In vitro and in vivo single-agent efficacy of checkpoint kinase inhibition in acute lymphoblastic leukemia
- Ilaria Iacobucci†1Email author,
- Andrea Ghelli Luserna Di Rorà†1,
- Maria Vittoria Verga Falzacappa2,
- Claudio Agostinelli1,
- Enrico Derenzini1,
- Anna Ferrari1,
- Cristina Papayannidis1,
- Annalisa Lonetti3,
- Simona Righi1,
- Enrica Imbrogno1,
- Silvia Pomella1,
- Claudia Venturi1,
- Viviana Guadagnuolo1,
- Federica Cattina1, 4,
- Emanuela Ottaviani1,
- Maria Chiara Abbenante1,
- Antonella Vitale5,
- Loredana Elia5,
- Domenico Russo4,
- Pier Luigi Zinzani1,
- Stefano Pileri1,
- Pier Giuseppe Pelicci2 and
- Giovanni Martinelli1Email author
© Iacobucci et al. 2015
Received: 9 July 2015
Accepted: 28 September 2015
Published: 5 November 2015
Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2).
The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B-/T-ALL cell lines. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent n-ethyl-n-nitrosourea.
Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment strategy efficiently triggered the Chk1-Cdc25-Cdc2 pathway resulting in a dose- and time-dependent cytotoxicity, induction of apoptosis, and increased DNA damage. Moreover, treatment with PF-00477736 showed efficacy ex vivo in primary leukemic blasts separated from 14 adult ALL patients and in vivo in mice transplanted with T-ALL, arguing in favor of its future clinical evaluation in leukemia.
In vitro, ex vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation.
KeywordsAcute lymphoblastic leukemia DNA damage Checkpoint kinase Drug-sensitivity New targets
Acute lymphoblastic leukemia represents a biologically and clinically heterogeneous group of B/T-precursor-stage lymphoid cell malignancies arising from genetic insults that block lymphoid differentiation and drive aberrant cell proliferation and survival. Survival rates for children are approximately 80–85 % with current risk-oriented treatment protocols in contrast to less than 40 % for adults [1–3]. Although the outcome of children with relapsed ALL is heterogeneous, many achieve lasting second remissions. In contrast, survival after relapse in adult ALL is short [4, 5]. New therapeutic strategies are therefore needed to improve remission rates in adults and to prevent relapse both in adult and pediatric ALL patients.
Recently, in order to enhance DNA-damaging effects inflicted by cytotoxic drugs or radiation, different checkpoint kinase (Chk) inhibitors have been developed and assessed alone or in combination with DNA-damaging agents in preclinical studies and in phase I/II trials for cancer therapy [6–14]. Some of them inhibit both Chk1 and Chk2; others have a higher specificity for Chk1. The biological background underlying the development of Chk1 inhibitors and preliminary data deriving from early clinical trials have been recently well reviewed in . Following DNA damage, multiprotein complexes recruit the transducers ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) which activate Chk2 and Chk1, respectively, despite an existing extensive crosstalk between the ATR-Chk1 and ATM-Chk2 pathways. Once activated by phosphorylation at S317/S345 in response to DNA single strand breaks, Chk1 phosphorylates the phosphatase Cdc25A at the G1/S and S phase checkpoints preventing cells from entering S phase and Cdc25A and Cdc25C at the G2/M checkpoint avoiding the entry in mitosis [16–19]. If Chk1 is induced by single strand breaks, Chk2 activation is largely restricted to DNA double strand breaks via ATM . Once activated, they mediate cell cycle delay, DNA repair, and apoptosis in response to DNA damage. Moreover, Chk1 is required for proper mitotic spindle assembly and maintenance of chromosomal stability during mitosis .
PF-00477736 is a potent, selective, ATP-competitive, small-molecule inhibitor of Chk1, synthesized by Pfizer, which is selective for Chk1 and shows general selectivity over other kinases . Preclinically, PF-00477736 enhanced docetaxel activity in tumor cells and xenografts by abrogating the mitotic spindle checkpoint, as well as the DNA damage checkpoint . The checkpoint abrogating and cytotoxic activities attributed to PF-00477736 in combination with chemotherapy agents (e.g., gemcitabine and carboplatin) showed selectivity for p53-defective cancer cell lines over p53-competent normal cells in vitro . In xenografts, PF-00477736 enhanced the antitumor activity of gemcitabine in a dose-dependent manner. PF-00477736 combinations were well tolerated with no exacerbation of side effects commonly associated with cytotoxic agents .
In this study, we investigated the activity of PF-0477736, as a single agent, in B-/T-ALL by the following: assessment of the in vitro efficacy in ALL cell lines and primary blast cells, assessment of the in vivo efficacy in mouse models, and identification and validation of potential biomarkers of functional inhibition. Results demonstrated that in vitro treatment of B-/T-ALL cell lines and primary blast cells with PF-0477736 resulted in inhibition of cell viability, induction of DNA damage, and apoptosis. Moreover, in vivo studies confirmed the efficacy of Chk1 inhibition, suggesting that this therapeutic strategy may be promising in leukemia.
Chk1 and Chk2 are overexpressed in acute lymphoblastic leukemia
PF-00477736 reduces cell viability in a dose-dependent manner
PF-00477736 induces apoptosis at 24 and 48 h in B-/T-ALL cells
In order to assess whether cytotoxicity was correlated to increased susceptibility to apoptosis, B-/T-ALL cell lines were incubated with increasing concentrations of drug (0.1, 0.5, and 1 μM and 0.05, 0.1, and 0.2 μM only for BV-173 and RPMI-8402, respectively) for 24 and 48 h. Consistent with the viability results, Annexin V/propidium iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 h in B- and T-ALL cells proportional to drug dose and drug exposure times (Additional file 1: Figure S3-A). The induction of apoptosis by PF-00477736 was also assessed by the detection of poly (ADP-ribose) polymerase (PARP) cleavage by Western blot analysis . The PARP-1 cleavage band at 89 kDa was observed in lysates from leukemia cell lines after 24 h of drug exposure, while it was undetectable in cells treated with only DMSO 0.1 % (Fig. 2c).
PF-00477736 perturbs the cell cycle profile in B-/T-ALL cells
The effect of the inhibition of Chk1 pathway on cell cycle progression was evaluated in RPMI-8402, BV-173, SUP-B15, and NALM-6 cell lines. Cells were incubated with increasing concentrations of PF-00477736 for 6 and 24 h and then stained with propidium iodide to quantify the DNA amount. The effect of PF-00477736 on the cell cycle progression was very weak after 6 h of incubation (data not showed) and very heterogeneous among the different cell lines after 24 h. In RPMI-8402, BV-173, and SUP-B15 cell lines, the treatment induced a progressive reduction of the number of cells in S and G2/M phase and a concomitant increment of cell debris. In the less sensitive cell line, NALM-6, the inhibition of Chk1 progressively increased the percentage of cells in G2/M phase and reduced the percentage of cells in G1 phase (Additional file 1: Figure S3-B).
PF-00477736 efficiently targets Chk1 pathway and induces DNA damage
The phospho-H2A.X accumulates and forms characteristic nuclear foci where the DNA is damaged . Both NALM-6 and BV-173 cell lines show an increased number of γ-H2A.X foci when treated with PF-00477736 compared to untreated controls (Fig. 3c). However, evaluating the mean fluorescence intensity of γ-H2A.X positive cells, BV-173 cells seem to be more damaged than NALM-6. Actually, BV-173 treated cells have five-fold change difference of mean value intensity of γ-H2A.X compared to BV-173 untreated cells, while NALM-6 treated cells have 1.4-fold change difference of mean value intensity of γ-H2A.X compared to their untreated counterparts. In addition to these changes in the overall amount of γ-H2A.X foci among the two different cell types, we observed the presence of hyper-γ-H2A.X-positive cells specifically in BV-173 cells treated with PF-00477736. The so-called hyper-γ-H2A.X-positive cells loose the typical foci signal of γ-H2A.X and emit a much higher and diffuse signal of γ-H2A.X positivity. Almost 6.5 % of the γ-H2A.X-positive cells are hyper-γ-H2A.X-positive in BV-176-treated cells compared to 1.8 % in the untreated controls. These data suggest a higher induction of DNA damage upon PF-00477736 treatment in BV-173 than NALM-6 cells as expected by the fact that BV-173 cells are more responsive to PF-00477736. However, the increase of hyper-γ-H2A.X-positive cells only in BV-176-treated cells would suggest the existence of a specific effect that causes a hyper DNA damage only in a restricted, but significant, population of leukemic cells.
Interestingly, upon treatment, we observed a strong reduction of Chk1. As already hypothesized, this may be the result of cleavage by caspase during apoptosis induced by genotoxic stress [26, 27]. Persisting protein levels of Cdc25, pChk1, Ser345, pCdc2 (Tyr 15), and Cdc2 after 48 h of exposure to PF-00477736 differentiated less sensitive leukemia cells from more sensitive ones (Additional file 1: Figure S3c).
Gene expression profiling results
PF-00477736 reduces viability in primary ALL blast cells
PF-00477736 impairs survival of leukemic mice
We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibitor in mice transplanted with T-ALL.
Leukemic mice were generated by the usage of the tumorigenic agent n-ethyl-n-nitrosourea (ENU). The obtained leukemia has been immunophenotyped and characterized as a T-ALL. Leukemic blasts coming from the spleen of leukemic animals were transplanted into C57BL/6Ly5.1-recipient mice. Three days after transplantation, a time sufficient for leukemic blasts to home the bone marrow of the host, we started to treat the animals. A concentration of 40 mg/kg of PF-00477736 given with a q3dx4 schedule seems not to be toxic for the animals; however, it significantly affects the overall survival of the treated animals (five mice) in comparison to the untreated ones (seven mice) (p value =0.0009). Actually, control animals (intraperitoneally injected with PBS) die all at day 17 post-transplantation while the animals treated with PF-00477736 live longer (up to 59 days post transplantation). The leukemia used for the experiments shown here was very aggressive (untreated animals die with massive infiltration of the spleen and the liver by leukemic blasts at day 17) suggesting a reason why the effect of the Chk inhibitor is significant though quite variable among treated mice (Fig. 5c).
The results obtained on cell viability and induction of apoptosis have been confirmed by genome-wide studies evaluating global gene expression changes upon treatment and by functional studies performed by Western blot analysis. Interestingly, by GEP analysis, the majority of genes downregulated were involved in the DNA damages response and in particular in DNA repair mechanisms, while the genes upregulated were involved in chromatin assembly, nucleosome organization, DNA packaging, and apoptosis. The efficacy of Chk inhibition has been evaluated and confirmed in terms of reduction of cell viability in primary ALL blast cells but not in normal bone marrow precursor cells. Furthermore, assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, we demonstrated that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016).
In conclusion, in vitro, ex-vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation. The checkpoint kinase inhibitor have been synthetized to increase the effectiveness of conventional chemotherapy, preventing cells to arrest cell cycle, and to repair the DNA damages caused by the exposure to genotoxic agents. Here, we highlight that even the treatment with a checkpoint kinase inhibitor alone associated with the high genetic instability can be enough to kill cancer cells. Indeed, we believe that leukemic cells, thanks to a higher activation of the ATR-Chk1/ATM-Chk2 pathways, can better tolerate high genetic instability. Switching off these mechanisms of survive, we can kill leukemic cell by overcoming the cell cycle checkpoint, by inhibiting the mechanisms of DNA damages repair and by inducing massive damages that cannot be tolerated (Fig. 6).
Inhibition of Chk1/2 represents a novel therapeutic strategy to overcome genetic instability and to promote selective killing of leukemia cells in ALL.
Leukemia cell lines
Human B- (BV-173, SUP-B15, REH, NALM-6, and NALM-19), T-ALL (MOLT-4, RPMI-8402, and CCRF-CEM) cell lines were obtained from Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Germany). Cells were cultured in RPMI-1640 medium (Invitrogen, Paisley, UK) with 1 % l-glutamine (Sigma, St. Louis, MO, USA) and penicillin and streptomycin (Gibco, Paisley, UK) supplemented with 10–20 % fetal bovine serum (Gibco) in a humidified atmosphere of 5 % CO2 at 37 °C. Online databases have been interrogated to molecularly characterize leukemia cell lines: International Agency for Research on Cancer (IARC) TP53 database (http://www-p53.iarc.fr/) and the Catalogue of Somatic Mutations in Cancer, (COSMIC, http://www.sanger.ac.uk/genetics/CGP/cosmic/). The Chk1 inhibitor PF-00477736 was purchased by Sigma-Aldrich (Sigma-Aldrich Co. St. Louis, MO, USA).
The Chk inhibitor PF-00477736 was purchased from Sigma-Aldrich (Sigma-Aldrich Co. St. Louis, Missouri 63103 United States). qPCR analysis of Chk1 and Chk2 mRNA expressions was evaluated in 41 (76 %) newly diagnosed BCR-ABL1-positive (median age 56 years, range 26–81; ratio male to female 20/21) and 13 (23 %) newly diagnosed BCR-ABL1-negative ALL cases (median age 41 years, range 18–69; ratio male to female 5/8). One microgram of RNA was reverse transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). PCR analysis was performed using HS00967506_m1 (Chk1) and HS00200485_m1 (Chk2) assays (Applied Biosystems) and the Fluidigm Dynamic Array 48 × 48 system, a real-time qPCR assay which enables to automatically assemble 48 samples and 48 assays to create individual TaqMan reactions of a final volume of 6.75 nl each (Fluidigm, San Francisco, CA, USA; http://www.fluidigm.com/). RNA integrity was confirmed by PCR amplification of the GAPDH mRNA (Hs99999905_m1), which is expressed ubiquitously in human hematopoietic cells. Results were expressed as 2exp(−ΔΔCt). GraphPad Prism 5 software (GraphPad, Avenida de la Playa La Jolla, CA, USA) was used to plot the data. The basal mRNA expression of Chk1 and of Chk2 was evaluated in all the cell lines using the same assays (Applied Biosystems: HS00967506_m1 Chk1 and HS00200485_m1 Chk2) used for the primary samples, and the statistical validity of the results were confirmed using ANOVA multiple comparisons test (GraphPad Prism 5 software). In addition, reverse transcription and quantitative PCR analysis for GADD45a, PLK3, CDK4, and CHK2 in treated cell lines and on their untreated counterpart was performed as described above using the following assays from Applied Biosystems: Hs00169255_m1 (GADD45a), Hs00177725_m1 (PLK3), Hs00262861_m1 (CDK4), and HS00200485_m1 (Chk2).
TP53 mutation screening
Total cellular RNA was extracted using the RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA). One microgram of total RNA was reverse transcribed using the M-MLV Reverse Transcriptase (Invitrogen, San Diego, CA, USA). Three overlapping shorter amplicons [amplicon 1 (491 bp) exons 1–5; amplicon 2 (482 bp) exons 5–8; amplicon 3 (498 bp) exons 8–11)] covering the entire TP53 coding sequence [GenBank:NM_000546.4] were amplified with 2 U of FastStart Taq DNA Polymerase (Roche Diagnostics, Mannheim, Germany), 0.8 mM dNTPs, 1 mM MgCl2, and 0.2 M forward and reverse primers (Additional file 5: Table S3) in 25 μl reaction volumes. PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and then directly sequenced using an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) and a Big Dye Terminator DNA sequencing kit (Applied Biosystems). All sequence variations were detected by comparison using the BLAST software tool (www.ncbi.nlm.nih.gov/BLAST/) to reference genome sequence data [GenBank:NM_000546.4].
Studies evaluating Chk1, phosphorylated Chk1 (Ser345), Chk2, phosphorylated Chk2 (Thr68) Cdc25C, phosphorylated Cdc25C (Ser216), and phosphorylated H2A.X (Ser139) (γ-H2A.X) protein expressions were performed on FFPE samples corresponding to three thymuses, three reactive lymph nodes, and to primary tumors collected from 60 ALL patients at diagnosis, including 36 B-ALL (median age 50 years, range 5–86; ratio male to female 18/18) and 24 T-ALL (median age 38 years, range 2–76; ratio male to female 15/9). All the cases were retrieved from the archives of the Haematopathology Unit, Department of Experimental, Diagnostic and Specialty Medicine—DIMES, University of Bologna. The study was conducted according to the principles of the Declaration of Helsinki after approval of the Internal Review Board. Two different tissue microarrays (TMAs) were constructed from these paraffin-embedded blocks as previously reported. TMA sections were investigated by antibodies raised against fixation resistant epitopes. The antibody reactivity and sources as well as the antigen retrieval protocols, dilutions, and revelation systems are detailed in Additional file 5: Table S4. A cutoff of staining >30 % of the examined cells was assigned as positive score, according to formerly defined criteria . Immunohistochemical preparations were visualized, and images were captured using Olympus dotSlide microscope digital system equipped with the VS110 image analysis software.
Primary blast cells from 14 newly diagnosed ALL cases were obtained, upon written informed consent, from bone marrow and peripheral blood samples by density gradient centrifugation over Lymphoprep (Nycomed UK, Birmingham, UK). ten (71 %) samples were from adults with BCR-ABL1-positive ALL (median age 51 years, range 25–74 years; median blast percentage 92 %, range 60–100 %) and four (29 %) from patients with BCR-ABL1-negative ALL (median age 34 years, range 18–43 years; median blast percentage 93 %, range 90–100 %). Main patients’ characteristics are given in Additional file 5: Table S1.
Normal bone marrow progenitors were harvested from bone marrow aspirations performed in lymphoma patients undergoing initial staging procedures, which then resulted negative for lymphoma infiltration. Normal peripheral blood progenitors were harvested from healthy donors.
Cell viability assay
In order to assess the cell viability after treatment with PF-00477736 (Pfizer), ALL cell lines were seeded in 96-well plates at 50,000 cell/100 μl/well with increasing concentrations of drug (0.005–2 μM) for 24 and 48 h and incubated at 37 °C. Cell viability was assessed by adding WST-1 reagent (Roche Applied Science, Basel, Switzerland) to the culture medium at 1:10 dilution. Cells were incubated at 37 °C, and the optical density was measured by microplate ELISA reader at λ = 450 after 3 h. The amount of the formazan formed directly correlates to the number of metabolically active cells. All viability experiments were performed in triplicates and repeated in least two separated experiments. In ex-vivo primary leukemia cells, the effects on cell viability were assessed by counting viable and non-viable cell numbers by the trypan blue dye exclusion method. Cells were seeded in six-well plates at 500,000 cell/1 ml with increasing concentrations of drug (0.1, 0.5, and 1 μM) for 24 h and incubated at 37 °C. Cellular viability was calculated as a percentage of the viable cells compared to the untreated controls (DMSO 0.1 %).
Annexin V staining of apoptotic cells
According to the WST-1 results, three different increasing concentrations of PF-00477736 were used to treat leukemia cells lines in order to detect and discriminate apoptotic, necrotic and dead cells. Cell lines were seeded in 12-well plates at 500,000 cell/1 ml with increasing concentrations of drug (0.1, 0.5 and 1 μM and 0.05, 0.1 and 0.2 μM only for BV-173 and RPMI-8402) for 24 and 48 h and incubated at 37 °C. Following the treatment, cells were harvested and stained with Annexin V/PI according to the manufacturer’s instruction (Roche). The percentage of Annexin V-PI-positive cells was determined within 1 × 104 cells of the population by flow cytometry (FACSCanto II, BD Biosciences Pharmingen, San Jose, CA, USA). The mean percentage of Annexin V-PI-positive cells and standard error measurement was calculated from at least two separate experiments.
Western blot analysis
To gain insight into the molecular mechanisms responsible for cell death following treatment, functional analyses by Western blot were performed. Leukemia cell lines were plated in six-well plates at 500,000 cell/1 ml with increasing concentrations of drug for 24 h and incubated at 37 °C. After the treatment, the cells were collected and lysate using a specific buffer made of KH2PO4 0.1 M (pH 7.5), Igepal 1 % (NP-40), β-glicerofosfato 0.1 mM, and complete protease inhibitor cocktail 1× (Roche Diagnostics). For each sample, 30 μg of protein was fractioned on Mini-PROTEAN TGX stain-free precasted gels, blotted to nitrocellulose membranes (Bio-Rad Trans-blot turbo transfer pack), and incubated overnight with the following antibodies: ATM (#2873S), phosphorylated ATM (Ser1981)(#5883S), ATR (#2790S), phosphorylated ATR (Ser428)(#2853S), Chk1 (#2345S), phosphorylated Chk1 (Ser317)(#2344S), phosphorylated Chk1 (Ser296)(#2349) and Chk1 (Ser345)(#2348S), Chk2 (#2662S), phosphorylated Chk2 (Thr68)(#2661S), Cdc25c (#4688S), phosphorylated Cdc25C (Ser216)(#9528S), PARP-1 (#9542), Cdc2 (#9112S), phosphorylated Cdc2 (Tyr15)(#4539S), and phosphorylated H2A.X (Ser139) (γ-H2A.X) (#2577S) from Cell Signaling. Antibody to β-actin came from Sigma (St. Louis, MO, USA). Finally, all these antibodies were detected using the enhanced chemiluminescence kit ECL (GE) and the compact darkroom ChemiDoc-It (UVP).
NALM-6 and BV-173 cells were seeded to poly-d lysine-coated slides, fixed with 4 % PFA (paraformaldehyde), and stained at 37 °C with a mouse anti-H2A.X-Phosphorylated Alexa 647 conjugated antibody (BioLegend). Then, they were treated with DAPI (4.6 diamidino-2-phenylindole; Sigma Aldrich), and the slides were mounted with Mowiol (Calbiochem). Images were acquired with wide field fluorescence microscope Olympus BX61 fully motorized driven by Metamorph software, and the analysis was performed using the freeware ImageJ software.
Gene expression profiling
Gene expression profiling on treated and untreated cells (DMSO 0.1 %) after 24 h of exposure to PF-0077736 was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix Inc. Santa Clara, CA, USA) and following manufacturers’ instructions. Raw data were normalized by using the RMA algorithm and filtered. Genes differentially expressed were selected by analysis of variance (ANOVA) (p value threshold =0.05) using the Partek Genomics Suite software (Partek Incorporated Saint Louis, MO, USA; http://www.partek.com). The most significantly involved process networks were defined by the Metacore software (GeneGo Inc., www.genego.com).
Cell cycle analysis
In order to evaluate the effect of PF-00477736 on cell cycle progression, NALM-6, SUPB-15, RPMI-8402, and BV-173 cell lines were seeded in a 24-well plate at 500,000 cell/1 ml with increasing concentrations of PF-00477736 (0.1, 0.5, and 1 μM for NALM-6 and SUP-B15; 0.05, 0.1, and 0.2 μM for RPMI-8402 and BV-173). After 6 and 24 h, cells were harvested and fixed overnight using ethanol at 70 %. Then, cells were stained using PI/RNase Staining Buffer according to the manufacturer’s instruction (BD Pharmingen), and the cell cycle profile was detected using FACSCanto II instrument. The percentage of the different cell cycle phases was performed using the DNA cell-cycle analysis software for flow cytometry data, ModFit LT (Verity).
In vivo studies
Experiments involving mice were performed in agreement with Italian guidelines and after the approval of the Institutional Review Board of the European Institute of Oncology. Leukemic mice were generated by a single administration of the tumorigenic agent n-ethyl-n-nitrosourea (Sigma, 50 mg/kg intraperitoneally). Spleen cells from leukemic C57BL6/Ly5.2 mice were injected intravenously (2 × 106 cells/mouse) into non-irradiated, recipient C57BL6/Ly5.1 mice. PF-0077736 was administered intraperitoneally starting from day 3 after the transplant of leukemic cells. Mice received doses of PF-0077736 (40 mg/kg each dose) every 3 days for four treatments (q3dx4). Kaplan-Meyer rank test was used to compare the survival rate between treated and control mice.
This study is supported by the European LeukemiaNet, AIL, AIRC research grant (IG 2010 10336), AIRC research grant (5×1000 10007), Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of Integrated Program (PIO), Programma di ricerca Regione—Università 2010–2012, and Fondazione Carisbo, Regione Emilia-Romagna, Bando “Ricerca Innovativa” (Prof. L. Bolondi).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Conter V, Aricò M, Basso G, Biondi A, Barisone E, Messina C, et al. Long-term results of the Italian Association of Pediatric Hematology and Oncology (AIEOP) studies 82, 87, 88, 91 and 95 for childhood acute lymphoblastic leukemia. Leukemia. 2010;24:255–64.View ArticlePubMedGoogle Scholar
- Pui C-H, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;371:1030–43.View ArticlePubMedGoogle Scholar
- Moorman AV. The clinical relevance of chromosomal and genomic abnormalities in B-cell precursor acute lymphoblastic leukaemia. Blood Rev. 2012;26:123–35.View ArticlePubMedGoogle Scholar
- Bailey LC, Lange BJ, Rheingold SR, Bunin NJ. Bone-marrow relapse in paediatric acute lymphoblastic leukaemia. Lancet Oncol. 2008;9(9):873–83.View ArticlePubMedGoogle Scholar
- Fielding AK. Current therapeutic strategies in adult acute lymphoblastic leukemia. Hematol Oncol Clin North Am. 2011;25(6):1255–79.View ArticlePubMedGoogle Scholar
- Blasina A, Hallin J, Chen E, Arango ME, Kraynov E, Register J, et al. Breaching the DNA damage checkpoint via PF-00477736, a novel small-molecule inhibitor of checkpoint kinase 1. Mol Cancer Ther. 2008;7:2394–404.View ArticlePubMedGoogle Scholar
- Kortmansky J, Shah MA, Kaubisch A, Weyerbacher A, Yi S, Tong W, et al. Phase I trial of the cyclin-dependent kinase inhibitor and protein kinase C inhibitor 7-hydroxystaurosporine in combination with fluorouracil in patients with advanced solid tumors. J Clin Oncol. 2005;23:1875–84.View ArticlePubMedGoogle Scholar
- Zabludoff SD, Deng C, Grondine MR, Sheehy AM, Ashwell S, Caleb BL, et al. AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies. Mol Cancer Ther. 2008;7:2955–66.View ArticlePubMedGoogle Scholar
- Sha S-K, Sato T, Kobayashi H, Ishigaki M, Yamamoto S, Sato H, et al. Cell cycle phenotype-based optimization of G2-abrogating peptides yields CBP501 with a unique mechanism of action at the G2 checkpoint. Mol Cancer Ther. 2007;6:147–53.View ArticlePubMedGoogle Scholar
- Zhang C, Yan Z, Painter CL, Zhang Q, Chen E, Arango ME, et al. PF-00477736 mediates checkpoint kinase 1 signaling pathway and potentiates docetaxel-induced efficacy in xenografts. Clin Cancer Res. 2009;15:4630–40.View ArticlePubMedGoogle Scholar
- Syljuåsen RG, Sørensen CS, Nylandsted J, Lukas C, Lukas J, Bartek J. Inhibition of Chk1 by CEP-3891 accelerates mitotic nuclear fragmentation in response to ionizing radiation. Cancer Res. 2004;64:9035–40.View ArticlePubMedGoogle Scholar
- Syljuåsen RG, Sørensen CS, Hansen LT, Fugger K, Lundin C, Johansson F, et al. Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage. Mol Cell Biol. 2005;25:3553–62.PubMed CentralView ArticlePubMedGoogle Scholar
- Parsels LA, Morgan MA, Tanska DM, Parsels JD, Palmer BD, Booth RJ, et al. Gemcitabine sensitization by checkpoint kinase 1 inhibition correlates with inhibition of a Rad51 DNA damage response in pancreatic cancer cells. Mol Cancer Ther. 2009;8:45–54.PubMed CentralView ArticlePubMedGoogle Scholar
- Karp JE, Thomas BM, Greer JM, Sorge C, Gore SD, Pratz KW, et al. Phase I and pharmacologic trial of cytosine arabinoside with the selective checkpoint 1 inhibitor Sch 900776 in refractory acute leukemias. Clin Cancer Res. 2012;18:6723–31.PubMed CentralView ArticlePubMedGoogle Scholar
- Maugeri-Saccà M, Bartucci M, De Maria R. Checkpoint kinase 1 inhibitors for potentiating systemic anticancer therapy. Cancer Treat Rev. 2013;39(5):525–33.View ArticlePubMedGoogle Scholar
- Mailand N, Falck J, Lukas C, Syljuâsen RG, Welcker M, Bartek J, et al. Rapid destruction of human Cdc25A in response to DNA damage. Science. 2000;288:1425–9.View ArticlePubMedGoogle Scholar
- Kuntz K, O’Connell MJ. The G2 DNA damage checkpoint: could this ancient regulator be the achilles heel of cancer? Cancer Biol Ther. 2009;8(15):1433–9.View ArticlePubMedGoogle Scholar
- Sørensen CS, Syljuåsen RG, Falck J, Schroeder T, Rönnstrand L, Khanna KK, et al. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell. 2003;3:247–58.View ArticlePubMedGoogle Scholar
- Chen M-S, Ryan CE, Piwnica-Worms H. Chk1 kinase negatively regulates mitotic function of Cdc25A phosphatase through 14-3-3 binding. Mol Cell Biol. 2003;23:7488–97.PubMed CentralView ArticlePubMedGoogle Scholar
- Bartek J, Lukas J. Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell. 2003;3(5):421–9.View ArticlePubMedGoogle Scholar
- Stolz A, Ertych N, Bastians H. Tumor suppressor CHK2: regulator of DNA damage response and mediator of chromosomal stability. Clin Cancer Res. 2011;17:401–5.View ArticlePubMedGoogle Scholar
- Rhodes DR, Yu J, Shanker K, Deshpande N, Varambally R, Ghosh D, et al. ONCOMINE: a cancer microarray database and integrated data-mining platform1. Neoplasia. 2004;6:1–6.PubMed CentralView ArticlePubMedGoogle Scholar
- Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res. 1993;53:3976–85.PubMedGoogle Scholar
- Peng CY, Graves PR, Thoma RS, Wu Z, Shaw AS, Piwnica-Worms H. Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science. 1997;277:1501–5.View ArticlePubMedGoogle Scholar
- Xu Y, Price BD. Chromatin dynamics and the repair of DNA double strand breaks. Cell Cycle. 2011;10:261–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Matsuura K, Wakasugi M, Yamashita K, Matsunaga T. Cleavage-mediated activation of Chk1 during apoptosis. J Biol Chem. 2008;283:25485–91.View ArticlePubMedGoogle Scholar
- Landau HJ, McNeely SC, Nair JS, Comenzo RL, Asai T, Friedman H, et al. The checkpoint kinase inhibitor AZD7762 potentiates chemotherapy-induced apoptosis of p53-mutated multiple myeloma cells. Mol Cancer Ther. 2012;11(8):1781–8.View ArticlePubMedGoogle Scholar
- Wisdom R, Johnson RS, Moore C. c-Jun regulates cell cycle progression and apoptosis by distinct mechanisms. EMBO J. 1999;18:188–97.PubMed CentralView ArticlePubMedGoogle Scholar
- Went P, Agostinelli C, Gallamini A, Piccaluga PP, Ascani S, Sabattini E, et al. Marker expression in peripheral T-cell lymphoma: a proposed clinical-pathologic prognostic score. J Clin Oncol. 2006;24:2472–9.View ArticlePubMedGoogle Scholar