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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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RAC-independent AKT-induced ROS caused oxidative DNA damage resulting in accumulation of imatinib-resistant clones(a) BCR-ABL1-transformed 32Dcl3 cells transfected with AKT(K179M) and Rac(T17N) dominant-negative mutants or empty plasmids (E) 8,10, and Lin−CD34+ CML-CP cells treated with 10 μM AKT inhibitor perifosine, 25 μM NSC23766 or diluent (C) 8,11 were tested for activation of AKT and RAC. Western analyses detect AKT phosphorylated on serine 473 (AKT-pS473) and Rac bound to GTP as described before 8,15; total levels of AKT and RAC were also determined as loading controls. (b) ROS were measured with DCFDA in BCR-ABL1 -32Dcl3 cells transfected with empty plasmid (black bar) and AKT(K179M) mutant (grey bar). (c–f) Lin−CD34+ CML-CP cells were left untreated (black bars) or incubated with 10 μM perifosine (grey bars) in the presence of growth factors.8 (c) ROS were measured with DCFDA in annexin V-negative cells as described before 5,8. (d) ROS were detected by DCFDA in G1, S and G2/M phase determined by Vybrant DyeCycle Orange live cell staining (Invitrogen/Molecular Probes) as described before. 8 ROS measurements are at the left sides, and percentages of cells in cell cycle phases are indicated at the bottom. (e) 8-oxoG and (f) γ-H2AX detected by specific immunofluorescence as described before.8 (g, h) BCR-ABL1 –positive 32Dcl3 cells transfected with AKT(K179M) mutant or empty plasmid (g) and untreated (Control) or treated with 1 μM perifosine (h) were cultured for 10 weeks. The frequency of TKI resistant (TKIR) clones was determined as described before.8 *p<0.05.
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Figure 1: RAC-independent AKT-induced ROS caused oxidative DNA damage resulting in accumulation of imatinib-resistant clones(a) BCR-ABL1-transformed 32Dcl3 cells transfected with AKT(K179M) and Rac(T17N) dominant-negative mutants or empty plasmids (E) 8,10, and Lin−CD34+ CML-CP cells treated with 10 μM AKT inhibitor perifosine, 25 μM NSC23766 or diluent (C) 8,11 were tested for activation of AKT and RAC. Western analyses detect AKT phosphorylated on serine 473 (AKT-pS473) and Rac bound to GTP as described before 8,15; total levels of AKT and RAC were also determined as loading controls. (b) ROS were measured with DCFDA in BCR-ABL1 -32Dcl3 cells transfected with empty plasmid (black bar) and AKT(K179M) mutant (grey bar). (c–f) Lin−CD34+ CML-CP cells were left untreated (black bars) or incubated with 10 μM perifosine (grey bars) in the presence of growth factors.8 (c) ROS were measured with DCFDA in annexin V-negative cells as described before 5,8. (d) ROS were detected by DCFDA in G1, S and G2/M phase determined by Vybrant DyeCycle Orange live cell staining (Invitrogen/Molecular Probes) as described before. 8 ROS measurements are at the left sides, and percentages of cells in cell cycle phases are indicated at the bottom. (e) 8-oxoG and (f) γ-H2AX detected by specific immunofluorescence as described before.8 (g, h) BCR-ABL1 –positive 32Dcl3 cells transfected with AKT(K179M) mutant or empty plasmid (g) and untreated (Control) or treated with 1 μM perifosine (h) were cultured for 10 weeks. The frequency of TKI resistant (TKIR) clones was determined as described before.8 *p<0.05.

Mentions: 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) 8,10, respectively, and in Lin−CD34+ CML-CP cells by AKT activation inhibitor perifosine 11 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.


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)

RAC-independent AKT-induced ROS caused oxidative DNA damage resulting in accumulation of imatinib-resistant clones(a) BCR-ABL1-transformed 32Dcl3 cells transfected with AKT(K179M) and Rac(T17N) dominant-negative mutants or empty plasmids (E) 8,10, and Lin−CD34+ CML-CP cells treated with 10 μM AKT inhibitor perifosine, 25 μM NSC23766 or diluent (C) 8,11 were tested for activation of AKT and RAC. Western analyses detect AKT phosphorylated on serine 473 (AKT-pS473) and Rac bound to GTP as described before 8,15; total levels of AKT and RAC were also determined as loading controls. (b) ROS were measured with DCFDA in BCR-ABL1 -32Dcl3 cells transfected with empty plasmid (black bar) and AKT(K179M) mutant (grey bar). (c–f) Lin−CD34+ CML-CP cells were left untreated (black bars) or incubated with 10 μM perifosine (grey bars) in the presence of growth factors.8 (c) ROS were measured with DCFDA in annexin V-negative cells as described before 5,8. (d) ROS were detected by DCFDA in G1, S and G2/M phase determined by Vybrant DyeCycle Orange live cell staining (Invitrogen/Molecular Probes) as described before. 8 ROS measurements are at the left sides, and percentages of cells in cell cycle phases are indicated at the bottom. (e) 8-oxoG and (f) γ-H2AX detected by specific immunofluorescence as described before.8 (g, h) BCR-ABL1 –positive 32Dcl3 cells transfected with AKT(K179M) mutant or empty plasmid (g) and untreated (Control) or treated with 1 μM perifosine (h) were cultured for 10 weeks. The frequency of TKI resistant (TKIR) clones was determined as described before.8 *p<0.05.
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Related In: Results  -  Collection

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Figure 1: RAC-independent AKT-induced ROS caused oxidative DNA damage resulting in accumulation of imatinib-resistant clones(a) BCR-ABL1-transformed 32Dcl3 cells transfected with AKT(K179M) and Rac(T17N) dominant-negative mutants or empty plasmids (E) 8,10, and Lin−CD34+ CML-CP cells treated with 10 μM AKT inhibitor perifosine, 25 μM NSC23766 or diluent (C) 8,11 were tested for activation of AKT and RAC. Western analyses detect AKT phosphorylated on serine 473 (AKT-pS473) and Rac bound to GTP as described before 8,15; total levels of AKT and RAC were also determined as loading controls. (b) ROS were measured with DCFDA in BCR-ABL1 -32Dcl3 cells transfected with empty plasmid (black bar) and AKT(K179M) mutant (grey bar). (c–f) Lin−CD34+ CML-CP cells were left untreated (black bars) or incubated with 10 μM perifosine (grey bars) in the presence of growth factors.8 (c) ROS were measured with DCFDA in annexin V-negative cells as described before 5,8. (d) ROS were detected by DCFDA in G1, S and G2/M phase determined by Vybrant DyeCycle Orange live cell staining (Invitrogen/Molecular Probes) as described before. 8 ROS measurements are at the left sides, and percentages of cells in cell cycle phases are indicated at the bottom. (e) 8-oxoG and (f) γ-H2AX detected by specific immunofluorescence as described before.8 (g, h) BCR-ABL1 –positive 32Dcl3 cells transfected with AKT(K179M) mutant or empty plasmid (g) and untreated (Control) or treated with 1 μM perifosine (h) were cultured for 10 weeks. The frequency of TKI resistant (TKIR) clones was determined as described before.8 *p<0.05.
Mentions: 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) 8,10, respectively, and in Lin−CD34+ CML-CP cells by AKT activation inhibitor perifosine 11 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.

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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