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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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Analyzed samples and their bioenergetics' organizationA.Left panel: Dasatinib IC50 distribution of 30 CLL patient's primary B-cell lymphocytes samples. We distinguished a set of Dasatinib sensitive (<0.1nM-1000nM) and a set of Dasatinib resistant samples (6-52μM). Right panel: The bioenergetics' profile of 22 samples of CLL primary lymphocytes cells. To calculate the Glycolysis (black boxes) and OXPHOS (grey boxes) level in the response to OXPHOS inhibitor, Oligomycin (2uM), we used lactate concentration ratios in the non treated/treated samples after 24h incubation. In high glucose media, Oligomycin increased significantly lactate production, 2.9 ± 0.5 folds (p=0.011) and OXPHOS contributes to the 70% in average to the bioenergetics need of the samples tested (t-Test p<0.001). The plotted differences in bioenergetics organization are not ordered following the samples' Dasatinib sensitivity. B.Left panel: Effect on ATP levels by Rotenone; Rotenone significantly decrease (*) ATP levels by 2.1 fold (Paired t-Test p<0.008) in Dasatinib sensitive samples (black shapes) vs. no effect (1.2 fold) on Dasatinib resistant samples (white shapes). The scatter plot shows fold changes in Log 10. Right panel: we assessed the effect of Rotenone at a toxic concentration of 500nM which induced cell death in sensitive and resistant samples nonetheless the effect was only significant (*) in sensitive samples (Paired t-Test p<0.001) (black shapes). C.Scatter plot showing that 2-DG affected by increasing lactate production in a set of representative Dasatinib sensitive and resistant samples (Dasatinib resistant samples-white shapes and sensitive samples-black shapes) and the values correspond to ratios fold differences (2-DG/Vehicle treatments).
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Figure 1: Analyzed samples and their bioenergetics' organizationA.Left panel: Dasatinib IC50 distribution of 30 CLL patient's primary B-cell lymphocytes samples. We distinguished a set of Dasatinib sensitive (<0.1nM-1000nM) and a set of Dasatinib resistant samples (6-52μM). Right panel: The bioenergetics' profile of 22 samples of CLL primary lymphocytes cells. To calculate the Glycolysis (black boxes) and OXPHOS (grey boxes) level in the response to OXPHOS inhibitor, Oligomycin (2uM), we used lactate concentration ratios in the non treated/treated samples after 24h incubation. In high glucose media, Oligomycin increased significantly lactate production, 2.9 ± 0.5 folds (p=0.011) and OXPHOS contributes to the 70% in average to the bioenergetics need of the samples tested (t-Test p<0.001). The plotted differences in bioenergetics organization are not ordered following the samples' Dasatinib sensitivity. B.Left panel: Effect on ATP levels by Rotenone; Rotenone significantly decrease (*) ATP levels by 2.1 fold (Paired t-Test p<0.008) in Dasatinib sensitive samples (black shapes) vs. no effect (1.2 fold) on Dasatinib resistant samples (white shapes). The scatter plot shows fold changes in Log 10. Right panel: we assessed the effect of Rotenone at a toxic concentration of 500nM which induced cell death in sensitive and resistant samples nonetheless the effect was only significant (*) in sensitive samples (Paired t-Test p<0.001) (black shapes). C.Scatter plot showing that 2-DG affected by increasing lactate production in a set of representative Dasatinib sensitive and resistant samples (Dasatinib resistant samples-white shapes and sensitive samples-black shapes) and the values correspond to ratios fold differences (2-DG/Vehicle treatments).

Mentions: We used primary CLL lymphocytes from 30 CLL cases followed at the Jewish General Hospital Hematology Clinic upon informed consent. Samples positive for Del17p and Del11q (deficient for p53 and ATM respectively) were excluded from this study. By their sensitivity to Dasatinib obtained by MTT essay, 17 of the samples fall in the sensitive range IC50s (1nM-1000nM) and 13 of the samples fall in the resistant range (6-52μM) (Table 1 and FIGURE 1A left panel). We confirmed Dasatinib IC50's cytotoxicity by AnnexinV/PI staining and FSC/SSC analysis. There were no significant associations between the IgVH mutational status or CD38 expression and Dasatinib resistance (p>0.05).


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)

Analyzed samples and their bioenergetics' organizationA.Left panel: Dasatinib IC50 distribution of 30 CLL patient's primary B-cell lymphocytes samples. We distinguished a set of Dasatinib sensitive (<0.1nM-1000nM) and a set of Dasatinib resistant samples (6-52μM). Right panel: The bioenergetics' profile of 22 samples of CLL primary lymphocytes cells. To calculate the Glycolysis (black boxes) and OXPHOS (grey boxes) level in the response to OXPHOS inhibitor, Oligomycin (2uM), we used lactate concentration ratios in the non treated/treated samples after 24h incubation. In high glucose media, Oligomycin increased significantly lactate production, 2.9 ± 0.5 folds (p=0.011) and OXPHOS contributes to the 70% in average to the bioenergetics need of the samples tested (t-Test p<0.001). The plotted differences in bioenergetics organization are not ordered following the samples' Dasatinib sensitivity. B.Left panel: Effect on ATP levels by Rotenone; Rotenone significantly decrease (*) ATP levels by 2.1 fold (Paired t-Test p<0.008) in Dasatinib sensitive samples (black shapes) vs. no effect (1.2 fold) on Dasatinib resistant samples (white shapes). The scatter plot shows fold changes in Log 10. Right panel: we assessed the effect of Rotenone at a toxic concentration of 500nM which induced cell death in sensitive and resistant samples nonetheless the effect was only significant (*) in sensitive samples (Paired t-Test p<0.001) (black shapes). C.Scatter plot showing that 2-DG affected by increasing lactate production in a set of representative Dasatinib sensitive and resistant samples (Dasatinib resistant samples-white shapes and sensitive samples-black shapes) and the values correspond to ratios fold differences (2-DG/Vehicle treatments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926848&req=5

Figure 1: Analyzed samples and their bioenergetics' organizationA.Left panel: Dasatinib IC50 distribution of 30 CLL patient's primary B-cell lymphocytes samples. We distinguished a set of Dasatinib sensitive (<0.1nM-1000nM) and a set of Dasatinib resistant samples (6-52μM). Right panel: The bioenergetics' profile of 22 samples of CLL primary lymphocytes cells. To calculate the Glycolysis (black boxes) and OXPHOS (grey boxes) level in the response to OXPHOS inhibitor, Oligomycin (2uM), we used lactate concentration ratios in the non treated/treated samples after 24h incubation. In high glucose media, Oligomycin increased significantly lactate production, 2.9 ± 0.5 folds (p=0.011) and OXPHOS contributes to the 70% in average to the bioenergetics need of the samples tested (t-Test p<0.001). The plotted differences in bioenergetics organization are not ordered following the samples' Dasatinib sensitivity. B.Left panel: Effect on ATP levels by Rotenone; Rotenone significantly decrease (*) ATP levels by 2.1 fold (Paired t-Test p<0.008) in Dasatinib sensitive samples (black shapes) vs. no effect (1.2 fold) on Dasatinib resistant samples (white shapes). The scatter plot shows fold changes in Log 10. Right panel: we assessed the effect of Rotenone at a toxic concentration of 500nM which induced cell death in sensitive and resistant samples nonetheless the effect was only significant (*) in sensitive samples (Paired t-Test p<0.001) (black shapes). C.Scatter plot showing that 2-DG affected by increasing lactate production in a set of representative Dasatinib sensitive and resistant samples (Dasatinib resistant samples-white shapes and sensitive samples-black shapes) and the values correspond to ratios fold differences (2-DG/Vehicle treatments).
Mentions: We used primary CLL lymphocytes from 30 CLL cases followed at the Jewish General Hospital Hematology Clinic upon informed consent. Samples positive for Del17p and Del11q (deficient for p53 and ATM respectively) were excluded from this study. By their sensitivity to Dasatinib obtained by MTT essay, 17 of the samples fall in the sensitive range IC50s (1nM-1000nM) and 13 of the samples fall in the resistant range (6-52μM) (Table 1 and FIGURE 1A left panel). We confirmed Dasatinib IC50's cytotoxicity by AnnexinV/PI staining and FSC/SSC analysis. There were no significant associations between the IgVH mutational status or CD38 expression and Dasatinib resistance (p>0.05).

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