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.2013 Apr 4;32(14):1784-93.
doi: 10.1038/onc.2012.203. Epub 2012 May 28.

Targeting abnormal DNA double-strand break repair in tyrosine kinase inhibitor-resistant chronic myeloid leukemias

Affiliations

Targeting abnormal DNA double-strand break repair in tyrosine kinase inhibitor-resistant chronic myeloid leukemias

L A Tobin et al. Oncogene..

Abstract

Resistance to imatinib (IM) and other tyrosine kinase inhibitors (TKI)s is an increasing problem in leukemias caused by expression of BCR-ABL1. As chronic myeloid leukemia (CML) cell lines expressing BCR-ABL1 utilize an alternative non-homologous end-joining pathway (ALT NHEJ) to repair DNA double-strand breaks (DSB)s, we asked whether this repair pathway is a novel therapeutic target in TKI-resistant disease. Notably, the steady state levels of two ALT NHEJ proteins, poly-(ADP-ribose) polymerase 1 (PARP1) and DNA ligase IIIα, were increased in the BCR-ABL1-positive CML cell line K562 and, to a greater extent, in its imatinib-resistant (IMR) derivative. Incubation of these cell lines with a combination of DNA ligase and PARP inhibitors inhibited ALT NHEJ and selectively decreased survival with the effect being greater in the IMR derivative. Similar results were obtained with TKI-resistant derivatives of two hematopoietic cell lines that had been engineered to stably express BCR-ABL1. Together our results show that the sensitivity of cell lines expressing BCR-ABL1 to the combination of DNA ligase and PARP inhibitors correlates with the steady state levels of PARP1 and DNA ligase IIIα, and ALT NHEJ activity. Importantly, analysis of clinical samples from CML patients confirmed that the expression levels of PARP1 and DNA ligase IIIα correlated with the sensitivity to the DNA repair inhibitor combination. Thus, the expression levels of PARP1 and DNA ligase IIIα serve as biomarkers to identify a subgroup of CML patients who may be candidates for therapies that target the ALT NHEJ pathway when treatment with TKIs has failed.

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Conflict of interest statement

Conflict of interest

AET is a co-inventor on a patent application that covers the use of DNA ligase inhibitors as anti-cancer agents.

Figures

Figure 1
Figure 1. Steady state levels of NHEJ proteins in IMS and IMR cell lines
(A) Representative immunoblot and(B–C) relative steady state levels of DNA ligase IIIα (LIG3, white bars), PARP1 (dark grey bars), DNA ligase IV (LIG4, light grey bars) and Ku70 (KU70, black bars) and β-actin (ACTIN) as loading control, in(B) IMS(K562) and IMR derivatives (K562 IMR) of a BCR-ABL1-positive human CML cell line, K562, compared to a lymphoblastoid cell line established from normal lymphocytes (NC10) and(C) IMS (Mo7e-P210 and Baf3-P210) and IMR derivatives (Mo7e-P210 IMR1, Mo7e-P210 IMR2 and Baf3-P210 IMR) of hematopoietic cell lines, Mo7e and Baf3 that had been engineered to stably express BCR-ABL1 compared to the parental cells Mo7e and Baf3, respectively.
Figure 2
Figure 2. Effect of DNA ligase and PARP inhibitors on the survival of IMS and IMR cell lines
(A–B) Colony survival after a 10-day treatment with L67 (0.3 μM, dark grey Bars), NU1025 (50 μM, light grey bars), L67+NU1025 (black bars) compared to vehicle control (CTRL, white bars) of(A) K562, K562 IMR compared to NC10,(B) Mo7e-P210, Baf3-P210, Mo7e-P210 IMR1, Mo7e-P210 IMR2 and Baf3-P210 IMR compared to Mo7e and Baf3 respectively.(C) Immunoblotting for DNA ligase IIIα (LIG3) and β-actin (ACTIN) in extracts from Mo7e, Mo7e-P210 and Mo7e-P210 IMR1 cells after treatment with either control (−) or DNA ligase IIIα siRNA.(+).(D) Colony survival after siRNA knockdown of DNA ligase IIIα (dark grey bars) and/or a 10-day treatment with NU1025 (50 μM, black bars) compared to siRNA CTRL (white bars) and/or a 10-day treatment with NU1025 (light grey bars). Results are representative of three independent experiments ± SEM, *p<0.05 based on TTEST from IMS versus control or IMR versus IMS cells.
Figure 3
Figure 3. Effect of DNA ligase and PARP inhibitors on the steady state levels of DSBs in IMS and IMR cell lines
(A) Representative nuclei (blue, DAPI) from K562, K562 IMR and NC10 cells immunostained for γH2AX (red). (B and C). Percentage of cells with more than 3 endogenous γH2AX foci/cell following a 24-hour treatment with L67 (0.3 μM) and NU1025 (50 μM, black bars) or CRTL (white bars);(B) K562, K562 IMR and NC10 cells;(C) Mo7e-P210, Baf3-P210, Mo7e-P210 IMR1, Mo7e-P210 IMR2, Baf3-P210 IMR, Mo7e and Baf3 cells. Results are representative of three independent experiments ± SEM, *p<0.05 based on TTEST from IMS versus control or IMR versus IMS cells.
Figure 4
Figure 4. Repair of DSBs by NHEJ in IMS and IMR: Effect of DNA ligase and PARP inhibitors
(A) Plasmid-based NHEJ repair assay was carried out as described in Materials of methods. The number of blue (accurate repair, dark bar) and white (inaccurate repair, white bars) recovered from the NC10, K562, K562IMR, Mo7e-P210, Mo7e-P210IMR1, Mo7e-P210IMR2, Baf3, Baf3-P210 and Baf3P210IMR cell lines. The results of 3 independent experiments are shown.(B–E)NHEJ repair assays following a 24-hour treatment without (white diamonds or −) or with L67 and NU1025 (0.3 μM and 50 μM, black diamonds or + in(B–C) K562, K562 IMR and NC10 cells;(D–E) Mo7e-P210, Baf3-P210, Mo7e-P210 IMR1, Mo7e-P210 IMR2, Baf3-P210 IMR, Mo7e and Baf3 cells.(B and D)Size of DNA deletions within the DSB region of repaired plasmids in white colonies. Average (AVG) size of DNA deletions is represented by black bars.(C and E) Ratio of the mean number of microhomologies (≥2 bp, black bars) versus no microhomology (white bars) within repaired plasmids in white colonies.
Figure 5
Figure 5. Genome-wide view of DNA copy number variation on Agilent 1M array CGH in IMS and IMR derivatives of Mo7e
(A) Log2 ratio of Mo7e-P210 (Cy5) versus Mo7e (Cy3) and(B) log2 ratio of Mo7e-P210 IMR1 (Cy5) versus Mo7e-P210 (Cy3). The numbers labeled on the horizontal axis indicate different chromosomes. Log2 ratios of signal intensities are plotted with horizontal central line equal to zero. Alterations above the line (dotted black arrows) indicate amplifications and below the line indicate deletions (black arrows). Because of the way the genome view figure is drawn, some of the genomic alterations encompassing regions at the ends of chromosomes, are not visible. Nevertheless, we have indicated these genomic alterations with arrows. The copy number variations are shown as trend lines with 2MB base pair moving average. The log2 ratio on the vertical axis ranges from +4 (top) to -4 (bottom).(C) Summary of the deletion and amplifications found in Mo7e-P210 compared to Mo7e and in Mo7e-P210 IMR1 compared to Mo7e-P210.
Figure 6
Figure 6. Steady state levels of DNA ligase IIIα and PARP1 in IMS and IMR samples from BCR-ABL1-positive CML patients: effect of DNA ligase and PARP inhibitors on the survival of IMS and IMR samples from BCR-ABL1-positive CML patients
(A) Relative steady state levels of DNA ligase IIIα (LIG3, White bars) and PARP1 (Black bars) mRNA transcripts in BCR-ABL1-positive CML patient samples compared to normal bone marrow (NBM). Results are presented graphically with the patient samples shown in the following groups; LIG3 and PARP1, significant (*p<0.05 based on TTEST) increases in both DNA ligase IIIα and PARP1 mRNA transcripts; LIG3 or PARP1, significant (*p<0.05 based on TTEST) increases in either DNA ligase IIIα or PARP1 mRNA transcript; Neither, no significant change in either DNA ligase IIIα or PARP1 mRNA transcript.(B) Colony survival assays after a 10-day growth of BCR-ABL1-positive CML patient samples and NBM in the absence (white bars) or presence of L67 and NU1025 (0.3 μM and 50 μM, black bars). Results are presented graphically with the patient samples grouped according to response to the combination of DNA repair inhibitors; L67 + NU1025 Sensitive, patient samples that were sensitive (**p<0.005 based on TTEST); Partially sensitive patient samples that showed partial sensitivity (*p<0.05 based on TTEST); Insensitive, patient samples that were insensitive.
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