RET Phosphorylation Research Articles

Abstract 4048: GFRα1 is required for GDNF-induced viability, migration, and signaling through RET in breast cancer cells

Abstract Hormone resistance is a major challenge in treating breast cancer patients. Although anti-estrogens, such as tamoxifen, are initially successful controlling disease in estrogen receptor alpha positive (ERα+) tumors, patients with metastatic disease and as many as 40 % of patients receiving adjuvant tamoxifen acquire resistance to treatment, relapse, and die. The receptor tyrosine kinase, RET, its co-receptor, GFRα1, and their ligand, GDNF, are proteins that are emerging as potential contributors to hormone resistance in breast cancer. RET and GFRα1 were recently shown to be overexpressed in a subset of ERα+ breast cancer tumors, and GFRα1 expression was associated with lymphovascular invasion. In vitro, silencing RET with siRNA increased MCF7 cells' sensitivity to tamoxifen and, in the absence of estrogens, treatment of MCF7 cells with GDNF induced upregulation of ERα target genes. Additionally, GDNF stimulation of MCF7 cells enhanced proliferation, survival, and cell scattering. Together, clinical and preclinical data suggest that this pathway plays an important role in driving breast cancer invasion, metastasis, and hormone resistance. However, the role of GFRα1 has not been examined in tumor cell invasion and metastasis. In this study we tested the functional consequences of silencing GFRα1 in ERα + MCF7 cells on tumor cell migration, invasion, and RET signaling. We showed that targeted reduction of GFRα1 significantly inhibited GDNF-induced migration in both a transwell (p=<0.001) and scratch wound assay (p=<0.001 at 24 hours). In addition, GDNF-induced increases in viability were significantly decreased when MCF7 cells were treated with neutralizing antibodies against GFRα1 (p=<0.001). Silencing GFRα1 in GDNF-treated MCF7 cells led to reduced invasion through collagen I, although this difference was not significant. Lastly, siRNA targeted reduction of GFRα1 in MCF7 cells inhibited GDNF-induced phosphorylation of RET. Together the data demonstrate that GFRα1 is important in migration and cell viability as well as RET signaling in breast cancer cells. Citation Format: Catherine A. Ferrante, Nikki DeAngelis, Raluca Verona. GFRα1 is required for GDNF-induced viability, migration, and signaling through RET in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4048. doi:10.1158/1538-7445.AM2014-4048

Abstract 2726: RET fusions identified in colorectal cancer PDX models are sensitive to the potent RET inhibitor ponatinib

Abstract Background: Chromosomal translocations resulting in formation of activating RET fusion genes have been identified in papillary thyroid cancer and 1-2% of NSCLC. We have previously shown that ponatinib, a pan-BCR-ABL and multi-targeted tyrosine kinase inhibitor (TKI), potently inhibits the most common NSCLC fusion variant, KIF5B-RET, at clinically-achievable concentrations. Here we used patient-derived (PDX) tumor models to explore the potential involvement of RET fusions, and the efficacy of ponatinib, in other cancers. Results: To identify potential RET fusion-positive PDX tumor models, we examined RET mRNA levels in 273 PDX tumors (Crown Bioscience) from various cancer types, looking for models with outlier RET expression. To search for RET fusions, we performed RNAseq on 4 tumors that had the highest RET levels. Interestingly, RET fusions were detected in tumors from 2 colorectal cancer (CRC) patients (pts) and not in tumors from 2 NSCLC pts that expressed similar levels of RET. The fusions detected in CRC pts, NCOA4-RET and CCDC6-RET, had previously been observed in NSCLC pts. Both CRC tumors were negative for other major hotspot mutations, including KRAS, BRAF and PI3K, suggesting that these RET-fusions might be primary oncogenic drivers in these tumors. To evaluate the cellular activity of ponatinib, we generated cell lines that were dependent on the activity of these fusions. Ponatinib potently inhibited the viability of Ba/F3 cells expressing NCOA4-RET and CCD6-RET with IC50s of 6 and 22 nM, respectively. Consistent with these effects being due to inhibition of RET, ponatinib inhibited RET phosphorylation with similar potency. Other TKIs with RET activity, vandetanib (IC50s: 564-1000 nM), cabozantinib (60-386 nM), sunitinib (277-584 nM), sorafenib (105-494 nM), and lenvatinib (68-257 nM), also inhibited viability of the Ba/F3 lines, but with potencies substantially reduced compared to ponatinib. Finally, we examined the efficacy of ponatinib in the RET fusion positive PDX colorectal models, compared to 2 RET fusion negative colorectal models. In the RET-negative models, daily oral dosing of ponatinib (20 mg/kg) inhibited tumor growth by 24-41%. In contrast, ponatinib exhibited much greater efficacy in the RET-fusion positive models, inhibiting tumor growth by 79% in the NCOA4-RET model and inducing near complete regression in the CCDC6-RET model. Conclusion: We identified, for the first time, oncogenic RET fusions in CRC patient samples, suggesting RET may be a driver in a subset of CRC patients. Ponatinib effectively inhibited these fusions with potency substantially exceeding that of other RET inhibitors and demonstrated significant anti-tumor activity in PDX models. These results provide strong support for the clinical evaluation of ponatinib in patients with RET-fusion positive cancers, including colorectal cancer. Citation Format: Joe M. Gozgit, Tzu-Hsiu Chen, Tim Clackson, Victor M. Rivera. RET fusions identified in colorectal cancer PDX models are sensitive to the potent RET inhibitor ponatinib. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2726. doi:10.1158/1538-7445.AM2014-2726

Antitumor activity of lenvatinib (e7080): an angiogenesis inhibitor that targets multiple receptor tyrosine kinases in preclinical human thyroid cancer models.

Inhibition of tumor angiogenesis by blockading the vascular endothelial growth factor (VEGF) signaling pathway is a promising therapeutic strategy for thyroid cancer. Lenvatinib mesilate (lenvatinib) is a potent inhibitor of VEGF receptors (VEGFR1–3) and other prooncogenic and prooncogenic receptor tyrosine kinases, including fibroblast growth factor receptors (FGFR1–4), platelet derived growth factor receptor α (PDGFRα), KIT, and RET. We examined the antitumor activity of lenvatinib against human thyroid cancer xenograft models in nude mice. Orally administered lenvatinib showed significant antitumor activity in 5 differentiated thyroid cancer (DTC), 5 anaplastic thyroid cancer (ATC), and 1 medullary thyroid cancer (MTC) xenograft models. Lenvatinib also showed antiangiogenesis activity against 5 DTC and 5 ATC xenografts, while lenvatinib showed in vitro antiproliferative activity against only 2 of 11 thyroid cancer cell lines: that is, RO82-W-1 and TT cells. Western blot analysis showed that cultured RO82-W-1 cells overexpressed FGFR1 and that lenvatinib inhibited the phosphorylation of FGFR1 and its downstream effector FRS2. Lenvatinib also inhibited the phosphorylation of RET with the activated mutation C634W in TT cells. These data demonstrate that lenvatinib provides antitumor activity mainly via angiogenesis inhibition but also inhibits FGFR and RET signaling pathway in preclinical human thyroid cancer models.

Abstract 1529: Endoneurial macrophages induce perineural invasion of pancreatic cancer cells by secretion of GDNF and activation of RET tyrosine kinase receptor.

Abstract Perineural invasion of cancer cells (CPNI) is found in most patients with pancreatic adenocarcinomas (PDAs), prostate or head and neck cancers. These patients undergo palliative rather than curative treatment due to dissemination of cancer along nerves, well beyond the extent of any local invasion. Although CPNI is a common source of distant tumor spread and a cause of significant morbidity, its exact mechanism is undefined. Immunohistochemical analysis of specimens excised from patients with PDAs showed a significant increase in the number of endoneurial macrophages (EMΦs) which lie around nerves invaded by cancer compared to normal nerves. Video microscopy and time-lapse analysis revealed that EMΦs are recruited by the tumor cells in response to colony stimulated factor-1 secreted by invading cancer cells. Conditioned medium (CM) of tumor-activated EMΦs (tEMΦs) induced a 5-fold increase in migration of PDA cells compared to controls. Compared to resting EMΦs, tEMΦs secreted higher levels of glial-derived neurotrophic factor (GDNF), inducing phosphorylation of RET and downstream activation of extracellular signal-regulated kinases (ERK) in PDA cells. Genetic and pharmacologic inhibition of the GDNF receptors GFRA1 and RET abrogated the migratory effect of EMΦ-CM and reduced ERK phosphorylation. In an in-vivo CPNI model, CCR2-deficient mice which have reduced macrophages recruitment and activation showed minimal nerve invasion, while wild-type mice developed complete sciatic nerve paralysis due to massive CPNI. Taken together, our results identify a paracrine response between EMΦs and PDA cells which orchestrates the formation of cancer nerve invasion. Citation Format: Oren Cavel, Richard J. Wong, Olga Shomron, Moran Amit, Ziv Gil. Endoneurial macrophages induce perineural invasion of pancreatic cancer cells by secretion of GDNF and activation of RET tyrosine kinase receptor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1529. doi:10.1158/1538-7445.AM2013-1529 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5139: Molecular docking exploration of potential RET tyrosine kinase inhibitors at non ATP-binding sites.

Abstract RET (REarranged during Transfection) tyrosine kinase receptor is a transmembrane protein required for the development of neural-crest-derived cells, the urogenital system, and the central and peripheral nervous systems, notably the enteric nervous system. The RET protein has an extracellular domain with a cysteine-rich region and four cadherin-like domains, a transmembrane domain, and an intracellular tyrosine kinase domain, required for RET phosphorylation and downstream signaling. The structure of the RET kinase shares with other tyrosine kinases many conserved functional motifs and regulatory residues that are important for the kinase enzyme function. RET activation requires binding of a glial cell-line-derived neurotrophic factor (GDNF) and a co-receptor of the GDNF family receptors α. Germline mutations of RET, leading to uncontrolled activation, are associated with thyroid cancer, and recently with colorectal and lung cancers, and chronic myelomonocytic leukemia. RET mutations that result in decreased receptor function have been linked to developmental defects, such as Hirschsprung disease and kidney anomalies. The design of receptor tyrosine kinase (RTK) inhibitors has traditionally targeted the enzymes’ highly conserved ATP binding pocket. This approach resulted in the discovery of potent small molecule inhibitors, but with relatively low selectivity. To date there are no RET-specific inhibitors available for therapy, although few small molecule inhibitors are undergoing clinical evaluations as potential RET inhibitors. Since there are important critical regions within the RET molecule (e.g., the substrate binding pocket and activation loop) that contain unique amino acids and structural features, we used the method of molecular docking to virtually screen a diverse library of compounds that potentially target non-ATP binding sites of RET. Known ligands of RET were used to test the success of docking before the library of compounds was screened, docking solutions were inspected and ranked, and best compounds selected for novel structural features and future testing. Multiple comparisons with other known structures of RTK-ligand complexes have been performed in order to identify molecular features that would characterize the discovery of potential RET-specific inhibitors. Citation Format: Adrian C. Nicolescu, Taranjit S. Gujral, Jordan DS Zelt, Lois M. Mulligan. Molecular docking exploration of potential RET tyrosine kinase inhibitors at non ATP-binding sites. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5139. doi:10.1158/1538-7445.AM2013-5139 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2091: Identification of a highly potent, selective and orally available RET inhibitor with antitumor efficacy in RET-dependent tumor models.

Abstract The RET proto-oncogene encodes a receptor tyrosine kinase that is mainly expressed in neural crest-derived tissues where it plays an important role in cell differentiation, growth and survival. Germline activating point mutations of RET are associated with multiple endocrine neoplasia type 2 (MEN2), an inherited cancer syndrome characterized by development of medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid hyperplasia and are present in circa 50% of sporadic cases of MTC. More recently, a chromosomal rearrangement of the RET gene was identified in a subset of lung adenocarcinomas (1-2%) which is reported to result in expression of a fusion protein containing constitutively active RET kinase domain fused to the N-terminal portion of the kinesin KIF5B. We recently presented data on NMS-173, a very potent RET inhibitor characterized by an excellent in vivo activity profile upon i.v. administration. Here we describe the identification and the preclinical characterization of NMS-616, a novel analogue belonging to the same chemical class, suitable for oral administration. NMS-616 is a highly potent (IC50: 2 nM), ATP competitive RET inhibitor, characterized by high selectivity when tested against a panel of more than 50 kinases. NMS-616 potently blocked proliferation of MTC cell lines, such as the TT human cell line, which endogenously harbours constitutively activated RET, concomitant with abrogation of RET autophosphorylation and signaling pathway activation. The compound also inhibited the IL-3 independent proliferation of RET-driven Ba/F3 cells, with down-modulation of RET autophosphorylation and downstream signalling pathways. NMS-616 is characterized by a good in vitro ADME profile and in vivo pharmacokinetic parameters in the mouse, including oral bioavailability. When tested in vivo in a murine subcutaneous xenograft model employing TT cells, NMS-616 displayed dose-dependent tumor growth inhibition following daily oral administration at 50 and 100 mg/Kg, with tumor regression observed at both dose levels. In addition ex vivo analysis demonstrated dose-dependent target modulation following a single administration and the maintenance of RET phosphorylation inhibition for at least 24 hours. Citation Format: Elena Ardini, Patrizia Banfi, Francesca Quartieri, Paolo Polucci, Nilla Avanzi, Dario Ballinari, Laura Mancini, Edward Felder, Daniele Donati, Arturo Galvani, Enrico Pesenti, Antonella Isacchi, Maria Menichincheri. Identification of a highly potent, selective and orally available RET inhibitor with antitumor efficacy in RET-dependent tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2091. doi:10.1158/1538-7445.AM2013-2091

Abstract 2084: Ponatinib is a highly potent inhibitor of activated variants of RET found in MTC and NSCLC .

Abstract Background: RET kinase is dysregulated by activating mutations or fusion gene formation in multiple cancers, including medullary thyroid cancer (MTC) and non-small cell lung cancer (NSCLC). RET mutations are found in 50-95% of MTCs, with mutations at C634R (extracellular domain) and M918T (kinase domain) predominating. Recently, a translocation that results in formation of a KIF5B-RET fusion gene has been identified in 1-2% of NSCLCs. Ponatinib (AP24534) is a multi-targeted tyrosine kinase inhibitor (TKI) with potent activity against BCR-ABL being investigated in patients with chronic myeloid leukemia. Previously, ponatinib has been shown to potently inhibit the in vitro kinase activity of RET and the viability of Ba/F3 cells transformed to IL-3 independence through expression of a RET kinase domain artificially-activated via fusion to the TEL dimerization domain. Here, the activity of ponatinib was examined in Ba/F3 cells transformed with the most common, naturally-occurring activated variants of RET found in MTC and NSCLC. In addition, the activity of ponatinib was compared to that of 4 other multi-targeted TKIs with anti-RET activity in clinical development: vandetanib, cabozantinib, sunitinib and sorafenib. Results: Ponatinib potently inhibited viability of Ba/F3 cells expressing RETC634R, RETM918T and KIF5B-RET, with IC50s of 2, 3, and 11 nM, respectively. Consistent with these effects being due to inhibition of RET, ponatinib inhibited RET phosphorylation with similar potency in each respective cell line (IC50s of 4, 2, and 9 nM). The TKIs vandetanib (IC50: 448, 357 and 773 nM, respectively), cabozantinib (226, 100 and 292 nM), sunitinib (299, 312 and 570 nM) and sorafenib (>1000, 372 and 861 nM) also inhibited viability of Ba/F3 cells expressing RETC634R, RETM918T and KIF5B-RET, however the potency of all 4 TKIs was substantially reduced compared to that of ponatinib. Importantly, trough ponatinib concentrations (64 nM) observed in patients treated once daily with ponatinib (45 mg) substantially exceed IC50s for inhibition of each of the mutationally-activated RET variants. Additional studies to further evaluate the anti-RET activity of ponatinib and other TKIs, including their susceptibility to mutation-based resistance, are ongoing and will be presented. Conclusion: Ponatinib is a highly potent inhibitor of activated variants of RET found in MTC and NSCLC. The potency of ponatinib substantially exceeds that of other TKIs with anti-RET activity that are being evaluated in clinical trials. These results provide strong support for the clinical evaluation of ponatinib in patients with RET-driven cancers. Citation Format: Joseph M. Gozgit, Tzu-Hsiu Chen, Tim Clackson, Victor Rivera. Ponatinib is a highly potent inhibitor of activated variants of RET found in MTC and NSCLC . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2084. doi:10.1158/1538-7445.AM2013-2084

Ponatinib (AP24534) Is a Novel Potent Inhibitor of Oncogenic RET Mutants Associated With Thyroid Cancer

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. We tested whether ponatinib inhibited RET kinase and oncogenic activity. 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. 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. Ponatinib is a potent inhibitor of RET kinase and has promising preclinical activity in models of RET-driven medullary thyroid carcinoma.

Characterization of Drosophila GDNF Receptor-Like and Evidence for Its Evolutionarily Conserved Interaction with Neural Cell Adhesion Molecule (NCAM)/FasII

BackgroundGlial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-β superfamily. In mammals, they bind to the GDNF family receptor α (Gfrα) and signal through the Ret receptor tyrosine kinase. In order to gain insight into the evolution of the Ret-Gfr-Gdnf signaling system, we have cloned and characterized the first invertebrate Gfr-like cDNA (DmGfrl) from Drosophila melanogaster and generated a DmGfrl mutant allele.ResultsWe found that DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediated neither Drosophila Ret phosphorylation nor mammalian RET phosphorylation. In situ hybridization analysis revealed that DmGfrl is expressed in the central and peripheral nervous systems throughout Drosophila development, but, surprisingly, DmGfrl and DmRet expression patterns were largely non-overlapping. We generated a DmGfrl null allele by genomic FLP deletion and found that both DmGfrl null females and males are viable but display fertility defects. The female fertility defect manifested as dorsal appendage malformation, small size and reduced viability of eggs laid by mutant females. In male flies DmGfrl interacted genetically with the Drosophila Ncam (neural cell adhesion molecule) homolog FasII to regulate fertility.ConclusionOur results suggest that Ret and Gfrl did not function as an in cis receptor-coreceptor pair before the emergence of GDNF family ligands, and that the Ncam-Gfr interaction predated the in cis Ret-Gfr interaction in evolution. The fertility defects that we describe in DmGfrl null flies suggest that GDNF receptor-like has an evolutionarily ancient role in regulating male fertility and a previously unrecognized role in regulating oogenesis.SignificanceThese results shed light on the evolutionary aspects of the structure, expression and function of Ret-Gfrα and Ncam-Gfrα signaling complexes.

Novel withanolides target medullary thyroid cancer through inhibition of both RET phosphorylation and the mammalian target of rapamycin pathway

Despite development of current targeted therapies for medullary thyroid cancer (MTC), long-term survival remains unchanged. Recently isolated novel withanolide compounds from Solanaceae physalis are highly potent against MTCs. We hypothesize that these withanolides uniquely inhibit RET phosphorylation and the mammalian target of rapamycin (mTOR) pathway in MTC cells as a mechanism of antiproliferation and apoptosis. MTC cells were treated with novel withanolides and MTC-targeted drugs. In vitro studies assessed cell viability and proliferation (MTS; trypan blue assays), apoptosis (flow cytometry with Annexin V/PI staining; confirmed by Western blot analysis), long-term cytotoxic effects (clonogenic assay), and suppression of key regulatory proteins such as RET, Akt, and mTOR (by Western blot analysis). The novel withanolides potently reduced MTC cell viability (half maximal inhibitory concentration [IC(50)], 270-2,850 nmol/L; 250-1,380 nmol/L for vandetanib; 360-1,640 nmol/L for cabozantinib) with induction of apoptosis at <1,000 nmol/L of drug. Unique from other targeted therapies, withanolides suppressed RET and Akt phosphorylation and protein expression (in a concentration- and time-dependent manner) as well as mTOR activity and translational activity of 4E-BP1 and protein synthesis mediated by p70S6kinase activation at IC(50) concentrations. Novel withanolides from Physalis selectively and potently inhibit MTC cells in vitro. Unlike other MTC-targeted therapies, these compounds uniquely inhibit both RET kinase activity and the Akt/mTOR prosurvival pathway. Further translational studies are warranted to evaluate their clinical potential.

Endoneurial Macrophages Induce Perineural Invasion of Pancreatic Cancer Cells by Secretion of GDNF and Activation of RET Tyrosine Kinase Receptor

Perineural invasion of cancer cells (CPNI) is found in most patients with pancreatic adenocarcinomas (PDA), prostate, or head and neck cancers. These patients undergo palliative rather than curative treatment due to dissemination of cancer along nerves, well beyond the extent of any local invasion. Although CPNI is a common source of distant tumor spread and a cause of significant morbidity, its exact mechanism is undefined. Immunohistochemical analysis of specimens excised from patients with PDAs showed a significant increase in the number of endoneurial macrophages (EMΦ) that lie around nerves invaded by cancer compared with normal nerves. Video microscopy and time-lapse analysis revealed that EMΦs are recruited by the tumor cells in response to colony-stimulated factor-1 secreted by invading cancer cells. Conditioned medium (CM) of tumor-activated EMΦs (tEMΦ) induced a 5-fold increase in migration of PDA cells compared with controls. Compared with resting EMΦs, tEMΦs secreted higher levels of glial-derived neurotrophic factor (GDNF), inducing phosphorylation of RET and downstream activation of extracellular signal-regulated kinases (ERK) in PDA cells. Genetic and pharmacologic inhibition of the GDNF receptors GFRA1 and RET abrogated the migratory effect of EMΦ-CM and reduced ERK phosphorylation. In an in vivo CPNI model, CCR2-deficient mice that have reduced macrophage recruitment and activation showed minimal nerve invasion, whereas wild-type mice developed complete sciatic nerve paralysis due to massive CPNI. Taken together, our results identify a paracrine response between EMΦs and PDA cells that orchestrates the formation of cancer nerve invasion.

Translational application of novel withanolides for cancer treatment from Physalis species and other Solanaceae

Natural products have been the most significant source of drugs and drug leads in history. Plant biodiversity provides a resource of unlimited structural diversity for drug lead and molecular probe discovery programs such as those currently underway at the University of Kansas. Technology is gradually overcoming the traditional difficulties encountered in natural products research by improving access to biodiverse resources and ensuring compatibility of samples with high throughput procedures. Our drug lead discovery effort is aimed at uncovering bioactive molecules from plants of the U.S. Great Plains. Approaches utilized in the selection of samples consist of biodiversity-based collections and ethnobotanical information on the medicinal uses in the areas of study. In order to identify antiproliferative compounds, a library of over 1,200 plant extracts was tested for antiproliferative activity against an array of cell lines [melanoma cell lines (B16F10, SKMEL28); human head and neck squamous cell carcinomas (HNSCC) cell lines (MDA1986, JMAR, UM-SCC-2, JHU011); medullary thyroid carcinoma (MTC) cell lines (TT and DRO81–1), and a non-malignant cell line (MRC5)]. In vitro experiments included evaluation of anticancer activity and effects on cell proliferation by MTS assay and trypan blue exclusion. Apoptosis and cell cycle arrest were characterized by annexin V-propidium iodide (PI) flow cytometry and confirmed by Western Blot analysis for activation of caspase 3 and cleavage of PARP as well as cell-cycle specific proteins. Analysis of specific pathways including modulation of Akt, MAP-kinase and RET phosphorylation were evaluated in vitro as well as ex vivo in treated mice using Western blot analysis. Physalis longifolia Nutt. and several related members in the Solanaceae were identified as the most active of the species tested in this study. This led to the discovery of over 30 withanolide-type steroidal lactones, 16 of which are new compounds. In addition to withaferin A (WA), 14 withanolides showed IC50 values in the range between 0.067 and 9.3µM in vitro by MTC with selectivity of 3 to 20 fold compared to normal cells. A mechanistic study showed that WA induced apoptosis and cell death in HNSCC and MTC cells as well as modulation of the cell cycle from G0/G1 arrest to a shift to G2/M and S-phase. Western blot analysis demonstrated that cancer cells treated with WA exhibited inhibition of Akt and caspase-3 expression, and cleavage of PARP. WA was further evaluated in vivo in a metastaic murine model of MTC. All treated animals were alive, showing tumor regression and growth delay without toxicity or weight loss at six weeks post-treatment. Tumor cells treated with WA demonstrated inhibition of total and phospho-RET levels by Western blot analysis in a dose dependent manner (almost complete inhibition with treatment of 5µM WA) as well as potent inhibition of phosphor-ERK and phosphor-Akt levels. WA was shown to be a novel natural-product RET-inhibitor with efficacy in a metastatic murine model of MTC. This investigation constitutes the first report of the antiproliferative activity of WA and other withanolides against MTC and HNSCC. Procurement and screening protocols, isolation and structure determination, lead optimization, and antiproliferative activities of withanolides will be presented.

Evaluation of expression and function of vascular endothelial growth factor receptor 2, platelet derived growth factor receptors-alpha and -beta, KIT, and RET in canine apocrine gland anal sac adenocarcinoma and thyroid carcinoma

BackgroundToceranib phosphate (Palladia) has a reported objective response rate of 25% in both canine apocrine gland anal sac adenocarcinoma (AGASACA) and thyroid carcinoma (TC), with stable disease occurring in an additional 50-60% of dogs. The basis for the observed responses to toceranib is not known. The purpose of this study was to evaluate AGASACA and TC samples for the expression and activation of VEGFR2, PDGFRα, PDGFRβ, KIT and RET to assess whether dysregulation of these receptor tyrosine kinases (RTKs) may contribute to the biologic activity of toceranib.ResultsmRNA for VEGFR2, PDGFRα/β, KIT and RET was detected in all AGASACA samples. mRNA for VEGFR2, PDGFRα/β, and KIT was detected in all TC samples, while mRNA for RET was amplified in 10/15 samples. No phosphorylation of VEGFR2, PDGFRα/β, or KIT was observed on the arrays. However, phosphorylation of RET was detected in 54% of the primary AGASACA and 20% of TC. VEGFR2 was expressed in 19/24 primary and 6/10 metastatic AGASACA and 6/15 TC samples. KIT was present in 8/24 primary and 3/10 metastatic AGASACA and 9/15 TC samples. PDGFRα expression was noted in all tumor samples. In contrast PDGFRβ expression was found in only a few tumor samples but was evident in the stroma of all tumor specimens.ConclusionsKnown targets of toceranib are expressed in both AGASAC and TC. Given the observed expression of VEGFR and PDGFRα/β and phosphorylation of RET, these RTKs merit investigation as to their roles in the biology of AGSACA and TC and their contribution to toceranib’s activity.

Discovery of recurrent KIF5B-RET fusions and other targetable alterations from clinical NSCLC specimens.

7510 Background: Many NSCLCs have driving oncogenic alterations including in EGFR, KRAS, ERBB2, BRAF, ALK and ROS1. Clinically effective drugs are approved for EGFR and ALK and clinical trials are underway for other genomic targets. Thus having a means of identifying genomic alterations in routine formalin fixed paraffin embedded (FFPE) clinical specimens is critical. Methods: We sequenced 24 FFPE NSCLC specimens with a next generation sequencing (NGS) assay that captures and sequences 2574 coding exons of 145 cancer relevant genes plus 37 introns from 14 genes often rearranged in cancer. Tumors from 643 additional patients were genotyped for KIF5B-RET. Results: We identified 50 alterations in 21 genes with at least one in 83% (20/24) of tumors (range 1-7). In 72% (36/50) of NSCLCs, at least one alteration was associated with a current clinical treatment or targeted therapy trial, including mutations in KRAS, BRAF, EGFR, MDM2, CDKN2A, CCNE1, CDK4, NF1 and PIK3CA. We also found an 11,294,741 bp pericentric inversion on chromosome 10 generating a novel gene fusion joining exons 1-15 of KIF5B to exons 12-20 of RET (K15:R12) in a Caucasian never smoker. This fusion gene contains the kinesin motor and coiled-coil domains of KIF5B and the entire RET tyrosine kinase domain. In 643 additional tumors we identified 11 fusion positive patients who were all wild type for known oncogenes (frequency of 6.3% (10/159)). Four unique KIF5B-RET variants were found: 8 K15:R12, 3 K16:R12, 1 K22:R12 and 1 K15:R11. We introduced K15:R12 into Ba/F3 cells and observed IL-3 independent growth consistent with oncogenic transformation. KIF5B-RET Ba/F3 cells were sensitive to sunitinib, sorafenib and vandetinib, multi-targeted kinase inhibitors that inhibit RET, but not gefitinib, an EGFR kinase inhibitor. Sunitinib, but not gefitinib, inhibited RET phosphorylation in these cells. Conclusions: We identified both known and novel genomic alterations from NSCLC FFPE specimens using a single test. Our findings suggest that RET inhibitors should be tested in prospective clinical trials in NSCLC patients bearing KIF5B-RET rearrangements and that NGS is a feasible approach to stratifying patients for treatment based on their genomic profiles.

Preparation of 3-Substituted-1-Isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines as RET Kinase Inhibitors

A series of 3-substituted-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines have been designed, synthesized, and evaluated as RET protein kinase inhibitors. On the basis of docking results, a small library of pyrazolopyrimidine compounds with an extended hydrophobic side arm was synthesized. The most promising of the compounds (7a) displayed efficient inhibition in vitro and good selectivity when tested on a panel of kinases. Furthermore, 7a inhibited GDNF-induced RET phosphorylation of ERK1/2 in MCF-7 breast cancer cells at concentrations as low as 100 nM.

Abstract 3757: CR8, a novel CDK inhibitor, enhances vandetanib activity in a human medullary thyroid carcinoma model

Abstract Introduction: CR8 (ManRos Therapeutics) is a novel inhibitor of cyclin-dependent kinases (CDKs) that selectively targets CDK1/2/3/5/7/9. CDK inhibitors have already shown potent antitumoral activity. Recently, vandetanib (Astra Zeneca), a tyrosine-kinase inhibitor, was approved in the U.S. for the treatment of adult patients with metastatic Medullary Thyroid Carcinoma (MTC). The absence of complete tumor responses justifies the development of new therapeutic options. The aim of this study was to investigate the preclinical activity of CR8 alone and in combination with vandetanib in a MTC model. Materials and Methods: The human MTC TT cell line, bearing a RETC634W activating mutation, was cultured in the absence or the presence of CR8 (10-10 000 nM) and/or vandetanib over 6 days. Cell proliferation was assessed by WST-1 assay. Activation of RET, PP1α and oncogenic pathways (PI3K/AKT, MEK/p44-42) was analyzed by western-immunoblotting. Calcitonin secretion in cell culture media was measured by ELISA. Results: Both CR8 and vandetanib when used alone strongly inhibit TT cell proliferation at day 3 of treatment. Addition of CR8 (10 nM) enhances the antiproliferative activity of low vandetanib concentrations. As expected, vandetanib alone inhibits RET, MAPK and AKT phosphorylation and CR8 inhibits PP1α. Interestingly, CR8 alone induces activation of RET, MAPK and AKT phosphorylation, and the addition of vandetanib (&amp;gt;100 nM) inhibits RET signalling pathways. At day 3 of treatment, vandetanib (&amp;gt;100 nM) and CR8 (100 nM) reduced and induced calcitonin secretion, respectively. Conclusion: CR8 displays an antiproliferative effect on TT cells and enhances the antiproliferative activity of vandetanib. CR8 acts by inhibiting PP1α and, surprisingly, induces an activation of RET and stimulates calcitonin production. These results suggest that the combination of vandetanib with CDKs inhibitors such as CR8 may be of value to improve therapeutic response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3757. doi:1538-7445.AM2012-3757

Abstract LB-88: Identification of recurrent oncogenic KIF5B-RET rearrangements in non-small cell lung cancer

Abstract Background: A large number of non-small cell lung cancer (NSCLC) cases from never/former light smokers can be explained by driving oncogenic alterations in EGFR, KRAS, ERBB2, BRAF, ALK and ROS1. EGFR and ALK targeted tyrosine kinase inhibitors are effective clinical therapies for EGFR mutant and ALK rearranged NSCLC, respectively. We wished to study cancers that do not harbor a known genomic alteration in order to identify novel therapeutic targets that may increase treatment options for NSCLC patients. Methods and Results: We initially sequenced a NSCLC specimen from a 44 year old Caucasian never smoker in a CLIA certified lab (Foundation Medicine) using a next generation sequencing assay that captures and sequences 2574 coding exons representing 145 cancer relevant genes plus 37 introns from 14 genes frequently rearranged in cancer. We identified an 11,294,741 bp pericentric inversion on chromosome 10 generating a novel gene fusion joining exons 1-15 of KIF5B to exons 12-20 of RET (K15:R12). This fusion gene contains the kinesin motor and coiled-coil domains of KIF5B and the entire tyrosine kinase domain of RET. Expression of RET was confirmed by immunohistochemistry (IHC) and cDNA sequencing detected a 7.3-fold RET expression increase beginning at exon 12 relative to exons 1-11. Screening of additional 117 NSCLC patients using RET IHC identified 22 positive cases; sequencing of 15/22 of these identified 1 additional patient with a KIF5B-RET fusion. We also evaluated 526 tumors from never/former limited smokers (121 Caucasian and 405 Asian) and identified 10 additional (1/121 Caucasian (0.8%) and 9/405 Asian (2%)) positive patients. All fusion positive tumors were wild type for known oncogenes (frequency of 6.3 % (10/159) in WT patients). Four unique KIF5B-RET variants were identified: 8 K15:R12 (variant 1), 3 K16:R12 (variant 2), 1 K22:R12 (variant 3) and 1 K15:R11 (variant 4). We introduced K15:R12 (variant 1) into Ba/F3 cells and observed IL-3 independent growth consistent with oncogenic transformation. These KIF5B-RET Ba/F3 cells were sensitive to sunitinib, sorafenib and vandetinib, multi-targeted kinase inhibitors that inhibit RET, but not gefitinib, an EGFR kinase inhibitor. Sunitinib, but not gefitinib, inhibited RET phosphorylation in the KIF5B-RET Ba/F3 cells. Conclusions: We identify a novel oncogenic KIF5B-RET fusion gene in a subset of NSCLC patients lacking other known driver mutations. Our findings suggest that RET kinase inhibitors should be tested in prospective clinical trials for therapeutic benefit in NSCLC patients bearing KIF5B-RET rearrangements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-88. doi:1538-7445.AM2012-LB-88

GAS1 induces cell death through an intrinsic apoptotic pathway

Growth Arrest Specific 1 (GAS1) is a protein expressed when cells are arrested and during development. When ectopically expressed, GAS1 induces cell arrest and apoptosis of different cell lines, and we have previously demonstrated that the apoptotic process set off by GAS1 is caused by its capacity inhibiting the GDNF-mediated intracellular survival signaling. In the present work, we have dissected the molecular pathway leading to cell death. We employed the SH-SY5Y human neuroblastoma cell line that expresses GAS1 when deprived of serum. We observed, as we have previously described, that the presence of GAS1 reduces RET phosphorylation and inhibits the activation of AKT. We have now determined that the presence of GAS1 also triggers the dephosphorylation of BAD, which, in turn, provokes the release of Cytochrome-c from the mitochondria to the cytosol activating caspase-9, prompting the activity of caspase-3 and resulting in apoptosis of the cells. The apoptotic process is intrinsic, because there is no activation of caspase-8, thus this is consistent with apoptosis induced by the lack of trophic support. Interestingly, in cells where GAS1 has been silenced there is a significant delay in the onset of apoptosis.

The insulin resistance Grb14 adaptor protein promotes thyroid cancer ret signaling and progression

The growth factor receptor-bound protein (Grb) 14 is an adapter molecule of the Grb7/10/14 family with characteristic BPS domains serving to avidly bind tyrosine kinases. Grb14 inhibits insulin receptor (IR) catalytic activity through interaction with the BPS domain and impedes peptide substrate binding. Members of this Grb family have also been shown to interact with other kinases through their SH2 domain. Here we examined the functional role of Grb14 in thyroid cancer using loss- and gain-of-function approaches. Stable knockdown of Grb14 in thyroid cancer cells facilitated insulin receptor signaling. In contrast, RET phosphorylation was diminished in concert with reduced activation of Akt and STAT3. Loss of Grb14 also resulted in diminished cell proliferation and invasion both in vitro and in mouse flank xenografts. In complementary studies, forced expression of Grb14 interrupted insulin receptor signaling but facilitated RET activation, STAT3, and Akt phosphorylation. Consistent with these findings Grb14 over-expression enhanced cell invasion and resulted in striking metastases in an orthotopic thyroid cancer mouse xenograft model. Primary human thyroid cancer microarrays revealed a positive correlation between Grb14 expression and invasive behavior. Our findings uncover a new role for Grb14 in finely tuning receptor signaling and modulating thyroid cancer progression.

Functional analyses of RET mutations in Chinese hirschsprung disease patients

Hirschsprung disease (HSCR) is a congenital disease characterized by the absence of ganglion cells in various length of distal digestive tract. The rearranged during transfection gene (RET) is considered the major gene in HSCR. Although an increasing number of HSCR-associated RET coding sequence (CDS) mutations have been identified in recent years, not many have been investigated for functional consequence on the RET protein. We examined the functional implications of the de novo RET-CDS mutations V145G, Y483X, V636fsX1, and F961L that we first identified in sporadic Chinese patients with HSCR. The V145G disrupted RET glycosylation and F961L RET phosphorylation. Presumably, the truncation mutations would affect the translocation or the anchoring of the RET protein onto the cellular membrane. The study of RET-CDS mutations that appear de novo is essential not only for understanding the mechanistic of the disease but also for penetrance and recurrence risk estimations, being the ultimate goal for the improvement in disease management and counseling.