CHK1 plays a critical role in the anti-leukemic activity of the wee1 inhibitor MK-1775 in acute myeloid leukemia cells

To investigate the effects of MK-1775 in AML cells, first we determined the protein levels of Wee1, p-CDK1, CDK1, p-CDK2, CDK2 and MYT1 in six AML cell lines. The proteins were expressed at variable levels (Figure 1A). Next, we tested drug sensitivity by MTT assays. The IC₅₀s ranged from about 200-400 nM after 72 h treatment (Figure 1B). To determine if MK-1775 induces cell death, we treated the cell lines with up to 500 nM MK-1775 for 48 h and assessed viability using the trypan blue exclusion assay. As shown in Figure 1C, there was a concentration-dependent increase in dead cells for all six cell lines tested. Annexin V/PI staining and flow cytometry analysis revealed that MK-1775 caused concentration-dependent increase in apoptotic cells (Figure 1D). This was accompanied by decreased p-CDK1 and p-CDK2 and increased total CDK1 and CDK2 (Figure 1E). There was also a concentration-dependent increase of phosphorylated H2AX (γH2AX), indicating increased DNA double-strand breaks (DSBs) [γH2AX is an established biomarker for DNA DSBs [20]]. The CTS and U937 cell lines were chosen as representative AML cell lines for further mechanistic studies.

Next, we determined ex vivo MK-1775 sensitivity in freshly isolated AML blast samples (n = 29). MK-1775 IC₅₀s ranged from 217 nM (AML#16) to 6.4 μM (AML#27, Table 1). Similar to the cell lines, we detected a concentration-dependent increase in apoptosis for three patient samples after MK-1775 treatment (Figure 2A). Interestingly, the median MK-1775 IC₅₀ for the diagnostic (n = 23) and relapse samples (n = 6) were similar (1176 and 896.1 nM, respectively, p = 0.936, Figure 2B). In addition, we found that patient samples harboring t(15;17) translocation (n = 5) were significantly more sensitive to MK-1775 than non-t(15;17) samples (n = 24, p = 0.007, Figure 2C). Next, we generated a cytarabine resistant cell line (HL60/Ara-C) to determine if they would exhibit cross-resistance to MK-1775. Despite being approximately 600-times more resistant to cytarabine than the parental HL-60 cells (Figure 2D), HL-60/Ara-C cells were more sensitive to MK-1775 treatment (Figure 2E). There was a concentration-dependent increase in apoptotic cells for the HL60/Ara-C cell line, whereas the parental cell line remained relatively unaffected by MK-1775 concentrations up to 500 nM. A concentration-dependent decrease in p-CDK1 and p-CDK2 accompanied by increase of γH2AX was detected in cells from patient AML#10 as well as in HL60/Ara-C (Figure 2F). HL60 cells treated with 500 nM MK-1775 had a small increase of γH2AX and no change in p-CDK1 or p-CDK2, probably due to very low levels of expression prior to drug treatment.

Next, we investigated the effects of MK-1775 on cell cycle progression in both CTS and U937 cells. Treatment with MK-1775 for 48 h revealed a concentration-dependent decrease of the G2/M population accompanied by concentration-dependent increase of the sub-G1 population (Additional file 1: Figure S1). Time course experiments revealed a time-dependent increase of the sub-G1 population and abrogation of the G2 checkpoint for both cell lines (Figure 3A&C and Additional file 2: Table S1). These changes were accompanied by a time-dependent increase of γH2AX as well as a decrease of p-CDK1 and p-CDK2 (Figure 3B&D). Increased total CDK1 levels were detected following MK-1775 treatment. Increased p-CHK1 was detected as early as 4 h following MK-1775 treatment.

To determine if CDK activity is required for MK-1775 induced DNA damage and apoptosis, we treated AML cells with Roscovitine, a CDK inhibitor. There was a concentration-dependent decrease in viable cells after Roscovitine treatment for both CTS and U937 cells, as measured by MTT assays (Additional file 1: Figure S2A). Increased γH2AX and p-CHK1 was observed following 8 h MK-1775 treatment, which was substantially abolished by the addition of Roscovitine (Figure 4A and Additional file 1: Figure S3A). MK-1775 induced apoptosis at both 8 h and 24 h in both CTS and U937 cell lines. Combined MK-1775 and Roscovitine treatment abolished MK-1775 induced apoptosis (Figure 4B&C). MTT assays revealed clearly antagonistic anti-leukemic interactions as shown in the standard isobolograms (Figure 4D&E). Similar results were obtained with primary AML patient samples (Figure 4F-H). These results demonstrate that CDK activity is required for MK-1775 anti-leukemic activity in AML cells.

CTS and U937 cells were treated with MK-1775 plus the selective CHK1 inhibitor LY2603618 for 8 h. There was a concentration-dependent decrease in viable cells following LY2603618 treatment for both CTS and U937 cells (Additional file 1: Figure S2B). γH2AX levels were increased after MK-1775 treatment, which was further increased by the addition of LY2603618 (Figure 5A and Additional file 1: Figure S3B). CDK1 and CDK2 phosphorylation was lower after MK-1775 treatment compared to vehicle control and was further decreased in the combined MK-1775 and LY2603618 treatment. MK-1775-induced apoptosis was significantly enhanced by the addition of LY2603618 (Figure 5B&C). MTT assays revealed synergistic anti-leukemic interactions from the combined MK-1775 and LY2603618 treatment as displayed by the standard isobolograms (Figure 5D&E). Similar results were obtained with primary AML patient samples (Figure 5F-H).

MK-1775 (MK), Roscovitine (Rosc), and LY2603618 (LY) were purchased from Selleck Chemicals (Houston, TX, USA). Cytarabine (ara-C) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The THP-1, MV4-11, U937, HL-60 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). The OCI-AML3 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). MOLM-13 cells were purchased from AddexBio (San Diego, CA, USA). The CTS cell line was a gift from Dr. A Fuse from the National Institute of Infectious Diseases, Tokyo, Japan. The cell lines were cultured in RPMI 1640 (except OCI-AML3, which was cultured in alpha-MEM) with 10-15% fetal bovine serum (Hyclone, Logan, UT, USA), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. All cells were cultured in a 37°C humidified atmosphere containing 5% CO2/95% air.

AML blast samples obtained either at diagnosis or at relapse were purified by standard Ficoll-Hypaque density centrifugation, then cultured in RPMI 1640 with 20% fetal bovine serum supplemented with ITS solution (Sigma-Aldrich) and 20% supernatant of the 5637 bladder cancer cell line [as a source of granulocyte-macrophage colony-stimulating factor [27],[28]].

HL-60 cytarabine resistant cells (designated HL-60/Ara-C) were generated by stepwise selection of HL-60 cells in the presence of cytarabine, until they could be maintained in the presence of 600 nM cytarabine.

Diagnostic blast samples were obtained from the First Hospital of Jilin University. Written informed consent was provided according to the Declaration of Helsinki. This study was approved by the Human Ethics Committee of the First Hospital of Jilin University. Clinical samples were screened for FLT3-ITD, NPM1, C-kit, CEBPA, IDH1, IDH2 and DNMT3A gene mutations. The samples were also screened for the following fusion genes by real-time RT-PCR: PML-RARα, BCR-ABL, AML1-MDS1, MLL-AF10, MLL-AF4, MLL-ELL, SET-CAN, TLS-ERG, NPM-RARα, E2A-PBX1, AML1-EAP, MLL-AF17, MLL-AF6, MLL-ENL, SIL-TAL1, HOX11, PLZF-RARα, TEL-AML1, DEK-CAN, MLL-AF1p, MLL-AF9, NPM-ALK, TEL-ABL, EIP1L1-PDGFRA, AML1-ETO, CBFB-MYH11, E2A-HLF, MLL-AF1q, MLL-AFX, NPM-MLF1, dupMLL, and TEL-PDGFB.

In vitro cytotoxicities of the AML cells were measured by using MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazoliumbromide, Sigma-Aldrich) assays, as previously described [29],[30]. Briefly, the cells were treated with variable concentrations of MK-1775 for 72 hours. MTT was added to a final concentration of 1 mM and cells were incubated for 4 hours at 37°C. The cells were lysed overnight using 10% SDS in 10 mM HCL and plates were read at 590 nm using a microplate reader. IC₅₀ values were calculated as drug concentrations necessary to inhibit 50% growth compared to vehicle control treated cells. The cell line IC₅₀ values are presented as mean values ± standard errors from at least three independent experiments. The IC₅₀ values for the patient samples are means of duplicates from one experiment, due to limited sample. Patient samples for the combined drug treatments were chosen based on sample availability.

Cells were lysed in the presence of protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN, USA). Whole cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Inc., Rockford, IL, USA) and immunoblotted with anti-p-CDK1 (Y15), -CDK1, -p-CDK2 (Y15), -CDK2, -Wee1, -MYT1, -γH2AX, -CHK1, -p-CHK1 (S345), -p-CDC25C (S216) (Cell Signaling Technology, Danvers, MA, USA), or -β-actin (Sigma-Aldrich) antibody, as previously described [31],[32]. Immunoreactive proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE, USA), as described by the manufacturer. Western blots were repeated at least three times and one representative blot is shown. Only one patient sample was used for MK-1775 treatment and subsequent Western blot analysis due to the limited amount of sample available. Densitometry measurements were made using Odyssey V3.0 (Li-Cor), normalized to β-actin, and then fold change relative to no drug control was calculated.

AML cells were treated with MK-1775, Roscovitine, LY2603618, or the indicated combinations and subjected to flow cytometry analysis to determine drug-induced apoptosis using an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis Kit (Beckman Coulter; Brea, CA, USA), as previously described [29],[33]. Results are expressed as percent of annexin V + cells. Experiments with AML cell lines were performed 3 independent times in triplicates and data presented are from one representative experiment, while patient sample experiments were performed once in triplicates. Data are presented as mean values ± standard errors from one representative experiment. Due to limited sample, only three patient samples were evaluated for MK-1775-induced apoptosis by flow cytometry.

Cells were treated with the indicated drugs for up to 48 h. The cells were harvested and fixed with ice-cold 80% (v/v) ethanol for 24 h. The cells were pelleted, washed with PBS, and resuspended in PBS containing 50 μg/mL propidium iodide (PI), 0.1% Triton X-100 (v/v), and 1 μg/mL DNase-free RNase. DNA content was determined by flow cytometry analysis using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) as previously described [34]. Cell cycle analysis was performed using Multicycle software (Phoenix Flow Systems, Inc., San Diego, CA, USA). Histograms were created using FlowJo v7.6.5 (Tree Star, Ashland, OR, USA).

Differences in cell apoptosis between treated (individually or combined) and untreated cells were compared using the pair-wise two-sample t-test. The p value for the differences between MK-1775 IC₅₀s for the groups of patient samples was calculated using the Mann-Whitney U test. Statistical analyses were performed with GraphPad Prism 5.0.