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Novel Pyrazolopyrimidine Derivatives as Tyrosine Kinase Inhibitors with Antitumoral Activity in Vitro and in Vivo in Papillary Dedifferentiated Thyroid Cancer Alessandro Antonelli, Guido Bocci, Concettina La Motta, Silvia Martina Ferrari, Poupak Fallahi, Anna Fioravanti, Stefania Sartini, Michele Minuto, Simona Piaggi, Alessandro Corti, Greta Alì, Piero Berti, Gabriella Fontanini, Romano Danesi, Federico Da Settimo, and Paolo Miccoli Departments of Internal Medicine (A.A., S.M.F., P.F.), Pharmaceutical Science (C.L.M., S.S., F.D.S.), Surgery (M.M., G.A., P.B., G.F., P.M.), and Experimental Pathology (S.P., A.C.) and Division of Pharmacology and Chemotherapy (G.B., A.F., R.D.), University of Pisa, School of Medicine, I-56100 Pisa, Italy

Aim: We have studied the antitumoral activity of two new pyrazolo[3,4-d]pyrimidine compounds (CLM3 and CLM29) in primary papillary dedifferentiated thyroid cancer (DePTC) cells. Methods: The antiproliferative effect was tested in DePTC cells obtained at reoperation from patients with recurrence of the tumor. The concentrations of CLM3 and CLM29 used in the in vitro experiments were 1, 10, 30, and 50 ␮M. Results: Proliferation assays in DePTC cells showed a significant reduction of proliferation by CLM3 and CLM29, which was by 12% with CLM3 (the most potent compound) 10 ␮M, 43% with CLM3 30 ␮M, and 60% with CLM3 50 ␮M. CLM3 and CLM29 increased the percentage of apoptotic cells in DePTC cells dose dependently (P ⬍ 0.001) and inhibited migration (P ⬍ 0.001). A DePTC cell line (AL) was injected sc in CD nu/nu mice, and tumor masses became detectable 10 d after xenotransplantation. CLM3 (40 mg/kg 䡠 die) significantly inhibited tumor growth and weight, and the therapeutic effect was significant starting on the 19th day after cell implantation (4 d after the beginning of treatment). The CLM3-treated group of animals did not show any appreciable toxicity. CLM3 and CLM29 increased thrombospondin-1 expression in the AL cell line. A significant reduction of microvessels and in the percentage of antivascular endothelial growth factor antibody immunoreactivity was observed in the CLM3 treated tumors, with a simultaneous increase of the percentage of necrosis. Conclusion: The antitumoral activity of two new pyrazolo[3,4-d]pyrimidine compounds (CLM3, CLM29) in vitro and CLM3 in vivo in DePTC has been shown, opening the way to a future clinical evaluation. (J Clin Endocrinol Metab 96: E288 –E296, 2011)

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apillary thyroid cancer (PTC) is usually curable by the combination of surgery, radioiodine ablation, and thyroid-stimulating hormone suppressive therapy; recurrence occurs in 20 – 40% of patients (1, 2). During tumor progression, cellular dedifferentiation occurs in up to 5% of cases, and it is usually accompanied by more

aggressive growth, metastatic spread, and loss of iodide uptake ability, making the tumor resistant to the traditional therapeutic modalities and radioiodine [papillary dedifferentiated thyroid cancer (DePTC)] (1–3). In cases, RET/PTC rearrangements are found in 30 – 40%, RAS mutations in about 10%, and BRAF mutations in

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/jc.2010-1905 Received August 13, 2010. Accepted October 22, 2010. First Published Online December 8, 2010

Abbreviations: Ct, Threshold cycle; DePTC, papillary dedifferentiated thyroid cancer; PP1, pyrazolo-pyrimidine; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine; PTC, papillary thyroid cancer; TSP, thrombospondin; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

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approximately 40 –50%, with no overlap among these mutations in PTC, whereas a higher prevalence of BRAF mutations (up to 70%) has been observed in DePTC (4 – 6). Activated oncogenes are highly attractive targets for the development of new specific anticancer agents. To date a number of structurally different compounds have been reported as ATP-competitive inhibitors of the tyrosine kinase activity of RET. They include quinazolines, such as ZD6474 (7), indolin-2-ones, i.e. RPI-1 (8) and SU5416 (9), and pyrazolo[3,4-d]pyrimidines, like pyrazolo-pyrimidine (PP1) (10) and 4-amino-5-(4chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP2) (11), all showing various degrees of efficacy and specificity with respect to other tyrosine kinases. The clinical activity of several compounds, including motesanib diphosphate (AMG 706), BAY 43–9006, ZD 6474, and AG-013736, in DePTC is being studied in phase II trials (12–15), with preliminary studies recording partial response and stabilization (16, 17). These molecules inhibit several targets, including RET tyrosine kinase, vascular endothelial growth factor receptor (VEGFR-1, VEGFR-2, and VEGFR-3), and have an antiangiogenic effect. BAY 43–9006 also inhibits BRAF kinase. Axitinib, a selective inhibitor of VEGFR-1, -2, and -3, has antitumor activity in all histologic subtypes of advanced thyroid cancer (16). AMG 706 (a novel oral inhibitor of VEGFR, platelet-derived growth factor receptor, and KIT) can induce partial responses in patients with advanced or metastatic differentiated thyroid cancer (18). Sorafenib (which inhibits a spectrum of kinases including Raf kinase, VEGFR, platelet-derived growth factor receptor, and RET tyrosine kinases) has clinically relevant antitumor activity in patients with metastatic, iodine-refractory thyroid carcinoma (17, 19). Progress is being made toward effective targeted DePTC therapy. However, additional efforts are needed to finally identify therapies able to control and to cure this disease (20). Researches aimed at the discovery of novel ATP-competitive tyrosine kinase inhibitors led to the development of different chemical templates. One of the most explored is certainly the pyrazolo[3,4-d]pyrimidine heterocyclic core, which proved to be a useful scaffold for the obtainment of effective compounds. Actually, derivatives belonging to this structural class show a large spectrum of activity against both cytoplasmatic and receptor ATP-competitive tyrosine kinases, thus standing out as multitarget agents (21–23). Here we report the in vitro and in vivo antitumoral activity of two novel pyrazolo[3,4-d]pyrimidine derivatives, which showed a remarkable efficacy in DePTC.

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Patients and Methods Patient source for thyroid tissue Thyroid tissue was obtained from five patients with PTC. The diagnosis was established on commonly accepted clinical, laboratory, and histological criteria (24). Tumors were classified according to the thyroid malignancy World Health Organization classification and staged according to the sixth edition of tumor node metastasis staging (variant: classic, 2; follicular, 1; columnar, 2) (pathological tumor node metastasis; T3mN1, T3N1, T2N1, T3N1a, T2N1). Immunohistochemistry showed the presence of expression of TSH receptor, thyroperoxidase, thyroglobulin, and sodium/iodide symporter. All subjects were reoperated for recurrence of thyroid cancer, which was not able to uptake 131-I. The thyroid cancer tissue samples were obtained at the reoperation. In addition, normal thyroid tissues were obtained from five patients (five undergoing parathyroidectomy). The study subjects gave their informed consent to the study, which was approved by the local ethical committee. Microdissection and DNA extraction were performed using conventional methods previously described (24). The detection of BRAF mutation by PCR-single-strand conformation polymorphism and direct DNA sequencing were performed using conventional methods previously described (24). The detection of RET/PTC1 and RET/PTC3 by real-time PCR was performed on a LightCycler (Roche, Indianapolis, IN), as previously described (25) in DePTC.

Thyroid follicular cells Control thyrocytes were prepared as reported previously (26).

Drugs Two novel pyrazolo[3,4-d]pyrimidine derivatives were used for the study, named CLM3 and CLM29 (Fig. 1). The synthesis of the new target compounds was accomplished (by C.L.M., S.S., F.D.S., Department of Pharmaceutical Science) starting from the commercially available 3-amino-4-pyrazolecarbonitrile (SigmaAldrich Co., St. Louis, MO), following a previously reported procedure (23). Briefly, alkylation with 2-bromoethylbenzene at position N1 of the pyrazole nucleus followed by cyclization with boiling formic acid provided the key intermediate 1-phenethyl1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one. Treatment with phosphoryl trichloride gave the corresponding 4-chloro derivative, which led to the target inhibitors, CLM3 and CLM29, by reaction with (R)-(⫹)-1-phenylethanamine and 3-fluorobenzenamine, respectively, in the presence of triethylamine. Stock

FIG. 1. Two novel pyrazolo[3,4-d]pyrimidine derivatives have been used, named CLM3 and CLM29. The structure of each compound is given in figure.

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solution of the tested compounds were made in sterile dimethyl sulfoxide and stored in aliquots at ⫺80 C. CLM3 and CLM29 inhibit several targets, including RET, epidermal growth factor receptor, and VEGFR and have an antiangiogenic effect (23). The concentrations of CLM3 and CLM29 used in the in vitro experiments were 1, 10, 30, and 50 ␮M.

Cell viability and proliferation assay To determine cell proliferation, we used a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (used in the dimethylthiazoldiphenyltetra-zoliumbromide) assay (WST-1; Roche Diagnostics, Almere, The Netherlands), as previously reported (24, 27, 28). To determine the concentration that causes 50% growth inhibition (IC50) of CLM3 or CLM29, a concentration range of CLM3 or CLM29 has been added to the wells (quadruplicates), and IC50 has been determined by using linear interpolation. All experiments were performed in triplicate.

Proliferation assay: cell counting Because 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide measures mitochondrial cell activity and it has already been demonstrated that there is not always a direct relationship with cell number, the proliferation also has been evaluated using cell number counting, as previously reported (24, 27, 28).

Apoptosis determination: Hoechst uptake DePTC cells were seeded in a 96-well microtiter plate at a concentration of 35,000 cells/ml in a final volume of 100 ␮l in each well. Then cultures were incubated for 48 h with CLM3 and CLM29 in a humidified atmosphere (37 C, 5% CO2). After 48 h

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of treatments, the cells were stained with Hoechst 33342 as previously described (24). The apoptosis index (ratio between apoptotic and total cells) ⫻ 100 was calculated.

Apoptosis determination: annexin V binding assay The cells were plated in Lab-tek II chamber slide system (Nalge Nunc International, Rochester, NY), treated with CLM3 or CLM29 for 48 h, and processed as previously described (24).

Migration assays To evaluate the effects on cell migration, DePTC has been cultured subconfluent and treated with increasing concentrations of CLM3 or CLM29 for 24, 48, 72, and 96 h, and migration was tested in Transwell chambers (Corning Life Sciences, Corning, NY), according to manufacturer instructions.

AL cell line From the five primary DePTC cells, one could be passed over 50 times. This line (AL cell line) had the V600EBRAF mutation and was able to grow in nu/nu mice when inoculated sc (see below).

Real-time PCR analysis of thrombospondin (TSP)-1 gene expression on tumor cells To evaluate the expression of the TSP-1 gene, 2 ⫻ 104 AL cells were grown in their respective media and treated with CLM3 (30, 9, 5 ␮M), or CLM29 (30, 15, 5 ␮M), or vehicle alone for 72 h. Briefly, RNA (1 ␮g) was reverse transcribed as previously described (29), the resulting cDNA was diluted (2:3), and then amplified by quantitative RT-PCR with the Applied Biosystems 7900HT sequence detection system (Applied Biosystems, Foster City, CA). The TSP-1-validated primer was purchased from Applied Biosystems (assay ID Hs00173626_m1). The PCR thermal cycling conditions and optimization of primer concentrations were followed as per the manufacturer’s instructions. Amplifications were normalized to glyceraldehyde 3-phosphate dehydrogenase, and the quantitation of gene expression was performed using the ⌬⌬Ct calculation, where Ct is the threshold cycle; the amount of target, normalized to the endogenous control and relative to the calibrator (vehicle-treated control cells), is given as 2⫺⌬⌬Ct.

In vivo studies Animals

FIG. 2. WST-1 assay (at 2 h from the start of tetrazolium reaction) (mean ⫾ SEM of all samples) in DePTC cells (A) or control thyroid cells (C), treated with CLM3 for 24 h. A significant reduction of proliferation with respect to the control was shown with CLM3 10, 30, or 50 ␮M (A) or 30 or 50 ␮M (C). WST-1 assay (at 2 h from the start of tetrazolium reaction) (mean ⫾ SEM of all samples) in DePTC cells (B) or control thyroid cells (D), treated with CLM29 for 24 h. A significant reduction of proliferation with respect to the control was shown with CLM29 30 or 50 ␮M (B) or 50 ␮M (D). Bars are mean ⫾ SEM. *, P ⬍ 0.05 or less vs. control (Ctrl).

The CD nu/nu male mice, weighing 20–25 g, were supplied by Charles River (Milan, Italy) and were allowed unrestricted access to food and tap water. Housing and all procedures involving animals were performed according to the protocol approved by the Academic Committee for the animal experimentation of the University of Pisa, in accordance with the European Community Council Directive 86609, recognized by the Italian government, on animal welfare. Each experiment employed the minimum number of mice needed to obtain statistically meaningful results.

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AL xenografts in nu/nu mice and drug treatments AL cell viability was assessed by trypan blue dye exclusion and on d 0, 1.3 ⫻ 106 ⫾ 5% cells/mouse were inoculated sc between the scapulae in 0.2 ml/mouse of culture medium without fetal bovine serum. Animal weights and appearance of a sc tumor were monitored; the tumor was measured every 2 d in two perpendicular directions using calipers. Tumor volume (cubic millimeters) was defined as follows: [(w1 ⫻ w2 ⫻ w2) ⫻ (␲/6)], where w1 and w2 were the largest and smallest tumor diameter (millimeters), respectively. The mice were randomized into groups of six animals. To treat an established tumor (⬃35 mm3), on d 15 from cell inoculum CLM3 was administered ip at the dose of 40 mg/kg 䡠 die for 40 d. The control group was injected ip with vehicle alone (saline solution). The experimental period ended 55 d after the cell inoculum when mice were killed by an anesthetic overdose.

Immunohistochemistry and microvessel density on AL tumor tissue samples After surgical resection, tumor tissue samples from all the different treatment groups were weighed and then fixed in 10% neutral-buffered formalin for 12–24 h and embedded in paraffin for histology and immunohistochemistry. Sections of the tumor (5 ␮m thick) were stained with hematoxylin and eosin. Immunostainings were performed by a Benchmark immunostainer (Ventana Medical Systems, Tucson, AZ) using the avidin-biotin-peroxidase complex method and counterstained with hematoxylin. Negative controls were carried out by omitting the primary antibodies. Immunohistochemistry was evaluated independently by two pathologists (G.A. and G.F.) blinded to the treatment code. For the vascular endothelial growth factor (VEGF) expression, an anti-VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:50 dilution. The expression of VEGF was evaluated as a percentage of positive cells over a total of at least 1000 tumor cells. Microvascular count was determined using anti-FVIII polyclonal antibody (Ventana Medical Systems). A single microvessel was defined as any immunostained endothelial cell separated from adjacent microvessel, tumor cells, and other connective tissue elements. Samples were examined by each pathologist, who identified the area with most intense vascularization (hot spot) under low microscopic power (⫻100). A region of 0.74 mm2/field was then scanned at ⫻250 microscope magnification. Five fields were analyzed, and for each of them, the number of stained blood vessels was counted. For individual tumors, microvascular count was scored by averaging the five field counts. The presence of a lumen was not required for scoring as a microvessel.

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ferroni-Dunn test. Data about apoptosis were analyzed by oneway ANOVA with Newman-Keuls multiple comparisons test.

Results In vitro studies Cell proliferation assays The results of the WST-1 assay (Roche Diagnostics) in the DePTC cells showed a significant reduction of proliferation with respect to the control with CLM3 at 1 and at 2 h (P ⬍ 0.01, for both, ANOVA) (Fig. 2A). The cell counting confirmed the above-mentioned results at 2 h. In DePTC the cell number was 18236 ⫾ 710/100 ␮l/well; 16010 ⫾ 1260 (88%) with CLM3 10 ␮M; 10460 ⫾ 1320 (57%) with CLM3 30 ␮M; 7294 ⫾ 1245 (40%) with CLM3 50 ␮M; (P ⬍ 0.01, ANOVA). The results of WST-1 assay (Roche Diagnostics) in DePTC cells showed a significant reduction of proliferation with respect to the control with CLM29 at 1 h and at 2 h (P ⬍ 0.01, for both, ANOVA) (Fig. 2B). In DePTC the cell number was 18565 ⫾ 809/100 ␮L, per well; 16830 ⫾ 1310 (91%) with CLM29 10 ␮M; 14552 ⫾ 1360 (78%) with CLM29 30 ␮M; 12996 ⫾ 1320 (70%) with CLM29 50 ␮M; (P ⬍ 0.01, ANOVA).

Data analysis Values are given as mean ⫾ SD for normally distributed variables (in text), or mean ⫾ SEM (in figures), otherwise as median and (interquartile range). The experiments were repeated three times with the cells from each donor. The mean of the experiments in the five specimens from different donors, for normal and PTC samples, is reported. The mean group values were compared by one-way ANOVA for normally distributed variables, otherwise by the Mann-Whitney U or Kruskal-Wallis test. Proportions were compared by the ␹2 test. Post hoc comparisons on normally distributed variables were carried out using the Bon-

FIG. 3. Apoptosis in DePTC cells treated with CLM3 or CLM29 48 h (mean ⫾ SEM of all samples). Apoptosis index was determined by Hoechst staining (see Patients and Methods). The percentage of apoptotic cells increased markedly in a dose-dependent manner with CLM3 10, 30, or 50 ␮M (A) or CLM29 30 or 50 ␮M (B). Data are expressed as means ⫾ SD (n ⫽ 5). Data were analyzed by one-way ANOVA with Newman-Keuls multiple comparisons test and a test for linear trend. P ⬍ 0.001 vs. control (Ctrl).

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The results of WST-1 assay (Roche Diagnostics) in normal thyroid follicular cells with CLM3 showed a slight but significant reduction of proliferation with respect to the control both at 1 and at 2 h (P ⬍ 0.01, for both, ANOVA) with CLM3 30 and 50 ␮M (Fig. 2C). The results of the WST-1 assay in normal thyroid follicular cells with CLM29 showed a slight but significant reduction of proliferation with respect to the control both at 1 and at 2 h (P ⬍ 0.01, for both, ANOVA) only with CLM29 50 ␮M (Fig. 2D). The cell counting confirmed the above-mentioned results (data not shown). The IC50 values determined by using linear interpolation, were 33 ␮M for CLM3 and 68 ␮M for CLM29. BRAF and proliferation The V600EBRAF mutation was observed in three PTCs; RET/PTC1 and RET/PTC3 by real-time PCR were not detected in primary cells from DePTC. The results about the inhibition of proliferation by CLM3 and CLM29, obtained in PTC from tumors with V600EBRAF mutation,

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were similar to those from tumors without BRAF mutations (data not shown). Apoptosis determination The percentage of apoptotic cells in DePTC cells increased dose dependently: after treatment with CLM3 10 ␮M, 5.3% of the cells were apoptotic, and this percentage increased up to 14.1 and 18.6% with CLM3 30 or 50 ␮M, respectively (P ⬍ 0.001; by ANOVA; Fig. 3A). At the lower dose of CLM29, 4% of the cells were apoptotic, and this percentage increased up to 12 and 13% with CLM29 30 or 50 ␮M, respectively (P ⬍ 0.001; by ANOVA; Fig. 3B). Annexin V was used to further confirm the induced cell apoptosis (data not shown). Migration assays To evaluate the effects on cell migration, DePTC has been cultured subconfluent and treated with increasing concentrations of CLM3 or CLM29 for 24, 48, 72, and 96 h, and migration was tested in Transwell chambers (Corning Life Sciences), revealing an inhibition of migration with CLM3 (Fig. 4A) or CLM29 (Fig. 4B). CLM3 and CLM29 modulate the expression of TSP-1, an endogenous inhibitor of angiogenesis, in cancer cells After 72 h of exposure, CLM3 and CLM29 increased TSP-1 expression in the AL cell line; in particular, CLM3 significantly increased TSP-1 at lower concentrations (P ⬍ 0.05; Fig. 5A), whereas CLM29 enhanced TSP-1 expression in a concentration-dependent manner (Fig. 5B), with the maximum increase at the highest concentration. In vivo studies

FIG. 4. Migration (tested in Transwell chambers; Corning Life Sciences) in DePTC treated with increasing concentrations of CLM3 (A) or CLM29 (B) for 24, 48, 72, and 96 h. An inhibition of migration and invasion was revealed with CLM3 or CLM29. Bars are mean ⫾ SD. *, P ⬍ 0.05 for CLM29 and *, P ⬍ 0.01 for CLM3 with respect to fetal calf serum (FCS) 10% (FCS10%, medium ⫹ FCS 10%) by Newman-Keuls test.

CLM3 inhibits significantly AL tumor growth in absence of toxicity AL cells were injected sc in CD nu/nu mice and tumor masses became detectable 10 d after xenotransplantation. Tumors in control animals showed a progressive but slow enlargement of their dimensions. At d 40 the animals of the control group and treated group were killed (Fig. 6A). CLM3 (40 mg/ kg 䡠 die) inhibited significantly the tumor growth, and the therapeutic effect was significant starting on the 19th day

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Discussion Two novel pyrazolo[3,4-d]pyrimidine derivatives have been disclosed (CLM3 and CLM29), which were able to inhibit the proliferation of primary cells of DePTC in vitro by increasing apoptosis. Furthermore, CLM3 and CLM29 inhibited the migration of DePTC cells. The inhibitory effect of CLM3 and CLM29 was independent from the presence of V600EBRAF mutation. A DePTC cell line (AL), with V600E BRAF mutation, was produced, which was able to grow in nu/nu mice when inoculated sc. CLM3 and CLM29 increased TSP-1 expression in the AL cell line. Furthermore, CLM3 significantly inhibited AL tumor growth in CD nu/nu mice in the absence of toxicity and decreased the microvessel density in AL tumor tissues. FIG. 5. TSP-1 gene expression after CLM3 (A) and CLM29 (B) treatment for 72 h (mean ⫾ SEM of all samples). Bars are mean ⫾ SEM. **, P ⬍ 0.001; #, P ⬍ 0.05. C, Representative To date, other pyrazolo[3,4-d]pyrimicroscopic image of sc AL tumor xenograft in nude mice. D, Microscopic image of FVIII midines, like PP1 (10) and PP2 (11), immunoreactivity of AL tumor xenograft. Arrowhead, tumor vessel. Magnification, ⫻20. have been investigated in thyroid cancer, which exerted potent inhibitory efafter cell implantation (4 d after the beginning of treat- fects on RET kinase (10) and inhibited the proliferation ment) compared with controls (Fig. 6A). The CLM3- and the invasive phenotype of human thyroid carcinoma treated group of animals did not show any appreciable cells sustaining RET/PTC1 rearrangements (11). Howtoxicity (Fig. 6B), as demonstrated from their weights. ever, PP2 was not selective for RET, being also a good Moreover, when the animals were killed at the end of the inhibitor of c-Src and related kinases (30). Therefore, it experiment, tumors were explanted and weighed showing was not possible to exclude additional indirect effects of a significant reduction of CLM3-treated tumor weights if PP2 mediated in vivo by the inhibition of other kinases. compared with controls (10.9 ⫾ 5.5 vs. 37.2 ⫾ 5.3 mg, Interestingly, the primary DePTC cells that we used were respectively; P ⬍ 0.05; Fig. 6C). all RET/PTC negative. Moreover, the antiproliferative action of CLM3 and CLM29 was observed in all primary DePTC cells, in presence or absence of V600EBRAF mutation. CLM3 significantly decreases the microvessel These results are in agreement with the concept that CLM3 density in AL tumor tissues The sc injection of AL cancer cells produced a tumor and CLM29 are proposed for a multiple signal transduction whose histological picture, after staining with hematox- inhibition (including RET, epithelial growth factor receptor, ylin and eosin, was consistent with undifferentiated PTC VEGFR), and they have an antiangiogenic effect (23). The CLM3 IC50 value for cell proliferation (in primary (Fig. 5C). A well-defined FVIII immunoreactivity was loDePTC cells) was 33 ␮M, similar to the IC50 of 2-indolicalized in endothelial cells inside the control tumor mass (Fig. 5D). A significant (P ⬍ 0.05) reduction of microvessel done derivatives (about 30 ␮M) (8) and higher than that of density was observed in the CLM3-treated tumors com- ZD6474 (1 ␮M) (7) or PP1 (10) and PP2 (11) (⬍5 ␮M) pared with the control group (8.2 ⫾ 2.7 vs. 17.0 ⫾ 8.1). (evaluated in human thyroid cancer cell lines). Interestingly, CLM3 significantly inhibited AL tumor In addition, a significant (P ⬍ 0.05) decrease in the percentage of anti-VEGF antibody immunoreactivity in the growth in CD nu/nu mice in the absence of toxicity, tumor mass was also observed in the treated group of whereas other compounds have a broad spectrum of adanimals (41.7 ⫾ 2.8 vs. 65.0 ⫾ 7.1), with a simultaneous verse effects both in animals and humans (31). There are still some limitations in the selective use of increase (P ⬍ 0.05) of the percentage of necrosis (20.0 ⫾ novel compounds. A lack of response can occur, for in17.0 vs. 5.0 ⫾ 0.1), with respect to control.

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FIG. 6. AL cells were injected sc in CD nu/nu mice, and tumor masses became detectable 10 d after xenotransplantation. CLM3 (40 mg/kg 䡠 die) inhibited significantly the tumor growth compared with controls (A). The CLM3-treated group of animals did not show any appreciable toxicity (B), as demonstrated from their weights. When the animals were killed, a significant reduction of CLM3-treated tumor weights, if compared with controls, was observed (C). Bars are mean ⫾ SEM. *, P ⬍ 0.05 vs. control.

stance, because target inhibition raises the activity of compensatory signal pathways, which, in turn, rescue tumor cell growth. However, the possibility of testing the sensitivity of primary DePTC cells from each subject to different tyrosine kinase inhibitors could increase the effectiveness of the treatment. In fact, in vitro chemosensitivity tests are able to predict in vivo effectiveness in 60% of cases (32), whereas it is well known that a negative chemosensitivity test in vitro is associated with a 90% of ineffectiveness of the treatment in vivo (32), allowing the administration of inactive chemotherapeutics to these patients to be avoided (6, 27, 33). Interestingly, our results show an antitumoral effect of CLM3 and CLM29, not on continuous cell lines that are quite different from the tumor of the patients themselves but directly on primary DePTC of patients refractory to the radioiodine therapy. Moreover, we have produced a new cell line (AL) derived from primary DePTC cells. This line (AL) had the V600E BRAF mutation and was able to grow in nu/nu mice when inoculated sc. To our knowledge, there is only one other study in the literature that has shown the establishment of xenografts from human PTC in mice (34); however, no effect of antitumoral drugs was demonstrated. The AL cell line could be a valuable tool, in the near future, for the evaluation of the antineoplastic activity of other drugs, both in vitro and in vivo.

The antineoplastic activity of CLM3 and CLM29 may result from the combination of an antiproliferative effect associated with the increase of apoptosis in the tumoral cells and the inhibition of the migration and the neoplastic neovascularization. This last effect has been shown in vivo. In fact, a significant reduction of microvessels was observed in the CLM3-treated tumors. In addition, a significant decrease in the percentage of anti-VEGF antibody immunoreactivity in the tumor mass was also observed in the CLM3-treated group of animals. The mechanisms uderlying the inhibition of the neoplastic neovascularization are probably related to the up-regulation of the main endogenous inhibitor of the angiogenesis, i.e. TSP-1; in fact, CLM3 and CLM29 increased TSP-1 expression in the AL cell line. TSP-1 has many antiangiogenic effect: 1) inducing apoptosis of endothelial replicating cells (35); and 2) interacting with many extracellular proteins involved in the angiogenic process, such as VEGF (36, 37). Other antiangiogenic therapies have shown an up-regulation of TSP-1, such as the metronomic therapy (29), and also molecular targeted drugs such as herceptin (monoclonal antibody against the cell surface receptor HER2 for human epidermal growth factor receptor-2) (38). Tumor cells often devise strategies to bypass the effects of antineoplastic agents, and selection of therapy-resistant clones is frequently the reason for treatment failure. The STI 571-treated chronic myeloid leukemia patients relapse

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is due to accumulation of very specific point mutations within the catalytic domain of the abl kinase (tyrosine kinase encoded by the Abelson murine leukemia viral oncogene homolog 1-ABL1 - proto-oncogene) (39). In this frame, the availability of a panel of compounds with different antineoplastic activity could be an important step in bypassing the development of treatment resistance. In conclusion, the antitumoral activity of two new pyrazolo[3,4-d]pyrimidine compounds (CLM3 and CLM29) in DePTC has been shown, opening the way to a future clinical evaluation.

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Note Added in Proof The authors would like to acknowledge previous work in the field of pyrazolopyramidine derivatives (40 – 42).

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Acknowledgments Address all correspondence and requests for reprints to: Alessandro Antonelli, M.D., Department of Internal Medicine, University of Pisa, School of Medicine, Via Roma 67, I-56100, Pisa, Italy. E-mail: [email protected]. Disclosure Summary: The authors have nothing to disclose.

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