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Supplementary Materials and Methods
Enzyme Kinetics
Assays for enzyme kinetic studies measured the CHK1-catalyzed phosphorylation of a 23-amino
acid peptide corresponding to amino acids 205-225 of CDC25C isoform A. In this assay CHK1
was added to a solution containing 20 µM peptide, 4 µM ATP and 0.4 µC [γ 32P]-ATP in 20 mM
HEPES, pH 7.5, 5 mM MgCl2 and 0.1% NP-40. Following a 10 minute incubation at 23ºC, the
kinase reaction was stopped by the addition of H3PO4 to 130 mM, the precipitate captured on
filter plates and incorporated γ32P measured by liquid scintillation counting. The inhibition
constant (Ki) was calculated by the Dixon method (1).
Kinase selectivity assays
Protein kinase selectivity assays were performed for 224 protein kinases at Upstate/Millipore.
Time-lapsed Microscopy
Time-lapsed microscopy was conducted as previously described (2).
Additional antibodies
pCHK1 (S345) (#2348), CHK2 (#2662), pCHK2 (S516) (#2669), pS6 Ribosomal Protein
(S235/S236) (#4857), pRSK (S380) (#9335), pRSK (T573) (#9346), GAPDH (#2118) and p44/42
Map Kinase (T202/Y204) (#9101) antibodies were from Cell Signaling Technology. Other
antibodies were to pCHK1 (S296) (Epitomics, #EPR915) and CHK1 (Santa Cruz, #SC-8408).
Supplementary References
1.
Nakamura Y, Oka M, Soda H, Shiozawa K, Yoshikawa M, Itoh A, et al. Gefitinib ("Iressa", ZD1839),
an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance
protein/ABCG2-mediated drug resistance. Cancer research. 2005;65:1541-6.
2.
Van Horn RD, Chu S, Fan L, Yin T, Du J, Beckmann R, et al. Cdk1 activity is required for mitotic
activation of aurora A during G2/M transition of human cells. The Journal of biological chemistry.
2010;285:21849-57.
3.
King C, Diaz H, Barnard D, Barda D, Clawson D, Blosser W, et al. Characterization and preclinical
development of LY2603618: a selective and potent Chk1 inhibitor. Investigational new drugs.
2014;32:213-26.
Supplementary Figure Legends
Figure S1. LY2606368 is an ATP competitive inhibitor of CHK1. The Ki was determined for
LY2606368 and CHK1 with ATP and a CDC25C peptide. The kinetic data were charted in a Dixon
plot with the linear equations of y = 0.0369x + 0.0369 for 1 µM ATP and y = 0.0035x + 0.0063
for 10 µM ATP. The x intercept for these two equations was -0.92 nM, giving a Ki of ~ 0.9 nM. ◊
= 1 µM ATP; □ = 10 µM ATP.
Figure S2. LY2606368 is a potent catalytic and phenotypic inhibitor of CHK1. A. The ability of
LY2606368 to inhibit DNA damage-induced CHK1 autophosphorylation was tested in HeLa,
Calu-6 and HT-29 cell lines. Calu-6 and HT-29 cells were treated with 60 nM gemcitabine and
HeLa cells with 100 nM doxorubicin. 22 hours following drug treatment, LY2606368 was added
to the cells and lysates prepared following two hours of treatment. CHK1 autophosphorylation
was measured as described (3). Chk1 activity was reduced with an IC50 < 1.0 nM in all three celllines. □ = HeLa cells with 100 nM doxorubicin ; Δ = Calu-6 with 60 nM gemcitabine; ∇ = HT-29
cells with 60 nM gemcitabine. B. The ability of LY2606368 to prevent the CHK1-dependent
G2/M checkpoint was determined in HeLa cells treated with 125 nM doxorubicin (3). After 24
hours incubation the cells were arrested at the G2/M checkpoint and dilutions of LY2606368
were added for seven hours. Cells were then fixed and immunostained for the pH3(S10) mitotic
marker. The mean intensity of pH3(S10) staining cells was measured and plotted.
Figure S3. LY2606368 inhibits DNA damage-dependent autophosphorylation of CHK2. To
determine if LY2606368 inhibits CHK2 kinase in cells, HT-29 cells were pre-treated with
different concentrations of LY2606368 for 15 minutes followed by the addition of 200 nM of
the DNA damaging agent, SN-38. After six hours, cells were collected, lysed and analyzed by
immunoblot for phosphorylation of DNA damage and checkpoint proteins. In particular the
relative levels of CHK2 autophosphorylated on serine 516 were measured (arrow).
Figure S4. LY2606368 does not inhibit RSK activity in cells. In order to determine if LY2606368
inhibits RSK in cells, HeLa cells were treated in a manner to cause the RSK-dependent
phosphorylation of ribosomal protein S6 on serines 235 and 236. Growing HeLa cells were
washed of 10% serum-containing medium and cultured overnight in medium containing 0.5%
FBS. Cells were then treated with either 0.4% DMSO, 100nM LY2606368, 10uM BI-D1870 (RSK
inhibitor) or 20uM U0126 (MEK inhibitor) for five hours total. RSK activity was than stimulated
by the addition of phorbol 12-myristate 13-acetate (PMA) for the final 30 minutes of the
compound incubation. Cells were then collected, lysed and analyzed by immunoblot for
phosphorylation of key sites involved in PMA-RSK signaling. Activated ERK phosphorylates RSK
on threonine 573 to activate the C-terminal catalytic domain which then autophosphorylates
RSK on serine 380, a PDPK1 docking site. PDPK1 then phosphorylates the N-terminal kinase
domain of RSK which then phosphorylates substrates such as S6 (S235/S236). RSK amino acid
numbering in this figure is taken from RSK1.
Figure S5. Treatment of Calu-6 lung cancer cells with LY2606368 results in rapid accumulation
of DNA strand breaks in early S-phase. Calu-6 cells were treated with 0.4% DMSO or 100 nM
LY2606368 for seven hours. The plates were fixed and stained for DNA, pH2AX and DNA strand
breaks by TUNEL assay (see Materials and Methods). Analysis was by high content imaging
(Thermo Scientific Cell Insight NXT). After seven hours of compound treatment, there was a
large decrease in G2M cells and a corresponding increase in G1 and early S-phase cells.
Additionally, significant DNA damage had occurred as indicated by increased pH2AX and TUNEL
staining. In order to determine when during the cell cycle LY2606368 was causing DNA damage,
the intensity of pH2AX and TUNEL staining for each cell was plotted as a function of DNA
content. The cells staining most intensely for DNA damage were those in early to mid-S phase.
The grey bar along the X-axis designates cells in S-phase. X and Y axis are x105 units of intensity.
Figure S6. Timelapse photomicroscopy of LY2606368 treated HeLa cells confirms that S-phase
transit is required for aberrant mitosis and cell death. HeLa cells were treated with 100 nM of
LY2606368 and imaged in culture over 24 hours with DIC optics. Representative timepoints are
shown at the time noted after addition of compound as elapsed hours:minutes. Normal mitotic
chromosome alignment is seen for up to 3.5 hours with mitotic defects visible by seven hours
followed by cell death. Non-dividing cells remain viable for at least 24 hours. Not shown are
0.5% DMSO treated control cells with normal cell division occurring throughout the time of
culture. P- prophase. M- metaphase, A- anaphase, T- telophase.
Figure S7. Staurosporine causes rapid appearance of apoptotic TUNEL staining, but not H2AX
serine 139 phosphorylation . As a control for the apoptotic induction of double stranded break
formation in the experiment represented in figure 2 in the text, HeLa cells were treated with
500 nM Staurosporine in the presence or absence of 20 µM Z-VAD-FMK for seven hours.
Following fixation the plate was stained for DNA (Hoechst 33342) and double-stranded DNA
breaks (DSB) using TUNEL and an antibody specific for pH2AX (S139). A. Representative visual
fields of Staurosporine or Staurosporine plus Z-VAD-FMK treated cells were photographed with
a 10x objective using the appropriate filter to capture staining as labeled above the figure. B.
The relative intensity for each stain was measured on a single cell basis as described in
Methods. DNA content (top row) is shown by plotting DNA intensities (x-axis) verses a sliding
average for the number of cells staining at that intensity (y-axis). pH2AX (S139) (middle row) or
TUNEL (bottom row) staining intensity was plotted (y-axis) versus the relative DNA content (xaxis) for each cell. No compound treatment controls are shown in figure 2 of the main text.
Supplementary table descriptions
Table 1. Protein kinases tested for inhibition by LY2606368. These 224 protein kinases were
tested for sensitivity to LY2606368. The species of enzyme origin are listed in parentheses
following the name of each protein kinase as are any oncogenic amino acid substitutions.
Table 2. Protein kinases sensitive to LY2606368 with an IC50 less than 10,000 nM. 24 protein
kinases out of the 224 listed in Table 1 had IC50 values below 10 µM for LY2606368.
Table 3. Induction of DNA damage by LY2606368 is not caused by caspase-dependent
apoptotic DNA fragmentation. The percentage of cells staining positive for TUNEL and pH2AX
(S139) staining following drug combination treatments was determined for the samples
described in figure 2 in the main text and supplementary figure 7. In addition the mean nuclear
area was determined for each sample as a means for scoring condensed mitotic and apoptotic
nuclei.
Table 4. LY2606368-dependent chromosome fragmentation is not caused by caspasedependent apoptosis. The number of nuclei with prometaphase and metaphase chromosomes
was determined for the drug treatment groups described in figure 4 of the main text and again
for the duplicate experiment described in supplementary figure 7 in which the apoptosis
inhibitor Z-VAD-FMK was also included. Each nucleus was labeled as having intact, broken or
shredded chromosomes, and the percentage of category was determined for each treatment
group.