Molecular targeting of the RET kinase in thyroid cancer

Papillary thyroid carcinoma (PTC) is the most prevalent thyroid cancer subtype. PTC is treated by surgical resection, adjuvant radioiodine treatment and thyroid hormone replacement to suppress thyroid-stimulating hormone. However, some PTC patients may have persistent or recurrent disease with loss of responsiveness to 131I therapy. Medullary thyroid carcinoma (MTC) accounts for 5%-10% of all thyroid cancers. MTC is sporadic in about 75% of cases, and in the others it is a component of the Multiple endocrine neoplasia type 2 (MEN 2) autosomal dominant cancer syndrome. MEN 2-associated hereditary MTC is bilateral and multicentric and it is usually preceded by multifocal C-cell hyperplasia. Early surgery in carriers of RET gene mutations has significantly improved the prognosis of familial MTC. However, MTC patients, particularly those with sporadic tumors, are often incurable because the cancer has already metastasized before being diagnosed.The RET receptor tyrosine kinase could be prime target for the molecular therapy of thyroid cancer. The RET receptor is rearranged (about 30% of the cases) in PTC, generating the chimeric RET/PTC oncogenes. Moreover, germline point mutations in RET cause MEN 2 syndromes and somatic mutations in RET, mainly targeting V804, M918 and E768, are also found in sporadic MTC. RET/PTC and RET/MEN 2 are constitutively active tyrosine kinases able to induce the formation of transformed foci, anchorage independent growth and tumorigenicity in nude mice when introduced in NIH 3T3 cells. Moreover, they initiate thyroid carcinogenesis when introduced in transgenic mice. We have searched for RET inhibitors by analysing by an in vitro RET kinase assay several known ATP-competitive tyrosine kinase inhibitors (pyrazolo-pyrimidines, cinnamomalonitrile derivaties, quinoxalines, quinazolines and anilino-quinazolines). Some pyrazolo-pyrimidines scored able to inhibit RET/PTC and RET/MEN 2 autophosphorylation in a dose dependent manner. ZD6474 (Zactima, vandetanib), an anilino-quinazoline, and BAY439006 (Sorafenib), an aryl-urea, also inhibited RET with an IC50 of roughly 100 nM. ZD6474 is a KDR and EGFR inhibitor that is already in advanced stage of clinical study for its antiangiogenic activity. BAY439006 (Sorafenib) is a multi-target kinase inhibitor approved for renal carcinoma able to affect besides RET also VEGFR, PDGFR and KIT kinases. ZD6474 had cytostatic effects in human thyroid carcinoma cells spontaneously harbouring oncogenic RET alleles and had significant activity in xenografted RET-mutation positive medullary thyroid carcinoma TT cells. X-ray structure analysis confirmed that ZD6474 binds the ATP-pocket of the RET kinase.One problem that could emerge with the use of these inhibitors is molecular resistance formation secondary to primary or secondary mutations in the RET kinase domain. Indeed, RET mutants in valine 804, corresponding to threonine 315 in Abl (the “gate-keeper”: a residue important for resistance formation against imatinib), exert significant resistance against ZD6474. Both ZD6474 and BAY439006 are currently undergoing phase II testing in patients with thyroid cancer. Clinical evaluation coupled with measurement of RET phosphorylation levels or RET-pathway activity under inhibitor treatment will be crucial to assess the capability of the compounds to hit the target in human patients and to establish the clinical value of RET inhibition for MTC and PTC treatment. In this context, the development of surrogate markers of RET inhibition will be of great help to assess the efficacy of the compounds.