Article Figures & Data
Figures
- 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.
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.
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 (▪).
- 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.
- 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.
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.
Tables
- Table 1.
Summary of leukemia subtype, MGMT activity, and MSI status in leukemia cell lines
Cell line Leukemia subtype MGMT activity (fmol/mg protein) MSI Group* U937 AML Absent Stable S0 HEL AML Absent Stable S0 THP1 AML Low (370 ± 11) Stable S1 JM1 Pre-B ALL Low (450 ± 17) Stable S1 Raji B ALL Average (380 ± 89) Stable S1 HL-60 AML Average (750 ± 94) Stable S2 Daudi B ALL Average (902 ± 17) Stable S2 KG1 AML High (1,170 ± 34) Stable S3 HSB2 T-ALL Low (320 ± 20) Unstable U1 Molt4 T-ALL Average (560 ± 37) Unstable U2 Jurkat T-ALL High (1,290 ± 36) Unstable U3 Reh Pre-B ALL High (1,300 ± 14) Unstable U3 ↵*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 ID Leukemia subtype MSI MGMT activity (fmol/mg protein) PARP activity (fmol/μg protein)* ABT-888 PF† p115 AML S 2,580 ND 4.6 p151 AML ND 1,200 670 3.2 p120 AML S UD 370 1.2 BM1261 AML ND 151 UD 1 p145 Pre-B ALL ND 1,690 1,320 1 p152 Pre-B ALL S 325 350 1 p157 Pre-B ALL S 1,260 1,180 1 p158 Pre-B ALL ND 3,250 1,860 1 BM1169 Pre-B ALL ND 310 40 1 BM1339 Pre-B ALL ND 1,110 90 1.2 BM1350 Pre-B ALL ND 550 70 1.5 p153 T-ALL ND 1,580 430 1 p154 T-ALL ND 1,760 310 1
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Volume 8, Issue 8
