Supplementary Figure legends

Fig. S1. Summary of EGFR status and sensitivity to erlotinib of NSCLC cell lines used in this study.

(A) Table summarizing the EGFR mutation status and IC50 values to erlotinib for NSCLC cell lines used in this study. (B) Sensitivity of EGFR-mutant NSCLC cell lines to erlotinib measured by 5 day-dose response curves. Cells were plated in triplicate one day before compound addition and incubated for 5 days with various concentrations of erlotinib. Cell viability was determined by Cell Titer Glo, and the inhibition of viability relative to DMSO-treated cells was calculated.

Fig. S2. Knockdown of CK1a inhibits proliferation of PC9 cells more than proliferation of HCC827 or HCC4006 cells under DMSO treatment.

Representation of the abundance of shRNAs at gene level in DMSO-treated condition versus baseline. RSA-score versus Q1-z-score are plotted for each individual gene. Red dots highlight CSNK1A1, blue dots CSNK1D and green dots CSNK1E.

Fig. S3. Performance of individual shRNAs against members of the CK1-family in shRNA screens.

Representation of the relative abundance of the shRNA barcode sequences from the shRNA screens as outlined in Figure 1. (A) The log2 counts in DMSO-treated versus erlotinib-treated condition for each shRNA are plotted. Highlighted are the 17 individual shRNAs against CSNK1A1. (B) As (A), but for CSNK1D. (C) As (A) but for CSNK1E.

Fig. S4. Knockdown of CK1a does not sensitize NSCLC cells to doxorubicin or cisplatin.

(A) 5 day-dose response curves with doxorubicin or cisplatin in HCC827 cells expressing doxycycline-inducible CK1a- or NTC-shRNAs. Cells pretreated for 72 h +/- doxycycline were plated in triplicate, and treatment with various doses of doxorubicin or cisplatin was started the following day. After 5 days, cell viability was assessed by Cell Titer Glo, and inhibition of viability relative to DMSO-treated cells was calculated. Data represent one of two independent experiments. (B) As (A), but for HCC4006 cells expressing doxycycline-inducible CK1a- or NTC-shRNAs.

Fig. S5. Suppression of CK1a attenuates acquired resistance to erlotinib in EGFR-mutant NSCLC cells.

Barplots represent the quantification of colony formation assays as presented in Figure 3. Colony areas were quantified using Odyssey CLx. Data are represented as fold change to - dox condition +/- STDV.

Fig. S6. Suppression of CK1a attenuates the outgrowth of drug tolerant persisters (DTPs) to resistant clones and inhibits proliferation of resistant PC9 cells in the presence of erlotinib.

(A) Left: HCC827, HCC4006 and PC9 cells expressing doxycycline-inducible CK1a- or NTC-shRNAs were treated continuously +/- doxycycline and +/- 2 mM erlotinib. Right: DTPs of HCC827, HCC4006 and PC9 cells expressing doxycycline-inducible CK1a- or NTC-shRNAs were generated by 8 d treatment with 2mM erlotinib, followed by continuous treatment with +/- doxycycline and +/- 2 mM erlotinib. Cells were fixed, stained and photographed at the indicated time points. (B) Resistant PC9 cells expressing doxycycline-inducible CK1a- or NTC-shRNAs were treated +/- doxycycline and +/- 2 mM erlotinib. Cell proliferation over time was assessed by confluency measurements every 12 hours using an Incucyte Kinetic Imaging System.

Fig. S7. The CK1 inhibitor D4476 can attenuate acquired resistance to erlotinib in NSCLC cell lines.

(A) Co-treatment of HCC827, HCC4006 and PC9 cells with indicated concentrations of CK1 inhibitor D4476 and +/- 2 mM erlotinib. At the indicated time points cells were fixed, stained and photographed. Data represent one of two independent experiments. (B) Barplots representing the quantification of colony formation assays as displayed in (A). Colony areas were quantified by Odyssey CLx.

Fig. S8: Knockdown of CK1a does not influence EGFR signaling.

Immunoblot analysis of components of EGFR signaling pathway. HCC827, HCC4006 and PC9 cells expressing doxycycline-inducible CK1a- or NTC-shRNAs were treated for 72 h +/- doxycycline followed by 24 h treatment +/- 2 mM erlotinib. Erlotinib resistant PC9 cells cultured in the presence of 2 mM erlotinib were treated +/- doxycycline and +/- 2 mM erlotinib for 96 h. Immunoblot analysis of total cell lysates was performed.

Fig. S9: Knockdown of CK1a does not activate WNT-signaling.

(A) WNT-responsive Super-Topflash (STF) luciferase reporter is activated by GSK3b-inhibitor, CHIR118637, in EGFR-mutant NSCLC cell lines HCC827 and PC9. HCC827 and PC9 cells expressing doxycycline-inducible CK1a- or NTC-shRNAs were transduced with WNT-responsive Super-Topflash (STF) luciferase reporter plasmid. Cells were treated for 24 h with 5 mM CHIR118637. Luciferase signal was normalized to cell viability assessed by Cell Titer Glo. Data are represented as fold change relative to DMSO treated condition +/-STDV. Data represent one of three independent experiments. (B) Knockdown of CK1a does not activate WNT-signaling in HCC827 and PC9 cells. Cells were treated for 72 h +/- doxycycline followed by 24 h treatment +/- 2 mM erlotinib. Luciferase signal was normalized to cell viability assessed by Cell Titer Glo. Data are represented as fold change relative to - dox-condition +/-STDV. Data represent one of three independent experiments.

Fig. S10: Erlotinib treatment activates NF-kB-signaling and CK1a-knockdown decreases expression of NF-kB-target genes.

(A) NF-kB-signaling is activated in erlotinib-treated versus DMSO-treated HCC827 and PC9 cells. Microarray gene expression data of HCC827 and PC9 cells treated for 8 days +/- 2 mM erlotinib. GeneGO pathway enrichment analysis was performed in erlotinib- versus DMSO-treated condition and the top 10 pathways are shown. Pathways referring to NF-kB-signaling are highlighted in red. (B) As (A), but for resistant PC9 cells treated for 30 days with 2 mM erlotinib. (C) Knockdown of CK1a downregulates expression of the two NF-kB-target genes TNFa and Serpine1. Quantitative real-time PCR was used to assess gene expression of cells treated for 72 h +/- doxycycline followed by treatment +/- 2 mM erlotinib for 24 h. Data are represented as fold change relative to - dox-condition +/-STDV. The data represent one of two independent experiments.

Fig. S11: Inhibition of NF-kB-signaling attenuates resistance to erlotinib while only slightly affecting proliferation in the absence of erlotinib.

(A) Expression of IkBa-superrepressor does not strongly affect proliferation of HCC827 and PC9 cells in the absence of erlotinib. HCC827 and PC9 cells expressing doxycycline-inducible IkBa-superrepressor were cultured +/- doxycycline. Cell proliferation was assessed over time by confluency measurements every 12 hours using an Incucyte Kinetic Imaging System. (B) Expression of IkBa-superrepressor does attenuate resistance to erlotinib while not strongly affecting proliferation of HCC827 and PC9 cells in the absence of erlotinib. Quantification of colony formation assays shown in Figure 4B. Colony areas were quantified using Odyssey CLx. (C) Treatment with IKKb-inhibitor AFN700 modestly affects proliferation of parental HCC827, HCC4006 and PC9 cells. Parental HCC827, HCC4006 and PC9 cells were cultured with different concentrations of AFN700. Cell proliferation was assessed over time by confluency measurements every 12 hours using an Incucyte Kinetic Imaging System. (D) Treatment with the IKKb-inhibitor AFN700 prevents resistance to erlotinib while only modestly affecting proliferation of HCC827, HCC4006 and PC9 cells in the absence of erlotinib. Quantification of colony formation assays shown in Figure 4C. Colony areas were quantified using Odyssey CLx. (E) Treatment with the IKKb-inhibitor AFN700 inhibits proliferation of resistant PC9 cells in the presence of erlotinib. Resistant PC9 cells were cultured +/- 2.5 mM AFN700 and +/- 2 mM erlotinib. Cell proliferation was assessed over time by confluency measurements every 12 hours using an Incucyte Kinetic Imaging System.