BCR/ABL and other kinases from chronic myeloproliferative disorders stimulate single-strand annealing, an unfaithful DNA double-strand break repair

Abstract

Myeloproliferative disorders (MPD) are stem cell-derived clonal diseases arising as a consequence of acquired aberrations in c-ABL, Janus-activated kinase 2 (JAK2), and platelet-derived growth factor receptor (PDGFR) that generate oncogenic fusion tyrosine kinases (FTK), including BCR/ABL, TEL/ABL, TEL/JAK2, and TEL/PDGFbetaR. Here, we show that FTKs stimulate the formation of reactive oxygen species and DNA double-strand breaks (DSB) both in hematopoietic cell lines and in CD34(+) leukemic stem/progenitor cells from patients with chronic myelogenous leukemia (CML). Single-strand annealing (SSA) represents a relatively rare but very unfaithful DSB repair mechanism causing chromosomal aberrations. Using a specific reporter cassette integrated into genomic DNA, we found that BCR/ABL and other FTKs stimulated SSA activity. Imatinib-mediated inhibition of BCR/ABL abrogated this effect, implicating a kinase-dependent mechanism. Y253F, E255K, T315I, and H396P mutants of BCR/ABL that confer imatinib resistance also stimulated SSA. Increased expression of either nonmutated or mutated BCR/ABL kinase, as is typical of blast phase cells and very primitive chronic phase CML cells, was associated with higher SSA activity. BCR/ABL-mediated stimulation of SSA was accompanied by enhanced nuclear colocalization of RAD52 and ERCC1, which play a key role in the repair. Taken together, these findings suggest a role of FTKs in causing disease progression in MPDs by inducing chromosomal instability through the production of DSBs and stimulation of SSA repair.

Figure 1. FTKs enhance the production of ROS and DSBs

Results in BCR/ABL (B/A), TEL/ABL (T/A), TEL/JAK2), and TEL/PDGFβR (T/P) – transformed cells were compared with results in G418-resistant control cells (P). Results in Lin⁻CD34⁺ CML CP and BC cells were compared with Lin⁻CD34⁺ peripheral blood cells isolated from healthy volunteers (H). (A) ROS levels were measured by fluorescence. (B,C) γ-H2AX nuclear foci were detected by immunofluorescence; the numbers indicate (B) the percentage of cells with 4–20 and >20 γ-H2AX foci/cell and (C) the mean number of foci/nucleus in cells containing ≥ 4 foci. (D) Representative nuclei containing γ-H2AX foci; nuclei borders are marked in blue. p<0.05 in comparison to other groups (*) and to BC (**).

Figure 2. FTKs stimulate SSA

(A) The structure of SA-GFP reporter cassette is shown before (upper panel, GFP⁻ cells) and after (lower panel, GFP⁺ cells) I-SceI cleavage and SSA. The cassette consists of the 5’GFP and SceGFP3’ fragments, which have 266 bp of homology and intervening sequence encoding puromycin-resistance (puroR). The black strip represents the I-SceI site in the SceGFP3’ and the large black triangle depicts the 3’ end of the cassette. Repair of the I-SceI generated DSB in SceGFP3’ by SSA results in a functional GFP gene when a DNA strand from SceGFP3’ is annealed to the complementary strand of 5’GFP, followed by appropriate DNA-processing steps. As a result, SSA between the homologous sequences in the GFP gene fragments produces a 2.7-kb deletion in the chromosome. The SA-GFP reporter can also be repaired by HR and NHEJ, but without restoration of a functional GFP gene (14). (B) I-SceI and Red1-Mito were expressed in parental (P) and FTK-transformed (BCR/ABL non-mutated = B/A-nm, BCR/ABL-T315I mutant = B/A-T315I, TEL/ABL = T/A, TEL/JAK2 = T/J, and TEL/PDGFβR = T/P) cells containing SA-GFP reporter cassette and cultured in the presence of IL3 and imatinib (IM) when indicated. SSA activity was determined as the number of GFP⁺/Red1⁺ cells in 10⁵ Red1⁺ cells. * p<10⁻⁸, <10⁻⁸, <10⁻⁸, 10⁻² and <10⁻⁷ in comparison to B/A-nm, B/A-T315I, T/A, T/J and T/P, respectively; and ** p<0.03 in comparison to B/A. (C) PCR products from genomic DNA of GFP⁺ and GFP⁻ cells.

Figure 3. Non-mutated and imatinib-resistant BCR/ABL kinase mutants stimulate SSA in a dose-dependent manner

(A) Similar high levels of non-mutated (nm), Y253F, E255K, T315I and H396P BCR/ABL kinase proteins, and (B) low (L) and high (H) levels of nm, Y253F, and E255K BCR/ABL kinase proteins were expressed in parental cells (P) containing the SA-GFP reporter cassette (lower panels). Cells were transfected with I-SceI and Red1-Mito and maintained in the presence of IL-3. SSA activity was determined as the number of GFP⁺/Red1⁺ cells in 10⁵ Red1⁺ cells (upper panels). * p<10⁻⁷ in comparison to other groups, ** p<10⁻², <10⁻⁴, and <10⁻³ in comparison to L groups of nm, Y253F, and E255K, respectively; and *** p=0.02, 0.006, and 0.02 in comparison to corresponding H groups.

Figure 4. BCR/ABL facilitates RAD52-ERCC1 co-localization

(A) Western analysis of RAD52 and ERCC1 expression in parental and FTK-transformed cells cultured in the presence of IL-3. Actin served as loading control. (B) Detection of RAD52 (green), ERCC1 (red) and co-localizing (yellow) foci in parental and BCR/ABL-positive cells presented as percentage of RAD52 + ERCC1 staining/RAD52 staining. * p<10⁻⁵ in comparison to B/A. (C) Representative nuclear staining for RAD52 and ERCC1; white arrows indicate co-localization sites; nuclei borders are marked in blue.