Year in Thyroidology: Basic Science

Atlas of TH responsive genes in tissues

As TH has broad influence on gene expression in many cell types, knowing its target genes is important for understanding the role of TH in developmental and physiology. ¹ Having a compilation of gene expression in multiple tissues would be an important tool, and previous data bases were small in scope and information. ² The atlas reported by Zekri et al ¹ is a compilation of published and unpublished mouse RNA-Seq (8) and ChIP-Seq (7) raw data from brain, heart, skeletal muscle, brown and white adipose tissues, and liver into a single database.

The authors reported that the number of differently expressed genes varied extensively between tissues/cell types and that TH responsive genes were not shared by all tissues. Only three upregulated and no downregulated genes were common to all data sets. Furthermore, in most tissues, an equivalent number of genes was up- or downregulated after triiodothyronine (T3) treatment. While 45% of upregulated genes possessed a TH receptor binding site within 30 kb of the translation start site, this was the case for <10% of downregulated genes. This atlas represents a valuable information source on specific genes pertaining to T3 regulation or TH receptor binding.

TH receptor beta phosphorylation

TH facilitates metabolic responses to environmental cues such as nutrient deficiency, as an adaptive mechanism. ^3,4 Minakhina et al ³ reported on how phosphorylation of the TH receptor beta 2 isoform at serine position 101 is an important mechanism for downregulating the HPT axis after T3 binding. This site is not found on other TH receptor isoforms. Fasting also promotes phosphorylation at this site, utilizing the same pathway as T3 to suppress the HPT axis. This physiologically relevant TH receptor phosphorylation function represents the convergence of nutritional and TH signaling pathways, using a common mechanism for acute suppression of the HPT axis.

Development of synthetic compounds to treat severe resistance to TH alpha

Resistance to TH (RTH) alpha is caused by heterozygous mutations in THRA gene, which were first reported in 2012. ^5,6 The very mild abnormalities in the thyroid function tests in this syndrome explain why RTH alpha has remained elusive. The phenotype of RTH alpha comprises hypothyroid features that reflect the expression of THRA in skeleton, brain, and gut, with growth delay, skeletal dysplasia, neurocognitive impairment, and constipation. ⁷ Of the known 19 different THRA defects, 8 manifest a severe phenotype and are caused by frameshift or early termination mutations that disrupt the C-terminus of the TR alpha 1 protein.

In vitro studies find these mutants to be transcriptionally inactive with impaired corepressors (CoRs) dissociation and result in a severe dominant negative effect on the normal TR alpha 1 isoform. Romartinez-Alonso et al ⁵ reported that these TR alpha mutants can bind T3, and, therefore, they hypothesized that some TH analogues could be more efficient in binding the mutant TR alpha 1 protein and dissociating the CoRs. They identified a compound, ES08, that when given to a zebrafish model of RTH alpha was able to rescue the typical developmental anomalies. It also resulted in higher TH gene expression when added to the culture medium of skin fibroblasts from a patient with a deleterious THRA mutation. This compound gives great hope for the treatment of patients with severe RTH alpha phenotype.

The protective role of deiodinase 3 against premature exposure to T3

Deiodinases are selenoenzymes that metabolize TH. They play an essential role in development by controlling the timing and intensity of TH signaling through their tightly regulated expression. ^4,8,9 Deiodinase 3 (D3) is the main TH inactivating enzyme and its deficiency results in increased TH availability. Furthermore, the coordinated expression of Dio2 and Dio3 is required during embryogenesis. Fonseca et al ⁸ demonstrated that inactivation of Dio3 in mouse brown adipose tissue (BAT) causes premature expression of the thermogenic program genes. This earlier exposure to T3 signaling led to an adult BAT that had a modified transcriptome. The observed long-term consequences illustrate the protective role of D3 in embryonic BAT development.

Similarly, Dio3^{− /−} mice were used by Martinez et al ⁹ as a model of persistent developmental thyrotoxicosis to study the effect of maternal hyperthyroidism on the development of congenital abnormalities. ⁹ This group previously reported that neonatal mice lacking D3 exhibited elevated levels of T3, which lead to an array of severe growth, endocrine and neurological abnormalities later in life, and thereby underscore the importance of timely TH action during development. ¹⁰ Mice lacking Dio3 had reduced neonatal viability with severe growth retardation. Surviving mice had brain and cranial dysmorphisms and heart defects. The observation that transient thyrotoxicosis during fetal development manifests with congenital abnormalities implies that TH may be involved in their etiology and deserves further investigations.

Animal models of MCT8 deficiency used for preclinical gene therapy in Allan–Herndon–Dudley Syndrome

Inactivating mutations in the X-linked TH transmembrane transporter MCT8 causes Allan–Herndon–Dudley syndrome (AHDS), which manifests as psychomotor disability in affected males. ^{11 –14} The characteristic thyroid phenotype was previously replicated in two Mct8 knockout mouse models. The double knockout (dKO) mct8^−/y;oatp1c1^−/− mice manifested disease-relevant phenotypes, including an impaired TH transport into the CNS, and were used for gene therapy. Liao et al ¹¹ demonstrated that systemic IV delivery of normal MCT8 using the adeno associated virus 9 (AAV9) at a young postnatal age, P30, resulted in improved locomotor and cognitive function in adulthood.

Furthermore, there was a near normalization of T3 content and improvement in expression of positively regulated TH genes in several brain regions. Similarly, Sundaram et al ¹² used the AAV-BR1-Mct8 to deliver normal Mct8 to brain endothelial cells. This increased T3 levels in the brain that resulted in long-lasting improvement in motor coordination. These preclinical studies suggest that gene therapy may be a promising approach for the future treatment of AHDS.

Insights into thyrotropin receptor Abs

Increased and decreased signaling through the thyrotropin receptor (TSHR) results in autoimmune hypo- and hyperthyroidism, respectively. ^{15 –17} However, the mechanism of antibody-mediated TSHR activation and inactivation remains poorly understood. Two research groups undertook the modeling of the physiological and pathological activation of the TSHR using cryogenic electron microscopy structures of full length active and inactive TSHR.

Faust et al ¹⁵ showed that stimulating antibodies and TSH pushed the extracellular domain (ECD) of the TSHR into an upright active conformation, while blocking antibodies prevented the transition of ECD orientation into the upright conformation. There was a key structural role for TSH glycosylation at position Asn52 for TSHR activation. A conserved 10-residue fragment from the hinge C-terminal loop mediated ECD interactions with the TSHR transmembrane domains. Duan et al ¹⁶ demonstrated the mechanism through which TSH and agonist antibodies activate TSHR. This newly characterized mechanism for stimulation and inhibition of the TSHR by autoantibodies may be further explored for future drug discovery.

Subtypes of Graves' disease

Both Graves' hyperthyroidism and the associated orbitopathy involve TSH receptor (TSHR) stimulatory autoantibodies that bind to and activate TSHRs on thyrocytes and orbital fibroblasts. ^18,19 Krieger et al ¹⁸ demonstrated the selectivity of TSHR-stimulating Abs from patients with Graves hyperthyroidism in stimulating TG from thyrocytes compared with Abs from patients with Graves' orbitopathy in stimulating hyaluronic acid from orbital fibroblasts. These findings illustrated that although clinical assays for TSHR-activating Abs can measure stimulation of the cAMP-PKA pathway, specific intracellular readouts may differentiate between thyroidal and extrathyroidal pathogenesis in Graves' disease (GD).

Faustino et al ¹⁹ explored another differential aspect of GD, specifically the existence of several common haplotypes at the CD40 locus. CD40 is expressed on antigen presenting cells and functions as a costimulatory molecule, associated with autoimmune diseases. A previous study has shown clinical remission only in ∼50% of GD patients when treated with the anti-CD40 monoclonal Ab Iscalimab. ²⁰ In this study, the investigators demonstrated that genetic polymorphisms in the CD40 gene drive its expression levels with a specific haplotype being associated with lower CD40 mRNA levels and no clinical response to Iscalimab. These results may be used in the future to inform implementation of precision medicine in the treatment of GD.

Distinct thyroiditis with immune checkpoint inhibitors

Immune checkpoint inhibitors (ICIs) such as the monoclonal antibodies blocking cytotoxic T lymphocyte antigen-4 (CTLA-4) or programmed death receptor-1 (PD-1) are commonly used in cancer treatment. ²¹ Thyroid dysfunction secondary to ICIs is the most common immune-related adverse event. ²² Ippolito et al ²¹ developed a mouse model of ICI-related thyroiditis, NOD-H2h4 mice treated with anti-PD-1 and/or anti-CTLA-4 checkpoint inhibitors. When assessing the respective clinical, hormonal, and cytokine profiles, the authors observed that thyroiditis was more severe when blocking PD-1, but it was more prevalent and more extensive when blocking CTLA-4.

Serum IL-6 was markedly increased with PD-1 blockade and it mirrored thyroiditis severity. Serum IL-6 was not increased in anti-CTLA-4-induced thyroiditis. These distinctive histopathological, sonographic, hormonal, and immunological features could potentially inform in the clinical management of specific ICI-induced thyroiditis in the future. For example, theoretically early treatment with corticosteroid in PD-1-induced thyroiditis could block the pathogenetic mechanism, thereby reducing the risk of long-term thyroid dysfunction.

Mechanisms of resistance in BRAF-positive thyroid cancer

Many papillary thyroid cancers (PTCs) have the common genetic variation BRAF^V600E, which activates MAPK signaling. ^23,24 BRAF^V600E-positive thyroid cancers may become treatment resistant, and dedifferentiation has been observed in some tumors, ²⁵ possibly through BRAF inhibition. Lee et al ²³ analyze NGS data from advanced thyroid cancers, some recurrent or metastatic, including cases treated with BRAF inhibition, and in some of them, samples were obtained before, during, or after treatment.

Genetic alterations in several pathways were highly prevalent in more dedifferentiated thyroid cancers, such as the SWI/SNF chromatin remodeling complex, PI3K-AKT, mTOR, MAPK, and JAK–STAT pathways. The authors concluded that there are many potential mechanisms that can lead to thyroid cancer resistance and/or dedifferentiation. The identification of multiple pathways of resistance suggests that combination therapies may deserve further study.

Recruitment of myeloid-derived suppressor cells

Zhang et al ²⁶ examined the types of immune cells present in BRAF^V600E-positive advanced PTCs. These authors found an abundance of myeloid-derived suppressor cells (MDSCs), and their presence played a critical role in cancer progression. The transcriptional reactivation of T-box transcription factor 3 (TBX3) was demonstrated to be an essential molecular event in the initiation and progression of the cancer. The activation of the BRAF/MAPK pathway was thus linked to the MDSCs infiltration through the TBX3 activation and the ligands of the C-X-C motif chemokine receptor 2 (CXCR2).

The authors also evaluated the therapeutic effects of combination therapies using BRAF/MAPK and MDSCs inhibition. These adjuvant treatments in a preclinical mouse model resulted in recognition and elimination of PTC cells by the immune system. These results could inform future clinical trials on selective use of combination therapy.