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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 knockdown inhibited camptothecin-induced HPRT mutations in cancer cells(a) Effects of SIRT1 knockdown on de novo HPRT mutations in CML cells. Top: HAT-selected SCR or shSIRT1 knockdown KCL-22 cells were treated with 0.5 μM CPT. After recovery, cells were seeded at 1 million/plate for clonogenic assay with 6-thiaguinine selection for HPRT mutations. HAT treated cells were also grown in normal medium without CPT for one month, and then used for clonogenic assay to detect newly occurring spontaneous mutations. Bottom: plating control with 500 cells/plate seeded without 6-thiaguinine. (b) Effects of SIRT1 knockdown on de novo HPRT mutations in PC3 cells. Top panel, SIRT1 knockdown using shSIRT1-1. Middle panel, five million HAT-selected and CPT treated SCR or shSIRT1 knockdown PC3 cells per plate were analyzed for 6-thioguanine resistance. Bottom panel, plating control with 200 cells/well seeded without drug. (c) HAT-selected PC3 cells were treated DMSO, 2.5 μM tenovin-6, 25 μM sirtinol or 15 mM NAM for 6h, followed by exposure to 0.5μM CPT for 1h. Cells were then cultured with DMSO, tenovin-6, sirtinol or NAM, respectively, for two days, followed by recovery without drugs for two weeks. HPRT mutations (left) and plating control (right) were analyzed as in b. (d) Flow cytometry analysis of γH2AX in SCR or shSIRT1 knockdown KCL-22 cells with or without CPT treatment. (e, f) Western blot analysis of γH2AX in KCL-22 (e) and PC3 (f) cells after 24h treatment with drugs indicated.
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Figure 3: SIRT1 knockdown inhibited camptothecin-induced HPRT mutations in cancer cells(a) Effects of SIRT1 knockdown on de novo HPRT mutations in CML cells. Top: HAT-selected SCR or shSIRT1 knockdown KCL-22 cells were treated with 0.5 μM CPT. After recovery, cells were seeded at 1 million/plate for clonogenic assay with 6-thiaguinine selection for HPRT mutations. HAT treated cells were also grown in normal medium without CPT for one month, and then used for clonogenic assay to detect newly occurring spontaneous mutations. Bottom: plating control with 500 cells/plate seeded without 6-thiaguinine. (b) Effects of SIRT1 knockdown on de novo HPRT mutations in PC3 cells. Top panel, SIRT1 knockdown using shSIRT1-1. Middle panel, five million HAT-selected and CPT treated SCR or shSIRT1 knockdown PC3 cells per plate were analyzed for 6-thioguanine resistance. Bottom panel, plating control with 200 cells/well seeded without drug. (c) HAT-selected PC3 cells were treated DMSO, 2.5 μM tenovin-6, 25 μM sirtinol or 15 mM NAM for 6h, followed by exposure to 0.5μM CPT for 1h. Cells were then cultured with DMSO, tenovin-6, sirtinol or NAM, respectively, for two days, followed by recovery without drugs for two weeks. HPRT mutations (left) and plating control (right) were analyzed as in b. (d) Flow cytometry analysis of γH2AX in SCR or shSIRT1 knockdown KCL-22 cells with or without CPT treatment. (e, f) Western blot analysis of γH2AX in KCL-22 (e) and PC3 (f) cells after 24h treatment with drugs indicated.

Mentions: We have shown that acquisition of BCR-ABL mutations under imatinib therapeutic stress is distinct from formation of spontaneous HPRT mutations that are not required for cell survival.12 To determine if SIRT1 is involved in regulating mutations under a different stress condition, we examined effects of SIRT1 knockdown on camptothecin (CPT)-induced de novo HPRT mutations. CPT is a chemotherapeutic agent that induces DNA damage by inhibiting topoisomerase I.36 Mock or SIRT1 knockdown KCL-22 cells were cultured with HAT (hypoxanthine aminopterin thymidine) to remove cells bearing pre-existing HPRT mutations. HAT-selected KCL-22 cells were then treated with CPT to induce DNA damage. We found that SIRT1 knockdown using shSIRT1-1 or shSIRT1-2 vector drastically suppressed CPT-induced de novo HPRT mutations in KCL-22 cells (Figure 3a). SIRT1 knockdown also reduced, but to a lesser extent, spontaneous HPRT mutations when these cells were allowed to grow continuously for one month in culture (Figure 3a). Similarly, SIRT1 knockdown robustly suppressed CPT-induced de novo HPRT mutations in prostate cancer PC3 cells (Figure 3b), in which SIRT1 is over-expressed.37,38 Furthermore, we found that transient exposure of PC3 cells to tenovin-6, sirtinol or nicotinamide for two days was sufficient to reduce CPT-induced de novo HPRT mutations (Figure 3c). These results indicate that SIRT1 promotes HPRT mutations under acute CPT-induced stress in a similar manner to its promotion of BCR-ABL mutations under imatinib therapeutic stress, and that SIRT1-mediated mutagenesis is independent of cancer cell types.


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 knockdown inhibited camptothecin-induced HPRT mutations in cancer cells(a) Effects of SIRT1 knockdown on de novo HPRT mutations in CML cells. Top: HAT-selected SCR or shSIRT1 knockdown KCL-22 cells were treated with 0.5 μM CPT. After recovery, cells were seeded at 1 million/plate for clonogenic assay with 6-thiaguinine selection for HPRT mutations. HAT treated cells were also grown in normal medium without CPT for one month, and then used for clonogenic assay to detect newly occurring spontaneous mutations. Bottom: plating control with 500 cells/plate seeded without 6-thiaguinine. (b) Effects of SIRT1 knockdown on de novo HPRT mutations in PC3 cells. Top panel, SIRT1 knockdown using shSIRT1-1. Middle panel, five million HAT-selected and CPT treated SCR or shSIRT1 knockdown PC3 cells per plate were analyzed for 6-thioguanine resistance. Bottom panel, plating control with 200 cells/well seeded without drug. (c) HAT-selected PC3 cells were treated DMSO, 2.5 μM tenovin-6, 25 μM sirtinol or 15 mM NAM for 6h, followed by exposure to 0.5μM CPT for 1h. Cells were then cultured with DMSO, tenovin-6, sirtinol or NAM, respectively, for two days, followed by recovery without drugs for two weeks. HPRT mutations (left) and plating control (right) were analyzed as in b. (d) Flow cytometry analysis of γH2AX in SCR or shSIRT1 knockdown KCL-22 cells with or without CPT treatment. (e, f) Western blot analysis of γH2AX in KCL-22 (e) and PC3 (f) cells after 24h treatment with drugs indicated.
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Figure 3: SIRT1 knockdown inhibited camptothecin-induced HPRT mutations in cancer cells(a) Effects of SIRT1 knockdown on de novo HPRT mutations in CML cells. Top: HAT-selected SCR or shSIRT1 knockdown KCL-22 cells were treated with 0.5 μM CPT. After recovery, cells were seeded at 1 million/plate for clonogenic assay with 6-thiaguinine selection for HPRT mutations. HAT treated cells were also grown in normal medium without CPT for one month, and then used for clonogenic assay to detect newly occurring spontaneous mutations. Bottom: plating control with 500 cells/plate seeded without 6-thiaguinine. (b) Effects of SIRT1 knockdown on de novo HPRT mutations in PC3 cells. Top panel, SIRT1 knockdown using shSIRT1-1. Middle panel, five million HAT-selected and CPT treated SCR or shSIRT1 knockdown PC3 cells per plate were analyzed for 6-thioguanine resistance. Bottom panel, plating control with 200 cells/well seeded without drug. (c) HAT-selected PC3 cells were treated DMSO, 2.5 μM tenovin-6, 25 μM sirtinol or 15 mM NAM for 6h, followed by exposure to 0.5μM CPT for 1h. Cells were then cultured with DMSO, tenovin-6, sirtinol or NAM, respectively, for two days, followed by recovery without drugs for two weeks. HPRT mutations (left) and plating control (right) were analyzed as in b. (d) Flow cytometry analysis of γH2AX in SCR or shSIRT1 knockdown KCL-22 cells with or without CPT treatment. (e, f) Western blot analysis of γH2AX in KCL-22 (e) and PC3 (f) cells after 24h treatment with drugs indicated.
Mentions: We have shown that acquisition of BCR-ABL mutations under imatinib therapeutic stress is distinct from formation of spontaneous HPRT mutations that are not required for cell survival.12 To determine if SIRT1 is involved in regulating mutations under a different stress condition, we examined effects of SIRT1 knockdown on camptothecin (CPT)-induced de novo HPRT mutations. CPT is a chemotherapeutic agent that induces DNA damage by inhibiting topoisomerase I.36 Mock or SIRT1 knockdown KCL-22 cells were cultured with HAT (hypoxanthine aminopterin thymidine) to remove cells bearing pre-existing HPRT mutations. HAT-selected KCL-22 cells were then treated with CPT to induce DNA damage. We found that SIRT1 knockdown using shSIRT1-1 or shSIRT1-2 vector drastically suppressed CPT-induced de novo HPRT mutations in KCL-22 cells (Figure 3a). SIRT1 knockdown also reduced, but to a lesser extent, spontaneous HPRT mutations when these cells were allowed to grow continuously for one month in culture (Figure 3a). Similarly, SIRT1 knockdown robustly suppressed CPT-induced de novo HPRT mutations in prostate cancer PC3 cells (Figure 3b), in which SIRT1 is over-expressed.37,38 Furthermore, we found that transient exposure of PC3 cells to tenovin-6, sirtinol or nicotinamide for two days was sufficient to reduce CPT-induced de novo HPRT mutations (Figure 3c). These results indicate that SIRT1 promotes HPRT mutations under acute CPT-induced stress in a similar manner to its promotion of BCR-ABL mutations under imatinib therapeutic stress, and that SIRT1-mediated mutagenesis is independent of cancer cell types.

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