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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 specific inhibition suppressed acquisition of BCR-ABL mutations(a) SIRT1 protein levels in KCL-22 cells after knockdown with 3 sets of SIRT1 shRNA. SCR, scrambled shRNA for control. (b) Effect of apoptosis induction in KCL-22 cells after SIRT1 knockdown using shSIRT1-2 or shSIRT1-3 with or without imatinib (STI). (c) Left, three days after shRNA transduction, one million SCR or shSIRT1 knockdown KCL-22 cells per plate were seeded in soft agar in triplicate with 5μM imatinib. At day 21, resistant colonies were scored. Right, plating control with 500 cells per well seeded in soft agar without imatinib. (d) Three days after shRNA transduction, one half million of SCR or shSIRT1 knockdown KCL-22 cells were treated with 5μM STI in triplicate and viable cells were counted at indicated days. (e) Over-expression of wild type or H363Y mutant SIRT1 in KCL-22 cells. The transduced cells were enriched by puromycin selection. (f) Left, Wild type or H363Y SIRT1 transduced cells were analyzed for BCR-ABL mutation frequency on imatinib by clonogenic assay as in c. Right, plating control as in c. pBabe was an empty vector control.
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Figure 2: SIRT1 specific inhibition suppressed acquisition of BCR-ABL mutations(a) SIRT1 protein levels in KCL-22 cells after knockdown with 3 sets of SIRT1 shRNA. SCR, scrambled shRNA for control. (b) Effect of apoptosis induction in KCL-22 cells after SIRT1 knockdown using shSIRT1-2 or shSIRT1-3 with or without imatinib (STI). (c) Left, three days after shRNA transduction, one million SCR or shSIRT1 knockdown KCL-22 cells per plate were seeded in soft agar in triplicate with 5μM imatinib. At day 21, resistant colonies were scored. Right, plating control with 500 cells per well seeded in soft agar without imatinib. (d) Three days after shRNA transduction, one half million of SCR or shSIRT1 knockdown KCL-22 cells were treated with 5μM STI in triplicate and viable cells were counted at indicated days. (e) Over-expression of wild type or H363Y mutant SIRT1 in KCL-22 cells. The transduced cells were enriched by puromycin selection. (f) Left, Wild type or H363Y SIRT1 transduced cells were analyzed for BCR-ABL mutation frequency on imatinib by clonogenic assay as in c. Right, plating control as in c. pBabe was an empty vector control.

Mentions: We designed three sets of SIRT1 shRNA to knock down the gene in KCL-22 cells (Figure 2a). The shSIRT1-3 vector exhibited the most robust SIRT1 knockdown that increased cell apoptosis in the absence or presence of imatinib, whereas shSIRT1-1 and shSIRT1-2 vectors did not (Figure 2a, b and not shown). By clonogenic assay, all three sets of SIRT1 shRNA suppressed formation of resistant colonies from KCL-22 cells treated with imatinib and the efficiency of suppression was proportional to the levels of gene knockdown, with shSIRT1-3 being the most effective (Figure 2c). Mutant colonies derived from scrambled shRNA, shSIRT1-1 or shSIRT1-2 knockdown were sequenced and confirmed to harbor T315I mutation (Supplementary Figure 3). In liquid culture, KCL-22 cell relapse on imatinib was delayed by shSIRT1-2 and completely blocked by shSIRT1-3 (Figure 2d). These findings are in line with the effect of SIRT1 inhibitors described above and suggest SIRT1 inhibition suppresses acquisition of BCR-ABL mutations and CML cell relapse from imatinib treatment.


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 specific inhibition suppressed acquisition of BCR-ABL mutations(a) SIRT1 protein levels in KCL-22 cells after knockdown with 3 sets of SIRT1 shRNA. SCR, scrambled shRNA for control. (b) Effect of apoptosis induction in KCL-22 cells after SIRT1 knockdown using shSIRT1-2 or shSIRT1-3 with or without imatinib (STI). (c) Left, three days after shRNA transduction, one million SCR or shSIRT1 knockdown KCL-22 cells per plate were seeded in soft agar in triplicate with 5μM imatinib. At day 21, resistant colonies were scored. Right, plating control with 500 cells per well seeded in soft agar without imatinib. (d) Three days after shRNA transduction, one half million of SCR or shSIRT1 knockdown KCL-22 cells were treated with 5μM STI in triplicate and viable cells were counted at indicated days. (e) Over-expression of wild type or H363Y mutant SIRT1 in KCL-22 cells. The transduced cells were enriched by puromycin selection. (f) Left, Wild type or H363Y SIRT1 transduced cells were analyzed for BCR-ABL mutation frequency on imatinib by clonogenic assay as in c. Right, plating control as in c. pBabe was an empty vector control.
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Related In: Results  -  Collection

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Figure 2: SIRT1 specific inhibition suppressed acquisition of BCR-ABL mutations(a) SIRT1 protein levels in KCL-22 cells after knockdown with 3 sets of SIRT1 shRNA. SCR, scrambled shRNA for control. (b) Effect of apoptosis induction in KCL-22 cells after SIRT1 knockdown using shSIRT1-2 or shSIRT1-3 with or without imatinib (STI). (c) Left, three days after shRNA transduction, one million SCR or shSIRT1 knockdown KCL-22 cells per plate were seeded in soft agar in triplicate with 5μM imatinib. At day 21, resistant colonies were scored. Right, plating control with 500 cells per well seeded in soft agar without imatinib. (d) Three days after shRNA transduction, one half million of SCR or shSIRT1 knockdown KCL-22 cells were treated with 5μM STI in triplicate and viable cells were counted at indicated days. (e) Over-expression of wild type or H363Y mutant SIRT1 in KCL-22 cells. The transduced cells were enriched by puromycin selection. (f) Left, Wild type or H363Y SIRT1 transduced cells were analyzed for BCR-ABL mutation frequency on imatinib by clonogenic assay as in c. Right, plating control as in c. pBabe was an empty vector control.
Mentions: We designed three sets of SIRT1 shRNA to knock down the gene in KCL-22 cells (Figure 2a). The shSIRT1-3 vector exhibited the most robust SIRT1 knockdown that increased cell apoptosis in the absence or presence of imatinib, whereas shSIRT1-1 and shSIRT1-2 vectors did not (Figure 2a, b and not shown). By clonogenic assay, all three sets of SIRT1 shRNA suppressed formation of resistant colonies from KCL-22 cells treated with imatinib and the efficiency of suppression was proportional to the levels of gene knockdown, with shSIRT1-3 being the most effective (Figure 2c). Mutant colonies derived from scrambled shRNA, shSIRT1-1 or shSIRT1-2 knockdown were sequenced and confirmed to harbor T315I mutation (Supplementary Figure 3). In liquid culture, KCL-22 cell relapse on imatinib was delayed by shSIRT1-2 and completely blocked by shSIRT1-3 (Figure 2d). These findings are in line with the effect of SIRT1 inhibitors described above and suggest SIRT1 inhibition suppresses acquisition of BCR-ABL mutations and CML cell relapse from imatinib treatment.

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