Published OnlineFirst September 18, 2017; DOI: 10.1158/2159-8290.CD-17-0281 RESEARCH ARTICLE A Transposon Screen Identifies Loss of Primary Cilia as a Mechanism of Resistance to SMO Inhibitors Xuesong Zhao1,2, Ekaterina Pak1,2, Kimberly J. Ornell1,2, Maria F. Pazyra-Murphy1,2, Ethan L. MacKenzie1,2, Emily J. Chadwick1,2, Tatyana Ponomaryov1,2,3, Joseph F. Kelleher4, and Rosalind A. Segal1,2 Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. 15-CD-17-0281_p1436-1449.indd 1436 11/17/17 2:34 PM Published OnlineFirst September 18, 2017; DOI: 10.1158/2159-8290.CD-17-0281 ABSTRACT Drug resistance poses a great challenge to targeted cancer therapies. In Hedgehog pathway–dependent cancers, the scope of mechanisms enabling resistance to SMO inhibitors is not known. Here, we performed a transposon mutagenesis screen in medulloblastoma and identifi ed multiple modes of resistance. Surprisingly, mutations in ciliogenesis genes represent a fre- quent cause of resistance, and patient datasets indicate that cilia loss constitutes a clinically relevant category of resistance. Conventionally, primary cilia are thought to enable oncogenic Hedgehog sig- naling. Paradoxically, we fi nd that cilia loss protects tumor cells from susceptibility to SMO inhibitors and maintains a “persister” state that depends on continuous low output of the Hedgehog program. Per- sister cells can serve as a reservoir for further tumor evolution, as additional alterations synergize with cilia loss to generate aggressive recurrent tumors. Together, our fi ndings reveal patterns of resistance and provide mechanistic insights for the role of cilia in tumor evolution and drug resistance. SIGNIFICANCE: Using a transposon screen and clinical datasets, we identifi ed mutations in ciliogenesis genes as a new class of resistance to SMO inhibitors. Mechanistically, cilia-mutant tumors can either grow slowly in a “persister” state or evolve and progress rapidly in an “aggressive” state. Cancer Discov; 7(12); 1436–49. ©2017 AACR. See related commentary by Goranci-Buzhala et al., p. 1374. INTRODUCTION 2012 and 2015 respectively, vismodegib and sonidegib (NVP- LDE225) became the fi rst FDA-approved SMO inhibitors for Aberrant Hedgehog (HH) signaling is implicated in many anticancer treatment in advanced BCC. Several clinical trials in cancers ( 1 ) and is particularly critical in medulloblastoma, the medulloblastoma are in progress. Despite the initial success of most common malignant brain tumor in children, and in basal SMO inhibitors in mouse models and subsequently in patients, cell carcinoma (BCC), the most common human cancer overall long-term effi cacy is limited by the emergence of drug resistance ( 2–4 ). Hyperactivity of the HH pathway is frequently caused ( 9–13 ). Thus far, preclinical and clinical studies have uncovered by inactivating mutations in Patched (Ptch) , which encodes a number of point mutations in SMO that confer resistance. the receptor for HH ligands ( 5–7 ). In the absence of PTCH Although most mutations located in the drug-binding pocket function, Smoothened (SMO), a G-protein-coupled receptor– of SMO directly impair drug binding, an additional group like molecule, traffi cs into the primary cilia, a distinctive, of mutations distally located to drug-binding sites may have microtubule-based signaling organelle. SMO functions within allosteric effects for drug binding or promote a constitutively the primary cilia to inhibit the negative regulators Suppres- activated state of SMO that is less sensitive to drug inhibition sor of fused (SUFU) and protein kinase A (PKA), triggering a ( 9, 10 ). Alterations that activate HH signaling downstream of signaling cascade that culminates in the nuclear import of GLI SMO, such as loss of SUFU or amplifi cation of GLI2 , as well as transcription factors, which activates an HH transcriptional mutations that activate intersecting oncogenic pathways such program that drives proliferation and tumor growth. as RAS/MAPK, PI3K, or aPKC, have also been reported ( 14–16 ). The importance of the HH pathway in human cancers has However, the scope of resistance mechanisms to SMO inhibi- stimulated great interest in developing targeted therapy that tors and the process by which HH pathway–dependent tumors antagonizes HH signaling ( 1, 8 ). These efforts have resulted evolve and recur following therapy are not yet known. in the approval of SMO inhibitors as anticancer agents. In To identify resistance mechanisms, we used a genome-wide transposon mutagenesis screen in HH pathway–dependent medulloblastoma cells and identifi ed recurrent mutations in 1 Departments of Cancer Biology and Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. 2 Department of Neurobiology, the ciliogenesis gene Oral-facial-digital syndrome 1 (Ofd1 ) , in Harvard Medical School, Boston, Massachusetts. 3 Institute of Cardio- cells resistant to SMO inhibitors. We demonstrate that resist- vascular Sciences, College of Medical and Dental Sciences, University of ant tumors that lack primary cilia enter a “persister state” of 4 Birmingham, Birmingham, United Kingdom. Novartis Institutes for Bio- slow, GLI2-dependent growth and show that this mechanism medical Research , Cambridge, Massachusetts. of therapeutic resistance occurs in patients. Note: Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/). X. Zhao and E. Pak are co-fi rst authors of this article. RESULTS Corresponding Authors: Rosalind A. Segal, Dana-Farber Cancer Institute, Transposon-Mediated Drug Resistance Screen 450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-4737; Fax: 617-632-2085; E-mail: [email protected] ; and Xuesong Zhao, Identifi es Recurrent Mutations in Sufu and Ofd1 [email protected] To identify genetic changes capable of circumventing SMO doi: 10.1158/2159-8290.CD-17-0281 inhibition, we launched a genome-wide transposon mutagen- ©2017 American Association for Cancer Research. esis screen in SMB21 cells, a murine cell line derived from a December 2017 CANCER DISCOVERY | 1437 Downloaded from cancerdiscovery.aacrjournals.org on September 30, 2021. © 2017 American Association for Cancer Research. 15-CD-17-0281_p1436-1449.indd 1437 11/17/17 2:34 PM Published OnlineFirst September 18, 2017; DOI: 10.1158/2159-8290.CD-17-0281 RESEARCH ARTICLE Zhao et al. Ptch−/− medulloblastoma (16). SMB21 retains key character- Loss of OFD1 Causes Resistance to SMO Inhibitors istics of SHH-subgroup medulloblastoma, is dependent on Identification of recurrent insertions inSufu provides a HH signaling for proliferation, and is exquisitely sensitive to good validation of the screen, as SUFU is a well-known nega- SMO inhibition (16). The engineered piggyBac transposon sys- tive regulator of the HH pathway that functions by binding tem utilized in this screen relies on a transposon vector that and sequestering GLI transcription factors, and mutations can either enhance or disrupt gene expression in the vicinity in Sufu have been identified in clinical tumors resistant to of the insertion site (Fig. 1A). To deploy the transposon-medi- SMO inhibitors (9–11, 17). To establish a causal relationship ated screen, SMB21 parental cells were transfected with both between mutations in Ofd1 and the resistant phenotype, we piggyBac transposon and transposase vectors for mutagenesis, first conducted an Flp/FRT-based rescue experiment (Fig. and were then selected for clones of cells able to grow in the 2A). As the transposon is engineered with two FRT sites presence of 1 μmol/L sonidegib (Fig. 1B). From 30 million flanking the DNA cargo, lentivirus expressing Flp was used cells, 29 resistant clones were isolated and 27 clones were to remove the DNA cargo. In clones where the transposon successfully propagated. All resistant clones showed robust is inserted in introns, such as R25, this approach success- resistance to sonidegib in subsequent multidose survival fully re-expresses OFD1 and restores sensitivity to sonidegib, assays (Supplementary Fig. S1A). A barcoded splinkerette- as assessed by survival assays and by GLI1 expression (Fig. PCR approach coupled with parallel sequencing was utilized 2B–D). However, this strategy cannot revert disruptions in to identify transposon insertion loci in each clone (Sup- genes if transposons are inserted in exons, such as in clones plementary Fig. S1B). A total of 182 transposon insertions R14 and R16, because the remaining transposon terminal were identified (Supplementary Fig. S1C and Supplementary repeat sequence outside of the FRT sites still causes truncated Table S1). The two genes with the greatest number of recur- mRNA (Supplementary Fig. S3A and S3B). In a second res- rent transposon insertion sites were Sufu (13 clones) and Ofd1 cue experiment, we asked whether expression of a full-length (3 clones; Fig. 1C and D). The robust resistance effects in OFD1 rescues the resistant phenotype in Ofd1 mutants. Sufu- and Ofd1-mutant cells were evident by the dramatic shift Indeed, expression of a full-length OFD1 restored sensitiv- of the growth-inhibitory concentration of sonidegib (Fig. 1E) ity to sonidegib in HH signaling and in cell viability assays and were further validated using additional SMO inhibitors, (Fig. 2E and F). To independently demonstrate that loss of vismodegib and cyclopamine (Supplementary Fig. S1D). OFD1 mediates resistance to SMO inhibition, we specifically We next evaluated