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AKT-induced reactive oxygen species generate imatinib-resistant clones emerging from chronic myeloid leukemia progenitor cells.

Nieborowska-Skorska M, Flis S, Skorski T - Leukemia (2014)

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, and Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA.

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BCR-ABL1 fusion tyrosine kinase transforms hematopoietic stem cells (HSCs) and cause chronic myeloid leukemia in chronic phase (CML-CP), which is a stem cell (leukemia stem cell=LSC) -derived but a progenitor (leukemia progenitor cell=LPC)-driven disease... There are several possible explanations for persistent elevated levels of ROS and oxidative DNA damage in CML-CP cells surviving TKI treatment... For example, the effect of TKIs on BCR-ABL1 kinase-induced signaling pathways stimulating ROS production may be obscured by growth factors, usually resulting in incomplete inhibition or even stimulation of STAT5, AKT, RAC2, and MAPK... As described before AKT and RAC2 were inhibited in BCR-ABL1 –positive 32Dcl3 cells either by expression of specific dominant negative mutants AKT(K179M) and RAC(T17N), respectively, and in LinCD34 CML-CP cells by AKT activation inhibitor perifosine and RAC inhibitor NSC23766, respectively (Figure 1a)... Inhibition of AKT does not affect the activity of RAC and inhibition of RAC did not affect AKT activity clearly indicating that their activation status does not depend on each other... In the presence of growth factors AKT(K179M) mutant and perifosine diminished ROS levels in annexin V-negative living BCR-ABL1 -32Dcl3 cells and LinCD34 cells, respectively (Figure 1b, c)... Perifosine effectively downregulated ROS in LinCD34 CML-CP cells in the G0/G1, S and G2/M cell cycle phases (Figure 1d), which was associated with reduction of oxidative DNA lesions, 8-oxoG and DSBs (Figure 1e, f)... Finally, inhibition of AKT either by AKT(K179M) mutant or perifosine resulted in reduction of accumulation of TKIR clones in BCR-ABL1 -32Dcl3 cells (Figure 1g, h)... To determine if AKT is responsible for overproduction of ROS in imatinib-treated LSCs and LPCs, LinCD34 CML-CP cells were incubated with imatinib in the presence of growth factors and ABL1 and AKT activation and ROS levels were measured... Since accumulation of DNA lesions such as 8-oxoG and DSBs directly depends on ROS levels in LinCD34CD38 LSCs and LinCD34CD38 LPCs, we postulate that AKT kinase regulates oxidative DNA damage in LPCs, but not in LSCs... In conclusion, we postulate that in imatinib-treated CML-CP patients AKT serine/threonine kinase plays a prominent role in accumulation of TKIR clones emerging from LinCD34CD38 LPCs, but probably not form LinCD34CD38 LSCs... Although AKT remained active in imatinib-treated LinCD34CD38 LPCs and LinCD34CD38 LSCs, intrinsic differences between leukemic progenitor and stem cells may contribute to the selective AKT effect in LPCs.

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AKT elevated ROS in imatinib-treated LPCs but not in LSCs(a) Lin−CD34+cells from normal donor (N) and from CML-CP patient were untreated (CML) or treated with 1 μM imatinib (CML+IM) in the presence of growth factors. Total tyrosine phosphorylated proteins (P-Tyr), AKT phosphorylated on serine 473 (pAKT) and total AKT protein were detected by Western analysis. (b) Lin−CD34+ cells from 3–6 CML-CP patients were untreated (black bars) and treated with 1 μM imatinib (light grey bars), 10 μM perifosine (dark grey bars), or 1 μM imatinib + 10 μM perifosine (white bars) in the presence of growth factors. Phospho-ABL1 (pABL1; phospho-Y245), phospho-AKT (pAKT; phospho-T308) and ROS (DCFDA) were detected in annexin V-negative Lin−CD34+CD38− LSCs and Lin−CD34+CD38+ LPCs as described before 5,8. *p<0.05 in comparison to untreated cells.
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Figure 2: AKT elevated ROS in imatinib-treated LPCs but not in LSCs(a) Lin−CD34+cells from normal donor (N) and from CML-CP patient were untreated (CML) or treated with 1 μM imatinib (CML+IM) in the presence of growth factors. Total tyrosine phosphorylated proteins (P-Tyr), AKT phosphorylated on serine 473 (pAKT) and total AKT protein were detected by Western analysis. (b) Lin−CD34+ cells from 3–6 CML-CP patients were untreated (black bars) and treated with 1 μM imatinib (light grey bars), 10 μM perifosine (dark grey bars), or 1 μM imatinib + 10 μM perifosine (white bars) in the presence of growth factors. Phospho-ABL1 (pABL1; phospho-Y245), phospho-AKT (pAKT; phospho-T308) and ROS (DCFDA) were detected in annexin V-negative Lin−CD34+CD38− LSCs and Lin−CD34+CD38+ LPCs as described before 5,8. *p<0.05 in comparison to untreated cells.

Mentions: To determine if AKT is responsible for overproduction of ROS in imatinib-treated LSCs and LPCs, Lin−CD34+ CML-CP cells were incubated with imatinib in the presence of growth factors and ABL1 and AKT activation and ROS levels were measured. Despite inhibition of ABL1 kinase activity, AKT activation was mostly preserved in imatinib-treated Lin−CD34+ cells (Figure 2a) in concordance with other report 7. As expected from previous studies with PI3K inhibitors LY294002 or wortmannin and BCR-ABL1 kinase inhibitor imatinib, 13 targeting AKT, a downstream effector of PI3K, with perifosine enhanced the inhibitory effect of imatinib on clonogenic growth of Lin−CD34+ CML-CP cells (Supplemental Figure 1). This effect may depend on perifosine-mediated elevation of ROS (including mitochondrial ROS) in a sub-population of annexin V-positive leukemia cells committed to apoptosis (Supplemental Figure 2) in concordance with other report 12.


AKT-induced reactive oxygen species generate imatinib-resistant clones emerging from chronic myeloid leukemia progenitor cells.

Nieborowska-Skorska M, Flis S, Skorski T - Leukemia (2014)

AKT elevated ROS in imatinib-treated LPCs but not in LSCs(a) Lin−CD34+cells from normal donor (N) and from CML-CP patient were untreated (CML) or treated with 1 μM imatinib (CML+IM) in the presence of growth factors. Total tyrosine phosphorylated proteins (P-Tyr), AKT phosphorylated on serine 473 (pAKT) and total AKT protein were detected by Western analysis. (b) Lin−CD34+ cells from 3–6 CML-CP patients were untreated (black bars) and treated with 1 μM imatinib (light grey bars), 10 μM perifosine (dark grey bars), or 1 μM imatinib + 10 μM perifosine (white bars) in the presence of growth factors. Phospho-ABL1 (pABL1; phospho-Y245), phospho-AKT (pAKT; phospho-T308) and ROS (DCFDA) were detected in annexin V-negative Lin−CD34+CD38− LSCs and Lin−CD34+CD38+ LPCs as described before 5,8. *p<0.05 in comparison to untreated cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4262587&req=5

Figure 2: AKT elevated ROS in imatinib-treated LPCs but not in LSCs(a) Lin−CD34+cells from normal donor (N) and from CML-CP patient were untreated (CML) or treated with 1 μM imatinib (CML+IM) in the presence of growth factors. Total tyrosine phosphorylated proteins (P-Tyr), AKT phosphorylated on serine 473 (pAKT) and total AKT protein were detected by Western analysis. (b) Lin−CD34+ cells from 3–6 CML-CP patients were untreated (black bars) and treated with 1 μM imatinib (light grey bars), 10 μM perifosine (dark grey bars), or 1 μM imatinib + 10 μM perifosine (white bars) in the presence of growth factors. Phospho-ABL1 (pABL1; phospho-Y245), phospho-AKT (pAKT; phospho-T308) and ROS (DCFDA) were detected in annexin V-negative Lin−CD34+CD38− LSCs and Lin−CD34+CD38+ LPCs as described before 5,8. *p<0.05 in comparison to untreated cells.
Mentions: To determine if AKT is responsible for overproduction of ROS in imatinib-treated LSCs and LPCs, Lin−CD34+ CML-CP cells were incubated with imatinib in the presence of growth factors and ABL1 and AKT activation and ROS levels were measured. Despite inhibition of ABL1 kinase activity, AKT activation was mostly preserved in imatinib-treated Lin−CD34+ cells (Figure 2a) in concordance with other report 7. As expected from previous studies with PI3K inhibitors LY294002 or wortmannin and BCR-ABL1 kinase inhibitor imatinib, 13 targeting AKT, a downstream effector of PI3K, with perifosine enhanced the inhibitory effect of imatinib on clonogenic growth of Lin−CD34+ CML-CP cells (Supplemental Figure 1). This effect may depend on perifosine-mediated elevation of ROS (including mitochondrial ROS) in a sub-population of annexin V-positive leukemia cells committed to apoptosis (Supplemental Figure 2) in concordance with other report 12.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, and Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

BCR-ABL1 fusion tyrosine kinase transforms hematopoietic stem cells (HSCs) and cause chronic myeloid leukemia in chronic phase (CML-CP), which is a stem cell (leukemia stem cell=LSC) -derived but a progenitor (leukemia progenitor cell=LPC)-driven disease... There are several possible explanations for persistent elevated levels of ROS and oxidative DNA damage in CML-CP cells surviving TKI treatment... For example, the effect of TKIs on BCR-ABL1 kinase-induced signaling pathways stimulating ROS production may be obscured by growth factors, usually resulting in incomplete inhibition or even stimulation of STAT5, AKT, RAC2, and MAPK... As described before AKT and RAC2 were inhibited in BCR-ABL1 –positive 32Dcl3 cells either by expression of specific dominant negative mutants AKT(K179M) and RAC(T17N), respectively, and in LinCD34 CML-CP cells by AKT activation inhibitor perifosine and RAC inhibitor NSC23766, respectively (Figure 1a)... Inhibition of AKT does not affect the activity of RAC and inhibition of RAC did not affect AKT activity clearly indicating that their activation status does not depend on each other... In the presence of growth factors AKT(K179M) mutant and perifosine diminished ROS levels in annexin V-negative living BCR-ABL1 -32Dcl3 cells and LinCD34 cells, respectively (Figure 1b, c)... Perifosine effectively downregulated ROS in LinCD34 CML-CP cells in the G0/G1, S and G2/M cell cycle phases (Figure 1d), which was associated with reduction of oxidative DNA lesions, 8-oxoG and DSBs (Figure 1e, f)... Finally, inhibition of AKT either by AKT(K179M) mutant or perifosine resulted in reduction of accumulation of TKIR clones in BCR-ABL1 -32Dcl3 cells (Figure 1g, h)... To determine if AKT is responsible for overproduction of ROS in imatinib-treated LSCs and LPCs, LinCD34 CML-CP cells were incubated with imatinib in the presence of growth factors and ABL1 and AKT activation and ROS levels were measured... Since accumulation of DNA lesions such as 8-oxoG and DSBs directly depends on ROS levels in LinCD34CD38 LSCs and LinCD34CD38 LPCs, we postulate that AKT kinase regulates oxidative DNA damage in LPCs, but not in LSCs... In conclusion, we postulate that in imatinib-treated CML-CP patients AKT serine/threonine kinase plays a prominent role in accumulation of TKIR clones emerging from LinCD34CD38 LPCs, but probably not form LinCD34CD38 LSCs... Although AKT remained active in imatinib-treated LinCD34CD38 LPCs and LinCD34CD38 LSCs, intrinsic differences between leukemic progenitor and stem cells may contribute to the selective AKT effect in LPCs.

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