SUPPLEMENTARY MATERIALS AND METHODS
Reagents and drugs. All reagents were purchased from Sigma unless otherwise noted.
PD173955, GSK-690693, and vandetanib were synthesized according to published patent
literature. Axitinib, ABT-869, BMS-387032, CP 690,550, pazopanib, R406, and VX680
were synthesized by Park Place Research (Cardiff, UK). Staurosporine was purchased from
IRIS Biotech (Marktredwitz), Ro31-7549 from Enzo Life Sciences (Lörrach), sunitinib
from ACC (Rochester), dasatinib from Toronto Research Chemicals, bosutinib, erlotinib
and sorafenib from Selleck (London, Canada), TKI258 and PD184352 from Axon
Medchem (Groningen, Netherlands), and flavopiridol from Onicon Chemical Co.
(Hangzhou, China).
Patient samples and cell isolation. Fresh blood samples were enriched by applying BRosetteSep (Stem Cell Technologies) to aggregate unwanted cells with erythrocytes and
Ficoll-Hypaque (Seromed) density gradient purification resulting in purity >98% of
CD19+/CD5+ CLL cells. CLL cells were characterized for CD19, CD5, CD23, FMC7,
CD38, ZAP-70, slgM, slgG, CD79a and CD79b expression on a FACS Canto flow
cytometer (BD PharMingen) and the IgVH-hypermutational status of CLL patients was
analyzed as previously reported (1). Controls were isolated from healthy blood donors
using anti-CD19-MACS-beads (Miltenyi) resulting in at least 95% CD19+ B-cells.
Cell culture. Freshly purified CLL cells were cultured in RPMI media supplemented with
10 % heat-inactivated fetal calf serum (FCS) and antibiotics (100 Units/ml penicillin and
100 µg/ml streptomycin) at 37 °C in a humidified atmosphere containing 5 % carbon
1
dioxide (1). Compounds were dissolved in DMSO and applied for CLL inhibition using 1
nM to 100 µM concentrations. DMSO-treated cells were taken as baseline control, the final
DMSO concentration in all treatments was 0.1%.
Kinase expression profiling experiment. Kinobeads were prepared by immobilization of
ATP-mimetics on sepharose beads as described (2). BMS-387032 was immobilized via the
piperidine moiety using the same protocol. Cell extracts of CLL cells and Ramos cells were
prepared as described below and incubated for 60 min with a 17% bead suspension
containing either kinobeads or BMS-387032 beads. Subsequently, the beads were washed
with lysis buffer containing 0.2% Igepal-CA630, collected by centrifugation, and beadbound proteins were eluted with NuPAGE LDS buffer (Invitrogen) containing 50 mM DTT
for 30 minutes at 50°C followed by alkylation with 20mg/ml iodoacetamide for 30 minutes.
Samples were briefly run into 4-12% NuPAGE gels, stained with colloidal Coomassie blue,
digested with trypsin and subsequently labeled with the TMT isobaric tagging reagents
(ThermoFisher Scientific). Samples were then combined, dried in vacuo and reconstituted
in 10 µl of water containing 20 mM ammonium formiate (pH 11). Labeled peptides were
subsequently separated within 100 min using reversed phase chromatography at pH11 on a
1 mm X-bridge C-18 column (Waters, UK) at a flow rate of 30 µl/min. Peptide containing
fractions were collected and fractions containing early and late eluting peptides were
combined to yield a total of 17 combined fractions. Samples where then dried and
reconstituted in 0.1% formic acid for further analysis using LC-MS.
Kinobeads expression charting of CDK9 interacting proteins in B cells and CLL patient
cells was done as described above with the exception that BMS-387032 was directly added
to the kinobeads mix (see below).
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Kinobeads assays. Kinobeads were prepared by immobilization of ATP-mimetics on
sepharose beads as described (2). In addition to the previously reported set of seven ligands,
BMS-387032 was included on the beads by immobilization via the piperidine moiety. For
kinobeads profiling, CLL cells were pelleted and homogenized on ice in lysis buffer (50
mM Tris/HCl pH 7.5, 5% glycerol, 1.5 mM MgCl2, 150 mM NaCl, 25 mM NaF, 1 mM
Na3VO4, 1 mM DTT, 50nM Calyculin A and 0.8% Igepal-CA630 supplemented with
protease inhibitor and phosphatase inhibitor cocktails (Roche)). Lysates were cleared by
centrifugation, and adjusted to 5 mg/ml total protein concentration and 0.4 % IgepalCA630. Compounds were dissolved in DMSO and added at various concentrations to 1ml
lysate samples; the final DMSO concentration was 0.5%. After 45 minutes at 4°C, 30 µl
(100 µl for the “expression profiling” experiment) of a 17% kinobeads suspension in lysis buffer
containing 0.4% Igepal-CA630 was added to each sample and incubated for additional 60
minutes. Subsequently, the beads were washed with lysis buffer containing 0.2% IgepalCA630, collected by centrifugation, and bead-bound proteins were eluted with NuPAGE
LDS buffer (Invitrogen) containing 50 mM DTT for 30 minutes at 50°C. Eluates were
alkylated with 20mg/ml iodoacetamide for 30 minutes, separated on 4-12% NuPAGE gels
(Invitrogen) and stained with colloidal Coomassie blue. For live cell profiling, compounds
were added at concentrations of 10, 1.0, and 0.1 µM (final DMSO 0.1%) to 5 x 108 CLL
cells per experiment in RPMI/10% FCS medium for 6 hours. Subsequently cells were
harvested by centrifugation, washed twice with PBS buffer and lysed as described above.
For kinobeads profiling 800 µl cell lysate corresponding to 1.7 mg total protein was mixed
with 50 µl kinobeads suspension and analyzed as described above. For the geldanamycin
experiment 2.5 x 108 cells per data point were used. Kinobeads profiles were established
using 1.1 mg protein in 400 µl cell lysate with 25 µl beads suspension.
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1.
Pallasch CP, Schwamb J, Königs S, Schulz A, Debey S, Kofler D, et al. Targeting lipid
metabolism by the lipoprotein lipase inhibitor orlistat results in apoptosis of B-cell chronic
lymphocytic leukemia cells. Leukemia. 2008 Mar;22(3):585-92.
2.
Bantscheff M, Eberhard D, Abraham Y, Bastuck S, Boesche M, Hobson S, et al.
Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase
inhibitors. Nat Biotechnol 2007 Sep;25(9):1035-44.
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