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Purpose:Most aggressive thyroid cancers are commonly associated with a BRAFV600E mutation. Preclinical and clinical data in BRAFV600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAFV600E thyroid cancer than in single-agent or BRAF and MEK inhibitors.Experimental Design:The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs.Results:The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAFV600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAFV600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control.Conclusions:Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAFV600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

SNAI2 promoter mutation decreased the reporter activity

Supplementary figures and tables

Angiogenesis-related proteins and VEGF secretion in cancer cells

IMPACT II

Regulation of SNAI2 by LOX in MCF-7 and BCPAP cells

Cellular invasion and migration in cells transfected with siC or siTIMP4

TCGA normal vs tumor raw data

Purpose: There is currently no standard therapy for anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC), which account for two-thirds of thyroid cancer–related deaths. Driver mutations in the PI3K/AKT and RAF/RAS/MEK/ERK pathways are common in ATC and PDTC. Histone deacetylases (HDAC) regulate cancer initiation and progression. Our aim was to determine the therapeutic efficacy of simultaneously targeting these pathways in thyroid cancer with a single agent and to evaluate biomarkers of treatment response.Experimental Design: CUDC-907 is a first-in-class compound, functioning as a dual inhibitor of HDACs and the PI3K/AKT pathway. We investigated its antiproliferative effect in vitro and in vivo.Results: CUDC-907 significantly inhibited cellular proliferation in thyroid cancer cell lines, induced G2–M arrest with decreased levels of the checkpoint regulators cyclin B1, AURKA, AURKB, PLK1, and increased p21 and p27. Treatment induced apoptosis with increased caspase-3/7 activity and decreased survivin levels and decreased cellular migration and invasion. CUDC-907 treatment caused H3 hyperacetylation and decreased HDAC2 expression. HDAC2 was upregulated in ATC and other thyroid cancer histologic subtypes. CUDC-907 treatment reduced both p-AKT and p-ERK1/2 levels. Finally, CUDC-907 treatment, in a metastatic mouse model of thyroid cancer, showed significant inhibition of growth and metastases, and tumors from treated mice had decreased HDAC2 expression, suggesting that this may be a useful biomarker of response.Conclusions: Dual inhibition of HDAC and the tyrosine kinase signaling pathways with CUDC-907 is a promising treatment strategy for advanced, metastatic thyroid cancer. Clin Cancer Res; 23(17); 5044–54. ©2017 AACR.

Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive cancer, and no current effective therapy is available for locally advanced and metastatic ACC. Drug repurposing is an emerging approach for identifying new indications for existing drugs, especially for rare cancers such as ACC. The objective of this study was to use quantitative high-throughput screening to identify agents with antineoplastic activity against ACC.Experimental Design: A screening of 4,292 compounds was performed on three ACC cell lines: BD140A, SW-13, and NCI-H295R.Results: Twenty-one active compounds were identified, with an efficacy of >80% in all three cell lines. Of these, niclosamide showed higher efficacy and lower IC50 than established anti-ACC drugs. We then validated niclosamide-inhibited cellular proliferation in all three ACC cell lines. Next, we investigated the mechanism by which niclosamide inhibited ACC cell proliferation, and found that it induced caspase-dependent apoptosis and G1 cell-cycle arrest. Niclosamide also decreased cellular migration and reduced the level of mediators of epithelial-to-mesenchymal transition, such as N-cadherin and vimentin. Furthermore, niclosamide treatment resulted in decreased expression of β-catenin. We also evaluated the effect of niclosamide on energy metabolism in ACC cell lines and found it resulted in mitochondrial uncoupling. Niclosamide treatment inhibited ACC tumor growth with no observed toxicity in mice in vivo.Conclusions: Our findings suggest that niclosamide has anti-ACC activity through its inhibition of multiple altered cellular pathways and cellular metabolism in ACC. Our results provide a preclinical rationale for evaluating niclosamide therapy in a clinical trial for ACC. Clin Cancer Res; 22(14); 3458–66. ©2016 AACR.

Table S1. Summary of mutations present in the thyroid cancer cell lines and expression levels of differentiation markers. The table depicts known mutations in the six thyroid cancer cell lines we have utilized in our study. It also indicates the presence or absence (based on Ct values > 28) of thyroid differentiation markers in normal thyroid tissue, such as sodium-iodide symporter (NIS), thyroglobulin (Tg), and thyroid peroxidase (TPO) based on mRNA expression in these cell lines.

Supplementary method and data

This file contains the legends for the supplementary figures

Purpose: Mitochondrial glycerophosphate dehydrogenase (MGPDH) is the key enzyme connecting oxidative phosphorylation (OXPHOS) and glycolysis as well as a target of the antidiabetic drug metformin in the liver. There are no data on the expression and role of MGPDH as a metformin target in cancer. In this study, we evaluated MGPDH as a potential target of metformin in thyroid cancer and investigated its contribution in thyroid cancer metabolism.Experimental Design: We analyzed MGPDH expression in 253 thyroid cancer and normal tissues by immunostaining and examined its expression and localization in thyroid cancer–derived cell lines (FTC133, BCPAP) by confocal microscopy. The effects of metformin on MGPDH expression were determined by qRT-PCR and Western blot analysis. Seahorse analyzer was utilized to assess the effects of metformin on OXPHOS and glycolysis in thyroid cancer cells. We analyzed the effects of metformin on tumor growth and MGPDH expression in metastatic thyroid cancer mouse models.Results: We show for the first time that MGPDH is overexpressed in thyroid cancer compared with normal thyroid. We demonstrate that MGPDH regulates human thyroid cancer cell growth and OXPHOS rate in vitro. Metformin treatment is associated with downregulation of MGPDH expression and inhibition of OXPHOS in thyroid cancer in vitro. Cells characterized by high MGPDH expression are more sensitive to OXPHOS-inhibitory effects of metformin in vitro and growth-inhibitory effects of metformin in vitro and in vivo.Conclusions: Our study established MGPDH as a novel regulator of thyroid cancer growth and metabolism that can be effectively targeted by metformin. Clin Cancer Res; 24(16); 4030–43. ©2018 AACR.

Supplemental Table 1 shows compounds that are pan-active in all three adrenocortical carcinoma cell lines (NCI-H295R, BD140A and SW-13).

Purpose: Epithelial-to-mesenchymal transition (EMT) is important in cancer progression and metastasis. We and others have previously reported that lysyl oxidase (LOX) is overexpressed in aggressive cancers, is associated with increased mortality, and regulates EMT. However, the mechanism by which LOX mediates EMT is unknown. In this study, we investigated the effect of LOX on mediators of EMT.Experimental Design: We used chromatin immunoprecipitation and promoter luciferase assays to determine the target gene of LOX. To determine the effects of SNAI2 in vivo, we used our metastatic anaplastic thyroid cancer (ATC) mouse model. To investigate the effects of LOX and SNAI2 on MMPs and TIMPs, protein arrays were used. Primary tumors from patients with metastatic, breast and colon cancer, and tissue array for thyroid cancer were assessed for SNAI2 and TIMP4 expression by immunohistochemistry.Results: We found that LOX knockdown decreases SNAI2 expression in cancer cell lines. Furthermore, knockdown of LOX reduced SNAI2 expression in a metastatic mouse model of thyroid cancer. We also demonstrated that LOX binds and transactivates the SNAI2 promoter. We found a direct correlation in thyroid and breast cancer samples between LOX and SNAI2 expression. To understand how LOX/SNAI2 axis mediates these effects, we performed a comprehensive analysis of MMPs/TIMPs. LOX and SNAI2 depletion reduced TIMP4 secretion. Analysis of SNAI2 and TIMP4 expression showed overexpression of both proteins in aggressive thyroid, colon, and breast tumors.Conclusions: Our findings provide new evidence that LOX regulates SNAI2 expression and that SNAI2-mediated TIMP4 secretion plays a role in cancer progression. Clin Cancer Res; 22(17); 4491–504. ©2016 AACR.

STR PROFILE

TIMP4 expression in cancer cells

Supplemental Figure 1: RT-PCR assay for miR-30a expression in transfected cells by either miR-30a mimic (A) or miR-30a inhibitor (B) and theirand its corresponding controls.

Supplemental Figure 3. Effect of LOX inhibition on metastasis in vivo. Treatment of mice before and after tail vein injection with 8505c cells reduced metastasis in vivo. BAPN treatment was initiated either 24 hours before (upper panel, four mice) or after (lower panel, four mice) tail vein injection. On the left are representative tumor luminescence images of untreated and BAPN-treated mice. B, Representative images of cellular migration and invasion in ATC cell lines transfected with siLOX.

Legends for supplemental figures

Supplemental Figure 2. Images for each cell line shows cells that penetrated the Matrigel (invasion) and those that penetrated the insert's membrane (migration). 72 hours after transfection with miR-30a (A). LOX inhibition reduces cellular migration and invasion in ATC cell lines. Transwell migration and invasion assays were performed after 24 hours of treatment with and without 100 µM BAPN (B).

Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase ATM using a metabolism-centered CRISPR/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In the TCGA pan-cancer datasets, we found that ATM levels strongly correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.

Purpose: Most aggressive thyroid cancers are commonly associated with a BRAFV600E mutation. Preclinical and clinical data in BRAFV600E cancers suggest that combined BRAF and MEK inhibitor treatment results in a response, but resistance is common. One mechanism of acquired resistance is through persistent activation of tyrosine kinase (TK) signaling by alternate pathways. We hypothesized that combination therapy with BRAF and multitargeting TK inhibitors (MTKI) might be more effective in BRAFV600E thyroid cancer than in single-agent or BRAF and MEK inhibitors. Experimental Design: The combined drug activity was analyzed to predict any synergistic effect using high-throughput screening (HTS) of active drugs. We performed follow-up in vitro and in vivo studies to validate and determine the mechanism of action of synergistic drugs. Results: The MTKI ponatinib and the BRAF inhibitor PLX4720 showed synergistic activity by HTS. This combination significantly inhibited proliferation, colony formation, invasion, and migration in BRAFV600E thyroid cancer cell lines and downregulated pERK/MEK and c-JUN signaling pathways, and increased apoptosis. PLX4720-resistant BRAFV600E cells became sensitized to the combination treatment, with decreased proliferation at lower PLX4720 concentrations. In an orthotopic thyroid cancer mouse model, combination therapy significantly reduced tumor growth (P < 0.05), decreased the number of metastases (P < 0.05), and increased survival (P < 0.05) compared with monotherapy and vehicle control. Conclusions: Combination treatment with ponatinib and PLX4720 exhibited significant synergistic anticancer activity in preclinical models of BRAFV600E thyroid cancer, in addition to overcoming PLX4720 resistance. Our results suggest this combination should be tested in clinical trials.

Genomic stability is essential for organismal development, cellular homeostasis, and survival. The DNA double-strand breaks are particularly deleterious, creating an environment prone to cellular transformation and oncogenic activation. The histone variant H2AX is an essential component of the nucleosome responsible for initiating the early steps of the DNA repair process. H2AX maintains genomic stability by initiating a signaling cascade that collectively functions to promote DNA double-strand breaks repair. Recent advances have linked genomic stability to energetic metabolism, and alterations in metabolism were found to interfere with genome maintenance. Utilizing genome-wide transcripts profiling to identify differentially-expressed genes involved in energetic metabolism, we compared control and H2AX-deficient metastatic breast cancer cell lines, and found that H2AX loss leads to the repression of key genes regulating glycolysis, with a prominent effect on hexokinase-2 (HK2). These observations are substantiated by evidence that H2AX loss compromises glycolysis, effect which was reversed by ectopic expression of HK2. Utilizing models of experimental metastasis, we found that H2AX silencing halts progression of metastatic breast cancer cells MDA-MB-231. Most interestingly, ectopic expression of HK2 in H2AX-deficient cells restores their metastatic potential. Using multiple publicly available datasets, we found a significantly strong positive correlation between H2AX expression levels in patients with invasive breast cancer, and levels of glycolysis genes, particularly HK2. These observations are consistent with the evidence that high H2AX expression is associated with shorther distant metastasis-free survival. Our findings reveal a role for histone H2AX in controlling the metastatic ability of breast cancer cells via maintenance of HK2-driven glycolysis.

Phosphorylation of histone H2AX is a major contributor to efficient DNA repair. We recently reported neurobehavioral deficits in mice lacking H2AX. Here we establish that this neural failure stems from impairment of mitochondrial function and repression of the mitochondrial biogenesis gene PGC-1α. H2AX loss leads to reduced levels of the major subunits of the mitochondrial respiratory complexes in mouse embryonic fibroblasts and in the striatum, a brain region particularly vulnerable to mitochondrial damage. These defects are substantiated by disruption of the mitochondrial shape in H2AX mutant cells. Ectopic expression of PGC-1α restores mitochondrial oxidative phosphorylation complexes and mitigates cell death. H2AX knockout mice display increased neuronal death in the brain when challenged with 3-nitropronionic acid, which targets mitochondria. This study establishes a role for H2AX in mitochondrial homeostasis associated with neuroprotection.

Thyroid cancer is the most common endocrine cancer. The discovery of new biomarkers for thyroid cancer has significantly improved the understanding of the molecular pathogenesis of thyroid cancer, thus allowing more personalized treatments for patients with thyroid cancer. Most of the recently discovered targeted therapies inhibit the known oncogenic mechanisms in thyroid cancer initiation and progression such as MAPK pathway, PI3K/Akt-mTOR pathways, or VEGF. Despite the significant advances in molecular testing and the discoveries of new and promising therapeutics, effective treatments for advanced and metastatic, iodine-refractory thyroid cancer are still lacking. Here, we aim to summarize the current understanding of the genetic alterations and the dysregulated pathways in thyroid cancer and to discuss the most recent targeted therapies and immunotherapy for advanced thyroid cancer with a promising anti-tumor activity and clinical benefit.

Drug repurposing is an effective approach to identify active drugs with known toxicity profiles for rare cancers such as ACC. The objective of this study was to determine the anticancer activity of combination treatment for ACC from previously identified candidate agents using quantitative high-throughput screening (qHTS). In this study, we evaluated the anticancer activity of flavopiridol and carfilzomib in three ACC cell lines in vitro and in vivo. Human ACC samples were analyzed for drug-target analysis, and cancer-related pathway arrays were used to identify biomarkers of treatment response. Because flavopiridol is a potent cyclin-dependent kinase (CDK) inhibitor, we found significantly higher CDK1 and CDK2 mRNA expression in three independent cohorts human ACC (p

Purpose: Mitochondrial glycerophosphate dehydrogenase (MGPDH) is the key enzyme connecting oxidative phosphorylation (OXPHOS) and glycolysis as well as a target of the antidiabetic drug metformin in the liver. There are no data on the expression and role of MGPDH as a metformin target in cancer. In this study, we evaluated MGPDH as a potential target of metformin in thyroid cancer and investigated its contribution in thyroid cancer metabolism. Experimental Design: We analyzed MGPDH expression in 253 thyroid cancer and normal tissues by immunostaining and examined its expression and localization in thyroid cancer–derived cell lines (FTC133, BCPAP) by confocal microscopy. The effects of metformin on MGPDH expression were determined by qRT-PCR and Western blot analysis. Seahorse analyzer was utilized to assess the effects of metformin on OXPHOS and glycolysis in thyroid cancer cells. We analyzed the effects of metformin on tumor growth and MGPDH expression in metastatic thyroid cancer mouse models. Results: We show for the first time that MGPDH is overexpressed in thyroid cancer compared with normal thyroid. We demonstrate that MGPDH regulates human thyroid cancer cell growth and OXPHOS rate in vitro. Metformin treatment is associated with downregulation of MGPDH expression and inhibition of OXPHOS in thyroid cancer in vitro. Cells characterized by high MGPDH expression are more sensitive to OXPHOS-inhibitory effects of metformin in vitro and growth-inhibitory effects of metformin in vitro and in vivo. Conclusions: Our study established MGPDH as a novel regulator of thyroid cancer growth and metabolism that can be effectively targeted by metformin. Clin Cancer Res; 24(16); 4030–43. ©2018 AACR.

BACKGROUND Nonfunctioning pancreatic neuroendocrine tumors (NFPanNETs) may be sporadic or inherited because of germline mutations associated with von Hippel-Lindau disease (VHL) or multiple endocrine neoplasia type 1 (MEN1). The clinical behavior of NFPanNETs is difficult to predict, even in tumors of the same stage and grade. The authors analyzed genotype-specific patterns of transcriptional messenger RNA (mRNA) levels of NFPanNETs to understand the molecular features that determine PanNET phenotype. METHODS Thirty-two samples were included for genome-wide mRNA gene expression analysis (9 VHL-associated, 10 MEN1-associated, and 9 sporadic NFPanNETs and 4 purified normal islet cell [NIC] samples). Validation of genes was performed by real-time polymerase chain reaction analysis and immunohistochemistry. Gene expression profiles were analyzed by tumor genotype, and pathway analysis was curated. RESULTS Consensus clustering of mRNA expression revealed separate clustering of NICs, VHL-associated NFPanNETs, and MEN1-associated NFPanNETs; whereas some sporadic tumors clustered with MEN1. Four of 5 MEN1-like sporadic PanNET subtypes had loss of heterozygosity at the MEN1 gene locus. Pathway analysis demonstrated subtype-specific pathway activation, comprising angiogenesis and immune response in VHL; neuronal development in MEN1; protein ubiquitination in the new MEN1/sporadic subtype; and cytokinesis and cilium/microtubule development in sporadic NFPanNETs. Among many genes, platelet-derived growth factor receptor β (PDGFRB), lymphoid enhancer-binding factor-1 (Lef-1), cyclin-dependent kinase 4 (CDK4), and CDK6 were upregulated in VHL or MEN1 NFPanNETs, providing potential subtype-specific treatment targets. CONCLUSIONS Distinct mRNA expression patterns were identified in sporadic-associated, VHL-associated, and MEN1-associated NFPanNETs. The current results uncover new pathways involved in NFPanNETs that are subtype-specific and provide potential new diagnostic or therapeutic targets based on tumor subtype. Cancer 2018;124:636-47. © 2017 American Cancer Society.

Introduction Adrenocortical carcinoma (ACC) is a rare and aggressive disease with high mortality rate, lack of effective medical therapy, and high recurrence rate even with complete resection. Prognostic biomarkers are needed to guide treatment. The aim of this study was to identify prognostic tissue metabolites and dysregulated pathways to guide treatment. Patients and methods In this study seventeen tumors from patients with advanced metastatic ACC were analyzed through an Ultra High-Performance Liquid Chromatography-Tandem Mass Spectroscopy to detect and identify known and unknown metabolites. Kaplan Meier survival analyses were performed separating patients with high and low levels of each metabolite by median values. A pathway and enrichment analysis utilizing the top 100 small molecules associated with survival was performed through MetaboAnalyst software. Results 841 metabolites were detected. Survival analysis identified 15 metabolites that were prognostic. Eleven known metabolites were identified including N-acetylglutamine, 1-methylhistamine, palmitoyl-linleoyl-glycerol, corticosterone, 2-palmitoyl-GPC, pregnen-diol, androstenediol, and 5,6-dihydrouracil. Three metabolites were found to be independently prognostic in a multivariable analysis: 3-methoxytyrosine (hazard ratio [HR] 36.0, p=0.005), taurocholenate (HR 48.3, p=0.004), and 21-hydroxypregnenolone (HR 7.0, p=0.02). Four adrenocortical steroid synthesis metabolites were significantly different between patients alive vs. dead at the end of follow-up. A pathway and enrichment analysis identified the linoleic acid metabolism pathway to be significantly enriched (p=0.025) with the highest impact coefficient (0.66). Four metabolites in the Linoleic acid pathway were detected: lecithin, linoleic acid, gamma-Linolenic acid and 12,13-dihydroxyoctadec-9-enoic acid (12,13-DiHOME). Among them lecithin, linoleic acid and gamma-Linolenic acid levels were lower while 12,13-DiHOME levels were higher among patients with a higher risk to die of their disease during follow-up (fold change values of 0.73, 0.13, 0.44 and 1.52, respectively). Conclusions In conclusion, an analysis of the tissue metabolome identified a metabolomic signature that was prognostic for overall survival. Furthermore, a metabolomic analysis of adrenocortical carcinoma suggests a pivotal role for the linoleic acid pathway in determining patients’ prognosis.

Thyroid cancer is the most frequent endocrine tumor. BRAF mutation is the most frequent alteration in thyroid cancer. The metabolic consequences of these mutations is unknown. Recent studies have suggested a pro-metastatic role of the metabolic processes in several solid malignancies. The aim of this study is to identify the metabolic pathways dysregulated in thyroid carcinogenesis with oncogenic mutations. BRAFV600Einduced HEK293 cells underwent a metabolomic profiling via mass spectrometry, which showed an increase in glutamine metabolism. Glutaminolysis, a dysregulated metabolic pathway is initiated by glutaminase (GLS), which converts glutamine to glutamate. Analysis of GLS expression in thyroid cancer tissue revealed an increase of GLS within the tumor compared to normal tissue. GEO dataset analysis revealed higher GLS expression in undifferentiated thyroid tumors compared to differentiated cancer and normal tissue (p=0.0001, p

Purpose: Adrenocortical carcinoma (ACC) is a rare and aggressive cancer, and no current effective therapy is available for locally advanced and metastatic ACC. Drug repurposing is an emerging approach for identifying new indications for existing drugs, especially for rare cancers such as ACC. The objective of this study was to use quantitative high-throughput screening to identify agents with antineoplastic activity against ACC. Experimental Design: A screening of 4,292 compounds was performed on three ACC cell lines: BD140A, SW-13, and NCI-H295R. Results: Twenty-one active compounds were identified, with an efficacy of >80% in all three cell lines. Of these, niclosamide showed higher efficacy and lower IC50 than established anti-ACC drugs. We then validated niclosamide-inhibited cellular proliferation in all three ACC cell lines. Next, we investigated the mechanism by which niclosamide inhibited ACC cell proliferation, and found that it induced caspase-dependent apoptosis and G1 cell-cycle arrest. Niclosamide also decreased cellular migration and reduced the level of mediators of epithelial-to-mesenchymal transition, such as N-cadherin and vimentin. Furthermore, niclosamide treatment resulted in decreased expression of β-catenin. We also evaluated the effect of niclosamide on energy metabolism in ACC cell lines and found it resulted in mitochondrial uncoupling. Niclosamide treatment inhibited ACC tumor growth with no observed toxicity in mice in vivo. Conclusions: Our findings suggest that niclosamide has anti-ACC activity through its inhibition of multiple altered cellular pathways and cellular metabolism in ACC. Our results provide a preclinical rationale for evaluating niclosamide therapy in a clinical trial for ACC. Clin Cancer Res; 22(14); 3458–66. ©2016 AACR.

Anaplastic thyroid cancer (ATC) is one of the most aggressive human malignancies, with no effective treatment currently available. Previously, we identified agents active against ATC cells, both in vitro and in vivo, using quantitative high-throughput screening of 3282 clinically approved drugs and small molecules. Here, we report that combining two of these active agents, carfilzomib, a second-generation proteasome inhibitor, and CUDC-101, a histone deacetylase and multi-kinase inhibitor, results in increased, synergistic activity in ATC cells. The combination of carfilzomib and CUDC-101 synergistically inhibited cellular proliferation and caused cell death in multiple ATC cell lines harboring various driver mutations observed in human ATC tumors. This increased anti-ATC effect was associated with a synergistically enhanced G2/M cell cycle arrest and increased caspase 3/7 activity induced by the drug combination. Mechanistically, treatment with carfilzomib and CUDC-101 increased p21 expression and poly (ADP-ribose) polymerase protein cleavage. Our results suggest that combining carfilzomib and CUDC-101 would offer an effective therapeutic strategy to treat ATC.

Background: The BRAF(V600E) 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 BRAF(V600E) 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

To gain insight into the pathogenesis of adrenocortical carcinoma (ACC) and whether there is progression from normal-to-adenoma-to-carcinoma, we performed genome-wide gene expression, gene methylation, microRNA expression and comparative genomic hybridization (CGH) analysis in human adrenocortical tissue (normal, adrenocortical adenomas and ACC) samples. A pairwise comparison of normal, adrenocortical adenomas and ACC gene expression profiles with more than four-fold expression differences and an adjusted P-value < 0.05 revealed no major differences in normal versus adrenocortical adenoma whereas there are 808 and 1085, respectively, dysregulated genes between ACC versus adrenocortical adenoma and ACC versus normal. The majority of the dysregulated genes in ACC were downregulated. By integrating the CGH, gene methylation and expression profiles of potential miRNAs with the gene expression of dysregulated genes, we found that there are higher alterations in ACC versus normal compared to ACC versus adrenocortical adenoma. Importantly, we identified several novel molecular pathways that are associated with dysregulated genes and further experimentally validated that oncostatin m signaling induces caspase 3 dependent apoptosis and suppresses cell proliferation. Finally, we propose that there is higher number of genomic changes from normal-to-adenoma-to-carcinoma and identified oncostatin m signaling as a plausible druggable pathway for therapeutics.

Purpose: Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy without any effective therapy. The aim of this study is to use a high-throughput drug library screening to identify a novel therapeutic agent that targets dysregulated genes/pathways in ATC. ExperimentalDesign: We performed quantitative high-throughput screening (qHTS) in ATC cell lines using a compound library of 3,282 drugs. Dysregulated genes in ATC were analyzed using genome-wide expression analysis and immunohistochemistry in human ATC tissue samples and ATC cell lines. In vitro and in vivo studies were performed for determining drug activity, effectiveness of targeting, and the mechanism of action. Results: qHTS identified 100 active compounds in three ATC cell lines. One of the most active agents was the first-in-class survivin inhibitor YM155. Genome-wide expression analysis and immunohistochemistry showed overexpression of survivin in human ATC tissue samples, and survivin was highly expressed in all ATC cell lines tested. YM155 significantly inhibited ATC cellular proliferation. Mechanistically, YM155 inhibited survivin expression in ATC cells. Furthermore, YM155 treatment reduced claspin expression, which was associated with S-phase arrest in ATC cells. In vivo, YM155 significantly inhibited growth and metastases and prolonged survival. Conclusions: Our data show that YM155 is a promising anticancer agent for ATC and that its target, survivin, is overexpressed in ATC. Our findings support the use of YM155 in clinical trials as a therapeutic option in advanced and metastatic ATC. Clin Cancer Res; 21(18); 4123–32. ©2015 AACR.

// Lisa Zhang 1 , Yaqin Zhang 2 , Amit Mehta 1, 3 , Myriem Boufraqech 1 , Sean Davis 4 , Jing Wang 5 , Ze Tian 5 , Zhiya Yu 6 , Matthew B. Boxer 2 , Jeffrey A. Kiefer 7 , John A. Copland 8 , Robert C. Smallridge 8, 9 , Zhuyin Li 2 , Min Shen 2 , Electron Kebebew 1 1 Endocrine Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA 3 Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA 4 Cancer Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 5 Curis, Inc., Translational Science MA, USA 6 Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 7 Division of Information Sciences, Translational Genomics Research Institute, Phoenix, AZ, USA 8 Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA 9 Endocrinology Division, Internal Medicine Department, Mayo Clinic, Jacksonville, FL, USA Correspondence to: Electron Kebebew, e-mail: kebebewe@mail.nih.gov Keywords: CUDC-101, anaplastic thyroid cancer, quantitative high-throughput screening, EGFR, HDAC Received: January 26, 2015 Accepted: January 31, 2015 Published: April 13, 2015 ABSTRACT Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies that currently has no effective therapy. We performed quantitative high-throughput screening (qHTS) in three ATC cell lines using 3,282 clinically approved drugs and drug candidates, and identified 100 active agents. Enrichment analysis of active compounds showed that inhibitors of EGFR and histone deacetylase (HDAC) were most active. Of these, the first-in-class dual inhibitor of EGFR, HER2 and HDACs, CUDC-101, had higher efficacy and lower IC 50 than established drugs. We validated that CUDC-101 inhibited cellular proliferation and resulted in cell death by inducing cell cycle arrest and caspase-dependent apoptosis. CUDC-101 also inhibited cellular migration in vitro . Mechanistically, CUDC-101 inhibited MAPK signaling and histone deacetylation in ATC cell lines with multiple driver mutations present in human ATC. The anticancer effect of CUDC-101 was associated with increased expression of p21 and E-cadherin, and reduced expression of survivin, XIAP, β-catenin, N-cadherin, and vimentin. In an in vivo mouse model of metastatic ATC, CUDC-101 inhibited tumor growth and metastases, and significantly prolonged survival. Response to CUDC-101 treatment in vivo was associated with increased histone 3 acetylation and reduced survivin nuclear expression. Our findings provide a preclinical basis to evaluate CUDC-101 therapy in ATC.

Anaplastic thyroid cancer (ATC) is rare but it is one of the most lethal human malignancies with no effective therapy. There is a pressing need to identify new therapeutic agents for ATC. We performed quantitative high-throughput screening (qHTS) in ATC cell lines using a compound library of 3,282 drugs. qHTS identified 100 pan-active agents. Enrichment analysis of qHTS data showed drugs targeting mTOR were one of the most active drug categories, and Torin2 showed the highest efficacy. We found mTOR to be upregulated in ATC. Treatment of multiple ATC cell lines with Torin2 showed significant dose-dependent inhibition of cellular proliferation with caspase-dependent apoptosis and G1/S phase arrest. Torin2 inhibited cellular migration and inhibited the phosphorylation of key effectors of the mTOR-pathway (AKT, 4E-BP1 and 70S6K), as well as claspin and survivin expression, regulators of cell cycle and apoptosis. In our in vivo mouse model of metastatic ATC, Torin2 inhibited tumor growth and metastasis and significantly prolonged overall survival. Our findings suggest that Torin2 is a promising agent for ATC therapy and that it effectively targets upregulated pathways in human ATC.

This file contains supplementary materials and methods, supplementary figures and figure legends, and supplementary tables.

Purpose: There is currently no standard therapy for anaplastic thyroid cancer (ATC) and poorly differentiated thyroid cancer (PDTC), which account for two-thirds of thyroid cancer–related deaths. Driver mutations in the PI3K/AKT and RAF/RAS/MEK/ERK pathways are common in ATC and PDTC. Histone deacetylases (HDAC) regulate cancer initiation and progression. Our aim was to determine the therapeutic efficacy of simultaneously targeting these pathways in thyroid cancer with a single agent and to evaluate biomarkers of treatment response. Experimental Design: CUDC-907 is a first-in-class compound, functioning as a dual inhibitor of HDACs and the PI3K/AKT pathway. We investigated its antiproliferative effect in vitro and in vivo. Results: CUDC-907 significantly inhibited cellular proliferation in thyroid cancer cell lines, induced G2–M arrest with decreased levels of the checkpoint regulators cyclin B1, AURKA, AURKB, PLK1, and increased p21 and p27. Treatment induced apoptosis with increased caspase-3/7 activity and decreased survivin levels and decreased cellular migration and invasion. CUDC-907 treatment caused H3 hyperacetylation and decreased HDAC2 expression. HDAC2 was upregulated in ATC and other thyroid cancer histologic subtypes. CUDC-907 treatment reduced both p-AKT and p-ERK1/2 levels. Finally, CUDC-907 treatment, in a metastatic mouse model of thyroid cancer, showed significant inhibition of growth and metastases, and tumors from treated mice had decreased HDAC2 expression, suggesting that this may be a useful biomarker of response. Conclusions: Dual inhibition of HDAC and the tyrosine kinase signaling pathways with CUDC-907 is a promising treatment strategy for advanced, metastatic thyroid cancer. Clin Cancer Res; 23(17); 5044–54. ©2017 AACR.

Aim: The study's aim was to determine the utility of 68-Gallium DOTATATE positron emission tomography (PET)-CT scanning in patients with carcinoid-like symptoms and negative anatomical imaging. Methods: Retrospective analysis of 22 of 196 patients with carcinoid-like symptoms and no evidence of primary neuroendocrine tumor (NET) based on anatomical imaging and endoscopy who underwent 68-Gallium DOTATATE PET-CT as part of a prospective clinical trial. Results: Of the biochemically positive patients (n = 11), 18% (n = 2) had additional evidence of NETs based on 68-Gallium DOTATATE PET-CT. Of the patients identified by 68-Gallium DOTATATE PET-CT, 50% (n = 1) had a treatment change and 100% showed symptom improvement. Of the biochemically negative patients (n = 11), 68-Gallium DOTATATE PET-CT identified NETs in 64% (n = 7). Change in management occurred in 71% patients, and 57% of patients showed symptom improvement. Conclusion: 68-Gallium DOTATATE PET-CT imaging is useful in detecting NETs in symptomatic patients with negative anatomical imaging and changes the treatments in these patients.

Background Hyperparathyroidism-jaw tumor syndrome (HPT-JT) is a rare autosomal dominant disease secondary to germline inactivating mutations of the tumor suppressor gene HRPT2/CDC73. The aim of the present study is to determine the optimal surgical approach to parathyroid disease in patients with HPT-JT.

Identification of molecular factors that promote thyroid cancer progression have important clinical implications for therapy and prognostication in patients with papillary thyroid cancer (PTC). The aim of this study was to validate and determine the function of dysregulated genes that were associated increased mortality in patients with PTC. Experiemental Design: We selected the cleavage and polyadenylation specificity factor subunit 2 (CPSF2) gene from the top 5 significantly dysregulated genes associated with PTC-associated mortality from our previous study. We used 86 PTC samples enriched for aggressive disease (recurrence and mortality) by quantitative RT-PCR (qRT-PCR). In vitro functional studies of the validated gene were performed.Decreased CPSF2 gene expression was associated with shorter disease-free survival (P = .03), large tumor size (T3 and T4) (P = .03), tumor recurrence (P.01), and mortality (P.01), independent of BRAF V600E mutation status. CPSF2 knockdown increased cellular invasion by 1.8- to 3.2-fold (P.01) and increased markers of thyroid cancer stem cells (CD44 and CD133 expression). Immunohistochemistry showed an inverse correlation between CD44 protein expression in PTC samples and CPSF2 expression.Decreased CPSF2 expression is associated with increased cellular invasion and cancer stem cell population, and more aggressive disease in PTC.