Ponatinib is a potent inhibitor of wild-type and drug-resistant gatekeeper mutant RET kinase Luca Mologni a,⇑ , Sara Redaelli a , Andrea Morandi b,1 , Ivan Plaza-Menacho c , Carlo Gambacorti-Passerini a a Dept. of Health Sciences, University of Milano-Bicocca, via Cadore 48, 20900 Monza, Italy b Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom c Structural Biology Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3LY, United Kingdom article info Article history: Received 25 March 2013 Received in revised form 11 June 2013 Accepted 17 June 2013 Available online 27 June 2013 Keywords: RET Thyroid cancer Ponatinib Tyrosine kinase Gatekeeper abstract RET kinase is aberrantly activated in thyroid cancers and in rare cases of lung and colon cancer, and has been validated as a molecular target in these tumors. Vandetanib was recently approved for the treat- ment of medullary thyroid cancer. However, vandetanib is ineffective in vitro against RET mutants car- rying bulky aminoacids at position 804, the gatekeeper residue, similarly to drug-resistant BCR–ABL mutants in chronic myeloid leukemia. Ponatinib is a multi-target kinase inhibitor that was recently approved for treatment-refractory Philadelphia-positive leukemia. We show here potent inhibition of oncogenic RET by ponatinib, including the drug-insensitive V804M/L mutants. Ponatinib inhibited the growth of RET+ and BCR–ABL+ cells with similar potency, while not affecting RET-negative cells. Both in biochemical and in cellular assays ponatinib compared favorably with known RET inhibitors, such as vandetanib, cabozantinib, sorafenib, sunitinib and motesanib, used as reference compounds. We sug- gest that ponatinib should be considered for the treatment of RET+ tumors, in particular those expressing vandetanib-resistant V804M/L mutations. Ó 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction RET (REarranged during Transfection) proto-oncogene is a transmembrane tyrosine kinase receptor expressed in central and peripheral nervous system and neural crest-derived cells that transduces proliferative and survival signals in response to GDNF-family neurotrophic factors. Aberrant RET kinase activity is involved in the onset of hereditary and sporadic thyroid cancer (Mologni, 2011) and in rare cases of colon and lung cancers (Wood et al., 2007; Lipson et al., 2012). Germline activating mutations affecting the extracellular and the catalytic domains have been de- scribed in 100% of multiple endocrine neoplasia type 2A (MEN2A) and MEN2B patients, respectively, as well as in familial medullary thyroid carcinoma (FMTC) (Borrello et al., 2013). In addition, differ- ent mutations spanning the entire receptor are found in a variable proportion of sporadic MTC (30–50%). Moreover, several rear- ranged forms of RET have been identified in up to 80% of papillary thyroid cancer (PTC) patients, depending on age, exposure to radi- ation, and histological tumor variant. In these cases, the intracellu- lar kinase domain is fused to the dimerization region of an activating gene. Whatever the mechanism, in all cases RET kinase activity is turned on independently of ligand binding and induces malignant transformation of cells. RET uncontrolled activity is both sufficient and necessary to cause neoplastic phenotype (Plaza- Menacho et al., 2006). Therefore, it represents an ideal target for molecular therapy. Several small-molecule inhibitors are currently under clinical investigation for selective RET inhibition (Mologni, 2011). Among them, vandetanib (ZD6474, Zactima™) is a rather potent inhibitor of rearranged RET and of oncogenic RET mutants observed in thy- roid cancer (Carlomagno et al., 2002; Vitagliano et al., 2011). After encouraging results of the ZETA trial (Wells et al., 2012), it was ap- proved in 2011 for metastatic MTC. Unfortunately, vandetanib is inactive against the V804M gatekeeper mutant of RET (Carlomagno et al., 2004), which has variable frequency in MEN2 families from different countries and is typically associated with FMTC and atyp- ical MEN2 (Pinna et al., 2007; Machens and Dralle, 2008; Shifrin et al., 2009). The gatekeeper residue is a key aminoacid within the active site of tyrosine kinases (Zuccotto et al., 2010). It controls access of small-molecules to a hydrophobic cavity also known as the selectivity pocket. Therefore, mutations at this position are 0303-7207/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.06.025 Abbreviations: CML, chronic myeloid leukemia; ALL, acute lymphoblastic leukemia; RET, REarranged during Transfection; GDNF, glial-derived neurotrophic factor; TKI, tyrosine kinase inhibitor; MTC, medullary thyroid carcinoma; FMTC, familial MTC; PTC, papillary thyroid carcinoma; MEN2, multiple endocrine neopla- sia type 2; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide. ⇑ Corresponding author. Tel.: +39 026448 8362; fax: +39 026448 8363. E-mail address: luca.mologni@unimib.it (L. Mologni). 1 Present address: Department of Experimental and Clinical Biomedical Sciences, University of Florence, Italy. Molecular and Cellular Endocrinology 377 (2013) 1–6 Contents lists available at SciVerse ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce