Jeffrey R. Peterson

Supplementary Table S3. List of primers used for qRT-PCR.

Supplementary Video S6. Time-lapse images of hTERT-RPE1-Arl13b-GFP cells starved in serum-free Opti-MEM for 48 hours, then incubated for 3 hours in 2.5 µM IRAK1/4 inhibitor + 20% FBS.

Supplementary Video S1. Time-lapse images of hTERT-RPE1-Arl13b-GFP cells starved in serum-free Opti-MEM for 48 hours.

Purpose:For many tumors, signaling exchanges between cancer cells and other cells in their microenvironment influence overall tumor signaling. Some of these exchanges depend on expression of the primary cilium on nontransformed cell populations, as extracellular ligands including Sonic Hedgehog (SHH), PDGFRα, and others function through receptors spatially localized to cilia. Cell ciliation is regulated by proteins that are themselves therapeutic targets. We investigated whether kinase inhibitors of clinical interest influence ciliation and signaling by proteins with ciliary receptors in cancer and other cilia-relevant disorders, such as polycystic kidney disease (PKD).Experimental Design:We screened a library of clinical and preclinical kinase inhibitors, identifying drugs that either prevented or induced ciliary disassembly. Specific bioactive protein targets of the drugs were identified by mRNA depletion. Mechanism of action was defined, and activity of select compounds investigated.Results:We identified multiple kinase inhibitors not previously linked to control of ciliation, including sunitinib, erlotinib, and an inhibitor of the innate immune pathway kinase, IRAK4. For all compounds, activity was mediated through regulation of Aurora-A (AURKA) activity. Drugs targeting cilia influenced proximal cellular responses to SHH and PDGFRα. In vivo, sunitinib durably limited ciliation and cilia-related biological activities in renal cells, renal carcinoma cells, and PKD cysts. Extended analysis of IRAK4 defined a subset of innate immune signaling effectors potently affecting ciliation.Conclusions:These results suggest a paradigm by which targeted drugs may have unexpected off-target effects in heterogeneous cell populations in vivo via control of a physical platform for receipt of extracellular ligands.

Supplementary Video S4. Time-lapse images of hTERT-RPE1-Arl13b-GFP cells starved in serum-free Opti-MEM for 48 hours, then incubated for 3 hours in 1 µM ganetespib.

Supplementary Figure S1. Additional analysis of siRNA effects on ciliation and target depleton. Supplementary Figure S2. Sorafenib is similar to sunitinib in inducing ciliary resorption. Supplementary Figure S3. Effect of compounds on AURKA catalytic activity. Supplementary Figure S4. Representative images demonstrating the effect of sunitinib (A) and ganetespib (B) on cilia resorption in hTERT-RPE1-Arl13b-GFP cells. Supplementary Figure S5. Cilia length upon treatment with indicated cilia-stabilizing compounds in hTERT-RPE1 (A) and NIH3T3 (B) cell lines. Supplementary Figure S6. Drug effects on ciliation correspond to changes in cell cycle over time. Supplementary Figure S7. Erlotinib increases ciliation in CFPAC-1 but not in PNX0010 cell line. Supplementary Figure S8. Representative Western blot images and quantitation of PDGF-AA-induced activation of PDGFR and MEK1/2 in NIH3T3 cells, related to Figures 3F, G.

Supplementary Video S2. Time-lapse images of hTERT-RPE1-Arl13b-GFP cells starved in serum-free Opti-MEM for 48 hours, then incubated for 3 hours in 20% FBS.

Supplementary Video S7. Time-lapse images of hTERT-RPE1-Arl13b-GFP cells starved in serum-free Opti-MEM for 48 hours, then incubated for 3 hours in 2.5 µM erlotinib + 20% FBS.

Supplementary Legend from Unexpected Activities in Regulating Ciliation Contribute to Off-target Effects of Targeted Drugs

Supplementary Table S1. Complete list of compounds screened.