Combination BRAFV600E Inhibition with the Multitargeting Tyrosine Kinase Inhibitor Axitinib Shows Additive Anticancer Activity in BRAFV600E-Mutant Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is uniformly lethal. BRAFV600E mutation is present in 45% of patients with ATC. Targeted therapy with combined BRAF and MEK inhibition in BRAFV600E-mutant ATC can be effective, but acquired resistance is common because this combination targets the same pathway. Drug matrix screening, in BRAFV600E ATC cells, of highly active compounds in combination with BRAF inhibition showed multitargeting tyrosine kinase inhibitors (MTKIs) had the highest synergistic/additive activity. Thus, we hypothesized that the combination of BRAFV600E inhibition and an MTKI is more effective than a single drug or combined BRAF and MEK inhibition in BRAFV600E-mutant ATC. We evaluated the effect of BRAFV600E inhibitors in combination with the MTKI axitinib and its mechanism(s) of action. Methods: We evaluated the effects of BRAFV600E inhibitors and axitinib alone and in combination in in vitro and in vivo models of BRAFV600E-mutant and wild-type ATC. Results: The combination of axitinib and BRAFV600E inhibitors (dabrafenib and PLX4720) showed an additive effect on inhibiting cell proliferation based on the Chou-Talalay algorithm in BRAFV600E-mutant ATC cell lines. This combination also significantly inhibited cell invasion and migration (p < 0.001) compared with the control. Dabrafenib and PLX4720 arrested ATC cells in the G0/G1 phase. Axitinib arrested ATC cells in the G2/M phase by decreasing phosphorylation of aurora kinase B (Thr232) and histone H3 (Ser10) proteins and by upregulating the c-JUN signaling pathway. The combination of BRAF inhibition and axitinib significantly inhibited tumor growth and was associated with improved survival in an orthotopic ATC model. Conclusions: The novel combination of axitinib and BRAFV600E inhibition enhanced anticancer activity in in vitro and in vivo models of BRAFV600E-mutant ATC. This combination may have clinical utility in BRAFV600E-mutant ATC that is refractory to current standard therapy, namely combined BRAF and MEK inhibition.

Detecting Immune Response to Therapies Targeting PDL1 and BRAF by Using Ferumoxytol MRI and Macrin in Anaplastic Thyroid Cancer.

Background Anaplastic thyroid cancer (ATC) is aggressive with a poor prognosis, partly because of the immunosuppressive microenvironment created by tumor-associated macrophages (TAMs). Purpose To understand the relationship between TAM infiltration, tumor vascularization, and corresponding drug delivery by using ferumoxytol-enhanced MRI and macrin in an ATC mouse model. Materials and Methods ATC tumors were generated in 6-8-week-old female B6129SF1/J mice through intrathyroid injection to model orthotopic tumors, or intravenously to model hematogenous metastasis, and prospectively enrolled randomly into treatment cohorts (n = 94 total; August 1, 2018, to January 15, 2020). Mice were treated with vehicle or combined serine/threonine-protein kinase B-Raf (BRAF) kinase inhibitor (BRAFi) and anti-PDL1 antibody (aPDL1). A subset was cotreated with therapies, including an approximately 70-nm model drug delivery nanoparticle (DDNP) to target TAM, and an antibody-neutralizing colony stimulating factor 1 receptor (CSF1R). Imaging was performed at the macroscopic level with ferumoxytol-MRI and microscopically with macrin. Genetically engineered BrafV600E/WT p53-null allografts were used and complemented by a GFP-transgenic derivative and human xenografts. Tumor-bearing organs were processed by using tissue clearing and imaged with confocal microscopy and MRI. Two-tailed Wilcoxon tests were used for comparison (≥five per group). Results TAM levels were higher in orthotopic thyroid tumors compared with pulmonary metastatic lesions by 79% ± 23 (standard deviation; P < .001). These findings were concordant with ferumoxytol MRI, which showed 136% ± 88 higher uptake in thyroid lesions (P = .02) compared with lung lesions. BRAFi and aPDL1 combination therapy resulted in higher tumor DDNP delivery by 39% ± 14 in pulmonary lesions (P = .004). Compared with the untreated group, tumors following BRAFi, aPDL1, and CSF1R-blocking antibody combination therapy did not show greater levels of TAM or DDNP (P = .82). Conclusion In a mouse model of anaplastic thyroid cancer, ferumoxytol MRI showed 136% ± 88 greater uptake in orthotopic thyroid tumors compared with pulmonary lesions, which reflected high vascularization and greater tumor-associated macrophage (TAM) levels. Serine/threonine-protein kinase B-Raf inhibitor and anti-programmed death ligand 1 antibody elicited higher local TAM levels and 43% ± 20 greater therapeutic nanoparticle delivery but not higher vascularization in pulmonary tumors. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Luker in this issue.

Probability of positive genetic testing in patients diagnosed with pheochromocytoma and paraganglioma: Criteria beyond a family history.

BACKGROUND: Genetic testing for germline pheochromocytoma and paraganglioma susceptibility genes is associated with improved patient management. However, data are currently sparse on the probability of a positive testing result based on an individual's clinical presentation. This study evaluates clinical characteristics for association with testing positive for known pheochromocytoma and paraganglioma susceptibility genes. METHODS: This retrospective analysis examined 111 patients with a diagnosis of pheochromocytoma and paraganglioma who underwent genetic testing. Logistic regression and receiver operating characteristic analyses were performed to identify factors associated with a positive genetic testing result. Probabilities were then calculated for combinations of significant factors to determine the likelihood of a positive test result in each group. RESULTS: Of 32 patients with a family history of pheochromocytoma and paraganglioma, 31 (97%) had a germline mutation detected. Of 79 patients without a family history, 24 (30%) had a pathogenic germline mutation detected. In multivariate analysis, a positive family history, aged ≤47 years, and tumor size ≤2.9 cm were independent factors associated with a positive genetic testing result. Patients meeting all 3 criteria had a 100% probability compared with 13% in those without any of the criteria. In addition to a positive family history, having either aged ≤47 years or tumor size ≤2.9 cm resulted in a 90% and 100% probability of a positive result, respectively. In the absence of a family history, the probability in patients who were aged ≤47 years and had a tumor size ≤2.9 cm was 60%. CONCLUSION: In addition to a family history of pheochromocytoma and paraganglioma, aged ≤47 years, and tumor size ≤2.9 cm are associated with a higher probability of testing positive for a pheochromocytoma and paraganglioma susceptibility gene mutation. Patients meeting all 3 criteria have a 100% probability of a positive genetic testing result.

Anti-PD-1/PD-L1 therapy augments lenvatinib's efficacy by favorably altering the immune microenvironment of murine anaplastic thyroid cancer.

Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite multimodal therapy with surgery and chemoradiation. Lenvatinib, a multi-targeted tyrosine kinase inhibitor, as well as checkpoint inhibitors targeting the programmed cell death pathway, have proven effective in some patients with advanced thyroid cancer. Combination of these therapies is a potential means to boost effectiveness and minimize treatment resistance in ATC. We utilized our novel immunocompetent murine model of orthotopic ATC to demonstrate that lenvatinib led to significant tumor shrinkage and increased survival, while combination therapy led to dramatic improvements in both. Lenvatinib monotherapy increased tumor-infiltrating macrophages, CD8+ T-cells, regulatory T-cells, and most notably, polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs). While both combination therapies led to further increases in CD8+ T-cells, only the lenvatinib and anti-PD-1 combination decreased PMN-MDSCs. PMN-MDSC expansion was also seen in the blood of mice and one patient receiving lenvatinib therapy for ATC. RNA-Seq of the ATC cell line used in our mouse model demonstrated that lenvatinib has multifaceted effects on angiogenesis, response to hypoxia, the epithelial-to-mesenchymal transition, and on multiple pathways implicated in inflammation and host immunity. Combination of lenvatinib with anti-Gr-1 antibody ameliorated lenvatinib's expansion of MDSCs and significantly improved lenvatinib's anti-tumor effect. These data suggest that MDSCs play a negative role in ATC's response to lenvatinib and support future study of their role as a potential biomarker and treatment target.

Lysyl Oxidase Is a Key Player in BRAF/MAPK Pathway-Driven Thyroid Cancer Aggressiveness.

BACKGROUND: The BRAFV600E mutation is the most common somatic mutation in thyroid cancer. The mechanism associated with BRAF-mutant tumor aggressiveness remains unclear. Lysyl oxidase (LOX) is highly expressed in aggressive thyroid cancers, and involved in cancer metastasis. The objective was to determine whether LOX mediates the effect of the activated MAPK pathway in thyroid cancer. METHODS: The prognostic value of LOX and its association with mutated BRAF was analyzed in The Cancer Genome Atlas and an independent cohort. Inhibition of mutant BRAF and the MAPK pathway, and overexpression of mutant BRAF and mouse models of BRAFV600E were used to test the effect on LOX expression. RESULTS: In The Cancer Genome Atlas cohort, LOX expression was higher in BRAF-mutant tumors compared to wild-type tumors (p < 0.0001). Patients with BRAF-mutant tumors with high LOX expression had a shorter disease-free survival (p = 0.03) compared to patients with a BRAF mutation and the low LOX group. In the independent cohort, a significant positive correlation between LOX and percentage of BRAF mutated cells was found. The independent cohort confirmed high LOX expression to be associated with a shorter disease-free survival (p = 0.01). Inhibition of BRAFV600E and MEK decreased LOX expression. Conversely, overexpression of mutant BRAF increased LOX expression. The mice with thyroid-specific expression of BRAFV600E showed strong LOX and p-ERK expression in tumor tissue. Inhibition of BRAFV600E in transgenic and orthotopic mouse models significantly reduced the tumor burden as well as LOX and p-ERK expression. CONCLUSIONS: The data suggest that BRAFV600E tumors with high LOX expression are associated with more aggressive disease. The biological underpinnings of the clinical findings were confirmed by showing that BRAF and the MAPK pathway regulate LOX expression.

Combinations of BRAF inhibitor and anti-PD-1/PD-L1 antibody improve survival and tumour immunity in an immunocompetent model of orthotopic murine anaplastic thyroid cancer.

BACKGROUND: Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite aggressive multimodal therapy. ATC has a high prevalence of BRAFV600E mutations and is associated with an immunosuppressive microenvironment; we previously demonstrated that the combination of BRAF inhibitor and checkpoint inhibitor immunotherapy synergistically reduce tumour volume in an immunocompetent mouse model of orthotopic ATC. METHODS: We again utilised our mouse model of ATC to assess the combination of BRAFV600E inhibitor PLX4720 and anti-PD-L1 or anti-PD-1 antibody on survival, and performed immune cell profiling of lymphoid and myeloid-lineage cells during maximal treatment response and tumour regrowth. RESULTS: Combination therapy dramatically improved mouse survival. Maximal tumour reduction was associated with increases in the number and cytotoxicity of CD8+ T cells and NK cells, as well as increases in mostly M1-polarised tumour-associated macrophages (TAM) and decreases in myeloid-derived suppressor-like cells. Regrowth of tumour occurred after 2-3 weeks of ongoing combination therapy, and was most significantly associated with decreased TAMs and a dramatic increase in M2-polarisation. CONCLUSIONS: Combination of PLX4720 and anti-PD-L1/PD-1 antibody dramatically reduced tumour volume, prolonged survival and improved the anti-tumour immune profile in murine ATC. Tumour growth inevitably recurred and demonstrated re-emergence of an immunosuppressive tumour microenvironment.

PD-L1 and IDO1 Are Expressed in Poorly Differentiated Thyroid Carcinoma.

Poorly differentiated thyroid carcinoma (PDTC) is an aggressive form of thyroid cancer that currently has limited effective treatment options. Immune checkpoint inhibitors (ICIs) have shown to be an effective treatment for a variety of carcinomas. In this study, we explore whether immune checkpoint pathways, such as programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1), are activated in a cohort of patients with PDTC to determine whether ICIs may be an effective therapy for these patients. PDTC from 28 patients were stained for IDO1, PD-L1, and CD8 using immunohistochemistry. Staining was scored using an H-score, and PD-L1 and IDO1 expression was correlated with clinicopathologic characteristics. Positivity for PD-L1 and IDO1 was set at an H-score cutoff of five. Twenty-five percent (n = 7/28) of the PDTC were positive for PD-L1 expression. Twenty-nine percent (n = 2/7) of the PD-L1 positive PDTCs also co-expressed IDO1. The expression of PD-L1 in PDTC was significantly associated with tumor size and multifocality, with a non-significant trend towards associations with older age, extrathyroidal extension, presence of metastasis, higher stage, increased number of CD8+ T cells, and decreased disease-free and overall survival. PD-L1 expression occurs in a subset of PDTC, and is associated with a subset of clinical features of aggressive thyroid disease. Given the limited effective treatments for this patient population, consideration for ICIs as monotherapy or in combination with an IDO1 inhibitor should be explored as a novel treatment modality for patients with PDTC.

Circulating BRAFV600E Levels Correlate with Treatment in Patients with Thyroid Carcinoma.

BACKGROUND: BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC) and can be associated with aggressive disease. Previously, a highly sensitive blood RNA-based BRAFV600E assay was reported. The objective of this study was to assess the correlation of BRAFV600E circulating tumor RNA levels with surgical and medical treatment. METHODS: Circulating BRAFV600E levels were assessed in (i) a murine model of undifferentiated (anaplastic) thyroid carcinoma with known BRAFV600E mutation undergoing BRAFV600E-inhibitor (BRAFi) treatment, and (ii) in 111 patients enrolled prior to thyroidectomy (n = 86) or treatment of advanced recurrent or metastatic PTC (n = 25). Blood samples were drawn for BRAFV600E analysis before and after treatment. Testing characteristics were assessed and positivity criteria optimized. Changes in blood BRAFV600E values were assessed and compared to clinical characteristics and response to therapy. RESULTS: In a murine model of anaplastic thyroid carcinoma with BRAFV600E mutation, blood BRAFV600E RNA correlated with tumor volume in animals treated with BRAFi. In tissue BRAFV600E-positive (n = 36) patients undergoing initial surgery for PTC, blood BRAFV600E levels declined postoperatively (median 370.0-178.5 fg/ng; p = 0.002). In four patients with metastatic or poorly differentiated thyroid carcinoma receiving targeted therapies, blood BRAFV600E declined following therapy and corresponded with radiographic evidence of partial response or stable disease. CONCLUSIONS: This study shows the correlation of blood BRAFV600E levels in response to treatment in both an established animal model of thyroid cancer and in patients with BRAFV600E-positive tumors with all stages of disease. This assay represents an alternative biomarker in patients with positive thyroglobulin antibodies, and tumors, which do not express thyroglobulin.

CDK4/6 Inhibition Augments Antitumor Immunity by Enhancing T-cell Activation.

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies.Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Jenkins et al., p. 196This article is highlighted in the In This Issue feature, p. 127.

Inhibition of MAPKinase pathway sensitizes thyroid cancer cells to ABT-737 induced apoptosis.

Bcl2 family proteins play an important role in the resistance of thyroid cancer cells to apoptosis induced by chemotherapeutic drugs and targeted therapies. BH3-profiling of seven fresh primary papillary thyroid cancer (PTC) tumors showed dependence for survival on Bcl-xL (2/7), Bcl2 (2/7), and Mcl-1 (2/7), while the majority of thyroid cell lines were mainly dependent on Bcl-xL. Targeting Bcl2 family proteins with the BH3 mimetic, ABT-737, while simultaneously inhibiting ERK pathway proteins with PLX4720 and PD325901 was shown to induce significantly high apoptosis in the majority of cell lines (8505c, SW1736, HTh7, BCPAP) and moderate apoptosis in the TPC-1 cell line. In orthotopic thyroid cancer mouse models of 8505c and BCPAP, treatment with the triple drug combination reduced the size of the tumors and showed significantly higher numbers of cells undergoing apoptosis. This treatment increased the expression of pro-apoptotic protein Bim, while decreasing anti-apoptotic protein Mcl-1. Our results suggest that analyzing the results of BH3-profiling along with the mutational status of tumor can reveal an effective therapy for targeted, personalized treatment of aggressive thyroid cancer.

Theranostic near-infrared fluorescent nanoplatform for imaging and systemic siRNA delivery to metastatic anaplastic thyroid cancer.

Anaplastic thyroid cancer (ATC), one of the most aggressive solid tumors, is characterized by rapid tumor growth and severe metastasis to other organs. Owing to the lack of effective treatment options, ATC has a mortality rate of ∼100% and median survival of less than 5 months. RNAi nanotechnology represents a promising strategy for cancer therapy through nanoparticle (NP) -mediated delivery of RNAi agents (e.g., siRNA) to solid tumors for specific silencing of target genes driving growth and/or metastasis. Nevertheless, the clinical success of RNAi cancer nanotherapies remains elusive in large part because of the suboptimal systemic siRNA NP delivery to tumors and the fact that tumor heterogeneity produces variable NP accumulation and thus, therapeutic response. To address these challenges, we here present an innovative theranostic NP platform composed of a near-infrared (NIR) fluorescent polymer for effective in vivo siRNA delivery to ATC tumors and simultaneous tracking of the tumor accumulation by noninvasive NIR imaging. The NIR polymeric NPs are small (∼50 nm), show long blood circulation and high tumor accumulation, and facilitate tumor imaging. Systemic siRNA delivery using these NPs efficiently silences the expression of V-Raf murine sarcoma viral oncogene homolog B (BRAF) in tumor tissues and significantly suppresses tumor growth and metastasis in an orthotopic mouse model of ATC. These results suggest that this theranostic NP system could become an effective tool for NIR imaging-guided siRNA delivery for personalized treatment of advanced malignancies.

Combining BRAF inhibitor and anti PD-L1 antibody dramatically improves tumor regression and anti tumor immunity in an immunocompetent murine model of anaplastic thyroid cancer.

The interaction of programmed cell death-1 and its ligand is widely studied in cancer. Monoclonal antibodies blocking these molecules have had great success but little is known about them in thyroid cancer. We investigated the role of PD-L1 in thyroid cancer with respect to BRAF mutation and MAP kinase pathway activity and the effect of anti PD-L1 antibody therapy on tumor regression and intra-tumoral immune response alone or in combination with BRAF inhibitor (BRAFi). BRAFV600E cells showed significantly higher baseline expression of PD-L1 at mRNA and protein levels compared to BRAFWT cells. MEK inhibitor treatment resulted in a decrease of PD-L1 expression across all cell lines. BRAFi treatment decreased PD-L1 expression in BRAFV600E cells, but paradoxically increased its expression in BRAFWT cells. BRAFV600E mutated patients samples had a higher level of PD-L1 mRNA compared to BRAFWT (p=0.015). Immunocompetent mice (B6129SF1/J) implanted with syngeneic 3747 BRAFV600E/WT P53-/- murine tumor cells were randomized to control, PLX4720, anti PD-L1 antibody and their combination. In this model of aggressive thyroid cancer, control tumor volume reached 782.3±174.6mm3 at two weeks. The combination dramatically reduced tumor volume to 147.3±60.8, compared to PLX4720 (439.3±188.4 mm3, P=0.023) or PD-L1 antibody (716.7±62.1, P<0.001) alone. Immunohistochemistry analysis revealed intense CD8+ CTL infiltration and cytotoxicity and favorable CD8+:Treg ratio compared to each individual treatment. Our results show anti PD-L1 treatment potentiates the effect of BRAFi on tumor regression and intensifies anti tumor immune response in an immunocompetent model of ATC. Clinical trials of this therapeutic combination may be of benefit in patients with ATC.

Genome-wide analysis of differentially expressed miRNA in PLX4720-resistant and parental human thyroid cancer cell lines.

BACKGROUND: Investigating BRAF((V600E)) inhibitors (BRAFi) as a strategy to treat patients with aggressive thyroid tumors harboring the BRAF((V600E)) mutant currently is in progress, and drug resistance is expected to pose a challenge. MicroRNAs (miRNAs) are involved in development of resistance to a variety of drugs in different malignancies. METHODS: miRNA expression profiles in the human anaplastic thyroid cancer cell line (8505c) were compared with its PLX4720-resistant counterpart (8505c-R) by the use of Illumina deep sequencing. We conducted a functional annotation and pathway analysis of the putative and experimentally validated target genes of the significantly altered miRNAs. RESULTS: We identified 61 known and 2 novel miRNAs whose expression was altered greatly in 8505c-R. Quantitative reverse-transcription polymerase chain reaction validated altered expression of 7 selected miRNAs in 8505c-R and BCPAP-R (PLX4720-resistant papillary thyroid cancer cell line). We found 14 and 25 miRNAs whose expression levels changed substantially in 8505c and 8505c-R, respectively, after treatment with BRAFi. The mitogen-activated protein kinase and phosphatidylinositol 3-kinase-AKT pathways were among the prominent targets of many of the deregulated miRNAs. CONCLUSION: We have identified a number of miRNAs that could be used as biomarkers of resistance to BRAFi in patients with thyroid cancer. In addition, these miRNAs can be explored as potential therapeutic targets in combination with BRAFi to overcome resistance.

Detection of Circulating BRAF(V600E) in Patients with Papillary Thyroid Carcinoma.

BRAF(V600E) is a common mutation in papillary thyroid carcinoma (PTC) correlated with aggressive features. Our objective was to assess the feasibility and accuracy of a novel RNA-based blood assay to identify individuals with a high-risk tumor mutation in patients with PTC. Patients with benign or malignant thyroid disorders were included between September 2013 and July 2014 before either thyroidectomy (n = 62) or treatment of recurrent or metastatic PTC (n = 8). RNA was isolated from peripheral blood lymphocytes and reverse transcribed and followed by two rounds of nested PCR amplification with a restriction digest specific for wild-type BRAF. BRAF(V600E) levels were quantified with standardization curves. Circulating BRAF(V600E) levels were compared with BRAF mutation status from surgical pathologic DNA-based tissue assays. Testing characteristics and receiving-operator curve using tissue results as the gold standard were assessed. Matched blood and tissue assays for BRAF(V600E) were performed on 70 patients with PTC (stages I to IV, n = 48) or other (n = 22) thyroid tumors. Sixty-three percent of PTC patients tested positive for BRAF(V600E) with conventional tissue assays on surgical specimens. The correlation between the RNA-based blood assay and tissue BRAF status was 0.71. PTC patients harbor detectable BRAF(V600E) circulating tumor cells. This blood assay is feasible and has potential as a biomarker for prognosis, surveillance, clinical decision making, and assessment of treatment response to BRAF-targeted therapies.

Combined BRAF(V600E)- and SRC-inhibition induces apoptosis, evokes an immune response and reduces tumor growth in an immunocompetent orthotopic mouse model of anaplastic thyroid cancer.

Anaplastic (ATC) and refractory papillary thyroid cancer (PTC) lack effective treatments. Inhibition of either oncogenic BRAF or SRC has marked anti-tumor effects in mouse models of thyroid cancer, however, neither drug induces notable apoptosis. Here we report that the SRC-inhibitor dasatinib further sensitizes BRAFV600E-positive thyroid cancer cells to the BRAFV600E-inhibitor PLX4720. Combined treatment with PLX4720 and dasatinib synergistically inhibited proliferation and reduced migration in PTC and ATC cells. Whereas PLX4720 did not induce robust apoptosis in thyroid cancer cells, combined treatment with dasatinib induced apoptosis in 4 of 6 lines. In an immunocompetent orthotopic mouse model of ATC, combined PLX4720 and dasatinib treatment significantly reduced tumor volume relative to PLX4720 treatment alone. Immune cell infiltration was increased by PLX4720 treatment and this effect was maintained in mice treated with both PLX4720 and dasatinib. Further, combined treatment significantly increased caspase 3 cleavage in vivo relative to control or either treatment alone. In conclusion, combined PLX4720 and dasatinib treatment induces apoptosis, increases immune cell infiltration and reduces tumor volume in a preclinical model of ATC, suggesting that the combination of these FDA-approved drugs may have potential for the treatment of patients with ATC or refractory PTC.

A potential role for immunotherapy in thyroid cancer by enhancing NY-ESO-1 cancer antigen expression.

BACKGROUND: NY-ESO-1 is one of the most immunogenic members of the cancer/testis antigen family and its levels can be increased after exposure to demethylating and deacetylating agents. This cytoplasmic antigen can serve as a potent target for cancer immunotherapy and yet has not been well studied in differentiated thyroid cancer cells. METHODS: We studied the baseline expression of NY-ESO-1 messenger RNA and protein before and after exposure to 5-aza-2'-deoxycytidine (DAC) (72 hours) in a panel of thyroid cancer cell lines using quantitative polymerase chain reaction and Western blot. HLA-A2+, NY-ESO-1+ thyroid cell lines were then co-cultured with peripheral blood lymphocytes transduced with NY-ESO-1 specific T-cell receptor (TCR) and assayed for interferon-gamma and Granzyme-B release in the medium. SCID mice injected orthotopically with BCPAP cells were treated with DAC to evaluate for NY-ESO-1 gene expression in vivo. RESULTS: None of the thyroid cancer cell lines showed baseline expression of NY-ESO-1. Three cell lines, BCPAP, TPC-1, and 8505c, showed an increase in NY-ESO-1 gene expression with DAC treatment and were found to be HLA-A2 positive. DAC-treated target BCPAP and TPC-1 tumor cells with up-regulated NY-ESO-1 levels were able to mount an appropriate interferon-gamma and Granzyme-B response upon co-culture with the NY-ESO-1-TCR-transduced peripheral blood lymphocytes. In vivo DAC treatment was able to increase NY-ESO-1 expression in an orthotopic mouse model with BCPAP cells. CONCLUSION: Our data suggest that many differentiated thyroid cancer cells can be pressed to express immune antigens, which can then be utilized in TCR-based immunotherapeutic interventions.

The next generation of orthotopic thyroid cancer models: immunocompetent orthotopic mouse models of BRAF V600E-positive papillary and anaplastic thyroid carcinoma.

BACKGROUND: While the development of new treatments for aggressive thyroid cancer has advanced in the last 10 years, progress has trailed headways made with other malignancies. A lack of reliable authenticated human cell lines and reproducible animal models is one major roadblock to preclinical testing of novel therapeutics. Existing xenograft and orthotopic mouse models of aggressive thyroid cancer rely on the implantation of highly passaged human thyroid carcinoma lines in immunodeficient mice. Genetically engineered models of papillary and undifferentiated (anaplastic) thyroid carcinoma (PTC and ATC) are immunocompetent; however, slow and stochastic tumor development hinders high-throughput testing. Novel models of PTC and ATC in which tumors arise rapidly and synchronously in immunocompetent mice would facilitate the investigation of novel therapeutics and approaches. METHODS: We characterized and utilized mouse cell lines derived from PTC and ATC tumors arising in genetically engineered mice with thyroid-specific expression of endogenous Braf(V600E/WT) and deletion of either Trp53 (p53) or Pten. These murine thyroid cancer cells were transduced with luciferase- and GFP-expressing lentivirus and implanted into the thyroid glands of immunocompetent syngeneic B6129SF1/J mice in which the growth characteristics were assessed. RESULTS: Large locally aggressive thyroid tumors form within one week of implantation. Tumors recapitulate their histologic subtype, including well-differentiated PTC and ATC, and exhibit CD3+, CD8+, B220+, and CD163+ immune cell infiltration. Tumor progression can be followed in vivo using luciferase and ex vivo using GFP. Metastatic spread is not detected at early time points. CONCLUSIONS: We describe the development of the next generation of murine orthotopic thyroid cancer models. The implantation of genetically defined murine BRAF-mutated PTC and ATC cell lines into syngeneic mice results in rapid and synchronous tumor formation. This model allows for preclinical investigation of novel therapeutics and/or therapeutic combinations in the context of a functional immune system.

Potential role of 5-aza-2'-deoxycytidine induced MAGE-A4 expression in immunotherapy for anaplastic thyroid cancer.

BACKGROUND: Melanoma antigen gene family (MAGE)-A4, a member of the cancer testis antigen family, has been reported in various cancers including melanoma, bladder, head and neck, oral, and lung, and is a potential target for T-cell-receptor-based immunotherapy. Baseline expression levels of the MAGE-A4 gene in thyroid cancer cell lines have not been previously studied thoroughly. METHODS: Human thyroid cancer cell lines (8505c, HTh7, BCPAP, and TPC-1) were treated with either 10 µmol/L 5'-azacytidine (Aza) or 10 µmol/L 5-AZA-2'deoxycytidine (DAC) and evaluated for various MAGEA gene expression. Later melanoma cell lines A375 and 8505c were treated with PLX4720 in combination with DAC and evaluated for MAGE-A4 expression. RESULTS: Only BCPAP cells expressed moderate levels of MAGE-A3 and MAGE-A6 at baseline. Treatment with DAC/Aza induced the expression of MAGE-A4 and MAGE-A1 in 8505c cells. PLX4720 treatment did not affect MAGE-A4 expression in 8505c cells, but increased its expression in A375 cells. In contrast, addition of PLX4720 to DAC-treated 8505c cells decreased the previously induced MAGE-A4 expression by DAC in these cells. A similar decrease in MAGE-A4 expression by DAC was also seen in 8505cBRAF(-/-) cells. Although DAC treatment resulted in demethylation of the MAGE-A4 promoter in 2 CpG sites, PLX addition to DAC did not affect the demethylation status. CONCLUSION: Demethylating agents increased the expression of MAGE genes in thyroid cancer cells. The effect of BRAFV600E inhibitors on MAGE-A4 expression suggest the role of downstream MEK/BRAF signaling in its expression apart from promoter demethylation being the sole requirement. Expression of MAGE-A4 may make immunotherapeutic intervention possible in selected patients with thyroid cancer.

Transcription factor Nrf2 regulates SHP and lipogenic gene expression in hepatic lipid metabolism.

Nuclear factor erythroid-2 related factor 2 (Nrf2) plays a pivotal role in cytoprotection against both endogenous and exogenous stresses. Here, we establish a novel molecular link between Nrf2, nuclear receptor small heterodimer partner (SHP; NROB2), lipogenic genes, and hepatic lipid homeostasis. Deletion of Nrf2 (Nrf2⁻(/)⁻) in mice resulted in a reduced liver weight, a decrease in fatty acid content of hepatic triacylglycerol, as well as concomitant increases in the levels of serum VLDL-triglyceride (TG), HDL cholesterol, and ketone bodies at 6 mo of age. Liver weight and hepatic TG content were consistently lower in Nrf2⁻(/)⁻ mice upon a high-fat challenge. This phenotype was accompanied by downregulation of genes in lipid synthesis and uptake and upregulation of genes in lipid oxidation in older Nrf2⁻(/)⁻ mice. Interestingly, SHP expression was induced with age in Nrf2(+/+) mice but decreased by Nrf2 deficiency. Forced expression and activation of Nrf2 by Nrf2 activators consistently induced SHP expression, and Nrf2 was identified as a novel activator of the SHP gene transcription. We also identified PPAR-γ, Fas, Scd1, and Srebp-1 as direct targets of Nrf2 activation. These findings provide evidence for a role of Nrf2 in the modulation of hepatic lipid homeostasis through transcriptional activation of SHP and lipogenic gene expression.

Insights into the binding mode and mechanism of action of some atypical retinoids as ligands of the small heterodimer partner (SHP).

The Small Heterodimer Partner (SHP) is an orphan nuclear receptor and an atypical member of the nuclear receptor superfamily Since its discovery, a growing body of evidences have pointed out a pivotal role for SHP in the transcriptional regulation of a variety of target genes involved in diverse metabolic pathways. While we have previously developed a homology model of the structure of SHP that was instrumental to identify a putative ligand binding pocket and suggest the possibility of the development of synthetic modulators, others reported that some atypical retinoids may represent the first synthetic ligands for this receptor. In this work, we report a combined computational approach aimed at shedding further lights on the binding mode and mechanism of action of some atypical retinoids as ligands of SHP. The results have been instrumental to design mutagenesis experiments whose preliminary data suggest the presence of a functional site in SHP as defined by residues Phe96, Arg138 and Arg238. While further experimental studies are ongoing, these findings constitute the basis for the design and identification of novel synthetic modulators of SHP functions.