## 5 Conclusions  
In this study, we identified CSNK1D as a critical positive regulator of oncogenic GLI activity and demonstrated that targeting CSNK1D interferes with the malignant properties of GLI-dependent TIC. Of note, genetic as well as pharmacologic inhibition of CSNK1D efficiently represses oncogenic GLI activity in cancer cells resistant to FDA-approved HH pathway inhibitors targeting the essential HH effector SMO. Targeting CSNK1D with compounds such as SR-3029 and CK1D008 may, therefore, be a promising future strategy to treat cancer patients with acquired or a priori resistance to SMOi, and to hopefully reduce severe side effects known to be caused by established anti-SMO drugs.  
**Supplementary Materials:** The following are available online at [[https://www.mdpi.com/article/10.3390/cancers13164227/s1](https://www.mdpi.com/article/10.3390/cancers13164227/s1)]([https://www.mdpi.com/article/10.3390/cancers13164227/s1](https://www.mdpi.com/article/10.3390/cancers13164227/s1)), Figure S1: S1 Targeting CSNK1D does not interfere with ciliogenesis in Daoy cells, Figure S2: Targeting CSNK1D interferes with oncogenic HH--GLI signaling in SMOi-resistant Ewing sarcoma cells, Figure S3: Effects of pharmacological targeting of CSNK1D on the proliferation of A549 and Daoy cells, Figure S4: Targeting of CSNK1D reduces HH target gene expression in SMOi-resistant cell lines, Figure S5: Pharmacologic and genetic inhibition of CSNK1D selectively abrogates clonal growth and impairs in vivo engraftment of GLI-dependent PANC-1 cancer cells, Figure S6: Original Western Blot, Table S1: qPCR cycling program, Table S2: Western Blot antibodies.  
**Author Contributions:** Conceptualization, E.P., W.G., S.H. and F.A.; data validation, formal analysis, investigation, E.P., W.G., T.P. and S.K.A.; methods and resources, A.R., S.G.-G. and F.R.; medicinal chemistry, conceptualization, compound screening and biological evaluation of CK1D008, M.Z., S.M. and S.H.; data curation, E.P., T.P., S.K.A. and F.V.; manuscript writing E.P., D.P.E., S.G.-G., S.H. and F.A.; visualization, E.P., D.P.E. and S.K.A.; supervision, project administration and funding acquisition F.A. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This research was funded by the Cancer Cluster Salzburg (project 20102-P1601064-FPR01-2017), Open Access Funding by the Austrian Science Fund (FWF) (projects W1213, P25629), the Biomed Center Salzburg (project 20102-F1901165-KZP) and the priority program ACBN by the University of Salzburg.  
**Institutional Review Board Statement:** Animal studies were approved by the institutional authorities and by the Federal Ministry of Science, Research and Economy (BMWFW-66.012/0016-WF/V/3b/2015 and BMWFW-66.011/0030-II/3b/2014).  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to patent and licensing reasons.  
**Acknowledgments:** The authors thank [PERSON] and [PERSON] for their excellent technical support. The graphical abstract has been designed with Biorender.com (accessed on 31 October 2020). We dedicate this publication to our dear colleague [PERSON], who sadly passed away during this study.  
**Conflicts of Interest:** [PERSON], [PERSON] and [PERSON] are employees of 4 SC AG. The other authors declare no conflict of interest.