Supplementarytable, Figures and Figure Legend

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LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity against MET, MST1R, and other oncoproteins, and displays anti-tumor activities in mouse xenograft models.Yan SB et al.

Corresponding Author: S. Betty Yan

Address: Lilly Research Laboratories, DC0522, 307 E. McCarty Street, Indianapolis, IN 46285

Email:

SupplementaryTable, Figures and Figure Legend

Table S1. Anti-proliferative activity of LY2801653 in MET expressing human tumor cell lines. Anti-proliferative activity of LY2801653 was tested using the CellTiter Glo viability/proliferation assay. The growth rate for each cell line was determined and the compound was incubated for duration of 2 cell doublings in each assay.

a - For U-87MG, the concentration response curve produced a plateau at 45% inhibition; thus the relative IC50 was plotted for this cell line.

Figure.S1. Determination of Ki (A) and Koff (B) of LY2801653 on the inhibition of MET.

The dissociation constant (Ki) and mode of action of LY2801653 were obtained by fitting theexperimental data to a mixed model inhibition equation using GraphPad Prism.A series of 8 ATPconcentrations (0-400 μM) and 10 inhibitor concentrations(0-250nM LY2801653). E (in Figure legend) is the assay control which represents the reaction rate of enzyme alone (no compound).

(A)

(B)

Figure.S2. Vessel normalizationand anti-apoptotic/anti-proliferative effects of LY2801653 in U-87MG xenograft model. Quantitative data for LY2801653-treated groups (1.3 mg/kg or 12 mg/kg daily dose) compared with vehicle controls in morphology of angiogenesis and proliferative parameters. Proliferation index is a ratio of the total area of proliferating cells to area of vessel in the tumor. Treatment groups (N=10 animals per group) were compared using the Dunnet’s analysis in JMP statistics software version 6.0.2 (SAS).

Figure. S3. Dendrogram of the in vitro cell-based activity of LY2801653 with IC50 less than 200nM.

Figure. S4.In vivo anti-tumor effect of LY2801653 in mouse xenograft of: (A) an acute myeloid leukemia line MV4-11 bearing FLT3 activating mutation; (B) orthotopic model of NSCLC line H1299 with over-expression of AXL. P-value (*) compared to vehicle control.(C) Effect of LY2801653 on reducing pAXL and pAKT measured in H1299 cultured cells in vitro. Cells were serum-starved overnight, incubated with LY2801653 for 4 hours, then stimulated with 200 ng/mL Gas6 for 15 minutes. Cells were lysed and analyzed for pAXL and pAKT by ELISA and Western blot respectively.

A.

B.

C.