Therapeutics, Targets, and Chemical Biology Small Molecule Inhibition of GDC-0449 Refractory Smoothened Mutants and Downstream Mechanisms of Drug Resistance Gerrit J. P. Dijkgraaf, Bruno Alicke, Lasse Weinmann, Thomas Januario, Kristina West, Zora Modrusan, Dan Burdick, Richard Goldsmith, Kirk Robarge, Dan Sutherlin, Suzie J. Scales, Stephen E. Gould, Robert L. Yauch, and Frederic J. de Sauvage Abstract Inappropriate Hedgehog (Hh) signaling has been directly linked to medulloblastoma (MB), a common malignant brain tumor in children. GDC-0449 is an Hh pathway inhibitor (HPI) currently under clinical investigation as an anticancer agent. Treatment of a MB patient with GDC-0449 initially regressed tumors, but this individual ultimately relapsed with a D473H resistance mutation in Smoothened (SMO), the molecular target of GDC-0449. To explore the role of the mutated aspartic acid residue in SMO function, we substituted D473 with every amino acid and found that all functional mutants were resistant to GDC-0449, with positively charged residues conferring potential oncogenic properties. Alanine scan mutagenesis of SMO further identified E518 as a novel prospective mutation site for GDC-0449 resistance. To overcome this form of acquired resistance, we screened a panel of chemically diverse HPIs and identified several antagonists with potent in vitro activity against these GDC-0449–resistant SMO mutants. The bis-amide compound 5 was of particular interest, as it was able to inhibit tumor growth mediated by drug resistant SMO in a murine allograft model of MB. However, focal amplifications of the Hh pathway transcription factor Gli2 and the Hh target gene cyclin D1 (Ccnd1) were observed in two additional resistant models, indicating that resistance may also occur downstream of SMO. Importantly, these HPI resistant MB allografts retained their sensitivity to PI3K inhibition, presenting additional opportunities for the treatment of such tumors. Cancer Res; 71(2); 435–44. Ó2010 AACR. Introduction MB is a primitive neuroectodermal tumor of the cerebel- lum, which is thought to originate from immature granule neural precursors (GNPs) located in the external granule layer (1). It is the most common brain malignancy among children 0–4 years old and accounts for nearly 18% of pediatric intracranial tumors, with 350 new cases being diagnosed each year in the United States (2). Many patients still succumb to this disease and a large number of survivors suffer from debilitating therapy-related side effects, highlighting the need for alternative therapies. Hedgehog (Hh) signaling is indispensable for normal embryonic development, yet this pathway is mostly dormant in healthy adults (see ref. 3 for a comprehensive review). Proper signaling is initiated by binding of 1 of 3 Hh ligands (Sonic [SHH], Indian [IHH] or Desert [DHH] Hedgehog) to the inhibitory receptor Patched (PTCH1), which somehow alleviates repression of SMO. Through a series of poorly understood events that at least partly take place in primary cilia, this G-protein coupled receptor (GPCR)–like signal transducer promotes activation of GLI transcription factors that facilitate transcription of Hh target genes. Suppressor of fused (SUFU) is a negative regulator of this pathway and acts by binding directly to GLI transcription factors, affecting both their nuclear trafficking and ability to transcribe DNA. Hh signaling was initially linked to cancer through the discovery that Gorlin Syndrome patients, who have a high incidence of sporadic MB, possess inherited inactivating mutations in PTCH1 (4). Constitutive Hh signaling has subsequently been shown in 25% of sporadic MBs, with loss of function PTCH1 or SUFU mutations occurring in approxi- mately half of these cases (5, 6). GDC-0449 is an orally bioavailable SMO antagonist that has produced promising antitumor responses in a Phase I study of patients with advanced basal cell carcinoma (BCC), a type of skin cancer also driven by mutations in Hh pathway compo- nents (7). Furthermore, GDC-0449 promptly regressed tumors and reduced symptoms in a 26-year-old man with metastatic MB that was unmanageable by conventional therapies (8). However, the dramatic response of this patient to GDC-0449 treatment was transient, as most of his metastatic tumors Authors' Affiliation: Genentech Inc., South San Francisco, California Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Frederic J. de Sauvage, Department of Molecular Biology, Genentech Inc., Mailstop 37, 460 Point San Bruno Blvd, South San Francisco, CA 94080. Phone: 650-225-5841; Fax: 650-225-6497. E-mail: sauvage@gene.com. doi: 10.1158/0008-5472.CAN-10-2876 Ó2010 American Association for Cancer Research. Cancer Research www.aacrjournals.org 435 Downloaded from http://aacrjournals.org/cancerres/article-pdf/71/2/435/2659330/435.pdf by guest on 20 June 2022