## 2 Results
### Targeting the CSNK1D-GLI Axis Inhibits CSC-Like Characteristics In Vitro and In Vivo  
Clonogenic and self-renewing spheroid growth in 3-dimensional (3D) in vitro cultures and the initiation of in vivo tumor growth are considered characteristics of TIC [60]. To functionally addressed a putative role of the CSNK1D-GLI axis in TICs, we first performed genetic and pharmacologic targeting of CSNK1D and measured its impact on the 3D spheroid growth properties of GL11-expressing A673 cells. Of note, shRNA-mediated knockdown of CSNK1D significantly diminished the clonogenic anchorage-independent growth capacity of A673 cells in 3D cultures (Figure 5A), while it did not affect anchorage-dependent growth under 2D culture conditions (Figure S5A). Accordingly, pharmacological inhibition of CSNK1D with CK1D008 or SR-3029 both drastically reduced clonogenic spheroid growth in 3D cultures at concentrations that had no impact on cell growth under planar 2D conditions (Figure 5B and Figure S5B). In addition, the inhibitory effect of CK1D008 on clonal growth was also evaluated in HH--GLI-dependent pancreatic (PANC1), NSCLC (A549), glioma (LNT-229) and CRC (HT29, HCT15, HCT116) tumor cells [6, 60, 61, 62]. To this end, tumor cells were pre-treated with inhibitor for 48 h and viable cells were seeded at limiting dilutions in an anchorage-dependent colony formation assay. CK1D008 efficiently reduced the colony formation ability of all tested cell lines at concentrations as low as 0.1-0.3 uM (Figure S5C). Figure 4: Pharmacological targeting of CSNK1D inhibits oncogenic HH—GLI signaling in SMOi-resistant cells. (**A**) Simplified schematic illustration of SMO-independent activation of HH—GLI signaling in Daoy medulloblastoma cells caused by the loss of the negative HH—GLI pathway regulator SUFU. (**B**) Representative Western blot analysis of GLII protein levels in Daoy-shSUFU cells treated with vismo [1 uM], HPI-1 [20 uM], CK1D008 [0.1-3 uM] or SR-3029 [0.01-1 uM]. (**C**) Representative Western blot analysis of GLII in A673 cells treated with vismo [1 uM], HPI-1 [20 uM], CK1D008 [0.1-1 uM] or SR-3029 [0.03-0.3 uM]. Relative quantification of Western blot bands was conducted via densitometric image analysis using Image Lab 5.0 software (Bio-Rad, Vienna, Austria). Relative protein levels normalized to the loading control tERK and to the Ctrl sample are shown above each protein band. GLII represents an important driver gene of proliferation and spheroid growth in Ewing sarcoma cells [29]. To further support that inhibition of CSNK1D decreases TIC properties via GLII inhibition, we performed a CRISPR/Cas-mediated _GLI1_ knockout in A673 cells (Figure S5D) and analyzed _GLI1_-deficient A673 cells for clonogenic spheroid growth in 3D and planar growth in 2D cultures. In line with our results on CSKN1D targeting, genetic deletion of _GLI1_ selectively abolished clonogenic spheroid formation in 3D, while it did not affect the 2D growth properties (Figure S5E,F), suggesting that CSNK1D encodes a crucial positive regulator of oncogenic GLI1 in TIC. To evaluate whether enforced overexpression of the GLI activator proteins GLII1 and GLI2 can attenuate the anti-tumor effect caused by CSNK1D inhibition, we overexpressed in A673 cells HA-tagged GLII and an active form of GLI2 [63], respectively (Figure S5G), and treated GLII, GLI2, or empty-vector control cells with CK1D008. In line with GLI  
Figure 5: Targeting CSNK1D reduces GLI-dependent 3D tumor spheroid formation in vitro as well as tumor engraftment in vivo. (**A**) A673 cells were lentivirusally transduced with shCSNK1D or control shRNA (shCtrl) and cultured under anchorage-independent conditions. Representative images of formed 3D spheroid colonies (left panel). The number of 3D spheroid colonies was counted (right panel) (\(n=8\)). (**B**) A673 cells were cultured under anchorage-independent conditions and treated with the GLI inhibitor HPI-1 [4 \(\mu\)M], CK1D008 [0.1–1 \(\mu\)M] or SR-3029 [0.03–0.3 \(\mu\)M]. The data represent the mean of six 3D culture experiments.