Corrigendum: Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.

[This corrects the article DOI: 10.3389/fimmu.2023.1213920.].

Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.

Introduction: The complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885. Methods: The interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers. Results: Zilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies. Conclusion: Our findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.

Prevention of Anti-HMGCR Immune-Mediated Necrotising Myopathy by C5 Complement Inhibition in a Humanised Mouse Model.

Introduction: immune-mediated necrotising myopathy (IMNM) is associated with pathogenic anti-signal recognition particle (SRP) or 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) antibodies, at least partly through activation of the classical pathway of the complement. We evaluated zilucoplan, an investigational drug, and a macrocyclic peptide inhibitor of complement component 5 (C5), in humanized mouse models of IMNM. Methods: purified immunoglobulin G (IgG) from an anti-HMGCR+ IMNM patient was co-injected intraperitoneally with human complement in C57BL/6, C5-deficient B10 (C5def) and Rag2 deficient (Rag2-/-) mice. Zilucoplan was administered subcutaneously in a preventive or interventional paradigm, either injected daily throughout the duration of the experiment in C57BL/6 and C5def mice or 8 days after disease induction in Rag2-/- mice. Results: prophylactic administration of zilucoplan prevented muscle strength loss in C5def mice (anti-HMGCR+ vs. anti-HMGCR+ + zilucoplan: p = 0.0289; control vs. anti-HMGCR+ + zilucoplan: p = 0.4634) and wild-type C57BL/6 (anti-HMGCR+ vs. anti-HMGCR+ + zilucoplan: p = 0.0002; control vs. anti-HMGCR+ + zilucoplan: p = 0.0939) with corresponding reduction in C5b-9 deposits on myofibres and number of regenerated myofibres. Interventional treatment of zilucoplan after disease induction reduced the complement deposits and number of regenerated myofibres in muscles of Rag2-/- mice, although to a lesser extent. In this latter setting, C5 inhibition did not significantly ameliorate muscle strength. Conclusion: Early administration of zilucoplan prevents the onset of myopathy at the clinical and histological level in a humanized mouse model of IMNM.

Heterogeneity of Acetylcholine Receptor Autoantibody-Mediated Complement Activity in Patients With Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Autoantibodies targeting the acetylcholine receptor (AChR), found in patients with myasthenia gravis (MG), mediate pathology through 3 mechanisms: complement-directed tissue damage, blocking of the acetylcholine binding site, and internalization of the AChR. Clinical assays, used to diagnose and monitor patients, measure only autoantibody binding. Consequently, they are limited in providing association with disease burden, understanding of mechanistic heterogeneity, and monitoring therapeutic response. The objective of this study was to develop a cell-based assay that measures AChR autoantibody-mediated complement membrane attack complex (MAC) formation. METHODS: An HEK293T cell line-modified using CRISPR/Cas9 genome editing to disrupt expression of the complement regulator genes (CD46, CD55, and CD59)-was used to measure AChR autoantibody-mediated MAC formation through flow cytometry. RESULTS: Serum samples (n = 155) from 96 clinically confirmed AChR MG patients, representing a wide range of disease burden and autoantibody titer, were tested along with 32 healthy donor (HD) samples. AChR autoantibodies were detected in 139 of the 155 (89.7%) MG samples through a cell-based assay. Of the 139 AChR-positive samples, autoantibody-mediated MAC formation was detected in 83 (59.7%), whereas MAC formation was undetectable in the HD group or AChR-positive samples with low autoantibody levels. MAC formation was positively associated with autoantibody binding in most patient samples; ratios (mean fluorescence intensity) of MAC formation to AChR autoantibody binding ranged between 0.27 and 48, with a median of 0.79 and an interquartile range of 0.43 (0.58-1.1). However, the distribution of ratios was asymmetric and included extreme values; 16 samples were beyond the 10-90 percentile, with high MAC to low AChR autoantibody binding ratio or the reverse. Correlation between MAC formation and clinical disease scores suggested a modest positive association (rho = 0.34, p = 0.0023), which included a subset of outliers that did not follow this pattern. MAC formation did not associate with exposure to immunotherapy, thymectomy, or MG subtypes defined by age-of-onset. DISCUSSION: A novel assay for evaluating AChR autoantibody-mediated complement activity was developed. A subset of patients that lacks association between MAC formation and autoantibody binding or disease burden was identified. The assay may provide a better understanding of the heterogeneous autoantibody molecular pathology and identify patients expected to benefit from complement inhibitor therapy.

Myasthenia gravis complement activity is independent of autoantibody titer and disease severity.

Acetylcholine receptor (AChR) autoantibodies, found in patients with autoimmune myasthenia gravis (MG), can directly contribute to disease pathology through activation of the classical complement pathway. Activation of the complement pathway in autoimmune diseases can lead to a secondary complement deficiency resulting in reduced complement activity, due to consumption, during episodes of disease activity. It is not clear whether complement activity in MG patients associates with measurements of disease activity or the titer of circulating pathogenic AChR autoantibodies. To explore such associations, as a means to identify a candidate biomarker, we measured complement activity in AChR MG samples (N = 51) using a CH50 hemolysis assay, then tested associations between these values and both clinical status and AChR autoantibody titer. The majority of the study subjects (88.2%) had complement activity within the range defined by healthy controls, while six patients (11.8%) showed reduced activity. No significant association between complement activity and disease status or AChR autoantibody titer was observed.

Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices.

Complement activation is key to anti-microbial defenses by directly acting on microbes and indirectly by triggering cellular immune responses. Complement activation may also contribute to the pathogenesis of numerous inflammatory and immunological diseases. Consequently, intense research focuses on developing therapeutics that block pathology-causing complement activation while preserving anti-microbial complement activities. However, the pace of research is slowed down significantly by the limitations of current tools for evaluating complement-targeting therapeutics. Moreover, the effects of potential therapeutic agents on innate immune cells, like neutrophils, are not fully understood. Here, we employ microfluidic assays and measure chemotaxis, phagocytosis, and swarming changes in human neutrophils ex vivo in response to various complement-targeting agents. We show that whereas complement factor 5 (C5) cleavage inhibitor eculizumab blocks all neutrophil anti-microbial functions, newer compounds like the C5 cleavage inhibitor RA101295 and C5a receptor antagonist avacopan inhibit chemotaxis and swarming while preserving neutrophil phagocytosis. These results highlight the utility of microfluidic neutrophil assays in evaluating potential complement-targeting therapeutics.

The Chemical Synthesis of Knob Domain Antibody Fragments.

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.

A REDD1/TXNIP pro-oxidant complex regulates ATG4B activity to control stress-induced autophagy and sustain exercise capacity.

Macroautophagy (autophagy) is a critical cellular stress response; however, the signal transduction pathways controlling autophagy induction in response to stress are poorly understood. Here we reveal a new mechanism of autophagy control whose deregulation disrupts mitochondrial integrity and energy homeostasis in vivo. Stress conditions including hypoxia and exercise induce reactive oxygen species (ROS) through upregulation of a protein complex involving REDD1, an mTORC1 inhibitor and the pro-oxidant protein TXNIP. Decreased ROS in cells and tissues lacking either REDD1 or TXNIP increases catalytic activity of the redox-sensitive ATG4B cysteine endopeptidase, leading to enhanced LC3B delipidation and failed autophagy. Conversely, REDD1/TXNIP complex expression is sufficient to induce ROS, suppress ATG4B activity and activate autophagy. In Redd1(-/-) mice, deregulated ATG4B activity and disabled autophagic flux cause accumulation of defective mitochondria, leading to impaired oxidative phosphorylation, muscle ATP depletion and poor exercise capacity. Thus, ROS regulation through REDD1/TXNIP is physiological rheostat controlling stress-induced autophagy.

mTOR activity under hypoxia.

The adaptive response to hypoxia, low oxygen tension, involves inhibition of energy-intensive cellular processes including protein translation. This effect is mediated in part through a decrease in the kinase activity of mammalian target of rapamycin complex 1 (mTORC1), a master regulator of protein translation. The principle mechanism for hypoxia-induced mTORC1 inhibition, however, was not elucidated until recently. Our work has demonstrated that the stress-induced protein REDD1 is essential for hypoxia regulation of mTORC1 activity and has further defined the molecular mechanism whereby REDD1 represses mTORC1 activity under hypoxic stress. Using our studies with REDD1 as an example, we describe in detail biochemical approaches to assess mTORC1 activity in the hypoxic response. Here, we provide methodologies to monitor signaling components both downstream and upstream of the hypoxia-induced mTORC1 inhibitory pathway. These methodologies will serve as valuable tools for researchers seeking to understand mTORC1 dysregulation in the context of hypoxic stress.

Feedback control of p53 translation by REDD1 and mTORC1 limits the p53-dependent DNA damage response.

Exquisite control of the level and activity of p53 are required in order to preserve cellular homeostasis following DNA damage. How this regulation is integrated with other key metabolic pathways in vivo is poorly understood. Here, we describe an endogenous feedback circuit for regulation of p53 through its transcriptional target gene, Redd1, a stress-induced inhibitor of TOR complex 1 (TORC1) activity. Cells and tissues of Redd1(-/-) mice exhibit enhanced sensitivity to ionizing radiation and chemotherapy treatment, which we demonstrate is attributable to abnormally increased p53 protein level and activity in the absence of Redd1. We find that deregulation of p53 in this setting is not due to failed DNA repair or to increased p53 stabilization but, instead, to increased p53 translation. We show that Redd1 loss leads to elevated mammalian TORC1 (mTORC1) activity, which explains the increased p53 translation and protein levels. Together, these findings suggest that REDD1-mediated suppression of mTORC1 activity exerts feedback control on p53, thereby limiting the apoptotic response and contributing to cellular survival following DNA damage. This work therefore defines a role for REDD1 in the control of p53 in vivo, with potential therapeutic implications for cancer and for the variety of genetic diseases involving TOR pathway signaling components.

A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma.

The p53 tumor suppressor, a central mediator of chemosensitivity in normal cells, is functionally inactivated in many human cancers. Therefore, a central challenge in human cancer therapy is the identification of pathways that control tumor cell survival and chemosensitivity in the absence of functional p53. The p53-related transcription factors p63 and p73 exhibit distinct functions­p73 mediates chemosensitivity while p63 promotes proliferation and cell survival­and are both overexpressed in squamous cell carcinomas (SCCs). However, how p63 and p73 interact functionally and govern the balance between prosurvival and proapoptotic programs in SCC remains elusive. Here, we identify a microRNA-dependent mechanism of p63/p73 crosstalk that regulates p53-independent survival of both human and murine SCC. We first discovered that a subset of p63-regulated microRNAs target p73 for inhibition. One of these, miR-193a-5p, expression of which was repressed by p63, was activated by proapoptotic p73 isoforms in both normal cells and tumor cells in vivo. Chemotherapy caused p63/p73-dependent induction of this microRNA, thereby limiting chemosensitivity due to microRNA-mediated feedback inhibition of p73. Importantly, inhibiting miR-193a interrupted this feedback and thereby suppressed tumor cell viability and induced dramatic chemosensitivity both in vitro and in vivo. Thus, we have identified a direct, microRNA-dependent regulatory circuit mediating inducible chemoresistance, whose inhibition may provide a new therapeutic opportunity in p53-deficient tumors.

The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha.

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

Negative feedback control of HIF-1 through REDD1-regulated ROS suppresses tumorigenesis.

The HIF family of hypoxia-inducible transcription factors are key mediators of the physiologic response to hypoxia, whose dysregulation promotes tumorigenesis. One important HIF-1 effector is the REDD1 protein, which is induced by HIF-1 and which functions as an essential regulator of TOR complex 1 (TORC1) activity in Drosophila and mammalian cells. Here we demonstrate a negative feedback loop for regulation of HIF-1 by REDD1, which plays a key role in tumor suppression. Genetic loss of REDD1 dramatically increases HIF-1 levels and HIF-regulated target gene expression in vitro and confers tumorigenicity in vivo. Increased HIF-1 in REDD1(-/-) cells induces a shift to glycolytic metabolism and provides a growth advantage under hypoxic conditions, and HIF-1 knockdown abrogates this advantage and suppresses tumorigenesis. Surprisingly, however, HIF-1 up-regulation in REDD1(-/-) cells is largely independent of mTORC1 activity. Instead, loss of REDD1 induces HIF-1 stabilization and tumorigenesis through a reactive oxygen species (ROS) -dependent mechanism. REDD1(-/-) cells demonstrate a substantial elevation of mitochondrial ROS, and antioxidant treatment is sufficient to normalize HIF-1 levels and inhibit REDD1-dependent tumor formation. REDD1 likely functions as a direct regulator of mitochondrial metabolism, as endogenous REDD1 localizes to the mitochondria, and this localization is required for REDD1 to reduce ROS production. Finally, human primary breast cancers that have silenced REDD1 exhibit evidence of HIF activation. Together, these findings uncover a specific genetic mechanism for HIF induction through loss of REDD1. Furthermore, they define REDD1 as a key metabolic regulator that suppresses tumorigenesis through distinct effects on mTORC1 activity and mitochondrial function.

Identification of in vivo phosphorylation sites and their functional significance in the sodium iodide symporter.

The Na+/I- symporter (NIS)-mediated iodide uptake activity is the basis for targeted radioiodide ablation of thyroid cancers. Although it has been shown that NIS protein is phosphorylated, neither the in vivo phosphorylation sites nor their functional significance has been reported. In this study, Ser-43, Thr-49, Ser-227, Thr-577, and Ser-581 were identified as in vivo NIS phosphorylation sites by mass spectrometry. Kinetic analysis of NIS mutants of the corresponding phosphorylated amino acid residue indicated that the velocity of iodide transport of NIS is modulated by the phosphorylation status of Ser-43 and Ser-581. We also found that the phosphorylation status of Thr-577 may be important for NIS protein stability and that the phosphorylation status of Ser-227 is functionally silent. Thr-49 appears to be critical for proper local structure/conformation of NIS because mutation of Thr-49 to alanine, aspartic acid, or serine results in reduced NIS activity without alterations in total or cell surface NIS protein levels. Taken together, we showed that NIS protein levels and functional activity could be modulated by phosphorylation through distinct mechanisms.

MEK signaling modulates sodium iodide symporter at multiple levels and in a paradoxical manner.

The Na(+)/I(-) symporter (NIS)-mediated iodide uptake is the basis for targeted radioiodine ablation of thyroid cancers. However, NIS-mediated radioiodide uptake (RAIU) activity is often reduced in thyroid cancers. As mitogen activated protein kinase (MAPK) signaling pathway is activated in about 70% of papillary thyroid carcinoma, we investigated whether MEK (MAPK kinase) inhibition will restore NIS protein levels and NIS-mediated RAIU activity in RET/PTC oncogene-transformed thyroid cells. We found that MEK inhibitor PD98059 increased NIS protein levels within 30 min of treatment. However, the increase of NIS protein level was not accompanied with an increase in NIS-mediated RAIU activity, particularly at early time points of PD98059 treatment. PD98059 also decreased RAIU activity mediated by exogenous NIS in non-thyroid cells. The transient decrease of RAIU activity by PD98059 in thyroid cells was not due to decreased NIS cell surface level, decreased NIS binding affinity for I(-) , or increased iodide efflux. While PD98059 moderately decreased Na(+)/K(+)-ATPase activity, ouabain titration indicates that the extent of decrease in Na(+)/K(+)-ATPase activity is much greater than the extent of decrease in RAIU activity. Additionally, a decrease of Na(+)/K(+)-ATPase activity was not accompanied with a decrease of biotin uptake activity mediated by Na(+)-dependent multivitamin transporter. Since PD98059 reduced V(max)- I(-) without decreasing NIS cell surface levels, it is most likely that PD98059 decreases the turnover rate of iodide transport with an yet to be identified mechanism.

Correlation of Na+/I- symporter expression and activity: implications of Na+/I- symporter as an imaging reporter gene.

UNLABELLED: The Na(+)/I(-) symporter (NIS) has been proposed as an imaging reporter gene to ascertain the expression of therapeutic genes in targeted tissues. In this study, we investigated whether posttranslational processing and cell-surface trafficking of NIS affect NIS-mediated radioiodide uptake in cells expressing exogenous NIS. METHODS: We established FTC133, HeLa, and PC12 cell lines with doxycycline-inducible NIS expression to investigate the correlation among total NIS protein levels, cell-surface NIS protein levels, and NIS-mediated radioiodide uptake in cells induced with various levels of NIS. RESULTS: We found that most exogenous NIS proteins were efficiently trafficked to the cell surface; thus, a possible deficiency in NIS cell-surface trafficking is not a concern for clinical applications of NIS gene transfer. The extent of radioiodide uptake correlated with cell-surface NIS protein level within a certain range, suggesting that the imaging signals can quantify levels of NIS expression only within a certain range in vivo. Finally, a moderate increase in NIS protein level significantly increased radioiodide uptake, indicating that a low level of NIS expression is sufficient to facilitate radionuclide imaging in vivo. CONCLUSION: Our study suggests that NIS will be useful as an imaging reporter gene to ascertain that the therapeutic gene is localized to the correct tissue and to monitor the expression levels and duration of the therapeutic gene.

Expression of sodium iodide symporter in the lacrimal drainage system: implication for the mechanism underlying nasolacrimal duct obstruction in I(131)-treated patients.

PURPOSE: Nasolacrimal outflow obstruction has been associated with high-dose (>150 mCi) radioactive iodine (I(131)) treatment. Commonly used for thyroid cancer treatment, I(131) is effectively transported in the targeted tissue by the Na(+)/I symporter (NIS). We hypothesized that NIS is expressed in the lacrimal sac and nasolacrimal duct and that active accumulation of I(131) is responsible for the clinical observations seen in these patients. METHODS: Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses were used to evaluate NIS expression in both archived and fresh human tissues RESULTS: Reverse transcriptase-polymerase chain reaction analysis showed that NIS mRNA is present in the lacrimal sac. Immunohistochemical analysis indicated that NIS protein is expressed in the stratified columnar epithelial cells of the lacrimal sac and nasolacrimal duct. NIS protein was undetectable in the lacrimal gland, Wolfring and Krause glands, conjunctiva, canaliculus, and nasal mucosa. NIS-expressing columnar epithelial cells were absent and fibrosis was evident in the lacrimal sacs from I(131)-treated patients undergoing dacryocystorhinostomy. CONCLUSIONS: NIS is present in the lacrimal sac and nasolacrimal duct of humans, correlating to the anatomic areas of clinical obstruction that develop in patients treated with greater than 150 mCi of I(131). This suggests that NIS may be the vector of radiation-induced injury to the lacrimal system. To our knowledge, this is the first report of any ion transporter in the nasolacrimal outflow system and raises new questions as to the role the lacrimal sac plays in the modification of tears and in lacrimal outflow pathology.

Variable expression of coxsackie-adenovirus receptor in thyroid tumors: implications for adenoviral gene therapy.

Adenoviral gene therapy represents a novel approach for the treatment of aggressive thyroid carcinomas. Both coxsackie-adenovirus receptor (CAR) and integrins have been shown to be the major determinants for adenoviral infectivity in many types of cancer cells, yet conflicting results have been reported. In this report we examine these factors mediating adenoviral infection in thyroid cells and to evaluate CAR expression in various types of thyroid cancer. We found that neither expression levels of CAR nor integrins are solely predictive of adenoviral infectivity in thyroid cells. However, the absence of CAR was associated with poor adenoviral infectivity in immortalized rat FRTL-5 cells. Moreover, preincubation with alpha-CAR antibody decreased infectivity in FTC 238 cells, a human thyroid tumor line. These results indicate that CAR does play a role in adenoviral infection of thyroid cells. Immunohistochemical analysis revealed that CAR is expressed at the cell surface in the majority of malignant thyroid tumors. We further show that adenoviral infectivity in some thyroid cancer cells can be improved by poly-L-lysine. Our study warrants a functional method to evaluate adenoviral infectivity should be developed and instituted prior to clinical trials of adenoviral gene therapy in patients with advanced thyroid cancer.

Inhibition of heat shock protein 90, a novel RET/PTC1-associated protein, increases radioiodide accumulation in thyroid cells.

RET/PTC1 is a rearranged form of the RET tyrosine kinase commonly seen in papillary thyroid carcinomas. It has been shown that RET/PTC1 decreases expression of the sodium/iodide symporter (NIS), the molecule that mediates radioiodide therapy for thyroid cancer. Using proteomic analysis, we identify hsp90 and its co-chaperone p50cdc37 as novel proteins associated with RET/PTC1. Inhibition of hsp90 function with 17-allylamino-17-demothoxygeldanamycin (17-AAG) reduces RET/PTC1 protein levels. Furthermore, 17-AAG increases radioiodide accumulation in thyroid cells, mediated in part through a protein kinase A-independent mechanism. We show that 17-AAG does not increase the total amount of NIS protein or cell surface NIS localization. Instead, 17-AAG increases radioiodide accumulation by decreasing iodide efflux. Finally, the ability of 17-AAG to increase radioiodide accumulation is not restricted to thyroid cells expressing RET/PTC1. These findings suggest that 17-AAG may be useful as a chemotherapeutic agent, not only to inhibit proliferation but also to increase the efficacy of radioiodide therapy in patients with thyroid cancer.