[Cell Cycle 8:11, 1720-1724; 1 June 2009]; ©2009 Landes Bioscience
Report
DNA damage response as a biomarker in treatment of leukemias H. Dorota Halicka,1 M. Fevzi Ozkaynak,2 Oya Levendoglu-Tugal,2 Claudio Sandoval,2 Karen Seiter,3 Malgorzata Kajstura,1 Frank Traganos,1 Somasunadaram Jayabose2 and Zbigniew Darzynkiewicz1,* 1Brander
Cancer Research Institute; Department of Pathology; 2Department of Pediatrics; 3Department of Medicine; New York Medical College; Valhalla, NY USA
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 Key words: histone H2AX phosphorylation, ATM activation, acute leukemias, apoptosis, DNA topoisomerase II inhibitors, mitoxantrone, doxorubicin, idarubicin, daunorubicin, cytometry
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-S1981P) 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-S1981P 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 *Correspondence to: Zbigniew Darzynkiewicz; Brander Cancer Research Institute at NYMC; Department of Pathology, BSB 438; Valhalla, NY 10595 USA; Tel.: 914.594.3780; Fax: 914.594.3790; Email:
[email protected] Submitted: 03/24/09; Accepted: 03/30/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/8598
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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 immunocytochemically17,23 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
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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-S1981P) 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-S1981P 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
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-S1981P or γH2AX. Note that neither right angle—nor forward—light scatter can discriminate these cells.
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-S1981P 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-S1981P 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-S1981P 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 4 illustrates the response of individual patients, in terms of both induction of ATM-S1981P and γH2AX, to the treatment. The data are presented as the percent increase in mean values of ATM-S1981P 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-S1981P 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. The present data demonstrate that the DNA damage response, reflected by ATM activation and H2AX phosphorylation of the www.landesbioscience.com
Figure 2. Effect of patients’ treatment with doxorubicin (DOX) on the level of ATM-S1981P 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-S1981P was measured in their blasts. The dashed horizontal lines show the upper threshold of ATM-S1981P 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.
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Figure 4. Induction of ATM-S1981P and γH2AX in blasts of individual leukemic patients after treatment with topo2 inhibitors. The bars show the percent increase in mean values of ATM-S1981P 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-S1981P; 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-S1981P and γH2AX response of individual patients.
Figure 5. Correlation between the drug-induced increase in expression of ATM-S1981P and γH2AX in blasts of the treated patients. The treatmentinduced percent increase in mean values of ATM-S1981P IF and γH2AX IF of blasts of the individual patients, as shown in Figure 4, is plotted as linear regression with 95% confidence limits.
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
