Thakker, Rajesh V.

Abstract O.A. Shariq: None. K.E. Lines: None. K.A. English: None. I. Rioja: Employee; Self; GlaxoSmithKline. A. Selberherr: None. H. Ferry: None. A.L. Oberg: None. R. Prinjha: Employee; Self; GlaxoSmithKline. D. Richards: None. R.V. Thakker: None. Background: Effective treatments for metastatic pancreatic neuroendocrine tumors (PNETs) are scarce. We previously showed that inhibiting bromodomain and extra-terminal (BET) epigenetic reader proteins via non-selective targeting of their two bromodomains (BD1 and BD2) reduces proliferation in preclinical PNET models. However, the clinical relevance of BET proteins in PNETs and functional differences between BD1/BD2 are unclear. Here, we characterised BET expression in human PNETs and performed efficacy and mechanistic studies using next-generation BD1- (BD1i) and BD2-selective (BD2i) BET inhibitors. Methods: BET expression was evaluated by immunohistochemistry in functioning and non-functioning human PNETs (n=52) vs normal adjacent islets (n=18). RNA sequencing (RNA-seq) and gene set enrichment analyses (GSEA) were performed in an independent PNET cohort (n=46) to identify biological pathways upregulated in metastatic vs non-metastatic tumors. Effects of BD1i and BD2i on viability, apoptosis, and cell cycle progression were assessed in three PNET cell lines (BON1, QGP1, MIN6). Early (6h) and late (48h) transcriptional responses to BET inhibition were examined in vitro using nascent RNA-seq (SLAM-seq) and standard RNA-seq, respectively. Epigenomic responses were explored using chromatin immunoprecipitation sequencing (ChIP-seq) for the histone mark H3K27ac. Effects of BD1i and BD2i on viability were evaluated in ex vivo neoplastic pancreatic islets derived from Men1L/L/RIP2-Cre mice. Results: BET proteins (BRD2, BRD3 and BRD4) were overexpressed by 1.5-2.5-fold in human PNETs vs islets (p<0.01). GSEA showed enrichment of BET-regulated gene sets in metastatic tumors (normalized enrichment score 1.31, adjusted p=0.04). BD2i marginally affected cell viability (IC50 55-90 µM), apoptosis, and cell cycle profile, while BD1i had a moderate impact on all phenotypes (IC50 1-1.5 µM). Synergistic effects were observed when BD1i+BD2i were combined (IC50 0.2-0.5 µM). RNA-seq and SLAM-seq revealed transcriptional changes that were minimal with BD2i (n=73 and n=10 differentially expressed genes, respectively), modest with BD1i (n=354 and n=60 genes), and greatest with BD1i+BD2i combined (n=1739 and n=375 genes). ChIP-seq showed a reduction in H3K27ac enrichment at promoters and super-enhancers of genes involved in BET protein-regulated pathways (e.g. cell cycle progression), with the greatest decrease observed with BD1i+BD2i combined. In ex vivo cultured neoplastic islets, viability was reduced by <30% with BD2i (p<0.005), ∼40% with BD1i (p<0.0001), and ∼60% with BD1i+BD2i (p<0.0001). Conclusions: Our studies confirm BET proteins as clinically-relevant targets in human PNETs and show that dual (BD1+BD2) inhibition has synergistic efficacy. These findings have important implications for the clinical application of next-generation epigenetic modulators. Sunday, June 2, 2024

AbstractGastroenteropancreatic neuroendocrine tumours (GEP‐NETs), which may be hormone secreting (e.g., gastrinomas and insulinomas) or non‐secreting (also known as non‐functioning NETs) are associated with severe morbidity and have a median overall survival of 75–124 months. Studies have highlighted the importance of epigenetic mechanisms in GEP‐NETs pathogenesis, with the most frequently mutated genes being the epigenetic regulators, MEN1, DAXX, and ATRX. However, the consequences of these aberrant epigenetic mechanisms are poorly understood. The calcium sensing receptor (CASR), a G protein coupled‐receptor, is epigenetically silenced in cancers, and therefore we examined its role in GEP‐NET subtypes. Using RNA‐Scope and quantitative PCR analyses in two independent tumour cohorts from Europe (n = 18 patients) and the USA (n = 46 patients) we showed that CASR mRNA is almost completely absent in gastrinomas, insulinomas and non‐functioning pancreatic NETs. Furthermore, immunohistochemical staining confirmed a significant reduction in CaSR protein expression in all GEP‐NET subtypes, compared to normal islets. DNA methylationEPIC and ATAC‐seq analyses in the pancreatic NET cell line QGP‐1 showed the CaSR promoter was both hypermethylated and in a region of closed chromatin. Furthermore, transfection of wild type CaSR into QGP‐1 cells decreased cell viability, in keeping with the CaSR having a role in cellular proliferation. In summary, our study reveals that CaSR expression is decreased in GEP‐NETs and that this reduced expression is likely due to DNA methylation and chromatin changes. Moreover, we demonstrate that transfection of the CaSR into a PNET cell line reduces cell viability, thereby indicating that the CaSR acts as a tumour suppressor in this tumour type.

ABSTRACT The last international guidelines on the evaluation and management of primary hyperparathyroidism (PHPT) were published in 2014. Research since that time has led to new insights into epidemiology, pathophysiology, diagnosis, measurements, genetics, outcomes, presentations, new imaging modalities, target and other organ systems, pregnancy, evaluation, and management. Advances in all these areas are demonstrated by the reference list in which the majority of listings were published after the last set of guidelines. It was thus, timely to convene an international group of over 50 experts to review these advances in our knowledge. Four Task Forces considered: 1. Epidemiology, Pathophysiology, and Genetics; 2. Classical and Nonclassical Features; 3. Surgical Aspects; and 4. Management. For Task Force 4 on the Management of PHPT, Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) methodology addressed surgical management of asymptomatic PHPT and non-surgical medical management of PHPT. The findings of this systematic review that applied GRADE methods to randomized trials are published as part of this series. Task Force 4 also reviewed a much larger body of new knowledge from observations studies that did not specifically fit the criteria of GRADE methodology. The full reports of these 4 Task Forces immediately follow this summary statement. Distilling the essence of all deliberations of all Task Force reports and Methodological reviews, we offer, in this summary statement, evidence-based recommendations and guidelines for the evaluation and management of PHPT. Different from the conclusions of the last workshop, these deliberations have led to revisions of renal guidelines and more evidence for the other recommendations. The accompanying papers present an in-depth discussion of topics summarized in this report. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

ABSTRACT In this narrative review, we present data gathered over four decades (1980–2020) on the epidemiology, pathophysiology and genetics of primary hyperparathyroidism (PHPT). PHPT is typically a disease of postmenopausal women, but its prevalence and incidence vary globally and depend on a number of factors, the most important being the availability to measure serum calcium and parathyroid hormone levels for screening. In the Western world, the change in presentation to asymptomatic PHPT is likely to occur, over time also, in Eastern regions. The selection of the population to be screened will, of course, affect the epidemiological data (ie, general practice as opposed to tertiary center). Parathyroid hormone has a pivotal role in regulating calcium homeostasis; small changes in extracellular Ca++ concentrations are detected by parathyroid cells, which express calcium-sensing receptors (CaSRs). Clonally dysregulated overgrowth of one or more parathyroid glands together with reduced expression of CaSRs is the most important pathophysiologic basis of PHPT. The spectrum of skeletal disease reflects different degrees of dysregulated bone remodeling. Intestinal calcium hyperabsorption together with increased bone resorption lead to increased filtered load of calcium that, in addition to other metabolic factors, predispose to the appearance of calcium-containing kidney stones. A genetic basis of PHPT can be identified in about 10% of all cases. These may occur as a part of multiple endocrine neoplasia syndromes (MEN1–MEN4), or the hyperparathyroidism jaw-tumor syndrome, or it may be caused by nonsyndromic isolated endocrinopathy, such as familial isolated PHPT and neonatal severe hyperparathyroidism. DNA testing may have value in: confirming the clinical diagnosis in a proband; eg, by distinguishing PHPT from familial hypocalciuric hypercalcemia (FHH). Mutation-specific carrier testing can be performed on a proband's relatives and identify where the proband is a mutation carrier, ruling out phenocopies that may confound the diagnosis; and potentially prevention via prenatal/preimplantation diagnosis. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). Abstract The prevalence and incidence of PHPT vary around the world and depend on a number of factors, the most important being availability of serum calcium (and parathyroid hormone) measurement for screening, which in the Western world has shifted its presentation to an asymptomatic disorder. Clonally dysregulated overgrowth of one or more parathyroid glands together with reduced expression of CaSRs is the most important pathophysiologic basis of PHPT, which may also occur as a part of inherited syndromes or as a nonsyndromic isolated endocrinopathy in 10% of cases. Genetic testing has roles in establishing the clinical diagnosis, although its value requires assessments using GRADE(176) methodology, which is recommended for future consideration.

Purpose: To provide practice recommendations for the diagnosis and management of hypoparathyroidism in adults. Methods: Key questions pertaining to the diagnosis and management of hypoparathyroidism were addressed following a literature review. We searched PubMed, MEDLINE, EMBASE and Cochrane databases from January 2000 to March 2018 using keywords ‘hypoparathyroidism, diagnosis, treatment, calcium, PTH, calcidiol, calcitriol, hydrochlorothiazide and pregnancy’. Only English language papers involving humans were included. We excluded letters, reviews and editorials. The quality of evidence was evaluated based on the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. These standards of care for hypoparathyroidism have been endorsed by the Canadian Society of Endocrinology and Metabolism. Results: Hypoparathyroidism is a rare disease characterized by hypocalcemia, hyperphosphatemia and a low or inappropriately normal serum parathyroid hormone level (PTH). The majority of cases are post-surgical (75%) with nonsurgical causes accounting for the remaining 25% of cases. A careful review is required to determine the etiology of the hypoparathyroidism in individuals with nonsurgical disease. Hypoparathyroidism is associated with significant morbidity and poor quality of life. Treatment requires close monitoring as well as patient education. Conventional therapy with calcium supplements and active vitamin D analogs is effective in improving serum calcium as well as in controlling the symptoms of hypocalcemia. PTH replacement is of value in lowering the doses of calcium and active vitamin D analogs required and may be of value in lowering long-term complications of hypoparathyroidism. This manuscript addresses acute and chronic management of hypoparathyroidism in adults. Main conclusions: Hypoparathyroidism requires careful evaluation and pharmacologic intervention in order to improve serum calcium and control the symptoms of hypocalcemia. Frequent laboratory monitoring of the biochemical profile and patient education is essential to achieving optimal control of serum calcium.

Abstract Objective: Hypoparathyroidism is a rare disorder characterized by hypocalcemia and absent or deficient PTH. This report presents a summary of current information about epidemiology, presentation, diagnosis, clinical features, and management and proposes guidelines to help clinicians diagnose, evaluate, and manage this disorder. Participants: Participants in the First International Conference on the Management of Hypoparathyroidism represented a worldwide constituency with acknowledged interest and expertise in key basic, translational, and clinical aspects of hypoparathyroidism. Three Workshop Panels were constituted to address questions for presentation and discussion at the Conference held in Florence, Italy, May 7–9, 2015. At that time, a series of presentations were made, followed by in-depth discussions in an open forum. Each Workshop Panel also met in closed sessions to formulate the three evidence-based reports that accompany this summary statement. An Expert Panel then considered this information, developed summaries, guidelines, and a research agenda that constitutes this summary statement. Evidence: Preceding the conference, each Workshop Panel conducted an extensive literature search as noted in the individual manuscripts accompanying this report. All presentations were based upon the best peer-reviewed information taking into account the historical and current literature. Consensus Process: This report represents the Expert Panel's synthesis of the conference material placed in a context designed to be relevant to clinicians and those engaged in cutting-edge studies of hypoparathyroidism. Conclusions: This document not only provides a summary of our current knowledge but also places recent advances in its management into a context that should enhance future advances in our understanding of hypoparathyroidism.