## 3 Discussion  
Hedgehog/GLI signaling has been associated with many human malignancies and aberrant pathway activation has been discovered in cancer stem cells with a critical role in tumor initiation, malignant growth, metastasis, and relapse [3; 4; 7; 8; 64]. Pharmacological targeting of oncogenic HH--GLI is therefore considered a promising therapeutic strategy. Three SMO inhibitors, vismodegib, sonidegib and glasdegib, have been approved for the treatment of locally advanced and metastatic basal cell carcinoma and for acute myeloid leukemia, respectively [20; 21; 23; 57]. Even though these inhibitors demonstrate remarkable therapeutic efficacy, treatment can cause frequent and severe side effects and result in acquired drug resistance. Additionally, several tumor entities are driven by SMO-independent GLI activity and thus display a priori SMOi resistance [25; 26; 30; 54]. Since, the GLI transcription factors can integrate with other oncogenic signaling cascades and promote cancer progression and malignant properties of cancer (stem) cells [5; 6; 34; 38; 39; 40; 60; 62; 65], oncogenic GLI transcription factors represent attractive therapeutic targets for patients with acquired and a priori resistance to SMOi. Although some previous studies have shown that direct targeting of oncogenic GLI proteins is feasible and promising [66; 67], small molecule-mediated direct inhibition of transcription factors is generally considered to be very challenging. The identification of druggable key regulators of GLI activity such as readily targetable kinases promoting the oncogenic activity of GLI transcription factors is therefore a critical requirement and of high medical relevance and need. In this study, we identify CSNK1D as a positive and druggable regulator of oncogenic GLI activity, both in canonical as well as SMO-independent settings of GLI activation. Furthermore, we describe the identification of the novel CSNK1D inhibitor CK1D008 and show that pharmacological targeting of CSNK1D with potent small molecule inhibitors including CK1D008 and SR-3029 [68] abolishes oncogenic HH--GLI signaling in distinct cancer entities with either SMO-dependent or SMO-independent oncogenic GLI activity. Of note, we provide evidence that the CSNK1D-GLI axis selectively promotes hallmarks of TICs including clonogenic growth in 2D- and 3D cultures and engraftment in immunocompromised mouse models, while not affecting the proliferation of non-TICs under standard 2D culture conditions. Therapeutic targeting of CSNK1D may therefore represent a promising approach to eradicate highly malignant GLI-driven TICs. Considering recent data on the immunosuppressive role of TICs/CSCs in several cancer entities [69] as well as the regulation of immunosuppressive factors by GLI [70; 71; 72; 73] combination therapy with CSNK1D and immune checkpoint inhibitors is an attractive therapeutic strategy to be evaluated in follow-up pre-clinical studies. CSNK1 family members interact with several oncogenic pathways such as the HH, Hippo and WNT, and their aberrant regulation closely linked to several human malignancies [74]. Small molecule inhibitors of CSNK1 family members have been developed, such as the CSNK1D/E inhibitor SR-3029 which is a potent ATP-competitor with high specificity for CSNK1D and CSNK1E [68]. SR-3029 displays striking therapeutic efficacy in triple negative as well as HER2\({}^{+}\) breast cancer models with WNT involvement [58]. Giventhe documented oncogenic role of HH--GLI in breast cancer development [75; 76; 77], it is possible that the therapeutic activity of this CSNK1 inhibitor also relies at least in part on the GLI inhibitory activity described in our study. Mechanistically, we propose that CSNK1D positively regulates the stability of oncogenic GLI proteins. In this context it is noteworthy that [PERSON] et al. have shown that members of the CSNK1 family phosphorylate GLI transcription factors at PKA-independent sites, thereby disrupting the interaction of GLI with SPOP.