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

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.

Figure 1. Pharmacological inhibition of SIRT1 blocked acquired resistance of CML cells on tyrosine kinase inhibitors

(a) KCL-22 cells were treated with 50 μM sirtinol or imatinib (STI) alone at the concentrations indicated or in combination of the two drugs. Cells for STI treatment alone all relapsed, and for simplicity, only the 2.5 μM curve was shown. (b) KCL-22 cells were treated with 15 mM nicotinamide (NAM) and 5 μM imatinib alone or in combination. (c) KCL-22 cells were treated with 1μM tenovin-6 and 2.5 μM imatinib alone or in combination. (d) KCL-22 cells were treated with 1μM trichostatin A (TSA) without or with STI at the concentrations indicated. (e) KCL-22 cell relapsed on 2.5 μM Nilotinib (Nil) with T315I mutation, but combination with tenovin-6 or sirtinol blocked relapse. (f) KCL-22 cell relapsed on 1 μM Dasatinib (Das) with T315I mutation. Combination with sirtinol or 5 μM tenovin-6 blocked relapse, and combination with 1 μM tenovin-6 delayed the relapse. (g) Left, sirtinol blocked clonal cells relapse on STI treatment. Right, relapse of clonal KCL-22 cells (L1, L7, Ag3 and Ag11) on STI plus TSA treatment.

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.

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.

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.

Figure 5. Influence of HR repair factors for CML acquired resistance

(a) NBS1 acetylation upon SIRT1 knockdown. NBS1 was immunoprecipitated and analyzed by Western blot with anti-acetylated lysine antibody, followed by NBS1 antibody. FLAG antibody was used for immunoprecipitation control. (b) NBS1 and RAD51 knockdown in KCL-22 cells. (c) Effects of RAD51 and NBS1 knockdown on KCL-22 cell growth. (d) NBS1 and RAD51 knockdown KCL-22 cells were enriched with puromycin selection and subjected to relapse assay on 2.5 μM imatinib. (e) RAD51 and NBS1 knockdown reduced BCR-ABL mutation by soft agar clonogenic assay. Left panel, resistant colonies with imatinib treatment. Right panel, plating control without imatinib.

Figure 6. SIRT1 altered functions of DNA damage repair in CML cells

(a) Flow cytometry analysis of HR and NHEJ repair after SIRT1 knockdown using stably integrated reporter constructs in KCL-22 cells. I-SceI was introduced by electroporation and DS-Red was used for transfection control. The repair rate was normalized to DS-Red. (b–d) NHEJ repair assay using inducible I-SceI expression. Gene knockdown was carried out in inducible I-SceI expressing EJ5-GFP KCL-22 cells. 48 hrs after gene knockdown, I-SceI expression was induced by doxycycline for 72 hrs, and GFP positive cells were analyzed by flow cytometry. b, effect of Ku70 and SIRT1 knockdown; c, effect of NBS1 knockdown; d, effect of RAD51 knockdown. ** indicates P<0.05.