## 2 Results  
Genetic Perturbation of CSNK1D Interfaces with Canonical, Oncogenic HH--GLI Signaling in Medulloblastoma Cells  
To analyze whether CSNK1D is involved in the regulation of oncogenic HH signaling, we performed an shRNA-mediated knockdown of CSNK1D in a HH responsive human medulloblastoma cell line (Daoy). In Daoy cells, canonical HH signaling can be activated and inhibited by treatment with a small molecule, Smoothened agonist (SAG), and SMO antagonists, respectively [51; 52] (Figure 1A). The GLI1 protein as well as mRNA levels of the HH target genes _GLI1_ and _HHIP_, whose expression significantly increases upon the addition of SAG, were used as readout parameter for HH pathway activity. In addition, Daoy cells express high levels of CSNK1D, which can be inhibited via shRNA-mediated knockdown (Figure 1B). Of note, knockdown of CSNK1D was sufficient to reduce HH target gene and GLI1 protein expression in Daoy medulloblastoma cells, suggesting that CSNK1D is required for canonical HH signaling and HH target gene activation (Figure 1B,C). Knockdown of CSNK1D showed no effect on primary cilia, suggesting that loss of CSNK1D directly interferes with GLI activation rather than via interfering with the formation of primary cilia, which serve as essential organelles in the reception and transduction of HH signaling upstream of GLI proteins (Figure S1A,B) [53]. Genetic Inhibition of CSNK1D Reduces HH--GLI Activity in SMOi-Resistant Tumor Entities Driven by Oncogenic GLI  
To expand our investigations towards SMOi-resistant cancer cells, we turned to Ewing sarcoma (EWS) cells, where SMO-independent GLI1 expression is driven by the EWS-FLI1I fusion oncogene [54] (Figure 2A). Further, in EWS cells, GLI1 acts downstream of EWS-FLI1I as oncogene to promote proliferation and 3D spheroid growth [55]. In line with SMOi resistance of the GLI1-expressing EWS cell lines A673 and MHH-ES-1, only treatment with the GLI inhibitor HPI-1 led to reduced GLI1 protein levels, while treatment with the FDA-approved SMOi vismodegib (vismo) did not affect GLI1 expression (Figure 2B and Figure S2A) [56; 57]. Figure 1: RNAi-mediated inhibition of CSNK1D interferes with canonical oncogenic HH—GLI signaling. **(A)** Simplified schematic illustration of the canonical HH—GLI signaling pathway with pathway activators in green and inhibitors in red. PTCH and SUFU inactivate the pathway, while it is turned on by the central pathway activator SMO causing activation of the GLI transcription factor. Pharmacologically, the SMO agonist SAG activates the pathway, while it is inhibited by the SMO inhibitor vismodegib. **(B)** Representative Western blot analysis of GLI1 and CSNK1D in Daoy medulloblastoma cells. Cells were lentivirusally transduced with shCSNK1D or control shRNA (shCtrl) and treated with or without SAG [100 nM]. 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 shCtrl + SAG sample are shown above each protein band. **(C)** qPCR analysis of _GLI1_ and _HHIP_ mRNA levels of Daoy cells treated as described in **(B)** (\(n=3\)). Student’s \(t\) test was used for statistical analysis (**\(p<0.01\)). To investigate whether CSNK1D regulates GLI activity also in SMO-independent cells, we performed shRNA-mediated knockdown of CSNK1D in A673 cells. As shown in Figure 2C and Figure S2B, genetic perturbation of CSNK1D reduced GLI1 protein levels, suggesting that CSNK1D also regulates SMO-independent GLI activity. To support these results, we inactivated CSNK1D by CRISPR-Cas9 in the EWS cell line MHH-ES-1 and confirmed that deletion of CSNK1D results in reduced GLI1 protein levels (Figure S2C).