RET therapeutic targeting in medullary thyroid carcinoma: molecular mechanisms of resistance

Medullary Thyroid Carcinoma (MTC) is a rare C cell-derived thyroid tumor secreting calcitonin. MTC is sporadic in about 75% of cases and it is a component of the autosomal dominant "multiple endocrine neoplasia type 2" (MEN2) syndrome in about 25% of the cases. MTC represents a challenging clinical problem, as most MTC patients show distant metastases at time of diagnosis and chemotherapy and radiotherapy have limited efficacy. MTC is commonly associated to germline or somatic point mutations causing a gain-of-function of RET receptor tyrosine kinase. Given the oncogenic role of RET, it is feasible that specific targeting of this kinase could block tumor growth. Therefore, tyrosine kinase small molecule inhibitors (TKI) have been studied as potential novel agents for MTC treatment. The clinically most advanced RET TKIs are vandetanib (ZD6474) and cabozantinib (XL184), both recently registered for the treatment of locally advanced or metastatic MTC. However, cancer patients may be refractory to TKIs or develop secondary resistance after an initial response. Two major mechanisms have been envisaged to allow cancer cells to escape treatment with TKIs: 1) target up-regulation or mutations impairing drug binding; 2) activation of alternative pathways that bypass drug-mediated block. Thus, it is worth studying in preclinical models, molecular mechanisms of resistance to TKIs, since these informations can be thereafter clinically applied. In this dissertation, we have addressed molecular mechanisms of resistance to RET TKIs in cultured MTC cells. Our results show that MTC cells can develop resistance to chronic vandetanib treatment. Vandetanib-resistant cells show increased proliferation rate, anchorage-independent growth and in vivo tumorigenicity. Despite the absence of secondary RET genetic lesions, resistant cells escape RET inhibition and, differently from parental cells, they are able to grow even when RET is inhibited. Importantly, resistant cells feature hyper-activation of p90RSK kinase, a component of the MAPK (mitogen activated protein kinase) signaling cascade, a fact that mediates RET inhibition bypass. Accordingly, resistant cells are sensitive to p90RSK chemical blockade. In conclusion, gain of MAPK cascade signaling seems to be able to mediate escape from RET inibition in vitro, thus suggesting p90RSK as a promising molecular target to overcome resistance formation to RET TKIs

Ponatinib (AP24534) is a novel potent inhibitor of oncogenic RET mutants associated with thyroid cancer.

CONTEXT: The RET tyrosine kinase encoding gene acts as a dominantly transforming oncogene in thyroid carcinoma and other malignancies. Ponatinib (AP24534) is an oral ATP-competitive tyrosine kinase inhibitor that is in advanced clinical experimentation in leukemia. OBJECTIVE: We tested whether ponatinib inhibited RET kinase and oncogenic activity. METHODS: Ponatinib activity was studied by an in vitro RET immunocomplex kinase assay and immunoblotting. The effects of ponatinib on proliferation of human TT, MZ-CRC-1, and TPC-1 thyroid carcinoma cells, which harbor endogenous oncogenic RET alleles, and of NIH3T3 fibroblasts transfected with oncogenic RET mutants were determined. Ponatinib activity on TT cell xenografted tumors in athymic mice was measured. RESULTS: Ponatinib inhibited immunopurified RET kinase at the IC₅₀ of 25.8 nM (95% confidence interval [CI] = 23.15-28.77 nM). It also inhibited (IC₅₀ = 33.9 nM; 95% CI = 26.41-43.58 nM) kinase activity of RET/V804M, a RET mutant displaying resistance to other tyrosine kinase inhibitor. Ponatinib blunted phosphorylation of point-mutant and rearranged RET-derived oncoproteins and inhibited proliferation of RET-transformed fibroblasts and RET mutant thyroid carcinoma cells. Finally, after 3 weeks of treatment with ponatinib (30 mg/kg/d), the volume of TT cell (medullary thyroid carcinoma) xenografts was reduced from 133 mm³ to an unmeasurable size (difference = 133 mm³, 95% CI = -83 to 349 mm³) (P < .001). Ponatinib-treated TT cell tumors displayed a reduction in the mitotic index, RET phosphorylation, and signaling. CONCLUSIONS: Ponatinib is a potent inhibitor of RET kinase and has promising preclinical activity in models of RET-driven medullary thyroid carcinoma

Ponatinib (AP24534) is a novel potent inhibitor of oncogenic RET mutants associated with thyroid cancer

Context: The RET tyrosine kinase encoding gene acts as a dominantly transforming oncogene in thyroid carcinomaandother malignancies. Ponatinib (AP24534) isanoral ATP-competitive tyrosine kinase inhibitor that is in advanced clinical experimentation in leukemia. Objective: We tested whether ponatinib inhibited RET kinase and oncogenic activity. Methods: Ponatinib activity was studied by an in vitro RET immunocomplex kinase assay and immunoblotting. The effects of ponatinib on proliferation of human TT, MZ-CRC-1, and TPC-1 thyroid carcinoma cells, which harbor endogenous oncogenic RET alleles, and of NIH3T3 fibroblasts transfected with oncogenic RET mutants were determined. Ponatinib activity on TT cell xenografted tumors in athymic mice was measured. Results: Ponatinib inhibited immunopurified RET kinase at the IC50 of 25.8 nM (95% confidence interval [CI] = 23.15-28.77 nM). It also inhibited (IC 50 = 33.9 nM; 95% CI = 26.41-43.58 nM) kinase activity of RET/V804M, a RET mutant displaying resistance to other tyrosine kinase inhibitor. Ponatinib blunted phosphorylation of point-mutant and rearranged RET-derived oncoproteins and inhibited proliferation of RET-transformed fibroblasts and RET mutant thyroid carcinoma cells. Finally, after 3 weeks of treatment with ponatinib (30 mg/kg/d), the volume of TT cell (medullary thyroid carcinoma) xenografts was reduced from 133 mm3 to an unmeasurable size (difference = 133 mm3, 95% CI = -83 to 349 mm3) (P < .001). Ponatinib-treated TT cell tumors displayed a reduction in the mitotic index, RET phosphorylation, and signaling. Conclusions: Ponatinib is a potent inhibitor of RET kinase and has promising preclinical activity in models of RET-driven medullary thyroid carcinoma. Copyright © 2013 by The Endocrine Society

p90RSK Regulates p53 Pathway by MDM2 Phosphorylation in Thyroid Tumors

: The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation