Eliminating Cancer Stem Cells by Inhibition of the Wnt Pathway via Small Molecule Kinase Inhibitors H. Kohlhof, S. Schrepfer, K. Kronthaler, M. Zaja, J. Krüger, R. Baumgartner, J. Leban 4SC AG, 82152 Martinsried, Germany Contact: [email protected] 10 9 8 Usually compounds < 1% of control are found to be potent in follow-up Ki determination 7 6 % of control 5 4 3 2 1 0 AAK1 ABL1 ABL1 ABL1 ABL2 ACVR2B AKT1 PRKAA1 MAP3K5 AURKC BMPR1A BRAF BRSK2 CAMK1G CAMK2G CASK CDC2L6 CDK4 CDK9 CDKL5 CLK1 CSF1R CSNK1D CSNK1G3 DAPK1 DCLK2 MAP3K12 STK17B EGFR EGFR EGFR EIF2AK1 EPHA4 EPHA8 EPHB4 ERBB4 MAPK4 PTK2 FGFR2 FGR FLT3 FLT3 GAK GRK7 HIPK1 MAP4K1 CHUK INSRR ITK JAK3 KIT KIT LATS1 LIMK2 LRRK2 MAK MAP3K3 MAP4K4 MARK1 MAST1 MAP2K4 MERTK MINK1 MYLK3 CDC42BPA STK3 MUSK MYO3A NEK1 NEK4 NEK9 MAPK14 PAK1 PAK6 PCTK3 PFB0815w PHKG1 PIK3CA PIK3CA PIK3CA PIK3CG PIM3 PIP4K2C PKN1 PLK2 PRKCE PRKD1 PRKG2 PTK2B RET RIOK2 RIPK4 ROS1 RPS6KA5 RPS6KA2 RPS6KB1 SIK1 SNRK SRPK2 STK35 MAP3K7 TBK1 TGFBR2 TLK2 TNNI3K TRPM6 TYK2 ULK2 WEE1 STK32C ZAK One hallmark of cancer stem cells (CSC) is their self renewal capacity driven by developmental pathways like Wnt, Hedgehog and Notch. To eliminate CSCs, we target the Wnt and the Hh pathway. A multitude of protein kinases is involved in positive and negative regulation of these pathways. We discovered several novel chemotypes of kinase inhibitors which inhibited the activity of Wnt signaling in a HEK293 reporter assay in the micromolar range (Fig.1). Some of these compounds have remarkable selectivity towards certain kinases in the low nanomolar range (Fig.2). Selected compounds potently inhibit colony formation of HCT116 Colon Carcinoma cells that exhibit constitutive active Wnt signaling due to a mutated ß-Catenin (Fig.3). Additionally, the compounds have a negative impact on spheroid formation of the embryonic carcinoma cell line NCCIT (Fig.4). 442 kinases of the kinome Cpd9 Fig.2 The selectivity towards the kinome was determined by an Ambit panel screen (competition assay) towards 442 kinases, tested at a concentration of 10 µM of Cpd9. Some of the compounds tested so far showed remarkable selectivity. We confirmed the activity of Cpd9 and others in a kinase inhibition assay and confirmed that we have identified specific kinases for Wnt inhibition by an RNAi approach. Concentration [M] Control VX-680 IC50 430 nM Fig.3 The compounds were tested for their potency to inhibit anchorage independent growth/colony formation in a soft agar assay with HCT116 cells. HCT116 cells show high levels of CD133 and CD44 expression, often described as markers for stemness, exhibit a constitutive active Wnt pathway, and form colonies very aggressively. Cpd9 was very potent to inhibit colony formation with an IC50 of 430 nM. % reporter activity/transfection efficiency Wnt Reporter Activity in HEK293 Cells 120 100 80 Cpd9 60 40 20 0 30 µM 12 µM 4.8 µM 1.9 µM 0.8 µM 0.3 µM 0.1 µM Low Contr. FH535 20µM High Contr. Unstim. Cells Fig.1 The capacity of the compound to inhibit the Wnt pathway was tested in a transient transfection WNT reporter assay in HEK293 cells after stimulation with 50mM LiCl. The IC50 of Cpd9 was determined at 1.5 µM. Fig.4 We use NCCIT cells as a model system for cancer stem cells. NCCIT is a pluripotent embryonal carcinoma cell line, that shows expression of markers described for cancer stem cells like OCT4, Nanog, SOX2, SOX9, CD133 and CD44 ,and are capable to form spheroids by induction via FGF. Incubation for 72h with Cpd9 led to dissolution of the tight structure of the spheroid and many dead cells, with an IC50 of 1.5 µM. The IC 50 was determined by the size of the spheroids and the viability of all cells by measurement of ATP levels. Cpd9 showed promising results in surrogate assays for stemness relevant read-outs like inhibition of anchorage independent growth and spheroid formation. Further in vitro and in vivo experiments to determine the effects on cancer stem cells are in progress. Compounds will be validated in animal models for prevention of metastasis and prolongation of time to relapse after treatment with conventional chemotherapy. Additionally, we will continue to validate kinase inhibitors of the Wnt and Hh pathway with the ultimate goal to develop therapeutical concepts against tumor initiating and metastasis forming cells.
© Copyright 2024 ExpyDoc
