## 2 Results
Pharmacological Targeting of CSNK1D Inhibits HH--GLI Signaling in Both Canonical and Non-Canonical Settings  
The critical role of CSNK1D in SMO-dependent and SMO-independent GLI activation prompted us to analyze the possible therapeutic effect of CSNK1D inhibitors by targeting oncogenic GLI transcription factors. In a collaborative approach, we performed a compound screen cascade in A549 non-small cell lung carcinoma (NSCLC) cells under proliferating and non-proliferating conditions to select for compounds interfering with quiescent and TIC properties. This approach identified a novel, highly effective CSNK1D inhibitor, termed CK1D008 (Figure S3A). To investigate whether chemical perturbation of CSNK1D can interfere with HH--GLI signaling, we treated HH-responsive Daoy cells with CK1D008 and for comparison with the known CSNK1D inhibitor SR-3029 [58]. As shown in Figure 3A, both CSNK1D inhibitors potently inhibited the activation of _GLI1_ mRNA expression in SAG-treated Daoy cells with IC\({}_{50}\) values in the nanomolar range (Figure 3A). Figure 2: RNAi-mediated inhibition of CSNK1D interferes with oncogenic HH—GLI signaling in SMOi resistant Ewing sarcoma cells. (**A**) Simplified schematic illustration of non-canonical, SMO-independent HH—GLI signaling in Ewing sarcoma with EWS–FLI1-driven transcription of _GLI1_. (**B**) Representative Western blot analysis of GLI1 in A673 cells treated with vismodegib [1 uM] or HPI-1 [20 uM]. (**C**) Representative Western blot analysis of GLI1 in A673 cells lentivirally transduced with shCSNK1D (#1, #2) or control shRNA (shCtrl). 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. Likewise, both inhibitors decreased SAG-induced GLI1 protein levels in Daoy cells in a dose-dependent manner (Figure 3B). Neither the protein levels of CSNK1D nor those of GLI2 were affected by pharmacological inhibition of CSNK1D (Figure S3B). Consistent with the proliferative role of HH--GLI in Daoy cells, both CSNK1D inhibitors also reduced cell proliferation and viability in a dose-dependent manner (Figure S3C). Furthermore, to mimic SMOi resistance, we generated Daoy cells with an shRNA-mediated knockdown of the GLI inhibitor SUFU [59], resulting in SMO-independent activation of GLI1 expression (Figure 4A,B). Of note, while vismodegib treatment failed to reduce GLI1 expression in Daoy-shSUFU cells, chemical targeting of CSNK1D with CK1D008 or SR-3029 efficiently reduced GLI1 protein expression and HH target gene expression in this SMO-independent model of GLI activation (Figure 4B and Figure S4A). To corroborate that CSNK1D inhibition is a potent strategy to block GLI activation in SMOi-resistant settings, we performed chemical inhibition of CSNK1D in A673 and MHH-ES-1 Ewing sarcoma cells both showing SMO-independent GLI1 expression. As shown in Figure 4C and Figure S4B, pharmacologic inhibition of CSNK1D with CK1D008 or SR-3029 efficiently reduced GLI1 expression in both cell lines. By contrast, vismodegib treatment did not affect GLI1 expression [60]. Figure 3: Pharmacological targeting of CSNK1D inhibits SMO-dependent GLI activation. (**A**) mRNA expression levels of _GLI1_ were analyzed by qPCR and expressed as percentage relative to the control sample (\(n=3\)). (**B**) Representative Western blot analysis of GLI1 protein levels in Daoy cells treated with SAG [100 nM] and increasing concentrations of CK1D008 [0.01–10 \(\upmu\)M] or SR-3029 [0.01–1 \(\upmu\)M]. 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 SAG-treated sample are shown above each protein band.