DNA Damage and DNA Damage Response in Chronic Myeloid Leukemia

DNA damage and alterations in the DNA damage response (DDR) are critical sources of genetic instability that might be involved in BCR-ABL1 kinase-mediated blastic transformation of chronic myeloid leukemia (CML). Here, increased DNA damage is detected by γH2AX foci analysis in peripheral blood mononuclear cells (PBMCs) of de novo untreated chronic phase (CP)-CML patients (n = 5; 2.5 γH2AX foci per PBMC ± 0.5) and blast phase (BP)-CML patients (n = 3; 4.4 γH2AX foci per PBMC ± 0.7) as well as CP-CML patients with loss of major molecular response (MMR) (n = 5; 1.8 γH2AX foci per PBMC ± 0.4) when compared to DNA damage in PBMC of healthy donors (n = 8; 1.0 γH2AX foci per PBMC ± 0.1) and CP-CML patients in deep molecular response or MMR (n = 26; 1.0 γH2AX foci per PBMC ± 0.1). Progressive activation of erroneous non-homologous end joining (NHEJ) repair mechanisms during blastic transformation in CML is indicated by abundant co-localization of γH2AX/53BP1 foci, while a decline of the DDR is suggested by defective expression of (p-)ATM and (p-)CHK2. In summary, our data provide evidence for the accumulation of DNA damage in the course of CML and suggest ongoing DNA damage, erroneous NHEJ repair mechanisms, and alterations in the DDR as critical mediators of blastic transformation in CML.

Tyrosine kinase inhibitors (TKIs) have revolutionized CML therapy, and induce high rates of deep or major molecular responses (DMRs or MMRs) [9]. However, TKI failure may occur, for example, by acquired point mutations in the BCR-ABL1 tyrosine kinase domain, clonal evolution, or BCR-ABL1 independent pathways resulting in loss of MMR and disease progression [10].
DNA double-strand breaks (DSBs) are serious DNA lesions that may accumulate during the course of CML. In response to DSB, the histone variant H2AX is phosphorylated at Ser139 in a region of several megabase pairs around the DSB, resulting in the formation of discrete γH2AX foci in the nucleus that are detectable by immunofluorescence microscopy [11]. γH2AX recruits additional proteins engaged in chromatin remodeling, DNA repair, and signal transduction [12][13][14][15]. One of these proteins is 53BP1 [16], which promotes ATM-dependent checkpoint signaling, regulates DSB repair pathway choice, and tethers DNA ends during non-homologous end joining (NHEJ) [17,18]. Importantly, 53BP1 triggers the repair of DSBs by erroneous NHEJ and microhomology-mediated end joining (MMEJ), which may aggravate genetic instability [19][20][21]. In this study, γH2AX and 53BP1 foci were analyzed by immunofluorescence microscopy in peripheral blood mononuclear cells (PBMCs) of CML patients at different stages.
The DNA damage response (DDR) is activated upon DNA damage and acts as an "anti-cancer barrier" [22]. In this signaling network, the ATM-CHK2 and ATR-CHK1 axes promote the repair of DSB and single-strand breaks, respectively. In addition, TP53 may induce apoptosis if apoptotic factors overwhelm DNA repair factors [23][24][25]. However, the frequency of additional chromosomal aberrations (ACAs) is about 5% in CP-CML and increases to about 80% in BP-CML [26,27], making a strong argument for the occurrence of DDR defects in the course of CML.
In summary, genetic instability is most likely involved in blastic transformation of CML. The goal of our project was to analyze mechanisms of genetic instability related to DNA damage, DSB repair, and DDR signaling in CML. For this purpose, immunofluorescence microscopy of γH2AX/53BP1 and Western blotting of (p-)ATM/(p-)CHK2 were applied in PBMC of CML patients at different disease stages in comparison to healthy controls.

Co-Localization of γH2AX and 53BP1 Foci
γH2AX and 53BP1 foci were analyzed in the PBMCs of healthy donors and all CML groups 2-5 ( Figure 2). All samples showed co-localizing γH2AX/53BP1 foci in similar patterns. Notably, the number of γH2AX/53BP1 foci in PBMCs increased across the spectrum from CP-CML towards BP-CML patients, suggesting the promotion of erroneous NHEJ and MMEJ during blastic transformation.

DNA Damage Response
Western blotting of (p-)ATM and (p-)CHK2 was performed in PBMCs of healthy donors and all CML groups 2-5 ( Figure 3). PBMCs of healthy donors, CP-CML patients with DMR or MMR, and CP-CML patients with loss of MMR demonstrated no or minor expression of p-ATM and variable expression of p-CHK2, which was in accordance with the presence of low levels of DNA damage in these cells as evidenced by γH2AX foci analysis. However, PBMC of de novo CP-CML patients and BP-CML patients demonstrated no expression of p-ATM and predominantly no expression of p-CHK2 (apart from strong expression of p-CHK2 in BP-CML#2), which was in contrast to the presence of relatively high levels of DNA damage in these cells as evidenced by γH2AX foci analysis. The results suggest an alteration of the DDR in transformed CML cells.

Discussion
DSBs continuously occur in each genome [28] and are elementarily involved in the blastic transformation of CML, as evidenced by the formation of t(9;22)(q34;q11) in CP-CML and a high degree of ACA in BP-CML. For the detection of DSBs, the immunofluorescence microscopy of γH2AX and 53BP1 foci was performed in PBMCs of healthy donors, CP-CML patients in DMR/MMR, CP-CML patients with loss of MMR, de novo CP-CML patients, and BP-CML patients. Analyses showed a gain in DSB in the course from CP-CML towards BP-CML, indicating a biological continuum to accumulate DNA damage across different CML stages. Moreover, the intersection of γH2AX foci levels between different CML stages indicated that γH2AX foci were a biological marker, rather than a stage-specific marker. Further, γH2AX foci levels were evaluated in CML patient samples in relation to ACA. The ACA-bearing BP-CML samples demonstrated increased γH2AX foci levels as compared to the non-ACA bearing CP-CML samples. This demonstrates that γH2AX foci and ACA increase concordantly in the process of blastic transformation in CML.
γH2AX and 53BP1 foci were analyzed semi-quantitatively in PBMCs of all healthy donors and CML patients. Analyses revealed similar patterns of γH2AX and 53BP1 foci co-localization in all samples. Furthermore, co-localizing γH2AX/53BP1 foci increased in the course from CP-CML patients towards BP-CML patients. This observation suggests the promotion of erroneous NHEJ and MMEJ as critical mechanisms of blastic transformation in CML. Considering the contribution of erroneous NHEJ and particularly MMEJ to the formation of chromosomal aberrations such as deletions, translocations, inversions, and other complex rearrangements [29], this data might, at least in part, explain the occurrence of ACAs in BP-CML.
γH2AX and 53BP1 foci were evaluated in different CML patients at various stages. One might ask for follow-up data in same CML patients; however, none of the CML patients in DMR or MMR (group 2), who were treated by TKI or were closely monitored in a TKI-free setting, demonstrated loss of MMR during study. Further, CML patients with loss of MMR (group 3) were changed promptly to another TKI according to BCR/ABL kinase domain mutations and patient characteristics. These patients achieved DMR or MMR shortly again, except for one patient who took the TKI irregularly and still demonstrated loss of MMR. Although our study lacks follow-up data on γH2AX and 53BP1 foci in same CML patients, the potential increases in the long-term course in these patients may be similar to the numbers of γH2AX and 53BP1 foci in different CML patients at various stages.
Western blotting of (p-)ATM and (p-)CHK2 was performed in the PBMCs of healthy donors and CML patients. Minor activation of the DDR proteins was detected in the PBMCs of healthy donors, CP-CML patients in DMR or MMR, and CP-CML patients with loss of MMR, which was in accordance with the detection of low levels of DNA damage in these cells, as evidenced by γH2AX foci analysis. However, in the PBMCs of de novo CP-CML patients and BP-CML patients, almost no activation of the DDR proteins was observed, too, despite of the presence of relatively high levels of DNA damage in these cells, as evidenced by γH2AX foci analysis and the presence of ACAs. The absence of p-ATM and the predominant absence of p-CHK2 in the PBMCs of de novo CP-CML and BP-CML patients might be explained by missing or minor expression of ATM and CHK2, respectively. Further, there may also have been defective phosphorylation of ATM and CHK2, aggravating DDR defects. Overall, our results implicate alterations of the DDR in CML cells and correspond well with the known function of the DDR as an "anti-cancer barrier" that becomes activated upon DNA damage in healthy cells and disarranged in cancer cells [22,30].
Our data may generate hypotheses for the development of genetic instability in CML, in which γH2AX and 53BP1 foci accumulate with ongoing DNA damage by intrinsic sources (e.g., reactive oxygen species, replication-stress-induced DNA damage), erroneous DSB repair (e.g., NHEJ and MMEJ), and alterations of the DDR (e.g., sensing and signaling of DNA damage) ( Figure 4). Importantly, genetic instability is a common feature in TKI-refractory CML. In consideration of the worse prognosis, the limited effectiveness of chemotherapy and the ineligibility of most of these patients for allogeneic bone marrow transplantation, novel treatment options are an unmet need at present. Here, DNA repair inhibitors (e.g., PARP or APE1 inhibitors) might be beneficial by inducing synthetic lethality in DNA repair defective TKI-refractory CML cells [31] and may constitute possible treatment options.

Immunofluorescence Staining of γH2AX and 53BP1
γH2AX and 53BP1 were detected in the PBMCs of healthy donors and CML patients using a mouse monoclonal anti-γH2AX antibody (clone JBW301; Merck Millipore, Darmstadt, Germany) and a polyclonal rabbit anti-53BP1 antibody (NB100-304; Novus Biologicals, Littleton, US), respectively. An Alexa Fluor 488-conjugated goat anti-mouse antibody and an Alexa Fluor 555-conjugated goat anti-rabbit antibody (Thermo Fisher Scientific) were used as previously described [38,39]. At least 50 PBMCs were analyzed for each measurement.

Western Blotting
Western blotting of (p-)ATM and (p-)CHK2 was conducted in the PBMCs of healthy donors, CP-CML patients in DMR or MMR, CP-CML patients with loss of MMR, de novo untreated CP-CML patients, and BP-CML patients as previously described [39]. (p-)ATM and (p-)CHK2 expressing SKM-1 acute myeloid leukemia cells (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany) served as positive control.

Statistics
Statistical calculations were performed using SAS software, release 9.4 (SAS Institute Inc., Cary, NC, USA). Quantitative variables were stated by mean values and standard errors. The number of foci in each sample were modeled by a Poisson regression model using the SAS procedure PROC GENMOD. Furthermore, the SAS "repeated" statement was applied with statistical consideration encompassing several cells, which were counted in each sample. p values < 0.05 were regarded statistically significant.

Conclusions
Our study reveals accumulation of DNA damage in the course from CP-CML towards BP-CML. In addition, our data suggest increase of DNA damage, erroneous DSB repair, and alterations of the DDR as critical mediators of blastic transformation in CML. Finally, γH2AX and 53BP1 foci might be useful markers for targeting genetic instability in TKI-refractory CML in future studies.