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Development and Characterization of a Differentiated Thyroid Cancer Cell Line Resistant to VEGFR-Targeted Kinase Inhibitors Crescent R. Isham, Brian C. Netzel, Ayoko R. Bossou, Dragana Milosevic, Kendall W. Cradic, Stefan K. Grebe, and Keith C. Bible Division of Medical Oncology (C.R.I., A.R.B., K.C.B.), Department of Oncology, and Department of Laboratory Medicine and Pathology (B.C.N., D.M., K.W.C., S.K.B.), and the Endocrine Malignancies Disease Oriented Group (C.R.I., A.R.B., S.K.G., K.C.B.), Mayo Clinic, Rochester, Minnesota 55905

Background: Vascular endothelial growth factor-targeted kinase inhibitors have emerged as highly promising therapies for radioiodine-refractory metastatic differentiated thyroid cancer. Unfortunately, drug resistance uniformly develops, limiting their therapeutic efficacies and thereby constituting a major clinical problem. Approach and Methods: To study acquired drug resistance and elucidate underlying mechanisms in this setting, BHP2–7 human differentiated thyroid cancer cells were subjected to prolonged continuous in vitro selection with 18 ␮M pazopanib, a clinically relevant concentration; acquisition of pazopanib resistance was serially assessed, with the resulting resistant cells thereafter subcloned and characterized to assess potential mechanisms of acquired pazopanib resistance. Results: Stable 2- to 4-fold in vitro pazopanib resistance emerged in response to pazopanib selection associated with similar in vitro growth characteristics but with markedly more aggressive in vivo xenograft growth. Selected cells were cross-resistant to sunitinib and to a lesser extent sorafenib but not to MAPK kinase (MEK1/2) inhibition by GSK1120212. Genotyping demonstrated acquisition of a novel activating KRAS codon 13 GGC to GTT (glycine to valine) mutation, consistent with the observed resistance to upstream vascular endothelial growth factor receptor inhibition yet sensitivity to downstream MAPK kinase (MEK1/2) inhibition. Conclusions: Selection of thyroid cancer cells with clinically utilized therapeutics can lead to acquired drug resistance and altered in vivo xenograft behavior that can recapitulate analogous drug resistance observed in patients. This approach has the potential to lead to insights into acquired treatment-related drug resistance in thyroid cancers that can be subjected to subsequent validation in serially collected patient samples and that has the potential to yield preemptive and responsive approaches to dealing with this important clinical problem. (J Clin Endocrinol Metab 99: E936 –E943, 2014)

D

ifferentiated thyroid cancer (DTC) incidence is rapidly rising worldwide (1– 6). In the United States, DTC is now the sixth most incident cancer in women and the eighth most incident cancer overall, with deaths increasing by almost 40% in the last decade (1). Although most DTC patients fare well in response to conventional

therapies including surgery and therapeutic radioactive iodine (RAI), a small portion develop widespread lifethreatening RAI-refractory metastatic disease that is largely resistant to cytotoxic chemotherapy (7). This observation of poor responsiveness to cytotoxic chemotherapy in most DTC patients who require systemic

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received June 27, 2013. Accepted February 28, 2014. First Published Online March 14, 2014

Abbreviations: DMSO, dimethylsulfoxide; DTC, differentiated thyroid cancer; gDNA, genomic DNA; KI, kinase inhibitor; MEK, MAPK kinase 1/2; PAX8, paired box transcription factor 8; PPAR, peroxisomal proliferator-activated receptor; RAI, radioactive iodine; STR, short tandem repeat; VEGFR, vascular endothelial growth factor receptor.

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therapy beyond RAI has led to a search for novel candidate therapeutic molecular targets and to the realization that signaling through the canonical MAPK pathway [eg, vascular endothelial growth factor receptor (VEGFR) ⬎ RAS ⬎ RAF ⬎ MAPK kinase (MEK1/2) ⬎ ERK] is often up-regulated in DTC (8 –10). Therapeutics targeting this pathway have consequently been subject to clinical evaluation in DTC (10 –12). Although VEGFR itself has been a target of particular interest (10 –14), activating mutations of several VEGFR-downstream protein kinases, including BRAF or KRAS, are common in DTC and, when combined with RET/PTC rearrangements, are found in greater than 70% of DTCs (8 –10). In this context, kinase inhibitors (KIs), particularly those targeting VEGFR, have emerged as highly promising therapeutics in this patient cohort (8 –11), with durable Response Evaluation Criteria In Solid Tumors response rates approaching 50% in DTC patients treated with pazopanib (8). Unfortunately, the therapeutic efficacies of these agents is severely limited by drug resistance, and no patients are cured using KI therapy (10 –14). The important problem of acquired resistance to VEGFR inhibitor therapy is very challenging to study in patients, making the derivation of preclinical approaches to defining mechanisms of acquired KI resistance in DTC attractive. In such an effort, we therefore undertook in vitro selection of DTC cells with the multitargeted and VEGFR-directed kinase inhibitor pazopanib. This initiative led to the generation of stable, pazopanib-resistant DTC cells that were characterized as reported in this manuscript, with the identification of an acquired novel activating KRAS mutation as apparently responsible.

Materials and Methods

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Selection of BHP2–7 cells with pazopanib BHP2–7 cells were selected in media containing 18 ␮M pazopanib for approximately 6 months, by which time substantive pazopanib resistance had developed; 18 ␮M pazopanib was used for selection because it represented the greatest concentration in which pazopanib was found to reliably remain solubilized in tissue culture media, and this concentration is easily clinically achievable (target patient pazopanib concentrations are ⬎ 40 ␮M) (16). Both wild-type and pazopanib-selected BHP2–7 cells were evaluated by STR analysis performed by the Mayo Clinic Genotyping Core to confirm lineage identity by comparison with the published BHP2–7 DNA microsatellite fingerprint (15). Pazopanib-selected sublines were created by subcloning to attain individual uniform pazopanib-resistant cell populations to avoid potential problems from studying mixed culture cell populations. This was done by seeding the already pazopanib-selected cell line sparsely on 100-mm tissue culture dishes and allowing individual colonies to develop over 7 days’ growth; colonies were picked up with cloning disk papers soaked in trypsin and put in individual wells of 24-well plates to allow expansion of the subcloned individual sublines. KRAS sequencing was performed on parental and on mixed cultured pazopanib cells and also on all 17 subcloned pazopanib-selected sublines; all but the wild-type cells shared an identical novel KRAS activating mutation as discussed in Results.

Colony-forming assay Colony-forming assays were used to assess drug effects on cell proliferation. Briefly, 400 cells obtained after trypsinizing subconfluent cell culture stocks were plated into each of triplicate sets of 35-mm tissue culture plates and allowed to adhere overnight. Plated cells were then treated with DMSO/diluent (0.1%) or the specified drugs at the indicated concentration ranges (continuous exposure) and allowed to proliferate to form colonies for 7 days; thereafter plates were washed (with PBS), stained with Coomassie blue, and colonies counted on the G:BOX imager system using GeneTools software (Syngene). Results were evaluated graphically using SigmaPlot software. Each presented figure is a representative of one of at least three independent experiments, each performed in triplicate.

Reagents and cell lines Pazopanib and GSK1120212 MEK1/2 inhibitor were kindly provided by GlaxoSmithKline, sunitinib was purchased from LC Labs, and sorafenib (Nexavar, Bayer, and Onyx Pharmaceuticals) was obtained from waste patient prescriptions. All drugs were diluted in dimethylsulfoxide (DMSO), and stock solutions were stored at ⫺20°C. BHP2–7, an established human DTC (papillary) cell line, was kindly provided by Dr John (Al) Copeland (Mayo Clinic Florida); short tandem repeat (STR) analysis was undertaken to confirm identity with the previously published DNA microsatellite fingerprint of the cell line (15). All BHP2–7 lines were cultured in RPMI 1640 with L-glutamine containing nonessential amino acids, sodium pyruvate, HEPES, sodium bicarbonate (media and supplements from CellGro, Fisher), 10% fetal bovine serum (Gemini Bio-Products), 100 U/mL penicillin G, and 100 ␮g/mL streptomycin. Cell lines were passaged twice weekly and maintained at 37°C in an atmosphere containing 95% air-5% CO2.

Mouse xenograft studies All mouse experiments followed institutional guidelines and were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Briefly, BHP2–7 wild-type and BHP2–7 pazopanib-selected/resistant cells were harvested, washed twice, and resuspended in PBS (10 million cells per 100 ␮L). Female athymic nu/nu mice (5– 6 wk old) purchased from Harlan Laboratories, Inc were anesthetized using isoflurane and injected sc in the flank with 100 ␮L of either BHP2–7 wt or pazopanib resistant cell inocula (10 animals per group). Tumors were measured with calipers one time weekly, with volumes calculated using the formula: volume ⫽A ⫻ B ⫻ B/2 (where A was the longest tumor dimension and B the smallest). The experiment was terminated when the tumor burden became too great to continue due to concerns over animal welfare, with tumor volumes increasing nearly 300% associated also with ulcerating tumors in the case of the pazopanib-selected cell line cohort.

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Gene mutational analyses

Immunoblotting

For all assessed cell lines, DNA and RNA were isolated sequentially from cells using Norgen’s all-in-one purification kit (Norgen catalog number 24200). Briefly, cells were lysed using provided lysis solution, ethanol was added, and lysates were applied separately to individual spin columns, with both RNA and DNA binding to columns. RNA was then washed (with provided RNA wash solution to remove impurities) and subsequently eluted with provided RNA elution buffer. The remaining column-bound genomic DNA was then washed (with provided genomic DNA (gDNA) wash solution to remove remaining trace RNA), and gDNA was then eluted with the provided gDNA elution buffer. Isolated gDNA was further used for BRAF, KRAS, NRAS, HRAS mutation analysis, with RNA converted to cDNA using Life Technologies SuperScript III first-strand supermix (catalog number 18080400) and used for KRAS, paired box transcription factor 8 (PAX8)/peroxisomal proliferator-activated receptor (PPAR)-␥, and RET/PTC fragment analysis as described below. Sequences of primers used are provided in listed references and Supplemental Tables 1 and 2. LightCycler system (Roche) screening assays were used to assess K-, N- and H-RAS gDNA sequences covering codons 12, 13, and 61 with primer sequences as previously published (17). A PCR was performed with a 95°C preincubation for 10 minutes, with 50 cycles of amplification (melting at 95°C for 5 sec, annealing at 50°C for 20 sec, extension at 72°C for 10 sec), followed by melting curve analysis (denaturation at 95°C for 30 sec and melting from 40°C-80°C with a slope of 0.15 °/sec). Once mutations were confirmed by LightCycler analysis, specimens were sequenced by Sanger sequencing to verify mutation identities. When the novel mutation in KRAS was found, mutation expression was verified by isolating RNA and performing the same LightCycler analysis and Sanger sequencing as above for KRAS codon 13. BRAF mutations were examined by allele-specific, real-time PCR (18). Briefly, LC-RED640 labeled primers designed for specificity to the mutation site were used for allele-specific extension as previously published. Specimens were split and amplified in parallel for each wild-type and mutant primers. PCR was performed with DNA denaturation at 95°C for 10 minutes, followed by 50 cycles (melting at 95°C for 15 sec, annealing at 58°C for 60 sec, and extension at 72°C for 1 min). Amplification curves obtained on each specimen were quantitated against standard curves (normal blood with serial dilutions of melanoma derived BRAFT1799A hemizygous A375 cells). All standard samples were carried through the same preparation in parallel with unknown specimens. The PAX8/PPAR␥ fusion protein (PPFP) and RET/PTC1, RET/PTC2, and RET/PTC3 rearrangements were detected via allele-specific, real-time PCR. cDNA was amplified by PCR using fluorescently labeled primers listed in Supplemental Tables 1 and 2. Denaturation was accomplished at 95°C for 10 minutes, followed with 40 cycles (melting at 95°C for 30 sec, annealing at 64°C for 30 sec, and elongation at 72°C 1 min), with a final elongation at 72°C for 7 minutes. Phosphoglycerate kinase and thyroid-specific gene PAX8 were assessed in parallel as controls. PCR products were analyzed by fragment analysis performed on the ABI 3130xl genetic analyzer (Applied Biosystems). Each fusion product was characterized and identified by the fragment size and the fluorescent dye color (19).

BHP2–7 cells in log-phase growth were treated with DMSO (0.1% final concentration), 10 nM MEK1/2 inhibitor GSK1120212, or 18 ␮M pazopanib for 2 hours, harvesting protein by scraping adherent cells off tissue culture plates maintained on ice, washing harvested cells twice with ice-cold PBS, and lysing cells in CelLytic M cell lysis reagent (Sigma Aldrich) containing complete miniprotease inhibitor cocktail (Roche) and Halt phosphatase inhibitor cocktail (Thermo Scientific). Bicinchoninic assays (Thermo Scientific) were performed to facilitate equal protein loading of 40 ␮g total protein per sample well. Samples boiled at 95°C in 4⫻ reducing sample buffer were loaded on duplicate 12.5% criterion Tris-HCl (BioRad Laboratories) gels for SDS-PAGE. Gels were transferred to nitrocellulose membranes (BioTrace NT; Pall Corp) and blotted for phospho-ERK1/2 Thr202/Tyr204, Thr185/Tyr187, and total ERK1/2 (catalog number 05–797R and 06 –182; Millipore). Detection was carried out by incubating with a horseradish peroxidase-conjugated secondary antibody, either to mouse or rabbit (catalog number 074 –1806 or 074 –1516; KPL) followed by the use of the detection system SuperSignal West Pico (Thermo Scientific) and imaging of the chemiluminescent signal on the G:BOX imager system (SynGene).

Results In vitro selection of BHP2–7 cells with pazopanib produced in vitro pazopanib resistance also associated with more aggressive in vivo xenograft behavior Continuous selection of the papillary thyroid cancer cell line BHP2–7 was undertaken using 18 ␮M pazopanib (dose chosen based on solubility constraints of pazopanib in cell media and because of the clinical achievability of this concentration) (16). Development of pazopanib resistance was periodically tested using colony-forming assays; after approximately 6 months of selection, stable pazopanib resistance developed as depicted in Figure 1A and was deemed sufficient to begin studies assessing potential responsible mechanism(s). Growth characteristics of pazopanib-selected/resistant BHP2–7 cells In vitro cell growth rate and colony forming efficiency of the pazopanib-selected cells were not appreciably different when compared with the parental unselected BHP2–7 cells. However, selected cells tended to be more adherent to tissue culture plates upon trypsinization. Importantly, assessment of tumor growth characteristics in mice demonstrated the pazopanib-selected/resistant cell line to be much more aggressive in comparison with BHP2–7 wt cells when subjected to sc xenotransplantation into nu/nu mice (Figure 1B).

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Table 1. Results of Mutational Analyses in Parental or Pazopanib-Selected BHP2–7 Cells Cell Line Assessed BHP2–7 WT Parental

BHP2–7 Pazopanib Selected

RET/PTC 1, -2, -3 rearrangements PAX8/PPAR␥ translocations BRAF (V600E) KRAS (codon 12, 13, 61)

RET/PTC1

RET/PTC1

Negative

Negative

Negative Negative

NRAS (codon 12, 13, 61) HRAS (codon 12, 13, 61)

Negative Negative

Negative Codon 13 GGC-⬎ GTT (G-⬎V) Negative Negative

Mutation Assessed

Figure 1. In vitro pazopanib selection of BHP2–7 cells led to in vitro pazopanib resistance and also to more aggressive in vivo xenograft behavior. A, 18 ␮M continuous in vitro pazopanib exposure led to pazopanib resistance as assessed via colony forming assays (continuous pazopanib exposures; WT, parental BHP2–7 cells; pazopanib selected, mixed culture pazopanib selected BHP2–7 cells). Figure 1B, subcutaneously implanted pazopanib selected/resistant cell nu/nu mouse flank xenografts demonstrated more rapid tumor growth compared to parental/unselected BHP2–7 cells (P ⬍ .01).

Pazopanib selection of BHP2–7 cells was associated with acquisition of a novel KRAS mutation In the initial characterization of pazopanib-selected, pazopanib-resistant BHP2–7 DTC cells, the line was first subjected to DNA profiling analysis to assure identity with parental/wild-type BHP2–7 cells and also when compared with published STR DNA microsatellite analysis results specific to the BHP2–7 cell line (12). Hypothesizing that a new mutation in the canonical VEGFR signaling pathway downstream of pazopanib inhibition of VEGFR (VEGFR ⬎ RAS ⬎ RAF ⬎ MEK1/2 ⬎ ERK) might be responsible for the noted pazopanib resistance in pazopanib-selected cells, BHP2–7 wt, and pazopanib-selected cells were subjected to mutational analyses to assess potential emergent differences in the most common mutations known to be involved in differentiated thyroid cancer pathogenesis (8 –10) including RET/PTC1, -2, and -3 rearrangements, PPFP translocations, and mutations of BRAF V600E and H,K,N-Ras codons 12, 13, and 61 (Table 1). Interestingly, we found a novel KRAS double-point mutation in codon 13 (GGC-⬎GTT) causing a glycine to valine coding substitution, which was found exclusively in

the resistant cell line, a mutation previously established as associated with constitutive KRAS activation (20). Although we initially detected the mutation upon gDNA analysis (data not shown), we thereafter isolated RNA, performed reverse transcription, and analyzed the cDNA for the same codon 13 region to establish that the KRAS activating mutation was indeed transcribed (Figure 2). Although the observed double-point mutation in our pazopanib-selected line was apparently unique, singlepoint mutations in the same region were previously described (20); both single and double mutations, however, yield the same coded constitutively activated KRAS protein kinase because the second-point mutation still codes for valine (21). All other sequencing results were identical across tested cell lines presenting as wild type, including the expected RET/PTC1 rearrangement already established for the BHP2–7 cell line (Table 1). Pazopanib-selected BHP2–7 cells maintained pazopanib resistance when subcloned and displayed cross-resistance to sunitinib but not to the MEK inhibitor GSK1120212 The pazopanib-resistant BHP2–7 cell line was subcloned into 17 individual sublines assure a uniform mutant population for further studies. All 17 sublines were sequenced and found to have the identical KRAS activating mutation (data not shown), suggesting that the pazopanib-selected cells were homogeneous, at least with respect to the KRAS mutational analyses. Sublines 7 and 14 were chosen for use in subsequent experiments upon demonstration of stable resistance to pazopanib in each of these subclonal lines using colony-forming assays (Figure 3, A and B). Based on the detection of an activating KRAS mutation in pazopanib-resistant BHP2–7 cells, we hypothesized that these cells might also be cross-resistance to other VEGFR inhibitors. We therefore assessed the effects of

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Figure 2. Mutational assessment of pazopanib-resistant BHP2–7 cells indicated acquisition of a novel KRAS mutation. A, Depiction of melting curve analysis from the LightCycler screen assay (Roche) for duplicates of a no reverse transcriptase (RT) control (green and pink), cDNA of a wild-type cell line transcript (green and blue), and the cDNA of the pazopanib-resistant cell line transcript (red and black). The abscissa represents the temperature of the capillary and the ordinate represents the fluorescence intensity as read by the LightCycler. B, Graph of the negative first derivative of the melting curve shown in panel A. Capillary temperature is again on the abscissa and the negative first derivative of the fluorescence [-d(F2/F1)/dT] is on the ordinate. The mutated form of KRAS codon 13 is responsible for the population of cDNA in which the melting curve peaks at 48.76°C. Wild-type cDNA is represented at the 62.09° peak in the curve. C, Sanger sequencing trace of the pazopanib-resistant BHP2–7 line cDNA against wild-type BHP2–7 cDNA. Highlighted in red is the KRAS codon 13 allele GGC showing the mutation to GTT in the resistant line.

sunitinib or sorafenib in analogous colony-forming assays, indeed finding cross-resistance to sunitinib and to a lesser extent also to sorafenib (Figure 3, C and D), potentially consistent with a resistance mechanism mediated by the identified activating KRAS mutation. Applying a similar rationale, we next reasoned that our pazopanib-resistant KRAS mut cell line might alternatively be equally sensitive to KRAS-downstream inhibition via the MEK1/2 inhibitor GSK1120212 when compared with the parental BHP2–7 (KRAS wt cell line), indeed also finding this to be the case (Figure 4A). This was again consistent with a mechanism of pazopanib resistance linked to the emergent activating KRAS mutation; inhibitors above the constitutively active KRAS are ineffective, whereas inhibitors below KRAS of MEK1/2 are effective. Pazopanib treatment attenuated ERK activation less effectively in pazopanib-selected BHP2–7 subclones in comparison with BHP2–7 wild-type cells The identified KRAS G13V mutation is a known activating mutation, resulting in constitutive GTP binding,

thereby activating the downstream MAPK pathway (20). We therefore next more closely evaluated the effects of MEK1/2 inhibition using GSK1120212 vs the effects of VEGFR inhibition using pazopanib on pazopanib-resistant subcloned lines 7 and 14 compared with parental BHP2–7 KRAS wild-type cells. Pharmacological MEK inhibition with GSK1120212 decreased ERK1/2 activating phosphorylation equally in all lines (Figure 4B, center of panel) as expected, given that MEK1/2 inhibition is acting downstream of KRAS. Also as expected, pazopanib treatment resulted in decreased phospho-ERK1/2 in the parental BHP2–7 KRAS wild-type cell line because the VEGFR downstream canonical signaling pathway is intact and wild type in this line. However, analogous pazopanib treatment instead resulted in maintained phosphoERK1/2 levels in the pazopanib-resistant KRAS (activating) mutant subclonal lines c7 and c14 (Figure 4B, right side of panel), consistent with prediction, given constitutive KRAS activation in these lines that is expected to be unaffected by upstream pazopanib inhibition of VEGFR.

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our model yielded more aggressive in vivo xenograft growth observed in concert with the acquisition of drug resistance (Figure 1B). In addition to mimicking the phenomenology of acquired KI resistance in patient DTCs, our model has also led to the definition of a candidate underlying mechanism of in vitro-acquired resistance (an acquired novel activating KRAS mutation) that can next be specifically assessed for relevance in patient DTCs; we are in the process of doing so presently in conjunction with an ongoing pazopanib clinical trial. We hypothesize that, by defining mechanisms of acquired kinase inhibitor DTC resistance, we may thereafter be able to act preemptively to correspondingly Figure 3. Pazopanib resistance was maintained in cloned sublines of pazopanib-selected/resistant BHP2–7 cells. A and B, Seenteen unique cloned sublines were develop means to circumvent said recreated from the BHP2–7 pazopanib-resistant/selected BHP, demonstrating maintained and sistance even before it occurs. In stable pazopanib resistance as assessed via in colony-forming assays (continuous pazopanib vitro selection of DTC cell lines may exposures, representative data from clones 7 and 14 shown). All 17 sublines were additionally thus represent a fruitful means of sequenced for KRAS mutations, and all contained the identical KRAS G13V mutation (see Figure 2). C and D, Cross-resistance to sunitinib and, to a lesser extent with respect to sorafenib, was studying the process of acquired demonstrated in pazopanib-selected/resistant BHP2–7 subline 7 (similar results were seen for drug resistance with potential transsubline 14, data not shown). lational relevance. Despite the apparent utility of the Discussion approach to studying acquired KI resistance presented herein, it has several inherent limitations. In particular, VEGFR-targeted kinase inhibitors including sorafenib, there exist means of resistance that cannot readily be insunitinib, pazopanib, axitinib, and vandetanib have terrogated using our in vitro approach, including those emerged as highly promising therapeutics in the setting of mediated by tumor stroma or by host pharmacokinetic/ progressive metastatic RAI-resistant differentiated thypharmacogenomic variability. Hence, the model used in roid cancers (10 –14). These agents can induce durable our study does not directly recapitulate the process operdisease regression in up to about 50% of treated DTC ative when the intact organisms are treated with the drug. patients (13). However, no cures result, and all patients ultimately develop resistant tumors that progress despite The limitations are, however, offset to some extent by the ongoing kinase inhibitor therapy (10 –14). Acquired re- simplicity of the approach. Interestingly, the pazopanib-selected and KRAS (actisistance to these agents is therefore now a major impedivating) mutant BHP2–7 cell lines appear to be more crossment to successful therapy in metastatic and progressive resistant to sunitinib than to sorafenib (Figure 3, C and D). RAI-refractory DTC. In the present manuscript, we describe our experience Because sorafenib was developed with intention to serve in in developing a DTC cell line with acquired pazopanib part as an inhibitor of the (KRAS-downstream) signaling resistance. The resulting pazopanib-resistant BHP2–7 kinase BRAF, it is possible that the observed lessened DTC cell line recapitulates some characteristics seen in sorafenib cross-resistance may thus be due in part to the patients who similarly develop KI-resistant tumors. In par- ability of sorafenib not just to inhibit VEGFR but also in ticular, patients developing KI-resistant DTC often benefit part to its ability to in parallel also inhibit BRAF downfrom second-line KI therapy, much as also seems the case stream of the acquired activating KRAS mutation. Furthermore, of note is that the similar activating sinin our model of pazopanib resistance, wherein resistance to pazopanib conferred incomplete cross-resistance to the gle-point mutation in KRAS codon 13 has also been reKI sorafenib (Figure 3). Furthermore, as has sometimes ported in colorectal carcinoma and found associated with been reported in patients who acquire KI-resistant DTC, poorer clinical outcomes as well as known to be activating

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may vary from those seen in hematological and lung cancers, something that will have to be better defined in the future. We conclude that in vitro models such as those presented herein have potential to be informative in providing testable hypotheses related to potential mechanisms of acquisition of kinase inhibitor resistance in thyroid cancer. We look forward to subsequently testing hypotheses so generated by interrogating acquired changes in serially collected samples from DTC patients who initially respond, but later become resistant, to KI therapy.

Acknowledgments

Figure 4. Pazopanib selected/resistant BHP2–7 cells were equally sensitive to the MEK1/2 inhibitor GSK1120212 compared with parental BHP2–7 wt cells, with activation of VEGFR downstream signaling through pERK demonstrated in pazopanib selected/resistant/ KRAS mutant BHP2–7 cells. A, Cross-resistance to GSK1120212 was not observed in subline 7 KRAS mutant cells compared with parental BHP2–7 KRAS wt cells (assessed by colony forming assays, continuous drug exposures). B, ERK1/2 phosphorylation was attenuated in BHP2–7 wt cells in response to 18 ␮M pazopanib exposure but not in KRAS mutant subclones 7 and 14. In contrast, ERK1/2 phosphorylation was similarly attenuated in response to MEK inhibition (10 nM GSK1120212) in parental and in KRAS mutant cell lines.

(20). The presently described double-point mutation developing in a pazopanib-resistant DTC cell line, albeit leading to the identical activated mutant KRAS protein, is to our knowledge novel. Moreover, recent studies have suggested that activating KRAS mutations, at least in codon 12, are apparently far more prevalent in DTC than previously suspected, especially in follicular-variant papillary thyroid cancer (12). It is also of interest that the observed mechanism of KI resistance in the case of our pazopanib-selected DTC cell line (that of an acquired activating KRAS mutation that activates the inhibited pathway downstream of the site of pathway inhibition by pazopanib) differs from mechanisms of KI resistance observed in the best-studied case of imatinib resistance in the setting of chronic myelogenous leukemia in which case resistance seems most frequently instead to arise from acquired mutations in the kinase ATP binding site that change binding site conformation (22– 24). Similarly, resistance to inhibition of activin receptorlike kinase in non-small cell lung cancer has also been reported to be commonly attributable to activin receptorlike kinase kinase (ALK1) mutations (25). Hence, it is possible that the means of acquired drug resistance in DTC

We are indebted to Ms Candy Kostelec for graciously providing administrative assistance, to Dr John (Al) Copland (Mayo Clinic Jacksonville) for providing the parental BHP2–7 cell line we used, and to the Genotyping Core at Mayo Clinic Rochester for providing the STR fingerprinting resources needed to validate cell lines as lineage identical. Address all correspondence and requests for reprints to: Keith C. Bible, MD, PhD, Division of Medical Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail: [email protected]. Disclosure Summary: The authors have nothing to disclose.

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Development and characterization of a differentiated thyroid cancer cell line resistant to VEGFR-targeted kinase inhibitors.

Vascular endothelial growth factor-targeted kinase inhibitors have emerged as highly promising therapies for radioiodine-refractory metastatic differe...
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