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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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The expression and phosphorylation status of metabolic sensors and transducers in primary CLL lymphocytes is correlated with Dasatinib responseA.Representative Western blots for Dasatinib sensitive and resistant samples showing significant differences (*) between sets for the expression of LKB1, activator of AMPK, was significantly decreased in sensitive samples (t-Test p=0.03) and correlated with Dasatinib in vitro resistance (IC50 values) (Spearman Corr. r=0.7, p= 0.03). Basal AMPK (Thr172) and ULK1/2 (Ser317) phosphorylation were significantly higher in resistant samples by 2 and 6.3 fold respectively (t-Test p=0.03 and p<0.001). Western blots analysis revealed similar expression of GLUT-4 in all samples tested and a significant lower expression of GLUT-1 in sensitive samples (Mann-Whitney U Statistic p=0.005). Resistant samples showed increased basal expression of TIGAR and was positively associated with Dasatinib resistance (Pearson Corr. r=0.75, p=0.02). PKM2, UCP2, PPARa, mTOR (Ser2448) and Hif-1a expression were variable and highly expressed in the samples tested but not associated with Dasatinib resistance. B.Left panel: Western blots showing Dasatinib in vitro treatment significantly (*) decreased mTor phosphorylation (Ser2448) and UCP2 protein levels by 5 fold (Paired t-Test p=0.004) and 2 fold (Paired t-Test p=0.008), respectively in all samples tested after 12h. While, Dasatinib-induced AMPKThr172/ULK1/2(Ser317) phosphorylation in Dasatinib resistant samples by 5 fold (Paired t-Test p=0.008), and 2.8 fold (Paired t-Test p=0.01). Right panel: scatter plot of the significant (***) effect in fold changes Log 10 of Dasatinib in Dasatinib sensitive (black shapes) and resistant samples (white shapes). C.Left panel: scatter plot showing ratios of co-treatment with Dasatinib and vehicle (CTL), 5μM compound C (Comp C) (an AMPK inhibitor) and 1μM Chloroquine (CQ) (an inhibitor of late autophagy). Both selective sensitized resistant samples (white shapes) to Dasatinib by 7 and 2 fold respectively (mean value) (Paired t-Test p<0.05). Right panel: scatter plot showing the effect on 4 drugs at non toxic concentrations on Dasatinib. Two mTORC1 inhibitors Rapamycin (100nM) and Metformin (1mM) as well as two downstream mTOR pathway inhibitors: CGP-57,380 (1μM) and 4EGI-1 (10μM) significantly (***) sensitized sensitive samples (black shapes) to Dasatinib with median sensitization values of 3.7, 5.3, 4.4 and 4.1 fold for Rapamycin, Metformin, CGP-57,380 and 4EGI-1 respectively (Paired t-Test p<0.01).
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Figure 3: The expression and phosphorylation status of metabolic sensors and transducers in primary CLL lymphocytes is correlated with Dasatinib responseA.Representative Western blots for Dasatinib sensitive and resistant samples showing significant differences (*) between sets for the expression of LKB1, activator of AMPK, was significantly decreased in sensitive samples (t-Test p=0.03) and correlated with Dasatinib in vitro resistance (IC50 values) (Spearman Corr. r=0.7, p= 0.03). Basal AMPK (Thr172) and ULK1/2 (Ser317) phosphorylation were significantly higher in resistant samples by 2 and 6.3 fold respectively (t-Test p=0.03 and p<0.001). Western blots analysis revealed similar expression of GLUT-4 in all samples tested and a significant lower expression of GLUT-1 in sensitive samples (Mann-Whitney U Statistic p=0.005). Resistant samples showed increased basal expression of TIGAR and was positively associated with Dasatinib resistance (Pearson Corr. r=0.75, p=0.02). PKM2, UCP2, PPARa, mTOR (Ser2448) and Hif-1a expression were variable and highly expressed in the samples tested but not associated with Dasatinib resistance. B.Left panel: Western blots showing Dasatinib in vitro treatment significantly (*) decreased mTor phosphorylation (Ser2448) and UCP2 protein levels by 5 fold (Paired t-Test p=0.004) and 2 fold (Paired t-Test p=0.008), respectively in all samples tested after 12h. While, Dasatinib-induced AMPKThr172/ULK1/2(Ser317) phosphorylation in Dasatinib resistant samples by 5 fold (Paired t-Test p=0.008), and 2.8 fold (Paired t-Test p=0.01). Right panel: scatter plot of the significant (***) effect in fold changes Log 10 of Dasatinib in Dasatinib sensitive (black shapes) and resistant samples (white shapes). C.Left panel: scatter plot showing ratios of co-treatment with Dasatinib and vehicle (CTL), 5μM compound C (Comp C) (an AMPK inhibitor) and 1μM Chloroquine (CQ) (an inhibitor of late autophagy). Both selective sensitized resistant samples (white shapes) to Dasatinib by 7 and 2 fold respectively (mean value) (Paired t-Test p<0.05). Right panel: scatter plot showing the effect on 4 drugs at non toxic concentrations on Dasatinib. Two mTORC1 inhibitors Rapamycin (100nM) and Metformin (1mM) as well as two downstream mTOR pathway inhibitors: CGP-57,380 (1μM) and 4EGI-1 (10μM) significantly (***) sensitized sensitive samples (black shapes) to Dasatinib with median sensitization values of 3.7, 5.3, 4.4 and 4.1 fold for Rapamycin, Metformin, CGP-57,380 and 4EGI-1 respectively (Paired t-Test p<0.01).

Mentions: In addition, we found no significant differences in the expression of UCP2, PPARa and CPT-1 (FIGURE 3A and Supplementary FIGURE 1B). UCP2 expression in all CLL samples suggested that primary CLL lymphocytes upon ROS induction may have the capacity to increase proton conductance of mitochondrial inner membrane [35, 36] while the expression of CPT-1 and PPARa could indicate a high lipid metabolism capacity in primary CLL lymphocytes [14].


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)

The expression and phosphorylation status of metabolic sensors and transducers in primary CLL lymphocytes is correlated with Dasatinib responseA.Representative Western blots for Dasatinib sensitive and resistant samples showing significant differences (*) between sets for the expression of LKB1, activator of AMPK, was significantly decreased in sensitive samples (t-Test p=0.03) and correlated with Dasatinib in vitro resistance (IC50 values) (Spearman Corr. r=0.7, p= 0.03). Basal AMPK (Thr172) and ULK1/2 (Ser317) phosphorylation were significantly higher in resistant samples by 2 and 6.3 fold respectively (t-Test p=0.03 and p<0.001). Western blots analysis revealed similar expression of GLUT-4 in all samples tested and a significant lower expression of GLUT-1 in sensitive samples (Mann-Whitney U Statistic p=0.005). Resistant samples showed increased basal expression of TIGAR and was positively associated with Dasatinib resistance (Pearson Corr. r=0.75, p=0.02). PKM2, UCP2, PPARa, mTOR (Ser2448) and Hif-1a expression were variable and highly expressed in the samples tested but not associated with Dasatinib resistance. B.Left panel: Western blots showing Dasatinib in vitro treatment significantly (*) decreased mTor phosphorylation (Ser2448) and UCP2 protein levels by 5 fold (Paired t-Test p=0.004) and 2 fold (Paired t-Test p=0.008), respectively in all samples tested after 12h. While, Dasatinib-induced AMPKThr172/ULK1/2(Ser317) phosphorylation in Dasatinib resistant samples by 5 fold (Paired t-Test p=0.008), and 2.8 fold (Paired t-Test p=0.01). Right panel: scatter plot of the significant (***) effect in fold changes Log 10 of Dasatinib in Dasatinib sensitive (black shapes) and resistant samples (white shapes). C.Left panel: scatter plot showing ratios of co-treatment with Dasatinib and vehicle (CTL), 5μM compound C (Comp C) (an AMPK inhibitor) and 1μM Chloroquine (CQ) (an inhibitor of late autophagy). Both selective sensitized resistant samples (white shapes) to Dasatinib by 7 and 2 fold respectively (mean value) (Paired t-Test p<0.05). Right panel: scatter plot showing the effect on 4 drugs at non toxic concentrations on Dasatinib. Two mTORC1 inhibitors Rapamycin (100nM) and Metformin (1mM) as well as two downstream mTOR pathway inhibitors: CGP-57,380 (1μM) and 4EGI-1 (10μM) significantly (***) sensitized sensitive samples (black shapes) to Dasatinib with median sensitization values of 3.7, 5.3, 4.4 and 4.1 fold for Rapamycin, Metformin, CGP-57,380 and 4EGI-1 respectively (Paired t-Test p<0.01).
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Figure 3: The expression and phosphorylation status of metabolic sensors and transducers in primary CLL lymphocytes is correlated with Dasatinib responseA.Representative Western blots for Dasatinib sensitive and resistant samples showing significant differences (*) between sets for the expression of LKB1, activator of AMPK, was significantly decreased in sensitive samples (t-Test p=0.03) and correlated with Dasatinib in vitro resistance (IC50 values) (Spearman Corr. r=0.7, p= 0.03). Basal AMPK (Thr172) and ULK1/2 (Ser317) phosphorylation were significantly higher in resistant samples by 2 and 6.3 fold respectively (t-Test p=0.03 and p<0.001). Western blots analysis revealed similar expression of GLUT-4 in all samples tested and a significant lower expression of GLUT-1 in sensitive samples (Mann-Whitney U Statistic p=0.005). Resistant samples showed increased basal expression of TIGAR and was positively associated with Dasatinib resistance (Pearson Corr. r=0.75, p=0.02). PKM2, UCP2, PPARa, mTOR (Ser2448) and Hif-1a expression were variable and highly expressed in the samples tested but not associated with Dasatinib resistance. B.Left panel: Western blots showing Dasatinib in vitro treatment significantly (*) decreased mTor phosphorylation (Ser2448) and UCP2 protein levels by 5 fold (Paired t-Test p=0.004) and 2 fold (Paired t-Test p=0.008), respectively in all samples tested after 12h. While, Dasatinib-induced AMPKThr172/ULK1/2(Ser317) phosphorylation in Dasatinib resistant samples by 5 fold (Paired t-Test p=0.008), and 2.8 fold (Paired t-Test p=0.01). Right panel: scatter plot of the significant (***) effect in fold changes Log 10 of Dasatinib in Dasatinib sensitive (black shapes) and resistant samples (white shapes). C.Left panel: scatter plot showing ratios of co-treatment with Dasatinib and vehicle (CTL), 5μM compound C (Comp C) (an AMPK inhibitor) and 1μM Chloroquine (CQ) (an inhibitor of late autophagy). Both selective sensitized resistant samples (white shapes) to Dasatinib by 7 and 2 fold respectively (mean value) (Paired t-Test p<0.05). Right panel: scatter plot showing the effect on 4 drugs at non toxic concentrations on Dasatinib. Two mTORC1 inhibitors Rapamycin (100nM) and Metformin (1mM) as well as two downstream mTOR pathway inhibitors: CGP-57,380 (1μM) and 4EGI-1 (10μM) significantly (***) sensitized sensitive samples (black shapes) to Dasatinib with median sensitization values of 3.7, 5.3, 4.4 and 4.1 fold for Rapamycin, Metformin, CGP-57,380 and 4EGI-1 respectively (Paired t-Test p<0.01).
Mentions: In addition, we found no significant differences in the expression of UCP2, PPARa and CPT-1 (FIGURE 3A and Supplementary FIGURE 1B). UCP2 expression in all CLL samples suggested that primary CLL lymphocytes upon ROS induction may have the capacity to increase proton conductance of mitochondrial inner membrane [35, 36] while the expression of CPT-1 and PPARa could indicate a high lipid metabolism capacity in primary CLL lymphocytes [14].

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