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. 2012 Oct 1;18(19):5364-73.
doi: 10.1158/1078-0432.CCR-12-0961. Epub 2012 Aug 6.

Effects of selective checkpoint kinase 1 inhibition on cytarabine cytotoxicity in acute myelogenous leukemia cells in vitro

Erin L Schenk  1 , Brian D Koh, Karen S Flatten, Kevin L Peterson, David Parry, Allan D Hess, B Douglas Smith, Judith E Karp, Larry M Karnitz, Scott H Kaufmann
Affiliations

Effects of selective checkpoint kinase 1 inhibition on cytarabine cytotoxicity in acute myelogenous leukemia cells in vitro

Erin L Schenk et al. Clin Cancer Res. .

Abstract

Purpose: Previous studies have shown that the replication checkpoint, which involves the kinases ataxia telangiectasia mutated and Rad3 related (ATR) and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical acute myelogenous leukemia (AML) during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro.

Experimental design: AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA, and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays.

Results: Immunoblotting revealed increased Chk1 phosphorylation, a marker of checkpoint activation, in more than half of Chk1-containing AMLs after 48 hours of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S-phase arrest but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the antiproliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors.

Conclusions: These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted.

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Figures

Figure 1
Figure 1. Effect of cytarabine on Chk1 phosphorylation in clinical AML samples in vivo
A, schematic of the ATR/Chk1 pathway. In response to stalled replication forks, ATR catalyzes the activating phosphorylation of Chk1 on Ser317 and Ser345. Chk1 in turns phosphorylates a number of substrates, including Cdc25A (which contributes to S phase progression but is degraded after phosphorylation), Cdc25C (which contributes to G2/M progression but is inhibited by phosphorylation), substrates that stabilize replication forks, a substrate leading to phosphatase PP2A activation (which leads to Chk1 dephosphorylation), and Chk1 Ser296, an autophosphorylation site. B, marrow mononuclear cells were obtained from patients 6 (lanes 1, 2), 8 (lanes 3, 4), 12 (lanes 5, 6) and 2 (lanes 7, 8) in Cohort 1 before treatment (−) and after 48 h of single-agent cytarabine (+) at 400 mg/m2/day by continuous infusion. All samples shown contained >80% blasts (median 91%). Whole cell lysates were prepared from each fresh sample, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies that recognize the activating phosphorylation of Chk1 on Ser317 or total Chk1. Hsp90β served as a loading control.
Figure 2
Figure 2. Effect of SCH 900776 on cytarabine-induced replication checkpoint activation
A, U937 cells were treated for 4 h with the indicated concentrations of SCH 900776 in the presence of diluent (lanes 1–5) or 50 nM cytarabine (lanes 6–10), solubilized in SDS sample buffer, subjected to SDS-PAGE and probed with antibodies to the indicated antigens. β-Actin served as a loading control. B, U937 cells were treated for 4 h with the indicated concentration of cytarabine in the absence or presence of 100 nM SCH 900776, with 3H-thymidine added for the last 20 min. At the completion of the incubation, incorporation of radiolabel into DNA was assayed. Error bars, ± SD of 3 independent experiments. C, U937 cells were treated for 24 h with diluent (0.2% DSMO), 50 nM cytarabine, 100 or 300 nM SCH 900776 or 50 nM cytarabine + SCH 900776 as indicated, stained with PI and subjected to flow microfluorimetry. Arrow, increased S phase population in cells treated with cytarabine alone (see also ref. 6). Double arrow, subdiploid population after cytarabine + SCH 900776. Numbers below each histogram indicate percentage of S phase cells observed in each sample. Additional analysis of DNA fragmentation in U937 cells is contained in Fig. S1.
Figure 3
Figure 3. SCH 900776 enhances cytarabine-induced apoptosis in human AML cell lines
A, HL-60 cells were treated for 24 h with diluent (0.2% DMSO), 300 nM cytarabine, 300 nM SCH 900776 or 300 nM cytarabine + 300 nM SCH 900776. After staining with PI, flow cytometry was performed as indicated in the METHODS. B, percentage of events with <2n DNA content calculated from histograms in panel A and additional samples from the same experiment. C, HL-60 cells were treated for 24 h with the indicated concentrations of cytarabine in the presence of diluent (lanes 1, 5, 9 and 13) or SCH 900776 at 100 (lanes 2, 6, 10 and 14), 300 (lanes 3, 7, 11 and 15) or 1000 nM (lanes 4, 8, 12 and 16). Samples in lanes 13–16 also contained the caspase inhibitor Q-VD-OPh at 5 μM. At the completion of the incubation, cells were lysed in 6 M guanidine hydrochloride under reducing conditions, prepared for SDS-PAGE and subjected to immunoblotting with antibodies to the indicated antigens. D, percentage of subdiploid events observed with ML-1 treated for 24 h with the indicated concentrations of cytarabine and SCH 900776.
Figure 4
Figure 4. Effect of SCH 900776 on colony formation in human AML cell lines
U937 (A) or HL-60 (B) were treated for 24 h with diluent or the indicated concentration of SCH 900776 in the absence or presence of the indicated concentration of cytarabine, washed and plated in 0.3% agar. After 10–12 days, colonies containing >50 cells were counted at low magnification. Error bars, ± SD of quadruplicate aliquots. Similar results were obtained in three independent experiments.
Figure 5
Figure 5. Effect of SCH 900776 and cytarabine on colony formation in primary specimens
A–D, marrow mononuclear cells from Cohort 2 AML patients 1, 6, 4 and 11 (Table 1) were plated in cytokine-containing Methocult® methylcellulose in the presence of diluent (0.1% DMSO) or 100 nM SCH 900776 and the indicated concentration of cytarabine. Leukemic colonies were counted as previously described (6). E, peripheral blood mononuclear cells from a normal volunteer were assayed for formation of erythroid and myeloid colonies (35), which were combined in this graph. Similar results were observed in samples from three additional normal volunteers. F, effects of 10 nM cytarabine (light grey bars), 100 nM SCH 900776 (white bars) or 10 nM cytarabine + 100 nM SCH 900776 (dark grey bars) on formation of erythroid (left) and myeloid (right) colonies in each of four normal volunteers. Error bars, range of duplicate aliquots at each drug concentration.
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