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SIRT1 deacetylase promotes acquisition of genetic mutations for drug resistance in CML cells.

Wang Z, Yuan H, Roth M, Stark JM, Bhatia R, Chen WY - Oncogene (2012)

Bottom Line: The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations.SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin.These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA.

ABSTRACT
BCR-ABL transforms bone marrow progenitor cells and promotes genome instability, leading to development of chronic myelogenous leukemia (CML). The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations. Mechanisms for acquisition of BCR-ABL mutations are not fully understood. Using a novel culture model of CML acquired resistance, we show that inhibition of SIRT1 deacetylase by small molecule inhibitors or gene knockdown blocks acquisition of BCR-ABL mutations and relapse of CML cells on tyrosine kinase inhibitors. SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin. Although SIRT1 can enhance cellular DNA damage response, it alters functions of DNA repair machineries in CML cells and stimulates activity of error-prone DNA damage repair, in association with acquisition of genetic mutations. These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

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SIRT1 regulated NHEJ repair for CML acquired resistance(a) Acetylation of Ku70 after SIRT1 knockdown in KCL-22 cells. Ku70 was immunoprecipitated from total cell lysate of mock or SIRT1 knockdown KCL-22 cells. Western blots were probed with anti-acetylated lysine antibody followed by Ku70 antibody. (b) Ku70 acetylation after exogenous expression of wild type or H363Y mutant SIRT1 was analyzed as in a. HA antibody was used for immunoprecipitation control. The numbers were densitometry results of acetylated Ku70 that was normalized to total Ku70 and compared to the vector control. (c) Moderate Ku70 knockdown in KCL-22 cells using a low MOI of 0.5. Cells were enriched by puromycin selection. (d) No change of KCL-22 cell apoptosis was observed after moderate Ku70 knockdown in the absence or presence of 2.5 μM imatinib. Cells were selected by puromycin for 4 days followed by recovery for 5 days in normal medium before analysis. (e) Moderate Ku70 knockdown blocked KCL-22 cells relapse from 5μM imatinib. (f) Left, moderate Ku70 knockdown eliminated BCR-ABL mutant soft agar colony formation on 5 μM imatinib. One million cells per well were seeded in triplicate in 6-well plates with imatinib. Right, plating control with 500 cells seeded per well.
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Figure 4: SIRT1 regulated NHEJ repair for CML acquired resistance(a) Acetylation of Ku70 after SIRT1 knockdown in KCL-22 cells. Ku70 was immunoprecipitated from total cell lysate of mock or SIRT1 knockdown KCL-22 cells. Western blots were probed with anti-acetylated lysine antibody followed by Ku70 antibody. (b) Ku70 acetylation after exogenous expression of wild type or H363Y mutant SIRT1 was analyzed as in a. HA antibody was used for immunoprecipitation control. The numbers were densitometry results of acetylated Ku70 that was normalized to total Ku70 and compared to the vector control. (c) Moderate Ku70 knockdown in KCL-22 cells using a low MOI of 0.5. Cells were enriched by puromycin selection. (d) No change of KCL-22 cell apoptosis was observed after moderate Ku70 knockdown in the absence or presence of 2.5 μM imatinib. Cells were selected by puromycin for 4 days followed by recovery for 5 days in normal medium before analysis. (e) Moderate Ku70 knockdown blocked KCL-22 cells relapse from 5μM imatinib. (f) Left, moderate Ku70 knockdown eliminated BCR-ABL mutant soft agar colony formation on 5 μM imatinib. One million cells per well were seeded in triplicate in 6-well plates with imatinib. Right, plating control with 500 cells seeded per well.

Mentions: BCR-ABL alters functions of both homologous recombination (HR) and error-prone, non-homologous end joining (NHEJ) DNA repair machineries. BCR-ABL increases expression of RAD51 that abnormally stimulates HR repair and CML drug resistance.40 BCR-ABL promotes DNA damage repair but increases genetic mutations in association with the compromised fidelity of both HR and NHEJ repairs.41 We have shown that acquisition of BCR-ABL mutations in KCL-22 cells depends on BCR-ABL expression,12 and that BCR-ABL activates SIRT1 expression.31 To determine if SIRT1 may act as a key downstream effecter of BCR-ABL to regulate repair machineries for CML acquired resistance, we first examined the roles of Ku70 that is a key component of NHEJ repair.42 Ku70 is deacetylated and activated by SIRT1, which promotes both cell survival under stress 25 and DNA damage repair.18 We found that Ku70 acetylation levels in CML cells was increased in proportion to the levels of SIRT1 knockdown (Figure 4a), which may inactivate Ku70 functions.25,43 Similarly, over-expression of H363Y deacetylase mutant SIRT1 increased Ku70 acetylation, whereas over-expression of wild type SIRT1 reduced Ku70 acetylation (Figure 4b).


SIRT1 deacetylase promotes acquisition of genetic mutations for drug resistance in CML cells.

Wang Z, Yuan H, Roth M, Stark JM, Bhatia R, Chen WY - Oncogene (2012)

SIRT1 regulated NHEJ repair for CML acquired resistance(a) Acetylation of Ku70 after SIRT1 knockdown in KCL-22 cells. Ku70 was immunoprecipitated from total cell lysate of mock or SIRT1 knockdown KCL-22 cells. Western blots were probed with anti-acetylated lysine antibody followed by Ku70 antibody. (b) Ku70 acetylation after exogenous expression of wild type or H363Y mutant SIRT1 was analyzed as in a. HA antibody was used for immunoprecipitation control. The numbers were densitometry results of acetylated Ku70 that was normalized to total Ku70 and compared to the vector control. (c) Moderate Ku70 knockdown in KCL-22 cells using a low MOI of 0.5. Cells were enriched by puromycin selection. (d) No change of KCL-22 cell apoptosis was observed after moderate Ku70 knockdown in the absence or presence of 2.5 μM imatinib. Cells were selected by puromycin for 4 days followed by recovery for 5 days in normal medium before analysis. (e) Moderate Ku70 knockdown blocked KCL-22 cells relapse from 5μM imatinib. (f) Left, moderate Ku70 knockdown eliminated BCR-ABL mutant soft agar colony formation on 5 μM imatinib. One million cells per well were seeded in triplicate in 6-well plates with imatinib. Right, plating control with 500 cells seeded per well.
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Related In: Results  -  Collection

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Figure 4: SIRT1 regulated NHEJ repair for CML acquired resistance(a) Acetylation of Ku70 after SIRT1 knockdown in KCL-22 cells. Ku70 was immunoprecipitated from total cell lysate of mock or SIRT1 knockdown KCL-22 cells. Western blots were probed with anti-acetylated lysine antibody followed by Ku70 antibody. (b) Ku70 acetylation after exogenous expression of wild type or H363Y mutant SIRT1 was analyzed as in a. HA antibody was used for immunoprecipitation control. The numbers were densitometry results of acetylated Ku70 that was normalized to total Ku70 and compared to the vector control. (c) Moderate Ku70 knockdown in KCL-22 cells using a low MOI of 0.5. Cells were enriched by puromycin selection. (d) No change of KCL-22 cell apoptosis was observed after moderate Ku70 knockdown in the absence or presence of 2.5 μM imatinib. Cells were selected by puromycin for 4 days followed by recovery for 5 days in normal medium before analysis. (e) Moderate Ku70 knockdown blocked KCL-22 cells relapse from 5μM imatinib. (f) Left, moderate Ku70 knockdown eliminated BCR-ABL mutant soft agar colony formation on 5 μM imatinib. One million cells per well were seeded in triplicate in 6-well plates with imatinib. Right, plating control with 500 cells seeded per well.
Mentions: BCR-ABL alters functions of both homologous recombination (HR) and error-prone, non-homologous end joining (NHEJ) DNA repair machineries. BCR-ABL increases expression of RAD51 that abnormally stimulates HR repair and CML drug resistance.40 BCR-ABL promotes DNA damage repair but increases genetic mutations in association with the compromised fidelity of both HR and NHEJ repairs.41 We have shown that acquisition of BCR-ABL mutations in KCL-22 cells depends on BCR-ABL expression,12 and that BCR-ABL activates SIRT1 expression.31 To determine if SIRT1 may act as a key downstream effecter of BCR-ABL to regulate repair machineries for CML acquired resistance, we first examined the roles of Ku70 that is a key component of NHEJ repair.42 Ku70 is deacetylated and activated by SIRT1, which promotes both cell survival under stress 25 and DNA damage repair.18 We found that Ku70 acetylation levels in CML cells was increased in proportion to the levels of SIRT1 knockdown (Figure 4a), which may inactivate Ku70 functions.25,43 Similarly, over-expression of H363Y deacetylase mutant SIRT1 increased Ku70 acetylation, whereas over-expression of wild type SIRT1 reduced Ku70 acetylation (Figure 4b).

Bottom Line: The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations.SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin.These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, Beckman Research Institute, City of Hope, Duarte, CA, USA.

ABSTRACT
BCR-ABL transforms bone marrow progenitor cells and promotes genome instability, leading to development of chronic myelogenous leukemia (CML). The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop because of BCR-ABL mutations. Mechanisms for acquisition of BCR-ABL mutations are not fully understood. Using a novel culture model of CML acquired resistance, we show that inhibition of SIRT1 deacetylase by small molecule inhibitors or gene knockdown blocks acquisition of BCR-ABL mutations and relapse of CML cells on tyrosine kinase inhibitors. SIRT1 knockdown also suppresses de novo genetic mutations of hypoxanthine phosphoribosyl transferase gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin. Although SIRT1 can enhance cellular DNA damage response, it alters functions of DNA repair machineries in CML cells and stimulates activity of error-prone DNA damage repair, in association with acquisition of genetic mutations. These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.

Show MeSH
Related in: MedlinePlus