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Molecular Cancer Therapeutics
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Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity

Terzah M. Horton, Gaye Jenkins, Debananda Pati, Linna Zhang, M. Eileen Dolan, Albert Ribes-Zamora, Alison A. Bertuch, Susan M. Blaney, Shannon L. Delaney, Madhuri Hegde and Stacey L. Berg
Terzah M. Horton
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Gaye Jenkins
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Debananda Pati
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Linna Zhang
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M. Eileen Dolan
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Albert Ribes-Zamora
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Alison A. Bertuch
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Susan M. Blaney
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Shannon L. Delaney
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Madhuri Hegde
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Stacey L. Berg
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DOI: 10.1158/1535-7163.MCT-09-0142 Published August 2009
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  • Figure 1.
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    Figure 1.

    Model of the effects of PARP inhibition on TMZ activity. TMZ creates O6-methylguanine adducts (CH2) and N7- and N3-methyl adducts (CH3 on either adenine or guanine), which are efficiently removed by BER. Model shows guanine as a representative nucleotide. TMZ resistance results from either (a) MMR mutations (left), which allow DNA replication in the presence of dinucleotide (mG-T) mismatches, or (b) elevated MGMT activity (right), which removes the O6-methyl group from guanine. PARP inhibition blocks the repair of N3- and N7-methyl adducts, resulting in apoptosis during cell division.

  • Figure 2.
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    Figure 2.

    TMZ and ABT-888 growth inhibition in leukemia cell lines. MTT cytotoxicity assays were done with either TMZ (A) or ABT-888 (B) at concentrations ranging from 1 to 2,000 μmol/L. Percentage cell survival is plotted as a function of drug concentration. Leukemia cells include the MMR-proficient JM1 (•), U937 (□), THP1 (◊), HEL (▵), Raji (•, dotted line), Daudi (▪, dotted line), and HL-60 (○) and the MMR-deficient Jurkat (♦), HSB2 (▴), Reh (▪), and Molt4 (▾) cell lines. Open symbols, AML cell lines; closed symbols, ALL cell lines.

  • Figure 3.
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    Figure 3.

    ABT-888 potentiation of TMZ in MMR-proficient (A) or MMR-deficient (B) cell lines. A, AML cell lines THP1 (S1, left), HL-60 (S2, middle), and KG1 (S3, right) treated with TMZ alone (○) or TMZ in combination with 0.5 μmol/L (•) or 5 μmol/L (▪) of ABT-888. Similar results were obtained with ALL cell lines JM1 (S1; Fig. 2D), Raji, and Daudi (S2; data not shown). B, T-cell ALL cell lines HSB2 (S1), Molt4 (S2), and pre-B ALL cell line Reh (S3) treated as above. Cell lines have low (S1, U1), average (S2, U2), or elevated (S3, U3) MGMT activity. C, summary of PFs (IC50 TMZ/IC50 TMZ + ABT-888) in leukemia cell lines that are MMR proficient (MSI stable; S) and MMR deficient (MSI unstable; U) treated with TMZ combined with either 0.5 μmol/L (□) or 5 μmol/L (▪) of ABT-888. Coefficients of variation for the modeled IC50s varied from 8% to 29%.D and E, MMR-proficient pre-B ALL cell line JM1 (D) and MMR-deficient T-cell ALL cell line Molt4 (E) treated with TMZ alone (○) and TMZ in combination with 0.5 μmol/L ABT-888 (□), 1 μmol/L O6-BG (◊), or both 0.5 μmol/L ABT-888 and 1 μmol/L O6-BG (▪).

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    Figure 4.

    Effect of MGMT overexpression on ABT-888 potentiation of TMZ in MMR-proficient and MMR-deficient leukemia cell lines. A, confirmation of increased MGMT expression by Western blot and quantitation of increased MGMT expression by densitometry. The MMR-proficient AML cell line THP1 (B) and the MMR-deficient T-cell ALL leukemia cell line HSB2 (C) were treated with TMZ alone (○) or TMZ with either 0.5 μmol/L (•) or 5 μmol/L (▪) of ABT-888. Left, transfected with pCDNA vector (EV); right, transfected with pCDNA vector containing the full-length coding sequence for MGMT.

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    Figure 5.

    Mechanisms of PARP potentiation in leukemia cell lines. A, pretreatment PARP, Ku70, and phospho-H2AX protein expression in four MMR-deficient (U) and three MMR-proficient (S) leukemia cell lines. Expression is normalized to actin expression. B, PARP activity for the leukemia cell lines described in A untreated (white columns) or treated with either 0.5 μmol/L (gray columns) or 5 μmol/L (black columns) of ABT-888. PARP activity (fmol/μg protein) was determined as described in Materials and Methods. C, PARP activity in established leukemia cell lines. Horizontal line, limit of detection for assay. D, dose-response curve of TMZ alone (○) and TMZ in combination with either 0.5 μmol/L (•) or 5 μmol/L (▪) of ABT-888 or 1 μmol/L O6-BG (×) in U937 AML cells. E, small-scale NHEJ in vitro assay. A 3-kb probe, designed to favor dimerization over recircularization or multimerization, was incubated with different leukemia cell line cell extracts. The presence of robust NHEJ can be detected by the formation of the 6-kb dimer as shown in the 293T control lane. Lane 1, DNA molecular weight ladder, lane 2, radiolabeled DNA fragment only (no cell lysate); lane 3, NHEJ-proficient control cell line 293T; lanes 4 to 6, leukemia cell lines THP1, JM1, and HL-60.

  • Figure 6.
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    Figure 6.

    PARP activity and ABT-888 potentiation of TMZ in primary leukemia samples. A to D, dose-response curve of TMZ alone (○) or TMZ in combination with either 0.5 μmol/L (•) or 5 μmol/L (▪) of ABT-888 in four representative primary leukemia patient samples. A, representative ALL patient samples in which ABT-888 did not potentiate TMZ. Left, 11-y-old Caucasian female with newly diagnosed pre-B ALL; right, 2-y-old Hispanic female with newly diagnosed T-cell ALL. Potentiation factors are listed in Table 2. B, AML patient samples in which ABT-888 potentiated TMZ. Left, 2-d-old Hispanic male with fulminant AML/myelodysplastic syndrome; right, 17-y-old Hispanic male with relapsed M0 AML (trisomy 8 translocation). C, PARP activity in 14 patient leukemia samples. Horizontal line, limit of detection for assay.

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  • Table 1.

    Summary of leukemia subtype, MGMT activity, and MSI status in leukemia cell lines

    Cell lineLeukemia subtypeMGMT activity (fmol/mg protein)MSIGroup*
    U937AMLAbsentStableS0
    HELAMLAbsentStableS0
    THP1AMLLow (370 ± 11)StableS1
    JM1Pre-B ALLLow (450 ± 17)StableS1
    RajiB ALLAverage (380 ± 89)StableS1
    HL-60AMLAverage (750 ± 94)StableS2
    DaudiB ALLAverage (902 ± 17)StableS2
    KG1AMLHigh (1,170 ± 34)StableS3
    HSB2T-ALLLow (320 ± 20)UnstableU1
    Molt4T-ALLAverage (560 ± 37)UnstableU2
    JurkatT-ALLHigh (1,290 ± 36)UnstableU3
    RehPre-B ALLHigh (1,300 ± 14)UnstableU3
    • ↵*Group assignment based on MGMT activity (0 = none, 1 = low, 2 = medium, 3 = high) and MSI status (S = stable, U = unstable).

  • Table 2.

    Summary of MGMT activity, PARP activity, and ABT-888 potentiation of TMZ in primary pediatric cells

    Cell IDLeukemia subtypeMSIMGMT activity (fmol/mg protein)PARP activity (fmol/μg protein)*ABT-888 PF†
    p115AMLS2,580ND4.6
    p151AMLND1,2006703.2
    p120AMLSUD3701.2
    BM1261AMLND151UD1
    p145Pre-B ALLND1,6901,3201
    p152Pre-B ALLS3253501
    p157Pre-B ALLS1,2601,1801
    p158Pre-B ALLND3,2501,8601
    BM1169Pre-B ALLND310401
    BM1339Pre-B ALLND1,110901.2
    BM1350Pre-B ALLND550701.5
    p153T-ALLND1,5804301
    p154T-ALLND1,7603101

    Abbreviations: S, MSI stable; ND, not determined; UD, undetectable.

    • ↵*MGMT activity <100 fmol/mg protein; PARP <40 fmol/μg protein.

    • ↵†IC50 TMZ/IC50 TMZ + ABT-888. Value of 1 indicates no potentiation.

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Molecular Cancer Therapeutics: 8 (8)
August 2009
Volume 8, Issue 8
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Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity
Terzah M. Horton, Gaye Jenkins, Debananda Pati, Linna Zhang, M. Eileen Dolan, Albert Ribes-Zamora, Alison A. Bertuch, Susan M. Blaney, Shannon L. Delaney, Madhuri Hegde and Stacey L. Berg
Mol Cancer Ther August 1 2009 (8) (8) 2232-2242; DOI: 10.1158/1535-7163.MCT-09-0142

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Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity
Terzah M. Horton, Gaye Jenkins, Debananda Pati, Linna Zhang, M. Eileen Dolan, Albert Ribes-Zamora, Alison A. Bertuch, Susan M. Blaney, Shannon L. Delaney, Madhuri Hegde and Stacey L. Berg
Mol Cancer Ther August 1 2009 (8) (8) 2232-2242; DOI: 10.1158/1535-7163.MCT-09-0142
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