples (Supplementary FIGURE 1F). Additionally, Dasatinib-induced AMPK and ULK phosphorylation in resistant samples by 5 fold (p=0.008), and 2.8 fold (p=0.01), respectively (FIGURE 3B, left panel and right panel). The results suggest that there are differences in regulation of the AMPK-mTor metabolic checkpoint between subsets. Thus, we assess the effect of non toxic concentrations of inhibitors of mTorc1 signaling or AMPK as well as autophagy in the resistance to Dasatinib in both subsets. In light of our results showing a differential dependency on OXPHOS between subsets, we also assess the effect of Metformin. Metformin is a partial mitochondrial complex I inhibitor used to treat diabetes [11, 40]. Co-treatment of Dasatinib with specific inhibitors of AMPK, mTORC1 signaling or late autophagy resulted in clearly differential response between subsets. We found that the combinations of Dasatinib with 5μM compound C (an AMPK inhibitor) or Chloroquine (1μM) (an inhibitor of late autophagy) selectively sensitized resistant samples to Dasatinib by 7 and 2 fold respectively (mean value) (p<0.05) (FIGURE 3C, left panel). In contrast, sensitive samples were sensitized to Dasatinib by Metformin (1mM) in 5.3 fold (median value) (p=0.004) (FIGURE 3C, right panel) or by other inhibitors of mTORC1 signaling pathway. We used Rapamycin as well as two downstream mTORC1 pathway inhibitors: CGP-57380 a MAP-kinase interacting kinase-1 (Mnk1, MNK1) inhibitor and a hinder of eIF4E phosphorylation [41] and the 4EGI-1, a polypeptide that inhibits cap-dependent protein translation by disruption of the eIF4E/eIF4G association [42]. The median sensitization values we obtained for Dasatinib sensitive samples were of 3.7 for Rapamycin, 4.4 for CGP-57380 and 4.1 fold for 4EGI-1 (p<0.05) (FIGURE 3C, right panel). Interestingly, the concentrations of CGP-57380 (1μM), 4EGI-1 (10μM) or Metformin (1mM) although not toxic induced AMPK (Thr172) phosphorylation selectively in sensitive samples (FIGURE 4A), supporting a higher dependency of sensitive samples on eIF4E mediated functions to maintain bioenergetics' homeostasis. Of note CGP-57380, 4EGI-1 or Metformin when used alone did not affect 4EBP-1 phosphorylation, suggesting that the sensitization effect of the drugs is not mediated by direct inhibition of mTorc1 (FIGURE 4A).


Resistance to Dasatinib in primary chronic lymphocytic leukemia lymphocytes involves AMPK-mediated energetic re-programming.

Martinez Marignac VL, Smith S, Toban N, Bazile M, Aloyz R - Oncotarget (2013)

Complex I and downstream mTor pathway in Dasatinib sensitizationA.Western blots showing that non toxic doses of Metformin (1mM), CGP-57,380 (1μM), Rapamycin (100nM) and 4EGI-1(10μM) increased AMPK (Thr172) in sensitive samples while they did not affecte 4EBP-1 (Thr37/46) phosphorylation, an mTor downstream target. B.Annexin V/PI staining and FSC-SSC assay showing 5mM Metformin effect after 48h treatment. The graphs are for representative samples and show that Metformin at 5mM induced cell death and apoptosis which were selectively in sensitive samples (t-Test p=0.035). C.Western blots showing 5mM Metformin effect on mTor and AMPK pathway. A higher concentration of 5mM Metformin treatments at 12h increased significantly (*) AMPK (Thr172) in resistant and sensitive samples together with a decrease in mTor-2448 in both sets however the inhibition of mTOR (Ser2448) as well as 4EBP-1 (Thr37/46) phosphorylation was significantly decreased (*) in sensitive samples compared to resistant samples by 2 folds for mTor (Paired t-Test p=0.029) and more than 3 folds in the case of 4EBP-1 (Paired t-Test p=0.011).
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Related In: Results  -  Collection

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Figure 4: Complex I and downstream mTor pathway in Dasatinib sensitizationA.Western blots showing that non toxic doses of Metformin (1mM), CGP-57,380 (1μM), Rapamycin (100nM) and 4EGI-1(10μM) increased AMPK (Thr172) in sensitive samples while they did not affecte 4EBP-1 (Thr37/46) phosphorylation, an mTor downstream target. B.Annexin V/PI staining and FSC-SSC assay showing 5mM Metformin effect after 48h treatment. The graphs are for representative samples and show that Metformin at 5mM induced cell death and apoptosis which were selectively in sensitive samples (t-Test p=0.035). C.Western blots showing 5mM Metformin effect on mTor and AMPK pathway. A higher concentration of 5mM Metformin treatments at 12h increased significantly (*) AMPK (Thr172) in resistant and sensitive samples together with a decrease in mTor-2448 in both sets however the inhibition of mTOR (Ser2448) as well as 4EBP-1 (Thr37/46) phosphorylation was significantly decreased (*) in sensitive samples compared to resistant samples by 2 folds for mTor (Paired t-Test p=0.029) and more than 3 folds in the case of 4EBP-1 (Paired t-Test p=0.011).
Mentions: We next assessed the effect of Dasatinib on significant targets twelve hours after Dasatinib treatment with equally cytotoxic concentrations meaning that each sample was treated with its own IC50. In this condition, in vitro treatment with the drug resulted in decreased mTor phosphorylation (Ser2448) and UCP2 protein levels by 5 fold (p=0.004) and 2 fold (p=0.008), respectively in all samples tested (FIGURE 3B, left panel). The decreased levels of UCP2 suggest a drug-induced increase in mitochondrial membrane potential. This was supported by Dasatinib induction of increased mitochondrial biomass staining with Mitotracker green in all the tested samples (Supplementary FIGURE 1F). Additionally, Dasatinib-induced AMPK and ULK phosphorylation in resistant samples by 5 fold (p=0.008), and 2.8 fold (p=0.01), respectively (FIGURE 3B, left panel and right panel). The results suggest that there are differences in regulation of the AMPK-mTor metabolic checkpoint between subsets. Thus, we assess the effect of non toxic concentrations of inhibitors of mTorc1 signaling or AMPK as well as autophagy in the resistance to Dasatinib in both subsets. In light of our results showing a differential dependency on OXPHOS between subsets, we also assess the effect of Metformin. Metformin is a partial mitochondrial complex I inhibitor used to treat diabetes [11, 40]. Co-treatment of Dasatinib with specific inhibitors of AMPK, mTORC1 signaling or late autophagy resulted in clearly differential response between subsets. We found that the combinations of Dasatinib with 5μM compound C (an AMPK inhibitor) or Chloroquine (1μM) (an inhibitor of late autophagy) selectively sensitized resistant samples to Dasatinib by 7 and 2 fold respectively (mean value) (p<0.05) (FIGURE 3C, left panel). In contrast, sensitive samples were sensitized to Dasatinib by Metformin (1mM) in 5.3 fold (median value) (p=0.004) (FIGURE 3C, right panel) or by other inhibitors of mTORC1 signaling pathway. We used Rapamycin as well as two downstream mTORC1 pathway inhibitors: CGP-57380 a MAP-kinase interacting kinase-1 (Mnk1, MNK1) inhibitor and a hinder of eIF4E phosphorylation [41] and the 4EGI-1, a polypeptide that inhibits cap-dependent protein translation by disruption of the eIF4E/eIF4G association [42]. The median sensitization values we obtained for Dasatinib sensitive samples were of 3.7 for Rapamycin, 4.4 for CGP-57380 and 4.1 fold for 4EGI-1 (p<0.05) (FIGURE 3C, right panel). Interestingly, the concentrations of CGP-57380 (1μM), 4EGI-1 (10μM) or Metformin (1mM) although not toxic induced AMPK (Thr172) phosphorylation selectively in sensitive samples (FIGURE 4A), supporting a higher dependency of sensitive samples on eIF4E mediated functions to maintain bioenergetics' homeostasis. Of note CGP-57380, 4EGI-1 or Metformin when used alone did not affect 4EBP-1 phosphorylation, suggesting that the sensitization effect of the drugs is not mediated by direct inhibition of mTorc1 (FIGURE 4A).

Bottom Line: The contrasting metabolic features revealed by our strategy could be used to metabolically target CLL lymphocyte subsets creating new therapeutic windows for this disease for mTORC1 or AMPK inhibitors.Indeed, we report that Metformin, a drug used to treat diabetes was selectively cytotoxic to Dasatinib sensitive samples.Ultimately, we suggest that a similar strategy could be applied to other cancer types by using Dasatinib and/or relevant tyrosine kinase inhibitors.

View Article: PubMed Central - PubMed

Affiliation: McGill University, Lady Davis Institute and Segal Cancer Center, Jewish General Hospital, Montreal, Canada.

ABSTRACT
Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. Although promising new therapies for this incurable disease are being tested in clinical trials, the therapeutic relevance of metabolic rewiring in chronic lymphocytic leukemia (CLL) is poorly understood. The aim of this study was to identify targetable metabolic differences in primary CLL lymphocytes by the use of Dasatinib. Dasatinib is a multi-tyrosine kinase inhibitor used to treat chronic myelogenous leukemia (CML) and is being tested in clinical trials for several cancers including CLL. This drug has been shown to be beneficial to CML patients suffering from diabetes by reducing their glucose plasma levels. In keeping with this previous observation, we report that Dasatinib induced glucose use while reducing lactate production, suggesting that this tyrosine kinase inhibitor decreases aerobic glycolysis and shifts glucose use in primary CLL lymphocytes. Our results suggest that primary CLL lymphocytes (independently of traditional prognostic factors) can be stratified in two subsets by their sensitivity to Dasatinib in vitro. Increased glucose use induced by Dasatinib or by inhibition of mitochondrial respiration was not sufficient to sustain survival and ATP levels in CLL samples sensitive to Dasatinib. The two subsets of primary CLL lymphocytes are characterized as well by a differential dependency on mitochondrial respiration and the use of anabolic or catabolic processes to cope with induced metabolic/energetic stress. Differential metabolic reprogramming between subsets is supported by the contrasting effect on the survival of Dasatinib treated CLL lymphocytes with pharmacological inhibition of two master metabolic regulators (mTorc1 and AMPK) as well as induced autophagy. Alternative metabolic organization between subsets is further supported by the differential basal expression (freshly purified lymphocytes) of active AMPK, regulators of glucose metabolism and modulators of AKT signaling. The contrasting metabolic features revealed by our strategy could be used to metabolically target CLL lymphocyte subsets creating new therapeutic windows for this disease for mTORC1 or AMPK inhibitors. Indeed, we report that Metformin, a drug used to treat diabetes was selectively cytotoxic to Dasatinib sensitive samples. Ultimately, we suggest that a similar strategy could be applied to other cancer types by using Dasatinib and/or relevant tyrosine kinase inhibitors.

Show MeSH
Related in: MedlinePlus