Supplemental Data

250 Control 200 JNJ-28312141

150

100 ** ** macrophages/field + 50

F4/80 0 Csf-1r -/- -/+ +/+

Supplemental Figure 1. JNJ-28312141 did not reduce hepatic macrophage counts in

mice deficient in CSF-1R. Groups of four each of male CSF-1R homozygous -

deficient (-/-), heterozygotes (-/+) or wild-type (+/+) mice > 8 weeks of age were provided milled chow for five days without (open bars) or with (filled bars) JNJ-

28312141 in sufficient quantity to deliver approximately 100 mg/kg compound per day.

On the sixth day, the mice were sacrificed and sections of formalin-fixed paraffin- embedded were stained for F4/80 positive cells (macrophages) as described in

Materials and Methods. Positive cells per 100X-field were counted and summed from 5- fields/mouse. Values are group means and SEM. **p<0.01 vs. corresponding compound-free control.

Table 1: Evaluation of JNJ-28312141 for inhibition of fifty-nine : Invitrogen SelectScreenTM Profiling Servicea Kinases inhibited >50% at 0.1 µM JNJ-28312141 CSF-1R, FLT3, TRKA, LCK Kinases inhibited >50% at 1 µM but <50% at 0.1 µM JNJ-28312141 Blk, BMX, BTK, FES, Fgr, Fyn, PDGFR-β, Yes Kinases not inhibited at least 50% at 1 µM JNJ-28312141 Abl1, Akt1, Akt2, Akt3, Arg, Brk, CaMKII-α, CHK1, CHK2, CK2-α1, MET, Csk, EGFR, EphA3, EphB4, ERK1, ERK2, FGFR1, FGFR2, FGFR3, FGFR4, GSK3-α, GSK3-β, Hck, Hyl, IGF1R, IRAK4, Lyn A, Lyn B, MAPKAP-K2, MAPKAP-K3, MAPKAP-K5, NEK2, PHKG2, PKA, PKC (alpha, beta I, beta II, delta, epsilon, eta, gamma, iota, theta, zeta), RSK2, Src a Kinase reactions were performed in the presence of 100 μM ATP and inhibition was determined for each kinase in the presence of 1 and 0.1 μM JNJ-28312141.

Table 2: Evaluation of JNJ-28312141 for inhibition of fifty-seven kinases: Millipore KinaseProfiler Assay Service Kinases inhibited >50% at 0.1 µM KIT, AXL Kinases inhibited >50% at 1 µM but <50% at 0.1 µM ALK, PDGFRα, PKD2, TrkB, Kinases not inhibited at least 50% at 1 µM AMPK, Aurora-A, CaMKIV, CDK/cyclinB, CDK2/cyclinA, CDK2/cyclinE, CDK3/cyclinE, CDK5/p35, CDK6/cyclinD3, CDK7/cyclinH, CK1, CK1d, c-RAF, CSK, EphB2, IKKα, IKKβ, IR, JNK1α1, JNKα2, JNK3, MAPK1, MAPK2, MEK1, MKK4, MKK6, MKK7b, MSK1, MST2, NEK2, p70S6K, PAK2, PAR-1Ba, PDK1, PKBα, PKBβ, PKBγ, PRAK, PRK2, ROCK-II, Ros, Rsk1, Rsk3, SAPK2a, SAPK2b, SAPK3, SAPK4, SGK, Syk, Tie, ZAP-70 a Kinase reactions were performed in the presence of 100 μM ATP and inhibition was determined for each kinase in the presence of 1 and 0.1 μM JNJ-28312141.

2 Table 3. Inhibitory activity of JNJ-28312141 in cellular assays

a Cells/cell-line Target Stimulus Endpoint IC50 (μM)

Mouse bone marrow- CSF-1R CSF-1 proliferation 0.003 derived macrophagesb CD14+ monocytesb CSF-1R CSF-1 Induced MCP-1 0.003

CSF-1R/HEKb CSF-1R CSF-1 Phospho-CSF-1R 0.005

MV-4-11b ITD-FLT None proliferation 0.021

Mo7eb KIT SCF proliferation 0.041

FLT3/Baf3b FLT3 FLT3-ligand Phospho-FLT3 0.076

TF-1b TRKA NGF proliferation 0.15

Jurkatc LCK Anti-CD3, PMA Induced IL-2 protein 3.9

AXL/HEKb AXL GAS6 Phospho-AXL 2.6

H460, MDA-MB-231, General None proliferation >5.0 A375d

3 a Means of at least three independent determinations except for FLT3/Baf3, Jurkat, H460, MDA-MD-

231, and A375 assays which were determined once. b Assays described in Materials and Methods of accompanying manuscript.

c Inhibition of cellular LCK was determined as described (Maier JA, Brugel TA, Sabat M, et al. Development of N-4,6-pyrimidine-N-alkyl-N’-phenyl ureas as orally active inhibitors of lymphocyte specific tyrosine kinase. Bioorg Med Chem Lett 2006;16:3646-3650.).

Jurkat cells (105/well) (ATCC) with graded concentrations of JNJ-28312141 were plated into wells pre-coated with CD3ε antibody (R&D Systems). PMA was added to a final concentration of 10 ng/ml and IL-2 secreted into 24 h culture supernatants was measured by

ELISA (R&D Systems). d H460 lung adenocarcinoma, MDA-MB-231 mammary carcinoma and A375 melanoma cells

(1000/well) were cultured in DMEM media containing 10% FCS and 0 or 5 µM JNJ-28312141 in 96 well dishes. On days 0, 1, 2, 3, 4, and 7, replicate plates were assayed for cell number using CellTiter-

Glo® reagent (Promega). One hundred μL of CellTiter-Glo® reagent was added to each well and luminescence (relative light units, RLU) was measured using a Berthold Orion microplate luminometer. In vitro growth curves were not different in the presence of 0 or 5 μM JNJ-28312141.

4 Table 4. CSF-1, MCP-1 and macrophage content of human tumor xenografts

ng cytokine/gram tumora Macrophage scoreb

Human Mouse Human Mouse Xenograft Stoma tumor CSF-1 CSF-1 MCP-1 JEc MDA-MB-231 breast 80 0.6 59 4.5 3 1 carcinoma SKOV3 breast 69 0.3 0.2 2.6 2 1 carcinoma H460 non-small cell lung 35 0.6 0.1 1.1 3 1 carcinoma A375 11 0.6 1.2 3.6 2 1 melanoma SW620 colon 1.7 0.4 0.7 4.2 2 1 carcinoma a Tumors were grown to a mass of approximately 0.5- 1 g. Three to five tumors per xenograft type were pooled and 10% lysates in 1% NP40 in saline were prepared. Cleared lysates were assessed for cytokine content using specific ELISAs purchased from R&D Systems. b Sections prepared from formalin-fixed, paraffin embedded tumors (0.5 – 1 g) were stained for F4/80 positive macrophages as described in Materials and Methods and a three point method was used to score macrophage content, viz., 0 = no macrophages, 1 = few macrophages, 2 = moderate numbers of macrophages, and 3 = abundant macrophages. Separate scores were assigned to regions of the tumors primarily consisting of host stroma and tumor cell areas. c JE is a mouse homolog of human MCP-1.

5