DNA damage response as a biomarker in treatment of leukemias

Abstract

Early assessment of cancer response to the treatment is of great importance in clinical oncology. Most antitumor drugs, among them DNA topoisomerase (topo) inhibitors, target nuclear DNA. The aim of the present study was to explore feasibility of the assessment of DNA damage response (DDR) as potential biomarker, eventually related to the clinical response, during treatment of human leukemias. We have measured DDR as reported by activation of ATM through its phosphorylation on Ser 1981 (ATM-S1981^P) concurrent with histone H2AX phosphorylation on Ser139 (γH2AX) in leukemic blast cells from the blood of twenty patients, 16 children/adolescents and 4 adults, diagnosed with acute leukemias and treated with topo2 inhibitors doxorubicin, daunomycin, mitoxantrone or idarubicin. Phosphorylation of H2AX and ATM was detected using phospho-specific Abs and measured in individual cells by flow cytometry. The increase in the level of ATM-S1981^P and γH2AX, varying in extent between the patients, was observed in blasts from the blood collected one hour after completion of the drug infusion with respect to the pre-treatment level. A modest degree of correlation was observed between the induction of ATM activation and H2AX phosphorylation in blasts of individual patients. The number of the studied patients (20) and the number of the clinically non-responding ones (2) was too low to draw a conclusion whether the assessment of DDR can be clinically prognostic. The present findings, however, demonstrate the feasibility of assessment of DDR during the treatment of leukemias with drugs targeting DNA.

Introduction

There is undisputable need to have the means for early assessment of the effectiveness of cancer treatment, to be able to change as soon as possible, the drug repertoire if the treatment is ineffective. The imaging techniques, including PET scanning, that reveal the solid tumors immediate response in terms of shrinkage, have already found application in clinical oncology.^1–4 Attempts have been also made to detect induction of apoptosis in leukemic marrow or blood blasts as early reporter of the response to the chemotherapy.^5,6 Likewise, the induction of apoptosis in cancer was assessed by measuring release of cytochrome c from the affected cells that led to its abundance in patients’ plasma.^7,8 However, because apoptosis is a transient and asynchronous (stochastic) event and apoptotic cells are being rapidly cleared it is difficult to optimally synchronize blood or marrow collection at the time of maximal response, that otherwise would have prognostic value.⁹ Hence, these approaches did not find widespread clinical utility as yet.

Most anticancer drugs target DNA in tumor cells, inducing various kinds of DNA damage. Among the most effective drugs are DNA topoisomerase (topo) I or II inhibitors which stabilize otherwise transient DNA-topo1 or DNA-topo2 associations forming so called “cleavable complexes”; collisions of the replication forks or the progressing along the DNA template RNA polymerase machinery with “cleavable complexes” convert them into DNA double-strand breaks (DSB).^10–13 Induction of DNA damage and in particular DSBs triggers complex and highly coordinated series of events generally characterized as DNA damage response (DDR).^14–20 Activation of ATM by its phosphorylation on Ser1981,^17,21 and phosphorylation of histone H2AX on Ser 139,^22,23 are the two key events of DDR. Both these events can be detected immunocytochemically^17,23 and measured rapidly in large cell populations by flow cytometry.^24–27 While studying the in vitro effect of topo1 (topotecan) or topo2 inhibitors [mitoxantrone (MXT), etoposide and daunorubicin (DNR)] on leukemia cell lines we observed that the induction of ATM activation and H2AX phosphorylation was rapid, peaking at 1–2 h upon cells exposure to the drugs.^24,26,27 The aim of the present study was to explore whether activation of ATM and H2AX phosphorylation can be detected and measured in blasts of leukemic patients undergoing routine chemotherapy with topo2 inhibitors. Under an assumption the level of DDR reports the extent of the drug-induced DNA damage and the latter is correlated with cytotoxicity, this approach is expected to be of potential prognostic value. In this preliminary study we have measured the level of ATM activation (ATM-S1981^P) and H2AX phosphorylation (γH2AX) in leukemic blasts from the blood of 20 patients diagnosed with acute lymphocytic leukemia (ALL) and acute myeloblastic leukemia (AML) collected prior to the infusion,—and one hour post infusion—of the drugs. A variable degree of DDR, both represented by induction of ATM-S1981^P as well as γH2AX, was observed in all patients providing thus the evidence of feasibility of the exploration of DDR as a potential prognostic biomarker.

Results and Discussion

The gating strategy for selection of blast cells from peripheral blood of the leukemic patients is presented in Figure 1. The population of blast cells was defined as CD45-dim (R1 gate) and the analysis of expression of ATM-S1981^P or γH2AX was subsequently restricted to this population. The percent of blasts varied between individual patients from 29 to 98%, with the mean for all patients 76.8%. Figure 2 presents an example of the raw data of ATM-S1981^P expression in blasts of two patients, pre- and post-treatment with DOX, one showing high degree of response in terms of ATM activation (patient A) and another minimal response (patient B). The dashed horizontal lines are drawn to define the upper threshold of ATM-S1981^P expression for 97% blasts, for each patient, prior to the treatment. This “background” level of ATM phosphorylation seen in the untreated patients to large extent represents the constitutive ATM activation in response to DNA damage by endogenous, metabolically generated oxidants.^28,29 Figure 3 presents similar examples of the raw data but regarding phosphorylation of H2AX. As is evident the response of one patient (A) was very pronounced whereas the other showed essentially no response.

Figure 1.

Identification of blast cells in blood of the leukemic patients. Following separation of mononuclear cells by gradient density centrifugation the cells were labeled with the CD45 Ab-tagged with PerCP. The population of CD45-dim cells (R1), presumed to be blasts, was subsequently analyzed with respect to expression of ATM-S1981^P or γH2AX. Note that neither right angle—nor forward—light scatter can discriminate these cells.

Figure 2.

Effect of patients’ treatment with doxorubicin (DOX) on the level of ATM-S1981^P expression in their blasts. The blood of patients A and B was collected prior to—and 1 h post—infusion of DOX and expression of ATM-S1981^P was measured in their blasts. The dashed horizontal lines show the upper threshold of ATM-S1981^P expression prior to treatment, for 97% of the cells for each patient. Insets present DNA content frequency histograms of the respective blasts population.

Figure 3.

Effect of patients’ treatment with doxorubicin (DOX) on the level of γH2AX expression in blasts. The blood of patients A and B was collected prior to—and 1 h post—infusion of DOX and γH2AX expression was measured in their blasts. The dashed horizontal lines show the upper threshold of γH2AX expression prior to treatment, for 97% of the cells for each patient. Insets present DNA content frequency histograms of the respective blasts population.

Figure 4 illustrates the response of individual patients, in terms of both induction of ATM-S1981^P and γH2AX, to the treatment. The data are presented as the percent increase in mean values of ATM-S1981^P IF or γH2AX IF of blasts after drug administration in relation to mean values of their IF before the treatment. Diagnosis, patients’ age and name of the topo2 inhibitor used for the treatment are listed under bars representing the response of individual patients. As it is evident in Figure 4 the drug-induced increase in either γH2AX IF or ATM-S1981^P IF, or both, was seen in blasts of almost all patients. The extent of the increase varied significantly between the individual patients. However, there was a modest degree of correlation between the induction of γH2AX and ATM activation (Fig. 5). On the linear regression plot the data of 4—among 16—patients, were outside of 95% confidence limits of the regression line.

Figure 4.

Induction of ATM-S1981^P and γH2AX in blasts of individual leukemic patients after treatment with topo2 inhibitors. The bars show the percent increase in mean values of ATM-S1981^P IF or γH2AX IF in blasts of each of individual patients one hour after drug infusion, related to the pre-treatment level. Four patients were tested only with respect to γH2AX and not to ATM-S1981^P; right side of the panel. Two patients marked NR (left) were clinical non-responders. The data of the remaining patients were set up with the increasing level of γH2AX induction. Leukemia type, patients’ age and name of the drug used for treatment are shown under the bars representing ATM-S1981^P and γH2AX response of individual patients.

Figure 5.

Correlation between the drug-induced increase in expression of ATM-S1981^P and γH2AX in blasts of the treated patients. The treatment-induced percent increase in mean values of ATM-S1981^P IF and γH2AX IF of blasts of the individual patients, as shown in Figure 4, is plotted as linear regression with 95% confidence limits.

The present data demonstrate that the DNA damage response, reflected by ATM activation and H2AX phosphorylation of the ALL patients’ blasts, can be measured following the treatment with DNA topo2 inhibitors. No attempts have been made to correlate this DNA damage response with clinical response. The complete remission (CR) was defined as peripheral blood counts rising toward normal, a mildly hypocellular to normal bone marrow with <5% blasts and no clinical signs or symptoms of the disease at the end of 1 month of induction therapy. Among of the 20 studied patients 18 underwent complete clinical remission. Of the two non-responding patients (Fig. 4, NR) one showed no induction of γH2AX and had minimal induction of ATM-S1981^P and another had relatively low level γH2AX induction and modest degree of ATM activation. Clearly, this low number of total-patients and particularly paucity of the clinically non-responding ones, does not allow us to draw any conclusion on the possible usefulness of the analysis of DNA damage response in predicting the clinical response.

It has been proposed that the induction of H2AX phosphorylation by the drugs targeting DNA can be predictive of cells death, serving thus as a surrogate for the estimate of cell viability after treatment with antitumor drugs.²⁵ Thus far, however, the analysis of induction of γH2AX during cancer treatment, as a potential prognostic biomarker, was limited. Karp et al. have reported DNA damage reflected by the induction of γH2AX in 12 adult patients with refractory acute leukemias following treatment with clofarabine and cyclophosphamide.³⁰ The number of patients in these studies was inadequate to draw any conclusion about the predictive value of γH2AX induction. In more recent studies a weak correlation between the clinical response and the induction of γH2AX when combined with the induction of apoptosis was seen in patients with AML treated after the farnesyltransferase inhibitor tipifarnib and etoposide.³¹

Our present findings reveal that activation of ATM concurrent with phosphorylation of H2AX can be measured in blasts of leukemic patients following the treatment with topo2 inhibitors. The advantage of analysis of ATM activation combined with H2AX phosphorylation is that it can provide a more specific reporter of induction of DSBs, which are the most severe and potentially lethal lesions, than the analysis of γH2AX alone.^14,15 H2AX phosphorylation alone, in the absence of ATM activation (e.g., when mediated by ATR or DNA-dependent protein kinase), can be a marker of less severe, potentially less lethal DNA damage, such as represented by ssDNA lesions³² or replication stress.³³ More extensive studies, however, involving significant groups of clinically responding and non-responding patients are needed to assess the clinical prognostic value of the analysis of DNA damage response to the treatment.

A point should be made that induction of apoptosis triggers ATM activation and H2AX phosphorylation.^24,26,33 However, the intensity of H2AX phosphorylation during apoptosis is an order of magnitude higher compared with the phosphorylation induced by the primary DSBs caused by topo1 or topo2 inhibitors.²⁶ In the present study we observed that the level of γH2AX expression of the treated patients’ blasts was relatively low, distinctly lower compared to that seen during apoptosis.²⁶ Furthermore, apoptosis of leukemic cells treated with topo1 or topo2 inhibitors is induced after 2–4 h of cell exposure to these drugs.^24,26,33 Thus, considering that presently we have analyzed blasts obtained 1 h after drug administration and the level of ATM activation and H2AX phosphorylation was much below that seen in apoptotic cells, it is most likely that we have measured blasts’ DDR to the primary DNA lesions induced by the studied drugs and not to the secondary DSBs generated upon DNA fragmentation during apoptosis.

Material and Methods

Among sixteen pediatric patients there was one diagnosed with AML, 14 with ALL and one with bi-lineage mature T-ALL + AML. Four pediatric ALL patients were in relapse. Among four adult patients two were diagnosed with ALL, one newly diagnosed with AML and one with AML post-MDS. Among all patients 8 were males 12 females. The percent of blast cells in the blood varied between 29 to 98%, with the mean for all patients 77%. Of all 20 treated patients 18 had clinical response which manifested as absence of blasts in blood and bone marrow one month after the treatment. One patient had no clinical response and one had partial response. The patients were treated with the anthracycline family DNA topoisomerase II inhibitors mitoxantrone (MXT), doxorubicin (DOX) or idarubicin (IDA) or daunorubicin (DNR). Upon giving informed consent to be a part of this study the peripheral blood samples (one tube containing 3 ml) was collected twice expressly for this study: (i) prior to the treatment and (ii) one hour after completion of the drug infusion (1^st dose of the drug). The mononuclear cells from the pretreatment and post-treatment samples were isolated by density gradient centrifugation on ficollhypaque. The cells were then fixed by placing them in 1% methanol-free formaldehyde (Polysciences, Warrington, PA) in phosphate buffer saline (PBS) on ice for 15 minutes, centrifuged (300 g, 5 min), resuspended in 70% ethanol and stored at least overnight at −20°C. The cells were washed twice in PBS and were split into two samples one for γH2AX and another for ATM-S1981^P detection. Next, the cells were suspended in 100 μl of 1% (w/v) BSA in PBS containing either 1:100 diluted γH2AX mAb (Upstate, Biotechnology Inc., Lake Placid, NY) or 1:100 diluted ATM-S1981^P mAb (Upstate, Biotechnology Inc., Lake Placid, NY), respectively for 1 h at room temperature. The cells were then rinsed and incubated with 1:100 diluted Alexa Fluor 488 secondary Ab (Invitrogen-Molecular Probes, Eugene, OR) in PBS containing 1% BSA for 30 min at room temperature. Cellular DNA was then counterstained by suspending cells in solution of PBS containing 10 μg/ml of propidium iodide (PI; Molecular Probes) and 10 μg/ml of RNase A (Sigma Chemical Co., St. Louis, MO) for 10 min. Cellular green (γH2AX or ATM-S1981^P) and red (PI) fluorescence was measured using FACScan flow cytometer (Becton Dickinson, San Jose, CA). The gating strategy using selection of blasts based on expression of CD45 and right-angle light scatter was also used to restrict the analysis to blast cells.³⁴ Toward this end the cells were initially labeled with the peridinin-chlorophyll-protein complex (PerCP)-tagged CD45 (Becton Dickinson), fixed with 1% formaldehyde, then labeled with γH2AX or ATM-S1981^P Ab and PI and the analysis was restricted to the PerCP-dim, low scatter blast cells (Fig. 1). Other details of the γH2AX or ATM-S1981^P analysis were presented before^35,36 and are given in Figure legends.

Abbreviations

ATM

ataxia telangiectasia mutated

DDR

DNA damage response

DOX

doxorubicin

DNR

daunorubicin

DSB

DNA double-strand break

IDA

idarubicin

MDS

myelodysplastic syndrome

MXT

mitoxantrone

PBS

phosphate buffered saline